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Synthetic nucleic acid delivery systems: present and perspectives Bogdan Draghici, and Marc A. Ilies J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500330k • Publication Date (Web): 06 Feb 2015 Downloaded from http://pubs.acs.org on February 9, 2015

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Journal of Medicinal Chemistry

Synthetic nucleic acid delivery systems - present and perspectives

Bogdan Draghici1 and Marc A. Ilies1,2*

1

Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research,

Temple University School of Pharmacy, 3307 North Broad Street, Philadelphia, PA-19140, USA. 2

Temple Materials Institute, 1803 N Broad Street, Philadelphia, PA 19122, USA

1 ACS Paragon Plus Environment


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Abstract Self-assembled synthetic gene delivery systems represent the “bottom up” approach to gene delivery and gene silencing, in which scientists are designing novel cationic and procationic amphiphiles that can pack, transport and deliver nucleic acids to various targets in the body in a controlled manner. These supramolecular assemblies are safer than viruses, but are lagging behind them in efficiency. We are presenting recent progresses that narrowed this gap through better understanding of the delivery barriers and incorporation of this knowledge in the design of novel synthetic amphiphiles, formulations and revolutionary screening and optimization processes. Structure-properties and structure-activity relationships were drawn within each amphiphile class, presenting the cellular and animal models used to generate them. We are also revealing pertinent in vitro/in vivo correlations that emphasize promising amphiphiles and successful formulation optimization efforts for efficient in vivo nucleic acid delivery, together with main organ targets and diseases treatable with these revolutionary technologies.

Keywords: amphiphile, self-assembly, nucleic acid, lipoplex, transfection, RNAi


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1. Introduction

Gene therapy is a revolutionary form of therapy that promises to correct/treat hereditary or acquired diseases by means of foreign genetic material introduced into affected cells, tissues or organs. The technology pioneers the use of DNA as therapeutic entity (drug) to alter the function of specific cells, tissues, or organs. Since the foreign genetic material is translated into proteins needed for homeostasis or suppresses the production of specific proteins, it can be considered that gene therapy represents a new form to modulate the protein production inside the targeted cells. Completion of the human genome charting, together with progress in understanding the role of various genes in different diseases including cancer confers tremendous clinical potential to this new therapeutic approach.1-4 Another very promising therapeutic strategy relaying on nucleic acid delivery is gene silencing via RNA interference. In this process foreign RNA molecules destroy specific mRNA molecules in the target cells/tissues/organs, effectively inhibiting the gene expression at that particular location.5-8 Despite the simple, straightforward, principle, implementation of the concept proved to be rather difficult. After decades since inception, the success of both technologies still relies on finding appropriate delivery vehicles (vectors) that would pack, transport and deliver the genetic material safely, efficiently and selectively to the target cells while displaying reduced cytotoxicity and immunogenicity, allowing repeated administrations and prolonged expression of the biological effect. However, this time seems rather insignificant if we consider the multiple protection mechanisms embedded into living organisms to ensure their genome integrity, which continuously evolved throughout time.3, 4



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The most efficient nucleic acid delivery systems are viruses, which evolved in parallel to their potential hosts. Their high transfer and expression (transfection) efficiency is due to controlled nucleic acid packing and unpacking in/from the capsid, as well as to the ability of the virus the overcome various extra-cellular and intracellular delivery barriers or defense mechanisms of the targeted cells.4, 9 They have the time advantage against man-made systems, with their supramolecular structures optimized for the delivery process, but they have the drawbacks of inducing strong immune responses that usually prevent repeated administration, mutagenicity and of a limited size of the nucleic acid that can be incorporated into the virion, especially significant for DNA delivery. Therefore, the top-down approach to gene delivery systems involves deletion of immunogenic features from the structure of known viruses in order to reduce immunogenicity while retaining key structural elements responsible for transfection efficiency.3, 4 An alternative bottom-up approach to gene delivery systems involves the use of synthetic systems - chemical entities that are able to mimic the main features of viral vectors, being able to compact and deliver nucleic acids in a similar manner. Since the artificial design is usually not recognized (immediately) by the immune system, these systems are much safer, can be administered repeatedly without significant immune response, can incorporate plasmids of practically unlimited size and, importantly, can be manufactured under GMP-compliant norms using platforms already existent in the pharmaceutical industry.10-12 However, their efficiency must be improved through better control towards packing/releasing of the nucleic acid cargo and by adapting the structure of the amphiphile/nucleic acid supramolecular assemblies to the delivery barriers encountered by the plasmid on its way to the target cell.13 The use of non-viral


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vectors and nucleic acid delivery methods in clinical trials increased constantly over time, mainly due to their superior safety.14

2. Delivery methods for nucleic acids Higher organisms evolved intricate ways to protect their genome integrity and temporal expression, which makes genetic reprogramming/silencing of cells and tissues a difficult task. Simple (naked) DNA or RNA is rapidly degraded in vivo by nucleases and has poor cellular uptake. However, nucleic acids are robust macromolecules and several physical delivery methods exploiting their physical stability were developed such as muscle and skin (micro)injection,15 electroporation,16

sonoporation,17 nucleic acid-coated gold particle

bombardment – the ‘gene gun’ technology,18 magnetofection19 and hydrodynamic injection.20 Accessibility issues limit their applications, despite recent development that increased their transfection efficiency to levels matching viral vectors.21, 22 On the other hand, both synthetic and biological delivery systems for nucleic acids modify their pharmacokinetics, simultaneously masking their negative charge and reducing their (relaxed) size. In the case of DNA, compaction is achieved via electrostatic association with cationic or procationic chemical entities such as positively charged ions like Ca2+ in calcium phosphate – mediated transfection, polyamines and positively charged proteins in sperm, selfassembled cationic amphiphiles in synthetic gene delivery systems and via specialized capsids in most of viruses.22, 23

3. Lipoplexes: formation, stability and delivery barriers



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Cationic amphiphiles of different packing parameters24 can self-assemble in aqueous media either alone or together with other lipids (co-lipids) such as cholesterol (Chol) or dioleoylphosphatidylethanolamine (DOPE) and can spontaneously associate with the DNA/RNA through electrostatic interactions forming cationic lipid-DNA/RNA complexes (lipoplexes).25 Lipoplex formation involves the nucleic acid-mediated fusion of liposomes with extensive tridimensional lipid rearrangement, driven by co-operative hydrophobic association between the hydrophobic tails of the amphiphiles and by counterion release from both lipids and DNA/RNA.6, 26 Amphiphilic cationic polymers follow a similar mechanism for compaction of genetic material, yielding polyplexes.25 The delivery system enhances the mechanical properties of the genetic cargo and protects it from the action of DNAses/RNAses, being able to maintain the structural integrity of the nucleic acids in the complex and dynamic environment encountered from the point of administration to the target cells, tissue or organ. Delivery systems can exploit existing cellular pathways or artificial ones and can provide targeting via ligands with affinity to specific target determinants. Importantly, they can optimize spatial-temporal delivery for optimal therapeutic effect.13, 27-29 The delivery barriers encountered by foreign DNA en-route to the nucleus of targeted cells are very diverse and depend highly on the administration route. Generally they can be classified into extracellular and intracellular delivery barriers. The first category refers to barriers that have to be passed before the target tissue is reached, and includes penetration through the skin and mucus, interaction with plasma proteins and figurative elements of the blood, hepatic/splenic/renal clearance, passage through the endothelium lining the blood vessels and through the extracellular matrix.27, 28, 30 The second category of delivery barriers addresses the ones that have to be surpassed within the targeted cells in order to elicit the desired biological 6 ACS Paragon Plus Environment

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effect, and include uptake by target cells with the passage through the plasma membrane, avoidance of endocytic recycling, endosomal escape, trafficking in the cytoplasm and nuclear import (for DNA).27, 28, 30-33 Consequently, the structure of self-assembled delivery systems has to be adapted to resist in different environments to be transited by the lipoplexes (which depends on the administration route) and to the specific tasks that have to be performed to generate the desired biological response. Obviously the nature of the genetic cargo that self-assembled systems have to deliver, especially the type (DNA, siRNA, oligonucleotides, etc.) and its specific size (since for example DNA is usually much bigger in size than siRNA) constitute other important issues to be taken into consideration when designing nucleic acid delivery systems. Last but not least, the development of efficient supramolecular systems for nucleic acid delivery strongly depends on the intended application and its specific organ/tissue target(s). The packing parameter of the individual amphiphiles forming the lipoplex has a great impact on their self-assembling and consequently on the lipoplex structure and dynamics. The molecular packing parameter is defined as vo/aelo, where vo is the amphiphile hydrophobic tail(s) volume, lo is the tail(s) length, and ae is the equilibrium area per molecule at the aggregate surface.24 One can distinguish a lamellar structure of lipoplexes (LαC) when the average packing parameter of lipid mixture is approximately 1.34 Increasing this packing parameter through the use of co-lipids like DOPE at elevated molar ratios was found to induce the formation of an inverted hexagonal structure for the corresponding lipoplexes (HIIC) that was shown to have superior DNA delivery properties as compared with the LαC one.35 Interestingly, a gyroid cubic structure with space group Ia3d (QIIG) was recently identified for siRNA complexes with cationic lipid-based amphiphile mixtures, besides the classical LαC and HIIC structures. This new phase



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was able to promote higher silencing efficiency than LαC phase due to its ability to mediate enhanced fusion between the lipoplexes and the endosomal membrane.36 In this perspective, we are reviewing the supramolecular systems formed through selfassembling of amphiphilic compounds of low and medium molecular weights that can compact and deliver nucleic acids. We emphasize how the structure and packing parameter of these amphiphiles/mixtures of amphiphiles is influencing the interaction with nucleic acid and the transfection

activity

of

the

corresponding

lipoplexes.

For

polyamine-,

dendrimers

(polydispersed)-, and polymer-mediated gene delivery systems or their conjugates with small amphiphilic moieties, we are referring the reader to excellent reviews covering these synthetic gene delivery systems.30, 37-41

4. Novel cationic self-assembling amphiphiles for nucleic acid delivery

4.1. Single-chain surfactants The highest charge/mass ratio of cationic amphiphiles can be achieved using positively charged surfactants, which can also display a high molecular curvature. This combination of properties makes them extremely efficient towards membrane poration, which can translate into a high toxicity associated with their use, hence the relatively small number of studies assessing their transfection properties. The use of surfactants for gene delivery was pioneered by Huang and collaborators, who assessed combinations of cetyltrimethylammonium bromide (CTAB 1c) and inferior congeners DTAB 1a and TTAB 1b. The surfactants alone were too toxic to be used as transfection agents; however, when coformulated with DOPE, their toxicity decreased significantly. In terms of chain length, CTAB 1c was found to be less toxic than 1b and 1a. 8 ACS Paragon Plus Environment

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Lipoplexes derived from CTAB 1c/DOPE at 1/4 molar ratio were able to successfully deliver PSV2 CAT plasmid DNA into L929 cells.

42

Even when the cationic charge is delocalized on a

heterocyclic pyridinium ring, the toxicity of the cationic amphiphiles remains elevated. Pyridinium surfactants 2 introduced by Ilies’s group were not able to compact DNA alone; they required colipids cholesterol or DOPE in order to fully compact and deliver DNA to NCI-H23 cell lines. The maximum transfection efficiency was observed for hexadecyl representative 2c coformulated with DOPE or cholesterol (1/1 molar ratio), at a cationic amphiphile/pGL3 DNA +/- charge ratio of 5. The transfection efficiency decreased in the order 2c > 2d > 2b > 2a when amphiphiles were formulated in same way.26 It was observed that increasing the hydrophilicity of the polar head decreases the cytotoxic effect, with amphiphile 3 being less cytotoxic than congener 2b, while retaining its transfection efficiency when coformulated with DOPE at 1/1 molar ratio and +/- charge ratio of 2.26, 43 In this context, it is relevant to note that Bhattacharya’s group synthesized surfactants bearing multiple cationic head groups and investigated their selfaggregation and the properties of the micellar supramolecular assemblies. Their results suggested that the micelles derived from multi-head surfactants are more hydrated as compared with single headed congeners and that the micropolarity of micelles increased with the increase in the number of head groups.44,

45

More recently, Bradley’s group revealed via DFT calculations that

multivalent cationic amphiphiles 4a-e can adopt a tripod-like structure ideally suited to form strong Coulombic interactions with two contiguous phosphate groups from the DNA backbone, thus recommending them as gene delivery agents. Various hydrophobic tails with lengths ranging from 4 to 22 carbon atoms, either saturated or unsaturated, were tested in conjunction with this polar head (4a-d). The cholesteryl moiety was also investigated (4e). Best transfection results were obtained for tetraeicosanamide derivative (4a, n = 22) when co-formulated with



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DOPE at 1/1.5 molar ratio and at an amphiphile/pLux DNA N/P ratio of 1.5 in CHO, COS7 and 16HBE14o- cell lines.46 In order to increase the safety profile, the same group also introduced the related biodegradable amphiphiles 5 and 6. Within series 5, the amphiphiles containing a bisarginine polar head attached to a tetraeicosan hydrophobic tail through a β-alanyl linker (5b, m = 2, n = 2) showed efficient transfection activity of pEGFP-Cl in HeLa, HEK293T and B16F10 cell lines when co-formulated with DOPE at 1/1 molar ratio and N/P charge ratio of 5.47 Interestingly, within series 6 the oleoyl derivative 6b co-formulated with DOPE at 1/1.5 molar ratio was the most efficient transfection agent for pEGFP-Cl in HeLa cells at N/P of 12. This formulation also gave good results in vivo, when administered via intratracheal instillation in a pulmonary murine model using pLux DNA.48 Mention must be made that many of the above mentioned formulations equaled or surpassed the transfection power of LFM2000 while displaying low cytotoxicity, thus proving the possibility to generate efficient transfection systems using simple surfactants. These findings proved that molecular design and the extensive use of natural building blocks and biodegradable linkers can substantially reduce the cytotoxicity of this category of amphiphiles believed to be very toxic, thus opening new avenues for research in this field. This class of amphiphiles possessing multivalent polar heads can be considered minidendrons and compounds 4-6 are representative for the transition between simple surfactants and minidendrons (vide infra). Pungente at al. designed cationic amphiphiles based on a rigid C30-carotenoid hydrophobic structural motif linked to a variable ammonium headgroup (7a-d). The new amphiphiles were investigated for their ability to transfect GL2 siRNA in HR5-CL11 cells expressing Luciferase. Lipoplexes generated from lipids 7a-b having either a tertiary or a quaternary ammonium head group, coformulated with DOPE at 3/2 molar ratio, showed


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significant knockdown efficiency at cationic lipid/siRNA charge ratios +/- of 2.5 and 7.5 respectively. Lipid 7a was found to be significantly less toxic than 7b. Introduction of hydroxyethyl residue(s) in the polar head (lipids 7c, 7d and 7e) improved the transfection efficiency. Lipoplexes of 7c and 7e were the most efficient but slightly more cytotoxic than congeners 7a and 7b. The overall cytotoxicity of these formulations was better than toxicity of DC-Chol 28 and equal or lower than EDMPC 64a (vide infra).49

4.2. Gemini (dimeric) surfactants



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Another promising category of cationic amphiphiles is represented by dimeric surfactants, defined as two simple surfactants linked at the level of polar head or very close to the polar head. Also known as gemini surfactants (GSs), they were investigated for more than four decades.50-52 Their extensive study was initiated in early 90’s by Menger and Zana’s groups,53-55 revealing special self-assembling and physicochemical properties for this class of amphiphiles.56, 57

The use of GSs for gene delivery is well documented and was periodically reviewed.58-68 Thus,

Zana introduced the ammonium gemini surfactants of type 8 and studied their self-assembling properties in solution as a function of chain and spacer length.55 MacDonald and co-workers later found that gemini surfactants were able to transfect pCMV-β-gal DNA into BHK cells in vitro either alone or when co-formulated with DOPE at 1/2 molar ratio. The same authors found that the linker had a strong impact on the biological properties of GSs 8 (n = 17, m = 2, 3, 6), with a maximum transfection efficiency observed for m = 6. The congeners having unsaturated alkyl chains were found less efficient.69 Interestingly, if one C16 chain is replaced by a phytanyl moiety such as in compounds of type 9 (n = 11) the transfection can be doubled, as shown recently by Wettig and collaborators in OVCAR-3 cells using pVGtelRL plasmid.70 More recently Bhattacharya, Aicart and collaborators reinvestigated the influence of spacer length of GS of type 8 on the lipoplex formation using a combination of experimental methods including zeta potential, gel electrophoresis, small angle X-ray scattering (SAXS), cryo-TEM, confocal fluorescence microscopy in conjunction with transfection and viability/cytotoxicity experiments. Their results revealed that GS C16C2C16 (8, n = 15, m = 2) having an ethylene linker was the most effective in compacting pEGFP-C3 DNA when co-formulated with DOPE at 1/1 molar ratio in HEK293T, H460, CHO, HeLa and U343 cell lines.

The authors investigated the

transfection efficiency of the lipoplexes as a function of the effective charge ratio of the lipoplex


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(ρeff) which was shown by the same group to depend on the nature of the plasmid and consequently on the effective negative charge of the DNA molecule.71 The C16C2C16/DOPE formulations equaled or surpassed the transfection efficiency of LFM2000 at ρeff values of 2 and 4, while displaying low cytotoxic effects.72 The efficiency of GSs of type 8 bearing a short ethylene linker was recently confirmed by Jurado and collaborators who investigated the tetradecyl congener (8 m = 2, n = 13). Best transfection results were obtained when the cationic amphiphiles were co-formulated with both cholesterol and DOPE at equimolar ratio and an N/P charge ratio of 8/1, as revealed by transfection assays in TSA cells.73 The efficiency of short hydrophobic linkers in the design of GSs for gene delivery was also confirmed in the case of GS with pyridinium polar heads.74, 75 A detailed study on the linker impact was done more recently by Balaban, Ilies and collaborators,43 who showed that the transfection efficiency of GSs 10a-h bearing tetradecyl hydrophobic chains co-formulated with cholesterol at 1/1 molar ratio dramatically decreases when linker is elongated from 2 to 3 methylene units. Further elongation of the linker from 3 to 8 carbon atoms rescues the transfection efficiency of these formulations, with the GS bearing an octamethylene linker being similarly active to the ethylene congener. The plasmid used in this study was pGL3. Interestingly, Zanda and coworkers recently confirmed the efficiency of GSs having an n-hexyl linker in combination with lauryl/tetradecyl hydrophobic chains in the design of triazine GSs 11 with polar heads decorated with aminopropyl pendant moieties in transfecting A549, U87MG, Bristol 8 cells and DRG neurons using a plasmid DNA containing maxGFP reporter gene.76 Another conclusion of the study of Ilies et al. was that polar, non-cationic/procationic linkers in compounds 12 and 13 can substantially enhance the transfection efficiency of pyridinium GSs.43



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Foldvari and coworkers confirmed the above-mentioned trends in their study involving a series of novel ammonium GSs bearing procationic linkers. Best transfection efficiency in COS7 cells was obtained with GS 14 having an aza-heptan-1,7-diyl linker and two lauryl hydrophobic chains using pMASIA.Luc plasmid. The amino group in the linker is not charged significantly at physiological pH and its methylation yields GSs with much lower gene delivery efficiencies due to an increase of basicity of the linker which favors its protonation.68 However, attachment of a polar glycine moiety on the N atom yielded GS 15 with increased transfection efficiency, as shown by Badea and collaborators using pGT•IFN-GFP plasmid against COS7, PAM212, Sf1Ep cell lines.77 Replacement of glycine unit in GS 15 by a larger amino acids or dipeptides sequence on the N atom of the linker yielded GSs with lower transfection efficiencies. Moreover, the efficiency of this type of amphiphiles bearing polar linkers was subsequently confirmed by Bhattacharya and collaborators,78 who compared the transfection efficiencies of three types of GSs derived from thiocholesterol

having either hydrophobic

flexible 16a, hydrophobic rigid 16b, or hydrophilic flexible 16c linkers. The authors found that 16c, having an 3-oxapentane-1,5-di-yl spacer, was the most efficient compound when coformulated with DOPE at 1/5 molar ratio, as revealed by GFP and MFI in HeLa, HT1080, PC3AR, HaCaT cell line using pEGFP-c3 DNA. The new GS formulation was also able to completely condense plasmid DNA at a charge ratio of 1. Similar results were reported recently by our group for the pyridinium GSs 17 (n = 9 – 16), when co-formulated with DOPE at 1/2 molar ratio using gWiz-Luc ™ plasmid DNA.79 Within pyridinium GS 17 series the transfection efficiency increased monotonously with the elongation of chain length and was strongly correlated with the nature of the counter ion. Maximum transfection efficiency was displayed by the chloride counter ion (borderline chaotropic/ kosmotropic), which surpassed the chaotropic


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PF6- , Br- and the kosmotropic H2PO4-. 79 Our study confirmed the superiority of 3-oxapentane-1, 5-diyl-linker proposed by Bhattacharya and his collaborators in the design of GSs 18. Thus 18f (n = 1) was the most transfection efficient GS when co-formulated with DOPE at 1/4 molar ratio, while homologues 18g-i (n = 2, 3, 5) with longer oligooxyethylene spacers were found less efficient.80 Importantly, formulations based on GSs having oligoethylene spacers maintained their transfection efficiencies in the presence of elevated levels of serum – an important feature for translation to in vivo applications.78-81 Bajaj et al. investigated GSs having benzyl-ammonium backbone substituted at position 3 and 4 with C14 and C16 saturated oxyalkyl tails, linked via ethylene oxide spacer 19a-f. GSs 19a-f were able to compact pEGFP-c3 DNA at charge ratio of 1; however, in the transfection assays, the best results were observed at +/- charge ratio of 0.75 (90% compaction). Moreover, GSs 19a-f displayed good transfection activity in HeLa cells when formulated with DOPE at 1/6 molar ratio for GSs with short ethyleneoxide spacers (19a, 19d) and at 1/8 molar ratio for GSs with long ethylene oxide spacers (19b, 19c, 19e, 19f) respectively. Within 19a-c series, the transfection activity decreased with the increase of ethylene oxide spacer length, while within 19d-f series the transfection efficiency increased with the increase of oxyethylene spacer length. Amphiphile 19f was found to be the most effective within 19a-f series. In the presence of serum, GSs 19a-c were able to transfect 20-30% cells and their efficiency decreased with an increase in the serum concentration. Congener 19f displayed 50% cell transfection in the presence of 10% serum, although only at a charge ratio of 4.5. Within GS series 19 the cytotoxicity decreased in the order 19f > 19a = 19b = 19c > 19e > 19f, with a general cell viability higher than 70%.81 Recently, Maslov et. al introduced pH sensitive gemini cholesterol spermine conjugates 20a-c having the spermine cationic headgroup attached to cholesteryl fragments via



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biodegradable ester or carbamate and an oligomethylene spacer of variable lengths. These amphiphiles were tested for pEGFP-C2 DNA delivery against HEK293 and siRNA-EGFP delivery against BHK IR780 cell lines. The authors found that transfection efficiency is influenced by linker type and spacer length. The Gemini conjugate 20c, having six methylene units and a carbamate linker, was found to be the most efficient when coformulated with DOPE (1/1 molar ratio) and at a charge ratio N/ P ratio of 4/1 and 6/1 lipid to nucleic acid. Importantly, these formulations displayed good serum stability and higher transfection efficiency than their monomeric analogues (vide infra), also surpassing the efficiency of LFM2000. The same trends were confirmed for siRNA–EGFP delivery into EGFP-BHK and IR780 cells. The silencing performance of 20c displayed stronger inhibition of EGFP expression than LFM2000. Interestingly, the variation of the spacer length and linker type did not influence the cytotoxicity of the amphiphilic GSs. All representatives of type 20 appeared to be less toxic than their monomeric congeners, discussed in the lipid section (vide infra).82


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CH3 H3C

n

8a-d m = 2, 3, 5, 12 n = 13, 15, 17

CH3 CH3

Bu-O

O

N

3 13

CH3

H3C

O

11

NH3

3 Cl

n

3

O

N

N

N H

N H

H3C N

11 CH3

Chol

15

H3C

CH3

R

N CH3

R

11CH3

CH3 O

N H3C

S S

16a-c

N

n

n

CH3

CH3

17

2X CH3

X- = PF6-, Br-, Cl-, H2PO4n = 9 - 16

Chol:

6

H2N O

16c: R =

O Chol

n

CH3 N CH3

Where:

18f-i

2 Br

14

16b: R =

18a-i

R 18a-e

N NH2 H n

N CH3

2Br

O

16a: R =

N

CH3

n

m

O n

n = 1 - 3, 5

n

CH3

O O n

X O

N H2

4

4 Cl

20a-c

N H2

3

NH2

X O

n

Where:

19a-f

n = 3 -6, 12

O

O

CH3 N CH3

3

Chol

H3C N

CH3

3 CH3

N

CH3 N CH3

N H

11

Where: CH3

N H

6

CH3 H3C N

CH3

2Br

S S

N

11a-e: n = 7, 9,11,13,15

2 PF6 CH3

13

HN 8TFA

N

13 CH3

CH3 N CH3

N

N

4

O

12 H3C H3C N

H2N

CH3

H3C

CH3

13

CH3

CH3

13 CH3 CH3

NH

10a-h: n = 2 - 8

N

2 PF6

2 PF6

13

3

N

Chol

13

CH3

N

m

t

H3C

n

H3C

Chol

N

CH3

N

CH3

CH3 n

H3C

3

9a-c: m = 11, 15, 17

CH3 N

CH3

N

Bu-O

H3C

CH3 N CH3

t

n

2 Br CH3

CH3 N m CH3 2 Br

Bu-O

CH3 m N CH3

t

H3C H3C N

Chol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O n

CH3 19a-c: n = 13, m = 1-3 CH3 19d-f: n = 15, m = 1-3

n CH3

20a: X = C(O), n = 4 20b: X = C(O)NH, n = 4 20c: X = C(O)NH, n =6

The boundaries between cationic surfactants and gemini surfactants became less obvious after pioneering work of Behr and his collaborators.83,

84

Thus, Blessing at al. showed that

positively charged guanidinium surfactants bearing mercapto groups such as (C10CG+)2 21, displaying low binding cooperatively and fast exchange rates, are capable of collapsing pCMVluc plasmid (5.6 kb), with a linear phage λ (48kb) and phage T4 (166 kb) DNA into small lipoplexes of about 25 nm in diameter that contained one single DNA molecule. Further in situ oxidation of the mercapto groups into disulfide bonds stabilized the structure of the lipoplex and formed the corresponding GSs. Interestingly, the oxidation rate of the mercapto groups was enhanced by the presence of the DNA molecule, proving the cooperatively of the process and its dependence of supramolecular interactions. Importantly, the lipoplexes formed under these conditions (N/P ratio around 1) are negatively charged and could not transfect cells. The lipoplex 17 ACS Paragon Plus Environment


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formation process is reversible: the addition of dithiothreitol converts the disulfide GS into individual mercapto surfactant molecules with the simultaneous unwrapping of the pDNA.83, 84 Further developments from the same group using biodegradable cysteinyl surfactants with various chain lengths and cationic headgroups derived from either ornithine or spermine revealed that at higher N/P charge ratios the corresponding lipoplexes were transfection efficient. The ornithine polar head was superior to the spermine one. In the case of ornithine mercapto surfactants, the efficiency increased with the elongation of the chain length, with the C16 representative C16Con 22b being the most efficient in transfecting 3T3 murine fibroblast cells using pCMV-luc plasmid (5.5 kp).85 More recently, Zuber et al. used the C14 congener C14CO 22a in combination with DOPE (4:1 molar ratio) and cyclic pentapeptide RGDfK-lipid 23 conjugate to compact pCMV-EGFP-luc plasmid (6.4 kp) into virus-like particles of about 45 nm in diameter and to deliver it to HUVEC and KB cell lines. The presence of RGDfK moiety, targeting αvβ3 integrin receptors, was found to increase the transfection efficiency. Further increase in transfection power was achieved through the use of endosomolytic agent chloroquine.86


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4.3. Lipids From the sections presented above, it can be easily understood that DNA condensation in aqueous solution depends heavily on the ability of the amphiphilic compounds to self-assemble into robust supramolecular aggregates, with stability of the assembly depending on the magnitude of the hydrophobic effect induced by the individual amphiphilic molecules. Classical glycerophospholipids and cholesteryl derivatives have extremely low critical aggregation concentrations (CACs) and, therefore, their cationic congeners were the first ones used for DNA delivery. Almost three decades after the introduction of the first synthetic transfection system based on cationic lipid DOTMA 24a11 by Felgner’s group, a huge number of cationic lipids have been synthesized and investigated as transfection agents by the scientific community, with the field being periodically reviewed. 58-62, 67, 87-90 Important trends that persisted throughout this time were the incorporation of natural building blocks and biodegradable linking groups, yielding quaternary ammonium lipid like DOTAP 25,91 procationic (poly)amine-lipids DOSPA 26,92 DOGS 27,93 DC-Chol 28,94 CDAN 29a95 that are commercially available either alone or in formulations such as DOTMA 24a/DOPE (Lipofectin™), DOSPA 26/DOPE 3/1 w/w formulation (Lipofectamine®, LFM), DOGS 27 (Transfectam®), being frequently used as reference(s) in transfection assays. Another trend was to “soften” the positive charge of the ammonium headgroups (cationic or procationic) through the use of larger polar heads such as phosphonium and arsonium (compare lipids 30-32 96) or through the use of heterocyclic positively charged polar heads such as imidazolium (e.g. lipid 33 97) or pyridinium (e.g. SAINT-2 3474, 75, SPYRIT lipids 35-3743, 98101

).



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Other important trends were represented by the introduction of stimuli-responsive building blocks as well as other biodegradable connecting linkers (esters, vinyl ethers, carbamates) in the structures of cationic amphiphiles, which can be used in conjunction with colipids having targeting moieties (such as sugar moieties, sugar mimics, RGD motifs, etc.) to enhance the specificity of delivery towards desired tissues/organs.

Thus, some time ago,

Thompson et al. proposed a cationic analogue of diplasmenylcholine BCAT 38 and compared it with the saturated analogue DCAT 39. The authors showed that the glyceryl bis-vinyl ether moieties that can undergo hydrolysis under acidic conditions encountered in the endosome, liberating the corresponding fatty aldehydes and the glycerol backbone. The hydrolysis process promotes the disassembling of the lipoplex and the simultaneous endosomal rupture and release of genetic cargo. Interestingly, BCAT 38 was able to transfect psoralen-labeled plasmid pGFPDNA into NIH-3T3 cells without any colipid, and the addition of DOPE actually decreased the transfection efficiency. In contrast, the saturated analog DCAT 39 required 20 mol % DOPE for reaching its biological activity peak (albeit still inferior to pure BCAT 38) under similar 20 ACS Paragon Plus Environment

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conditions. BCAT 38 lipoplexes showed superior transfection efficiency and lower toxicity compared to DCAT 39 and DOTMA 24a/Chol lipoplexes in NIH-3T3, SCCVII and RENCA cell lines at 4/1 +/- charge ratio. Moreover, BCAT lipoplexes were well tolerated in mice after administration via tail vein injection, while DCAT formulations displayed significant toxicity.102 Recently, the same group combined the unique features of BCAT lipoplexes with receptor targeting. Lipoplexes of BCAT/DOPE having 10% mannose-PEG3000-DSPE 40 ligand (generated through click chemistry) successfully delivered gWIZ GFP plasmid to dendritic cells (DC) with efficiencies superior to LFM and comparable cell viability. Interestingly, when the targeting ligand was added to LFM in the same proportion (10%), the transfection efficiency of the corresponding lipoplexes almost doubled.103 With the same objective of DNA delivery to dendritic cells and genetic immunization, Chaudhuri group introduced the mannosyl cationic lipid 41 and the quinic- and shikimic acid conjugates 42a and 42b respectively. The quinic and shikimic targeting are mannose bioisosteres and were previously shown by Grandjean at al. to be effective as DC receptors binders.104-106 Lipoplexes generated from cationic amphiphiles 42a, 42b having mannose-mimicking moieties were found to be more efficient then mannosylated lipid 41 in transfecting mouse bone marrow derived dendritic cells (mbmDCs) using α5-GFP DNA plasmid. Furthermore, in a genetic immunization experiment, lipoplexes derived from the cationic lipid 42b having a shikimic headgroup and the pCMV-MART1 plasmid DNA were found to be the most effective in inhibiting the growth of B16 melanoma in C57BL/6 mice challenged with aggressive B16F1 tumor cells, surpassing both mannosylated and quinylated congeners. 106 Long-lasting dendritic cell was achieved by second generation cationic lipids 43ac with a lysine residue inserted in between the core amphiphiles structure and the mannose/mannose mimicking moiety using pCMV-SPORT-β-gal, p-CMV-MART1 and pα5GFP



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DNA plasmids. Lipoplexes derived from quinoyl and shikimoyl lipids (43a and 43b) conferred long lasting protective immunity (300 days post tumor challenge) with significant memory response in 80% of the immunized mice, a step forward towards DC-based vaccines.107 The same group investigated the possibility of liver targeting using cationic glycolipids 44a-e and 45a-e, bearing either cyclic or open D-galactose targeting moieties attached to the cationic head via hydrophobic linkers of various lengths. Their results showed that lipids 44, having the cyclized sugar on the headgroup, required a longer spacer (six methylene units, 44c (n = 5)) for optimal efficiency than their open analogues 45, where two methylene units were found to give the best results (lipid 45a). Interestingly, these glycolipids were able to fully compact pCMV-SPORT-β-gal DNA at a lipid/DNA +/- charge ratio of 2 when coformulated with cholesterol at equimolar ratio. Their lipoplexes, generated at charge ratios of 4, 2 and 1, were able to transfect HepG2 and A549 cell lines, with a maximum transfection efficiency observed at a lipid/DNA +/- charge ratio of 1. However, in the case of hepatocytes a charge ratio of 4 was required for maximum transfection power. The efficiency of lipoplexes derived from lipids 44c and 45a was retained in vivo towards selective liver transfection, the two lipids being equally potent.108


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In an effort to improve biocompatibility profile of lipoplexes, Chaudhuri et al.109 also investigated the influence of inserting biodegradable ester linkers in the structure of the amphiphilic lipids he previously developed.67 The authors also investigated the structural orientation of the biodegradable group, synthesizing ester containing lipids isomers 46a and 46b. Biological testing revealed that only lipid 46a was efficient in transfecting COS1, HepG2, and A549 cells at lipid/pCMV-SPORT-β-gal DNA plasmid charge ratio of 1 and CHO cells at a charge ratio of 2. The isomer 46b was much less efficient under same experimental conditions. Unfortunately, lipoplexes generated form 46a and 46b were not efficient in vivo, which prompted the same group to investigate the two amide congeners 47a and 47b respectively. Lipoplexes generated from lipid 47a were significantly more efficient than the ones generated from lipid 47b towards transfecting A549, B16F10, CHO cell lines, at a lipid/DNA charge ratio of 2 and towards transfecting HepG2 cells at a +/- charge ratio of 4. In the same study it was also shown that serum resistance of lipoplexes generated from lipid 47a was three times higher than lipoplexes obtained from isomer 47b. Subsequent gene delivery experiments in Balb/C mice



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revealed that lipoplexes of 47a retained their efficiency in vivo while 47b based lipoplexes were totally devoid of transfection activity. The main organ targeted was the lung, with new cationic amphiphiles being compared to DOTAP/Chol standard transfection system.110 Towards targeted delivery of lipoplexes, Chaudhuri at al. compared the therapeutic activity of two series of lipoplexes derived from cationic lipids, 48a and 49a, displaying RGDK targeting moieties against α5β1 integrin receptors. Lipopeptides 48b and 49b having subtle structural differences in the peptide unit were also synthesized in order to test the contribution of the targeting moiety and its specificity to overall transfection efficiency of the DNA complexes. In the first study, the authors showed that DNA complexes of lipopeptide 48a/Chol (2/1 molar ratio) presented superior transfection activity to congener 48b/Chol (2/1) lipoplex formulations in 3T3 mouse fibroblast cells and A549 lung cancer cell lines. Subsequent subcutaneous administration of rhPDGF-B plasmid via RGDK-lipopeptide 48/Chol (2/1) lipoplexes healed chronic wounds in streptozotocin-induced diabetic Sprague-Dawley rats.111 In a subsequent study, the same group investigated the pharmacological activity of lipoplexes generated from RGDGWK-lipopeptide 49a/Chol and anticancer p53 gene for inhibiting tumor growth in C57BL/6J mouse model bearing aggressive B16F10 tumors. The RGDLFK-lipopeptide (49b) was also evaluated in parallel. The therapeutic activity of lipoplexes derived from 49a and 49b was evaluated 23 days post-administration, when it was observed that tumors treated with RGDGWK 49a/p53 lipoplexes showed higher regression rate as compared with RGDLFK 49b/p53 lipoplexes. Tumor growth was inhibited via apoptosis induced by p53 gene using pCMV-p53. In the absence of p53 only modest tumor regression was observed. Moreover, immunohistochemical staining of tumor cryosections with vasculature markers, combined with


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monitoring expression of GFP in the same tumor cryosection, revealed that the RGDGWKlipopeptide 49a lipoplexes primary target the tumor vasculature.112 Within the same class of lipids, Liberska et al. introduced two new series of argininebased double tailed cationic lipopeptides having either succinyl (50a-ad) or maleinyl (51a-ad) linkers.113 The transfection performance of these systems was evaluated in HeLa and HEK293T cell lines using the pEGFP-Cl plasmid. Transfection activity of lipoplexes was strongly influenced by the number of arginine units present within the amphiphile, with amphiphiles having two arginine units found to be more efficacious than congeners having one or three arginine units. Amphiphiles having short tails (palmitoyl) combined with a short diethylenetriamine spacer (m = 1) (50a and 51a) displayed the best transfection efficiency within 50a-ad and 51a-ad series, surpassing LFM2000 when formulated as lipid/DOPE at 1/1 molar ratio and a +/- charge ratio lipid/pEGFP-Cl of 5 and 10. Introduction of oleyl tails did not improve the transfection efficiency of these formulations. Introduction of an unsaturated linker had also only a minor impact on the transfection activity. In addition, lipopeptides 50a-ad and 51a-ad were relatively non-toxic, preserving 90% cell viability 24h post transfection as determined by MTT cell proliferation assay.113



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4.3.1. Cationic lipids bearing ether linkages

As previously mentioned, the field of synthetic transfection systems was initiated by Felgner who introduced DOTMA 24a as the first cationic lipid used in gene delivery;11 its glycerol-like backbone, together with trimethylammonium polar heads and two oleyl chains makes it relatively biocompatible and the design remained a standard one in the field. DOTMA 24a formulation with DOPE at 1/1 molar ratio gained widespread use as a standard transfection system (vide supra). Further SAR studies were conducted at the level of the polar head and hydrophobic chains by Felgner114 and at the level of linker by Ren and Liu.60, 115 More recently, Bajaj et al. introduced lipids having benzyl-ammonium backbone substituted at position 3 and 4 26 ACS Paragon Plus Environment

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with C14 and C16 saturated oxyalkyl tails 52a, 52b. These lipids were able to fully compact pEGFP-c3 DNA at +/- charge ratio of 1. Lipoplexes derived from lipids 52a and 52b achieved similar levels of GFP expression in HeLa cells in the absence of serum when formulated with DOPE at 1/2 molar ratio. However, in the presence of serum lipoplexes from 52a were slightly more stable. Compared with GS congeners, DNA complexes generated from lipid 52a displayed similar transfection efficiency with 19a-c (within C14 aliphatic tail series), while congeners made out of lipid 52b were less effective than lipoplexes obtained from GS 19f. In addition, lipid 52a was slightly more cytotoxic than 52b.81 Dobbs et al. introduced a new series mesomorphic imidazolium lipids 53a-d, 54a-d and investigated the knockdown efficiencies of their corresponding GL3Luc siRNA complexes in A549 Luc cells. Derivatives 53b and 54b were able to compact 700 ng siRNA at 5 nmol lipid loadings, while 54d was able to compact siRNA at 10 nmol lipid loadings. The authors compared the particle size and zeta potential of lipoplexes generated from lipid 53b and 53d when coformulated with DOPE at 1/2 molar ratio and 700 ng siRNA by DLS measurements. Lipoplexes of 53b displayed a diameter of 69 nm and a zeta potential of +61 mV, while lipoplexes of 53d displayed a diameter of 593 nm and zeta potential of 28 mV. Thus, lipoplexes of 53b were found to be more suitable for cellular uptake than 53d. In addition, lipoplexes of 53b and 54b produced 80% knockdown in A549 Luc cells; while, lipoplexes of 53d produced only 20% knockdown when loaded with 10 nM siRNA. Under these conditions, LFM2000 displayed up to 98% knockdown efficiency. These results suggested that imidazolium lipids having C12 aliphatic tails were more efficient than congeners having C18 aliphatic tails; moreover, the binding affinity for siRNA was not dependent on the counter ion used.116



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Recently, our team reported a new series of bio-compatible pyridinium cationic lipids 55a-f based on a dopamine backbone and hydrophobic aliphatic tails of different lengths and degrees of unsaturation. In addition, we also investigated the effect of the counterion and we proved the superiority of Cl- counterion versus more lipophilic PF6- in both liposomes and lipoplexes. Interestingly, lipids having the same chemical structure but different counterions displayed similar gel/liquid crystalline transition temperatures in hydrated form despite major differences in the self-assembling of the same amphiphiles in bulk (non-hydrated). All new cationic amphiphiles were able to fully compact plasmid DNA at +/- charge ratio of 3 alone when coformulated with either DOPE or cholesterol at 1/1 molar ratio. In addition, corresponding lipoplexes were able to transfect gWiz-Luc into NCI-H23, MCF7, PC-3, DU-145, A549 and Caco-2 cell lines when formulated with DOPE at 1/1 molar ratio. Within 55a-f series, the transfection efficiency decreased in the order: 55a > 55f > DOTAP 25 > 55b > 55c = 55d in NCI-H23 cells. Moreover, these lipids displayed low cytotoxicity except 55a. Formulations of 55f/DOPE were found to be the most efficient within 55a-f series irrespective of the cell line used, retaining the efficiency in the presence of up to 20% serum. Importantly, we revealed that 55f was able to transfect selectively primary rat dopaminergic neurons harvested form nucleus accumbens and neurons from frontal cortex, and these features recommend 55f as potential neuronal reprogramming agent in vivo.117 Recently, Kudsiova et al. synthesized new tetradecyl-DOTMA analogues 56a-e and conducted a SAR study at the level of hydrophobic chains. Thus, the British team investigated the effect of repositioning the double bond at C9 or C11, synthesizing and analyzing both E and Z isomers for each position of the double bond within the C14 chain. Lipid 56a, having a triple bond in position C9, was also included in the same series for comparison. All new lipids were


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co-formulated with DOPE at 1/1 weight ratio and were able to condense gWIZ-Luc plasmids at several lipid/DNA weight ratios. The transfection results of 56a-e lipoplexes in MDA-MB 231 breast cancer cell line and 1HAEo- human alveolar cells revealed that the formulations of lipids 56c and 56e, possessing E double bonds into the hydrophobic chains, were superior to corresponding formulations of the corresponding Z isomers, surpassing DOTMA 24a/DOPE at 4/1 lipid to DNA weight ratio. Within E isomer series, formulations based on lipid 56c (that has ∆11 double bonds) were superior to the ones derived from the lipid 56e having (bearing ∆9 double bonds). Lipoplexes based on alkyl lipid 56a were less efficient than lipoplexes based on lipids 56e and 56c bearing E-∆9 and E-∆11 double bonds but more efficient that lipoplexes derived from lipids 56d and 56b bearing Z-∆9 and Z-∆11 double bonds. Cytotoxicity of lipoplexes was negligible at 4/1 lipid to DNA weight ratio. Confocal microscopy revealed that E lipids had a higher internalization capacity and a more diffuse cellular distribution than Z-lipids, a fact that was attributed to a greater degree of endosomal escape and /or nuclear import. Interestingly, small angle neutron scattering (SANS) revealed negligible differences in the thickness of the bilayer and on d-spacing for lipoplexes generated from lipids 56a-e when coformulated with DOPE at 1:1 weight ratio. Thus, the addition of DOPE in the supramolecular assemblies of these cationic lipids had a leveling effect, compensating for the differences in individual packing parameters generated by structural induced differences in double/triple bond position and geometry.118 The same lipids were used in conjunction with a bifunctional targeting peptide 56f possessing a lysine-rich (K16) region designed to condense DNA and a motif targeting α5β1 integrin receptors. The ternary lipid-gWIZ-Luc-DNA-peptide (LPD) complexes were found more efficient that the parent lipoplexes revealing a potentiation effect of the targeting peptide on lipoplex activity.119



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Moreover, in order to stabilize the lipoplexes against plasma proteins and uptake by the reticuloendothelial (RES) system, the same team introduced lipids 57a-d bearing oligoethylene glycol (oligoEG) moieties in the structure of the polar head and other structurally related congeners.

120

The best transfection efficiencies against 1HaEo- and SCFTE29o- cell lines were

observed for lipoplexes generated from lipids 57a and 57b coformulated with DOPE at a weight ratio of 1/1 and at total lipid/pClLux DNA ratio of either 2/1 or 4/1. Lipids 57a and 57b possessing four ethylene glycol units attached to the polar head were found to be superior in transfection efficiency when compared to their congeners 57c and 57d having six ethylene glycol units in the hydrophilic domain. This trend was found to be true for lipoplexes generated form lipids alone and also for lipoplexes obtained from lipids coformulated with DOPE at 1/1 weight ratio. Introduction of a second quaternary ammonium group in the hydrophilic domain (lipid 57e) was not beneficial for transfection efficiency. Importantly, lipoplexes generated form lipid 57a were found superior to DOTMA 24a when transfection was performed in the presence of serum.

120

Building on the success of cationic lipids 57a and 57b, Mustapa et al. proposed

analogues 57f and 57g in which a biodegradable ester group was inserted in between the oligoEG moiety and the tetraalkylammonium polar head. The cleavable ester bond was designed to ensure that detachment of oligo EG moiety in the endosome after internalization of the DNA complex, leading to the disassembly of the complexes and endosomal release of nucleic acid. Lipoplexes generated from these new lipids had modest transfection of pCl-Luc, even when coformulated with DOPE at 1/1 weight ratio. Addition of polylysine positively charged peptides incorporating integrin targeting sequence CRDGCLG in the structure of congeners 57i, 57j rescued the transfection efficiency. Thus, the ternary LPD complexes based on 57f/DOPE were more efficient as compared with the corresponding complexes generated from lipid 57g in Neuro


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2A, AJ3.1, 16HBE14o-, PVSMC cell lines. Both LPD complexes were equally efficient in transfecting bEND.3 cells. However, LPD complexes of lipid 57f were equally efficient with LPD complexes of non-cleavable lipid 57a in Neuro 2A, AJ3.1, 16HBE14o- cell lines and surpassed these control LPD complexes in bEND.3 and PVSMC cell lines.121 LPD complexes derived from lipid 57f and pCl-Luc (5.7kb), pAB11 (7.7 kb) and, respectively, pEGFP-N1 (4.7 kb) were used successfully in vivo to selectively deliver luciferase genes into tumors. The oligoEG-containing complexes were 5-fold more efficient than non-EG analogues. Cleavability of the lipid was without significant effect in vivo.122 Another strategy to improve transfection efficiency of LPD type complexes described above was proposed by Wong et. al who introduced lipids 58a-f having a cleavable acetal group inserted in between two oligo EG units and cationic polar head. Acetals are prone to hydrolysis under mild acidic conditions (pH between 5.0 and 3.0) typically encountered in the endosomal compartment, thus triggering the disassembling of the complex and pCl-Luc DNA (5.7 kb) release from the endosome. The sensitivity of lipids 58a-f in acidic media decreased in the order 58a > 58c > 58e > 58b > 58d ~ 58f. A good correlation was observed between hydrolytic sensitivity and transfection performance of corresponding LPD complexes generated with peptide 56f or 58h and DOPE. Best transfection results were obtained for LPD complexes generated from lipid 58a, 58c and 58e against 16HBE14o- ΣCFTE29- and bEND.3 cell lines. Interestingly, in the case of PVSMC, the best transfection efficiency was observed for LPD complex of lipid 58g while similar transfection efficiencies were observed for LPD complexes of lipids 58a-c and 58f. The transfection efficiency reported for LPD systems having peptide 58h was in general higher than transfection of similar systems based on peptide 58f alone due to receptor availability on the investigated cell lines.123



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4.3.2. Phosphonate/phosphoramidate (pro)cationic lipids

A structural alternative to the glycerol scaffold in the design of cationic lipids for gene delivery is the use of phosphonate/phosphoramidate functional group.124 A comprehensive structure-activity study within the class of phosphonate cationic lipids 59a-i was reported by Floch and collaborators, who investigated the impact of cationic headgroup, spacer and hydrophobic tails on their transfection efficiency against K562, CFT1 and HT29 cancer cell lines. Their results showed that decreasing the charge density by increasing the volume of the headgroup [-X(CH3)3]+ in the series N < P < As improve the transfection efficiencies of the corresponding lipoplexes regardless of the cell line used. In terms of linker, the best transfection efficiencies of pTG11033 plasmid DNA were observed for ethylene and propylene linkers when Z = P (59e) and As (59g), and for methylene linkers when Z = N (59a), irrespective of


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hydrophobic chains used. Interestingly, the unsaturated oleyl chains were less efficient than tetradecyl ones.96, 125 The safety concerns associated with the use of arsonium lipids in biological systems were addressed in earlier reports of Stekar at al. who showed that arsonium-based amphiphiles have a lower cytotoxicity as compared with their corresponding phosphonium and ammonium analogues.126 These lipophosphonates contain a robust phosphorous-carbon bond which is not biodegradable. In an attempt to improve the toxicity profile of these cationic amphiphiles, the Brest team introduced cationic lipophosphoramidates having a weaker phosphorous-nitrogen bond that can be degraded in vivo. Thus, Clement et al. and Montier et al. introduced new series of lipophosphoramidates (60a-f) and investigated the effect of cationic headgroup, spacer and hydrophobic tail on transfection efficiency against HeLa, CHO cell lines. As in the previous case, the transfection performance, using the same pTG11033 plasmid DNA was mainly governed by the diameter of the cationic headgroup (N < P < As), with arsonium-based lipids (60d-f) being superior to phosphonium-based ones (60b-c) and to ammonium ones (60a), respectively.

127,

128

Moreover, lipophosphoramidates with small cationic headgroups

(ammonium and phosphonium) were more effective in the presence of long spacers (n = 3), while bulky cationic headgroups (arsonium) were more effective with short spacers (n = 2). In addition to the cationic headgroup and spacer, the nature of the hydrophobic tail influenced the transfection performance. For example, short saturated tails such as C14H29 had been proven to be less effective than mono-unsaturated (oleyl) and diunsaturated (linoyl), a trend that was opposite to the one observed in lipophosphonates (vide supra). In general, the presence of polyunsaturated chains increased the membrane fluidity and fusogenicity. Indeed, lipids such as BSV4 (60f) containing linoyl hydrophobic tails were found to be better gene delivery vectors



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than oleyl ones such as KLN47 (60e). Compounds with branched aliphatic hydrophobic tails such as phytanyl, known to lower even more further the gel/liquid crystalline transition temperature such as BSV18 (60g), were found to be even more effective. Thus, within the arsonium lipid series, the transfection efficiency increased in the order 60e < 60f < 60g. Moreover, the cytotoxicity in A549, A375, SKMEL28 cell lines followed the same trend, BSV18 being the least cytotoxic amphiphile.127, 129 In vivo results showed good transfection activity for BSV18-based lipoplexes in a mouse model of lung delivery aimed at cystic fibrosis treatment.130 Surprisingly, in vivo 60e was significantly more efficient than 60f. Using anisotropy measurements, the authors explained these contradictory results by showing that 60f liposomes were more fluid but significantly less fusogenic than 60e-based ones.124 In another recent study, Clement, Jaffres and collaborators investigated novel phosphoramidates having resonance stabilized cationic headgroups such as guanidyl (61a, 61b), imidazolium (61c), or dicationic headgroups such as ammonium-phosphonium (61d). Biological testing revealed that dicationic lipid (50d) surpassed the transfection efficiencies of monocationic derivatives (61a-c, 61e) in A549 and HeLa cell lines, while displaying lower cytotoxic effects at similar +/- charge ratio. 124, 131 Within the guanidyl series, the ethylene spacer (61a) was more effective than butylene spacer 61b as revealed by testing in HECK293-T7 cell lines using pTG11033, pCMV Luc and pCMV-EGFP plasmid DNA. Further biological testing of lipids bearing an ethylene spacer showed that guanidyl headgroup was less efficient than imidazolium one (61c).132 In an effort to overcome the intercellular delivery barriers, the same team designed pHresponsive helper-lipids having imidazole polar head (61e, 61f) which may act as a proton sponge in the acidic media of the endosome (pKa around 6.0), preventing the degradation of


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nucleic acid. It was showed that the transfection efficiency of imidazolium cationic lipid 61c coformulated with imidazole helper lipid 61e was similar to the formulation of arsonium lipophosphoramidates 61e coformulated with the same helper lipid in C2C12 cells using pCMVluc, p3NF-luc-3NF, Cy5-pDNA, Rab5-EGFP, Rab7-EGFP, Cav1-EGFP plasmids. However, the cytotoxicity profile of 61c/61e was better than the one displayed by formulation 60e/61e.

133

Addition of an ester moiety in the spacer of 61e, thus generating helper lipid 61f, decreased the overall transfection performance of 61e-based formulations.133 The Brest team also proposed the insertion of two disulfide units into the hydrophobic chains of lipophosphoramidates, which upon reduction in cytosol can disassemble and release pDNA from lipoplexes. The best transfection efficiency of stimuli-responsible cationic lipids 62a-c was observed for lipoplexes generated with lipid 62b at a +/- charge ratio of 4 against A549, HeLa and 16HBE14o(-) cell lines. Again, the phytanyl lipids 62b and 62c were more efficient than lipid 62a containing n-alkyl hydrophobic chains and the phosphonium lipid 62b was more efficient than ammonium lipid 62c, thus confirming the conclusions of the previous SAR studies (vide supra).134 The ability of lipophosphoramidates to compact and deliver DNA relies in part on strong intermolecular hydrogen bonds formed by phosphoramide functional group. It was anticipated that replacement of an oxygen atom in the phosphoramidate group by sulfur atom, thus forming thiophosphoramidates may affect the dynamic properties of the lipoplexes generated from these new cationic lipids. Moreover, the difference in polarity between the P=S versus P=O bonds should affect (decrease) the hydration level of liposomal formulations. Thus, Jaffres and collaborators compared the transfection efficiencies of phosphoramides 63a, 63b with thiophosphoramidates 63d, 63e with a propylene spacer. The authors found that replacing P=O



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group with P=S group slightly improved the transfection efficiencies in A549 and 16HBE14o(-) cell lines, the most efficient being derived from cationic lipid BSV44 63d. N-Methylation of the linker decreased the transfection efficiencies, BSV25 (63e) being inferior to BSV44 63d in the same cell lines.135

4.3.3. Bioresponsive deciduous-charge cationic lipids

Another cationic lipid design was proposed by Gorman et al. who introduced the ethyl ester of dimyristoyl phosphatidylcholine (EDMPC) 64a and demonstrated that liposomal formulations of 64a with either cholesterol or DOPE at 1: 1 molar ratio can efficiently deliver pMB10 DNA in vitro in HEK293, COS1, A549, H441 cell lines, p4119 plasmid DNA in 293 cells and in vivo via intralobal instillation into rodent lungs of p4119 CAT plasmid.136 Two years later MacDonald’s group prepared and assessed various EDMPC congeners bearing different hydrophobic tails. The most efficient was found to be the dioleoyl derivative EDOPC 64c which 36 ACS Paragon Plus Environment

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was able to compact and deliver pCMV-β-Gal DNA without colipid, reaching the transfection performance of LFM.137 Interestingly, the transfection activity of EDMPC 64a lipoplexes was maintained in media with high concentration of serum.138 The good results obtained with EDOPC prompted the same group to extend the series, synthesizing the methyl-, ethyl-, npropyl-, n-hexyl, n-decyl and n-octadecyl derivatives 64b-g. The new cationic lipids behaved very differently upon hydration. Thus, O-methyl, O-ethyl- and O-propyl- DOPC (64b-d) hydrated readily and formed unilamellar vesicles at room temperature. The O-decyl-DOPC 64f and O-octadecyl- DOPC 64g did not form liposomes in either water or salt solutions, assuming an inverted hexagonal phase in water. The O-hexyl-DOPC 64e hydrated in water forming vesicles whereas in salt solution it remained a viscous nonbirefringent liquid, assumed to be a cubic liquid crystalline phase. The triester lipids 64b-g did not require DOPE in order to mediate DNA transfection. The most efficient in transfecting BHK cells were lipoplexes generated from O-ethyl-DOPC 64c and O-propyl-DOPC 64d, with the ethyl derivative 64c being efficient over a wide range of lipid/DNA ratios. This behavior was attributed to the ability of 64c and 64d to form lamellar phase with DNA. The long chain lipids 64e and 64f formed hexagonal phase complexes with DNA that were devoid of any transfection activity.139 More recently, Lebeau’s group investigated a new series of cationic DOPC ester conjugates having either PEG (64h) or Triton X-100 (TX100) (64i) moieties as siRNA delivery systems against U87-Luc cell line. Efficient siRNA transfection was obtained with 64i conjugate at N/P ratios ranging from 25 to 100. Formulations based on PEG conjugate 64h and EDOPC 64c were devoid of any knockdown efficiency. However, an equimolar mixture of EDOPC and TX100 had some knockdown efficiency, but significantly smaller than the one displayed by 64i, showing that covalent immobilization of the detergent molecule on the lipid carrier is required



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for efficient luciferase knockdown. Toxicity generated via TX100-mediated membrane poration was assessed using sheep erythrocytes. No hemolytic activity was found for DOPC-TX100 conjugate 64i, while the parent TX100 detergent was significantly porating the erythrocyte membranes. Furthermore, DOPC-TX100 64i exhibited only moderate levels of toxicity in 16HBE cells, determined by lactate dehydrogenase assays.140 In an effort to improve the cytotoxicity profile and the siRNA delivery properties of these phospholipid conjugates, the same group inserted biodegradable/hydrolysable acetal groups between the cationic lipid and the detergent moiety. The knockdown efficiency of the carbonate conjugates 64j and 64k, determined in the same U87-Luc cell line, matched the efficiency of ester conjugate 64i and surpassed their succinyl congeners 64l, 64m. The new generation of TX100 conjugates 64j-m was less cytotoxic than ester 64i as revealed by MTT and LDH assays.141 The later design was subsequently expanded to acetal conjugates 64n-v, in an effort to obtain bioresponsive charge-deciduous amphiphiles which can be cleaved under acidic or enzymatic conditions. The stability of these lipids against hydrolysis at physiologically relevant pH values (7.5 and 4.5) was found to depend on the nature of the acetal scaffold as shown by 31

P-NMR. Thus, lipids 64q, 64o, 64t containing a methyl-acetal scaffold hydrolyzed faster than

formaldehyde-derived congeners 64s, 64r, 64p, 64v. Replacement of the methyl acetal with an isopropyl one in lipid 64u strongly decreased the hydrolysis rate. Lipids having short alkyl tails hydrolyzed faster than congeners with longer aliphatic tails. The overall hydrolysis order was 64q > 64o > 64t > 64n > 64p ~ 64v > 64r > 64s > 64u. The hydrolysis results were in good agreement with ethidium bromide intercalation assay experiments which revealed that lipids 64o and 64q offered the fastest kinetics of nucleic acid release. Transfection assays using pCMVLuc-DNA against BHK-21cells in media containing 10% FBS showed that these lipids were


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efficient alone and that coformulation with DOPE at 1/1 molar ratio decreased the efficiency of DNA transfection of the corresponding lipoplexes. The transfection efficiency order did not correlate with the hydrolysis rate sequence and was found to decrease in the order 64t > 64s > 64n =64p > 64r > 64u =64v = 64c (EDOPC) > 64o > 64q, while the cytotoxicity increased in the order 64c (EDOPC) = 64v = 64q < 64o < 64n < 64p = 64r < 64s < 64t < 64u at an optimal N/P charge ratio of 3. Transfection assays in A549, Calu-3, NCI-H292 cell lines confirmed that lipids 64n, 64p, 64r and 64u were the most efficient representatives. However, significant cytotoxic effects were reported for these members. Lipids 64u and 64t were also the most efficient for siRNA delivery in U87-Luc and A549 cell lines, eliciting up to 80% gene knockdown.142 This study emphasized the importance of maintaining a good stability of liposomes and lipoplexes in buffer and serum while preserving the charge deciduous design. Fast hydrolyzing lipids such as 64q and 64o were probably not stable enough to ensure minimum lipoplex stability prior to cell delivery.



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4.3.4. pH-Sensitive and procationic lipids

Another commonly used strategy to improve transfection efficiency is the use of lipids with pH sensitive/ionizable polar heads such as DC-Chol 28 ,94 or the polyamine lipids DOSPA 26,92 DOGS 2793 and CDAN 29a.95 These lipids proved to be efficient for siRNA delivery in vitro and in vivo, and represent an important class of transfection agents. Early reports of MacLachlan et al. introduced pH sensitive procationic lipids 65a-d and showed that lipids 65b-c bearing unsaturated groups on the aliphatic tails were more effective as RNAi transfection vectors than saturated congener 65a. The pKa of the amino group is another important factor that affects the endosomal buffering and facilitates the endosomal escape. Procationic lipids 65c and 65d having a pKa around 6.7 were found efficacious in RNA silencing both in vitro and in vivo. Thus,

when

formulated

as

SNALPs

(Stable

Nucleic

Acid

Lipid

Particles)

as

cholesterol/lipid/DSPC/PEG-C-DMA(54e) at 48/40/10/2 molar ratio and a lipid/siRNA charge ratio of +/- 6, the knockdown efficiency in Neuro2A Luc cells using siLuc decreased in the order DLen-DMA 65d = DLin-DMA 65c > DODMA 65b > DSDMA 65a.143 Formulations based on lipid DLinDMA 65c were effective in vivo to silence apolipoprotein B in non-human primates.144 Tekmira and Alnylam introduced biodegrable procationic lipids having ketal units linked to procationic dimethylamine via a methylene spacer of different lengths, such as DLinKC2-DMA 66a and congeners, which were previously reviewed.64 Recently, Basha et al. showed that lipid nanoparticles based on 66a were effective towards silencing GAPDH in antigen presenting cells in the spleen and peritoneal cavity following systemic administration in a mouse model.145 Lee et al. showed that 66a was also effective in silencing of the androgen receptor (AR) in human prostate cancer cells CWR22Rv1, LNCaP and LAPC-4 cells lines using shRNA.


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Moreover, lipid nanoparticles of 66a, formulated as DSPC/PEG-C-DOMG(66l)/cholesterol/SPDiOC18(66n)/66a at molar ratio 10/10/39.8/0.2/40, were able to silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels post injection in a mouse xenograft model.146, 147 In a related study, Jayaraman, Hope and collaborators investigated the role of amine headgroup on the transfection efficiency within a series of 53 procationic (ionizable) lipids. It is claimed that these lipids enable siRNA to escape the endosomal compartment and access the cell cytoplasm more efficiently than cationic lipid, with the pKa of the headgroup playing a key role in the release profile of the nucleic cargo. Indeed, the authors found a strong correlation between the pKa value of the polar head and the siRNA silencing efficiency of this class of lipids. Lipid D-Lin-MC3-DMA 66d having a pKa value of 6.44 gave smallest ED50 (median effective dose) of factor VII in C57BL/6 mouse model when administrated as LNPs (vide infra). Structural modification of 66d by variation of substituents on the amino headgroup (lipids 66g-j) or variation of methylene spacer length between dimethyl amino headgroup and ester unit (lipids 66b, 66c, 66e, 66f) did not improve the siRNA silencing efficiency of 66d. The pKa of the amino groups in these congeners ranged from 4.17 to 7.16; only lipids having pKa values between 6.2 – 6.5 were found effective as siRNA cargos. The transfection efficiency decreased in the order: 66d > 66a > 66e > 66g > 66i > 66c = 66f > 66j > 66h > 66b. Another important parameter is the volume of the ionizable amino group. Lipids bearing dimethyl amine polar heads displayed the best siRNA silencing performance, while introduction of bulky amine groups decreased the transfection performance of these lipids. Procationic lipids 66a-h were effective as LNPs when formulated as lipid/DSPC/Chol/PEG-C-DMA 66k at 40/10/40/10 molar ratio and LNP/DNA 1/1 weight ratio, at doses of 0.3 mg/kg in mice and cynomolgus monkeys.148



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Building on the success of DLin-MC3-DMA 66d, Maier et al. incorporated biodegradable ester groups within the aliphatic tails and investigated the efficiency of a new series of lipids (66o-t) for systemic delivery of RNAi therapeutics as LNPs. Their results showed that LNPs of 66q containing FVII targeting siRNA (siFVII) generated up to 75% knockdown of serum FVII at 0.01 mg/kg doses in mouse model and up to 90% at 0.1 mg/kg in rat model when administrated via tail vein injection. The authors correlated the transfection efficiency of these lipids with the position of ester group within the aliphatic tail; a shift of ester group toward the headgroup linker decreased the transfection efficiency (66q ~ 66r > 66p > 66o). The authors reported similar in vivo efficiencies for LNPs based on 66q, 66r, 66s, 66t. Moreover, LNPs of 66q displayed a rapid distribution and clearance from plasma, tissue and liver. Lipid 66s displayed similar clearance profile with 66q, while substitution of the methyl group with a tbutyl one decreased the elimination rate from plasma and liver.4 In another related study, Harashima and collaborators introduced the “programmed packaging” - Multifunctional Envelope-type Nano Device (MEND) concept which had been reviewed by the same group.149 Recently, the same team introduced pH sensitive cationic lipid YSK05 67b which proved to be effective for siRNA delivery in HeLa cells as compared with DODAP 67a and DOTAP 25 when formulated as MEND particles containing 67b/POPE 67c/cholesterol

in 50/25/25 molar ratio.150 Within this series, the RNA silencing efficacy

decreased in the order: 67b > 67a > 25, with 67b displaying double the efficiency of 67a. The authors investigated the uptake mechanism and the siRNA release from these formulations and they observed that the release of siRNA form 67b-MEND lipoplexes was dependent on the endosomal acidification. Introduction of endosomal buffering additives such as chloroquine or ammonium chloride suppressed the gene silencing activity of 67b as compared with 25-based


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MEND. Based on these results, the authors concluded that the cellular uptake takes place via endocytosis, and escape from endosome depends on the endosomal buffering efficiency induced by these lipids. Lipid YSK05 67b (pKa 6.6) was found more efficient than DODAP 67a (pKa 6.0) towards siRNA delivery. Considering the pKa of the headgroup, this result is in agreement with data reported by Jayaraman and Hope. 148 Fusogenic lipids displayed enhanced transfection efficiencies. Thus, introduction of linoleyl tails improved the membrane fusogenicity, with 67bbased MEND complexes being more fusogenic than 25–based MEND complexes as shown by hemolysis assays.150 These results are in agreement with MacLachlan’s earlier report.143

4.3.5. Cationic lipids with programmed self-assembling/disassembling



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Gardner et al. introduced a new series of lipophilic polyamines linked to 3,4bis(oleyloxy)benzyl motif (68a-g) and evaluated their eGFP- and Alexa Fluor-488- plasmid DNA delivery and transfection efficiency in CHO and K1 cell lines. Lipids 68a-f were able to transport plasmid DNA into cells with high efficiency, which was found to be dependent on number of amine groups per polar head unit. Thus, within 68a-g series, the transfection efficiency of lipoplexes formulated as 5µg/mg decreased in the order: 68e > LFM > 68f > 68d > 68a > 68c > 68b. In terms of toxicity, lipids 68a-f were relatively non-toxic, with observed cell viabilities higher than 90%. A notable exception is represented by compound 68g, which displayed severe cytotoxic effects under the investigated transfection conditions.151 An interesting method to improve liposome and lipoplex stability in buffer and serum is through enhancement of amphiphile self-assembling via fluorophobic effect.152 Thus, Vierling et al. introduced perfluoroalkylated glycerophosphoetanolamides lipids and showed that fluorinated liposomes displayed lower membrane permeability, enhanced blood, buffer and serum stability as compared with nonfluorinated derivatives. Moreover fluorinated liposomes were able to retain much more efficiently their entrapped content.153 Recently, Lebeau’s group introduced polycationic lipospermine semifluorinated lipids (69a-c), having a polar head similar to DOGS 27. These fluorinated lipids coformulated with DOPE at 1/2 – 1/4 molar ratios were able to deliver DNA, as CMV-luciferase plasmid (pCMV-Luc, 7.6 kb), into HepG2 and siRNA into 911 cell lines with high efficiency while displaying good cell viability at +/- charge ratios between 4 and 8. Under these conditions, the transfection efficiencies of 69a-c were relatively similar despite different sizes of hydrophobic chains and fluorinated blocks used and reached the efficiency of DOGS 27. Lipoplexes of 69a/DOPE at 1/4 molar ratio presented enhanced stability and preserved their transfection efficiency even at 75% fetal calf serum in the transfection


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medium, being up to 1000 times more efficient than DOGS under similar conditions. The transfection efficiency of these lipids decreased dramatically in the absence of DOPE. For siRNA delivery (si911-Luc), the formulation 69a/DOPE 1/2 molar ratio gave the best luciferase knockdown in 911 cells at a +/- charge ratio of 8 and surpassed the transfection efficiencies of DOGS 27, DOTAP 25 and LFM2000 under the investigated conditions.154 The same group investigated fluoroalkyl DOTAP analogues 70a-c together with their congeners 71a-c produced via HF elimination under basic conditions. Since purification was difficult, the team converted 70a-c into 71a-c and these new lipids were subsequently tested for their transfection efficiency using the HepG2 cell line. Both classes required DOPE for maximizing the transfection efficiency, which was retained even at 25% fetal calf serum in the transfection media. Formulations of 70c and 71c with DOPE at 1/2 molar ratio reached and surpassed the efficiency of similar DOTAP 25 formulations at charge ratios between 3 and 6. This trend remained valid in BHK21 and HeLa cell lines where pSMD2-Luc∆ITR (7.6 kb) and pEGFP-Luc (6.4 kb) expression plasmids were used. All formulations based on lipids 70 and 71 were equal or less cytotoxic than DOTAP 25 ones, irrespective of the cell line used. Replacement of DOPE with a fluorinated analogue 72 (also synthesized in the study) failed to generate better transfection systems.155 Another strategy to improve the transfection efficiency of DOTAP-like cationic lipids was the charge reversal concept introduced by Grinstaff and collaborators.156 Thus, cationic lipids 73a-g, decorated with peripheral cleavable ester or amide groups, were able to condense pVax-LacZ1 DNA, when the net charge of the cationic lipid was (+1) via electrostatic interactions. Upon acid or enzymatic hydrolysis of peripheral ester or amide groups, the net charge of the lipid is reversed to (-1) with subsequent release of the nucleic acid through negative



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charge repulsion. Lipid 73a, bearing hydrophobic chains containing hydrolysable benzyl ester groups, was the best performer of the series, with transfection properties superior to DOTAP in CHO, HEK293, K562 cell lines while displaying low cytotoxicity. Interestingly, coformulation with DOPE did not improve the transfection efficiency.156-159 The same group extended the utility of charge reversal concept to zwitterionic phospholipid 74, which enhanced the transfection efficiency of DOTAP when formulated at 2/1 molar ratio.160 With the aim of improving the binding efficiencies between cationic lipid and nucleic acids, Grinstaff’s team introduced charge-reversible lipids 75a-f, having basic lysine amino acids either directly linked or separated by tryptophan or glycine units from the amphiphile backbone. The transfection results showed that KWK-C16 75c bearing a Lys-Trp-Lys oligopeptide was the most effective in expressing β-gal reporter gene in NIH 3T3 cells at +/- charge ratio of 4, surpassing LFM2000. Inferior homologue KWK-C14 75b was less efficient that 75c at low charge ratios but more efficient at higher charge ratios. Further shortening or elongation of the hydrophobic chains (lipids75a/75d) translated in decreased transfection efficiency. Substitution by tryptophan with glycine KGK-C14 75e also decreased the transfection efficiency. Interestingly, within the C14 lipopeptides, the KGG-C14 75f was the most efficient, revealing that the total cationic charge in the molecule is not the sole contributor to transfection power. A similar trend was observed in CHO cells, where lipopeptide KGG-C14 75f was the most effective lipopeptide, at a +/- charge ratio of 16.161 Building on these conclusions, the same group investigated C14-lipopeptides 75j-75m having di- and tripeptide headgroups with different (pro)cationic amino acids separated by a mono/di- glycine spacer. Biological testing in CHO and NIH 3T3 cell lines revealed that lipopeptides having a (pro)cationic amino acid separated by a


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bis-glycine spacer 75f-75i displayed much higher DNA transfection activity that their congeners 75j-75m having single glycine spacers. The transfection efficiency increased with the increase in basicity of the amino acid 75m 81e).167 Miller’s group conducted extensive research in the field of polyamine cholesteryl cationic lipids, yielding efficient representatives such as CDAN 29a. The transfection efficiency of these amphiphiles was found to be dependent on the length of the spacers separating the amino moieties within the polyamine headgroup, which influenced their pKa and protonation state. These physicochemical parameters were directly correlated with the endosomal buffering capacity of the amphiphiles and the ability of the lipid to facilitate endosomal escape.58, 95, 168 In an effort to find cationic lipids with further improved and/or more versatile properties, the same group recently introduced cholesteryl lipids having amine-arginine motifs CEAG 82a and CAPG 82b, together with related lipids based on aminodecanoylglycine backbone DODAG 83a or DOAG 83b. Lipoplexes of DODAG 83a coformulated with either DOPE or MM27 61e at 1/1 molar ratio and combined with pEGFP-Luc DNA at 4/1 weight ratio were the most efficient and surpassed the transfection efficiency of LFM2000 in OVCAR-3 and HeLa cell lines in the presence of serum. Under similar conditions, DODAG 83a lipoplexes were followed in transfection efficiency by CAPG 82b, CDAN 29a and CEAG 82a. However, CAPG 82b was the most efficient cationic lipid followed by DODAG 83a, LFM2000, CDAN 29a, and CEAG 82a in IGROV-1 cell line. Lipoplexes of DOAG 83b were devoid of any transfection activity under similar conditions irrespective of the cell line used. The results proved that the presence of guanidine cationic polar head in this design was less efficient than the polyamine one. The above mentioned lipids had a different co-lipid preference: CAPG 82b and CEAG 82a performed better when coformulated with MM27 61e, while CDAN 29a was more efficacious when coformulated with DOPE. DODAG 83a was efficacious with both colipids. Lipoplexes formulated with DOPE


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were found less cytotoxic than MM27 61e -based based ones. Importantly, DODAG 83a-based lipoplexes were also able to transfect anti-hepatitis B siRNA to liver in a murine mouse model with subsequent infection parameters (virion and hepatic mRNA levels) equivalent or better to those generated after lamivudine treatment.169 Sheng et al. introduced a new series of cationic cholesteryl lipids 84a-d with cationic or procationic headgroups and bioreducible disulfide-containing linkers connected to cholesteryl moiety via carbonate groups. All these lipids were able to bind Cy3-labeled pEGFP-N1 DNA at +/- charge ratios of 3 and higher and were able to release it after exposure to dithiothreitol (DTT). Transfection efficiency was assessed using COS-7 cell line and either the luciferase or GFP reporter plasmid. It was found that lipoplexes of 84a and 84c were efficient starting with a +/- charge ratio of 1. At charge ratio of 5 and in the absence of serum the transfection efficiency decreased in the order 84c = 84a > 84b > 84d, with the best representatives matching the transfection efficiency of PEI 25K while displaying much lower cytotoxicity. Intracellular uptake capability of CHOSS lipoplexes with Cy3-labelled pDNA were found much higher than those of lipoplexes generated from LFM2000.170 Maslov et. al investigated cholesteryl polycationic lipids 85 having a spermine cationic headgroup attached to the cholesteryl unit via a variable length linker and a biodegradable ester or carbamate groups. Gemini amphiphiles containing two cholesteryl moieties were also synthesized (compounds 20a-c, vide supra). The new lipids were co-formulated with DOPE at 1/1 molar ratio and they were able to transfect pEGFP-C2 DNA with efficiencies reaching or surpassing LFM2000 under serum free conditions. Cholesteryl polyamines 85a and 85b having shorter linkers were more efficient than 85c. The carbamate linker was found to be superior to the ester one. Interestingly, the transfection efficacy of lipoplexes generated from 85a-c



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decreased 2 to 7 fold in the presence of serum, while their Gemini congeners 20a-c retained their transfection efficiency even in these conditions (vide supra). The same trend was observed for siRNA delivery, with the GSs 20a-c being 4 times more efficient than 85a-c in the presence of serum. In the absence of serum, lipoplexes of 85c/DOPE achieved up to 80% gene knockdown in EGFP-BHK IR780 cells, equaling the efficiency of LFM2000, followed by lipoplexes of 85a and 85b generated under same conditions.82 One major drawback of the efficiency of non-viral vectors is represented by their instability in biological fluids with respect to aggregation (especially in serum), robustness and target-specificity. Therefore, more complex formulations are often required to address these issues usually containing the nucleic acid (A) complexed by the cationic amphiphile (B) and surrounded by a PEGylated layer as stealth /biocompatible layer (C) decorated with a targeting ligands (D) (the ABCD nanoparticle paradigm).61 A recent example of this design was proposed by Miller’s group via incorporation in stealth lipoplexes of lipopeptide U11 86 displaying a urokinase plasminogen activator receptor (uPAR) targeting ligand, known to induce receptor mediated endocytosis (RME). These nanoparticles were formulated as CDAN 29a/DOPE at 1/1 molar ratio or as CDAN 29a/DOPE and MeO-PEG2000-DSPE 87a at 49.75/49.75/0.5 molar ratios and lipid/pDNA weight ratio of 12, and were tested on DU145 cells. ABC-type formulations containing PEGylated lipid displayed enhanced serum stability, therefore they were selected for further uPAR –mediated transfection studies. The ABCD formulation containing 1% targeting lipopeptide 86 was found optimal for efficient delivery of nucleic cargo into DU145 cells. Increasing the amount of lipopeptide 86 to 5 mol % resulted in a decrease in the transfection efficiency, possible due to particle aggregation.171 The example above proves that ABCD-type nanoparticles can act efficiently towards cell


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targeting while displaying enhanced serum stability. However, the presence of the stealth polymer PEGylated layer (C-layer) impairs to some extent the intracellular delivery of the nucleic acid. To address this problem, the same group introduced pH-triggered nanoparticles that were able to release siRNA cargo under endosomal acidic conditions. Thus, the siRNA was initially condensed with CDAN 29a/DOPE (~ 1/1 molar ratio) and aminooxy cholesteryl lipid (CA5 89a or CPA 89b). The resulting AB lipoplexes decorated with aminooxy groups were reacted with PEG2000-(CHO)2 polymer 88 (C-component) to generate an acid-liable PEG oxime on the inner face of the stealth layer of the PEGylated siRNA-ABC nanoparticles (siFECTplus nanoparticles). Authors subsequently showed that these nanoparticles tend to accumulate in the mouse liver following systemic administration and were able to suppress HBV replication with a therapeutic efficiency comparable with lamivudine standard treatment. The best transfection efficiencies were observed for the ABC system CDAN 29a/DOPE/CPA 89b formulated at 40/50/10 molar ratio and containing 5 % PEGylated lipid. CA5 89a-based formulations displayed better reproducibility of the biological effect, while CPA-based formulations (89b) were less prone to degradation and displayed improved shelf-life.172 Miller’s team also showed that AB lipoplexes of DODAG 83a/DOPE at 1/1 molar ratio were shown to be efficacious in expressing pDNA (pCH-9/3091 expressing β-galactosidase) into HeLa, A549 and IB-3 cell lines, while ABC nanoparticles formulated from DODAG 83a/DOPE/PEG4600-Chol 90 at 43/43/14 molar ratio and lipid/pDNA weight ratio of 4 were able to transfect lung airways in a BALB/c murine model.173 Recently, the same group showed that siRNA-ABC systems containing DODAG 83a/DOPE/Chol/CPA 89b, formulated at 20/50/10/20 molar ratio and at a lipid/siRNA +/charge ratio of 4 were able to mediate β-Gal and GAPDH knockdown in HepG2, HepG2 and



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Huh7 cell lines at 1 mol% PEGylayed lipid. However, lipoplexes were relatively unstable in the presence of serum. Increasing the percentage of PEGylated lipid 89b to 5 mol % translated into lipoplexes with enhanced transfection efficiency in HepG2 cells and stable in the presence of serum. It is worth mentioning that DODAG 83a- based formulations were found superior to CDAN 29a-based ones. Moreover, DODAG-based formulations had a tendency to accumulate in liver when administrated via tail vein injection in a mouse model. These formulations were able to deliver anti-iresHCV sshRNA, generating a 2-3 fold knockdown of luciferase protein in the liver after systemic administration. However, since 100-fold knockdown is required for clinical applications, further optimizations of these systems are required.174 Bhattacharya et al. showed that cholesteryl-based lipid loaded on a single walled carbon nanotube (SWCNT) displayed high DNA compaction efficiency at low charge ratios and enhanced stability in the presence of 10% FBS in media. In addition, cholesteryl lipids-SWCNT formulations equalled and surpassed the performance of LFM2000 in expressing pEGFP-C3 DNA in A549 and HeLa cells lines, and they were superior to cholesteryl lipids alone irrespective of the polar head used.175


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O Chol

N N

N CH3 Br

O Chol

Br

O

O

81c: CvII

80d: Chol-PYR

N

O Chol

N

N

81f: CmMB

O Chol

O

O

H3C 16

O

H3C 16

O

16

CH3

O

H2N

H

88: PEG2000-(CHO)2

N H2

4 CF3CO2

N H

Where:

O

O O P O

N H

O

O

O Me

H

45

O

O

O

105

O

Chol

90: PEG4600-Chol

O O

O

O

O O P O

N H

O

O O

O

45

N H

O

H N

N H

N H OH

O

OH OH O

6

87b: DSPE-PEG2000-MAL-(DSS)6

H O

O

H2 N

OH

HN 86: U11-Peptide Lipid

N H2

87a: MeO-PEG2000-DSPE

O

O

N H

Chol =

O

16

N Br CH3 80h: Chol-PR+

NH3

84d: CHOSS-4N

O H3C

44-45

N X H2 n

H3N

O Chol

85a: X = C(O), n = 4 85b: X = C(O)NH, n = 4 85c: X = C(O)NH, n = 6

16

O

2 CF3CO2-

Where:

H3C

O

17

85a-c

O Chol

Ac-GWHINSFYKNSV NH

O H3N

CH3

N H2

4 Cl

O Chol

80g: Chol-PR

HN

17

84c: CHOSS-Lys

H2 N

H3N

Br

N Br CH3 80f: Chol-DMAP N

N

O

I 84b: CHOSS-N+

83b: DOAG

O Chol

O

O CH3

CH3 N CH3

H3C

NH2 Cl

Br 81e: CvPB

80e: Chol-NMe

CH3

17

H2N

O

O

17

HN N H2

HN

81d: CcHBP

H3C O Chol N H3C CH3 Br

H3C

O Chol

O Chol

84a: CHOSS-N

CH3

O

83a: DODAG

6

Br

O

N H2

O

S

CH3 HN CH3 CF CO 3 2

Where: R=

O Chol

N

H N

N

H N

3 Cl

H3N

S

O

84a-d

N

4

I

80c: Chol-NMP

H3C

NH2

R N H

O

N H

O Chol

N

O Chol

82b: CAPG

O

O Chol

H N

H2N Cl

N

O

Cl H N

N H 82a: CEAG

81b: CvMI

O Chol

N

O Chol 4

I

Br

H2N

81a: CvPB

N

80b: Chol-MOR N

NH2

4

O

Br 80a: Chol-DABCO

O

O Chol

N

O

H N O 89a: CA

O N H

O Chol

H2N

H N

O O

O

O 89b: CPA

O

O

H N O 2

O N H

4.4. Oligomeric amphiphiles

In 1995 Talmon’s group introduced trimeric positively charged surfactant 91, and characterized their self-assembling properties in solution revealing the formation of branched


O Chol


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Page 58 of 140

tread-like micelles due to their particular molecular shape.176 A decade later, Ilies, Balaban and collaborators introduced amphiphilic pyridinium trimers and tetramers 92, 93a, 93b and 94 and assessed the transfection efficiency of their lipoplexes against NCI-H23 lung cancer cell line when the amphiphiles were formulated alone or co-formulated with DOPE or cholesterol at different molar ratios, using pGL3 plasmid. The most efficient lipoplexes were derived from amphiphiles 92 and 94, co-formulated with cholesterol or DOPE at 1/1 molar ratio, which reached or surpassed the transfection efficiency of LFM. Interestingly, both amphiphiles 92 and 94 contained procationic tertiary amino groups in the structure of the spacers connecting the pyridinium moieties. Notably, trimeric amphiphile 92 was able to transfect DNA alone, at a +/charge ratio of 2.43 More recently, Anderson, Langer and collaborators introduced a new series of oligomeric amphiphiles, termed lipidoids, which were synthesized through a combinatorial approach involving Michael addition of di- and polyamines to lipophilic acryl amides and esters of chain lengths between 9 and 18 carbon atoms. A series of 1200 compounds such as 95a-r and congeners bearing different core structures were generated using this synthetic strategy. Screening the amphiphiles for siRNA delivery into HeLa cells revealed that the most efficient lipidoids contained amide linkages and usually had more than two alkyl tails with an optimum tail length between 8 and 12 carbon atoms. In terms of tail numbers, the maximum efficiency was observed for lipidoids having one tail short of maximum substitution possible at the amine reactive centers, therefore possessing a secondary amine moiety. When the most efficient lipidoids were tested in vivo for Factor VII - targeting siRNA delivery to the liver in a mouse model best results were obtained with lipidoid 98N12-5 95d, having 5 aliphatic tails of 12 carbon atoms each attached on a triethylenetetramine core (“Core 98”). Mention must be made that 95d


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was slightly more efficient than its isomer 96d which does not have any free secondary N atom on triethylenetetramine core. 177 Lipidoid nanoparticles based on 95d have also been proven effective for silencing of liver-specific proprotein convertase subtilisin/kexin type 9 (PCSK9) in mice and cynomolgus monkeys, as a potential cross-species therapeutic regimen against hyperlipidemia. Thus, decreased levels of plasma cholesterol / LDL cholesterol were observed in rodents and nonhuman primates following systemic administration of 95d lipoplexes.178 Moreover, related siRNA nanoparticles with sizes between 70 and 100 nm were generated from lipidoid 95d co-formulated with cholesterol and polyethylene glycol (PEG)ceramide, with an encapsulation efficiently reaching 90%. These nanoparticles were used successfully in a Swiss mouse model to suppress PR8 strain influenza A virus replication in the lungs of the animals.179, 180 Following a significant formulation effort, nanoparticles of 95d/Chol/mPEG2000-C14 glyceride (42/48/10 molar ratio) “LNP01” were able to encapsulate anti-factor VII siRNA (lipid/siRNA weight ratio of 7.5) and to efficiently deliver it in a C57BL/6 mouse model.181 Following the initial success of amide lipidoids 95d and 96d, Anderson and collaborators attempted to improve the efficiency of this class of amphiphiles through systematic variation of side chains, their structure and length, in conjunction with amide and ester linkages and the most efficient backbones previously found - triethylenetetramine core (95 and 96) and 1,3propylenediamine -“core 100” (97). Compounds 96a, 96d, 96i, 96j, 96l, 96r and compounds 97a, 97d, 97i, 97j, 97l, 97r were synthesized together with compounds 96s-ad and 97s-ad also prepared for this study. Lipidoids were coformulated with cholesterol, mPEG2000-ceramideC16 at a molar ratio amphiphile/cholesterol/mPEG2000-ceramideC16 of 42/48/10 and nanoparticles were



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Page 60 of 140

formed through nanoprecipitation from ethanol. Addition of siRNA in 35% ethanol at pH = 5 followed by incubation at 37 oC yielded the siRNA complexes. Screening in HeLa cells at fixed siRNA amount (50 ng) and variable lipidoid/siRNA weight ratio (2.5, 5, 10, 15) and then at fixed lipidoid/siRNA weight ratio of 15 and variable siRNA amounts (0.5, 2.5, 5, 15, 50) revealed that the most efficient lipidoids were 96ac, 96ad and 96y, together with 97j, 97l, which were able to knockdown expression of luciferase more than 80% of control, surpassing LFM2000. Viability assays done in parallel showed the toxicity increasing in the order 96ac < 96y < 97j < 97l < 96ad. In vivo silencing of Factor VII in mice through administration of a single dose of 2.5 mg/kg siRNA complexed with selected library members from series 96 and 97 formulated with cholesterol and mPEG2000-ceramideC16 as presented above revealed the superiority of lipidoids 96 over their congeners 97. The most efficient representatives proved to be lipidoids 96ac and 97y, formulated at 12/1 weight ratio to siRNA, which were able to induce over 80% knockdown of factor VII in mice 72h post injection of 4 mg/kg siRNA.182 Anderson’s team also optimized these types of lipidoids to deliver immunostimulatory RNA (isRNA) into human peripheral blood mononuclear (PBMC) immune cells via toll-like receptors (TLR) TLR7 and TLR8 in order to mimic the immune responses triggered by viral infection through efficient and localized activation of innate immune responses. A number of 96 lipidoids based on various cores were generated in the same way as presented above.

177

These

lipidoids were used to complex RNA at five different lipidoid/RNA weight ratios (1, 2.5, 5, 10 and 15) and were screened in PBMC cells using a high-throughput assay that detected the γ-type interferon (IFN-γ). Out of over 900 combinations tested, 14 lipidoids displayed activities equal or greater than LFM2000, with the most efficient containing 1,3-propylenediamine core (“Core 100”), an amide linkage and dodecyl, tetradecyl and pentadecyl chain lengths. Thus, at lipidoid:


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siRNA weight ratio of 2.5, the transfection efficiency decreased in the order 98f > 98g > 98d > 98n. On the other hand, the C12 chain length gave efficient lipidoids with various cores. Consequently, a second library of heterogeneous lipidoids (98-101) based on 1,3propylenediamine (“Core 100”), C9/C12 acrylates and/or acrylamides, were synthesized and tested in the same in-vitro TLR7/8 PBMC cell model. Results revealed the superiority of lipidoids 99 and 102 over congeners 100 and 102 and over lipidoid 95d, through a wide range of lipidoid/isRNA weight ratios. Moreover, lipidoids 99 and 101 prove more efficient than 98d. However, in vivo testing of nanoparticles generated from lipidoids 99-102 coformulated with cholesterol and PEG revealed compounds 100, 102 at lipidoid: isRNA ratio of 15 as the most efficient formulations, which outperformed DOTAP and 95d. The lipidoid 101 and 102 complexes with isRNA were subsequently found efficacious in suppressing A/PR8 influenza virus replication in a mouse model. This strategy has potential utility for the development of a variety of therapeutics such as antiviral immunotherapy and vaccine adjuvants.183 Besides Michael addition, the same team used an alternative synthetic strategy that involved efficient ring opening of lipophilic epoxides with various chain structure and length by oligo/polyamine cores, thus generating lipidoids of type 103–106 and related congeners (126 lipidoids). Structure-activity relationship correlations within the lipidoid library have been performed after screening in a luciferase expressing HeLa-derived cell line, at lipidoid/antifirefly luciferase siRNA weight ratios of 2.5, 5, 10 and 15. Seven out of the top fifteen performing lipidoids had tails of 14 C atoms while lipidoids with tails shorter than 12 C atoms displayed silencing efficiencies less than 30%. In terms of core structure, the most efficient lipidoids were derived from 4-methyl-diethylenetriamine (“Core 113”) (102) followed by congeners obtained from “core 120” 104, “Core 96” 105 and “Core 200” 106. Thus, at low



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Page 62 of 140

siRNA doses and a constant lipidoid/siRNA weight ratio of 5, the transfection efficiency decreased in the order 103b > 103a > 104 > 105a > 105b > 106c. For in vivo translation twelve of the most efficient lipidoids in vitro were coformulated with DSPC and mPEG2000-DMG 107 and were administrated in mice as a single 3 mg/kg dose via tail vein injection. The most efficient formulation in this case was based on lipidoid 106, which was found to be two orders of magnitude more potent than 111d. This formulation was proved to provide durable single gene silencing, as well as efficient multigene (PCSK9, ApoB, FVII, Xbp1 and SORT1) silencing in the same mouse model. Moreover, nanoparticles based on lipidoid 106 were able to knockdown transthyretin (TTR) protein in non-human primates with exceptional efficiency.184 In addition, nanoparticle formulations of 106/DSPC/mPEG2000-DMG 107 were able to transfect anti-CCR2 siRNA to a murine model of myocardial infarction with established atherosclerosis, to pancreatic islets in diabetic C57BL/6 mice and to a murine model of lymphoma, with beneficial effects towards reduction of infarct size, inflammatory atherosclerosis, treatment of diabetes and reduction of lymphoma-associated macrophages.185 The mechanism of lipidoid internalization was examined in detail recently.32 The combinatorial synthetic approach introduced by Anderson and collaborators and described above was extended by Xu’s group, who introduced two new series of lipidoids having N,N-bis(2-hydroxyethyl)ethylene

diamine

(“core

86”,

lipidoids

108a-ab)

and

N-(3-

aminopropyl)diethyleneamine (“core 87”, lipidoids 109a-ab) backbones. These lipidoids were found efficacious in transfecting p-CMV β-gal DNA in HeLa cells, with lipidoids having amide linkers generally displaying better transfection efficiencies than congeners having either ester linkers or just hydroxyhydrocarbonate tails. The transfection of the most efficient lipidoids followed the sequence 14N-87 109c > 14N-86 108c > 15N-86 108d > 14C-86 108u > 16C-87


Page 63 of 140

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109z > 13N-86 108b > LFM2000. For the same backbone, the amines proved superior to hydroxy alkyls and esters, with 14N-87 109c > 16C-87 109l > 14O-87 109l, irrespective of the lipidoid/DNA charge ratio used (0.5, 1, 2, 5, 8, 10, 15, pCMV β-gal DNA). An optimal charge ratio of 5 was found for 14N-87 109c and 14O-87 109l, while the transfection efficiency of lipidoid 16C-87 109z increased with the increase of lipidoid/DNA charge ratio. Lipidoid 14N-87 109c was able to encapsulate up to 70% DNA, while 14O-87 109l and 16C-87 109z were able to encapsulate 82% and respectively 55%, as revealed by a picogreen assay.

186

These optimized

parameters were further used to test the efficiency of amide lipidoids of type 108, 109 and congeners 110 and 111 having unsaturated tails, versus their saturated congeners. It was found that unsaturated lipidoids 108ab, 109ab, 110a and 111a displayed superior transfection efficiency of both EGFP-DNA and GFP mRNA in HeLa cells as compared with their corresponding saturated congeners 108g, 109g, 110b and 111b. Moreover, the same unsaturated lipidoids were found at least 50% more efficient than LFM2000 for mRNA delivery into HeLa cells, but were found less potent than the commercial transfection system in delivering DNA in the same cell line. In general, the magnitude of mRNA expression was higher than that of DNA expression in HepG2, MCF-7, MDA-MB-231 and BJ cells, whereas in NIH-3T3 cells, the efficiency order was reversed.187 The Anderson – Langer team showed that microfluidic formulation technique relying on multi dilution process is a fast and efficient preparation method of well-defined LNPs (Lipid Nano Particles). Dilution can be used to control lipid self-aggregation into LNPs. The resulting LNPs were able to entrap negatively charged siRNA via electrostatic interactions. Aggregation of freshly formed siRNA-LNPs was further prevented using buffer solution which dilutes the ethanol content using an aqueous solution of nucleic acid. The freshly prepared siRNA-LNPs are



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Page 64 of 140

transferred into a rapid mixing microfluidic channel equipped with trenches and ridges where siRNA-LNPs of narrow distribution diameter are generated (60-90 nm) with a final composition of cationic lipid/DSPC/Chol/mPEG2000-DMG 2.0/0.28/0.52/0.13 mg/mL. The efficiency of LNPbased formulations was tested in HeLa cells and in a mouse model of Factor VII knockdown where they were effective at doses of 1 mg/kg.188 Similar LNP microfluidic formulation devices were reported by Wheeler et al. and by Zhigaltzev et al.189, 190 Very recently the same team described a new series of bioinspired

lipopeptide

nanoparticles (LPN), based on lipoamino acid derivatives synthesized from a library of amino acids upon condensation with fatty aldehydes, acrylates or epoxides bearing 12 C in the hydrocarbon chain. The new lipidoids (60 compounds) were assessed for their ability to encapsulate siRNA and the most promising candidates were further assessed in vivo for their ability to knockdown FVII in mice at a dose of 1 mg siRNA/kg. Products derived from C12 epoxide (E12) proved more active than congeners with lysine yielding the most active representative. The same fatty reagents were reacted with oligo and polylysine peptides generating a second library of lipidoids (43 compounds) that were screened in the same way for siRNA delivery efficiency. The most efficient amphiphiles were again the E12 derivatives represented by lipidoid cKK-E12 112b generated from a dilysine-derived diketopiperazine core. Thus, lipidoid cKK-E12 112b, formulated as 112b/Chol/DSPC/mPEG2000-DMG 107 (50/10/38.5/1.5 mol ratio), displayed high FVII RNA silencing effect in mice (ED50 = 0.002 mg/kg), rats (ED50 < 0.01 mg/kg) and nonhuman primates (over 95% silencing of TTR at 0.3 mg/kg siTTR). In addition, formulations derived from cKK-E12 showed excellent in vitro (HeLa cells)/ in vivo correlation (rodents and nonhuman primates, see entry 8 Table S2). A SAR study revealed that lipidoids derived from the same diketopiperazine core having peripheral alkyl


Page 65 of 140

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chains decorated with OH groups (112a-d) were consistently more potent than congeners having peripheral alkylated groups (112e-h) or peripheral esters (112i-l) derivatives (efficiency in vivo decreased in the order: cKK-E12 112b > cKK-A12 112f > cKK-O12 112j. The tail length proved also important, with derivatives having 12-14 C atoms being more potent than C16 and C10 congeners. Thus, the silencing efficiency of similar formulations decreased in the order cKK-E12 112b > cKK-E14 112c > cKK-E10 112a > cKK-E16 112d in the presence of ApoE in HeLa cells and these results translated in mouse model were they were shown to be effective at doses of 0.1 mg/kg for FVII silencing. Lipidoid cKK-E12 112b, formulated as described above (cKK-E12 LPN), was 500-fold more selective toward liver parenchymal cells, and required significant lower doses to induce selective silencing in hepatocytes compared with endothelial cells and immune cells from different from spleen, peritoneal cavity and bone marrow. LPN of cKK-E12 was well-tolerated in vivo at doses of 1 mg/kg (over 100-fold higher than ED50) as proved by the rat model. The same group showed that LPN of cKK-E12 112b surpassed the efficiency of C12-200 106 (effective at 0.005 mg/kg) and 98N12-5 95d (2 mg/kg) in silencing FVII in mouse model. In addition, LPN of cKK-E12 were able to silence CD45 in spleen macrophages and Tie2 in liver endothelial cells when administrated at 0.1 mg/kg.191 More recently, Whitehead et al. investigated the knockdown efficiency of lipidoids having biodegradable ester linkers which may be hydrolyzed by esterases present in liver. A large series (more than 1400) ester lipidoids derived from different linkers were tested in HeLaLuc cells, formulated as lipidoid/Chol/DSPC/PEG2000-DMG 107 at 50/38.5/10.5/0.75 molar ratio and at 10/1 lipid/siRNA-Luc weight ratio. Results revealed that lipidoids having tertiary or secondary amine cores linked to C12 and C13 aliphatic tails displayed over 95% knockdown efficiency in HeLa-Luc cells, surpassing C11, C14, and C10 congeners. The study also shown



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Page 66 of 140

that the maximum knockdown efficiency peaked at a substitution number of 4, with the branched cores containing tertiary and secondary amines being the most efficient (compounds 113 -117). The leading LNPs identified in HeLa cells were further investigated for FVII silencing in C57BL/6 mice at 5 mg siRNA/kg, identifying lipidoids that produced over 95% FVII protein inhibition. The EC50 of these compounds was subsequently found to vary from 0.05 to 1.5 mg/kg, with silencing efficiency decreasing in the order 116b > 113c =114c = 115c > 116a >> 117a, when coformulated with cholesterol, DSPC and mPEG2000-DMG 107 (50/38.5/(11.5-x)/x, mol ratio, x = 1.5). The amount of PEG in formulation was found to greatly influence the silencing efficiency, with a maximum knockdown achieved with formulations containing between 0.5 and 1 mol % mPEG2000-DMG 107. The leading formulation 304O13 114c/Chol/DSPC/mPEG2000-DMG 107 (50/38.5/10.75/0.75 mol ratio) produced 100% FVII knockdown at 0.1 mg/kg, with FVII protein levels returned to 98% of initial value after 18 days. Similar formulations of lipidoids 304O13 114c and 306O13 116c produced high efficiency CD45 silencing in immune cells from peritoneal cavity (monocytes, macrophages and dendritic cells) and spleen, especially in macrophages and dendritic cells. Formulations of 304O13 114c matched or surpassed the efficiency of congener 306O13 116c formulations in both cells (up to 90% CD45 knockdown in macrophages from peritoneal cavity). The surface pKa of these lipidoids played an important role towards in vivo efficacy, with all materials displaying in vivo potency having a surface pKa of 5.5 or higher. The most efficient lipidoid 304O13 114c had a pKa value of 6.8, which is in agreement with previous reports dealing with ionizable lipids.

148, 150

Toxicity of

novel lipidoids was found superior as compared with the previously established benchmark C12200 106 congener. Lipoplexes of LNP 114c accumulated in liver and spleen and were cleared from circulation within 20 min. The biodistribution and potency of these two lipidoids was found


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to be near identical, with both having an ED50 ~ 0.01 mg/kg. In general, LNPs are well tolerated in mouse model at therapeutically relevant doses; however, at high doses innate immune responses associated with liver necrosis and pancreatic islet inflammations were observed for lipidoid 114c and 106 formulations when administrated at 7.5 and 10 mg/kg as a single dose. Under repeated systemic administration (1 injection /week for 4 consecutive weeks) 106 LNP (C12-200) produced liver necrosis and inflammation at higher than 1 mg/kg doses, while 114c LNP did not display any liver toxicity in mice even at 5 mg/kg siRNA. In addition, these LNPs did not display any toxic effect in mice when administrated at doses at least two orders of magnitude above the ED50 (see also Table S2 entries 7 and 9).192



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CH3

CH3 N

H3C

N

N

11

H3C

11

H3C

N

H3C

CH3

3

3

11

H3C

3 Br

91

N

13

H3C

CH3

H3C

CH3

CH3

13

3 PF6

n

CH3

O

R1

N

N H

O

O

O

N

N

95

R

O

O

N O

O

H N

a-i:

CH3 n = 8-15, 17

O

j-r:

n = 8, 10-15,17

CH3

CH3 N CH3

O

u:

n

O

v:

O

n

s:

O

OH

O

x:

O

t:

O

O

N H

O

z:

CH3

3

aa:

O

NH

N

R1 N

O

ab:

O

ac:

O

ad:

CH3

CH3 N CH3 H

O

7

H3C CH3 O O

O

3

HN

HN H3C

HN

O

H3C

HN

O

11

HN

11

H3C

N

O

11

H3C

N HN

O II-3 = ND(2)LD(1)-100 101

11

11

O

O

N

I-4 = ND(2)NA(2)-100 100

CH3 O

HN

HN H3C

11

O

11

O

N

O I-3 = ND(2)NA(1)-100 99

11

N

O

8

11

O

CH3

CH3 HN

O

N HN

O

8

11

H3C

CH3

CH3

8

O

HN H3C

R1 O

98

CH3

CH3

O

N

CH3

O

CH3

CH3

HN HN

2 PF6

Core 100

97

CH3 CH3 CH3 CH3

O

CH3

13

CH3CH3

O

R1

CH3

3

~7

CH3

N

O

O

14

R1

R1

Core 100 O

R2

R1

O

y:

O H

O

O N H

96(a, d, i, j, l, r, s-ad)

R1

R1

14

CH3

N

3

94

R1

O

N

N

N

4

13

2 PF6

CH3

CH3 CH3

R1

Core 98

R1

1

13

N

3

CH3 H3C

93a: n = 12 93b: n = 14

R1

R1

Core 98

n

CH3

N

N

3

O

CH3 R1

N

4

O

92

13

H3C

13

CH3

N

3

H3C

CH3

CH3

CH3

N

CH3

N

N

Page 68 of 140

H3C

11

II-4 = ND(2)LD(2)-100 102

O

H3C H3C H3C

n

OH

N

HO

CH3

n

CH3 N

n

N

Core 113

OH

N

HO OH 103a, 103b

H3C

H3C

H3C

HO

R3 3 N

N

HO

O

n

CH3

j-r: n = 11 - 19

CH3 n OH s-aa: n = 11 - 19

O

N R3 R4

N CH3 113(a-c)

R4 N

R4 H C 3

N

N N 4 R 114(a-c)

7

8

H3C

13 O

H3C

13

n

a:

O R2

H N

CH3 8

N R4

CH3

CH3

N R4 115(a-c)

R4

R

R4 4 N

CH3 N 116(a-c)

R4

R

CH3

O O

n

CH3

112i: n = 8 cKK-O10 112j: n = 10 cKK-O12 112k:n = 12 cKK-O14 112l: n = 14 cKK-O16

N N 117(a-c)

N R4

R4

Where: R4 =

O O a: n=10 b: n=11 c: n=12


R2

17

R3 =

4

R 4 N

H N

b:

7

112e: n = 8 cKK-A10 112f: n = 10 cKK-A12 112g: n = 12 cKK-A14 112h: n = 14 cKK-A16

R4 N

CH3 N O

N

111a, 111b

CH3 R4 N R4

CH3

OMe O 50 O 107: mPEG2000-DMG H3C

R3 = -CnH2n+1

CH3

9

H N

O

O

R2

N

R2 =

CH3

OH 112a: n = 8 cKK-E10 112b: n = 10 cKK-E12 112c: n = 12 cKK-E14 112d: n = 14 cKK-E16

R3

n

O 110a, 110b O

N H ab

R3 =

NH

HO 106

9

N R2

9

HO

CH3

R2

O

Where:

HN

105a-c

N N

OH H3C

CH3

Core 200 N

HO

N

HO

N

105a: n = 11 C14-96 105b: n = 13 C16-96 105c: n = 15 C18-96

Core 87 R2 N

OH

CH3

N

109(a-ab)

O

112

R

HO

N

n

CH3

O

N CH3 n H a-i: n = 11 - 19

R4 4 N

n

n

HO Core 96

OH

104a: n = 11 C14-120 104b: n = 9 C12-120

Core 86 R2 N 2 108(a-ab) R

O

R

HO

N

HO

H3C H3C

104a, 104b

n

n

103a: n = 11 C14-113 103b; n = 9 C12-113

CH3

N

O

OH CH3

n

R2 =

HO

Core 120 O

n

9 9

n

CH3

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4.5. Dendrons and dendrimers

Dendrons and dendrimers are tree-like molecules that fill an intermediate position between small molecular weight compounds and polymers, combining the multivalency specific to polymers with a well-defined, precise structure (monodispersity) characteristic of small molecules. Specific covalent bonding and molecular connectivity allows precise tuning of molecular size, shape and curvature, thus generating unique molecular architectures and aggregation properties that recommends them as attractive materials for biotechnological applications, including genetic material delivery.38, 193-201 The dendritic architecture can be considered to be first used for gene delivery with the pioneering work of Jean Paul Behr who introduced the lipid DOGS 27 in 1989.93 More recently, Safinya’s group introduced multicationic dendron-like lipid MVL5 (118a) in an attempted to deliver pGL3 DNA at lower molar lipid/DNA charge ratios (ρchg). The authors showed that lipoplexes of 118a with DOPC displayed higher transfection activity in mouse fibroblast L cells than DOTAP 25/DOPC lipoplexes formulated at the same charge ratio (2.85) and containing the same percentage of colipid. The difference in transfection efficiency between the two systems increased one-to-three orders of magnitude when the amount of cationic lipid was reduced for 50 to 20 mol %. Safinya and collaborators also showed that multivalent lipids such as 118a allowed higher membrane charge densities than monovalent lipids such as DOTAP 25; therefore, a smaller number of multivalent lipids were required to form stable complexes.202 For siRNA



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delivery, the same group found that siRNA complexes required a molar charge ratio an order of magnitude higher than is normally required by pDNA complexes. The best transfection efficiency for 118a/DOPC-siRNA complex in mouse fibroblast L cells was observed at a molar charge ratio of 15. In addition, cationic lipid-siRNA complexes displayed lower toxicity and superior silencing efficiency over a large range of lipid/DNA molar ratios than monovalent DOTAP corresponding complexes. Overall, 118a could achieve higher total knockdown of the target gene in lipoplexes regimes of low toxicity.203 The same group introduced MVLBG2 118b, another cone-shaped cationic lipid with dendritic architecture bearing 16 positive charges in the polar head group. Lipoplexes generated from 118b/DOPC displayed different phases as a function of molar fraction of 118b in the lipid mixture: at 1/9 molar ratio (10 mol % 118b) the classical34, 204 lamellar phase LαC was observed, while at 1/3 molar ratio (25 mol% 118b) a new dual lattice structure (HIC) was observed in which hexagonally arranged tubular lipid micelles are surrounded by DNA rods forming a three dimensionally continuous substructure with honeycomb symmetry. This structure is different from the inverted hexagonal structure (HIIC) revealed by the same group for DOTAP/DOPE (at approx. 1/3 molar ratio) lipoplexes.35 Moreover, lipoplexes based on MVLBG2 118b/DOPC were found more transfection efficient than DOTAP 25/DOPC ones over a wide range of cationic lipid mole fraction at ρchg of 4.5 and 7 in mouse L cells, mouse embryonic fibroblasts, as well as in HeLa and 293 human tumor cell lines using pGL3 plasmid.205-207 In an effort to improve the cytotoxicity profile of their cationic lipids, the same group introduced multicationic lipids (CMVLn) 118d-g containing a disulfide group which can be cleaved by glutathione inside the cytoplasm. At a weight ratio lipid/DOPCDNA of 10/1 lipoplexes of CMVL5 (118g)/DOPC at 6/4 molar ratio displayed the highest transfection efficiency within CMV series and were equally efficient but significantly less


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cytotoxic than 118a and LFM2000. The transfection efficiency decreased in order 118g > 118f > 118d > 113e. However, at a higher weight ratio lipid/DNA of 20/1, the transfection efficiency followed the order 118f > 118g = 118e = 118d. Lipids 118e-g displayed above 90% cell viability at weight ratio of 20/1 lipid/DNA when they displayed the highest transfection efficiency and were considerable less cytotoxic than DOTAP and LFM2000. The cytotoxicity of the whole disulfide-containing CMVL series 118d-g in mouse L cells was lower as compared with MVL5 118a. The hydrodynamic diameter of lipoplexes generated from CMVL/DOPC (6/4 mol ratio) and DNA at +/- charge ratio lipid/DNA of 4, ranged from 100 - 300 nm. The diameter of these complexes increased with the increase in the molar fraction of CMVL from 0.4 to 1.

208

Moreover, the same group showed via small-angle and wide-angle X-ray scattering (SAXS and WAXS) a change in morphology of CMVL4 118f/DOPC-DNA complexes from LαC lamellar to a loosely organized phase upon exposure to reducing agents such as DTT and glutathione (GSH). The hydrophobic part rearranged into a tilted chain-ordered Lβ phase upon incubation at 37 oC, while the cationic part formed DNA bundles. Lipoplexes of 118h congener formulated in the same conditions as 118f were insensitive to reducing agents, proving that disulfide reduction of 118f is responsible for the change in the morphology.209 Malhotra et al. introduced a new series of multivalent oligoglycerol amphiphilic dendrons 119a-119d for gene delivery. The binding efficiency of these amphiphiles for DNA decreases in the order 119a = 119d > 119b > 119c when formulated at an optimal charge ratio +/- of 10. Therefore, within this series, tetraamine dendrons 119c, 119d gave the best DNA and siRNA compaction at charge ratios of 10, and respectively 70. Formulations of 119a and 119b displayed severe cytotoxic effects, assigned via WST-1 and xCELLigence assays, as compared with 119c and 119d congeners which displayed similar cytotoxic effects with LFM2000 at charge ratio +/-



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of 100. Polyglycerol amphiphiles 119a-119d were found efficacious for luciferase knockdown and GAPDH activity in HeLa Luc and HeLa cells, surpassing the efficiency of LFM2000 when formulated at +/- charge ratio of 100.210 Takahashi et al. showed that amphiphiles having a polyamidoamine (PAMAM) dendron and two dodecyl chains are efficacious as gene delivery vectors and their efficiency increased with the increasing generation of the dendron (120a < 120c 90%) and relatively modest transfection performances; the cytotoxicity increased in the order 121i < PEI < 121b < 121d.216 In an extended study, the same group compared the transfection efficiencies of hydrophobically modified dendrons 121c, 121e-121h. From the first dendron generation, the best transfection efficiencies were obtained for Chol-G1 121d and C12Lys-G1 121e when formulated as dendron/DNA at 4/1 and, respectively 5/1weight ratio in



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HEK 293 cells. From the second dendron generation, the best transfection efficiencies were obtained for Chol-G2 121b and C12-G2 121c, when formulated as dendron/pGL3 DNA at 4/1 and, respectively 6/1 weight ratio in the same cell line. First generation dendrons 121d-h required chloroquine to enhance the endosomal escape; however, second generation dendrons 121b and 121d did not require chloroquine, presumably due to higher number of amine groups per dendron which might provide some buffering effect. These systems displayed relatively modest transfection efficiencies and addition of small amounts of PEI did not improve significantly the transfection performances of these amphiphiles.217 In a related study, the same group introduced self-assembling dendrons having degradable ester linkers (122a-e). These amphiphiles were 10-fold more effective then PEI in transporting DNA into HEK293 cell line; however, they displayed only 10-20% of the transfection efficiency of PEI. For example, amphiphile C22-G2 122d, which self-assembled into micellar aggregates, was the most effective in up-taking pDNA into HEK293 cells, while it was devoid of any transfection activity, possible due to inefficient release of DNA from these dendriplexes. The highest transfection activity was observed for Chol-G2 (122a) and Chol2-G2 122e at dendron/pGL3 DNA weight ratio of 2/1, and respectively 10/1. In addition, cholesterylbased amphiphiles Chol-G2 122a and Chol2-G2 122e displayed lower cytotoxicities than singlechained aliphatic dendrimers, C12-G2 122b, C16-G2 122c and C22-G2 122d, preserving a cell viability of 100% at 60 µg/mg and respectively, 10% at 10 µg/mg. The degradation of these dendrons was investigated in detail by the same group; for example Chol-G2 122a was cleaved after 10h at pH = 7 but it remained stable at pH = 5.0. Unfortunately, the resulting degraded dendrons were still able to self-assemble and bundle DNA thus limiting the therapeutic effect.218


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OH O

O

H3C H3C

7

8

7

8O

O

H N

N H

O H3N

118a: MVL5 O

H3C H3C

7

8

7

8

H3N

NH2

NH H3N

H3C

8

7

8O

OH CF CO 3 2

O

HN

NH2

N H

O

NH2

NH3

OH O

NH3

H2N

NH2

NH3

N N H3C

O

O

H3C

7 7

8

O

S

S

O

N N

NH3

H3C

O

O

S

S

8

7

O 8

O

O

O

NH2

H3N

H3C

8

NH2

n

N

H3C

NH3

NH

n

7

8O

S

S

O

118g: CMVL5

N H

H3N

NH

120a-f

NH

H3C

8

7

8O

7

O

O

O

118h: TMVL4

O 120d: n= 17, R =

NH3

N H 121a-c

NH2

NH2

N H

O

O O O H3C O H3C

N N

3

H N

O

O N N R1

NH

O

H N

N

4 CF3CO2

H O N

O

O

O

O

122a-e

R2

122b: R1 = H- ; R2 = C11H23 122c: R1 = H- ; R2 = C15H31

O

C12-G2

H N

H O N

NH2

O

N H

H N

NH

O

NH2

NH

NH NH

NH

NH2

NH NH2

NH2 121a: R = C6H5-CH2121b: R = Chol-O121c: R = C11H23-

NH2

N H H N

N H H N

NH2 NH2

121d-i

CH3 121h: R =

121d: R = Chol-O121e: R = C11H23121f: R =

C22-G2 121g: R = Chol2-G2

N H

HN

Chol-G1 C12-G1 H N

C11H23

H3C O

NH

NH2

O

NH2

O

122e: R1 = R2 = Chol-O

O

C16-G2

122d: R1 = H- ; R2 = C21H43

N H

O

O

O

NH

NH2

O

H N

H N

O

O

NH N CH3 CH3 N

122a: R1 = H- ; R2 = Chol O

NH

NH2

O

NH

O

N H

N H

N H

O

CH3 N

R

N H

HN

O

O

H N

N

NH2

O O

O H3C

O

NH2

NH2

O O

NH H N

N H

O

O N H

120f: n = 17, R =

O O

NH N H

NH3 120e: n = 11, R =

H N

N H

NH O

O

O

NH2

O

N CH3

O

O

R

O

NH2

NH

NH2

N H

N H

NH

O

O

N H

H3N

R N R

O

O

H N

O O

O

NH3

NH2

NH

O

120c: n = 11, R =

O

R O

N H

HN

O

O

5 CF3CO2 H3C

N R

N

N H

NH2

O

4 CF3CO2

O

H N

N H

NH

O

120a: n = 11, R = -H 120b: n = 17, R = -H 7

NH2

NH3

O

H3C

N H

H3N NH3

NH3

O

O

8 CF3CO2

4 CF3CO2

NH3

O S

S

O

17

NH2 3 CF CO 3 2

O

H3C

O O

NH3

H3N

H N

118f: CMVL4

O

O

O

N H

O 7

O

NH3

O

O 119d:

NH3

NH

H3C

O

O

N

NH3

O O

O

O

O H3C

O

O

NH3

O

O

118e: CMVL3

H3C

O

N

O

NH3 2 CF CO 3 2

H N

N N

NH3

OH O

NH3

N H

O O

17

119b:

O

O

H3N

O

NH3

O 2 CF3CO2 O

N

O 8

O

O

O

H N

NH3

O

OH

119c: H3C

NH3

O

16 CF3CO2

NH

NH2

118d: CMVL2

NH3

O

NH2

O 7

O

O

N

119a:

17

118b: MVLBG2

H3C

17

NH2

H3N

O

O

O N H

N H

H3N

H3C

NH3 5 Cl O

N H O O

O

NH3

NH2

O

H N

O

NH

N N

C12Lys-G1 4 NH

O

H3C H3C

HN

H3C

D2Gly-G1 O

H3C

2

H3C

HN

H3C H3C CH3 O

C11H23 O

H3C H3C

N

O N

NH HN D1Gly-G1

N

Chol-O 121i: R = Chol-O

N

Chol2-G1 3

NH HN O

O


NH2 NH2


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Focusing on covalent dendrimers, one must mention the early reports of Haensler and Szoka who showed that polyamidoamine dendrimers (PAMAM) can mediate pCLUC4 and pCMV-β-Gal transfection into a variety of cell lines (CV-1, HeLa, HepG2, K-562, EL-4 and human T) cells.219 Later on, other covalent dendrimers based on PAMAM or related structures have been successfully applied for DNA delivery220, 221 and this field was constantly reviewed.38, 199, 222

Only nucleic acid delivery systems based on self-assembled dendrons and monodisperse

covalent dendrimers are reviewed in this section. Thus, recently, How et al. showed that third generation lysine-capped bis-dendrons 123a, 123b, were able to compact pEGFP-N1 at weight ratios of 10/1. Dendrimer 123b displayed remarkable pEGFP-N DNA expression into HEK293T, ND7 and B16F10 cells lines, where it equaled the transfection efficiency of Superfect™ and surpassed the efficiency of EFT when formulated with pEGFP at 10/1 weight ratio. In addition, both 123a and 123b were found to be non-toxic in these formulations, by displaying more than 90% cell viability by MTT assays.223 Simanek and his collaborators investigated triazine-based dendrimers for gene delivery and their research progress has been periodically reviewed.224, 225 The same group investigated recently the transfection efficiencies within a series of triazine dendrimers having rigid cores G11 (124), G2-1 (125a) and G3-1 (126), semi-flexible (bow-tie) B2-1 (127), or flexible cores F2-1 (128) in L929 and MeWo cell lines using pCMV-Luc DNA. Triazine dendrimers having flexible chains may provide a promising platform for gene delivery systems as they displayed significant improvements in the transfection efficiency in 3T3, L929 and MeWo cell lines and lower erythrocyte aggregation at N/P of 5, contrary to rigid cores which displayed erythrocyte aggregation. In terms of DNA complexation efficiency, higher dendrimer generation improves the compaction properties 124 < 125a < 126.226 Simanek et al. showed that flexible dendrimers


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such as 127 were able to self-aggregate into larger morphologies and peripheral amine groups linked to flexible dendrimers were more exposed to interact with nucleic acids as compared with amines linked to rigid dendritic systems. In addition, the hydrodynamic diameter of dendriplex formed by 128 (second generation) was higher than for the ones generated from rigid 126, suggesting that flexible dendrimers were more prone to self-aggregation than rigid ones. Moreover, authors proposed that flexible dendrimers were able to adopt conformations which enhanced the interactions with nucleic acids.227 The same group also investigated the effect of peripheral groups on transfection performance.228 Dendrimers decorated with twelve amine/guanidine peripheral groups were able to compact DNA better than congeners having a lower number of amine or guanidine peripheral headgroups; dendrimer 125a was more efficacious in binding DNA as compared with 125c and 125e. Introduction of guanidyl peripheral groups (dendrimer 125d) appeared to improve the packing as compared to analogues 125c containing primary amino groups, and this feature is important because electrostatic complexes having small diameter (less than 200 nm) are endocytosed by most cells.229 Interestingly, decoration of one peripheral triazine ring with one alkyl chain improved the DNA complexation efficiency, using pTRE2hygLuc plasmid. The highest transfection efficiency in MeWo cells was observed for 125a and 125f at +/- a charge ratio of 7.5. Within the series 125f, 126, 128 dendrimer 125f was the most efficient in knocking down luciferase expression in HeLa cells, using siRNA targeting firefly luciferase. The authors explained this result by the ability of n-hexyl chain to induce a favorable destabilization of the cell membrane.230 These dendrimers displayed lower cytotoxicity than PEI 25kDA, and within triazine core series dendrimers having flexible chains such as 128 were less cytotoxic then rigid congeners.227 The toxicity of these dendrimers appeared to depend more on the surface groups



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than on interior of the molecule. A recent study in this direction performed in C3H mice showed that dendrimers decorated with peripheral PEG2000 can be administrated via injection at doses up to 2.56 g/kg with no renal or hepatic acute toxicity observed; however, cationic triazine dendrimers induce death within 6 – 12h post-administration at doses of 160 mg/kg. These results suggest that the toxicity of triazine based dendrimers can be efficiently decreased by surface passivation using PEGylated peripheral chains.231, 232


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5. In vitro in vivo translation and selected clinical trials involving synthetic DNA and siRNA delivery systems

As presented above, the introduction of DOTMA 24a as the first synthetic cationic lipid for DNA delivery was followed by the invention of a large number of other cationic lipids and their formulations that were screened for efficiency and toxicity using in vitro cellular assays. Promising representatives with good transfection efficiency and very low cytotoxicity were also assessed in vivo, using experimental animal models, aiming for treatment of various types of cancer, genetic disorders and diseases.58-60 Unfortunately, the translational efforts in vitro/in vivo proved quite difficult due to the complexity of external delivery barriers encountered by lipoplexes in vivo.27 Very few formulations proved to be able to overcome these delivery barriers while maintaining an acceptable safety profile. Thus, DMRIE 24b-based lipoplexes were extensively used in human clinical trials due to high efficiency and low toxicity. For example, Leuvectin plasmid encoding human interleukin 2 (IL-2) gene was successfully delivered as DNA IL-2 cDNA/DMRIE 24b/DOPE lipoplexes in patients having metastatic renal cell carcinoma via intratumoral injection. A long lasting PR/CR clinical response was observed in 14% patients in phase II/III clinical trials while maintaining a good safety profile. Intravenous administration of these lipoplexes showed no toxicity even at high doses, without adverse immune responses being observed.233, 234 Clinical responses were observed at doses higher than 750 µg pDNA / patient and the detection of plasmid DNA and the increased IL-2 expression in tumor cells was observed at least 6 to 8 weeks form initiation of treatment in the phase I/II trial, with a disease reversion rate of 14%.234



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In the same context, Nabel et al. demonstrated for the first time that the intratumoral injection of DC-Chol 28/DOPE-pHLA-B7 (encoding a foreign major histocompatibility complex protein, deficient in melanoma patients) lipoplexes is safe and effective against stage IV skin melanoma in human clinical trials. One patient displayed regression of the injected nodules 60 days post treatment. These early results demonstrated the feasibility, safety and therapeutic potential of non-viral gene therapy in the management of melanoma.235 About the same time, in another clinical proof of concept, DC-Chol 28/DOPE system was used for delivery of E1A gene (encoding an epidermal growth factor (EGF) receptor-related protein) into patients with both HER-2/neu–overexpressing and low HER-2/neu–expressing breast and ovarian cancers. The E1A gene expression was detected in tumor cells and was accompanied by HER-2/neu downregulation, increased apoptosis, and reduced proliferation. However, due to the advanced disease stage, with bulky tumors known to have low prognosis, the evaluation of overall therapeutic/toxic effect of DC-Chol-E1A therapy was challenging.236,

237

The same DC-Chol

28/DOPE delivery system was used for transferring CFTR complementary cDNA into the airways of cystic fibrosis human subjects, with limited success.238 On the other hand, DOTAP 25/Chol formulation was found effective in the treatment of cystic fibrosis patients. It was administrated to the nasal epithelium via inhalation at doses of 400 µg CMV-CFTR complementary DNA/patient, with no evidence of nasal inflammation. The transgene DNA was detected up to one month post administration in seven out of eight subjects and partial sustained correction of CFTR toward normal values was achieved in two out of eight patients.239 DOTAP 25/Chol was also used in the management of advanced stage small cell lung cancer (SCLC) that has low prognosis. Thus, Ito et al. transfected FUS1 gene into FUS1deficient tumors, using DOTAP 25/Chol-FUS1 lipoplexes and a murine model. Administration


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of nanoparticles decreased the number of metastatic tumor nodules and increased the survival rate after intratumoral administration of these lipoplexes.240 These promising results prompted more recently a phase I clinical trial using FUS1-mediated molecular therapy in patients having lung cancer stage IV, at the University of Texas M. D. Anderson Cancer Center and prompted other research teams to focus on DOTAP 25-based lipoplexes as safe and effective gene delivery systems.241 More recently, DOTAP 25/Chol system was successfully used to deliver plasmid pLJ143-KB2-TUC (TUSC2 gene), in patients with lung tumors. TUSC2 gene is known to mediate apoptosis in cancer cells but not in normal cells. After systemic administration of lipoplexes, the disease was stabilized in 5 out of 31 patients for 2.6 - 10.8 months, with two patients displaying a 14% reduction in primary tumor size. However, the disease progression could not be stopped despite the fact that TUSC2 mRNA expression was observed in 5 out of 6 patients by RT-PCR. Minimal toxic effects, including hypophosphatemia, myalgia and fever, were observed at doses of 0.06 and 0.09 mg plasmid/kg.242 Another lipid investigated in human clinical trials for CFTR treatment was lipid 67 29a. Thus, Alton et al. successfully mediated p-CF1-cDNA gene transfer to the lungs and nose of patients having cystic fibrosis using lipid 67 29b lipoplexes (GL-67 29b/DOPE/DMPE-PEG5000 1/2/0.05 mol ratio), nasally administrated. About 25% restoration in chlorine channel function towards normal values was achieved in this study.12 Lipoplexes were well tolerated in general, with seven out of eight patients developing flu-like symptoms, myalgia and headache 6h post nebulization and returning to normal state 30h post-treatment. Ruiz et al. investigated the efficiency of lipid 67 29b-based lipoplexes in another CF human clinical trial. Physical examination, pulmonary function testing and chest CT proved that CF airway was not altered by lipid 67 29b-based lipoplexes, which was well tolerated in humans at concentrations as high as



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21.12 mg pCF1-CFTR/ml. Treated patients expressed CFTR transgene 2-7 days post administration, with side effects such as fever, myalgias and arthralgia being observed 6 h post administration. No changes in blood, serum or urine composition were observed posttreatment.243 We would like to emphasize at this point that the host-immune responses against delivery vectors or plasmids are responsible for inflammatory side effects associated with the use of synthetic gene delivery systems in vivo. Strategies to reduce inflammation and toxic side effects while preserving and even improving the transgene activity included the use of COX-2 inhibitors such as naproxen,244 and the use of high-quality ultra-purified plasmids devoid of endotoxin contamination or other E. coli derived products used to amplify the plasmids.243, 245-249 These early successes towards DNA delivery in humans via synthetic delivery systems prompted the expansion of the technology towards siRNA delivery. Many synthetic systems were used for successful delivery of both DNA (gene therapy) and siRNA (gene silencing) in vitro and in vivo as presented above. However, since the delivery barriers and the mechanism of action for the two nucleic acids differ, the in vitro/in vivo translation of DNA delivery and siRNA delivery was performed differently. As a consequence we analyzed these translational efforts in separate sections.

5.1. In vitro/in vivo translation for DNA delivery

The process of selecting promising in vivo synthetic DNA delivery systems continued to improve in both diversity of the representatives and in sophistication of the studies. In order to provide a better understating of structural and physicochemical parameters important in


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overcoming internal and external delivery barriers, we selected pertinent studies containing both in vitro and in vivo data (Table S1), hoping to provide an overview of current successful translational efforts in the field of DNA delivery. As one may observe, the targeted organ and the preferred route of administration of lipoplexes impact significantly on the physicochemical properties and composition of efficient lipoplexes. An analysis of studies presented in Table S1 reveals several general trends that can be useful for future formulation design, including the selected cationic component, colipid(s) and their molar ratio, the +/- charge ratio (N/P), size, ζ potential and surface chemistry of lipoplexes. Thus, liver targeting was achieved via i.v. delivery. Mukthavaram et al. showed that lipoplexes containing glycolipids targeting motifs such as 44c or 45a, with a diameter of 200-500 nm and a ζ potential ranging from - 0.1 ± 5.6 to + 8.9 ± 0.5 mV (pre-optimized in HepG2 and A549 cells), can selectively deliver genes to this organ.108 (Entry 1 of Table S1). For lung targeting two major route of administration were used: instillation/aerosolization and i.v. delivery. Focusing on aerosolization, Boomer et al.102 and Aissaoui et al. 173 showed that lipoplexes of 6b/DOPE and, respectively, DODAG 83a/DOPE/PEG4600-Chol 90, having a diameter of 200 - 450 nm and a ζ potential of +25 ± 5 mV, can selectively target and transfect the lungs of a mouse model for cystic fibrosis therapy. These lipids were also found efficient in vitro against several lung specific cells including IB-3, A549, mES and other cancer cell lines COS, HEK293, HeLa, B6F10 (entries 2, 3 of Table 1). Mention must be made that DODAG 83a/DOPE-pDNA lipoplexes were formulated with PEG4600 –Chol 90 in order to make the lipoplexes stable enough to resist in lung mucus and to avoid mucocilliary clearance mechanisms.173 (Entry 3 of Table S1)



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When i.v. delivery was used to target lungs, successful nanoparticles contained either simple formulations based on lipids BCAT 38, BSV4 60f or BSV18 60g and pDNA at +/- charge ratios between 4 and 6,102, 127, 130 or complex formulations using both cationic lipids and colipids such as 47a/Chol/DNA at a +/- charge ratio of 2.110 Thus, inclusion of colipid in the formulation improved nanoparticle’s robustness, which translated in an improved stability at lower +/- charge ratios where the particles are less immunogenic and the nucleic acid delivery is more effective. Efficient nanoparticles sizes were in the range of 150 to 790 nm, with the most effective representatives having a diameter of 150-300 nm and a ζ potential ranging from +10 to +45 mV (entries 2, 5, 6, 7 of Table S1) . More complex formulations such as DOTAP 25/Chol/DSPEPEG2000 87a, preoptimized in H1299 and NCI-H69 cell lines, with an average diameter of 350 nm, accumulated in lungs when administrated i.v.. However, for lipoplexes without targeting moieties aimed at lung cancer, the tumor selectivity was low irrespective of the DSPE-PEG2000 87a molar ratio used (Entry 8 of Table S1).250 Notably, lipoplexes formulated with PEGylated lipids having C18 aliphatic tails were less efficient than formulations containing PEGylated lipids with shorter aliphatic tails (C14, C16) or even cholesterol. One can observe that liver targeting was achieved with lipoplexes displaying low ζ potential while lung targeting required lipoplexes with much higher ζ potential. A similar formulation was used by Grosse et al. for targeting neuroblastoma following i.v. injection of lipoplexes derived from lipids ME42 57f and CH300 57a bearing oligo-ethylene oxide moieties attached to the lipid polar head. Formulations of these cationic lipids with DOPE and cleavable integrin-targeting peptide ME27 57i were preoptimized in bEND.3, neuro-2A, PVSMC, 16HBE14o- cell lines at N/P = 4 – 6, d ~ 100 nm, ζ = + 45 – 70 mV, and were subsequently injected in mice bearing neuro-2A xenografts. Both lipoplexes were able to reach


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tumor site selectively, with complexes based on biodegradable lipid ME42 57f displaying similar transfection efficiency with lipoplexes based on non-biodegradable congener CH300 57a.122 (Entry 9 of Table S1) Systemic administration of 48a/Chol and 49a/Chol lipoplexes, at 2/1 mol ratio, encapsulating the p53 pro-apoptotic gene, were found efficacious in animal models of melanoma.112, 251 In this case, lipoplexes were preoptimized on A549, L27 and S180 cell lines, and were having diameters around 300 nm, a +/- charge ratio of 6 and were containing the RGD targeting motif aimed at α5β1 integrin receptors overexpressed on the surface of the tumor. Importantly, formulations based on cationic lipids 48a and 49a were more efficient than similar formulations based on congeners 48b and 49b and generated significant tumor reduction 23 days post treatment (Entry 11 and 12, Table S1). Another strategy used for melanoma treatment was the administration of dendritic cells (DCs) pre-transfected with lipoplexes encapsulating plasmids encoding MART1 antigens normally displayed by melanoma cells (DNA vaccination). Thus, Srinivas et al. showed that lipoplexes of 42b/Chol, 43a/DOPC and 43b/DOPC, formulated with pDNA at N/P of 4 and 6, can efficiently transfect mbmDC cells via mannan receptors.106, 107 Subcutaneous immunization of rodents using these transfected mbmDC produced 8- to 10- fold smaller tumors than in control animals (entries 13 and 14 of Table S1). Topical delivery of lipoplexes was successfully applied in the treatment of scleroderma in rodents using lipoplexes of GS16-3-16 8/ DOPE/DPPC at 1/1/1 weight ratio and lipid/pDNA at N/P = 10. These complexes, optimized in PAM212 cells, displayed large diameters of 450-700 nm and ζ-potentials in the range of +45-50 mV. Formulations of GS C16-3-16 8 constantly outperformed the efficiency of similar formulations based on DC-Chol 28 while maintaining



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non-irritant properties when topically applied on melanoma animal models.252 (Entry 15 of Table S1)

5.2. In vitro/in vivo translation for siRNA delivery

Similarly to DNA delivery, a major challenge associated with the development of clinically viable siRNA delivery systems is the in vitro / in vivo translatability. However, there are major differences between the two technologies that impact the translation from in vitro efficiency assessment to in vivo biological models. Thus, the siRNAs typically used in gene silencing are relatively small (20-23 nucleotides), much smaller than the plasmids used for DNA delivery, thus allowing the routine generation of smaller nanoparticles as compared with DNA lipoplexes. Importantly, it is possible to finely tune the size of siRNA lipoplexes using microfluidic devices188, 189 for selective delivery to the desired tissue/organ, while simultaneously achieving high nucleic acid entrapment efficiency (~80%). Moreover, siRNA lipoplexes have to act in the cytoplasm and the foreign nucleic acid does not have to cross the nuclear membrane (an important delivery barrier), making the gene silencing process generally more efficient than gene delivery for the same amount of foreign nucleic acid administered. This explains the larger number of siRNA translational studies as compared with DNA-focused ones. We have selected representative examples of successful in vitro/in vivo translational studies for siRNA delivery, compiled in Table S2. One may observe while analyzing the data from Table S2 that, again, the targeted organ and the route of administration are key factors determining the physicochemical parameters of siRNA lipoplexes and also the successful in vitro in vivo translation. In this context, a major 86 ACS Paragon Plus Environment

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constraint is constituted by the natural fenestrations existent in different organs (liver, spleen, lungs etc.) or “the leaky” sites present in growing tumors. 253-256 The majority of these technologies targeted the liver, with the lipoplexes being administered systemically via i.v. injection. SiRNA delivery was used to induce potent anti-viral suppression of HBV and HCV and to target genes involved in metabolic pathways in cholesterol homeostasis and TTR (transthyretin) amyloidosis. PEGylated lipoplexes were used for all in vivo studies (entries 1-7 of Table S2), with sizes between 70 and 100 nm, which are slightly smaller than the fenestrations of the local blood vasculature existent in this organ. The “stealth” component used in majority of cases was PEG2000, conjugated with various hydrophobic anchors (C14-C18 phospholipids, lipid 107, 66l, etc. or cholesteryl derivatives such as 89b).

The

percentage of PEG lipid conjugate used in each case varied (1.4 to 10 mol%). The (pro)cationic lipids were always used in conjunction with colipids such as cholesterol, DOPE/POPE, and DPPC/DSPC. Another important feature in these studies was the use of ionizable lipids, which allowed the generation of stable and robust formulations at low ζ potentials, where the immunologic responses are minimal. In particular, HCV therapy is very challenging because the virus is prone to mutations and therapy becomes ineffective; therefore, cargos using multiple siRNA sequences were developed. Harashima et al. utilized a dicer-hunting strategy to identify effective siRNA sequences anti-HCV. They identified potent (low nM) anti-HCV siRNA targeting the most conserved sequences among different HCV genotypes such as the internal ribosome entry site (IRES) sequence. The authors subsequently used a mixture of three siRNAs (si197-1, si197-6 and si50-10) to target IRES sequence, which generated an inhibition of HCV genome replication. These results translated well form cell culture into BXP283-27 chimeric mice when YSK05-



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MEND, formulated as YSK05 67b/Chol/PEG2000-DMG 107 70/70/3 mol ratio with a diameter of ~50 nm, suppressed HCV replication for two weeks257 (Entry 1 of Table S2). Similar strategies have been used by other teams for HCV and HBV therapies172, 174 (Entries 2, 3 of Table S2). The development of the above-mentioned formulations was achieved using established cell cultures in vitro. Interestingly, in some cases the starting formulations did not contain a PEGylated lipid or colipid (cholesterol), known to be vital components for successful translation of the corresponding formulation in vivo145, 172, 177, 179, 184 (entries 3, 4, 5, 8, 12 of Table S2). Another important parameter for successful in vitro in vivo translation besides size and composition is the ratio between the (pro)cationic component and the nucleic acid, which also dictates the overall zeta potential of the nanoformulation. For in vivo applications, the most effective siRNA lipoplexes had a zeta potential close to neutral (0-15 mV) in order to maximize the circulation time and minimize the interactions with serum proteins and consequently the immunogenicity of the formulation (entries 1-7). Incorporation of biodegradable linkers in lipid design (entry 7 of Table S2) decreased the toxicity profile of the corresponding lipid while preserving high siRNA delivery efficiency.192 It is important to note that all these studies used PEGylated siRNA lipoplexes devoid of targeting systems, which depend essentially on physicochemical properties of the nanoparticles to ensure uptake in the liver. Size of the nanoparticles is the most important parameter and it is usually tuned to match the liver fenestration dimensions. Additionally, this optimum size, in conjunction with neutral zeta potential induced specific interactions with apolipoproteins circulating in blood, especially ApoB and ApoE. 184 258 The same general design principles and physicochemical properties are valid when targeting the lungs through systemic (i.v.) delivery in the antiviral therapies focused on treating


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influenza virus infections (entries 4, 8 of Table S2).177, 179 An important physicochemical feature influencing the efficiency of lung-targeted lipoplexes is constituted by their size, with lipoplexes of larger sizes (430 – 630 nm) proved to be more efficient than smaller ones (90 – 95 nm) as shown in entry 8 of Table S2.179 Interestingly, in the same study the authors proved that the siRNA sequence can generate immune responses that enhanced the antiviral response independently of its specific knockdown mechanism. Chemical modification of siRNA that blocked immune stimulation in vitro were shown to prevent activation of IFNα and TFNα in vivo in black Swiss mice using DOTAP 29 based formulations – known to be non-immunogenic. Furthermore, the use of lipoplexes derived from immunostimulatory and non-stimulatory siRNAs allowed the authors to decouple and assess the individual contribution of the two mechanisms of antiviral responses elicited by these siRNA lipoplexes. It is important to note that delivery of lipoplexes via aerosolization/inhalation requires much smaller (50 nm) lipoplexes (entry 4 of Table S2). Interestingly, kidney targeting was achieved with lipoplexes having a diameter of 110 120 nm, which is intermediate between optimized diameters of the complexes used for liver and for lung targeting (70-100 nm, 140-450 nm respectively), as presented in entry 9 of Table S2.150 An analysis of entries 10 - 12 of Table S2 reveals the fact that other organs such as ovaries, prostate and spleen can be targeted with lipoplexes of similar physicochemical and composition parameters as the ones used for liver targeting, with sizes around 80 nm, profiting from the specificity of siRNA against a particular mRNA target. Thus, Goldberg et al. used complexes of NC100 97c, having a diameter of ~75 nm, to deliver siRNAs against DNA repair enzyme poly(ADP-ribose) polymerase (Parp1).259 Optimization of lipoplexes in T22H (Brca1 wild type), BR5FVB1 (Brca-) and mT2K-Luc cells was followed by their assessment in BALB/c



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mice bearing T22H- and BR5FVB1 xenografts. Silencing of Parp1 improved the survival rate in mice bearing BR5FVB1 tumors (Brca- tumors), and showed no therapeutic advantage in mice bearing T22H tumors that have intact Brca1 gene, thus proving the selective tumor synthetic lethality. Co-delivery of siParp1 with siMyc (silencing the myc transcription factor) did not show additional therapeutic benefit.259 (Entry 10 of Table S2) In a relevant case of prostate cancer therapy, Lee et al. optimized formulations containing DLin-KC2-DMA 66a/DSPC/Chol/PEG-S-DMG 66m at 10/40/40/10 mol ratio in vitro from a series of lipoplexes containing different procationic lipids, using LNCaP, LAPC-4 and CWR22Rv1 cell lines. Another optimization round was performed in vivo using BALB/c mice bearing LNCaP xenografts, in order to find the optimum PEG-lipid conjugate and its percentage in the final formulation that will allow selective localization to prostate. Thus, DLin-KC2-DMA 66a/DSPC/Chol/PEG-C-DOMG 66l at 10/40/47.5/2.5 mol ratio, having a diameter of 86 nm, was able to reduce PSA levels, being 2-times more efficient than control (Entry 11 of Table S2).146 In all the examples provided in this category the siRNA nanoparticles were effective in the absence of any targeting moiety since tumor vasculatures are more permeable and display an enhanced permeability and retention effect (EPR).254-256 These siRNA complexes were designed to ensure long circulation life times, which in combination with the EPR effect translated into enhanced accumulation at tumor sites following i.v. injection. However, the efficiency of these tumor-aimed systems needs to be improved as it is currently several folds smaller as compared with liver-oriented complexes (compare dose of 10 mg siRNA/kg used for prostate cancer treatment vs. dose of 30 µg siRNA/kg used for achieving 50% gene silencing in the liver). The huge difference in overall efficiency was attributed by the authors to the targeting effects of ApoE lipoprotein recruited by the circulating siRNA complexes, which allows their uptake into


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hepatocytes via the LDL receptors.146 These findings suggest that one way to improve efficiency of tumor-aimed siRNA lipoplexes is through introduction of targeting moieties into their structure following the model of other related technologies. 146, 260 Importantly, tuning the size of siRNA complexes to 240-360 nm can induce their preferential accumulation to antigen presenting cells (APCs, macrophages and dendritic cells). These cells are capable of internalizing particles ranging from 50 nm to 10 µm, while the liver fenestrations are 100 -150 nm in diameter. Optimization and screening of various formulations in bmMϕ, bmDC APCs revealed DLin-KC2-DMA 66a/DSPC/Chol/PEG-C-DOMG 66l as the most efficient siRNA delivery system. These lipoplexes were further optimized for efficient in vivo delivery to APCs in terms of PEG-C-DOMG content and size and were subsequently able to selectively deliver model siRNA cargo into APCs from peritoneal cavity and spleen, for treatment of inflammatory and autoimmune diseases that requires silencing of genes in APCs.145 (Entry 12 of Table S2) A challenging target is constituted by metabolic skeletal disorders such as bone osteosarcomas. Zhang et al. compared DOTAP 25/DOPE/Chol/DSPE-MPEG2000 87a siPlekno1lipoplexes, targeting casein kinase-2 interacting protein-1 (Plekno1), with similar siRNA complexes having (AspSerSer)6 targeting units attached on the PEG-lipid conjugate 87b in hFOB 1.19 human-osteoblast and human osteoclast-like cells. In vitro optimization was followed by in vivo i.v. delivery. The authors revealed that (AspSerSer)6 targeting moiety could reroute PEGylated lipoplexes (140 nm) to the bones and induced a 2-fold increase efficiency for siPlekno1 delivery as compared with similar non-targeted lipoplexes into a bone osteosarcoma rat model. In addition, Plekno1-siRNA complexes were successfully restoring to bone density in



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bone deficient experimental animals, with efficiency comparable to jetPEI.261 (Entry 13 of Table S2) siRNA therapy is well advanced into human clinical trials (phase I and II), with many planned or ongoing programs being recently reported.262,263 Taberno et al. reported ALN-VSP lipoplexes encapsulating siRNAs against two different targets - VEGF-A and kinesin spindle protein (KSP). Their results suggested that ALN-VSP nanoparticles were safe and well-tolerated under systemic administration on human subjects at doses ranging from 0.1 to 1.5 mg siRNA/kg over a 4 month period treatment. In some cases, complete regression of liver metastases in endometrial cancer was observed after 2 cycles of ALN-VSP at 0.7 mg/kg and, after 6 cycles, partial response with complete regression of lymph node metastasis and reduction of liver tumor was achieved in certain patients.264 Significantly, one technology developed by Alnylam Pharmaceuticals (ALN-TTR02) reached the human clinical trial phase III for the treatment of transthyretin (TTR)-mediated amyloidosis265 using LNP of DLinMC3-DMA 66d.148

6. Conclusions and future directions

The above cited works reveal the fact that a large variety of amphiphiles with different packing parameters and molecular weights can be used for transfection purposes and show the critical impact of several factors that influence the formation and transfection efficiency of synthetic gene delivery systems. One can observe that these amphiphiles can rarely act alone and that they require variable amounts of colipids such as DOPE and cholesterol. Colipids can compensate for high molecular curvature of various classes of synthetic amphiphiles used for gene delivery, changing the packing parameter of the mixture and enhancing the stability of the


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supramolecular amphiphile self-assembly. Naturally, the amount of colipid required depends on the individual structure and packing parameter of the amphiphile used. The amount of colipid decreases with the increase of the packing parameter of the cationic amphiphiles and with the increase of stability of supramolecular assemblies. Colipids such as DOPE, DOPC have been shown to improve the fluidity, fusogenicity and colloidal stability of lipoplexes. As mentioned above, co-lipids can also control the structure of the lipoplex and can help overcome specific intracellular delivery barriers such as endosomal escape, thus being critical for the stability and efficiency of the delivery systems. An active research towards identifying better co-lipids for synthetic gene delivery systems is currently underway. Considering amphiphile design, one can observe that amphiphiles containing oleyl and linoleyl unsaturated hydrophobic tails or branched phytanoyl ones were more transfectionefficient than congeners having linear saturated aliphatic tails. Several SAR studies were done and were successful in identifying the influence of unsaturation(s) and their position in the hydrophobic chain on the transfection efficiency of the parent amphiphiles. Introduction of soft charge polar heads such as phosphonium, arsonium or imidazolium and pyridinium generated amphiphiles with remarkable transfection efficiencies in vitro and in vivo. Stimuli-responsive building blocks, biodegradable connecting linkers, targeting moieties and shielding (stealth) structural elements were constantly used to enhance the stability and specificity of delivery towards desired tissues/organs and to reduce the toxicity of the delivery systems. As presented above, enhanced transfection efficiency is the result of several events, namely the association of DNA to form the lipoplexes and their successful delivery to the target cells, followed by the dissociation of complexes inside the cell with the release of nucleic acid cargo. For DNA delivery the trafficking to the nucleus and passage through the nuclear pore



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constitute also very important barriers.33 The complexation event is favored by enhanced selfassembling of the cationic compound and by electrostatic association between the cationic polar heads and negatively charged nucleic acid backbone, which can be modulated through the increase of +/- charge ratio (N/P ratio). Cationic amphiphiles having enhanced self-assembling and high charge per mass (charge per mol) such as polycationic surfactants and lipids, gemini surfactants, lipidoids, are able to strongly bind and condense DNA forming stable lipoplexes at low charge ratios. However, in the DNA release process these highly positively charged cationic amphiphiles end up into the cytoplasm where they can cause significant cytotoxicity. The use of natural building blocks held together through biodegradable linkages, charge neutralization via programmed biodegradation (metabolic charge reversal) are common solutions to overcome these challenges. Another important trend that can be observed in the design of synthetic transfection systems is the adaptation of structure of lipoplexes and its cationic amphiphile component to the various delivery barriers. In this context, it must be emphasized that many delivery systems discussed in section 4 were developed for in vitro transfection and their translation towards in vivo applications is uncertain due to the additional delivery barriers. On the other hand, lipoplexes containing DSPC, cholesterol, ceramide, PEGylated lipids or other stealth layers, displayed improved robustness, enhanced shelf-time stability and conferred superior circulation time in vivo. The percentage of PEGylated lipid dictates the circulation time of the lipoplexes, with formulations having up to 10 mol% PEG2000-lipid being preferred for achieving long circulation times. In terms of aliphatic tails, PEGylated lipids having C14, C16 or cholesterol hydrophobic anchors were preferred over the C18 congeners since they were able to release the nucleic acid cargo more efficiently. Comprehensive optimization studies relating the nature and


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molar percentage of PEGylated lipid with circulation time, organ targeting and nucleic acid releasing properties were shown to be essential for achieving efficient delivery of both DNA and siRNA. It was recently shown by us that the use of combinations of amphiphiles from different classes (e.g. gemini surfactants and lipids) in the structure of lipoplexes can enhance the transfection efficiency through synergistic combination of the advantages conferred by each class of amphiphiles.266 Introduction of stimuli responsive groups such as procationic polar heads, having a pKa between 6 and 7, was found to be effective in enhancing transfection efficiency due to an endosomal buffering effect which facilitates nucleic acid release (endosomal escape). The use of cleavable, pH sensitive chemical groups such as esters (charge reversal), acetals (charge deciduous), disulfides and oximes in strategic position within the structure of these amphiphiles was also shown to be an efficient strategy towards releasing the nucleic acid cargo both in vitro and in vivo. Importantly, one has to be able to maintain a good stability of liposomes and lipoplexes in buffer and serum for these charge reversal/deciduous designs in order to ensure efficient cell delivery. In addition, incorporation of a targeting system such as sugar/sugar mimic units or peptides (RGD motifs) is an effective solution for optimizing biodistribution and maximizing the therapeutic index of the formulations. The diversity of (targeted) transfection systems available is important, as it was learned from the use of viral gene delivery systems that different tissues may require different transfection systems. In general, efficient gene delivery systems require high quality complex formulations. Tight control of the formulation process is critical to achieve optimum nucleic acid compaction and reproducible results in terms of physicochemical properties and composition of lipoplexes. Use of different formulation conditions, additives and excipients in the lipoplex generation



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process can change dramatically the nanoparticle properties and consequently the SAR within a series of related cationic amphiphiles since it affects the type of self-assemblies generated from the same amphiphile(s). Advanced microfluidic protocols for the preparation of LNP or SNALP developed by several teams allowed the generation reproducible formulations of controllable size, with high nucleic acid entrapment efficiency that can be used for rapid screening of various compositions and structural parameters. The tight control of particle size and polydispersity together with a neutral zeta potential can be used to manipulate the in vivo dynamics and fate (primary accumulation site) of the lipoplexes and induce a favorable interaction with natural circulating targeting apolipoproteins such as ApoE and ApoB. As mentioned above, nanoparticles able to recruit ApoE and ApoB are easily recognized by LDL receptors in hepatocytes. This particular en-route loading of targeting lipoproteins translate into a huge increase in overall efficiency of siRNA delivery systems targeting the liver. Consequently, the siRNA dose needed to achieve 50% gene silencing in this organ was significantly smaller as compared with other tissues, suggesting that one way to improve efficiency of siRNA lipoplexes is through introduction of specific targeting moieties into their structure. Notably, a major advantage of siRNA therapy (gene silencing) over the DNA delivery and gene therapy is the target selectivity induced by siRNA that restricts the impact of the technology to the cells where the mRNA target is expressed. Another advantage is the possibility to silence multiple genes using complexes containing several siRNAs, thus enhancing the therapeutic effect in terms of efficiency and selectivity. This strategy is particularly important in antiviral and anticancer therapies where viruses (such as hepatitis C and HIV) and tumors are undergoing frequent mutations. Recently, a proof-of-concept for RNAi therapeutics in humans was presented that constitutes a promising basis for further development of full therapeutic


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potential of gene silencing in human therapy. Similarly, DNA delivery using synthetic amphiphiles is progressing in human clinical trials for curing various forms of cancer and inherited genetic diseases such as cystic fibrosis, confirming the low immunogenicity of these vectors. Further advances in overcoming specific DNA delivery barriers will bring gene therapy closer to clinical use.

Associated Content Supporting information available: Representative in vitro/in vivo translation studies for DNA and RNA delivery, including formulations used and their physicochemical properties, targets, doses used, efficiency and toxicity. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding author *E-mail: [email protected]. Phone: +1 (215) 707-1749. Fax +1 (215) 707-5620.

Biographies

Dr. Bogdan Draghici earned his Ph.D. from the University of Florida, Department of Chemistry, under the supervision of Prof. Alan R. Katritzky in 2011, where he investigated the reactivity, rearrangement and tautomerism of several heterocyclic amphiphiles and prepared libraries of bioconjugates via benzotriazole mediated-coupling. After his graduation, he joined



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the group of Prof. Virgil Percec at the University of Pennsylvania, where he prepared and formulated self-assembling Janus dendrimers for drug delivery. Since 2013, he is a postdoctoral fellow of the School of Pharmacy, Temple University in the laboratory of Prof. Marc Ilies, affiliated to Moulder Center for Drug Discovery Research. His research interests include the design, preparation, formulation of small heterocyclic amphiphiles as memory enhancers and of medium size amphiphiles as gene/drug delivery vectors.

Dr. Marc A. Ilies was born and educated in Romania, receiving his B.Sc. and M.Sc. degrees from University of Bucharest and his Ph.D. in 2001 from Polytechnic University of Bucharest with Professor Alexandru T. Balaban on a thesis focused on the synthesis and biological properties of pyridinium salts. After post-doctoral studies at Texas A&M University and at University of Pennsylvania he joined the Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, where he is currently Associate Professor and member of Moulder Center for Drug Discovery Research. His research interests fit within the broadlydefined medicinal chemistry/chemical biology of membrane interfaces using pyridinium compounds, being focused on the development of chemical solutions for the efficient and selective delivery of drugs and genes for therapeutic purposes.

Acknowledgements


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This work was supported by Temple University School of Pharmacy Dean’s Office. The authors are acknowledging Dr. Peter H Doukas for his constant support towards this project within the framework of Temple University Drug Discovery Initiative.

Abbreviations Used APC – antigen presenting cells; AR– androgen receptor; ALT – alanine transaminase; Brca1 – breast cancer type 1 gene; CA – cholesteryl-aminooxy lipid; CD31 – protein that in humans is encoded by the PECAM1 gene found on chromosome 17; CD45 – leukocyte common antigen; CDAN - N'-1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine; cDNA – complementary DNA; CFTR– cystic fibrosis transmembrane conductance regulator; c–myc – gene which is activated in various animal and human tumors; CL – cationic lipid; CMVL – cleavable multivalent lipids; COX–2 – prostaglandin–endoperoxide synthase 2 (PTGS2); CPA – cholesteryl-PEG350-aminooxy lipid; DC-Chol – 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol hydrochloride; DFT – density functional theory; DLen-DMA - 1,2dilinolenyloxy-ketal-N,N-dimethyl-3-aminopropane; DLin-DMA – 1,2-dilinoleyloxy-ketal-N,Ndimethyl-3-aminopropane; DLin-KC2-DMA – 2,2-dilinoleyl-4-(2-dimethyl aminoethyl)-[1,3]dioxolane; D-Lin-MC3-DMA - [6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4(dimethylamino)butanoate]; DLS - dynamic light scattering; DNA IL–2 – interleukin–2 plasmid DNA; DODAP - 1,2-dioleoyl-3-dimethylammonium propane; DODMA – 1,2-dioleyloxy-N,Ndimethyl-3-aminopropane; DOGS – dioctadecyl amido glycyl spermine tetra trifluoroacetate; DOPC - 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE -1,2-di-(9Z-octadecenoyl)-snglycero-3-phosphoethanolamine; DOSPA - 2,3-dioleyloxy-N-[2(sperminecarbox amino)ethyl]N,N-dimethyl-1-propanaminium trifluoroacetate; DOTAP - 1,2-dioleoyl-3-trimethylammonium-



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propane (chloride salt); DOTMA – 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt); DRG neurons – posterior root ganglion neurons; DSDMA - 1,2-distearyloxyN,N-dimethyl-3-aminopropane; DSPC – 1,2-distearoyl-sn-glycero-3-phosphocholine; DTT – dithiothreitol; E1A – adenovirus early region 1A; EDMPC – 1,2-dimyristoyl-sn-glycero-3ethylphosphocholine (chloride salt); EGFP - enhanced green florescent protein; EFT – Effectene®; FBS – fetal bovine serum; FUS1 – tumor suppressor gene identified in the human chromosome 3p21.3 region; GAPDH – glyceraldehyde-3-phosphate dehydrogenase; GS Gemini surfactant; GFP – green fluorescent protein; HER–2 – human epidermal growth factor receptor 2 gene; IFN-α – interferon α; ires – internal ribosome entry site; isRNA – immunostimulatory RNA; KE – knockdown efficiency; KDalert – assay kit for the measurement of GAPDH enzyme activity in cultured human or murine cells; LFM – Lipofectamine®; LFM2000 – Lipofectamine 2000®; LDH – lactate dehydrogenase; LDL – lowdensity lipoproteins; LNP – lipid nanoparticle; LPD – lipopolyplex; LPN – Lipopeptide nanoparticle; LRNP – lipidoid-RNA nanoparticles; MEND – multifunctional envelope-type nanodevice; mES – mouse embryonic stem cells; MFI – mean fluorescence intensity; MTT – 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; MVL – multivalent lipid; MVLBG2 – N-1-2-[((1S)-1,4-Di[(1S)-1,4-di((1S)-1,4-di[(3-aminopropyl)amino]butylcarboxamido) butyl]carboxamidobutyl)-carboxamido]- ethyl-3,4-di[(Z)-9-octadecenyloxy]benzamide; NFX – NeoFX®; PBS – phosphate buffered saline; pCMV-MART1 – plasmid DNA encoding the human MelanA/MART-1 antigen; PCSK9 (mRNA) – proprotein convertase subtilisin/kexin type 9 (messenger RNA); pDNA – plasmid deoxyribonucleic acid; PEI (25kDa) – polyethylenimine (MW 25kDa); PEG2000–DMyrG – 1,2–dimyristoyl–sn–glycerol, methoxypolyethylene glycol; PEG-C-DOMG – R-3-[(ω-methoxy poly(ethylene glycol)2000) 100 ACS Paragon Plus Environment

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carbamoyl)]-1,2-dimyristyloxl- propyl-3-amine; PEG-DMP – 1-(mono-methoxypolyethylene glycol)-2,3-dimyrystoyl glycerol; PFU – plaque forming units; pHLA–B7 – histocompatibility complex protein plasmid– human melanoma treatment; plekno1-siRNA – siRNA that targets casein kinase–2 interacting protein–1 (also known as Ckip–1); PLK1 – Polo–like kinase 1; POPE – 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PR/CR – partial response / complete response; PSA – prostate specific antigen; PTEN – phosphatase and tensin homolog gene; PVSMC – porcine vascular smooth muscle cells; PXB283–27 – chimeric mice with humanized hepatocytes; RACE – rapid amplification of cDNA ends; RNAi – RNA interference, post transcriptional gene silencing; RSV – siRNA specific to respiratory syncytial virus; RT– PCR – reverse transcription polymerase chain reaction; SAINT-2 – 4-[(9Z,28Z)-heptatriaconta9,28-dien-19-y]-1-methylpyridin-1-ium chloride; SAXS – small–angle X–ray scattering; SC– siRNA – scrambled siRNA (control); siCCR(2) – short interfering chemokine receptor (2); siCon – siRNA control; Parp1 – poly(ADP–ribose) polymerase 1; siRNA – small interfering ribonucleic acid; SORT1 – sortilin 1 protein–coding gene; SNALP – stable nucleic acid lipid particles; SP-DiOC18 – 3,3'-dioctadecyl-5,5'-di(4-sulfophenyl)oxacarbocyanine, sodium salt; sshRNA – small synthetic stem-loop (or hairpin) RNAs; SWCNT – single-wall carbon nanotubes; TE – transfection efficiency; TFNα – tumor necrosis factor α; TTR – transthyretin; TUSC2 – tumor suppressor candidate 2; WAXS – wide–angle X–ray scattering; Xbp1 – X-box binding protein 1.



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