Polymers for siRNA Delivery: A Critical Assessment of Current

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Review

Polymers for siRNA delivery: A Critical Assessment of Current Technology Prospects for Clinical Application Bailey Mae Cooper, and David Putnam ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00363 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 29, 2016

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ACS Biomaterials Science & Engineering

Polymers for siRNA delivery: A Critical Assessment of Current Technology Prospects for Clinical Application

Bailey M. Cooper1 and David Putnam1,2*

1

Meinig School of Biomedical Engineering, 2Smith School of Chemical and Biomolecular Engineering,

Cornell University, Ithaca, NY 14853

*

To whom correspondence should be addressed: 147 Weill Hall, Cornell University, Ithaca, NY 14853.

Email: [email protected]

ACS Paragon Plus Environment


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Abstract The number of polymer-based vectors for siRNA delivery in clinical trials lags behind other delivery strategies, however, the molecular architectures and chemical compositions available to polymers make them attractive candidates for further exploration. Polymer vectors are extensively investigated in academic labs worldwide with fundamental progress having recently been made in the areas of high-throughput screening, synthetic methods, cellular internalization, endosomal escape and computational prediction and analysis. This review assesses recent advances within the field and highlights relevant developments from within the complementary fields of nanotechnology and protein chemistry with the intent to propose future work that addresses key gaps within the current body of knowledge – potentially advancing the development of the next generation of polymeric vectors.

Keywords: siRNA, DNA, polymer chemistry, structure-activity relationships


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Introduction Eighteen years following the first thorough explanation of the RNAi mechanism1 and ten years following the first presentation of clinical data for an siRNA-based therapeutic,2 only seven of the more than sixty national clinical trials (NCTs) studying siRNA delivery systems employed synthetic polymeric vectors - either in the form of surgically-implanted local drug eluters (LODERs) or systemicallyadministered cyclodextrin-based systems and dynamic poly-conjugates (which employ multi-functional, environmentally responsive polymers designed to facilitate separate aspects of delivery). Other in vivo strategies under consideration include self-delivering siRNAs, in which chemical modifications are used to incorporate drug-like properties into the nucleic acid compound, and GalNAc conjugates, which employ the small molecule triantennary N-acetylgalactosamine as a targeting ligand. In a field dominated by liposomal formulations and chemically modified-naked siRNA, the prospects of nonlipidoid synthetic polymers remain unclear. Customizability, stability, low cost and ease of manufacture of polymeric vectors make them attractive candidates for research; however, they have suffered from low efficiency, cytotoxicity, poorly controlled synthesis, and a general lack of mechanistic understanding of their cellular uptake, intracellular trafficking and payload release. An assessment of the potential for polymer vector viability in the clinic is needed. The use of high throughput library-based approaches aided by predictive computational models, innovative polymer structures, new chemistries and a growing understanding of key cellular mechanisms, have propelled the field forward substantially in recent years. But even as the field rapidly matures in response to cutting-edge experimental techniques and improved mechanistic insights into siRNA delivery, there remain non-trivial obstacles to greater adoption of polymer vectors in clinical trials. This review critically explores and summarizes the integration of the new methods, materials, chemistries and computational methods brought to bear in an effort to better understand and navigate the intracellular delivery of siRNA sequences.



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Polymeric libraries - rapid screening in vitro plus considerations for in vivo studies

Figure 1. High Throughput (HT) methods of analysis allow for the rapid assessment of multiple parameters, thereby enabling researchers to identify structure-activity relationships within large libraries of polymers. Reprinted with permission from ref 4. Copyright 2013 American Chemical Society.

The broad molecular parameter space available to polymeric vectors enables investigators to explore widely diverse molecular architectures and compositions in the search of siRNA delivery systems with enhanced capabilities. Additionally, the influence of polymer structure and composition on siRNA delivery can help to enhance or refine siRNA delivery. One approach to this end is through the parallel screening of polymer libraries to facilitate the rapid evaluation of structure-activity relationships (Figure 1).3 One challenge to the high-throughput screening approach is the reliable correlation between in vitro and in vivo efficacy, as well as reliable preclinical animal models to accurately predict clinical outcomes. The development of in vitro – in vivo correlations (IVIVCs) is particularly difficult for systemically administered materials where multiple cell types and boundaries are expected to contact the material. Two-dimensional cell culture alone does not have the capability to predict events such as the chelation of ions that can impact homeostasis, activation of intravascular coagulation cascades or agglomeration of particles following intravenous administration, etc.4 Furthermore, the most appropriate cell line(s) for


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in vitro studies might not be obvious. For example, one study found that the HeLa cell line (human cervical carcinoma) provided data that was more predictive of hepatocellular delivery to healthy mice than the Hepa1-6 cell line (mouse hepatoma). While freshly isolated mouse hepatocytes “retained native gene function for 1-2 days post-isolation” and provided the best correlation (R2=0.99), the investigators found that HeLa cells provided adequate selection pressure for in vitro screening.5 Threedimensional cultures of human cells and “lab-on-a-chip” microfluidic devices have not been widely utilized for developing IVIVCs in this field, but hold the potential to serve as more predictive screening systems.6 Whitehead et al. reviewed the correlation that exists between transfection and in vivo efficacy for a number of lipidoid vectors targeting hepatocellular siRNA delivery and established that variations in formulation technique significantly affect the IVIVC.5 A comparable study for synthetic polymers would be a useful and critical step to help facilitate the accurate identification of candidate polymers for in vivo efficacy while potentially providing insight that may lead to the development of more relevant in vitro models. Furthermore, standardization of screening practices across the field (e.g. a base set of cell lines for in vitro studies, uniform transfection SOPs (standard operating procedures), a uniform set of commercially available positive controls , etc.) would provide a foundation for the quantitative comparison of results from different studies. The development of in silico approaches, leading to predictive models,7 and the broader adoption of quantitative studies evaluating structure-activity relationships6 would both aid in the search for more accurate trends with increasing clinical significance. Zuckerman et al. recently reported on the correlation between preclinical, multispecies animal studies and the initial Phase I clinical trials of the first systemically administered cationic polymer-siRNA delivery system - CALAA-01 – where “allometric scaling across species best [correlated] with body weight rather than with body surface area”. The area under the curve (AUC) and maximum plasma concentration (Cmax) appeared linearly related in all species, and the animal studies were generally



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found to predict clinical outcomes, but the authors noted that the pharmacokinetics might deviate for systems not dependent upon electrostatic interactions since the primary clearance mechanism is renal clearance for polyplexes in contrast to the monocyte phagocytic clearance expected for other nanoparticles.8 Therefore, a comparable study for non-ionically associated polymer-siRNA complexes is needed within the field.

Advances in chemistry To explore the rich parameter space available to polymer-based vectors though the synthesis of libraries, a number of broadly applicable synthetic strategies have been reported. The early work of Lynn, Anderson and Langer established the utility of Michael-type additions to form libraries of poly(beta-amino esters) and introduced the concept of combinatorial libraries for nucleic acid delivery. As more labs begin to span the discovery-to-development timeline, where both the fundamental understanding of transfection and the practicality of product viability are equally important, the chemistries used to investigate new polymer structures and architectures have evolved to accommodate both facile synthesis as well as feasible scale-up. Perhaps most important to the synthesis of polymer libraries that can lead to an understanding of structure-activity relationships, as well as scalability for translation into the clinic, is the fidelity and reproducibility of the reactions. Small changes to polymer structure can have significant influence on transfection, and the presence of uncharacterized side-reaction products on the polymer can lead to incorrect conclusions with respect to how polymer structure correlates to siRNA delivery. One example of how a seemingly simple polymer-analogous conjugation can have unwittingly side reactions is the use of polymers containing side chains terminated with the N-hydroxysuccinimide (NHS) leaving group (Figure 2A). The activated ester of the NHS group allows amidation when incubated with amino groups.9–11 However, two unexpected side reactions can occur. One side reaction is the formation of a


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linear adduct generated in sterically constrained environments from attack of the amino group on one of the ring carbonyl NHS groups (Figure 2B). The other side reaction is the formation of a cyclic glutarimide through amide attack on a proximal NHS group (Figure 2C). There are ways to circumvent these specific side reactions, as well as others formed during library synthesis.12 But without first fully characterizing the conditions under which they are formed, failure to recognize their existence can lead to mischaracterization of structure-activity relationships, which can complicate robust scale-up and reproducibility for commercial or clinical applications.

A

B

C

Figure 2. (A) Polymer with NHS side chains used to make polymer libraries (B) Structure of ring addition side reaction product when amine attacks carbonyl on the NHS ring (C) Structure of glutarimide side reaction product when amine attacks NHS in close proximity

Click chemistry methods are reported for the synthesis of polymer libraries for the delivery of siRNA sequences. The Reineke group has investigated carbohydrate-based polymers and reported how polymer length and type of carbohydrate influence delivery potency using copper-catalyzed azide/alkyne cycloaddition. Trehalose and β-cyclodextrin were made part of the backbone though copolymerization of oligoethylenamine.13 The influence of carbohydrate type was nucleic aciddependent. Trehalose-containing polymers were superior for plasmid DNA whereas β-cyclodextrin was



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superior for siRNA. UV-initiated thiol-ene click chemistry is another nice approach to the synthesis of polymer libraries, although its use has yet to be reported for siRNA delivery (Figure 3). One example of its utility was reported by the group of Nystrom wherein histamine-functionalized polymers were investigated for their ability to deliver doxorubicin to in vitro 3D breast cancer architectures.14

Figure 3. UV-activated thiol-ene click chemistry used to functionalize polymer side chains

The addition of amine to epoxide groups is clean and robust. Polymers containing side chains terminated with epoxides were used as a crosslinking strategy to make nanoparticles containing amine crosslinking sequences (Figure 4).15 Each material made in this very large library, which contained over 1500 unique compositions, was thoroughly characterized for molecular weight, hydrodynamic diameter in solution, the ability to complex with nucleic acids, cellular internalization and the ability to deliver both plasmid DNA and siRNA. The result was a comprehensive understanding of how the material composition led to delivery, both in cell culture and in animal models. In particular, polymers crosslinked with piperazine and dimethylamine worked optimally, particularly when cholesterol was included as a way to facilitate transport to hepatocytes.


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Figure 4. Epoxide amine addition reaction (top) applied to the crosslinking of epoxideterminated polymers (bottom) toward the synthesis of polymer libraries.

Synthesis of more targeted and complex combinatorial libraries often rely on more traditional condensation reagents, like EDAC and maleimide, in order to give more flexibility and control over the final stoichiometry of components. A recent example is the work of Amiji wherein a combinatorial library, based on chitosan as a backbone, was generated varying the content of polyethylene glycol, a lipid chain, and the targeting agent (epithelial growth factor receptor binding peptide). The approach was used to investigate the delivery of both siRNA as well as cisplatin.16 A newer condensation reagent, DMTMM, has been used to create poly(acrylic acid)-based polymer libraries for siRNA delivery containing variations in side chain carbohydrate (galactose) and amine (agmatine) composition.17 Conjugation strategies like these are widely used and are simple and fairly robust for laboratory scale work to define structure-activity relationships, but are unlikely to be viable strategies for large-scale manufacturing owing to expense and purification challenges.

Complexation Strategies to package and deliver siRNA using synthetic polymers include physical entrapment within a nano- or microparticle matrix, covalent bonding, or electrostatic self-assembly. The latter is by



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far the most common but can result in thermodynamically pseudo-stable structures18 due to the short length (~6nm) and rigid nature of siRNA.19 Stability has been improved through the use of “gene-like” RNAs (formed when sticky ends enable self-assembly into longer structures),20 the addition of crosslinking elements, and by reducing the rigidity of the polymer.21–23 Although robust binding between polymers and nucleic acids (NA) favors structural stability and protection, these benefits must be balanced against the need for timely intracellular dissociation, and recently, both cellular internalization and subcellular distribution were shown to be influenced by binding affinity in a cell line-dependent manner.21 Current research supports an optimal balance between the size and rigidity of the polymer and that of the NA being condensed, with short siRNA molecules demonstrating favorable complexation with flexible 25kDa PEI and generation 7 TEA-core PAMAM dendrimers (attributed to the relative inability of complexes formed by rigid NAs to fold, physically rearrange or result in hydrophobic aggregation of complexes in solution), while less-flexible generation 5 dendrimers required 25 to 27 mer sticky siRNAS in order to achieve sufficiently strong binding.20,22 Naked, un-modified, siRNA has a circulation half-life on the order of minutes. When using a polymer carrier, the packaging must be sufficient to protect the siRNA from enzymatic degradation prior to cytosolic delivery, yet still able to fully un-package or dissociate from the carrier before being functionally incorporated into the RISC complex. As a result, the optimum complexation strength is material-dependent, but is seldom at the maximum.24 This balance is further complicated following administration into the circulation by physiological salt conditions and competing anionic biomacromolecules. Another interesting challenge is the kidney’s glomerular basal membrane (GBM); in circulation, particles formed primarily by electrostatic interactions are prone to transient decomplexation at the GBM due to interactions with negatively charged proteoglycans – leading to rapid clearance.25 Cyclodextrin-based nanoparticles (NPs) are one example of a material prone to this phenomenon. When formulated with a hydrodynamic radius less than 100nm with a positive zeta


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potential, Zuckerman et al. showed that the components of a cyclodextrin-based delivery system could self-assemble in circulation and potentially in urine (post excretion), but would “transiently accumulate in and be disassembled by the GBM”. This phenomenon was not observed in similarly-sized particles with anionic zeta potentials, which the authors attribute to charge repulsion at the GBM. Furthermore, they recommend heparin sulfate stability be considered when designing and validating future systems utilizing ionic complexation.26

Systemic Delivery As the field of polymeric siRNA delivery matures, an understanding of polymer-blood interactions is essential to elucidate the key structure-activity relationships needed to rationally design the next generation of vectors, as well as to identify ways to better correlate the outcome of in vitro studies to pre-clinical in vivo models. Toward this aim, strides have been made recently within the fields of drug delivery, nanotechnology and proteomics, but further work is needed to establish a meaningful understanding of the ways in which polymer architectures and polyplex parameters affect a wide range of interactions within the blood stream. Upon intravenous injection, polymeric vectors come into contact with a vast array of cells and proteins in the blood stream, which can cause the rapid formation of a complex protein corona.27 Until recently, little was known regarding the pharmacokinetics and pharmacodynamics involved with these interactions. In 2012 a systematic investigation of blood interactions at a molecular and cellular level revealed 41 plasma proteins that interact with PEI. By varying the molecular weight (from 0.6 - 25kDa) and structure (linear vs branched) of PEI, the investigators discovered this common delivery vector had a profound impact on the membrane structure of red blood cells, the conformation of albumin and blood coagulation components, and systemic oxygen delivery efficacy at higher doses (see Figure 5).28,29 A year later, proteomic analysis revealed nearly 300 different proteins that could rapidly adsorb onto the



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surface of silica and polystyrene nanoparticles, affecting not only cellular uptake but also cytotoxicity, hemolysis and thrombocyte activation of the vectors. Tenzer et al showed that these coronas vary in quantity but not composition, and recommended further quantitative proteomic analysis to predictively model serum protein binding kinetics (see Table 1).27 Since surface proteins may effect many aspects of polymeric delivery systems (i.e. immune response, transport, coagulation, etc.), similar quantitative and qualitative proteomic studies across a range of polymeric siRNA delivery vectors could provide valuable insights into materials selection parameters for future delivery systems.

Figure 5. SEM images showing the effects of PEI on both the morphology and aggregation of red blood cells. At 0.6kDa and 1.8kDa, branched PEI was observed to induce morphological changes in a concentration-dependent manner, with RBCs progressing from biconcave to crenated to spherical with spicules along the surface. At higher molecular weights (i.e. 25kDa), both linear and branched PEI induced aggregation as well as morphological changes when administered at 0.1-1mg/mL. Reprinted with permission from ref 28. Copyright 2013 Elsevier.


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Table 1. The 20 most prevalent proteins (measured in ppm) comprising coronas after nanoparticles had been exposed to plasma for 30 seconds. Reprinted by permission from Macmillan Publishers Ltd: Nature Nanotechnology, ref 27. Copyright 2013 No. Amorphous Silica Nanoparticles (commercial) 1 Serum albumin* 2 Apolipoprotein A-I* 3 Complement C3* 4 Ig γ-1 chain C region*

Negatively Charged Polystyrene Nanoparticles Serum albumin* Complement C3* Complement factor H β-2-glycoprotein 1

5 6

Complement factor H Kininogen-1*

7 8 9

Complement C4-A Ig κ chain C region* Serotransferrin

10

Gelsolin

Kininogen-1* Inter-α-trypsin inhibitor heavy chain H4* Ig γ-1 chain C region* Vitronectin Complement C1r subcomponent* Ig γ-3 chain C region*

11

Ceruloplasmin

12

Ig γ-3 chain C region*

Lipopolysaccharide-binding protein Gelsolin

13 14 15 16 17

Positively Charged Polystyrene Nanoparticles Serum albumin* Apolipoprotein A-I* Ig γ-1 chain C region* Inter-α-trypsin inhibitor heavy chain H4* Ig μ chain C region Ig γ-3 chain C region* Ig κ chain C region* Vitronectin Complement C3* Complement C1r subcomponent* Complement C4-A Complement C1s subcomponent α-2-macroglobulin Serotransferrin Apolipoprotein A-IV α-1-antitrypsin Keratin, type II cytoskeletal 1

α-2-macroglobulin Complement C5 Haemopexin Complement C1s subcomponent Ig γ-4 chain C region Apolipoprotein A-I* β-2-glycoprotein 1 Ig μ chain C region Complement C1r Complement C4-B subcomponent* 18 Ig α-1 chain C region Ig κ chain C region* Kininogen-1* 19 Inter-α-trypsin inhibitor heavy C4b-binding protein α chain Clusterin chain H4* 20 Haptoglobin Histidine-rich glycoprotein Ig α-1 chain C region * Denotes proteins that are among the top 20 for all indicated nanoparticles.

Polymers can increase the circulation half-life of siRNA by providing protection against endogenous ribonucleases, hindering opsonization and contributing to a particle size above the glomerular filtration limit, but a strong net cationic charge on the carrier can lead to erythrocyte aggregation and enhanced uptake by the reticuloendothelial system.28 As a result, amphiphilic polymer side chains have long been favored for their ability to increase circulation time while also improving



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cellular uptake. Perhaps the most widely used surface-modifying polymer for this application has been poly(ethylene glycol) (PEG), yet further quantitative studies are needed pertaining to siRNA delivery. In recent years, three studies have assessed the correlation between PEG molecular weight, surface density and polymer chain architecture without using siRNA. In one study, a rapid screening technique was developed that employed highly uniform hydrogel nanoparticles with varying PEGylation densities to study protein adsorption, macrophage uptake and circulation time in the absence of particle loading. Although all particles, regardless of size, shape or composition were ultimately filtered by the organs of the mononuclear phagocyte system, circulation time increased due to delayed phagocytosis, even when the distance between PEG grafts exceeded the Flory radius (RF), resulting in a mushroom conformation (see Figure 6).30 These findings are in contrast with earlier intravenous administrations in which dense PEG brushes were required for adequate shielding and many short PEG chains provided better protection than fewer, longer PEGs.31

Figure 6. Representation of functionalized PEG on the surface of a PRINT hydrogel nanoparticle showing two possible conformations - brush (A) and mushroom (B). Reprinted with permission from ref 30. Copyright 2012 American Chemical Society.

Zheng et al. have assessed the effect of graft density of biodegradable polycaprolactone-blockpoly(ethylene glycol) (PCL-PEG) on a 25kDa hyperbranched PEI (hy-PEI) backbone and found that higher graft densities of 3 and 5 enhanced circulation time and siRNA transfection efficiency. Although


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molecular weight was increased, micelle hydrodynamic diameter, polydispersity, and zeta potential decreased, which was attributed to the increasingly amphiphilic nature of the copolymer. At these two ratios, 95% of siRNA was completely condensed, greater stability against competing polyanions was observed, transfection rates improved (showing 72% and 84% knockdown of GAPDH, respectively), and pharmacokinetics improved (with 3-4 fold greater AUC compared to 25kDa hy-PEI complexes with free siRNA). The increased circulation time was attributed to greater polyplex stability and a decreased zeta potential theoretically resulting in a decreased rate of interaction with macrophages.32 Similarly, Zhao et al. showed that spermine densely grafted to a polyglutamate-derivative backbone yielded serumresistant complexes with weaker binding to both serum proteins and nucleotides, compared to PEI 25kDa, resulting in highly stable polyplexes capable of rapid cytosolic unpacking and transfection rates comparable to Lipofectamine 2000.33 The adsorption of serum proteins onto the surface of a delivery vehicle can be utilized as a “natural functionalization” with beneficial therapeutic effect27 or, alternatively, can mask or alter desired functionality.34 This complex phenomenon can influence both extracellular and intracellular interactions35 - further demonstrating the need for an enhanced understanding of the relationship between physiochemical properties and function in order to enable the optimization of polymer size, nanoparticle packing density, and composition leading to the rational design of clinically relevant delivery systems.

Immunological Considerations Naked siRNA can activate the innate immune system via toll-like receptors (TLRs) – especially TLRs 3 (dsRNA), 7 and 8 (ssRNA) – and cytoplasmic receptors.36 Delivery vectors are intended to shield the RNA from recognition by the immune system, and the relatively inert nature of polymers makes them attractive options. However, as previously discussed, the properties of the vector material (e.g.



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charge, hydrophobicity, rigidity and physical size of resulting complexes) will effect both the manner by which compounds enter the cell and into which intracellular compartments they are sorted, therefore dictating which TLRs will be encountered and potentially stimulated.36 Opsonin proteins adhered to the surface of a foreign object mark the object for phagocytosis. While the immunostimulatory effects of dsRNA are understood and can be mitigated or eliminated through 2’-O-methyl or 2’ fluoro ribonucleotide modifications, polymeric vectors can still have profound immunostimulatory effects both extra- and intracellularly. These effects are important for polymeric vectors that are often cationic and enter the cell via endocytosis where they are exposed to additional pattern recognition receptors (PRRs), thereby increasing the opportunity for innate immune recognition.36–38 To ascertain a structure-activity relationship between hydrophobicity and immune response, freshly-harvested murine splenocytes (comprised of B-lymphocytes, T cells and monocytes) were exposed to gold nanoparticles coated by PEG capped with head groups of varying degrees of hydrophobicity. A nearly linear relationship between the hydrophobicity of polymer head groups and the associated immune response of splenocytes in vitro was observed (see Figures 7 and 8). While increased hydrophobicity also correlated to increased immune response for polymers with LogP values less than 2.0 in vivo, the trend could not be observed in more hydrophobic polymers – possibly due to the poor in vivo biodistribution of such hydrophobic constructs and/or a maximum immune response being reached (see Figure 8B). Quantitative studies, such as this one, exploring the relationship between key material properties and biological function are essential for improving the efficacy of polymeric vectors for siRNA delivery.39 While much work has been done in this field in recent years,37,38 and databases now facilitate the exploration of various factors effecting innate immune response,40 the field will continue to grow as new, and increasingly intricate, polymers are developed. The advantageous or deleterious effects of


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immunostimulation by synthetic vectors must be considered when designing experiments and interpreting results.

Figure 7. Nine chemical structures with varying degrees of hydrophobicity (quantified by an estimate of Log P values via MacroModel – Maestro 8.0) were each conjugated to the ligand termini of tetra(ethylene glycol) spacers attached to gold nanoparticles via hydrocarbon linkers. The resulting library of particles was used to explore the structure-activity relationship between hydrophobicity and immune response. Reprinted with permission from ref 39. Copyright 2012 American Chemical Society.



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Figure 8. Normalized cytokine gene expression as a function of the calculated hydrophobicity (Log P) of headgroups conjugated to gold nanoparticles. Researchers reported on a representative proinflammatory cytokine, TNFα, measured in vitro (A) as well as a representative anti-inflammatory cytokine, IL-10, measured in vivo (B). Gene expression values are reported as a proportion of the expression measured after exposure to a positive control (LPS) under the same experimental set. No correlation was observed in vivo after 6 hours. Reprinted with permission from ref 39. Copyright 2012 American Chemical Society.

Targeting Targeting siRNA to specific organ and cell populations is preferred for systemic delivery as it minimizes the required dose of both polymer and nucleic acid, and reduces potential adverse off-target effects (e.g. cytotoxicity, off-target silencing, overwhelming the RNAi machinery). In the past, polymeric carriers have been designed to actively or passively target specific organs, tissues, disease states and cell types by exploiting unique environmental parameters (e.g. pH,41–44 temperature, redox potential,45–49 leaky vasculature,44,50 over expressed surface receptors,44,51–63 enzymes,64 etc.).65,66 Solid phase


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synthesis, click chemistry, and novel phosphoramidation reactions have enabled the synthesis of many new siRNA-peptide/carbohydrate/small molecule conjugations,67 yielding mono-disperse, targeted polymeric vectors.54 For example, the attachment of siRNA to an anti-Her2 single-chain fragmented antibody (ScFv) with C-terminal protamine peptide and His6 tag was recently heralded as a “bullseye for breast cancer” due to its remarkable targeting ability,68 but it is important to note that the conjugation site of antibodies is crucial to avoid steric interference of antigen/ligand binding and subsequent internalization by the target cell. Chemically defined, site-specific antibody-polymer conjugates (APCs) for siRNA delivery have recently been synthesized by genetically incorporating an unnatural amino acid with biorthogonal reactivity (i.e. p-acetyl L-phenylalanine (pAcF)) into an antibody then selectively coupling the ketone group of pAcF to the terminal aminooxy group of a cationic polymer via a stable oxime bond. The resulting APCs exhibited binding affinities comparable to that of their parent antibodies, selective delivery, and significant silencing at both the mRNA and protein levels. High yields, ease of formulation and mild reaction conditions make this approach an attractive prospect for pharmaceutical scale-up and clinical application.69 Transferrin (Tf) receptors are overexpressed on many types of cancer cells, and Tf has been used as a targeting ligand in several systems currently undergoing clinical trials. Excitingly, investigators recently discovered a way to improve this common targeting modality. Mathematical modeling of cellular trafficking was used to identify ligand-metal interactions as a novel design criterion for improving the cellular association of human transferrin protein (Tf). When nanoparticles were conjugated to oxalate Tf, with its slower iron release rate, in vitro and in vivo studies validated the model.70 Using a similar approach, a library of tandem peptides for dual tumor targeting and cellpenetration were designed then screened for their ability to complex and deliver siRNA in a receptor-



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specific manner. Computational regression analysis was used to identify the structural parameters key to cell type specificity (i.e. overall charge of the peptide and valence of the targeting ligand). Through this systemic approach, myristoylation was shown to not only strengthen hydrophobic interactions and peptide attraction to the cell membrane, but to also condense siRNA into multivalent nanocomplexes and facilitate targeted delivery. This combined computational/experimental approach could help to optimize peptides for cell-type specific delivery.71 Although this study did not employ synthetic polymers, the peptides and structure-activity relationship which were identified can help to inform the delivery of future polymeric delivery systems.

Cellular Uptake Crossing the cell membrane and entering the cytosol remains a critical challenge for polymerbased delivery systems. While all systems appear to undergo some form of endocytosis, some Crossing the cell membrane and entering the cytosol remains a critical challenge for polymer-based delivery systems. While all systems appear to undergo some form of endocytosis, some paths (e.g. caveolaemediated endocytosis and clathrin-independent endocytosis) hold the potential to expedite uptake and bypass the endosome (along with the associated TLRs) and eventual lysosomal degradation.37,72,73 The uptake pathway is polymer-dependent, and earlier reviews have discussed the relationships between the vector composition, internalization and intracellular trafficking for both polymer- and lipidbased strategies.74,75 Caveolar uptake has been shown to be the dominant pathway for a library of micelleplexes76 as well as a variety of polyplexes formed from cationic polymers,77–79 while clathrinmediated endocytosis was the primary pathway for G5 PAMAM dendrimer-based nanoparticles80 and PLGA-grafted, diamine-modified poly(vinyl alcohol) nanoparticles.81 Furthermore, the preferred pathway appears to be system-dependent with some studies showing improved outcomes after clathrinmediated endocytosis77 and others after caveolae-mediated delivery.74,76 Although earlier studies have


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suggested a relationship between most uptake mechanisms and either the size or charge of internalized particles,77,82,83 a new mechanism of macropinocytosis was recently proposed based on experiments showing that cationic polystyrene nanoparticles were endocytosed at a rate dependent upon total polymer mass rather than surface area or number of particles (Figure 9).84

Figure 9. Proposed internalization routes for cationic polystyrene particles of various sizes entering HeLa cells. An inverse correlation between particle size and number of particles per vesicle suggests an “excavator shovel like mechanism” in which the maximum volume of each vesicle is finite, and vesicles form along the cell membrane at a limited rate. Reprinted from European Journal of Pharmaceutics and Biopharmaceutics, 84 / 2, Lerch, et al. “Polymeric nanoparticles of different sizes overcome the cell membrane barrier”, (2013), with permission from Elsevier.84

Functionalization that alters the physiochemical properties of a polymer can also significantly impact the rate and mechanism of cellular uptake. For example, when facial amphipathic deoxycholic acid was conjugated to the terminal amine groups of 1.8kDa PEI, cellular uptake surpassed that of 25kDa PEI without a significant increase in toxicity, due to an apparently energy-independent, non-endocytotic pathway related to enhanced membrane permeability, which mimics the cell penetrating peptide (CPP)mediated transport of macromolecules across the cell membrane. Furthermore, the authors recommend this synthetic conjugation of molecular amphiphiles for use in future high throughput studies .85,86 Since the cell internalization pathway of a polymeric vector is dependent upon both the



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delivery system and cell type,87 there are clear advantages to testing new vectors across a variety of cell types to better understand the potential utility of each vector. Although favorable for masking polymers and stabilizing polyplexes in circulation, PEG can hinder internalization of cationic polymer systems. Interestingly, optimized PEGylation was recently shown to increase receptor-mediated transfection efficiency of anionic nanocomplexes.88 When attached by disulfide bonds or benzoic imine linkers, PEG can shield the system in circulation then be shed in reductive or acid microenvironments to facilitate uptake at the target site.89–92 Gelatin coatings,93,94 polymerized trehalose coatings,95 crosslinking of surface amines, hydrophobic modifications, disulfide bridges, pi-pi stacking, and tyrosine modifications have been used as alternative stabilization strategies for polyplexes. 96–100 Tyrosine modifications are particularly versatile as they have been used as a hydrophobic modification (e.g. partially modifying amines in PEI), as a functionalization for pi-pi stacking (as an aromatic amino acid), and as promotor of endosomal buffering (again due to the aromatic structural component). 96,99,101,102 Hydrophobic acrylates have been used to stabilize the core of polyamine-based structures, and the cross-linking of surface amines has improved the transfection ability of polyplexes utilizing a variety of amine-based polymers. 96,97,100,103,104 These strategies promote a steady rate of transfection over time by stabilizing the polyplex and protecting the siRNA from degradation. The same fusion peptides found within glycoproteins that enable viruses to escape from the endosome can provide endosomolytic function to polymeric vectors. Naturally derived and synthetic cell penetrating peptides (CPPs) are often used to enhance internalization.96,105–107 Although these peptides have been in use for decades,108,109 their mechanism of entry is not fully understood. Endocytosis appears to be the primary route when CPP concentrations are low, but route of entry, delivery efficiency, and toxicity have been shown to depend upon polymer concentration, binding method, cell type, and disease state. Rational vector design would benefit from studies systematically comparing


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CPP-mediated delivery systems within a common cell type to elucidate the nature of peptide-mediated delivery and toxicity patterns.105 Functionality is preserved when CPPs are non-covalently complexed to siRNA or conjugated to polymers, and the duration of action can be prolonged either by conformational stabilization or the use of unnatural amino acids. The cationic nature of CPPs typically necessitates shielding as well as targeting - either through environmentally responsive polymer design or the addition of targeting ligands - for in vivo applications.105 In one study, amphipathic CPPs provided silencing comparable to Lipofectamine 2000, vastly outperforming cationic CPPs.110 In many cases, CPPs have not elicited an innate immune response despite their frequent derivation from non-human proteins; however, there have been some notable exceptions (e.g. the innate immune response observed when a Penetratin-siRNA complex was administered intratracheally to mice).111,112 To address the risks of anti-CPP responses and immunological memory, researchers in the Langer lab recently employed a systematic proteomic study of 50 sequences and identified 3 novel human-derived CPPs that facilitated enhanced siRNA delivery and reduced immunostimulation in vivo.113

Endosomal Escape When endocytosis is the primary uptake mechanism, endosomal escape is a critical, and often rate limiting, step.114 Environmentally responsive systems,115 fusogenic peptides, membranedestabilizing polymers, and the “proton sponge” effect are frequently used to address this challenge. As previously discussed, some CPPs also exhibit a pH sensitive lytic transition (e.g. EB1, MALA, KALA) 105,116,117 when random coils transition to α-helices near pH 5, and this property has been further enhanced by cysteine conjugation resulting in disulfide bond formation,118 making them candidates for dual functionalization.119 Other cell-penetrating peptides (such as hCT) have been added to polymeric



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carriers solely for endosomolysis, and have even been modified (e.g. by the conjugation of fatty acids and peptide sequences) to serve as delivery vehicles in the absence of polymers. 61 If researchers continue to discover novel, human derived CPPs at the current rate, the list of pH sensitive peptides is likely to increase substantially in the years ahead. Maleic acid amide (MAA) has often been used as a linker which hydrolyzes rapidly at endosomal pH levels. Recently, Kataoka’s group used Click chemistry and this anionic moiety to link siRNA to an endosome-disrupting polyaspartamide derivative with two repeating units of aminoethylene per side chain (termed PAsp(DET)), and in so doing, converted cationic cites to anions - rendering the system biologically inert. Delivery of siRNA was enhanced while the IFN-α response was significantly reduced in the murine macrophage cell line (Raw264.7) to 24.3±3.5 pg/mL vs. 60.8±12.9 pg/mL for the same polycation lacking the MAA linkage, but in vivo tests are needed to confirm the in vitro findings (Figure 10).41 Similarly, ketal linkages rapidly hydrolyze under acidic conditions, and have been used in vitro with a poly(β-amino ester) backbone to form a non-toxic, non-immunogenic carrier that delivers siRNA more efficiently than PEI.42,120


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Figure 10. a) Illustration of releasable/enzyme-disrupting conjugate (REC) with the multifunctionality toward endosomal escape and release of mono-siRNA. b) Chemical structure of REC. c) Chemical structure of uREC. The PAsp derivative in this study has the mixed sequence of α and β isomers. Only α isomers are depicted in (b) and (c) for simplicity. Figure and caption reprinted with permission from Takemoto, et al.41 © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Although the validity and nature of the “proton sponge” effect is debated, amine groups are commonly incorporated into polymeric vectors with the goal of exploiting this method of endosomal escape.42,94,121–123 Histamines have been used, in combination with primary amines, to potentially



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increase the buffering capacity of micelles, cSCKs, and dynamic polyconjugates, resulting in improved endosomal escape. Notably, transfection efficiency was lost when histamine conjugations replaced half of the primary amines, and histamines could play a role in the fusion of lipid bilayers under acidic conditions, resulting in lysis in the absence of a “proton sponge” effect.124,125 Recently, nanoparticlebased pH sensors with a dynamic range from pH 3.2-7.0 have been developed and used to demonstrate a lack of endosomal acidification in the presence of PEI.126 As new sensors are developed and computational modeling becomes an increasingly powerful way to predict complex interactions, it is reasonable to expect a more thorough, mechanistic understanding of endosomal escape pathways to inform polymer design in coming years.

Unpackaging Finally, once in the cytosol, the carrier must dissociate from the siRNA for RISC to become active. Hydrolyzable esters, acetal bonds and reducible disulfide bonds are perhaps the most common means for ensuring cytosolic release, and when incorporated into the polymeric backbone, they enable large polymers to be cleaved into segments of a biocompatible molecular weight and charge density.125,127,128 In recent years, investigators tuned the bioreducibility of polymeric vectors along with other physiochemical properties (e.g. hydrophobicity) to design stable systems that commonly selfassemble and provide significant rates of protein knockdown with reduced cytotoxicity over a time span ranging from minutes to weeks.47,49,94,129,130 Interestingly, recent experiments with protein have shown that the redox-responsiveness of disulfide bonds is strongly influenced by the proximity of electrostatic charge as well as steric hindrance. Researchers have demonstrated the potential to manipulate these factors to control disulfide bond stability and optimize intracellular targeting and timed release based on pH. The ability to precisely alter disulfide stability (by approx. three orders of magnitude) within neutral pH environments and complex


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redox buffers (e.g. serum) is a significant achievement with the potential to be useful beyond proteins and into polymer vehicles.131 Other interesting advancements in cytosolic delivery include a recent study in which the 10-fold difference between intracellular and extracellular ATP levels was used to trigger the release of siRNA from polyplex micelles. Katakoa et al. used 3-fluoro-4-carboxyphenylboronic acid phenylboronic acid (FPBA) to quantitatively modify the cationic PEG-block-poly(L-lysine) copolymer. This yielded a delivery vector in which boronic acid formed a reversible covalent ester bond with the siRNA, and PEG enabled the formation of micelles upon complexation with siRNA. This system has provided a dose-dependent silencing of the proto-oncogene pol-like kinase 1 (PLK-1) in vitro.132 In recent years, quantum dot mediated Förster resonance energy transfer (QD-FRET) has been used to study the intracellular trafficking and unpacking of ionically associated constructs, in vitro. Using this approach, Lee et al. quantitatively analyzed the dissociation of siRNA from branched PEI, with and without conjugation to human transcriptional factor (Hph-1), within live cells under flow cytometry.133 Rather than using covalently labeling, Endres et al. used physical entrapment of hydrophobic QDs within the PCL core of PEG-PCL-PEI and demonstrated the feasibility of FRET-switching for the prospective study of polymer-siRNA dissociation within the cytosol.134 In the future, similar QD-FRET studies could be employed across a library of polymers and a variety of cell types in order to build a more robust structure-activity relationship between chemical structure and dissociation kinetics, thereby facilitating the design of the next generation of polymers for siRNA delivery.

Computational techniques for simulating polymer/siRNA interactions Despite an abundance of experimental work, many critical steps in siRNA delivery are not fully understood at a mechanistic level, including polymer/siRNA particle formation, cellular uptake, intracellular trafficking pathways and payload release. In recent years, innovative computational



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strategies have provided new ways to approach these problems as researchers have looked to multiscale modeling bolstered by uncertainty quantification and molecular field theory to elucidate key physical mechanisms for the purposes of designing more efficient carriers. In silico studies have been both validated and complemented by in vitro and in vivo experiments resulting in new mechanistic insights and model systems that can enable a more quantitative evaluation of the structure-activity relationships that might lead to of the next generation of multi-component polymer vectors. Atomistic simulations, coarse grained molecular dynamics, monomer-resolved simulations, and immersed molecular electrokinetic finite element method (IMEFEM) have been successfully combined in a multiscale computational modeling framework to lower computational costs for the exploration of key parameters (e.g. a wide range of sizes, shapes, surface properties, rigidities, flow parameters, physiological conditions, etc.) while quantitatively assessing polymer-siRNA complexation and dissociation, margination dynamics, extravasation, and cellular uptake. When followed by experimental validation, such simulations have become a powerful instrument to enhance mechanistic understanding and drive the rational design of more efficient delivery vectors.135,136 Recent advancements in computational modeling have been reviewed for dendrimeric vectors136 as well as polyplexes and lipoplexes.137,138 Purely in silico studies have shown lipid substitution on PEI impairs neither charge neutralization nor the formation of polyion bridges (critical for complexation), that longer lipid chains have the potential to further stabilize and compact polyplexes as a result of lipid associations139, and that longer ligands on nanoparticles can enhance attachment to cell membranes at the cost of internalization efficiency unless careful attention is paid to side chain density, rigidity and hydrophobic/hydrophilic balance.140 When combined with isothermal titration calorimetry, the importance of minimizing the N/P ratios to achieve efficient cationic polymeric vectors with reduced cytotoxicity has been highlighted,


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with a thermodynamic assessment of polyplex self-assembly revealing the optimum N/P ratio for cationic polymers to be lower than previously expected.22 The field has benefited from the integration of in silico studies within the traditional framework of polymeric delivery studies. When compared to prior in vitro studies, modeling revealed several energetic aspects of dendrimer-siRNA interactions with increased intermolecular hydrogen bonds affecting binding enthalpy and increased polymer flexibility decreasing the entropic penalty of complexation. Furthermore, the length, composition and flexibility of sticky siRNA overhangs greatly affected silencing ability with rigid (dA)5-7 overhangs enhancing binding capacity but flexible (dT)5-7 ends favoring the unbinding necessary for delivery.23,141 In vitro and in vivo studies supported the comparative studies of bolaamphiphiles in silico,142 and confirmed that the formation of tight complexes with proteins depended upon maintaining an arginine/phosphate charge ratio near unity rather than molar ratio or peptide length alone.143 The potential for such computational strategies remains under investigation. For example, time and length scales of atomistic simulations are currently limited by computational power; therefore, coarse grained simulations are used to model larger nanoparticles. Model scales and accuracy are expected to continue to improve as superior coarse grained simulations are developed and computational power continues to increase.137 Novel computational strategies promise to play a prominent role in resolving yet-poorly understood aspects of the delivery process in the years ahead.

Conclusion, Challenges and Future Directions In recent years, the field of polymeric siRNA delivery has made strides toward greater clinical viability largely due to the development of novel chemistries and a more thorough understanding of both cellular processes and polymer interactions at the biological interface, resulting in more nuanced understanding of structure/activity relationships. While polymers comprise only a small percentage of



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the delivery strategies currently undergoing clinical trials, the entire field of siRNA delivery is still maturing. Recent advances in polymer chemistry have enabled the design and synthesis of highly varied architectures with unprecedented consistency, and the computational capabilities available to research today make in silico screening of vast libraries possible. Additional experimental observations will be required in order to build predictive computational models, but imaging techniques are now allowing for more precise intracellular tracking, and 3D human cell culture is providing a superior means for conducting the in vitro studies needed when building and validating such models. These factors, combined with lessons learned from the fields of nanotechnology and protein chemistry - all make this an excellent time for researchers to more thoroughly evaluate the clinical viability of polymeric systems. The challenges are considerable, but so too are the possibilities to improve quality of life for many through effective and affordable siRNA delivery. As discussed previously, endosomal escape has been widely cited as a rate-limiting step, and systemic studies are still needed to elucidate the nature of peptide-mediated delivery and toxicity patterns. Further work is needed to quantitatively and qualitatively assess factors such as immunostimulatory effects, protein interactions, binding affinity, etc. A thorough assessment will require the collaborative efforts of many groups, but the use of in silico strategies and the adoption of standardized SOPs, cell lines and controls will reduce the number of hours required while facilitating the meta-analysis of resulting data. Only once polymeric vectors have been systematically studied on a large scale – with an eye toward structure-activity relationships, interactions at the biological interface, and novel design criteria – will we truly understand their potential.


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For table of contents use only

Strategies Used to Deliver siRNA in Clinical Trials Naked siRNA (n=25)

3.3% 1.6% 4.9% 4.9% 6.6%

41.0%

6.6%

Lipid Nanoparticles (n=19) Polymers (LODER & Cyclodextrin) (n=4) siRNA-GalNAc Conjugates (n=4) Ex Vivo (n=3) Dynamic PolyConjugates (n=3) Self-Delivering (n=2) Viral (n=1)

31.1% TOC Graphic: A breakdown of the number of national clinical trials that employ each siRNA delivery strategy – source: clinicaltrials.gov Manuscript Title: Polymers for siRNA delivery: A Critical Assessment of Current Technology Prospects for Clinical Application Authors: Bailey M. Cooper & David Putnam


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Polymers for siRNA Delivery: A Critical Assessment of Current

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