Abstract
The recent success of gene therapy during the COVID-19 pandemic has underscored the importance of effective and safe delivery systems. Complementing lipid-based delivery systems, polymers present a promising alternative for gene delivery. Significant advances have been made in the recent past, with multiple clinical trials progressing beyond phase I and several companies actively working on polymeric delivery systems which provides assurance that polymeric carriers can soon achieve clinical translation. The massive advantage of structural tunability and vast chemical space of polymers is being actively leveraged to mitigate shortcomings of traditional polycationic polymers and improve the translatability of delivery systems. Tailored polymeric approaches for diverse nucleic acids and for specific subcellular targets are now being designed to improve therapeutic efficacy. This review describes the recent advances in polymer design for improved gene delivery by polyplexes and covalent polymer-nucleic acid conjugates. The review also offers a brief note on novel computational techniques for improved polymer design. We conclude with an overview of the current state of polymeric gene therapies in the clinic as well as future directions on their translation to the clinic.
Keywords: Polymers, Polycations, Gene delivery, Nucleic acid delivery, RNA, Polyplexes
Graphical Abstract
The recent surge in the popularity of lipids, propelled by the approval of several LNP mRNA vaccines, overshadows polymers as gene delivery vectors, despite their strong potential. In the shadows of the LNP publicity, several polymeric systems are in clinical trials, with multiple early-stage companies actively developing polymeric gene delivery systems. This review highlights advances in polymer design for gene delivery via polyplexes and covalent polymer-nucleic acid conjugates, and briefly covers novel computational techniques for improved design.
1. Introduction
The recent success of the mRNA-based COVID-19 vaccines and recognition of the CRISPR-Cas9 technology with a Nobel Prize have propelled gene therapy into renewed spotlight [1]. Gene therapy, as defined by the American Society of Gene and Cell Therapy, is the use of genetic material, commonly DNA or RNA, to treat or prevent diseases [2]. Gene therapy relies heavily on the successful delivery of therapeutic DNA or RNA across multiple physiological barriers to the target tissues and cells. Although formulations of nucleic acids without the use of delivery systems remains an area of active research, their applications are restricted to local administration, and they demonstrate limited efficacy [3]. Systemically administered nucleic acids must overcome multiple extracellular and intracellular barriers before reaching their site of action [4]. Delivery systems help overcome these issues, thus improving therapeutic efficacy [5]. Currently, majority of approved gene therapies and those in clinical trials employ viral vectors, most commonly adeno-associated viruses (AAVs) and retroviruses, primarily due to their high transfection efficiency, arising from their natural ability to infect cells [6]. However, viral vectors are fraught with multiple concerns regarding their safety, immunogenicity, off-target effects, limited loading efficiencies, and risks of insertional mutagenesis [7, 8]. Moreover, high manufacturing and production costs translate to exorbitant treatment costs, thus limiting their widespread use [9]. Non-viral delivery systems, particularly polymers and lipid nanoparticles (LNPs), are increasingly explored as a welcome alternative.
Despite recent clinical approvals and the surging industry interest in LNPs, major bottlenecks still exist in their widespread use. These include challenging long-term storage stability, requirement of specialized ultra-cold temperature-controlled transport systems, difficulties in achieving extra-hepatic targeting, and limited cytosolic delivery [10, 11]. It was reported that less than 2% of endocytosed LNPs escape the endosome and reach the cytoplasm [12]. Similarly, current presentations of LNP vaccines require storage at ultracold temperatures (−80 °C) which is a major hurdle in their transport and distribution [13]. Moreover, poly(ethylene glycol) (PEG)-ylated lipids, which form a crucial part of the current clinical LNP formulations, have been reported to generate unwanted immune reactions [14-16]. The global surge in LNP development has resulted in a complex, crowded intellectual property landscape with an exponential rise in reports of patent infringement. Stringent intellectual property laws combined with a crowded market point to reduced operational freedom, thus restricting development [17].
Polymers offer a promising alternative with a strong potential to overcome many of these issues and expand the therapeutic landscape of gene therapies. The vast chemical space of polymers easily surpasses that of lipids due to the ability to control chemical composition by copolymerization of two or more comonomers, increased ability to control the monomer sequence in the polymers, ability to control molecular weight, and the possibility to synthesize polymers in multiple architectures, creating opportunities for the discovery of novel materials with desirable properties that are well-suited for gene delivery applications. Similarly, different nucleic acids by virtue of differences in their structure and site of action demand the development of payload- and tissue-specific delivery systems. Screening of polymer libraries offers an ability to choose polymer structures that are most effective for the specific type of genetic payload to be delivered [18, 19]. Advanced characterization and computational techniques have enabled the exploration of detailed structure-activity relationships of polymers, thus paving the way for further optimization. Novel design concepts have helped overcome limitations of cytotoxicity, unwanted activation of the immune system, and premature extracellular dissociation associated with traditional polymer-based systems [20-22]. Advanced polymerization techniques have also enabled the synthesis of covalent polymer-nucleic acid conjugates, offering a promising alternative to traditional nanoparticles based on self-assembled polyelectrolyte complexes (polyplexes) of polycations and nucleic acids.
The recent surge in the popularity of lipids propelled by the approval of several LNP vaccines has overshadowed polymers despite their strong potential as gene delivery vectors. Despite the lack of publicity, several polymeric systems are currently undergoing clinical trials, and multiple early-stage companies are actively working on the development of polymeric gene delivery systems. Ongoing efforts in the field suggest that polymeric gene therapies are on track for clinical translation. Moreover, expanding the applicability, polymeric gene delivery systems are also being actively explored for pulmonary delivery of mRNA vaccines for respiratory infections due to the relative simplicity and potential for improved patient compliance of this route [23, 24]. This review aims to highlight the vast design space and functionality of polymers by discussing recent advances in polymer design for enhanced gene delivery (Figure 1, Table 1). The first section discusses structural modifications of cationic polymers for improved polyplex formation, including optimization of polymer composition and architecture, development of non-cationic polyplexes, incorporation of hydrophobic moieties, and fabrication of polymeric supramolecular structures. The second section discusses recent advances in the development of polymer-nucleic acid bioconjugates. The review also offers a brief note on the utilization of advanced computational techniques for improved polymer design. Lastly, we conclude with an overview of the current state of polymeric gene therapies in the clinic as well as future directions on their translation to the clinic.
Figure 1 -. Advances in polymer design for improved gene delivery.
Overview highlighting some of the structural modifications of polymeric gene delivery systems (top panel) and the resulting areas of improvement across various sectors of gene delivery (bottom panel). As highlighted in this review, the vast design space and functionality of polymers creates opportunities for the discovery of novel materials with desirable properties that are well-suited for gene delivery applications. Created with BioRender.com.
Table 1.
Summary of polymer design strategies contributing to improved gene delivery.
| Design Strategies | Characteristics | References |
|---|---|---|
| Choice of nitrogenous and non-nitrogenous cationic groups |
|
[53, 57, 59, 60, 63, 65] |
| Incorporation of hydrophobic domains |
|
[72, 73, 77, 78, 80-84, 89] [96-99, 101-109, 112, 113, 115-118] |
| Zwitterionic polymers |
|
[127, 130-132, 135-139, 141-146] |
| Decationizable polymers |
|
[148-153] |
| Supramolecular polymers |
|
[163, 164, 167-169, 174, 175, 177-181] |
| Covalent polymer-nucleic acid conjugates |
|
[222, 224, 225, 227-229, 232, 236-238, 244] |
2. Polymer properties impacting polyplex-mediated gene delivery
Polyplexes are electrostatic complexes of anionic nucleic acids and cationic polymers, generally in the nanometer range. Flexibility in polymer synthesis allows fine-tuning of the polymer properties to enhance polyplex-mediated gene delivery. Advanced polymerization techniques have enabled the synthesis of polymers with well-defined structures and pre-determined molecular weights. Precise control on polymerization has allowed researchers to explore the effects of polymer structure and composition on various aspects of gene delivery, including stability, toxicity, and transfection efficiency in an effort to design polymers better optimized for successful gene delivery. Multiple factors, including polymer topology (arrangement of individual monomers in the polymer), charge density, monomer composition, and molecular weight can be fine-tuned to tackle specific hurdles in gene delivery. Based on their composition, polymers can be divided into homopolymers (synthesized from a single type of monomer) or hetero- or co-polymers (synthesized from two or more monomers) [25]. Heteropolymers have the advantage of combining monomers with different properties in a single polymer chain. Polymeric delivery systems, in general, are simple and employ a single chemical component (the polymer) in addition to the therapeutic payload, enabling ease of scale-up and manufacturing. In contrast, LNPs are complex and require a precise balance between multiple lipids to form a stable and effective delivery system. The following section discusses different polymer properties that can be fine-tuned to optimize polyplex-mediated gene delivery.
2.1. Polymer architecture
Based on their spatial arrangement, polymers can broadly be divided into linear and branched architectures. Linear polycations and dendrimers represent the first generation of polymers to be explored for gene delivery. In fact, JetPEI®, a commercially available linear polyethylenimine (PEI), is still routinely employed in clinical trials for gene delivery. However, toxicity concerns with native PEI have limited its applications and thus efforts have been directed towards safety-enhancing modifications [26]. Linear polymers have a simple architecture with a single backbone and can be designed to contain various cationic groups at different locations of their structure to improve polyplex stability and transfection efficiency. Similarly, linear copolymers can be synthesized with hydrophobic blocks or non-ionic hydrophilic blocks to form micelles and poly-ion complexes respectively. In contrast to the amphiphilic self-assembly seen with micelles, poly-ion complexes are formed by electrostatic interactions which place the therapeutic nucleic acid in the core with the hydrophilic polymer forming a protective corona around it [27].
The progression of polymer architecture from linear to more complex branched topologies opened up new avenues for gene delivery. Branched polymers can further form multiple structures depending on the type and extent of branching, including but not limited to dendritic, star-shaped, bottlebrush, and comb-shaped structures [28] (Figure 2). Branched polymeric structures enable intrinsic multifunctionality within a single polymer as individual arms may consist of different polymeric chains, a task difficult to achieve with lipids [29]. Branched polymers are said to have a higher potential for gene delivery because of their improved buffering capacity, three-dimensional structure, and multiple terminal groups, which allow for improved interactions with cell membranes during intracellular trafficking and subsequently enhanced transfection [30, 31]. Multiple studies have demonstrated enhanced transfection efficiencies of the branched architectures of several common polymers, including polylysines (PLLs), poly(β-aminoester)s (PBAEs), and poly(dimethylaminoethyl methacrylate)s (PDMAEMA)s, compared to their linear counterparts [30, 32, 33]. Additionally, Schallon et al. reported that polyplexes formed using branched PDMAEMA and PEI follow a different route of uptake and intracellular trafficking compared to their linear counterparts, further emphasizing the impact of polymeric architecture on different aspects of gene delivery.
Figure 2 -.
Schematic representation of topologically different polymers. Created with BioRender.com
With advances in polymerization techniques, novel polymeric architectures are being increasingly reported. Wei et al. reported the development of novel macrocyclic brush or sunflower-shaped polymers. These polymers were synthesized by controlled radical polymerization using a cyclic macroinitiator “core” from which “petals” were polymerized, radiating from the core [34]. Cheng et al. synthesized a sunflower-shaped PDMAEMA polymer and evaluated its ability to deliver pDNA compared to its linear and comb-shaped analogs. The sunflower-shaped polymers demonstrated improved stability, reduced cytotoxicity, and a higher transfection efficiency both in vitro and after intraventricular injection to the mouse brain. Experiments revealed faster unpacking of polyplexes in the presence of competing anions compared to the controls, suggesting that the sunflower architecture may have provided the desired balance between extracellular stability and intracellular release, leading to improved performance [35]. In a follow-up comparison study between linear, branched, comb, and sunflower-shaped polymers, only the comb and sunflower topologies successfully transfected an immortalized human T cell line (Jurkat cells), pointing to their potential use in CAR T cell manufacturing [36].
2.2. Monomer arrangement
In addition to the architecture, the arrangement of monomers also impacts the delivery performance of cationic polymers [37, 38]. Different arrangements can be fabricated based on the position of monomers in a heteropolymer, including statistical, block, gradient, and random copolymers (Figure 2). In contrast, lipid structures offer limited flexibility in composition and are restricted to specific arrangements of a headgroup, degradable linkers, and a non-polar tail. The choice and arrangement of monomers in a copolymer impact its interaction with the nucleic acid as well as with cellular components [39, 40]. Correia et al. explored the influence of cationic charge distribution on cytotoxicity while keeping other properties, such as monomer composition and molar mass constant. A panel of linear copolymers was synthesized by group transfer polymerization using DMAEMA and oligo(ethylene glycol) methyl ether methacrylate (OEGMA) as monomers. The architectures explored included AB diblock, BAB and ABA triblock, ABAB tetrablock, statistical, A-b-(A-co-B)-b-B, and gradient/tapered copolymers. The cytotoxicity of these polymers was assessed in multiple cell lines. Results demonstrated that block copolymers were less toxic than other types of copolymers. This lower toxicity is attributed to the reduced accessibility of the cationic charges to the cell membrane, thus reducing the likelihood of membrane disruption and leading to higher cell viability (Figure 3) [41]. These results contrasted with previous studies, which reported that random copolymers were less cytotoxic due to a lower charge density than block copolymers [40, 42]. A limitation of these studies is that they were conducted with polymers with different structures and properties, thus compromising the ability of direct comparison. Additionally, differences between the experimental conditions such as the molecular weight, composition of monomers, and even the cell lines used make it challenging to draw definitive conclusions from such comparisons. Overall, these results suggest that several factors in addition to the arrangement of monomers such as the type of monomer, charge density, and molecular weight influence polymer cytotoxicity.
Figure 3 –
A) Chemical structures and architectures of the co-polymers. OEGMA300 and DMAEMA units are represented in green and blue, respectively. B) Hypothesized illustration of the interaction of different polymer architectures with the cell membrane. A more spread-out distribution of positive charges in the gradient architecture may lead to increased interactions with the cell membrane, thus causing increased toxicity. Reproduced with permission from [41]. Copyright 2023, Royal Society of Chemistry
2.3. Molecular weight
The molecular weight of polymers has a significant impact on gene delivery, primarily affecting stability, cytotoxicity, and transfection efficiency. However, since each polymer has its own distinct chemical properties, the range of optimum molecular weights for gene delivery differs from polymer-to-polymer. Generally, with polycations, enhancing molecular weight leads to heightened transfection efficiency; however, this improvement often comes at the expense of increased cytotoxicity. Both these effects have been partially attributed to increased interactions with the cell membrane. Direct correlation of molecular weight to cytotoxicity and transfection efficiency has been well documented in multiple commonly used polymers, including PEI, chitosan, PLL, and PDMAEMA, across different architectures [43, 44]. However, with the development of novel polymers, these trends no longer hold true, and high molecular weight polymers with low toxicity are increasingly reported. Gao et al. reported an increase in pDNA transfection efficiency and a decrease in cytotoxicity with increasing molecular weights in highly branched PBAEs. Polyplexes formed with the low molecular weight polymers had a much larger size and aggregated morphology, leading to excessive interactions with the cell membrane, which was hypothesized to be a possible reason for the elevated cytotoxicity [45].
Lastly, the intracellular site of action as well as the structural differences in nucleic acid cargo demand polymeric delivery systems optimized for each therapeutic payload. Blakney et al. explored the impact of charge density and molecular weight on the transfection efficiency of mRNA, pDNA, and self-amplifying replicon RNA (RepRNA) using a library of poly(2-ethyl-2-oxazoline)/PEI copolymers with diverse charge densities and molecular weights. They reported that a combination of high molecular weight and high charge density was required for optimum transfection with pDNA and RepRNA. In comparison, a smaller molecular weight with a relatively lower charge density was sufficient for the transfection of the smaller mRNA [19]. In contrast to the well-established LNP template, the strategies discussed above, combined with additional techniques discussed in the upcoming sections, provide formulators with a large selection of tools and the independence to use them in designing new, improved polymeric gene delivery systems.
3. Chemical composition of polymers for enhanced polyplex-mediated gene delivery
Modulating the chemical composition of cationic polymers provides an opportunity to enhance various aspects of gene delivery, including complexation, stability, cellular uptake, and endosomal escape. Incorporating functionalities in the polymer structure that complement electrostatic interactions with additional non-covalent interactions, such as hydrogen bonding and hydrophobic interactions, is a strategy used to improve the stability of polyplexes. Moreover, hydrophobic segments can enhance interaction with cellular membranes, facilitating better cellular uptake of the genetic material. The type and nature of the cationic groups in the polymers offer further opportunities to enhance gene delivery. Nontraditional cationic groups such as phosphonium and sulfonium can improve the binding affinity and protection of nucleic acids, offering an alternative to the conventional nitrogenous cations [46, 47]. Slight variations in the chemical structure of a polymer can help optimize gene delivery to specific organs. For instance, Rotolo et al. employed a combinatorial synthesis approach to screen a library of 166 polyplexes formulated using PBAE based polymers with varying backbones, linkers, branching components, and end-capping structures to develop a species-agnostic polyplex for inhalable mRNA delivery to the lungs. Through this approach, they identified P76, a poly-β-amino-thioester, that enabled potent inhalable delivery of mRNA cargo irrespective of size to multiple animal species, including mice, hamsters, ferrets, cows, and rhesus macaques, thus exhibiting the species-agnostic behavior of this formulation [48]. Similarly, Dirisala et al. demonstrated that a single methylene group could impact the nuclease stability of mRNA polyplexes. Polyplexes formed using poly(L-ornithine), which has a trimethylene spacer, were more stable against nucleases than polyplexes formed using PLL, which has a tetramethylene spacer. Improved stability was attributed to tighter mRNA packaging by poly(L-ornithine) [49]. Systematic investigation of these parameters can aid the development of more effective and versatile polymer-based gene delivery vectors, paving the way for advanced therapeutic applications. The following section explores various strategies employed to aid gene delivery performance of cationic polymers through modulating their chemical composition.
3.1. Types of cationic groups
The most important feature of a polycation is the cationic group that binds electrostatically to the nucleic acid, forming a polyplex. Amines are by far the most employed cationic groups. However, other nitrogenous and non-nitrogenous groups are also being explored. Fine tuning the type and content of amine groups and incorporating non-traditional cationic groups in the structure of polymers offers an opportunity to improve the gene delivery performance of cationic polymers.
3.1.1. Nitrogenous Cationic Groups
Nitrogenous cationic groups explored for gene delivery include amines, imidazolium, and guanidium groups. Of these, amines are the most prominent. The type of amine group determines the charge density as well as the pKa of the polymer and hence the extent of protonation under different pH conditions. The presence of primary, secondary, and tertiary amines with different pKa values and thus a broader buffering range has been a major contributing factor to the performance of branched PEI, aiding its endosomal escape through the ‘proton sponge’ mechanism [50, 51]. While the mechanism has been questioned, fine tuning of the pKa values of a polymer has been a dominant technique often employed to aid efficient transfection. An important point to note is that the apparent pKa of the polymer is the average ratio of all ionized to unionized groups in the polymer and not the intrinsic pKa of individual groups in the polymer [52]. Du et al. synthesized a series of tri-block polymers with pKa values ranging from 5.2 to 7 to assess the impact of pKa on the efficiency of siRNA delivery. Different pKa values were achieved by varying the number and type of hydrophobic amine monomers. Results indicated that improved silencing efficiency was detected in polymers with pKa ranging from 5.8 to 6.2, both in vitro and in vivo, potentially due to improved siRNA release and endosomal escape [53]. Similarly, a computational study suggested that ideal polycations should display tandem pKas. The study indicated that a polymer should have one pKa between 10 and 8 for efficient binding and complexation and another between 7.25 and 6.25 to ensure efficient endosomal escape [54]. Similarly, the apparent pKa value of ionizable lipids was proven to be critical for the efficient delivery and transfection efficiency of LNPs [55]. Additionally, cationic polymers containing primary amines were reported to be more toxic than those with tertiary amines [56]. At physiological pH, primary amines are protonated and interact with the negatively charged cell membranes, resulting in elevated toxicity compared with tertiary amines. Complementing electrostatic complexation with non-covalent interaction like hydrogen bonding and hydrophobic interactions improves stability of the polyplexes. Aryl amines have shown strong performance by virtue of the additional non-covalent interactions. In a series of glycine, leucine, and tyrosine-modified PEG-poly(glycerol) block copolymers, tyrosine modification led to enhanced stability against nucleases, leading to improved blood circulation following intravenous injection in mice. The amines on the amino acids were responsible for electrostatic condensation of the nucleic acids. Improved performance for the tyrosine-modified polymer was attributed to π-π stacking between mRNA bases and tyrosine as indicated by measurement of tyrosine fluorescence quenching [57]. In another study, modification of low molecular weight PEI with tryptophan led to improved stability and transfection efficiency when compared to modification with leucine, an aliphatic amino acid [58]. Similarly, the guanidium group forms hydrogen bonds with the phosphate bases in nucleic acids, further improving stability of the polyplexes [63-65]. Miyazaki et al. compared effectiveness of amines and guanidines by synthesizing PEG-poly(glycidyl methyl amine) (PEG-PGMA) and PEG-poly(glycidyl methyl guanidine) (PEG-PGMG) block copolymers. They demonstrated that mRNA complexes formed with the guanidine containing polymers showed enhanced stability against polyanions, urea, and nucleases compared to the amine containing complexes [59].
3.1.2. Nontraditional cationic groups
In addition to the traditional nitrogenous polycations, non-nitrogenous phosphonium and sulfonium polycations have been explored as promising alternatives for gene delivery [60]. In some studies, the nontraditional cations were reported to have better transfection efficiencies and cytotoxicity profiles compared to the nitrogenous analogues. Differences in the electronegativity and electron density distribution in sulfur and phosphorous-based cations compared to nitrogen is thought to influence the polymer binding of nucleic acids. Several studies reported that phosphonium containing polymers exhibit better transfection efficiency combined with reduced cytotoxicity than ammonium containing analogues [61, 62]. Herma et al. compared the cytotoxicity and siRNA transfection efficiency of carbosilane dendrimers with phosphonium terminal groups to their structural analogues with traditional ammonium terminal groups. The results demonstrated that the phosphonium-containing dendrimers had a comparable transfection efficiency but significantly lower in vivo toxicity compared to the ammonium group containing dendrimers [63]. Sulfonium containing polymers are another alternative to nitrogenous cations. Despite improved stability of their polyplexes and low cytotoxicity, they tend to exhibit low transfection efficiency, constituting a major drawback for their use in gene delivery. This has been hypothesized due to the strong binding affinity between nucleic acids and sulfonium polycations leading to difficulties in the release of the therapeutic cargo after cellular uptake [64]. To overcome this drawback, Zhu et al. reported the development of a novel class of reactive oxygen species (ROS) responsive, disintegrable polysulfonium polymers. The polysulfoniums could efficiently condense DNA, forming stable polyplexes with improved transfection efficiency compared to the control PEI, both in vitro and in vivo [65].
3.2. Incorporation of hydrophobic domains
Incorporation of hydrophobic domains into cationic polymers is a popular strategy to improve their gene delivery properties. Recent theoretical and simulation studies revealed that the morphological reorganization and disruption of lipid bilayers, resulting from the interaction of nanoparticles with cell membranes is significantly influenced by the surface hydrophilicity [66]. Polymeric nanoparticles, when sufficiently hydrophilic, can rapidly extract lipid molecules from cell membrane. This process leads to the irreversible formation of "pores" or "holes" in the lipid bilayers [67-69]. In contrast, nanoparticles with higher hydrophobicity, like LNPs, despite being embedded in lipid bilayers, do not induce pore formation [70]. Based on these studies, hydrophobic moieties are widely incorporated in polymers to reduce pore formation and thus minimize cytotoxicity. Hydrophobic interactions between the polymer and the nucleic acids complement electrostatic interactions, thus enhancing polyplex stability [71]. Conversely, hydrophobic moieties lead to reduced electrostatic interactions which enhance transfection by promoting faster complex dissociation following cellular uptake. Gabrielson et al. reported that the partial acetylation of PEI improved its transfection efficiency through weakened polymer/DNA interactions leading to enhanced polyplex dissociation inside the cells [72]. However, these results are not universal and are impacted by the polymer structure. For example, Kravitz et al. reported reduced transfection efficiency with dodecylated polysuccinimide-based polymers due to incomplete polyplex dissociation resulting from a strong interaction of the pDNA with the alkylated polymer [73]. Additionally, hydrophobic moieties increase cell membrane interactions thus aiding in adsorptive endocytosis [71]. The extent of hydrophobicity has been reported to impact pathways of cellular uptake and intracellular trafficking [74-76]. Zhang et al. synthesized a series of polymers with similar structures but varying hydrophobicity using 2-(pyrrolidin-1-yl)ethyl methacrylate and 2-(N,N-di-iso-propylamino)ethyl methacrylate as monomers. They demonstrated that the partially hydrophobic cationic polymers were taken up by macropinocytosis, thus avoiding endosomal degradation leading to improved transfection even at low concentrations [77].
In the past decade, multiple hydrophobic moieties, including aliphatic chains, cholesterol and its derivatives, palmitic and fatty acids, have all been conjugated to cationic polymers to improve their delivery efficiency [78]. Rui et al. explored the impact of polymer backbone hydrophobicity using a series of PBAE polymers with varying hydrophobic monomer content. In general, increasing polymer backbone hydrophobicity increased uptake and transfection in vitro across the three types of nucleic acid modalities tested – siRNA, mRNA, and pDNA [79]. Kim et al. utilized a series of amphiphilic polyaspartamide derivatives with varying alkyl sidechains to explore the impact of the type of hydrophobic moiety on mRNA delivery. Linear alkyl amines with 5-10 carbons, phenylethyl amine, and cyclohexyl ethyl amine were used. Additionally, to quantify and assess the impact of hydrophobicity on stability, uptake, and transfection efficiency, the data obtained were plotted against the octanol-water partition coefficient (logP) values of the polymers. A direct correlation of the logP values to uptake, stability, and transfection was observed. In general, a threshold of logP > −2.31 was found to be essential for achieving high levels of mRNA expression. All polymers with logP > −2.31, had similar uptake; however, in this subgroup, the polyaspartamide derivative with alicyclic cyclohexylethyl side chain, which had the lowest logP had a much higher transfection efficiency. Further experiments revealed that this was due to the rapid unpackaging of the mRNA following cellular uptake [80]. A subsequent study with a series of alicyclic moieties revealed a distinct correlation of hydrophobicity with in vitro and in vivo mRNA expression, demonstrating the potential of fine-tuning the alkyl moieties for optimum design of mRNA polyplexes [81]. In addition to the type and content of hydrophobic moieties, recent studies have revealed that the distribution of hydrophobic groups in the polymer chain can also impact gene delivery [82, 83].
Majority of the studies till date have explored hydrophobization of either the polymer or the nucleic acid to improve delivery efficacy of the polyplexes. Taking a step forward, Sarett et al. explored a dual hydrophobization approach with a palmitic acid-conjugated siRNA and a partially hydrophobic polymer to explore the impact of combining electrostatic and hydrophobic interactions on the delivery efficiency (Figure 4). Increased van der Waals interactions between the hydrophobic polymer and the hydrophobized siRNA led to more efficient loading and increased stability which in turn translated to improved pharmacokinetics and an enhanced transfection efficiency of the polyplex [84]. Increasing the polymer content while keeping the siRNA amount constant often leads to improved circulation half-lives at the cost of increased hepatic toxicity. The same group later reported that the dual hydrophobization approach decreased the amount of polymer required to attain optimum pharmacokinetic profiles as well as reduced in vivo cytotoxicity thus improving therapeutic efficacy. Thus, the dual hydrophobization approach is a promising strategy for enhancing delivery efficacy while simultaneously reducing polymer associated toxicity [85].
Figure 4.
A) Synthesis and MALDI-TOF and GPC characterization of 50B polymer and palmitic acid conjugated siRNA. B) Dually hydrophobized polyplexes (siPA-NPs) were more stable in presence of heparin and had a longer circulation half-life than control polyplexes formed with unmodified siRNA (si-NPs) when injected intravenously. C) siPA-NPs showed a higher tumor accumulation as well as D) enhanced luciferase silencing in MDA-MB-231 breast cancer orthotopic model. Reproduced with permission from [84]. Copyright 2016, Elsevier
Depending on the architecture, the presence of hydrophobic moieties in the polymers can lead to the formation of core-shell nanostructures. This is especially true in case of amphiphilic block copolymers which can self-assemble in an aqueous medium to form micelles with a hydrophobic core and a hydrophilic shell [86]. Examples of polycationic micelles with multiple polycationic and hydrophobic blocks have been reported [87]. Micelle-forming amphiphilic block copolymers with a cationic group condense nucleic acids to form structures called micelleplexes. Studies from the Reineke lab have shown that micelleplexes outperform analogous polyplexes in the delivery of pDNA and antisense oligonucleotides ASO [88, 89]. Additionally, the hydrophobic core of the micelles offers an opportunity to co-deliver hydrophobic drugs in addition to the complexed nucleic acids to achieve synergistic effects [90-93]. A cross-linking agent can also be incorporated in micelleplexes, leading to the formation of a sustained delivery vehicle, a strategy yet to be reported for LNPs.
3.2.1. Perfluoroalkylated polymers
Polymers containing hydrophobic perfluoroalkyl groups (“fluorinated polymers” henceforth) form a unique subset of polymers containing hydrophobic domains. Perfluorocarbons (PFCs) are unique organofluorine compounds generally represented as CxFy. PFCs contain only C-C and C-F bonds, are biologically inert, and are widely used in biomedical applications [94]. The low polarizability and low surface energy of the C-F bond results in PFCs being both hydrophobic and lipophobic. PFCs exhibit phase-separation tendencies in both aqueous and organic solvents but a high tendency to associate with other fluorinated species [95]. This phenomenon is known as the fluorophilic effect. These special properties of perfluorinated compounds have been extensively applied to cationic polymers to develop their fluorinated analogues with superior properties. The low surface energy as well as the fluorophilic effect allows for excellent self-assembly, leading to improved polyplex stability. This in turn enables lowering the N/P ratio (N: protonatable nitrogen in a transfection reagent and P: anionic phosphate groups in a nucleic acid), which reduces toxicity of the polyplexes.
Fluorinated G5 PAMAM dendrimers were reported to form stable polyplexes and could transfect HEK293 and HeLa cells at an extremely low N/P ratio (N/P = 1.5) [96]. The inert, hydrophobic nature of fluorinated polymers prevents serum protein binding while the lipophobicity prevents fusion of the polymer with the phospholipids in the cell membrane, thus avoiding early unpacking of the complex, while enabling faster internalization. Shen et al. reported higher knockdown efficiencies for fluoroalkylated PEI, compared to alkylated and cycloalkylated analogues [97]. Fluorinated cationic polymers demonstrate significant transfection efficiencies even in the presence of serum which is often a drawback of non-fluorinated cationic polymers [98, 99]. Moreover, incorporating fluorinated moieties in the chemical structure of nucleic acids can improve their stability through the fluorophilic effect [100]. Fluorination of commonly employed polymers including PEI, PLL, Poly(amidoamine) (PAMAM), and PDMAEMA has demonstrated improved gene delivery in multiple studies [101-104]. Transfection efficiency of PEI is directly proportional to its molecular weight; however, its cytotoxicity also increases with molecular weight. Xiao et al. synthesized a series of low molecular weight (8 kDa) fluorinated PEIs with varying extents of fluorination and compared their delivery efficiency to the standard 25 kDa branched PEI. The fluorinated polymers demonstrated low cytotoxicity and a similar transfection efficiency compared to the control [105]. Interestingly, in addition to reducing their toxicity, it was found that fluorination of branched PEI changed the biodistribution of siRNA polyplexes from the lungs to the liver [106]. Fluorinated polymers can be synthesized by grafting fluoroalkyl moieties on the polymer chain postpolymerization or by incorporating fluorinated monomers in the polymerization reaction. In line with non-fluorinated polymers, fluorinated random copolymers demonstrated better efficiency than fluorinated block copolymers [107].
Compared to linear fluoroalkyl groups, aromatic fluorocarbons have demonstrated enhanced stability due to combined fluorophilic effect and π-π stacking interactions between aromatic rings. A recent study reported enhanced transfection efficiency of fluorinated PAMAM dendrimers modified with a guanidium group due to improved cellular uptake attributable to the hydrogen bonding of the guanidium group with its counterparts on the cell surface [108]. Wang et al. explored the structure-activity relationship of fluorinated dendrimers for the delivery of pDNA and siRNA. A series of heptafluorobutyric acid conjugated PAMAM dendrimers of G4-G7 generations and varying degrees of fluorination were synthesized. A minimum of 50% fluorination was found to be essential for efficient delivery. A higher fluorination amount was required for efficient transfection of siRNA compared to pDNA (64% vs 57%) [109]. Thus, the degree of fluorination needs to be optimized based on the therapeutic cargo to achieve optimum transfection efficiency.
Fluorinated polymers also possess high cell and tissue penetration, again attributable to their lipophobic and hydrophobic properties. Fluorinated polymers have demonstrated enhanced penetration capabilities in 3D tumor spheroids and solid tumors [103, 110]. Ge et al. reported that fluorinated peptides demonstrated a 240-fold enhanced mucus penetration following intratracheal delivery of siRNA resulting in efficient downregulation of the target gene (Figure 5). In contrast, the non-fluorinated peptides were trapped in the mucus layer. Fluorination enhanced mucus penetration by preventing adsorption of mucin glycoproteins on the polyplexes which helped prevent polyplex destabilization [111]. Fluorinated amphiphilic polymers have also been used as surfactants in the formulation of emulsion polyplexes [112-115]. Xiao et al. synthesized fluorinated, amphiphilic, low molecular weight PEI and explored its use as a surfactant to form cationic fluorinated perfluorodecalin emulsions. The fluorinated emulsions demonstrated higher transfection efficiency than their corresponding polyplexes, and this efficiency could be further improved by increasing the amount of perfluorodecalin in the emulsions. It was hypothesized that the emulsion core could help stabilize the polyplexes, leading to improved uptake and better transfection efficiency [116]. Additionally, Deng et al. reported that fluorinated PBAEs could achieve high transfection efficiencies in two difficult-to-transfect adherent and suspension cell lines, HepG2 and Molt4, compared to their non-fluorinated counterparts thus expanding the applicability of gene therapy [117].
Figure 5.
Fluorinated and guanidinated bifunctional polyplexes display enhanced stability and penetration through the mucus, and high cellular uptake leading to improved delivery of siTNF-α. Reproduced with permission from [111] Copyright 2020, American Chemical Society
Oral delivery of therapeutics offers a promising alternative to systemic delivery for chronic ailments requiring regular injections. However, the harsh gastrointestinal environment and thick intestinal mucosa are major hurdles in leveraging this route for the delivery of nucleic acids. Recently, fluorinated polymers in the form of fluorinated nanocapsules (F-NCs) were employed for the oral delivery of siRNA. The F-NCs were synthesized by in situ free radical interfacial polymerization forming a cross-linked polymer shell around the siRNA that could be cleaved in the presence of GSH. F-NCs demonstrated significant mucus penetration and transepithelial absorption. The best performing F-NC with 48% fluorination demonstrated high oral bioavailability of 20.4% compared to intravenous administration with efficient internalization in F4/80 positive macrophages. TNF-α siRNA-encapsulated F-NCs mediated significant TNF- α silencing in serum and in the target tissue alleviating inflammation in multiple models of acute and chronic murine inflammation across multiple organs (acute hepatic failure, acute kidney injury, acute pancreatitis, and rheumatoid arthritis) [118]. Collectively, fluorination of polymers is a promising strategy to develop better gene delivery systems. The unique fluorophilic effect leads to improved stability, enhanced uptake, endosomal escape, tissue penetration, and reduced cytoxicity. Additionally, perfluorocarbons have a unique ability to store and transport oxygen which can be employed to remodel hypoxic tissue environments leading to improved therapeutic outcomes [119, 120]. The fluorophilic effect also enables combination therapy with fluorine containing drugs. Lastly, utilizing imaging techniques such as 19F MRI and 18F PET has potential for the development of complex nanotheranostic systems. Fluorinated lipids have also been explored, albeit to a much lower extent. In contrast to the clear advantages seen with fluorinated polymers, fluorinated lipids have demonstrated comparable results to their nonfluorinated counterparts [95, 121]. However, despite these advantages, a few challenges still remain in achieving widespread use of fluorinated polymers. Primarily, their non-biodegradable nature demands detailed in vivo studies to critically elucidate toxicity profiles [122]. Secondly, the exact mechanism by which fluoropolymers enable endocytosis and endosomal escape is still unknown and warrants further studies. Lastly, accurate control on the extent of polymerization as well as rigorous in vivo structure-activity studies are essential to advance the development of fluorinated polymers for gene delivery.
3.3. Zwitterionic Polymers
Zwitterionic polymers contain equivalent number of cationic and anionic groups in their repeating units, thus imparting electroneutrality to the overall structure [123]. Similar to ionizable lipids, certain zwitterionic polymers regain their cationic nature in the acidic endosome or in the tumor microenvironment leading to enhanced uptake and endosomal escape while maintaining electroneutrality during circulation at the physiological pH. Moreover, in addition to pH, zwitterionic polymers can also be designed to undergo charge conversion in response to several endogenous and exogenous stimuli, creating possibilities for targeted delivery and controlled release of the therapeutic payload, a strategy that is yet to be achieved using LNPs [124]. Zwitterionic polymers are extremely hydrophilic and offer an alternative to PEG in aiding gene delivery with cationic systems. Compared to PEG, zwitterionic polymers have enhanced antifouling properties thus improving resistance to serum proteins, increasing serum stability, and in vivo circulation times. In contrast to hydrogen bonding driven hydration shells formed by PEG, zwitterions form denser, more stable hydration shells on account of their strong dipole interactions with water molecules [125, 126]. Such dipole interactions are more powerful than hydrogen bonding, leading to formation of stronger antifouling shells that can completely inhibit serum protein binding. In addition, PEG is non-biodegradable and can reduce, but not completely suppress protein adsorption, thus limiting its use. In contrast, Debayle et al. demonstrated that nanoparticles coated with polymeric sulfobetaine could completely suppress formation of the protein corona [127]. Moreover, repeat administration of PEGylated therapeutics induces the formation of anti-PEG antibodies leading to unwanted activation of the immune system [128]. Consequently, a late-stage clinical trial of PEGylated aptamer had to be discontinued due to severe PEG-related allergic reactions [129]. Zwitterionic polymers offer a promising, non-immunogenic alternative to PEG in enhancing gene delivery while maintaining all advantages attributed to PEG. Nanoparticles with zwitterionic coronas have demonstrated longer circulation and improved tumor accumulation compared to their PEGylated counterparts [130-132]. Depending on the structure, zwitterionic polymers can be divided into two main categories. The first category includes monomers that carry both a cationic and an anionic charge on the same molecule which can then be polymerized for delivery applications. Common zwitterionic compounds of this type include phosphorylcholine, sulfobetaine, and carboxybetaine (Figure 6A). Notably, distearoylphosphatidylcholine (DSPC) is a phospholipid containing the zwitterionic phosphorylcholine group that is employed as a helper lipid in two commercial LNP formulations [55]. The second group of zwitterionic polymers are polyampholytes. These are prepared by copolymerization of an equal amount of positively and negatively charged monomers thus forming electroneutral copolymers [133].
Figure 6.
A) Structures of common zwitterions (Phosphorylcholine, sulfobetaine, carboxybetaine) B) Liu et al. developed zwitterionic phospholipidated polymers (ZPPs) by conjugation of alkylated dioxaphospholane oxides to cationic polymers. The formed zwitterionic polymers enabled mRNA delivery to the spleen and lymph nodes. Reproduced with permission from [138]. Copyright 2021 American Chemical Society. C) pH responsive charge switching of PGlu(DET-Car) from neutral to cationic under acidic conditions. Reproduced with permission from [141]. Copyright 2018 John Wiley and Sons.
Grafting zwitterionic moieties on traditional polycations has led to improved serum stability and reduced cytotoxicity in multiple studies [134]. Jackson et al. reported that siRNA polyplexes formed with copolymers containing a zwitterionic phosphorylcholine-based polymer (PMPC) had comparable in vivo pharmacokinetics with polyplexes formed by PEG containing copolymers. However, the zwitterionic polyplexes had a significantly higher uptake in tumor-bearing mice, leading to improved gene silencing compared to the PEGylated polyplexes. Enhanced uptake could potentially be attributed to the similarity of the PMPC structure to the head groups of membrane phospholipids, thus increasing interactions of the polyplexes with the cell membrane [135]. In a follow up study, the group reported sustained biocompatibility of the PMPC-containing polyplexes even after repeat administration thus alleviating any concerns of immunogenic side effects [136]. The same copolymer also improved delivery of pDNA to glioblastoma cells, leading to an 18-fold increase in luciferase expression compared to the control PEI polyplexes [137].
Liu et al. developed a novel postpolymerization strategy to transform cationic polymers to zwitterionic phospholipidated polymers (ZPP) (Figure 6B). A library of 420 ZPPs was synthesized with different species of zwitterions including phosphate-quaternary amine and phosphate-tertiary amine zwitterions. In vitro transfection experiments revealed that the phosphate-tertiary amine zwitterions had better efficiency. The top-performing ZPP demonstrated a 39500-fold higher mRNA delivery efficacy compared to its cationic analogue as measured by an in vitro firefly luciferase mRNA delivery assay. Additionally, the ZPPs also demonstrated improved serum stability and enhanced uptake due to increased membrane fusion attributable to the phospholipid moieties. ZPPs also demonstrated in vivo mRNA transfection efficiency and selective delivery to the spleen and lymph nodes [138].
Despite improved serum stability and long circulation times, the zwitterionic nature of polymers limits cellular interaction and thus uptake. To overcome this issue, charge converting zwitterionic polymers have been developed that maintain their zwitterionic nature in circulation but convert to a cationic form in response to endogenous stimuli, most commonly pH, thus improving uptake [139, 140]. Ranneh et al. developed a pH-responsive polycarboxybetaine based zwitterionic polymer that undergoes charge conversion from neutral at pH 7.4 to cationic at an acidic pH of the tumor microenvironment and inside the endosomes. This charge conversion strategy improved uptake and endosomal escape and enhanced therapeutic efficacy using a polymer based on PEI and a previously developed zwitterionic polymer [141] (Figure 6C). Charge conversion behavior of the polymer was attributed to the unique protonation behavior of ethylenediamine in the zwitterionic structure, which has two distinct pKa values and maintains this property even after polymerization. The ethylenediamine moiety is monoprotonated at pH 7.4, and the degree of protonation increases with a fall in pH. The novel copolymer complexed pDNA to form micelleplexes and led to prolonged blood circulation, higher tumor accumulation, and enhanced gene transfection compared to a PEGylated counterpart in a subcutaneous tumor model [142]. Based on a similar strategy, Zhang et al. developed poly(2-hydroxyethyl methacrylate)-retinoic acid-poly(carboxybetaine) cell-penetrating peptide (PHEMA-RA-PCB-CPP) polymers. The PCB block of the polymer was protonated and condensed siRNA in acidic conditions while reverting back to neutral at the physiological pH [143]. Additionally, Ou et al. developed surface adaptive, zwitterionic shell micelles formed by the self-assembly of 2 block copolymers, poly(ε-caprolactone)-b-poly(2-methacryloyloxyethyl phosphorylcholine) (PCL-b-PMPC) and poly(ε-caprolactone)-b-poly(β-aminoester) (PCL-b-PAE) which present a neutral non-fouling zwitterionic shell in blood that converts to a positively charged shell in the tumor microenvironment. At physiological pH the PAE chains collapse to the interior of the nanoparticle due to deprotonation, thus improving circulation times while in acidic tumors PAE is protonated, thus improving uptake [144].
Zwitterionic polymers have also been utilized as a non-covalent shielding system to coat cationic polyplexes and enhance in vivo stability. Such shielding polymers are often stimuli responsive and undergo charge conversion to form polycationic polymers in response to endogenous stimuli such as pH or ROS. The positive charges then lead to electrostatic repulsion with the positively charged polyplexes underneath, thus leading to de-shielding [145, 146]. Chen et al. developed a pH responsive, biodegradable poly(l-glutamic acid)-based zwitterionic polymer that could electrostatically coat the surface of PEI/pDNA polyplexes forming negatively charged nanoparticles under physiological conditions. The polymer could undergo charge conversion to a cationic form under the acidic conditions of the tumor microenvironment, leading to enhanced uptake and improved gene transfection [146].
4. Decationizable Polymers
Polycation-associated cytotoxicity is a primary drawback of polymeric delivery systems. To overcome this, decationizable polyplexes were developed by the Hennick group [147]. The strategy employs interchain crosslinking through a thiol-disulfide exchange reaction to physically entrap electrostatically complexed nucleic acids, followed by hydrolytic cleavage of the cationic group to form neutral or slightly anionic polyplexes. Formation of decationized polyplexes occurs in three steps. In the first step, the nucleic acids undergo charge-driven electrostatic condensation with the cationic block copolymer poly(hydroxypropylmethacrylamide-dimethylaminoethyl-co-pyridyldithioethylamine-methacryl amide)-b-PEG) p(HPMA-DMAE-co-PDTEMA)-b-PEG. In the second step, a thiol-dithiol exchange reaction induces interchain disulfide crosslinking to form stable, cationic polyplexes. This crosslinking also ensures physical entrapment of the nucleic acid in the polyplex core, rendering electrostatic interactions redundant. In the last decationization step, the cationic sidechains of the polymer are removed by hydrolyzing the connecting carbonate ester bond to obtain disulfide crosslinked neutral polyplexes. Following uptake, the interchain disulfide crosslinks destabilize in the reductive environment of the cell, releasing the entrapped payload. These polyplexes were non cytotoxic as assessed by (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT) and lactate dehydrogenase release assays [148]. However, by virtue of their non-cationic nature, decationized polyplexes demonstrated very low non-specific cellular uptake compared to their cationic analogue as well as linear PEI. To overcome this and to impart cell specific targeting, the strategy was further improved by incorporating a targeting ligand. The developed folate containing decationized polyplexes demonstrated a higher uptake in a folate-overexpressing HeLa and OVCAR-3 cell lines, compared to the nontargeted polyplexes. A similar uptake for targeted and nontargeted polyplexes was seen in a non-folate-overexpressing cell line [149].
In vivo performance of decationized polyplexes was explored in a follow up study by assessing systemic toxicity, systemic stability, and biodistribution in tumor-bearing mice. Systemic toxicity of the polyplexes was assessed in a zebrafish embryo model. In contrast to cationic polyplexes, which showed high teratogenicity, decationized polyplexes were safe up to 1 mg/ml concentration. A stable particle size distribution without aggregation was observed for up to 48 h after incubation with human plasma. Additionally, the folate decorated decationized polyplexes demonstrated improved circulation half-life, improved tumor accumulation, and induced expression of a reporter transgene in A431 tumor bearing mice [150] (Figure 7A). Decationized polyplexes were originally developed for targeted delivery of pDNA. Compared to the large size of pDNA (>3000 bp), siRNA is significantly smaller (21 bp) with a stiffer backbone. Additionally, siRNA has a lower binding affinity than pDNA towards polycations. The polyplexes were optimized for siRNA delivery by tweaking the precursor monomer ratios, the degree of crosslinking, as well as the crosslinking agent. The formulated polyplexes were stable up to 24h at 37 °C in PBS. The polyplexes were nontoxic in vitro. However, transfection experiments revealed that an extremely high dose of siRNA (1000 nM) was required to achieve 25% gene silencing. In presence of human plasma, a burst release of about 60% was observed with the decationized polyplexes. Previous studies have shown that p(HPMA-DMAE-co-PDTEMA)-b-PEG-FA based decationized polyplexes lack endosomal escape abilities. Significant burst release coupled with poor endosomal escape could potentially explain the requirement of high siRNA concentration. Further studies are required to optimize this system for effective transfection at low siRNA concentrations [151].
Figure 7.
A) Novo et al. developed cross-linked decationized polyplexes with improved tumor targeting compared to their cationic counterparts. The polyplexes were developed by electrostatic complexation, followed by disulfide crosslinking to physically entrap the siRNA, and lastly hydrolysis to cleave off the cationic side chains. Reproduced with permission from [150]. Copyright 2014 Elsevier. B) Jiang et al. developed noncationic polyplexes by utilizing a cationic methylated pyridyl disulfide side chain moiety that performs dual functions. Initially it acts to provide the positive charges necessary to complex the RNA and after complexation, in presence of dithiothreitol (DTT) undergoes a thiol-disulfide exchange reaction releasing a N-methyl-2-pyridothione byproduct. This results in a disulfide crosslinked polyplex with simultaneous loss of the cationic moiety. Reproduced with permission from [152]. Copyright 2019 American Chemical Society.
In a novel strategy utilizing the thiol-disulfide exchange chemistry, Jiang et al. developed a methacrylate-based random copolymer with cationic methylated pyridyl disulfide (MPDS) (Figure 7B). MPDS serves a dual role. In the first step, it acts to complex the double stranded RNA (dsRNA). In the second step, it undergoes a thiol-disulfide exchange to release an unreactive N-methyl-2-pyridothione byproduct, forming inter-chain disulfide crosslinks and physically entrapping the dsRNA and forming a non-cationic complex. Gel electrophoresis data demonstrated that the formed non-cationic complex could still encapsulate dsRNA and was nontoxic in vitro compared to lipofectamine. Additionally, successful gene knockdown could be detected during mouse embryo development [152]. A follow up study reported on successful encapsulation of siRNA by tuning the degree of crosslinking and molecular weight of the polymer. In vivo applicability of this system is yet to be explored [153].
5. Supramolecular Structures
Supramolecular chemistry allows for a relatively simple, environmental friendly, and convenient method for developing complex nanostructures self-assembled via intermolecular noncovalent interactions such as hydrogen bonding, electrostatic interactions, Van der Waals forces, and π-π stacking interactions [154]. The noncovalent nature of supramolecular polymers endows them with the ability to undergo reversible changes in structure, morphology, and properties in response to internal and external stimuli, enabling controlled release of the therapeutic payload [155, 156]. Moreover, noncovalent interactions reduce the complexity of multi-step syntheses and purification procedures, thus reducing the cost and effort involved in the fabrication of delivery systems. Complexes formed by host-guest interactions and non-covalent self-assembly are the two major types of supramolecular polymeric structures employed for gene delivery.
5.1. Host-guest interactions
Host-guest interactions are a common strategy for fabricating supramolecular structures. Common supramolecular hosts include cyclodextrins, calixerenes, curcurbiturils, and pillararenes [157-160]. Host macromolecules trap the guest molecule in a cavity by multiple noncovalent interactions. β-Cyclodextrin (β-CD) and adamantane (Ad) is a widely explored host-guest pair that can self-assemble to form inclusion complexes [161, 162]. Recently, Wen et al. developed a β-CD – Ad based multifunctional system with reduction responsive disulfide bonds and zwitterionic phosphorylcholine for enhanced extracellular stabilization and cellular uptake [163]. The host polymer was a star-shaped cationic PDMAEMA with disulfide bonds linked to a β-CD core and the guest polymer was poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) end capped with Ad. PMPC improves serum stability by sterically inhibiting protein adsorption and enhances uptake due to its membrane-mimicking nature. The host-guest system self-assembled to form a supramolecular structure which complexed DNA with PMPC as the surrounding corona forming stable nanoparticles. Compared to non-reducible controls, and controls without PMPC or with PEG as the shielding moiety, the nanoparticles demonstrated significant serum stability and enhanced cellular uptake. Further, the nanoparticles delivered the therapeutic anti-p53 gene in MCF-7 cells and achieved high therapeutic efficiency [163] (Figure 8). The same group later expanded the use of the β-CD – Ad host-guest strategy for siRNA delivery by using the star-shaped cationic PDMAEMA host polymer and exploring a library of linear and comb shaped Ad-capped PEG guest polymers. The impact of PEG architecture on the shielding effect was assessed by fabricating a series of Ad-PEG polymers with different molecular weights and linear and comb-shaped architectures. With similar molecular weights, comb shaped PEG performed better than its linear counterpart [164]. CD based host-guest assemblies have been extensively explored with multiple polymers of varying composition and architecture for delivery of nucleic acids [165-167]. In a novel application of the host-guest strategy, Chen et al. fabricated a supramolecular core-shell dendrimer called ‘tecto-dendrimer’ for enhanced gene delivery. A β-CD modified G5 PAMAM dendrimer (G5-CD) acted as a core while an Ad-modified G3 PAMAM (G3-Ad) dendrimer acted as the shell. G5-CD/G3-Ad core-shell tecto-dendrimers were formed by supramolecular recognition between CD and Ad. The tecto-dendrimers mimic high generation dendrimers without the associated cytotoxicity. The dendrimers could complex pDNA at a N/P ratio of 1 forming stable complexes which demonstrated 20 times and 170 times higher pDNA transfection efficiencies than individual G3 and G5 dendrimers in HeLa cells. This was attributed to a higher cellular uptake, potentially due to increased interactions with the cell membrane [168]. The same group late reported the use of these tecto-dendrimers to co-deliver nucleic acids and anticancer drugs in multiple follow up studies [169-171].
Figure 8.
Wen et al. developed a β-CD – Ad based multifunctional system with reduction responsive disulfide bonds and zwitterionic phosphorylcholine for enhanced extracellular stabilization and cellular uptake A) Formation of βCD-SS-P/Ad-pMPC pseudo-diblock copolymer via host-guest interaction, followed by DNA condensation to form a polyplex. B) The anti-fouling properties of pMPC impart the extracellular stability of the polyplex. C) The zwitterionic phosphorylcholine structure of pMPC enables enhanced cellular association and uptake. D) The redox-sensitive disulfide linkages degrade in the cytoplasm leading to rapid intracellular dissociation of the polyplex. Reproduced with permission from [163]. Copyright 2014 John Wiley and Sons.
Polyrotaxanes are a subclass of supramolecular host-guest polymers with multiple host molecules threaded through their cavities on a long axial guest polymer chain [172]. The supramolecular hosts within the polyrotaxanes are commonly conjugated to cationic polymers, allowing condensation of genetic material. Such an assembly thus mimics a high molecular weight branched polycation, but without the associated cytotoxicity. Additionally, the long polymeric backbone can itself be cationic to aid complexation [173]. Interestingly, the host macromolecules on the polymer chains are known to be mobile and can freely rotate and move along the axial polymer chain. Modifying these macromolecules with targeting functional groups is therefore hypothesized to improve interaction with the target leading to improved uptake. Mohammed et al. explored this concept for a folate-targeted delivery of siRNA. An Ad-capped polyrotaxane was prepared with folate decorated CDs on a PEG chain (Figure 9A). The polyrotaxane was further complexed with CD containing PAMAM dendrimer-siRNA complexes. The resulting host-guest assembly resulted in surface decoration of the polyplex with mobile folate targeting ligands with a higher binding efficiency due to multivalent interactions with the receptors [174]. Yasen et al. fabricated a linear supramolecular block copolymer with a conventional hydrophilic CD conjugated PEG block and a main chain CD/ferrocene disubstituted cationic polymer. The resulting supramolecular polymer could condense pDNA forming stable complexes which could release the pDNA in cancer cells in response to H2O2 [175]. Polyrotaxanes can self-assemble via hydrogen bonding to from larger structures with altered hydrophilicities leading to the formation of supramolecular hydrogels [176]. Liu et al. fabricated an injectable supramolecular hydrogel system based on polyrotaxane formation between a large cationic triblock polymer ethoxy-poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(ethylene imine) (MPEG–PCL–PEI) and α-CD for sustained delivery of DNA. The cationic PEI segments condensed the DNA while the α-CDs were threaded on the PEG segments leading to formation of hydrogels by self-association [177]. Cucurbit[n]urils (CB[n]) are a type of host macrocycles that are made up of repeating glycoluril units. The size of its pumpkin shaped central host cavity depends on the number of repeating glycoluril units. CB[6] and CB[7] can bind in a 1:1 host-guest ratio, forming binary complexes. The larger size of CB[8] allows for 1:2 host – guest binding ratio, thus enabling formation of ternary complexes. Huang et al. utilized CB[7] to form polypseudorotaxanes with 25 kD branched PEI. The complex had reduced cytotoxicity with preserved transfection efficiency compared to native PEI [178]. Li et al. fabricated a supramolecular polymer nanocapsule with CB[8] as a host and a 3-armed viologen as a guest molecule and explored the efficacy of the formed complex for intracellular delivery of siRNA. The complex demonstrated comparable transfection efficiency to commercially available transfection agent – Lipofectamine 2000 [179].
Figure 9.
A)Mohammed et al. developed folate-appended polyrotaxanes (Fol-PRX) which stabilized cyclodextrin (CD) /siRNA polyplexes by intermolecularly joining CD molecules through host guest interactions between adamantane attached to the ends of Fol-PRX and CD of the polyplex. The long, connected Fol-PRX strings allowed for unrestricted movement and rotation of the folate decorated CDs on the PRX, resulting in enhanced interaction with the folate receptors and hence improved uptake. Reprinted with permission from [174]. Copyright 2022 American Chemical Society. B) Development of 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers for siRNA delivery. The supramolecularly assembled fibers contain a functionalizable hydrophilic external domain that can complex nucleic acids and an interior lipophilic core that can be used to encapsulate hydrophobic compounds. Reproduced with permission from [180]. Copyright 2016 American Chemical Society.
5.2. Non-covalent self-assembly
In addition to host-guest interactions, non-covalent self-assembly of monomeric building blocks is a common strategy to form supramolecular polymers. PEGylated 1,3,5-benzenetricarboxamide (BTA) can self-assemble in water by a mixture of hydrogen binding and hydrophobic interactions to form 1-dimensional aggregates. BTA polymers in water have a hydrophilic exterior which can be made cationic, and a hydrophobic core that can encapsulate hydrophobic small molecules. This supramolecular platform with varying amounts of cationic monomers was explored for the delivery of siRNA (Figure 9B). Cytotoxicity and uptake were dependent on the total charge as well as the distribution of charge density on the supramolecular structures which was in line with cytotoxicity effects of cationic covalent polymers. Similarly, a charge dependent knockdown efficiency was detected in HK-2 cells [180].
Ureidopyrimidinone (UPy) is another example of a building block that can form supramolecular polymers through a self-complementary quadruple hydrogen binding moiety. Monomers functionalized with the UPy moiety can self-assemble to form 1- dimensional columnar stacks via hydrogen bonds and π-π interactions. Bakker et al. fabricated a supramolecular structure using cationic monomers containing the UPy motifs and explored their use in the delivery of siRNA. Polymer composition could be tuned by modulating mixing ratios of individual monomers. siRNA complexes were formed in a single step by simultaneous injection of all monomers and siRNA in the aqueous solution. Formed supramolecular complexes with 100% cationic monomers or even 50% cationic monomers could transfect HK2 cells with a similar reduction of mRNA expression [181].
6. Recent advances in polymer synthesis with applications in gene delivery
Polymer science has advanced from refining polymerization techniques to synthesizing structurally well-defined polymers with unique physicochemical and biological properties. Recent innovations in click and photoinduced electron/energy transfer RAFT (PET-RAFT) polymerizations have played a major part in this progress [182, 183]. Click polymerization originated from click chemistry which offers efficiency, selectivity, versatility, bioorthogonality, and robustness [184]. Metal catalyst-based alkyne-azide click polymerization is a common example of this polymerization which maintains a 100% atom economy [185]. Despite considerable progress, safety remains a concern in click polymerizations due to the potential explosiveness associated with azide monomers. Therefore, the current focus has shifted to non-azide monomer-based click polymerization which includes thiol-yne, hydroxyl-yne, and amino-yne click polymerizations [186]. Each of these polymerizations employs the alkyne as one of the monomers, leading to the creation of thioether, ether, and amino functional groups in thiol-yne, hydroxyl-yne, and amino-yne click polymerizations, respectively. Careful selection of amine and alkyne monomers in amino-yne click polymerization can yield biodegradable cationic polymers with predefined topologies, offering enhanced prospects for gene delivery. Moreover, significant advances has also been made in radical polymerization where conventional RAFT polymerization is being replaced with PET-RAFT [187]. Compared to the conventional RAFT, PET-RAFT offers wide versatility in the polymerization of monomers from unconjugated (e.g. vinyl acetate) to conjugated (methacrylate ester) monomers [188]. Furthermore, these polymerizations exhibit excellent tolerance to environmental oxygen, eliminating the need for deoxygenation [189]. While RAFT polymerization has previously been employed in the synthesis of polymers, the advent of PET-RAFT further expands possibilities, enabling the synthesis of biodegradable cationic polymers suitable for efficient gene delivery.
Structurally well-defined polymers, in particular, sequence defined polymers (SDPs) and cyclic polymers have gathered significant attention due to their distinctive structural properties [190, 191]. Unlike conventional polymers, SDPs have a precisely defined sequences of monomeric units and monodisperse molecular weights. SDPs are inspired by the precise control of monomer sequences in biopolymers like DNA, RNA, and proteins and enable establishment of precise structure-property relationships, leading to optimized polymer design [192]. While nature utilizes enzymes for SDP synthesis, scientists rely on chemical reactions to conjugate monomers one by one sequentially. Commonly used solution-phase iterative and support-based syntheses are time-consuming and costly as they require deprotection, conjugation, and purification in each cycle. To overcome these limitations, DNA-guided synthesis and template polymerization have emerged as viable alternatives [193]. Current synthetic methods typically result in SDPs with low degrees of polymerization (2 to 20), prompting ongoing efforts to increase this limit [193]. Leveraging SDPs, scientists can control the density of cationic and ionizable groups to reduce toxicity, enhance polymer-gene complex stability, and achieve targeted delivery to specific cells in a controlled manner. Börner et al. presented early works on the synthesis and application of sequence-defined oligo(amido amine)s for DNA delivery [194, 195]. Similarly, Wagner et al. have emphasized the importance of SDPs in polyplex-mediated delivery of nucleic acids through multiple studies [196, 197].
Cyclic polymers are another unique class of polymers with a ring-like topology that has been inspired by the discovery of cyclic DNA [190]. Unlike linear polymers, cyclic polymers lack chain ends, limiting their dynamic movement and chain entanglement. This results in a small hydrodynamic radius, low intrinsic viscosity, and high glass transition temperature [198]. Cyclic polymers are synthesized using three distinct approaches namely, unimolecular ring-closure, bimolecular ring-closure, and ring-expansion. The ring expansion approach has been widely used due to its scalability and advantage of achieving a high molecular weight polymer with desirable purity [199]. It is worth mentioning that the ring expansion approach still produces non-cyclic polymers as a side product. Therefore, it is always recommended to evaluate the topological purity of cyclic polymers before validating their properties. Cationic cyclic polymers have been demonstrated as efficient gene delivery vectors [200]. Cyclic polymers form polyplexes with smaller hydrodynamic diameters, reduced toxicity, improved stability, enhanced permeability, and longer blood circulation half-lives compared to their linear analogs [199, 201]. Cyclic polymers with molecular weights over the renal filtration threshold were reported to have longer circulation half-lives compared to their linear counterparts due to reduced renal clearance [201, 202]. Cortez et al. reported better plasmid DNA (pDNA) transfection efficiencies and reduced cytotoxicity with cyclic PEI compared to its linear analogue. Improved transfection efficiency was attributed to a more compact structure due to higher charge density than the linear counterpart [203]. In contrast, a comparable or even reduced transfection efficiency at certain molecular weights was reported by Wei et al. for cyclized PDMAEMA, underscoring the importance of polymer structure, in addition to topology, on gene delivery [204].
7. Covalent polymer-nucleic acid conjugates
Polyplexes are typically formulated with an excess of polycations which leads to several limitations, including toxicity issues with free, excess polycations and potential for destabilization during circulation. Covalent conjugation of the nucleic acids to a polymer allows for the development of a highly regulated structure without the need for excess polycations, thus reducing cytotoxicity and avoiding the risk of polyplex dissociation in the bloodstream. Traditionally, nucleic acids have been extensively conjugated to cell-penetrating peptides and small molecules such as cholesterol, tocopherol, and carbohydrates to improve various aspects of gene delivery including targeting, stability, and uptake [205-208]. Cholesterol-conjugated siRNA demonstrated a significantly prolonged blood circulation compared to naked siRNA, leading to enhanced gene silencing [209]. Covalent conjugation of siRNA to galactose and galactose derivatives including N-acetylgalactosamine (GalNAc) has become especially popular for hepatocyte targeting with multiple GalNAc-siRNA conjugates already on the market or undergoing clinical trials [210]. Dynamic polyconjugate (DPC) technology is an siRNA-polymer bioconjugation strategy that was developed by Arrowhead Pharmaceuticals. DPCs were initially developed by conjugating siRNA to an endosomolytic polymer , poly(butyl amino vinyl ether) (PBAVE) through disulfide linkage. PBAVE was further conjugated reversibly with PEG and GalNAc which were designed to be cleaved off in the acidic environment of the endosomes after uptake leading to the conjugate recovering its endosomolytic activity [211]. The second generation of DPCs (DPC 2.0) involved co-injection of a siRNA-cholesterol conjugate and a hepatocyte targeting membrane-active peptide [212]. Although a significant reduction in biomarkers was seen in hepatitis B patients receiving a single dose of ARC-520, which employed the DPC 2.0 technology, clinical development was stopped due to unfavorable long term toxicity in non-human primates [213]. Currently, Arrowhead employs a technology called TRiM (Targeted RNAi Molecule), which comprises of targeting ligands covalently conjugated to siRNA with different linkers and additional structures that enhance pharmacokinetic performance. Several clinical trials are currently underway using the TRiM platform.
7.1. Synthetic approaches to bioconjugation
Nucleic acids need to be functionalized with suitable functional groups to enable conjugation to a polymer. Cleavable linkers are preferred to facilitate release of nucleic acids following cell uptake. Commonly employed linkers include those that are cleaved in acidic environment (e.g., hydrazone, β-thiopropionate) and those that are cleaved in reducing environment [e.g., succinimidyl 3-(2-pyridyldithio)propionate (SPDP), (N-succinimidyl S-acetylthioacetate) (SATA)]. Acid labile linkers may degrade in the acidic environment of the endosomes while the disulfide linkers are cleaved in the reductive environment of the cytoplasm [214-216]. Additionally, some bioconjugates rely on the action of an endogenous enzyme (dicer) to cleave the double-stranded RNA conjugate into its active form. A few commonly used conjugation chemistries include copper-free or copper-catalyzed azide-alkyne cycloaddition, amidation, or thiol-based conjugation [217-219]. The synthetic approaches to bioconjugation can broadly be divided into three major categories – grafting from, grafting to, and grafting through [220].
Multiple controlled radical polymerization techniques, including reversible addition–fragmentation transfer (RAFT) and atom-transfer radical polymerization (ATRP), have been explored in recent years for the synthesis of covalent polymer-oligonucleotide conjugates [221]. Despite growing popularity, the bioconjugation strategy is still in its infancy and has several limitations attributable to the innate differences in the nature of the two components – polymers and nucleic acids. Conjugation reactions performed in the solution phase are typically restricted to aqueous solvents and hydrophilic polymers due to solubility and stability limitations of the nucleic acids. Additionally, scale-up is often difficult due to rigorous purification and high cost of the nucleic acids.
7.1.1. Grafting to
Grafting to is a common strategy wherein, both the polymer and the nucleic acids are synthesized prior to the conjugation step. The conjugation of PEG represents one of the very first use cases employing the grafting to strategy in covalently conjugating nucleic acids to polymers [222-224]. Early studies revealed that the half-life of siRNA increased from 5 minutes for the non-PEGylated siRNA to 1 hour without significant degradation for the PEGylated analogue [225]. Despite advances in PEGylation strategies, PEG antigenicity can lead to the activation of the complement system and, in rare cases, severe allergic reactions and death [226]. To overcome these issues, Ozer et al. synthesized a novel PEG-like brush polymer poly[(oligoethylene glycol) methyl ether methacrylate)] (POEGMA) and conjugated it to an RNA-aptamer which was previously PEGylated but had to be discontinued due to severe allergic reactions in a late-stage clinical trial. The novel POEGMA-conjugated aptamer had no reactivity to PEG of various origins in an in vitro experiment. Additionally, the conjugate did not lead to de novo generation of anti-PEG antibodies in mice. This absence of immunogenicity was attributed to the hyperbranched structure of the novel polymer with tri(ethylene glycol) sequences on a polymethacrylate backbone instead of the long, repeating ethylene glycol chains of traditional PEG. The short chains were hypothesized to lack the binding epitopes for PEG antibodies [227].
Steric hindrance caused by the conjugation of large molecules such as PEG to siRNA limits exposure of siRNA to the cytosolic proteins and thus the gene silencing pathway. This has been proposed to contribute to ineffective gene silencing activity of PEGylated nucleic acids. To overcome this drawback, Harun et al. conjugated siRNA to poly(N-isoproprylacrylamide) (PNIPAM). PNIPAM changes its hydrodynamic size in response to temperature and shrinks from an extended coil to a spherical globule at its lower critical solution temperature (LCST) of 33°C. This transition exposes the attached siRNA molecule to the RNAi machinery in the cytoplasm, leading to improved transfection [228] (Figure 10A). Further optimization of the polymer’s thermo-responsiveness for in vivo applications utilized copolymers of NIPAM with hydrophilic N,N-dimethylacrylamide (DMAA). The augmented hydrophilicity increased the LCST, resulting in a conjugate where the gene silencing ability was suppressed at 37°C but recovered at slightly hyperthermic conditions of 41°C. The results indicated a potential for controlling the bioactivity of the conjugated siRNA to achieve selective gene silencing based on localized thermal cues. However, the studies required the use of a commercially available Lipofectamine RNAiMAX to aid transfection, suggesting that transfection efficacy of the conjugate needs further optimization [229]
Figure 10. -.
A) Harun et. al reported the development of thermo-responsive polymer-siRNA conjugates to achieve thermally controlled gene silencing around the body’s temperature. Structure of siRNA-PNIPAAm conjugates. The polymer undergoes a coil-globule transition above the LCST. Reproduced with permission from [228]. Copyright 2016 American Chemical Society B) Jeong et al. reported the development of a novel acylating agent that enables direct incorporation of an ATRP initiator at the 2′-OHs in the structure of RNA. Polymer chains could then be grafted from the resulting initiator-functionalized RNAs by photo-induced ATRP resulting in the formation of RNA-polymer hybrids. Reproduced with permission from [238]. (Open access)
Averick et al. designed polymer- siRNA constructs by conjugating polymers to the 2 terminal ends of the passenger/sense strand using click chemistry followed by simple annealing of the guide strand to the polymer-sense strand conjugate. The construct was synthesized using a copper-catalyzed azide–alkyne cycloaddition. Bisalkyne functionalized passenger RNA strands were reacted for 90 mins in Tris buffer (pH 7.5) with 0.6% acetonitrile as a cosolvent with azide-terminated polymers to form the polymer-sense strand conjugate. The final polymer-siRNA duplex construct was obtained by annealing a complementary guide strand to the synthesized conjugate. Resistance of the conjugate to exonucleases was assessed by incubating them with RNAse A followed by gel electrophoresis. Unmodified siRNA duplexes were completely degraded by the enzyme while the polymer-conjugated construct was intact even after 2 hours. Transfection efficacy of the polymer-conjugated construct was confirmed in HEK293 cells [230].
Spherical nucleic acids are a novel class of stable RNA delivery vehicles fabricated through self-assembly of amphiphilic polymer-nucleic acid conjugates with the nucleic acids conjugated to the hydrophilic portion of the polymer and displayed at the surface of spherical nanoparticles while the hydrophobic blocks of the polymer form the core [231]. Zheng et al. utilized the temperature-responsive behavior of PNIPAM to develop siRNAsomes. These vesicular structures were formed by the temperature-dependent self-assembly of siRNA–SS–PNIPAM conjugates. The structure consisted of a hydrophilic siRNA shell, a hydrophobic middle layer, and an aqueous core. The aqueous core and the hydrophobic layer could be utilized to load suitable therapeutics for combination delivery. In vitro and in vivo efficacy of the conjugates was demonstrated by co-delivering siRNA and doxorubicin-hydrochloride in a multidrug-resistant MC7 cancer model [232]. A similar spherical siRNA micelle, with a lower proportion of hydrophobic components in the PNIPAM polymer was later developed to co-deliver siRNA and small molecule therapeutics across the blood-brain barrier to treat glioblastoma [233]. Rush et al. reported a micellar strategy with polymer-nucleic acid conjugates synthesized via solid-phase coupling of an amine modified oligonucleotide with carboxyl terminated norbornyl polymer-controlled pore glass beads. The conjugates self-assemble in an aqueous medium, forming dense hydrophobic polymeric cores with solvated hydrophilic nucleic acid shells [234]. Receptor saturation limits receptor mediated siRNA uptake. To overcome this issue, Brunner et al. developed covalently conjugated, hyperbranched siRNA dendrimers with a single neurotargeting ligand. A copper catalyzed click reaction was employed to conjugate alkyne modified siRNA to the azide terminated dendritic structures. To evaluate the influence of branching, a reporter assay in RBL-2H3 cells was employed. Silencing efficiency of dendrimers with 1, 3 and 9 siRNA duplexes per ligand with the same total number of siRNA duplexes was assessed. Dendrimers with 3 duplexes showed maximum gene silencing compared to the monomeric siRNA. Additionally, despite low concentration and the relatively large size, dendrimers with 9 duplexes showed better efficacy compared to the monomer [235].
7.1.2. Grafting from
Grafting from is a relatively new strategy in gene delivery, where the nucleic acid acts as a macroinitiator, leading to in situ growth of the polymer. Lin et al. utilized this strategy in the synthesis of siRNA-polymer conjugates where the siRNA acted as a macroinitiator for the polymerization reaction. An ATRP initiator, pyridyl disulfide bromoisobutyrate, was attached to the siRNA using a disulfide linker to enable cleavage in the reducing environment of the cytoplasm. ATRP was used to grow the polymers from siRNA based on two monomers, PEG methyl ether methacrylate (PEGMA) and di(ethylene glycol) methyl ether methacrylate (DEGMA). The monomers were chosen based on their ability to improve nuclease stability of the siRNA [236]. A similar strategy was previously reported for DNA [237]. The grafting from technique circumvents possible steric hindrance and purification challenges that arise when utilizing pre-synthesized polymers. Recently, Jeong et al. expanded this strategy of ATRP polymerization using RNA as a macroinitiator by developing a novel universal acylating reagent that allows for easy incorporation of the ATRP initiator to the RNA strands (Figure 10B). The acyl imidazole reagent allowed for controlled, selective conjugation of 2′-OH ATRP initiators to short synthetic RNA oligonucleotide sequences. Successful polymerization was demonstrated using oligo(ethylene oxide) methyl ether methacrylate, PEG dimethacrylate, and N-isopropylacrylamide monomers to from conjugates with varied architecture and narrow molecular weight distributions. Although this technique represents an interesting future strategy to develop polymer-siRNA conjugates, no in vitro or in vivo results have been published till date [238].
Lueckerath et al. employed the grafting from strategy to synthesize a series of ssDNA-polymer conjugates with different monomers and chain lengths using solution-based photoinduced RAFT polymerization. Two commonly employed RAFT agents 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPADB) and 2-(butylthiocarbonothioyl)propionic acid (BTPA) were conjugated to ss-DNA sequences followed by polymerization with methacrylates, acrylates, and acrylamides as monomers. Successful DNA-polymer conjugates with varying molecular weights were synthesized. However, similar to the majority of the previously discussed examples, in vitro and in vivo data are yet to be published [239]. Overall, despite multiple advances in bioconjugation chemistry, a lot of work evaluating the bioconjugates in mammalian cell lines and animal models is necessary to fully unlock their potential as a novel strategy for gene therapy.
7.1.3. Grafting through
Grafting through employs nucleic acid macromonomers, i.e., nucleic acid strands terminated with a polymerizable unit. This approach ensures that every monomer unit contains a nucleic acid, thus achieving a much higher nucleic acid incorporation efficiency than the other two approaches. This approach is often used to synthesize polymer brushes with well-defined side chains. Compared to the other two approaches, grafting through is rarely used for the synthesis of polymer-nucleic acid conjugates. Liu et al. employed this approach to polymerize norbornene-functionalized DNA surfactant complex macromonomers to form DNA side chain homopolymers [240]. Tan et al. reported the synthesis of DNA grafted brush polymers utilizing a grafting through approach that employed norbornene modified, phosphotriester- and exocyclic amine-protected DNA. The protective groups allowed for a homopolymerization via ring-opening metathesis in organic solvents. A mild deprotection step after the polymerization provided the final polymer [241]. Recently, Arkinstall et al. reported the synthesis of DNA containing bottlebrush copolymers using a direct, graft-through, ring-opening metathesis polymerization. In a step up from previous efforts, the reaction was conducted in water without the requirement of any additional protection/deprotection steps for the DNA, leading to a quicker, more efficient synthesis [242].
6.2. Limitations of polymer-nucleic acid conjugation reactions
Despite growing popularity, bioconjugation strategies are still in their infancy and have several limitations attributable to the innate differences in the nature of the two components – polymers and nucleic acids. Conjugation reactions performed in the solution phase are typically restricted to aqueous solvents and hydrophilic polymers due to solubility and stability limitations of nucleic acids. Additionally, scale-up is often difficult due to rigorous purification requirements and high cost of the nucleic acids.
Steric hindrance significantly impacts the grafting to approach, as it involves preformed polymers and nucleic acids, both of which can shield the reactive sites. Additionally, removal of the unreacted polymer and/or the nucleic acid is difficult, requiring development of sophisticated separation techniques. Lastly, being a post polymerization conjugation reaction, there are limitations regarding the achievable nucleic acid grafting density.
Significant efforts are being directed to overcome current limitations of bioconjugation reactions. For instance, Liu et al. addressed the solubility restrictions of nucleic acids by utilizing a surfactant/DNA complex to enable hydrophobic functionalization in organic solvents, thereby expanding the scope of bioconjugation [240]. Similarly, polymerization-induced self-assembly (PISA) is a novel technique that helps in the synthesis of hydrophobic polymer bioconjugates [243]. PISA involves polymerization of hydrophilic monomers to eventually form an overall hydrophobic polymer. PISA has been successfully employed to produce hydrophobic polymer-DNA conjugates [244]. Solid-phase synthesis is another approach that has been developed to overcome solubility limitations and conjugate hydrophobic polymers to nucleic acids [245]. Phosphoramidite chemistry is commonly employed to add functional groups to nucleic acids via solid phase synthesis. Fully automated DNA synthesizers capable of solid phase synthesis of DNA conjugates with hydrophobic polymers are now available. Recently, Pan et al. reported the use of a fully automated DNA synthesizer for synthesis of DNA-polymer hybrids using photo-mediated ATRP [246]. These innovative techniques mark significant progress in the field of bioconjugation, addressing solubility challenges and enabling more efficient synthesis of polymer-nucleic acid conjugates. Continued advancements are essential to further overcome existing limitations and realize the full potential of the bioconjugation strategies in gene delivery.
8. New approaches for improved polymer design
Recent advances in automation, artificial intelligence, and computational approaches have ushered in a new era in the design of drug delivery systems. These technologies have enabled researchers to explore vast chemical spaces and gain an in-depth understanding of structure-property relationships without employing traditional time- and labor-intensive methods. With advances in inter-disciplinary research, these next-generation approaches promise to revolutionize the design and optimization of polymeric gene delivery systems.
8.1. Automated high throughput techniques
Unlike libraries of small molecules for drug discovery, currently there are no commercially available validated libraries of polymers, let alone polycations, to enable utilization of combinatorial approaches to polycation-mediated gene delivery. There has been considerable interest and advances in the synthesis of combinatorial polymer libraries and their use in high-throughput (HT) materials research [247, 248]. Anderson et al. provided an early impetus to combining automation, HT polymer synthesis, and HT screening in gene delivery. In an early study, a large library of 2350 degradable cationic PBAEs was synthesized in 96 well plates by semi-automated methods and screened for their transfection ability in mammalian cell lines using automated high-throughput fluid handling systems [249]. Advances in the development of air-tolerant polymerization and advanced robotics have enabled utilizing automated high throughput techniques to not only synthesize large polymer libraries but also screen them in cell lines to establish quantitative structure-property relationships. Recently, Mishra et al. reported the use of a high-throughput platform to synthesize and screen a library of 148 polymeric nanoparticles to identify structures that enable efficient transfection in difficult-to-transfect human retinal pigment endothelial cells. The polymers were synthesized in 96 well plates using DMSO as a solvent. pDNA was added to the wells containing the polymers leading to formation of nanoparticles by self-assembly. DMSO is tolerated by cells in low concentrations, allowing direct automated transfer of the nanoparticles to wells containing the cells. The transfection efficiency was assessed using an automated fluorescence-based imaging system for endogenous nuclear GFP and mCherry delivered by the pDNA [250]. Despite advances in automated synthesis, basic purification and characterization remains a significant bottleneck in the automated synthesis of polymer libraries. There is little benefit in creating extensive polymer libraries if the materials cannot be effectively purified and characterized with equally high-throughput methods. Additionally, while automated systems excel at small scale synthesis, scaling up these reactions for larger quantities is often difficult. High-throughput automated synthesis systems, being in their early stages, can be costly to purchase, operate, and maintain. This expense limits their accessibility for smaller research groups and organizations. Despite these hurdles, as automation and polymerization techniques continue to grow, it is unquestionable that the implementation of automated high throughput techniques will greatly accelerate the development of polymeric gene delivery systems.
8.2. Molecular modeling and simulations
Molecular dynamics (MD) simulation is a robust in silico method employed in computer-aided drug discovery to explore atomistic-level interactions within various systems, addressing questions beyond experimental detection [251]. MD simulations provide access to crucial data not accessible by experimentation, expand exploration of the chemical space, and complement experimental results by offering quantitative and microscopic insights into the underlying mechanisms. There is a significant interest in the use of simulations for the design of novel polymers since the computational cost associated with modeling a new polymer is less than the material cost associated with synthesizing and characterizing it. Utilizing high-throughput in silico methods for evaluating biomaterials can expedite the optimization and development of therapeutics, allowing rapid exploration of extensive chemical design spaces with cost-effectiveness [252]. MD simulations have been extensively employed to predict in vitro behavior of polymeric systems. For instance, in a recent study, MD simulations with coarse-grained (CG) MARTINI force fields were used to model nanoparticles with varying PEG densities translocating across an asymmetric lipid bilayer at different temperatures to assess the impact of temperature and PEG grafting density on the nanoparticles' ability to cross the cell membrane [253]. Similarly, Raman et al. utilized CG MARTINI force fields to simulate the permeation of self-assembled PEG-b-PCL copolymer micelles across a model lipid bilayer. Data obtained through the simulations demonstrated that permeation depended on the balance between hydrophobic and hydrophilic segments in the polymer [254]. In a recent study, Binder et al. developed a novel CG model based on the Martini 3 force field to investigate thermodynamics of siRNA complexation with PEI. The simulations revealed the molecular weight of PEI was a critical parameter in binding dynamics. These results were further validated by experimental analyses [255].
Simulations also help illuminate aspects like charge neutralization in local regions, the overall nanoparticle charge, and bridging interactions among constituent molecules [256]. Meneksedag-Erol et al. performed all-atom MD simulations of PEI-siRNA polyplexes with varying degrees of propionic acid substitution on PEI to elucidate the interactions between hydrophobically modified PEI and siRNA at the molecular level, providing mechanistic insights into how the level of hydrophobic substitution affects the assembly, surface properties, and delivery performance of the polyplexes [257]. Similarly, Li et al. utilized CG MD simulations to assess the impact of PEG chain length, molecular weight, and grafting density on the endocytosis of PEGylated nanoparticles [258, 259]. All-atom MD simulations have also been used to unveil the dynamic nature of the internalization process, capturing the entire journey from initial interactions between nanoparticles and cell membranes to penetration and release from the lipid-rich phase [256]. In another study, all-atom MD simulations were used to model the interactions between siRNA-PEI complexes and heparin. The simulations provided mechanistic insights into the role of glycosaminoglycans in the delivery of polymeric nucleic acid nanoparticles by revealing the atomistic details of heparin binding and its effects on nanoparticle stability and disassembly [260]. Further, MD simulations have been performed to model the interactions between branched PEI and DMPC lipid bilayers, and it was inferred that PEI induces stable pore formation in lipid bilayers by creating persistent water channels that could allow DNA transport into the nucleus. This proposed mechanism of PEI-mediated DNA nuclear entry via lipid pore stabilization can aid future optimization of PEI gene vectors by highlighting key aspects of the PEI-membrane interaction that can be tuned to enhance transfection efficiency [261]. Simulations aim to elucidate variations in polymer-DNA binding modes, considering factors such as polymer molecular weight, architecture, and chain flexibility. Specifically, the simulations depicted linear PEI interacting with DNA in a "cord-like" manner, preventing binding to other DNA molecules and promoting subsequent release. On the contrary, polyplexes formed by branched PEI-DNA exhibited a bead-like structure, allowing for multiple DNA bindings and aggregations which resulted in the formation of more stable polyplexes thus facilitating improved cell uptake [262]. Furthermore, simulations revealed that polymers with pendant oligolysines exhibit a lower binding free energy to DNA compared to linear PLL which can be attributed in part, to the hydrophobic backbone that constrains these interactions [263]. Another study employed atomistic molecular dynamics simulations to study complexes of DNA with different cationic polymers like PEI, PLL, polyvinylamine, and polyallylamine and provided insights into the impact of polymer concentration on the binding patterns and structural/electrostatic properties of the DNA-polycation complexes [264]. Molecular modeling and simulations thus have a tremendous potential in aiding development of novel polymers and uncovering mechanistic intricacies of gene delivery using polymeric systems. MD simulations are computationally intensive, requiring significant computational resources and time, especially for large systems or long simulation times. This can limit the feasibility of performing extensive simulations needed to explore the parameter space or to achieve statistically significant results. Similarly, timescale limitations associated with MD simulations limit the ability to fully capture the dynamic processes and long-term behaviors of polymer-gene interactions.
8.3. Artificial Intelligence
Artificial intelligence (AI) presents an unprecedented opportunity to bypass traditional, time and resource-consuming methods in polymer design. Predictive models based on machine learning (ML) and artificial intelligence (AI) can be broadly divided into two areas. First, models can be utilized to predict properties of interest given a specific polymer structure. Second, given properties of interest, AI models can be utilized to predict polymer structures that may demonstrate the desired properties. Borrowing from “big data” toolset, ML algorithms have been successfully employed recently to delineate nanoparticle attributes that are required for effective intracellular delivery in vitro [265-267]. These studies identified multiple properties of both the polymers and the nanoparticles, with balanced hydrophobicity being particularly important. Limited number of studies, however, use ML to understand nanoparticle behavior beyond in vitro cell uptake and transfection to the in vivo performance in distinct disease states and administration protocols. ML models, specifically a light gradient boosting machine (LGBM) model, were able to accurately predict fractional drug release from polymeric long-acting injectables and provide insights to guide the design of new optimized formulations with controlled release properties [268]. In addition to release profiles, ML has also been used to model other properties of polymeric systems, including the average size of nanoparticles and the extent of drug loading. Wang et al. utilized data for 445 PLGA formulations compiled from the literature to train machine learning models to predict the size of PLGA nanoparticles synthesized by electrospraying [269]. Powerful predictive models using robust statistical learning algorithms and deep neural networks have reduced the need for extensive de novo polymer synthesis by enabling accurate property predictions directly from chemical structures. Gong et al. utilized techniques such as random forest regression and XGBoost on a polymer dataset of 297 poly(beta-amino ester) (PBAE) formulations to train an ML model to predict transfection efficiency and toxicity of new PBAE polymers directly from chemical structures without requiring experimental testing [252]. Li et al. employed an ML approach to decipher the relationship between polymer component distribution and transfection efficiency in a library of PBAEs [270]. ML models can uncover complex relationships in the data to guide polymer design. Kumar et al. utilized machine learning algorithms to identify key polymer properties such as cooperative deprotonation and hydrophobicity, which facilitate efficient intracellular delivery of CRISPR ribonucleoprotein (RNP) payloads, guiding future optimization of polymer vectors for enhanced unpackaging and editing efficiency [271]. Similarly, Dalal et al. employed ML techniques like SHAP and Bayesian optimization to identify structure-activity relationships for polymer design, enhancing delivery efficiency for both pDNA and RNP cargos; the closed-loop optimization of 552 formulations resulted in three top performers exhibiting 1.7-fold improved pDNA delivery and prolonged gene expression in mouse liver over 20 days compared to controls [272]. AI methods like machine learning and deep learning can be used to predict polymer structures and properties based on training data. Models can be trained on databases of known polymers to generate and evaluate potential new polymer structures. Polymer generators create new candidate polymer structures, typically using statistical or machine learning models trained on existing polymers, but the key limitation of existing polymer generators is that they do not sufficiently consider synthetic feasibility. Ohno et. created Small Molecules into Polymers (SMiPoly) that uses a rule-based algorithm for classifying input monomers, matches them to applicable chemical reaction rules for polymerization, and generates polymers by applying those reactions, in order to address the above limitation. SMiPoly achieved the generation of over 169,000 unique, potentially synthesizable polymers spanning 7 polymer classes by implementing 22 chemical rules for common polymerization reactions and exhaustively applying them to 1,083 commercially available small molecule monomers [273].
A major hurdle to the field of AI in the context of polymeric gene delivery is the lack of accessible, high-quality data and public data repositories that encode chemical information from polymers into machine-readable formats. Current digital representation methods fall short in their ability to fully capture the complex 3D structures, chain architectures, and conformational variations that are characteristic of polymeric systems [274]. ML and AI require extensive datasets for experimental validation. However, such datasets currently do not exist in the public domain, restricting these efforts to individual institutions and research groups utilizing their internal datasets. Moreover, a lack of practical consensus on polymer nomenclature makes establishment of universal datasets a difficult task. Meaningful integration of these approaches in formulation development is often a complicated task due to the traditionally mutually exclusive nature of the two disciplines. Scientists with strong interdisciplinary knowledge in the fields of polymer chemistry and computer science are required to drive advances in this field.
In summary, machine learning and optimization fuel the generative "in-silico" phase, while automation and robotics support physical validation, establishing an integrated AI-driven polymer discovery pipeline that shifts from manual design to automated exploration of extensive virtual candidate libraries, with simulations offering preliminary assessments to expedite the overall pipeline. Table 2 offers a summary of MD simulators and recent AI/ML approaches for polymer design and property prediction.
Table 2.
Summary of MD simulators and recent AI/ML approaches for polymer design and property prediction.
| Simulator/ML Tools | Applications |
|---|---|
| All-Atom MD Simulations |
|
| Coarse-Grained (CG) MD Simulations | |
Predictive ML Models
|
|
Generative ML models
|
|
9. Clinical outlook for polymeric gene therapeutics
Gene therapies have received a tremendous boost in the recent past led by the phenomenal success enjoyed by the mRNA vaccines against COVID-19. Gene therapies offer an unmatched ability to precisely target the root cause of a disease by introducing or modulating the genetic material in the target cells. In general, gene therapies fall into two categories, ex-vivo and in vivo gene therapies. In vivo gene therapies, involving direct administration of the therapeutic to a patient generally require a delivery system or a physical delivery method such as electroporation to deliver the genetic payload. Despite concerns with safety and immunogenicity, viral vectors, led by adeno-associated viruses (AAVs), have dominated this space. LNPs have emerged as a promising delivery vector post approval of COVID 29 vaccine.
Despite promising pre-clinical results, very few polymers have advanced to clinical testing. PEG, utilized in the development of PEGylated lipids and PEGylated proteins, is the only polymer that has reached the stage of clinical translation pertaining to gene therapy [213, 275]. PEGylated lipids are extensively employed in the formulation of LNPs to form a hydrophilic PEG corona, thus improving stability and pharmacokinetic profiles [55]. Another promising polymeric system that underwent clinical trials but was eventually phased off was the DPC system developed by Arrowhead pharmaceuticals. First generation DPCs involved siRNAs covalently conjugated to an endosomolytic polymer (PBAVE) by disulfide linkage [276]. The Davies’ lab extensively reported on a cyclodextrin based siRNA delivery platform in primates as well as humans. The transferrin ligand decorated cyclodextrin based polyplex, CALAA-01, encapsulating a siRNA targeted to ribonucleotide reductase subunit M2 (RRM2) resulted in preferential, dose dependent accumulation in metastatic melanoma tumors in humans and inhibited melanoma-associated RRM2 expression. However, a phase Ib clinical trial was terminated due to low rate of response and nonspecific toxicity [277, 278]. Recently, in June 2022, a Phase II clinical trial (NCT02806687) for CYL-02, a complex of pDNA and linear PEI (jetPEI 22 kDa) in combination with gemcitabine for pancreatic ductal adenocarcinoma was completed. However, the results of the trial are not yet published [279]. Silexion (formerly Silenseed) developed a biodegradable polymeric matrix from PLGA called LOcal Drug EluteR (LODER) for local, sustained delivery of siRNA to solid tumors. siG12D-LODER is a G12D-mutated KRAS-targeting siRNA delivered locally using the LODER platform. siG12D-LODER demonstrated promising results in a phase 1/2a clinical trials (NCT01188785 and NCT01676259) in combination with chemotherapy for inoperable locally advanced pancreatic cancer patients [280]. Currently 7 clinical trials exploring polymeric systems for the delivery of multiple genetic cargo are underway (Table 3). The following section gives a comprehensive overlook of these trials.
Table 3.
Active clinical trials for polymer-based gene delivery systems
| Therapeutic | Polymer | Disease | Cargo | Additional Therapeutic |
Sponsor | Trial number |
|---|---|---|---|---|---|---|
| IMNN-001 | PEG-PEI-Cholesterol | Advanced ovarian, fallopian tube or primary peritoneal cancers | pDNA | Bevacizumab Paclitaxel Carboplatin | Immunon Therapeutics | NCT05739981 |
| Paclitaxel Carboplatin | NCT03393884 | |||||
| IFx-Hu2.0 | jetPEI | Advanced non-melanoma skin cancers | pDNA | N/A | TuHURA Biosciences, Inc. | NCT04160065 |
| BO-112 | jetPEI | Resectable soft tissue sarcoma | dsRNA (poly I:C) | Nivolumab | Jonsson Comprehensive Cancer Center | NCT04420975 |
| Unresectable malignant melanoma | Pembrolizumab | Highlight Therapeutics | NCT04570332 | |||
| Metastatic refractory non-small cell lung carcinoma | Radiotherapy Nivolumab | Clinica Universidad de Navarra, Universidad de Navarra | NCT05265650 | |||
| SRN-001 | Self-Assembled Micelles | Idiopathic Pulmonary Fibrosis | siRNA | N/A | siRNAgen Therapeutics Inc. | NCT05984992 |
| Stimotimagene Copolymerplasmid | polyethyleneimine (PEI) - polyethylene glycol (PEG) - TAT peptide) | Advanced-Stage Solid Tumors | pDNA | Ganciclovir | Gene Surgery LLC | NCT05578820 |
jetPEI (linear, 22 kDa) has been extensively explored in the past and in the current clinical trials for delivery of genetic material. IMNN-001 (Gen-1) by Immunon therapeutics is an immunotherapeutic formulation containing pDNA coding for IL-12. The pDNA is delivered by complexation with a PEG-PEI-Cholesterol copolymer. Multiple clinical trials with IMNN-001 monotherapy as well in combination with chemotherapy have been completed [281-283]. Currently, there are 2 active trials, with one actively recruiting participants. NCT05739981 is a Phase II clinical trial evaluating the effect of intraperitoneal IMNN-001 on second look laparoscopy in combination with bevacizumab and neoadjuvant chemotherapy (paclitaxel and carboplatin) in patients newly diagnosed with advanced ovarian, fallopian tube or primary peritoneal cancers. OVATION 2 (NCT03393884) is another Phase I/II study evaluating intraperitoneal IMNN-001 in combination with neoadjuvant chemotherapy without bevacizumab for the same clinical indications. Recent interim data reported that the combination therapy led to an improvement in progression free survival compared with the neoadjuvant platinum-based chemotherapy alone. About 33% delay in disease progression was reported with a trend towards improvement in overall survival. The study enrolled 110 patients above 18 years of age with suspected histologic diagnosis of stage III or IV epithelial ovarian, fallopian tube, or primary peritoneal carcinoma [284].
IFx-Hu2.0 is a novel personalized cancer vaccine currently undergoing Phase I clinical trials for intralesional immunotherapy in patients with advanced non melanoma skin cancers (NCT04160065). IFx-Hu2.0 is a pDNA encoding for an immunogenic bacterial protein, Emm55, formulated by complexation with the commercial in vivo-jetPEI. The formulation is supplemented with 5% dextrose for enhanced complex stability [285, 286]. BO-112 is a dsRNA (poly I:C) formulated with in vivo-jetPEI. BO-112 acts by making tumors more visible to the immune system. It activates TLR3, RIG-1 and MDA-5, causing immunogenic cell death and enhancing effects of immune-checkpoint inhibition [287, 288]. Currently, 3 clinical trials are ongoing, with BO-112 in combination with nivolumab for resectable soft tissue sarcoma (NCT04420975), intratumoral BO-112 in combination with pembrolizumab for unresectable malignant melanoma (NCT04570332), and BO-112 with radiotherapy plus nivolumab for metastatic refractory non-small cell lung carcinoma (NCT05265650). Additionally, a pDNA vaccine complexed with 20 kDa linear PEI is under clinical trials for neuroblastoma (NCT04049864).
Moving away from PEI, SRN-001 is the flagship therapeutic from siRNAgen Therapeutics Inc. based on their SAMiRNA platform. The platform is a modular micelle formed by self-assembly of individual RNA bioconjugates synthesized by conjugation of a hydrophilic polymer and lipid to the RNA [289]. SRN-001 utilizes siRNA against amphiregulin, which is a growth factor involved in the progression of idiopathic pulmonary fibrosis. SRN-001 is currently undergoing a first-in-human single ascending dose clinical trial to evaluate its safety, tolerability and pharmacokinetics in healthy participants (NCT05984992). In addition to these active clinical trials, multiple terminated or completed clinical trials for polymer-based gene delivery systems have been reported.
Despite promising results, the scale up and manufacturing of polymers has its own set of challenges. The large scale synthesis of polymers is challenging, as there is always a hurdle in controlling the polymerization reaction to achieve the polymer with the expected monomeric composition, topology, molecular weight, and charge density, which are crucial for gene delivery [290]. The variability in the starting materials can lead to irregularities in the above-mentioned parameters of the synthesized polymers [291]. In the industrial setting, managing the reactivity of cationic monomers can be another hurdle that requires complete control of the reaction conditions such as temperature, pH, monomer concentration [292]. Laboratory-to-industrial-scale polymer synthesis generally introduces batch-to-batch variability due to changes in equipment and the environmental conditions [293]. Maintenance of raw material consistency, polymerization conditions, employment of an advanced monitoring system, and adherence to quality control protocols help to overcome batch-to-batch variability. Finally, the synthesized polymer must meet stringent regulatory standards directed toward performing rigorous quality control testing [294].
10. Conclusions and Future Outlook
Gene therapy has experienced a remarkable surge in momentum and interest in the recent years, marking a transformative era in biomedical research. The approval of gene therapies for the treatment of inherited retinal dystrophy (Luxturna) and spinal muscular atrophy (Zolgensma) have exemplified the tangible impact of gene therapy in addressing previously untreatable medical conditions. Delivery systems play a pivotal role in the success of gene therapies by ensuring that the therapeutic genes reach their target tissues and cells. Despite the recent, well-deserved spotlight on LNPs, polymeric systems continue to hold significant promise in expanding the therapeutic landscape of gene therapies. The near infinite design space for monomers coupled with advances in polymerization techniques allow for precise customization of formulation properties to meet specific requirements of various nucleic acid cargo and target tissues. The extensive tunability offered by polymeric systems enables researchers to optimize biocompatibility, pharmacokinetics, stability, and targetability to ensure safe and effective delivery of the genetic payload. Additionally, polymeric systems offer a broader range of responsive mechanisms (such as pH, temperature, etc.) through the integration of smart monomers and architectures, allowing for more effective customization to exploit triggers in disease environments —a limitation currently associated with lipid-based vehicles. This review discussed recent advances in polymer design with the aim to highlight the fact that polymeric gene delivery systems continue to remain the focal point of research by academia and the industry with multiple preclinical/clinical studies currently ongoing and several biotechnology companies actively exploring this space. Additionally, as predictive artificial intelligence continues to evolve, the decades of data on polymeric gene delivery offers an unmatched opportunity to be leveraged in computational studies to unravel the intricate interplay between polymers, nucleic acids, and cellular processes providing valuable inputs in the design of novel polymers. Although considerable improvements in polymeric systems have been achieved, there remains a substantial space for improvement. (1) Academic research labs should consider translatability and scale-up aspects while designing novel delivery systems. Ideally, strategic partnerships between industry and academia should be initiated early on to account for translatability in the early stages of formulation design. (2) A synergistic approach with chemists and biologists working in tandem would enable leveraging the subtle aspects of cellular biology to develop cell-type and/or patient specific delivery systems. (3) Advanced physiologically relevant cellular and non-cellular models which better mimic disease pathology, physiological barriers, and target tissue microenvironment will enhance in vitro testing and improve chances of in vivo success. Similarly, importance should be given to assessing physicochemical stability of the genetic payload at the site of injection. As research in polymeric gene delivery systems continues to advance, their unique attributes position them as compelling candidates to complement lipid nanoparticles in expanding the therapeutic landscape of gene therapies.
Figure 11. Novel technologies for improved polymer design.
Various novel automated and computational technologies have emerged to provide insights in structure function relationships of polymers and guide design of well optimized polymers. High-throughput experimentation strategies and molecular dynamics simulations aid in the generation of comprehensive databases containing information about polymer structure-function relationships. Such databases can be used to facilitate the training of machine learning models to predict innovative polymer structures, thereby revolutionizing the field of polymer design by circumventing traditional time-consuming and labor-intensive approaches. Created with BioRender.com
Acknowledgments –
Partial support from the NIH through grants R01 DK120533, R01 CA235863, and R01 AA027695 is acknowledged.
Biographies
Dr. David Oupický
David Oupický is a Professor and Parke-Davis Chair in Pharmaceutics at the University of Nebraska Medical Center and the director of the Center for Drug Delivery and Nanomedicine. He obtained his Ph.D. in macromolecular chemistry with the late Prof. Karel Ulbrich at the Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic. He was a postdoc at the University of Birmingham where he worked with Prof. Len Seymour on systemic gene delivery. His research interests include synthesis of bioactive polymers and development of drug and nucleic acid delivery systems.
Chinmay M. Jogdeo
Chinmay M. Jogdeo received his Bachelor’s in Pharmacy from Maharashtra Institute of Pharmacy, Pune, India in 2019 and is currently a Ph.D. candidate at the University of Nebraska Medical Center. His doctoral research, under the guidance of Dr. David Oupický focuses on developing novel delivery systems for nucleic acids with a special interest in renal targeted delivery systems.
Kasturi Siddhanta
Kasturi Siddhanta earned her Bachelor's degree in Zoology from the University of Delhi in 2016 and a Master's in Genomics from Madurai Kamaraj University in 2018. She then worked as a Junior Research Fellow at Tata Memorial Center, Mumbai, focusing on medulloblastoma research. In 2021, she moved to the USA to pursue a Ph.D. in Pharmaceutical Sciences at the University of Nebraska Medical Center. Under the guidance of Dr. David Oupický, her doctoral research focuses on developing polymeric/lipid nanoparticle-based delivery systems for targeted pulmonary delivery of RNA therapeutics.
Footnotes
Conflict of Interests – The authors declare no conflicts of interest.
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