Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications

Abstract

Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.

I. INTRODUCTION

In modern medicine, gene therapy has become a promising way to treat diseases by inserting, deleting, or replacing genes.^1,2 To achieve successful gene therapy, the crucial step is to transport the therapeutic genes into host cells, which is also called gene delivery.³ However, genes, such as DNAs and RNAs, are negatively charged due to the abundance of phosphate groups on the molecular backbone and this nature makes it difficult, if not impossible for genes to attach and cross the cell membranes which are negatively charged as well. In addition, genes are vulnerable to the environment including oxidative reagents, enzymes (nuclease),⁴ and so on. During the gene delivery, the biological functions of genes should be preserved. Therefore, gene delivery has been the bottleneck for successful gene therapies.

Compared to the physical gene delivery approaches, such as electroporation, microinjection, and hydrodynamic method, using vectors to deliver genes is a safer and more efficient way.^3,5 Gene vectors are the vehicles to load and transport genes into the host cells.⁶ After getting into the cell, genes must be released from the vectors to achieve their biological functions. The entire process of introducing nucleic acids to cells via non-viral vectors, their subsequent release from the vectors, and reaching their gargets to regulate gene expression is called transfection. During the transfection, the vectors should not only facilitate the cell entry, but also protect the genes from the harmful environment.⁷ Commonly used gene vectors can be classified into viral and non-viral ones and the latter ones mainly refer to the lipid-based and polymer-based gene vectors.^8–10 Both viral and lipid gene vectors show merits in high transfection efficiency, however, their safety issues including immunological and biological concerns limit their applications.^11,12 For example, the immune system might have the antigen specific adaptive immune responses against certain viral vectors, leading to compromised transduction efficacy.¹³ The lipid-based vectors' colloidal stability, especially in in vivo applications, is a crucial limitation.¹⁴ In addition, both viral and lipid vectors are relatively expensive, which could negatively affect their market and commercialization.

Polymeric gene vectors are good candidates due to their unique advantages. In addition to good gene packing ability and biocompatibility,¹⁴ it is convenient to design and modify polymers to meet different requirements, for example, attaching functional groups on the polymer to achieve targeted delivery.¹⁵ In addition, the ease of commercial production makes polymeric vectors more accessible to meet the huge market demands. However, when compared with viral and lipid gene vectors, the polymeric ones need improvement in transfection efficiency. The cytotoxicity issue should also be well addressed without sacrificing the transfection efficiency. In the past decade, researchers developed various ways to improve polymeric vectors to pursue clinical applications. In this review, the authors first introduce the principle of polymer mediated transfection to clarify the challenges in the transfection process. Then, the recent progress in the molecular designs to overcome certain challenges is summarized, where more details of optimization (transfection and cytotoxicity) and functionalization rationality are discussed. Furthermore, the applications in gene editing, tissue engineering, and products on the market are also presented. Hopefully, the contents will benefit the researchers in this field to develop more advanced polymeric gene vectors for biomedical/clinical applications.

II. PRINCIPLE OF POLYMERIC GENE VECTORS

A. Mechanisms of gene delivery by polymeric gene vectors

The brief mechanism of polymer mediated gene delivery is illustrated in Fig. 1, including the formation of the polyplexes (gene packaging), entering cells via endocytosis, endosome/lysosome escape, gene release, entering the nucleus (for plasmid DNA), and expression. Since there are several steps before genes eventually realize their biological functions, any improvements in one of these gene delivery steps might contribute to better transfection/expression outcomes. So, it is important to understand the polymeric gene vector delivery mechanism step by step.

FIG. 1.

Brief illustration of gene packaging and transfection. (a) Gene packaging: positively charged polymers packaging negatively charged genes via electrostatic interaction to form the polyplex, and (b) transfection: the polymer/gene polyplex entering cells via endocytosis and escaping from endosome or lysosome. They either function in the cytoplasm (RNA) or enter the nucleus to function (DNA).

1. Gene packaging

To deliver genes, the polymeric gene vectors should first load genes, forming the so-called complexes or polyplexes.^16,17 Generally, there are two ways to load genes: via electrostatic interaction or physical encapsulation.^18–20 The electrostatic interaction happens between negatively charged genes and positively charged polymers (cationic polymers) to form the complex/polyplex. Most cationic polymers are positively charged due to the abundant protonated amine groups in their molecules, such as polyethyleneimine (PEI), poly(L-lysine) (PLL), polyamidoamine (PAMAM), poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA), and chitosan. The ratio between polymer and genes is referred to as N/P ratio where “N” represents the positive amine groups of the polymer and “P” represents the phosphate groups of the genes. It is obvious that the more positive groups, the stronger the gene packaging ability, which could be determined using agarose gel electrophoresis (AGE).²¹ AGE is a way to resolve gene fragments based on their molecular weight (MW) in the electric field: the lower the MW (with a fixed negative charge), the faster the genes traveling to the anode side in the gel.²² With increasing N/P ratios, more genes are complexed, resulting in more polyplexes and fewer free negative genes. Starting at certain N/P ratio, there are no free genes any more to travel to the anode (called retardation). By determining this specific N/P ratio, the gene packing ability can be determined: the lower N/P ratio required to reach retardation, the stronger the gene packing ability of the polymer vector. Though more polymer is good for gene packaging, too high N/P ratio (meaning too much positive polymer used) may cause higher cytotoxicity and also impair the gene release from the polyplex.⁴ As a result, the balance of N/P ratio is crucial for good gene packaging.

Alternatively, genes could also be physically embedded or encapsulated in the polymer vehicle such as poly(lactic-co-glycolic acid) (PLGA)^23,24 and alginate hydrogel.^25,26 Unlike the cationic polymer, the physical embedding methods result in weaker interaction between polymers and genes. After being delivered into cells, genes could be released via diffusion or vehicle degradation.

2. Cell uptake

By adjusting the N/P ratio, the polymer/DNA polyplex could be positively charged, which endows polyplex the affinity to the inherently negatively charged cell membrane.²⁷ When interacting with the polyplex, the cell membrane could deform to encapsulate and transport the polyplex into the cell, which is also referred to as the endocytosis.^28,29 Figure 2(a) shows five cell uptake mechanisms depending on different internalized particle sizes.¹⁶ For example, polyplexes with the size of around 200 nm mostly enter the cell via the “clathrin-mediated endocytosis” and stay in the formed endosome which eventually transforms into the acidic lysosome. Yet/However since there are numerous polymer-based polyplexes, there is no single theory to explain the cell entry pathway of all polyplexes. Monnery also shared the perspective in the endocytosis mechanisms of polyplexes.¹⁶ One of the explanations is that due to the fact that polyplexes do not internalize without free polycations, free polycations which disrupt cell membranes^30,31 and cause changes in membrane curvature facilitate the endocytosis of polyplexes. However, Vaidyanathan et al. pointed out that free cationic polymers cause long lasting cell plasma permeability, providing a plausible mechanism for the toxicity and inflammatory response.³² Based on these points of view, the free cationic polymer is helpful for endocytosis but may also contribute to the cytotoxicity. The amount of free cationic polymer must be balanced well for the benefit of both transfection efficiency and cytotoxicity.

FIG. 2.

(a) The mechanism of cellular uptake via endocytosis and endosome/lysosome formation. Reprinted with permission from Monnery, Biomacromolecules 22(10), 4060 (2021). Copyright 2021 American Chemical Society;¹⁶ (b) the possible endosome/lysosome escape mechanisms: proton sponge effect (increased osmotic pressure leading to the rupture of the endosome) or the membrane permeabilization (polymers intercalating into the endosomal membrane and causing defects and/or nano-holes for polyplex escape). Reprinted with permission from Kumar et al., Chem. Rev. 121(18), 11527 (2021). Copyright 2021 American Chemical Society.⁵¹

To understand the cell entry pathway, Sun et al. developed an amphiphilic polymer (PHML₃₀-b-PLLA₂₂-b-PHML₃₀) to deliver plasmid DNA to H1299 cells.³³ They found that when an inhibitor of the lipid-raft mediated gateways [methyl-b-cyclodextrin (M-b-CD)] was used, the cell uptake capability was inhibited by 90%, indicating that the lipid raft-mediated pathway is the favorable way for cell uptake.

Cell uptake is the first step for polymers to interact with and transfect cells. To improve the cell uptake, one possible strategy is the addition of a targeting ligand, which could improve the recognition between the polyplex and the cell membrane and may increase the endocytosis in specific cell types.^15,34–36

3. Endosome/lysosome escape

After cell uptake, the polyplexes are entrapped in the endosome which turns into the more acidic lysosome [as shown in Figs. 1 and 2(a)]. The polyplexes must escape from the endosome or lysosome. Otherwise, the complex could either be degraded in the lysosome or excluded out of the cell by the extracellular vehicles.³⁷ To some extent, the success of endosome/lysosome escape can be a decisive step for an efficient gene vector. This point was also emphasized by Iqbal et al. in their study of lysosome labeling.³⁸ The escape mechanism is a controversial topic and theories such as proton sponge, buffering effect, membrane break, and complex leakage were raised trying to explain the polyplex escape.^37,39 Figure 2(b) shows two examples of escape mechanisms: proton sponge effect and membrane permeabilization. The proton sponge effect theory depicts that the polymers/polyplexes make the endosome/lysosome osmotic pressure higher to swell and eventually break the endosome/lysosome, while the membrane permeabilization theory describes that the polymers/polyplexes could interact with the endosome/lysosome membrane structure to break the membrane. In both examples, the cationic polymers facilitated the escape process.

Recently, researchers tried various methods to enhance the endosome/lysosome escape. For example, Zhan et al. found that the instability and inability of polyplexes to escape from lysosomes at low concentrations are the key to low transfection efficiency. Slightly increasing the polycation hydrophobicity can facilitate the endocytosis, lysosome escape, and gene release, resulting in higher transfection and expression.⁴⁰ Another example to improve endosome/lysosome escape is from Shi et al. who constructed a core–shell spherical nucleic acid (SNA) by integrating an antisense oligonucleotide (OSAs) onto the surface of photosensitizer (PS) nanoparticles.⁴¹ This SNA could produce ¹O₂ to rupture lysosome under light irradiation to realize the polyplex escape.

4. Gene release

After the escape from endosome/lysosome, the polyplexes need to dissociate and to release the genes to achieve the biological functions. For example, mRNA⁴² and siRNA⁴³ should be released to the cytoplasm to either express protein or silence certain mRNA. Plasmid DNA needs to be released and then enter the nucleus via nucleus pores or during cell division followed by transcription and expression.^44,45 Well-controlled gene release systems usually have better transfection efficiency. For example, Rajendrakumar et al. formulated a PEI based dual responsive nanoassembly.⁴⁶ This assembly can either be dissociated by laser irradiation or via the breakage of disulfide bond, which made endosomal escape and gene release faster and resulted in four times higher transfection efficiency compared with the control.

The cationic polymer gene vectors might also have the merit to facilitate the release and nucleus entry for plasmid DNA. Vaidyanathan et al. proposed a hypothesis that cationic polymer associated with the nuclear membrane induces permeabilization and facilitates DNA transport into the nucleus.⁴⁷ Another example of enhanced gene release was achieved by using peptides. Wang et al.⁴⁸ reported that their PEGylated cationic peptide [a cell-penetrating peptide (CPP)] enhanced cellular uptake, endosome escape, and nucleus entry. Their polymer/pCas9 nanoparticle achieved up to up 47.3% gene editing efficiency in vitro. In cancer treatment study, the tumor growth was suppressed by >71% and animal survival rate at 60 days was increased to 60% when polymer/pCas9 was applied. They believed the cell penetrating peptide played a crucial role in endosome escape, gene release, or nucleus entry, and eventually enhanced the gene editing efficiency and the therapeutic results.

In summary, the transfection of polymeric gene vector consists of several steps including gene packaging, cell uptake, endosome/lysosome escape, gene release, and nucleus entry (for plasmid DNA). Any of these steps influences the ultimate transfection efficiency. Taking plasmid DNA as an example, after overcoming all these delivery steps, only 1%−5% of the internalized DNA reaches the nucleus for further gene expression.^16,45 Thus, any improvements in the above steps may increase gene delivery efficiency.

B. Commonly used polymers as gene vectors

Since polyethyleneimine (PEI) was used as the gene vectors in 1995,⁴⁹ various synthetic cationic polymers were employed as gene vectors such as polyamidoamine (PAMAM), poly-l-lysine (PLL), poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA), and aminoesters.⁵⁰ In addition, natural cationic polymers, such as chitosan, can also be utilized to deliver genes due to the positively charged groups (for example, amine groups) in their molecules. In addition, PLGA micro/nano spheres can physically envelop genes or polymer/gene polyplexes. The spheres encapsulating genes or polyplexes can either directly transfect cells or release the gene or polyplexes for transfection. The molecular structures of these mentioned conventional polymers are shown in Fig. 3.

FIG. 3.

Representative conventional polymers used as gene vectors (full names, abbreviations, and chemical structures). The cationic polymers (linear, branched, or dendritic) bearing abundant amine groups, including PEIs, PAMAM, PLL, and PDMAEMA, (or other cationic groups) can complex with genes via the electrostatic interaction to form the polyplex for transfection purpose. The PLGA can physically encapsulate genes or polyplexes to form nano or micro spheres which can either directly enter cells or release the polyplex for transfection purpose.

III. RECENT PROGRESS IN MOLECULAR DESIGN AND COMMERCIALIZATION OF POLYMERIC GENE VECTORS

A. Recent progress in polymer molecular design

One major advantage of the synthetic polymeric gene vectors is their capacity of being molecularly designed. Polymer gene vectors could be designed to overcome specific gene delivery challenges, such as poor degradability, high toxicity, or off targeting delivery.⁵¹ Herein, we introduce a few recently synthesized polymeric vectors aiming to overcome one or more of the challenges, such as cellular uptake, gene release, gene protection, serum stability, degradability, responsiveness (pH, temperature, redox, and radiation), or molecular targeting.^52–59

To enhance the cellular uptake, Evans et al. used an anionic polymer poly (propylacrylic acid) (PPAA) together with a cationic component to deliver genes or peptides.⁶⁰ This negatively charged PPAA can complex with cationic cell penetrating peptides (CPPs), where CPPs provide not only the positive charge for affinity to gene and cell membrane but also improve the transport crossing membrane into the cells, synergistically improve gene delivery efficiency. This point is also supported by other related publications.^54,58,61,62

To address the gene release challenge, Wang et al. developed a cationic polymer PADDAC which could be cleaved specifically by glutathione (GSH)⁶³ [Fig. 4(a)]. After binding with genes to form the polyplex and delivering the polyplex into cells, PADDAC can be cleaved by GSH, resulting in the charge inversion from positive to negative. Due to the charge inversion, the complexed genes could not bind the polymer anymore and got released from the polyplex. As a result, this delivery system showed improved transfection efficiency.

FIG. 4.

Representative molecular designs of polymers for gene delivery. (a) Glutathione (GSH)-specific polymer (PADDAC) chemical structure and its GSH-triggered charge reversal. Reprinted with permission from Wang et al., ACS Appl. Mater. Interfaces 12(13), 14825 (2020). Copyright 2020 American Chemical Society;⁶³ (b) the micelle self-assembled from poly (ethylene glycol)-block-PDMAEMA-block-poly (n-butylmethacrylate) (ODB) copolymer and the chemical structure of each component (PEG, PDMAEMA, and PnBMA). Reprinted with permission from Tan et al., J. Am. Chem. Soc. 141(40), 15804 (2019). Copyright 2019 American Chemical Society;⁶⁴ (c) the triblock copolymer composed of different hydrophilic heads, linker, and hydrophobic tail. The copolymer self-assembled with a surfactant DOPE and further complexed with genes. Reprinted with permission from Wu et al., Acta Biomater. 115, 410 (2020). Copyright 2020 with permission from Elsevier.⁶⁵

To better preserve the gene bioactivities, Tan et al. prepared a triblock copolymer containing a hydrophilic (PEG), a hydrophobic (PnBMA), and a cationic (PDMAEMA) segment⁶⁴ [Fig. 4(b)]. This triblock copolymer formed micelles via self-assembling to complex plasmid DNA into relatively “stretched” complexes rather than conventional dense spherical complexes. The authors stated that this innovative physical state of complexes preserved the pDNA secondary structure in its native B-form upon packaging and allows greater protein expression in comparison with polyplexes which tightly condense pDNA and significantly distort its helicity.

Wu et al. setup a useful database of 120 polymers (the polyalkylamines) and investigated all polymers' transfection efficiency and toxicity⁶⁵ [Fig. 4(c)]. They found that with appropriate particle size (around 200 nm) and zeta potential (+40–+50 mV), the polyalkylamines exhibited better transfection. They also found that the introduction of proper hydrophobicity greatly enhanced the transfection, which is in good accordance with the results from other research groups.^40,64 This study revealed how transfection efficiency of polymeric gene vectors was related to physical characteristics such as size, zeta potential, and hydrophobicity.

B. Commercial polymeric gene vectors/reagents

In addition to academic research, polymeric gene vectors or transfection reagents also succeeded on the market. In Table I, we summarized some commonly used, efficient polymer-based gene transfection reagents on the market. These reagents are either highly efficient in multiple cell lines in in vitro studies or can be used in in vivo applications (example: cancer therapy) for the delivery of DNA or siRNA. It is reasonable to anticipate that, with increasing development of polymeric gene vectors, more such products will be on the market to meet medical treatment needs.

TABLE I.

Commercially available polymeric gene vectors (reagents).

Reagent name	Based on	Nucleic acid can be delivered	Features/applications
jetPEI® and jetPRIME®	Linear polyethyleneimine	DNA, siRNA	Highly effective and Low amounts of nucleic acid
jetMESSENGER® and in vivo-jetRNA®	Cationic polymer	mRNA	mRNA in vitro or in vivo delivery vaccination/immunization
X-tremeGENE™ 360 transfection reagent	Cationic polymer	DNA, siRNA, miRNA and CRISPR/RNP	Transfection of a broad range of eukaryotic cells, including insect cells and hard-to-transfect cell lines (Sf9, U-2 OS, MEF, MCF7, Hep G2, PC-3, HeLa, HT-29, HT-1080, K-562, RAW 264.7, Jurkat, PC-12, and HCT 116)
FuGENE® 4K transfection reagent	Cationic polymer + lipid	DNA	Ideal for protein and virus production
TurboFect™ transfection reagent	Cationic polymer	DNA or RNA	Excellent transfection efficiency in the presence or absence of serum
Xfect transfection reagent	Biodegradable polymer	Plasmid DNA	Very low cytotoxicity profile and high transfection efficiency
Altogen's POLYMER in vivo transfection reagent	Biodegradable polymer	siRNA and plasmid DNA	In vivo delivery reagent via systemic intravenous (i.v.) injection or direct intratumoral (i.t.) injection

IV. RECENT APPLICATIONS OF POLYMERIC GENE VECTORS

In the past ten years, there are large amounts of literature in polymeric gene delivery every year. Among them are successful molecular modifications, improvements of transfection efficiency, and in vivo applications. Along with the thriving fields of gene editing and tissue engineering, polymeric gene vectors have shown their potential in these fast-evolving fields. Here, we discuss a few examples of successful applications of polymeric gene vectors in gene editing and tissue engineering.

A. Polymeric gene vectors for gene editing

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has been one of the most successful tools for genome editing due to its simplicity and high efficiency.^66–68 By adding, deleting, or replacing certain genetic sequences, the CRISPR/Cas9 has great potential in gene therapy.⁶⁹ For example, Tan et al. developed a block polymer (poly[ethylene oxide-b-2-(dimethylamino) ethyl methacrylate-b-n-butyl methacrylate] (PEO-b-PDMAEMA-b-PnBMA)) micelles to deliver the Cas9/sgRNA ribonucleoprotein.⁵⁷ The positively charged block copolymer micelles can complex with the negatively charged “Cas9 protein/sgRNA complex” (sgRNA are excessive) to deliver the CRISPR system into cells for gene editing purposes.

However, directly delivering the Cas9/sgRNA (protein and RNA) system to the cells encounters challenges, such as low efficiency, enzymatic degradation, and high cost.⁷⁰ Alternatively, delivering the plasmid DNA or mRNA encoding the Cas9 and sgRNA, is more feasible.⁷¹ The delivered DNA or RNA could express corresponding Cas9 protein and sgRNA to achieve the gene editing purpose. For example, Lyu et al. developed a photolabile semiconducting polymer (pSPN) composed of an O₂-generating backbone and grafted polyethyleneimine (through an O₂-cleavable linker) to deliver the CRISPR system plasmid.⁷² As shown in Fig. 5, the rationality of this molecular design is that after polymer packaging the plasmid and carrying it into the cell, the cleavable linker could be broken using the near-infrared radiation (NIR). As a result, grafted PEI chains are detached from the complex and the packaged plasmid DNA could be released, enter the cell nucleus, and express the Cas9/sgRNA for gene editing. Compared with the control which received no NIR, the NIR treated group showed 15- and 1.8-fold enhancement in repaired gene expression in cultured cells (i.e., in vitro) and mice (i.e., in vivo), respectively. Similarly, Li et al. developed a semiconducting polymer brush (named as SPPF)⁷³ by conjugating alkyl side chains, PEG chains, and fluorinated polyethyleneimine to a semiconducting polymer backbone. This positively charged SPPF could bind and delivery the CRISPR/Cas9 cassettes (plasmid DNA) to cells. Under laser irradiation, the polymer–DNA polyplex could generate heat via photothermal conversion to enhance the polyplex escape from the endosome/lysosome. The enhanced escape benefited the gene release and therefore the gene editing efficiency. The design of polymers which respond to certain stimuli (such as temperature, pH, and redox) for improved gene delivery can also be found in gene editing literature.^74–76 The growing new polymeric designs may open an avenue for the polymeric vectors to be used in the CRISPR-based gene editing applications.

FIG. 5.

The representative polymeric gene vectors applications in gene editing: a CRISPR/Cas9 plasmid DNA delivered by a photolabile polymer (chemical structure shown). The polymer self-assembled and complexed with genes for transfection, after which, near infrared radiation was applied to trigger the detachment of genes from the self-assembly to achieve gene release, nucleus entry, and gene editing effects. Reprinted with permission from Lyu et al., Angew. Chem. Int. Ed. Engl. 58(50), 18197 (2019). Copyright 2019 John Wiley and Sons.⁷²

For example, in vivo gene editing studies have been conducted using polymeric gene vectors to treat cancer.^{34,35,77–79} Wang et al. developed an α-helical polypeptide named PPABLG to deliver the Cas9 plasmid and sgRNA⁴⁸ in mice. This delivery system achieved 35% gene deletion in HeLa tumor tissue, 66.7% reduction of the polo-like kinase 1 (Plk1) protein, greater than 71% of tumor growth suppression, and increase in the animal survival rate to 60% within 60 days. Sun et al. delivered the CRISPR-Cas12a system via a pH-sensitive polymer.⁷⁵ They observed ∼48% Pcsk9 (proprotein convertase subtilisin/kexin type 9) disruption in vivo (mouse) and subsequently significant cholesterol control (∼45% of cholesterol reduction). These in vivo studies indicate that the polymeric gene vectors are promising candidates in gene editing for disease treatment.

B. Polymeric gene vectors for tissue engineering

Tissue engineering aims to regenerate biological alternatives to harvested tissues that restore, maintain, or improve tissue function by using the principles of engineering, physical and biomedical sciences.^80,81 Tissue engineering utilizes scaffolds, cells, and/or biological signals to achieve the desired living tissue regeneration. Our lab has developed numerous biodegradable and biomimetic scaffolds to support cells for tissue regeneration.^81–90 Our lab has also developed many delivery systems to deliver biological signals alone or in combination with scaffolds. These biological signals include growth factors,^91–93 peptides,^90–94 small molecule drugs,⁹⁵ and nucleic acids.⁹⁶ To accomplish the delivery of nucleic acids (RNA and DNA), novel hyperbranched polymers (HPs) consisting of a biodegradable core, cationic chains (PEI), and hydrophilic chains (PEG) were developed.^96–98 These HP polymers can package genes to form stable polyplexes which can be encapsulated in PLGA microspheres to achieve the controlled release of the polyplexes (stage 1 delivery). Subsequently, the released polyplexes carry genes into the cells (stage 2 delivery).⁹⁶ The polyplex-loaded PLGA microspheres were then attached onto a scaffold to achieve specially and temporarily controlled gene delivery for various tissue engineering applications.

One example is the two-stage delivery of a miRNA (miRNA-26a) for calvarial defect repair⁹⁶ (Fig. 6). In this study, the miRNA was complexed with the HP polymer to form polyplexes, which were then encapsulated in the PLGA microspheres for the sustained release of polyplexes. The microspheres were then seeded on a scaffold to be implanted in the calvarial defect of a mouse. The scaffold carrying the two-stage delivered miRNA showed significantly better bone regeneration when compared to the control groups (no miRNA group or bolus miRNA delivery). The delivery of miRNA via HP polymer platform also succeeded in the enrichment of regulatory T cells to reduce the periodontal bone loss⁹⁹ and in the delivery of an anti-miR-199a for nucleus pulposus regeneration and calcification prevention.⁹⁷

FIG. 6.

The representative polymeric gene vectors applications in tissue engineering: a miRNA delivered by a hyperbranched polymer for calvarial defect repair. The chemical structure of each component [polyethylene glycol (PEG), polyethyleneimine (PEI), and the hyperbranched polyester core (H20)] are shown. The complex formation and self-assembly is also illustrated. (b) The negative control (upper) and miRNA delivered (lower) mouse skulls are compared. The gene delivery system achieved better calvarial defect repair effect. Reprinted with permission from Zhang et al., Nat. Commun. 7, 10376 (2016). Copyright 2016 John Wiley and Sons.⁹⁶

In addition, we applied this HP polymer for plasmid DNA delivery. The HP polymer was used to package a plasmid DNA (NR4A1-based plasmid) to form polyplexes, which were subsequently encapsulated in PLGA nanospheres and seeded on injectable porous and nanofibrous microspheres.⁹⁸ The microspheres containing the HP/pDNA were injected in the nucleus pulposus (NP) of a rat tail. The two-stage delivery system released the polyplexes for more than 30 days and the delivered NR4A1 pDNA therapeutically reduced the pathogenic fibrosis of NP tissue in the rat model. These examples of gene delivery in tissue engineering constructs demonstrate the great potential of polymeric gene vectors in biomedical applications.

V. CONCLUSIVE REMARKS AND FUTURE PERSPECTIVES

Although great progress has been made in polymeric gene delivery,¹⁰⁰ there remain significant challenges, especially in their in vivo applications, which hindered the broad applications of polymeric gene vectors. Here, we would like to discuss a few important aspects such as transfection efficiency, cytotoxicity, the in vivo environment, and targeted delivery.

A. Transfection efficiency and cytotoxicity¹⁰¹

For cationic polymer vectors, transfection efficiency is largely determined by the positive charges of the polymers that both complex genes forming polyplexes and interact with the negatively charged cell membrane leading to crossing it into the cells. Here, the magnitude and distribution of the charge play important roles in both transfection efficiency and cytotoxicity. In general, more positive charges in the polymer lead to higher transfection efficiency and higher cytotoxicity. Appropriately choosing and designing the molecular structure of the polymer may optimize transfection efficiency and minimize cytotoxicity, including using degradable polymers,¹¹ varying types of cations, distributing the positive charge centers, balancing the hydrophobicity/hydrophilicity, incorporating nontoxic ingredients, and decorating with cell penetrating peptides.^102–105 In addition to the molecular design of the polymer, balancing between transfection efficiency and cytotoxicity can also be accomplished by varying the polymer/gene ratio (N/P ratio) when a polymer has been determined. Higher polymer content can improve gene packaging. However, too much polymer also elevates the cytotoxicity by impairing the cell membrane. Therefore, the optimal N/P ratio must be determined for the polymeric gene vector.

B. In vivo environment

The in vivo environment is a major challenge since it is more complicated than in vitro environment. The polymer–gene complexes (polyplexes) need to evade in vivo barriers, such as protein fouling (proteins in the body to form aggregates with polyplexes),^106,107 nonspecific clearance from the body,¹⁰⁸ and enzymatic degradation,¹⁰⁹ and to eventually reach and transfect cells. One way to circumvent such in vivo destruction is to decorate polymers with polyethylene glycol (PEG).^110–112 The superhydrophilic PEG makes the polymer and the complex/polyplex more protected in vivo to achieve prolonged circulation time. Zwitterionic polymer and polysaccharides are also used to achieve the same goal in vivo.

C. The size of polyplexes

The size of polyplexes can affect transportation and therefore transfection both in vitro and in vivo. However, because of significantly more diffusion limitations in vivo, the size of the polyplexes can become a critical barrier to overcome in vivo. The polyplexes should be able to penetrate the cell gaps so that they can migrate in tissues to transfect as many cells as possible. The cell gaps are usually in the range of nanometers,¹¹³ and this is the reason why too large sized polyplexes cannot penetrate easily. In another scenario, when administrated via intravenous injection, the polyplexes need to penetrate the blood vessel's cell gaps to transfect cells outside the blood vessel.^10,114 From this point, the size of polyplexes should be controlled in a reasonable range (most polyplexes are in the range of 100–200 nm).^65,115,116

D. Targeted delivery

Due to the heterogeneous nature in vivo, delivering genes to the specific cells is challenging. Generally, incorporating specific ligand onto polymeric gene vectors can promote the recognition and interaction of target cells so that the polyplexes preferentially accumulate in the aimed cells/tissues/organs.^15,62 The enrichment of polyplexes in the specific region in vivo not only enhanced the transfection efficiency but also reduce the off-target transfection, which is important especially in clinical applications.

To conclude, the polymeric gene vectors have drawn much attention due to their safety (in terms of immunologic response or genome integration), versatility in design, low cost, and ease of large-scale manufacturing. To better understand polymeric vectors, this review discussed the principle of polymer-based gene transfection and challenges in each transfection step. In addition, recent efforts and progress in polymer molecular designs were summarized: either to improve the performance (increasing transfection efficiency and reducing cytotoxicity) of polymeric vectors or to overcome the transfection challenges. With rational molecular design and synthesis, functionalized polymers with properties including degradability, stimulation responsiveness, targeting, and prolonged circulation time have been demonstrated in biomedical research such as cancer therapy, cardiovascular disease treatment, and bone regeneration. With deeper understanding of the gene delivery mechanisms and rational molecular designs, we can only expect more advanced polymeric gene vectors to be developed to meet clinical applications.

AUTHOR DECLARATIONS

Conflict of Interest

The authors have no conflicts to disclose.

Author Contributions

Kemao Xiu: Conceptualization (equal); Writing – original draft (lead); Writing – review & editing (equal). Jifeng Zhang: Funding acquisition (supporting); Writing – original draft (supporting); Writing – review & editing (supporting). Jie Xu: Funding acquisition (supporting); Writing – original draft (supporting); Writing – review & editing (supporting). Y. Eugene Chen: Funding acquisition (supporting); Writing – original draft (supporting); Writing – review & editing (supporting). Peter X. Ma: Conceptualization (lead); Funding acquisition (lead); Supervision (lead); Writing – original draft (lead); Writing – review & editing (lead).

DATA AVAILABILITY

Data sharing is not applicable to this article as no new data were created or analyzed in this study.