Synthetic Delivery Systems for DNA, siRNA, and mRNA Based on

Chapter 1

Synthetic Delivery Systems for DNA, siRNA, and mRNA Based on Pyridinium Amphiphiles Downloaded by 80.82.77.83 on November 21, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch001

Marc A Ilies,*,1,2 Uttam Satyal,1 and Vishnu D. Sharma1 1Department

of Pharmaceutical Sciences and Moulder Center of Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, Pennsylvania 19140, United States 2Temple Materials Institute, 1803 N. Broad Street, Philadelphia, Pennsylvania 19122, United States *E-mail: [email protected].

Self-assembled synthetic nucleic acid delivery systems represent the “bottom up” approach to gene delivery and gene silencing, in which novel cationic and procationic amphiphiles are used to 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. However, the use of synthetic vectors and nucleic acid delivery methods in clinical trials increased constantly over time, mainly due to their superior safety. In recent years a plethora of studies evidenced that heterocyclic cationic amphiphiles are superior to classical ammonium-based amphiphiles, with pyridinium representatives occupying a privileged spot within this class of amphiphilic compounds. The chapter presents recent progresses that narrowed the efficiency gap through better understanding of the delivery barriers and incorporation of this knowledge in the design of novel pyridinium amphiphiles and formulations. We are also revealing relevant structure-properties and structure-activity relationships that were drawn within each pyridinium amphiphile class, presenting the cellular and animal models used to generate them. Benchmarking of the efficiency of these delivery systems relative to established formulations used in human clinical trials was also performed whenever it was possible. © 2017 American Chemical Society Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by 80.82.77.83 on November 21, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch001

Introduction Paul Ehrlich’s concept of the “magic bullet” – the ability to treat diseases with highly selective agents – remained an elusive desiderate throughout the 20th century. However, in the beginning of the ’80s decade significant progresses in isolation, purification and cloning of DNA opened the possibility to use engineered nucleic acids to correct dysfunctions and diseases at the molecular level in a selective manner. Several teams reported successful transfer and expression (transfection) of plasmid DNA into different human cells and tissues, proving the possibility to use functional plasmid DNA (pDNA) as a new “drug” to treat hereditary and acquired diseases at cellular level through gene therapy (1–3). Since then, gene therapy and the use of DNA as a revolutionary way to deliver proteins into cells grew constantly, culminating in the first commercially-approved gene therapy products, Gendicine® and alipogene tiparvovec (Glybera®) (1, 4–6). Completion of human genome charting and the progresses made by genetics and proteomics towards involvement of genes in different diseases greatly expanded the potential impact of gene therapy. The discovery of RNA interference (7) at the end of the 90’s and the recent discovery and development of CRISPR-Cas9-mediated genome editing (8–10) enhanced and broadened the capabilities of nucleic acid-mediated therapy (11–14). All these technologies rely on finding efficient delivery vehicle(s) for each type of nucleic acid –pDNA, oligonucleotides, siRNA, mRNA, etc, as these macromolecules are membrane-impermeant due to their polyanionic nature and are rapidly degraded outside and inside the cells by nucleases (15, 16). Viruses were the delivery vehicles of choice since they evolved to avoid external and internal nucleic acid delivery barriers in order to perform this specific task. However, the immune systems of the hosts evolved in parallel to recognize these biological transfection systems and to fight viral infections efficiently. Viral vectors use was associated with severe immune reactions that usually prevented repeated administrations and proved fatal in certain instances. Consequently, researchers embarked in a “top-down” approach, by deleting immunogenic features from the structure of complex viruses or by using simpler viruses. This strategy usually translated into an increased safety profile of the biological transfection systems but also into a decrease of their transfection efficiency. Intense efforts to balance a high transfection efficiency with tolerable immune responses are underway (4, 15, 17, 18). Synthetic Nucleic Acid Delivery Systems An opposite, “bottom up”, approach to gene delivery and gene editing was initiated by chemists and pharmaceutical scientists to produce synthetic nucleic acid delivery systems. These artificial transfection systems are based on self-assembled cationic amphiphiles that could compact and transfect pDNA similarly to viral vectors. Many attempts were made, with partial success (19). In 1987 Felgner et al. introduced cationic lipid DOTMA 1, which became the prototype of cationic amphiphiles, comprising of a quaternary ammonium polar head, attached via a short linker to a dialkoxyglycerol hydrophobic tail. Several 2 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


synthetic variations at the level of these structural elements quickly followed, yielding quaternary ammonium (pro)cationic lipids DOTAP 2, DODAB 3, DOSPA 4, DC-Chol 5, EGX 6, together with its phosphonium and arsonium congeners 7 and 8. Other variations at the level of the cationic polar head included amidinium and guanidinium lipids such as Di-C14-amidine 9 and BGTC 10, imidazolium lipids such as 11 or pyridinium lipids such as SAINT-2 12 and SPYRIT-7 13 (Chart 1A). These early studies involving a large variety of cationic lipids quickly revealed that synthetic transfection systems have a relatively low immunogenicity and a high cargo-loading capacity, being suitable for the packing and delivery of diverse nucleic acids (16, 18, 20–22). Researchers also recognized quickly the impact of the molecular shape of the cationic amphiphile, represented best by packing parameter P (23), towards the amphiphile ability to self-assemble and produce cationic assemblies able to pack and deliver nucleic acids. Packing parameter P is defined as v0/ael0, where v0 represents the volume occupied by the hydrophobic tail of the amphiphile, ae depicts the equilibrium area of a molecule of amphiphile at the aggregate surface, and l0 is the tail length (23). Thus, during the early formulation attempts, co-lipids such as DOPE 14 and cholesterol (Chol) 15 (Chart 1B) that have a P > 1 were used to shape-compensate cationic lipids with P < 1 in order to produce tightly packed, robust, lipid bilayers with an average P around 1. This cationic amphiphile supramolecular co-assembly bilayer (usually self-closed into a liposome) can associate and subsequently compact the nucleic acid. The process is driven by the electrostatic attraction between the cationic polar headgroups of the positively charged amphiphile and the negatively charged phosphate of the nucleic acid, together with the release of counterions of both entities. This later process occurs with significant entropic gain and has a significant contribution to cationic lipid/nucleic acid complex (lipoplex) formation and stability (24). Lipoplex Formation, Structure, Stability, and Delivery Barriers Small angle X-ray scattering (SAXS) studies revealed that in the case of DNA, the nucleic acid is sandwiched between cationic lipid assemblies, in either a lamellar LαC (25), or inverted hexagonal HIIC structure (26), depending on the composition of the cationic amphiphile mixture used and on the packing parameter of the individual components. For siRNA-based lipoplexes a gyroid cubic structure QIIsiRNA was also identified (27), besides the LαsiRNA and HIIsiRNA structures (Figure 1A-C). The stability of a lipoplex generated from a given pDNA is directly related to the ΔG of lipoplex formation, which depends the nature of the polar head of the cationic component, its counterions, counterions of DNA, the size of the plasmid and the stability of the supramolecular amphiphile assembly. The later one is directly proportional with the overall hydrophobic effect generated by its amphiphilic components. Therefore all structural elements of the cationic amphiphile will impact the nucleic acid compaction process: polar head type and size, its counterions, the hydrophobic anchor nature and size, and the nature and size of the linker, all contributing to the overall amphiphile shape and packing parameter. Other important parameters are the molar ratio between the cationic 3 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


lipid and co-lipid, the nominal charge ratio between the cationic lipid assembly and the nucleic acid (oven denominated N/P ratio), the type and size of nucleic acid and the protocol used to bring these entities together (formulation protocol). All these parameters will influence the physicochemical properties (size, shape, zeta potential) of the lipoplex, which in turn will determine the lipoplex ability to overcome the delivery barriers, and also the stability, efficiency and toxicity of the lipoplex in vitro and in vivo (16, 19, 28–31).

Chart 1. Representative cationic lipids (A) and co-lipids (B) used in self-assembled systems for nucleic acid delivery.

4 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 1. The main structures found for lipoplexes: the lamellar Lα (A), inverted hexagonal HII (B) and the gyroid cubic structure QIIG (C) formed by DNA (A, B) and by siRNA (A-C) with cationic lipid-based assemblies. Adapted with permission from reference (26). Copyright 1998 American Association for the Advancement of Sciences; and from reference (27). Copyright 2010 American Chemical Society.

The delivery barriers encountered by the lipoplexes in vivo depend on the target tissue and on the route of administration selected for initial delivery of lipoplexes. One can separate them into extracellular barriers, namely the barriers needed to be overcome by lipoplexes till the targeted tissue is reached and the intracellular barriers that lipoplexes are facing before the packed nucleic acid can elicit its action while inside the target cells. External delivery barriers include lipoplex penetration through external protective cellular layers of skin and different mucosa and through additional layers of mucus and other natural polymers shielding them. If vasculature is reached or used as initial administration route, then lipoplexes should minimize their interaction with figurative elements of blood, plasma protein, complement system, and avoid the hepatic, splenic and renal clearance systems. The endothelium lining the blood vessels also constitutes a major delivery barrier. In the case of many tumors the endothelium is quite leaky due to poorly formed blood vessels, which favors the permeation and accumulation of lipoplexes at tumor site. Lipoplexes have to be able to pass intact the extracellular matrix before reaching the target cells, where they have to be internalized either passively, via poration of plasma membrane or actively, via endocytosis and related mechanisms of internalization. Intracellular barriers include endosomal escape, trafficking into the cytoplasm and nuclear import (for DNA) (32–34). 5 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Synthetic Cationic Lipids for Nucleic Acid Delivery Therefore, for successful nucleic acid delivery the cationic supramolecular assembly should be able to compact efficiently the nucleic acid into stable lipoplexes with optimum physicochemical properties, transport them over the above mentioned delivery barriers, and deliver their nucleic acid cargo inside the target cells (35, 36). Due to the antagonistic nature of these processes, many (pro)cationic lipids-based formulations were synthesized in the three decades of active research following the success of DOTMA 1, with cationic lipids having different (pro)cationic polar heads, linkers and hydrophobic chains, as outlined in Chart 1 (16, 19, 21, 30, 37–40). Thus, an examination of Chart 1 reveals the large diversity of cationic or procationic polar heads of alkylammonium type such as DOTMA 1 (41), its ester congener DOTAP 2 (42), the simple lipid DODAB 3, phosphonate-backboned lipid 6 and its phosphonium and arsonium congeners 7 and 8, monoamine DCChol 5 and polyamine DOSPA 4, together with amidinium Di-C14amidine 9, guanidinium BGTC 10 and heterocyclic imidazolium Im118 11 and pyridinium SAINT-2 12 and SPYRIT-7 13. Chart 1 also reveals the different cationic lipid designs, having either two alkyl chains or a cholesteryl moiety as hydrophobic structural elements. One may notice that the hydrophobic anchor is connected to the polar head either directly (as in the case of DODAB 3, DiC14amidine 9) or via a short, flexible linker as in the other lipids presented in Chart 1. Immediately after their introduction, many cationic lipids-based formulations became available: DOTMA 1/DOPE 14 (1/1 molar ratio) as Lipofectin®, DOSPA 4/DOPE 14 (3/1 w/w) as Lipofectamine®, DOTAP 2 alone or DOTAP 2/Cholesterol 15, DC-Chol 5 alone or DC-Chol 5/DOPE 14 and were frequently used as positive controls to benchmark future formulations (19, 43). Out of them, DOTAP 2/Cholesterol 15 1/1 (molar ratio) was successfully used in human subjects for gene therapy of cystic fibrosis (44, 45) and lung cancer (46, 47). Reporter gene plasmids encoding firefly luciferase, green fluorescent protein (GFP), β-galactosidase, etc were used to assess transfection efficiency in vitro and in vivo (16). Extensive structure-activity and structure-property studies within different classes of cationic lipids and related amphiphiles (16, 48, 49) revealed that when the charge density on the lipid cationic polar head is decreasing by either increasing the volume of the atom carrying the positive charge (N+ → P+ → As+) (48) and/or by delocalizing the charge within a heterocyclic polar head (N+ → Imidazolium+ → Pyridinium+) (50–54) the transfection activity of the corresponding cationic lipids is increasing. The arsonium, imidazolium and pyridinium bulky, polarizable, “softer” cationic species are believed to be able to optimally interact with the delocalized – hence also “soft”- negative phosphodiester groups in nucleic acids. They also seem to provide a better balance for the antagonistic processes of lipoplex assembly (outside the cell) and lipoplex disassembly (inside the cell, with nucleic acid delivery).



Pyridinium Amphiphiles for Nucleic Acid Delivery Pyridinium cationic lipids were introduced by Engberts’ team, who synthesized SAINT-2 12 (from Synthetic Amphiphile INTeraction) and congeners 16-18 via quaternization of corresponding lipophilic pyridines (Chart 2) (50, 55). In collaboration with Hoekstra and Ruiters these lipids were efficiently co-formulated with DOPE 14 (1/1 molar ratio) into liposomes, which were proved to be able to compact and transfect pDNA in vitro, on COS-7, CV-1, BHK, and 36C2.21 cell lines. In a detailed structure-activity relationships study, the team revealed that elongation of the two hydrocarbon tails from C12 to C16 (lipids 17→19) increases the transfection efficiency to levels comparable with commercial Lipofectin® when these pyridinium cationic lipids were co-formulated with DOPE 14 at 1/1 molar ratio. Further elongation of the tails to C18 (SAINT-5 20/DOPE 14) decreased the ability to deliver pDNA, probably due to increase stiffness of the resulting bilayer. Introduction of a double bond into each C18 hydrophobic chain of lipid decreased the gel to liquid crystalline transition temperature for the corresponding pyridinium amphiphile and increased bilayer fluidity, translating into a higher transfection efficiency of the amphiphile/DOPE 14 (1/1 molar ratio) formulation. Thus, lipoplexes made from pyridinium lipid SAINT-4 21, which has two trans double bonds in the C18 hydrocarbon chains, were four times more efficient than similar lipoplexes made from saturated C18 congener SAINT-5 20. Furthermore, lipoplexes formulated in identical conditions using the stereoisomer SAINT-2 12, having the double bonds in cis configuration, were almost three times more transfection efficient than double-trans SAINT-4 21 and almost 12 times more efficient than the saturated C18 congener SAINT-5 20 (Chart 2) (50, 55). SAINT-2 pyridinium cationic lipid 12 was the most efficient cationic lipid generated in these studies and formulations based on it constituted a benchmark standard ever since (vide infra). Attempts to combine a double cis-unsaturation in a shorter chain (C14 or C16) yielded lipids 22 and 23 that displayed a transfection efficiency higher than Lipofectin® but inferior to SAINT-2 12 (56). Interestingly, the addition of another positive charge in the cationic polar head, as in lipids SAINT-21 24, SAINT9 25 and their congeners, significantly decreased the transfection efficiency as compared with parent monocationic amphiphiles, irrespective of the nature of the second charge or of the spacer separating the two different positive charges in the polar head (Chart 2) (50, 55, 57). This was probably due to different packing and release characteristics of pDNA in/from the corresponding lipoplexes. SAXS studies revealed that lipoplexes generated from SAINT-2 12/DOPE 14 (1/1 molar ratio) and pDNA had a hexagonal structure, while lipoplexes formulated in similar conditions from SAINT-21 24 had a lamellar structure (57). In an attempt to make the pyridinium lipids more biocompatible, Engberts’ team inserted biodegradable ester bonds in between the pyridinium polar head and the hydrophobic anchors, generating pyridinium amphiphiles 26 – 30 (Chart 2) (56). Their transfection efficiency was 2-4 times higher than Lipofectin® when tested on COS-7 cells, still inferior to SAINT-2 12. The transfection studies also revealed that pyridinium lipids bearing cis-double bonds in the 7 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


hydrophobic chains 28 and 29 were less efficient than their saturated congeners 26 and 27, in an opposite trend to previous studies (vide supra, Chart 2) (56). Lipids 26 and 27 displayed good transfection efficiency, 3 times higher than Lipofectin®, doubled by an excellent cytotoxic profile, also better than Lipofectin®. Interestingly, the authors determined that the relative orientation of the pyridinium ring to the backbone had a significant impact on the transfection efficiency, with 3-pyridinium lipid 30 being almost 4 times more efficient than 4-pyridinium congener 27, while displaying the same excellent cytotoxic profile (56). The above findings clearly reveal that all structural elements of cationic lipids contribute to the self-assembling process and to the subsequent nucleic acid compaction and delivery.

Chart 2. Pyridinium cationic lipids synthesized through alkylation of pyridines.

Enberts’s team also proposed the biodegradable pyridinium lipids 31-36, in which the two chains are attached directly on the pyridinium ring via ester bonds (Chart 2). After formulation with equimolar amounts of DOPE 14, the cationic assemblies formed lipoplexes with reporter pDNA encoding GFP at a +/- charge ratio of 2.5, which were tested for transfection activity and 8 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


cytotoxicity on COS-7 and HepG2 cell lines. Transfection efficiency was found to be highly cell-dependent, with lipids 31-36 displaying a rather homogeneous transfection ability in COS-7 cells, about 60% of transfection efficiency of SAINT-2 12/DOPE 14 formulation, while being significantly less cytotoxic. This was a quite remarkable finding, considering that the phase transition temperatures for lipids 31-33 were between 46-72 °C, much higher than those of unsaturated congeners 34-36 (all below 0 °C). In the HepG2 cell line, formulation 34/DOPE 14 was found to be twice more efficient than SAINT-2 12/DOPE 14 formulation, but more cytotoxic. The other congeners either matched the transfection efficiency of SAINT-2 12/DOPE 14 formulation or had an inferior efficiency and also displayed a higher toxicity, despite the di-ester biodegradable design (58). More recently, Pungente’s team confirmed the good transfection efficiency of tetrafluoroborate analogs of 31-33 and unsaturated congeners 37 and 38 (also as tetrafluoroborates, Chart 2) when co-formulated with 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), DOPE 14 or Cholesterol 15 for reporter pDNA transfection into CHO-K1 cells (59, 60) Using the same pyridine alkylation procedure, Bhattacharya et al. synthesized pyridinium ether lipids 39 and 40, having cholesteryl moieties as hydrophobic anchors (Chart 2). Using the GFP reporter plasmid, his group revealed a maximum transfection efficiency when co-formulated with DOPE 14 at 1:1 molar ratio, at a +/- charge ratio around 3. Interestingly, pyridinium lipid 40 was able to compact pDNA alone, without colipids, and to maintain its transfection efficiency in the presence of serum, while displaying low cytotoxicity (61). Structurally-related lipids were synthesized by Zenkova et al., who also synthesized cholesteryl-based pyridinium lipids 41 and 42, bearing ester or carbamate moieties in conjunction with their cholesteryl hydrophobic anchor. Biological assessing using a GFP reporter plasmid in BHK cell line revealed that carbamate 42 was more transfection-efficient than DC-Chol 5 and that it preserved its transfection efficiency in presence of serum. Importantly, carbamate 42 was found to be more efficient than ester congeners of type 41 (62). Balaban, Ilies, and collaborators accessed pyridinium lipids through an original strategy, involving the condensation of pyrylium salts with primary amines. This strategy allows the generation of the pyridinium polar head and linker of the cationic lipid in a single, high yield step (19, 37, 51, 52, 63–65). The choice of primary amine dictates the molecular shape of the cationic amphiphile. Thus, our team was able to synthesize pyridinium cationic lipids 43-59 (coined SPYRIT, from Synthetic PYRIdinium for Transfection), bearing either aliphatic or aromatic linkers connecting the pyridinium polar head with two hydrocarbon tails of different lengths, saturated or unsaturated (Chart 3) (49, 51, 52). A substantial formulation effort, conducted in parallel with the synthesis and characterization of novel amphiphiles, established structure-activity and structure-property relationships within this set of pyridinium cationic lipids. Thus, DOPE 14 and cholesterol 15 were tested as co-lipids at different molar ratios to cationic species. Biological testing on NCI-H23 lung cancer cell line using a luciferase reporter plasmid proved that cholesterol 15 was the most efficient colipid when co-formulated with pyridinium cationic lipids 43-59 at 1:1 molar ratio. Pyridinium lipids 48-52, derived from 2-amino-1,3-propanediol were the 9 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


most efficient in transfecting pDNA, followed by lipids 43-47, synthesized from 1-amino-2,3-propanediol. Pyridinium amphiphiles 53-59, bearing a dopamine backbone, were the least efficient. Mention must be made that these pyridinium lipids were able to fully compact pDNA at a +/- charge ratio as low as 2. In terms of hydrophobic anchors, our studies revealed that for lipids 43-52 the myristoyl chains conferred the best balance between the two antagonistic processes of DNA compaction with lipoplex stabilization and lipoplex unwrapping with pDNA delivery inside the cell, followed by oleoyl chains. Interestingly, for lipids 53-59 a transfection maximum was found at lipid 55, bearing two dodecanoyl chains, probably due to the contribution of the phenyl ring to the overall hydrophobic effect generated by self-assembling of this type of amphiphiles (Chart 3). Dioleyl lipid 47 and especially myristoyl pyridinium lipids 49, co-formulated with Cholesterol 15 at 1/1 molar ratio proved superior to DOTAP 2/Cholesterol 15 1/1 in transfecting luciferase pDNA into NCI-H23 lung carcinoma, MCF-7 and MDA-Mb231 breast carcinoma, DU-145 prostate carcinoma and SWB-95 glioma cell lines, while displaying an excellent cytotoxic profile (49, 52).

Chart 3. Pyridinium cationic lipids synthesized via reaction of pyrylium salts with primary amines. 10 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


We used lipid 49 to conduct a counterion study in order to establish the best anion for pyridinium lipids in terms of transfection efficiency and associated cytotoxic effect (Figure 2) (52). As one can observe from data of Figure 2, the most efficient counterion for this pyridinium lipid was ClO4-, which was also the most toxic one. Best balance transfection efficiency versus toxicity was achieved with PF6- and Cl- counterions, therefore these counterions were used in all our subsequent studies (52). Lipid SPYRIT-7 13, with Cl- as counterion (Chart 1), emerged from these studies and remained a benchmark standard for our novel technologies (16).

Figure 2. Transfection efficiency (A) and associated cytotoxicity (B, determined via a WST-1 viability assay) in NCI-H23 lung cancer cells of pyridinium cationic lipid 49, bearing different counterions, against DOTAP 2 (as Cl-). All lipids were co-formulated with Cholesterol 15 at 1/1 molar ratio and interacted with the same amount of pDNA. Adapted with permission from reference (52). Copyright 2004 American Chemical Society. Another important finding was the ability of this class of cationic lipids to pack and deliver pDNA in vivo. Thus, contralateral subcutaneous NCI-H23 lung cancer tumors (5-7 mm in diameter) were established in 4–6 weeks old Harlan Sprague–Dawley nude mice. The animals were injected directly intratumor 11 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


with lipoplexes containing 4.5 µg of DsRed-Express-C1 red fluorescent reporter plasmid. Lipoplexes were generated either from lipid 47/Cholesterol 15 (1/1 molar ratio) formulation or from benchmark DOTAP 2/Cholesterol 15 (1/1 molar ratio) formulation. A nominal +/- charge ratio of 2 was used in both lipoplex formulations. Thirty-six hours post-injection the animals were sacrificed, the tumors were harvested, fixed in 4% formaldehyde, paraffin embedded, sliced and examined for the expression of red fluorescent protein (RFP) using a confocal microscope (Figure 3). Representative images shown in Figure 3 revealed a more intense and diffuse transfection achieved by the pyridinium cationic lipid-based lipoplexes, while transfection of DOTAP 2/Cholesterol 15 was mostly focused around the injection point (49). Meanwhile, advances in lipophilic pyrylium salts synthesis and purification allowed us to access pyridinium lipids with the two hydrophobic chains attached directly on the pyridinium cationic polar head (63). We have synthesized pyridinium cationic lipids 60-73 having chain lengths between C10 and C17 and we have tested them for their ability to transfect luciferase reporter pDNA in the same NCI-H23 lung cancer cell line used previously. The 14 pyridinium cationic lipids 60-73 were co-formulated with Cholesterol 15 at 1/1 molar ratio into liposomes, which were combined with pDNA at a +/- nominal charge ratio of 2. Lipoplexes generated from DOTAP 2/Chol 15 1/1 in similar conditions were used to benchmark the new series. Biological results showed a great dependence of transfection efficiency on the chain length, with a maximum at lipid 68, bearing two myristyl chains. Lipoplexes made out of 68/Cholesterol 15 1/1 were twice more efficient in transfecting pDNA than DOTAP 2/Chol 15 1/1. Even small decreases or increases in chain length caused significant drops in transfection efficiency, revealing the importance of detailed structure-activity relationship (SAR) studies (63). Mention must be made that the same lipophilic pyrylium salt precursors used in the synthesis of lipids 60-73 allowed us to access other classes of cationic amphiphiles (vide infra). The efficiency displayed by formulation 68/Cholesterol 15 1/1, based on pyridinium lipid 68 that does not have a linker between the pyridinium cationic head and the hydrophobic anchor prompted us to reengineer lipids 53-59 based on dopamine backbone. We hypothesized that a bent conformation of the flexible ethylene linker could allow the pyridinium polar head to be positioned near the water/oil interface situated in lipids 53-59 at the level of the polar ester groups attached on the phenyl ring (Figure 4A). Replacing the ester groups with more lipophilic ether ones could allow the aromatic linker to move into the hydrophobic phase and induce an extended conformation of the backbone of the novel amphiphiles, effectively moving the water/oil interface at the level of the pyridinium polar head. The extended conformation was expected to reduce the cross-section of the individual amphiphile and induce a packing parameter closer to 1, and to enhance its self-assembling into robust lamellar phases. The two anions, PF6- and Cl-, found to be give optimum transfection efficiency/cytotoxicity in conjunction with the pyridinium polar head (vide supra), were used in the study, thus yielding lipids 74-85 (Figure 4A).



Figure 3. Cross-section images of NCI-H23 lung cancer tumors in vivo (nude mice) transfected with DsRed-Express-C1 red fluorescent plasmid after direct tumor injection with lipoplexes containing 4.5 µg of pDNA, complexed by either lipid 47/Cholesterol 15 or DOTAP 2/Cholesterol 15. Note the more intense and diffuse transfection achieved by the pyridinium cationic lipid-based lipoplexes, while transfection of DOTAP 2/Cholesterol 15 is mostly focused around the injection point. Adapted with permission from reference (49). Copyright 2005 Elsevier.



Figure 4. Working hypothesis behind interfacial engineering of pyridinium lipids 53-59 into congeners 74-85 (A): replacement of polar ester groups connecting the hydrophobic chains with the phenyl ring with more lipophilic ether ones can induce an extended conformation of the backbone of the novel amphiphiles, can reduce the cross-section of the molecule and enhance its self-assembling, as proved by the appearance of stable liquid crystalline phases in lipids 76-83 evidenced by DSC (1st heating, panels B, C, 2nd cooling panels D, E), SAXS dynamic (for lipid 80, panel F) and TOPM texture (for lipid 80, panel G and lipid 81, panel H). Adapted with permission from reference (66). Copyright 2013 American Chemical Society. 14 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


An examination of the self-assembling in bulk of novel amphiphiles 74-85 by a combination of differential scanning calorimetry (DSC), transmission optical polarized microscopy (TOPM) and small angle X-ray scattering (SAXS) confirmed a robust ability to self-assemble, with the tendency to form liquid crystalline (LC) phases in congeners with saturated alkyl chains longer than 10 carbon atoms 76-83 (Figure 3B-3H). The stability and extent of LC phases was found to depend on the counterion with the more lipophilic PF6- templating better the LC phases than Cl- (Figure 4B-4E). SAXS and TOPM analysis of self-assemblies generated from lipid 82 revealed a smectic C (SC) phase, with a d-spacing of about 41 Å at low and medium temperatures, which increased to about 55 Å at 102 °C (Figure 4F, 4G). The LC phases of congener 83, bearing the Cl- counterion, were found to be less organized by both SAXS and TOPM (compare Figure 4G with Figure 4H), probably due to the smaller radius and lower lipophilicity of this counterion as compared with PF6-. This smaller lipophilicity of the Cl- translated into faster hydration of the individual amphiphiles bearing this counterion and easier generation of the lyotropic supramolecular assemblies as compared with PF6- congeners. However, the gel to LC transition temperature (Tc) of the hydrated lipids with PF6- and Cl- were almost identical, revealing a negligible contribution of the counterion to the supramolecular assembly in the lyotropic LC phase (66). Pyridinium lipids 74-85 were formulated either alone or co-formulated with colipids DOPE 14 or Cholesterol 15 at 1/1 molar ratio and were able to fully compact pDNA starting with a +/- charge ratio of 2, yielding lipoplexes about 175-450 nm in size, with a zeta potential between 25 and 45 mV, lamellar in structure as revealed by TEM. Representative results are shown in Figure 5A-5D for lipid 85. Assessment of transfection efficiency in NCI-H23 cancer cell line revealed a direct correlation of transfection efficiency with the fluidity of the pyridinium cationic lipids, indicated by the Tc. Thus, best results were obtained with lipids 75 and 85, which have the lowest Tc. Importantly, these lipids were also efficient when formulated alone, besides the formulations with colipids DOPE 14 or Cholesterol 15 at 1/1 molar ratio (exemplified for lipid 85, Figure 5E and 5F). Data from Figure 5E, 5F revealed that all lipoplexes made from these formulations surpassed similar lipoplexes generated from standard transfection systems DOTAP 2/Cholesterol 15 1/1 and Lipofectamine® in transfection efficiency, while displaying practically negligible cytotoxicity. Lipoplexes generated from formulations 85/DOPE 14 and especially 85/Cholesterol 15 were also able to efficiently transfect lung cancer cell lines MCF-7 and A549, prostate cancer PC-3 and DU-145, colon cancer Caco-2, surpassing several folds the above-mentioned standard transfection systems. Importantly, lipoplexes made out of formulations 85/Cholesterol 15 and 85/DOPE 14 (both 1/1 molar ratio cationic lipid/colipid) were able to maintain their transfection efficiency in the presence of elevated levels of serum in the transfection media (up to 40% for 85/DOPE 14 1/1), being clearly superior to DOTAP 2/Cholesterol 15 1/1 and Lipofectamine® (Figure 5G).



Figure 5. Size (A), zeta potential (B), aspect (by TEM, C), electrophoretic mobility (D) at different +/- charge ratios, transfection efficiency (E) with associated cytotoxic effect (F) and transfection efficiency in the presence of serum (G), for lipoplexes generated from lipid 85, either alone of co-formulated with Cholesterol 15 or DOPE 14 at 1/1 molar ratio, as compared with lipoplexes from DOTAP 2/Cholesterol 15 1/1 and Lipofectamine® made at the same charge ratio of 3. pDNA reporter plasmid encoding firefly luciferase and NCI-H23 cell line were used in all cases. Adapted with permission from reference (66). Copyright 2013 American Chemical Society. 16 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Moreover, we were able to confirm the excellent transfection efficiency, lack of toxicity and serum resistance displayed by lipoplexes generated from 85/DOPE 14 1/1 formulation in transfecting neurons, ex-vivo. Thus, neurons/glial cells co-cultures were established using cells harvested from nucleus accumbens (rich in dopaminergic neurons) and from prefrontal cortex (containing a mixed population of neurons) of rat neonates (1-2 days old). After 3 days in co-culture, cells were transfected with lipoplexes generated from formulation 85/DOPE 14 1/1 and reporter plasmid encoding green fluorescent protein (GFP), at the same +/- charge ratio used in previous experiments. After 48 h incubation time cultured were imaged for GFP expression and were tested for cell viability using an established Ca2+ stimulation assay (Figure 6). Results from Figure 6 revealed the selective transfection of neurons from nucleus accumbens and prefrontal cortex. Glial cells, although present in larger amounts than neurons, were not transfected. Ca2+ stimulation assay proved that both neurons and glial cells were viable (Figure 6A, 6B). The same lipoplexes were not able to transfect rat myometrial smooth muscle cells (Figure 6C), proving selectivity against dopaminergic neurons, which we believe is related with the presence of dopamine receptors on the surface of these CNS cells that can trigger receptor-mediated internalization of lipoplexes based on pyridinium lipid 85 bearing a dopamine backbone (66). Besides the successful transfection of pDNA in vivo and ex-vivo, pyridinium cationic lipids were shown to be able to pack and deliver siRNA. Thus, Kamps’s team co-formulated SAINT-2 12 with POPC, Chol 15, DSPE-PEG and DSPEmaleinimide (18:37:40:4:1 mol %) and subsequently encapsulated VE-cadherin siRNA with a 71 % encapsulation efficiency. The lipoplexes were post-decorated with thiolated anti-E-selectin and anti-VCAM-1 antibody via reactive maleinmide surface ligands. The targeted lipoplexes (termed SAINT-O-Somes) were stable in the presence of serum and were able to deliver siRNA to inflammation-activated primary endothelial cells (67). In vivo translation attempts with similarly targeted, stealth, formulations from the Dutch team, confirmed the feasibility of the concept (68) but also showed that these delivery systems must be further improved in terms of intracellular release of nucleic acid cargo (69). Recently, lipoplexes based on SAINT-2 12 and MIDGE-Th1 DNA vectors were shown to increase the immune response to encoded antigen in mice and rats, with the cationic lipid changing the biodistribution of the DNA in a significant manner. The vaccination procedure was well-tolerated by experimental animals – a great premise for translation towards human applications (70). Besides cationic lipids, our team investigated pyridinium amphiphiles with different charge/mass, shape and packing parameters, including simple surfactants (64), gemini surfactants (63, 71, 72), and lipophilic polycations (63) for nucleic acid delivery (16). Thus, we have shown that the same lipophilic pyrylium salts used in the synthesis of pyridinium cationic lipids 60-73 can provide access to gemini surfactants (GSs), trimeric and oligomeric amphiphiles 86-122 through reaction with a primary di- or triamine (Chart 4) (63, 71).



Figure 6. Images of co-cultured cells (neurons and glial cells) from rat nucleus accumbens (A), pre-frontal rat cortex (B) and of rat myometrial smooth muscle cells (C), 48 h post-transfection with 85/DOPE 14 1/1 lipoplexes encapsulating GFP reporter plasmid. Left: DIC image; center: GFP fluorescence image (488 nm excitation, 540 nm emission); right: basal calcium fluorescence image (ratio 340nm/380nm excitation and 520 nm emission). Only neurons were transfected with GFP, showing neuronal selectivity of the transfection agent. All neurons, glial cells and myometrial smooth muscle cells were viable post-transfection, showing Fura-2 calcium binder post-loading. Adapted with permission from reference (66). Copyright 2013 American Chemical Society.

Gemini surfactants have a higher charge/mass ratio than pyridinium lipids, being expected to generate smaller lipoplexes (72, 73). Safinya’s group revealed that endosomal escape of lipoplexes is favored by a high (positive) charge density of the lipoplex and by a high elastic modulus of the complex membrane (74), properties relatively easy to achieve with GS-based lipoplexes. Therefore, we conducted a detailed structure-activity relationship study involving GSs and lipophilic polycations 86-104 (63). We analyzed the influence of different structural elements of these amphiphiles on their ability to transfect pGL3 luciferase reporter plasmid DNA into NCI-H23 lung cancer cell line. We found that the spacer between the two cationic polar heads in GSs has a significant impact on the transfection efficiency of these amphiphiles. Thus, amphiphiles with polar linkers containing protonable secondary amine groups 93-96 were 18 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


less efficient as compared with their congeners bearing hydrophobic linkers 86-92, a fact that was associated with the strong complexation of pDNA by these multi-cationic species, with detrimental impact on the release of the plasmid into the cytoplasm after internalization of lipoplexes. Within the hydrophobic pyridinium GSs, transfection efficiency dropped abruptly when elongating the linker from 2 to 3 C atoms (86 → 87). The transfection efficiency was gradually restored with further elongation of the hydrophobic linker from 3 to 8 methylene units (87 → 92), being correlated with the tendency of the longer linkers to “dive” into the oil phase and to increase the hydrophobic effect in conjunction with the two hydrocarbon chains, helping the packing of DNA into the lipoplex. All these amphiphiles were co-formulated with Chol 15 at 1/1 molar ratio. The transfection efficiency of the 86/Chol 15 1/1 formulation was about the same as 92/Chol 15 1/1 and matched the efficiency of standard DOTAP 2/Chol 15 1/1 used for benchmarking (63).

Chart 4. Representative structures of efficient pyridinium cationic gemini surfactants and lipophilic polycations synthesized in our discovery program via reaction of lipophilic pyrylium salts with primary di- or triamines.

It was hypothesized that capping the protonable secondary amines in the hydrophic linkers of GSs 93-96 would rescue the transfection efficiency by allowing the efficient release of the nucleic acid from lipoplex. The hypothesis was confirmed when the precursors 98-101 proved several orders of magnitude 19 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


more efficient in transfecting the pDNA as compared with the final protonable GSs 93-96. Interestingly, the most efficient representative was 96, containing two hydrophobic t-butyloxycarbonyl (Boc) protecting groups, which proved twice more efficient than standard DOTAP 2/Chol 15 1/1 in transfecting pGL3 luciferase plasmid into NCI-H23 cell line when co-formulated with Chol 15 at 1/1 molar ratio. The increased potency of GS 96 also suggested the importance of enhancing the self-assembling capabilities of these amphiphiles in water, through additional contribution of the lipophilic Boc groups to the hydrophobic effect generated by the two hydrocarbon tails of the GS. Since a stronger self-assembling ability should translate into better pDNA compaction and lipoplex stabilization, we synthesized and tested the transfection efficiency of trimeric amphiphile 97 and tetrameric congeners 102-104 using the same reporter plasmid and cell line as above. The good transfection efficiency observed experimentally with all these lipophilic polycations confirmed this hypothesis too. Interestingly, these amphiphiles displayed a slightly higher transfection efficiency when co-formulated with DOPE 14 at 1/1 molar ratio than when Chol 15 was used a colipids at a similar formulation ratio. Lipoplexes made out of 97/DOPE 14 1/1 surpassed both DOTAP/Chol 15 1/1 and Lipofectamine® standard transfection systems (63). Also important was the observation that these amphiphiles were able to compact and transfect pGL3 plasmid alone, without any co-lipid (63), establishing the class of lipophilic polycations as a new class of synthetic transfection systems (16). Bhattacharya’s group independently confirmed our findings and introduced new efficient GSs with oligoethylene glycol (non-protonable) polar linkers (75). We have adopted this linker in the structure of pyridinium GSs 105-122, synthesized from lipophilic pyrylium salts and 1,5-diamino-3-oxapentane (71). The most efficient lipoplexes in terms of transfection efficiency were found those generated from GS 105-122/DOPE 14 at 1/2 molar ratio, probably due to the tapered shape of these amphiphiles, which confers them a low packing parameter. The ability to pack and transfect gWizLuc pDNA in the NCI-H23 lung cancer cell line proved to increase monotonously with the increase in chain length. It was also strongly influenced by the counterion of the GS, decreasing in order Cl- > Br- ~ H2PO4- > PF6- (GS 119 > 121 ~ 122 > 111, all co-formulated with DOPE 14 at 1/2 GS/colipid molar ratio). SAXS studies, coupled with DSC and TOPM investigations, of the self-assembling in bulk of the C16 GSs 111, 119, 121, 122 bearing different counterions revealed the clear preference of pyridinium polar head for the dihydrogenophosphate counterion. This preference explains the transfection activity order of these GSs, as the chloride counterion is expected to be quickly released within packing of nucleic acids with GSs and replaced with phosphodiester anions, forming a very stable lipoplex. The strong pyridinium-phosphodiester ionic interaction may be one important factor contributing to the ability of pyridinium amphiphiles to compact nucleic acids at very low +/- charge ratios (as low as 1 for pyridinium lipids, vide supra). Along the same line, it must be emphasized that the lipoplexes generated from GSs 113-120 were able to maintain their transfection efficiency in the presence of high serum levels in the transfection media, a promising feature for future in vivo translational efforts (71). Interestingly, the cytotoxic effect associated 20 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


with transfection via GSs 113-120 was minimum for C13 representative 116 and increased with the shortening or elongation of hydrophobic chains. Lipoplexes generated from C17 representative 120 had the highest transfection efficiency, but also displayed a significant cytotoxic effect associated with transfection. The cytotoxicity of pyridinium GSs is generally higher than cationic lipid (CL) congeners. This effect is generated partially by their higher charge/mass ratio as compared with CL, but it is also an effect of their tapered shape. The relatively high molecular curvature of GSs with hydrophilic linkers allows GSs to temporary porate external and internal membranes – beneficial for increasing the transfection efficiency but detrimental for cytotoxicity of the formulation (16, 40, 72, 73, 76–79). We proposed to circumvent this problem through the use pyridinium GSs in combination with pyridinium CLs. We co-formulated pyridinium cationic GS 120 and CL 49 (Cl- form), therefore exploiting the high charge/mass ratio and molecular curvature of GS 120, ideal for membrane poration but generating toxicity, with robust nucleic acid compaction of (safer) CL 49. We revealed that substitution of 5% (molar) positive charge in pyridinium CL 49-based lipoplexes with GS 120 generates a perfectly fluid mixed amphiphile bilayer that can compact pDNA at lower +/- nominal charge ratios than CL 49 alone (as low as 1), and can synergistically enhance the transfection efficiency while displaying negligible cytotoxicity. The excellent transfection efficiency of these mixed formulations was also confirmed in 3D cell cultures of tumor spheroids (Figure 7) (80). Further increase in the percentage of GS in the formulation had a detrimental effect on transfection efficiency, pointing towards the role of GS as a “catalyst” to porate external and internal membranes. This mechanism of action was proved via lipoplex cell uptake experiments using inhibitors against specific cell internalization pathways and using fluorescence microscopy in cells in which endosomes were labelled with green fluorescent protein (GFP) markers (e.g. GFP-Rab7, Figure 8) (80). Interestingly, similar synergistic results can be obtained when pyridinium cationic lipids were paired with pyridinium surfactants chemisorbed on the surface of curve Au nanoparticles (81). An alternative, synthetic, solution for combining the properties of GS and CL investigated by our team was the introduction of pseudo-gemini surfactants (PGSs). These amphiphiles are (mono)cationic lipids that have a tapered shape and polar topology similar to the GS when placed in water and water-based media (63, 65). The tapered shape is ensured by placing at the water-oil interface the pyridinium cationic polar head and another polar group capable of H-bonding (ester, amide), connected together via a short polar linker (Chart 5). Thus, supramolecular assemblies of the PGSs were expected to be more stable due to reduced repulsions between the (mono)cationic polar heads of PGSs vs dicationic GSs. We investigated two main designs, represented by PGSs 123-128 and 129-131 (Chart 5), via the same condensation of lipophilic pyrylium salts with amino-derivatives (63). The first set included the ester PGSs 123-125 and the amide congeners 126-128. Biological evaluation of the lipoplexes generated from these amphiphiles, co-formulated with either DOPE 14 or Chol 15 for delivery of pGL3 plasmid in NCI-H23 lung cancer cell line revealed an excellent transfection profile, which peaked at PGSs 124 and 127 with 14 C atoms in 21 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


both chains. Both PGS series preferred DOPE 14 as co-lipid at 1/1 molar ratio, behaving similarly to pyridinium GS described above. Lipoplexes generated from PGS 124/DOPE 14 1/1, PGS 127/Chol 15 1/1 and PGS 127/DOPE 14 1/1 surpassed the standard transfection systems DOTAP 2/Chol 15 1/1 and Lipofectamine® in transfecting lung carcinoma NCI-H23, breast carcinomas MCF-7 and MDA-Mb231, prostate carcinoma DU-145, and glioma SWB-95 (63). Mention must be made that the cytotoxicity of amide PGSs 126-128 was higher than cytotoxicity generated by the use of PGSs 123-125. We hypothesized that the toxicity may be generated by the positively charged pyridinium surfactant that PGS 126-128 generate upon biodegradation (Chart 5). Consequently, in order to preserve the transfection efficiency but reduce the cytotoxic effect, we recently synthesized the structurally related PGSs 129-131 in which we switched the position of amide. These new PGSs were expected to generate two neutral species upon hydrolytic (bio)degradation, and therefore display a much lower cytotoxicity (Chart 5). We shortened the chain attached to the pyridinium ring from 14 to 13 C atoms to allow a better packing of the new amphiphiles and also to match the minimum toxicity found for GS 113-120 (vide supra). Three chain lengths (12, 14 and 16 C atoms) were tested for the second hydrocarbon chain, in order to generate a different molecular curvature of respective PGSs. Biological testing for gWizLuc pDNA delivery in NCI-H23 cell line showed a similar behavior with pyridinium GS presented in the previous section. The amphiphiles formulated alone could not fully compact pDNA and had to be co-formulated with DOPE 14 or Chol 15, at 1/1, 1/2 and 1/4 molar ratio, when full compaction occurred at +/- charge ratios as low as 1. Lipoplexes from PGSs 129-131 achieved high transfection efficiencies, surpassing standard transfection systems DOTAP 2/Chol 15 1/1 and Lipofectamine® several times, in both 2D and 3D (tumor spheroids) cellular model. The transfection efficiency of lipoplexes generated from these PGSs was retained in the presence of serum, with PGS 131/Chol 15 1/2 being the most efficient, followed by PGS 131/Chol 15 1/4. This confirmed our initial working hypothesis that is possible to bridge synthetically the field of GS and CL and to preserve both characteristics of these classes of amphiphiles. Moreover, the cytotoxicity of the new PGSs was improved as compared with congeners 126-128, confirming our second work hypothesis. Motivated by these achievements, we tested PGS 131 and its formulations for their ability to transfect siRNA and mRNA, besides pDNA. We used a siRNA designed to knockdown luciferase in MDA-Mb-231-luc-D3 breast cancer cell line stably expressing this protein or an mRNA encoding the same protein, in the NCIH23 cell line. The biological results revealed an excellent transfection profile for these two nucleic acids, with 131/Chol 15 1/2, 131/Chol 15 1/4, and 131/DOPE 14 1/2 being the most efficient formulations (Figures 9 and 10). Using fluorescence microscopy on NCI-H23 cells expressing GFP-Rab7 endolysosomal protein, we proved that this performance is at least in part due to the ability of PGSs to promote endosomal escape of corresponding lipoplexes, through a poration mechanism similar to that displayed by GS (71, 82).



Figure 7. Pyridinium GS 120 can act synergistically with pyridinium CL 49 towards lipoplex stabilization and transfection efficiency, without generating cytotoxicity: lipoplex electrophoretic mobility (a), transfection efficiency and cytotoxicity (b and c, in 2D NCI-H23 cell culture), and 3D transfection efficiency in tumor spheroids. Adapted with permission from reference (80). Copyright 2013 American Chemical Society.



Figure 8. Fluorescent confocal imaging of NCI-H23 cells (excitation 488 nm), expressing the endolysosomal protein GFP-Rab 7, transfected with CL 49 or with GS 120/CL 49 lipoplexes. Pyridinium GS 120 promotes endolysosomal membrane poration and endosomal escape of GS 120/CL 49 lipoplexes irrespective of the co-lipid used and its molar ratio in lipoplex formulations, triggering a diffuse distribution of Rab-7 in the cytoplasm (right panels). Lipoplexes that do not contain GS do not change significantly Rab-7 intracellular distribution vs control cells (central panels vs left panels).



Chart 5. Pyridinium pseudo-gemini surfactants (PGSs) synthesized by our team and proposed (bio)degradation patterns for the two types of amide PGSs.

The transfection efficiency was found to be nucleic acid dependent, dose-dependent, and cell-dependent, as expected. Importantly, the charge ratio required for compacting siRNA (about 8) was much higher than the same parameter for mRNA (3-5) and pDNA (1-3), in agreement with literature data (16). Cell viability was very high, despite the very high charge ratios used, confirming the very good toxicity profile of these PGSs and recommending their use as new synthetic systems for nucleic acid delivery with great translational perspectives.



Figure 9. A) Luciferase knockdown activity of PGS 131-based lipoplexes encapsulating Luc-siRNA (7 pg/cell), generated at charge ratio 8/1, from formulations based on PGS 131 alone or co-formulated with Cholesterol 15 or DOPE 14, and the cytotoxic effect associated with the delivery process, in MDA-MB-231-Luc-D3 cell line; B) Effect of doubling the Luc-siRNA amount, from 7 pg/cell to 14 pg/cell, in the same knockdown experiment. Results presented represent the average of four transfection experiments ± one standard deviation. Adapted with permission from reference (82). Copyright 2017 American Chemical Society.



Figure 10. Transfection efficiency of mRNA with PGS 131-based formulations at charge ratios 3/1 and 5/1 (A) and their cytotoxicity profile (B), in the NCI-H23 cell line. Results presented represent the average of four transfection experiments ± one standard deviation. Adapted with permission from reference (82). Copyright 2017 American Chemical Society.



Conclusions Pyridinium cationic amphiphiles occupy a privileged space in the field of cationic amphiphiles for nucleic acid delivery due to their delocalized charge, lipophilicity and hydration properties. The chapter presented the main classes of pyridinium amphiphiles, reviewing cationic lipids, gemini surfactants, pseudo-gemini surfactants, and lipophilic polycations. Extensive structure-activity and structure-property relationship studies revealed their potential for delivery of nucleic acids of all types, including pDNA, siRNA and mRNA, either alone or in co-formulated with various co-lipids. Formulations containing different classes of pyridinium amphiphiles were also highlighted, together with their proved synergistic action towards enhancement of transfection efficiency. In parallel, we presented relevant translational efforts that were made to advance various technologies based on pyridinium amphiphiles from 2D and 3D cell cultures to ex-vivo and in vivo experiments. The unique compaction and delivery properties of these heterocyclic amphiphiles were presented in a comparative manner with classical synthetic transfection systems that were used in human clinical trials. Significant advantages were revealed for pyridinium lipoplexes in terms of transfection efficiency, cytotoxicity, serum stability, and tissue penetrability, thus paving the way towards the use of pyridinium amphiphiles in nucleic acid delivery in human subjects for therapeutic purposes, as well as in human gene therapy.

Acknowledgments Financial support from NSF (CHE-0923077), from Temple University School of Pharmacy – Dean’s Office and from Temple Undergraduate Research Program is gratefully acknowledged.

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