Intelligent nucleic acid delivery systems based on stimuli-responsive polymers

Abstract

Despite significant advances in the past two decades, gene therapy is still in the stage of clinical trials worldwide mainly due to the lack of safe and efficient delivery vehicles for therapeutic nucleic acids. Among the various attempts to develop clinically applicable gene therapy, polymer-based nucleic acid delivery systems have attracted great interest, especially for the exciting RNAi-based gene therapy. Regarding in vivo nucleic acid delivery, in particular via intravenous injection, there are many extra- and intracellular obstacles, some of which are conflicting. Virus-mimicking nucleic acid delivery systems that combine multiple and programmable functions are thought to be very promising for conquering these challenging barriers. In this review article, we highlight recent progress in stimuli-responsive polymers that have been applied in fabrication of non-viral multi-functional nucleic acid vehicles, which are categorized by the type of stimulus: reduction potential, pH, temperature, and others. In each section, intelligent pDNA delivery systems are introduced first, followed by summarizing various responsive polymer-based siRNA vehicles. Considering the great potential of RNAi-based gene therapy, we devote some space to the recent progress of multi-functional siRNA delivery systems. In addition, different requirements in designing polymer-based siRNA and pDNA carriers are also specified in this review.

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Fu-Sheng Du

Fu-Sheng Du is an associate professor in the Department of Polymer Science & Engineering at Peking University (PKU). He received a BSc (1989) and a MSc (1992) in chemistry from PKU. After four years working at the National Research Institute for Family Planning as a research scientist, he joined the Department of Polymer Science & Engineering of PKU where he earned his PhD in polymer chemistry in 2000. His current research interests include stimuli-responsive polymers, intelligent polymer-based drug/gene delivery and biodegradable polymers.

Yang Wang

Yang Wang obtained his BSc degree from the College of Chemistry and Molecular Engineering (CCME) of Peking University in 2006. In the same year, he entered the Department of Polymer Science and Engineering of CCME as a graduate student. Now he is pursuing his doctoral degree on the development of multi-functional stimuli-responsive gene carriers, under the guidance of Prof. Fu-Sheng Du and Prof. Zi-Chen Li.

Rui Zhang

Rui Zhang obtained his BSc in Chemistry from Shan-Dong University in 2006. In the same year, he started his PhD in the Department of Polymer Science and Engineering of CCME at PKU, under the guidance of Prof. Zi-Chen Li. His doctoral research focuses on the synthesis of stimuli-responsive polymers for biomedical applications, specifically as gene carriers.

Zi-Chen Li

Zi-Chen Li studied polymer chemistry and graduated from Shandong University in 1987, he received his MSc degree from the Institute of Chemistry CAS in 1990, and obtained his PhD from Peking University in 1995. From 1995 until 1997, he was a post-doctoral fellow at Peking University and Waseda University Japan. In 2002, he became a Professor in polymer chemistry at Peking University. His main research interests are: new polymer synthesis, bio-related polymers, drug carriers and controlled release.

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1. Introduction

The basic idea of gene therapy is to transfer therapeutic genetic materials (i.e., nucleic acids) into cells of an individual in order to cure diseases through the expression of helpful proteins or the knockdown of unwanted proteins. The concept of gene therapy was proposed about forty years ago, however, great interest in gene therapy worldwide did not emerge until 1990 when the first clinical trial was performed for curing a patient with severe combined immunodeficiency (SCID) using a retroviral vector.^1,2 In the past two decades, the field of gene therapy has undergone several cycles of ups and downs, with the successful cure of child patients who suffered X-linked SCID and the patient death or development of T-cell leukemia caused by viral vectors.^3,4 In particular, the discovery of RNA interference (RNAi) phenomenon has greatly spurred research interest in RNAi-based gene therapy.

RNAi is a naturally occurring phenomenon that regulates gene expression at the post-transcriptional stage, and is thought to play a significant role in protecting the host against viral attack.^5–7 In 1998, Fire, Mello and colleagues discovered that long double-stranded RNA (dsRNA) could silence gene expression in the nematode Caenorhabditis elegans.⁸ Soon after, in 2001, Tuschl et al. demonstrated that synthetic small interfering RNA (siRNA) with 21 nucleotide base pairs (bps) could function in various mammalian cells to mediate sequence-specific gene silencing (or knockdown) without inducing interferon synthesis.⁹ In the past decade, research on RNAi has significantly advanced both in basic and applied fields, as a laboratory tool^5,10 or a new therapeutic strategy.^6,7,11–15 For therapeutic applications, siRNAs with approximately 21–23 bps are generally practicable because longer (> 30 bps) dsRNAs may elicit harmful responses such as the initiation of antiviral interferon response and global protein expression shutdown.^16,17

Efficient and safe delivery is one of the key issues for the clinical application of nucleic acids as therapeutic agents. Both plasmid DNA (pDNA) and oligonucleotides including antisense oligonucleotide (AON), oligodeoxynucleotide (ODN), and siRNA are negatively charged biomacromolecules which are subject to enzymatic degradation by the endogenous nucleases and are too large to cross the cellular membranes in their naked form without using external force. Although viral vectors are highly efficient and remain the most popular tool for in vivo gene delivery and clinical trials, some issues such as bio-safety, non-specific features, cost of production, and loading capacity limit their clinical applications. In order to avoid the disadvantages of viral vectors, non-viral methods have been widely used as a significant alternative for gene delivery, especially for RNAi-based gene therapies.^13,18 Regarding non-viral delivery systems, when naked plasmids or siRNAs were used for in vivo tests by local administration, in some cases, effective gene expression or knockdown could be observed though the mechanism is unclear. However, for in vivo applications such as in liver and the remote tumor tissues, systemic injection of nucleic acids in an appropriate formulation is necessary to achieve an effective cure.^7,19,20

In the past fifteen years, polymer-based nucleic acid delivery systems have significantly advanced because of their advantages such as improved bio-safety, greater flexibility, and facile manufacturing and modification.^21–24 Recently, a number of polymeric siRNA delivery systems, especially the cationic polymer-based ones, have been developed based on the experiences from pDNA and ODN (or AON) delivery.^25–32 In general, nucleic acids can be complexed by cationic polymers to form polyplex nanoparticles. For systemic administration of these nucleic acid-containing nanoparticles, there are many extracellular and intracellular barriers that must be overcome. These barriers include effective circulation in blood, extravasation across the vascular endothelial membrane, diffusion through the extracellular matrix (ECM), cellular association and uptake, endosomal escape, unpacking of the polyplexes and release of the intact nucleic acid therapeutic in the cytoplasm or in nucleus (Fig. 1).^{7,17,19,33–36} Failure at any step of these obstacles will significantly reduce the efficacy of gene expression or knockdown. In addition, some conflicting and challenging demands are encountered by these nanoparticles after systemic injection. For example, during the circulation in blood, a neutral and hydrophilic corona on the outside of the nanoparticles is preferred, however, this shielding layer is generally considered to be unwanted for efficient cellular uptake and endosomal escape of the nanoparticles. While dense and stably formed polyplex nanoparticles are helpful for their circulation in blood and diffusion in the ECM, a weaker binding capability of a cationic carrier favors unpacking of the polyplexes and the subsequent release of free nucleic acid.^22,33 One of the practical strategies to solve these dilemmas is to use intelligent systems that mimic viruses, showing programmed changes in conformation and/or properties with the endogenous stimulus such as changes in pH, redox potential, enzyme concentration, or exogenous stimuli such as temperature change, light, and so forth.

Fig. 1 Various barriers encountered by the nucleic acid-containing nanoparticles upon intravenous injection.

Stimuli-responsive polymers have been extensively studied and widely used, alone or combined together with other materials, for smart drug delivery and tissue engineering.^37–48 Virus-mimicking gene delivery systems based on responsive polymers^35,49–52 or nanotechnological approaches^53,54 have also been summarized in some recent review articles. In this review, we focus on recent advances in the development of new stimuli-responsive polymers and their applications in non-viral intelligent nucleic acid delivery systems. In particular, we devote some space to the design protocols of multi-functional siRNA delivery systems based on the stimuli-responsive polymers. These polymers are categorized by the type of stimulus. In each section, we first introduce the responsive polymers used for pDNA, considering that there are some common obstacles for in vivo delivery of either siRNA or pDNA. Then, we summarize the different design protocols for polymer-based siRNA delivery, since siRNA and pDNA are quite different in many aspects, such as molecular weight, rigidity, sensitivity to enzymes, and intracellular action site.^30,55–57 Regarding ligand-mediated targeted delivery and in vivo biodistribution of the nucleic acid-containing nanoparticles, there are several recent review papers,^20,31,58 therefore, we do not pay special attention on these issues.

2. Reduction-sensitive systems

Generally speaking, polycations with large molecular weight (M_w) and high cationic density condense nucleic acids more efficiently to form stable polyplexes under physiologically conditions, and result in high transfection efficacies compared to the low-M_w oligomers with a similar structure. However, it is well known that high-M_w polycations such as poly(ethylenimine) (PEI) and poly(L-lysine) (PLL) are more cytotoxic than low-M_w polycations. Repeated administration of these non-degradable polymers may elicit a risk of accumulation in the body.^59,60 Furthermore, strong binding of the polycations to the nucleic acids may limit the intracellular unpacking of the polyplexes which is necessary for an efficient transfection. One of the solutions to these problems is to use degradable polycations.²³ Although the degradable polycations whose degradation is based on the hydrolysis of an ester or amide bond have been widely used as gene carriers with decreased cytotoxicities, it is difficult to control the degradation occurring in the cytoplasm where free siRNA should be released to take action.^61–63 Since the reduction potential in the cytoplasm is much higher (∼100 fold) than in the extracellular environments,^64,65 reduction-sensitive polyplexes are considered to be superior degradable candidates, especially for siRNA delivery.⁵² The chemistry involved in molecular design is the reductive cleavage of disulfide-containing polymers. As shown in Fig. 2, these sensitive polyplexes can be prepared by two approaches: preparation of disulfide-containing polymer first followed by polyplex formation,⁶⁶ and polyplex formation first followed by in situ crosslinking or template polymerization.⁶⁷

Fig. 2 Two general approaches for preparing the reduction-sensitive nucleic acid-containing polyplex nanoparticles: polymer first followed by polyplex formation (A) and polyplex first followed by in situ crosslinking or template polymerization (B).

2.1. Reducible cationic peptides and polymers

Most of the reported reduction-sensitive polyplexes were prepared by the first approach (route A in Fig. 2). Read et al. reported a type of linear reducible polycations (RPCs) by oxidative polycondensation of a peptide with ten lysine residues and two cysteine units at both the chain ends. These RPCs showed reduced toxicity and efficient pDNA delivery in the presence of cationic lipids or choloroquine.⁶⁸ In order to improve the endosomolytic properties, histidine residues were incorporated into the backbones of the RPCs. The imidazole group in histidine has a pK_a ∼6.0 which possesses a buffering capacity and may promote endosomal escape. The modified RPC with ∼70% histidine content (His-RPC) demonstrated a more efficient gene transfer in different cell lines than branched PEI (25 kDa), without requirement for other endosomolytic agents. His-RPC showed negligible cytotoxicity compared to high-M_w PEI or PLL, which was ascribed to the reducible cleavage in the cytoplasm of the high-M_w His-RPC backbones. In addition, His-RPC could mediate efficient transfer of other nucleic acids such as mRNA and siRNA.⁶⁹ Recently, these authors prepared a series of His-RPCs of different molecular weights, with or without a targeting ligand. These polycations were comparatively studied as carriers for EGFP plasmid and siRNA. It was found that the optimized molecular weights of His-RPCs for efficient plasmid and siRNA delivery were different. Furthermore, M_w elicited different effects on the condensing capability of His-RPCs and sizes of the formed polyplexes. While His-RPC with a high M_w (162 kDa) showed a stronger condensing capability for the plasmid than for siRNA, the middle-sized His-RPCs (M_w 38–80 kDa) were preferred for condensing siRNA into the smaller polyplexes.⁵⁵ Although the detailed mechanism for these differences has not been completely clarified, these results combined with other data demonstrate that the findings from polycations used for pDNA delivery can not be simply extrapolated for designing siRNA carriers.^70,71 A similar strategy has been used for the synthesis of other reducible linear polypeptides consisting of KALA,⁷² TAT,⁷³ or NLS⁷⁴ peptide residues in the backbones, which were used for plasmid or siRNA delivery.

It is known that PEIs with high M_w have high transfection efficiency and significant cytotoxicity while low-M_w PEIs are non or less cytotoxic but with little efficiency for pDNA.^75,76 Lee et al. crosslinked low-M_w PEI (PEI 800) using reducible crosslinking reagents. The crosslinked polymers showed much higher transfection efficiency compared to the intact PEI 800.⁷⁷ Although these polymers were not superior to the branched PEI (25 kDa) in terms of transfection efficiency, their cytotoxicity was greatly reduced, probably due to the reductive cleavage of the disulfide bonds in cytoplasm. This work also demonstrated that the crosslinking approach was a practical strategy for development of efficient and low-toxicity polymer-based gene carriers. In fact, low-M_w PEI or other polyamines have been crosslinked by other crosslinkers on the basis of different reactions, producing a number of polycations with various linkage structures including hydrolysable ester,^78–81 acid-labile imine or acetal,^82–84 disulfide,^85–87 and slowly degradable amide or urethane.^88–90 For example, Breunig and coworkers prepared a family of reducible polycations by crosslinking linear low-M_w PEIs (M_w 2.6–4.6 kDa) with disulfide-containing crosslinkers. Some of these materials have superior transfection efficacies and lower cytotoxicities compared to commercial transfection reagents. By treating CHO-K1 cells with duroquinone, a compound that oxidizes NADPH/H⁺ and subsequently lowers the reduction potential within the cells, the authors confirmed that the disulfide linkage in these polycations did play a role.⁹¹ Furthermore, some of these reducible polycations have been used for siRNA delivery. The results of flow cytometry and confocal microscopy showed that the branching structure of PEI was helpful for cellular uptake of siRNA/PEI polyplexes, and the release of free siRNA in the cytoplasm was promoted by reduction cleavage of the disulfide bond.⁹² However, these polycations were not well purified and a lot of low-M_w raw materials remained whose effect on the transfection was not clearly demonstrated.

Engbersen, Zhong and coworkers reported a family of linear reducible poly(amido amine)s (PAAs) having various chemical structures in the main chains and pendant groups of the polymers which were prepared by the Michael addition reaction of amines and bisacrylamides.^93,94 The effect of position and content of the disulfide bonds, type of amino groups and charge density, hydrophilic/hydrophobic balance, and intercalating groups in the polymers on their DNA binding capability and the physicochemical and biological properties of the DNA/polycation polyplexes were investigated in detail. Some of these reducible polymers showed improved transfection efficiency and reduced cytotoxicity compared to their counterparts without disulfide bonds and PEI (25 kDa) confirming the important role of the disulfide bond.⁹⁵ Although these reducible polymers have been used only for transferring pDNA at present stage, some of them are expected to be applicable for siRNA delivery. By the similar approach, Kim et al. prepared a series of branched PAAs with different charge densities, which showed great potential for plasmid and siRNA delivery.^96,97 They also synthesized the linear PAAs with pendant primary amino groups and arginine groups which have been used for in vitro siRNA delivery and may be useful for siRNA-based cancer therapy or inhibiting apoptosis in cardiovascular disease.^98,99 Recently, a reducible PAA based on spermine was reported to show excellent efficacy for both in vitro and in vivo EGFP pDNA delivery, as well as for Akt siRNA delivery into lung cancer cells.¹⁰⁰

The reducible polycations were also applied as carriers in layer-by-layer (LbL) film-based gene delivery, considering the presence of thiol groups in some proteins on the cellular plasma membranes.^101,102In vitro and in vivo results revealed that the exofacial reducing potential of plasma membranes did serve as a trigger for DNA release from the bioreducible LbL film which may have potential for localized gene delivery applications.¹⁰³ In recent years, reversible addition-fragmentation chain transfer polymerization has become an alternative approach for preparing reducible cationic polymers that can be used for pDNA or siRNA delivery.¹⁰⁴

2.2. Reduction-sensitive polyplexes formed by post-crosslinking or template polymerization

Trubetskoy et al. first reported reductive pDNA polyplex particles formed through template polycondensation using crosslinkers with a disulfide bond. Biological activity of the DNA in the polyplexes remained after crosslinking. The reduction-cleavable linkage enabled the polyplexes to deliver DNA into cells.⁶⁷ Soon after, McKenzie and coworkers prepared a new class of peptides consisting of ∼16 lysine units and 2–5 cysteine residues.¹⁰⁵ These peptides could bind pDNA to form polyplexes which were transiently stabilized by the subsequent formation of disulfide linkages upon oxidation. The stabilized polyplexes showed significantly enhanced transfection efficacies compared to the uncrosslinked ones. They further extended this approach to the preparation of reducible polyplexes with a poly(ethylene glycol) (PEG) shell and N-glycan as a targeting ligand (Fig. 3). The multi-functional polyplexes were applied to in vivo gene transfer targeting to hepatocytes.¹⁰⁶ These reducible carriers should be applicable for siRNA delivery. In a recent work, a surface-engineered targeted siRNA delivery system was reported using poly(propylenimine) (PPI) dendrimers as the polycation backbone. In this system, the polyplex nanoparticles formed from PPI dendrimers and siRNA were sequentially caged with a disulfide-containing crosslinker, surface-coated by PEG, and conjugated with a LHRH peptide at the telechelic end of the PEG chain. These finely tailored siRNA nanoparticles have good stability in the plasma and are capable of delivering siRNA into the cytoplasm of tumor cells in a receptor-mediated manner. In particular, high specific accumulation in tumor tissues was observed when the siRNA nanoparticles were applied in vivo in nude mice by systemic administration. This work demonstrates that the sequential layer-by-layer modification provides a promising approach for the development of siRNA vehicles.¹⁰⁷

Fig. 3 Disulfide crosslinked peptide DNA co-condensates with N-glycan ligands and PEG palisades. Adapted from ref. 106.

Kataoka et al. first reported a type of polyion complex (PIC) micelles with the core crosslinked by disulfide bonds, which were prepared from PEG-b-PLL with pendant thiol groups and poly(α,β-aspartic acid) [P(Asp)] by spontaneous electrostatic association and subsequent oxidation. These micelles were stable against high salt concentrations (∼0.5 M NaCl) due to the stealth effect of PEG corona and the core crosslinking, but dissociated in the presence of a reducing agent (Fig. 4).¹⁰⁸ On the basis of this concept, they developed a series of reduction-sensitive core crosslinked PIC micelles for gene delivery, using pDNA or siRNA instead of P(Asp) as the polyanions. It was found that an optimized balance between the cationic charge density and the disulfide crosslinking degree played a critical role for the efficient deliveries of pDNA¹⁰⁹ or siRNA.¹¹⁰ Incorporation of cyclic RGD peptide ligands onto the surface gave the reducible PIC micelles great potential for cancer gene therapy through intravenous injection.¹¹¹ The post crosslinking approach was also applied for PEI-based reductive polyplexes with or without hydrophilic shells, using either small-molecule crosslinkers¹¹² or in situ thiol oxidation.¹¹³

Fig. 4 Schematic illustration of the reduction-sensitive stabilization of a PIC micelle through the formation of disulfide bonds in the core. Adapted from ref. 108.

2.3. Reducible siRNA/ODN–polymer conjugates

ODN or siRNA can be synthetically prepared and their molecular weights are much smaller than that of pDNA, which affords them another delivery strategy: covalently conjugation. For example, after intravenous injection in mice, cholesterol-conjugated siRNA targeting the apolipoprotein B (ApoB) gene showed significantly improved biological activity compared to the intact siRNA.¹¹⁴ However, stable covalent linkage between siRNA and the carrier may hinder the interference activity of siRNA in the cytoplasm. The transiently covalent linkages that are cleavable upon biological or physical stimulus provide a useful solution. By combining the smart PEGylation strategy and the PIC micelle strategy, Nagasaki and his colleagues reported a type of reduction-sensitive supramolecular assembly for ODN delivery.¹¹⁵ The conjugates of ODN and PEG with or without the disulfide linkage, termed as PEG-SS-ODN and PEG-ODN, were synthesized. PIC micelles were spontaneously formed by condensation of these conjugates with branched PEI or PLL. PEG provides the stealth effect, which is beneficial for extracellular stabilization of the micelles. The presence of the disulfide linkage makes the PEG chain cleavable in reductive microenvironments such as in cytoplasm and probably in endosome,¹¹⁶ which would facilitate endosomal escape of the polyplexes and cytoplasmic release of free ODN. As expected, the micelles formed by PEI with PEG-SS-ODN showed a more efficient antisense effect than ODN itself and PEG-SS-ODN in the free form, as well as the PIC micelles of PEG-ODN without the disulfide linkage. Park et al. conjugated vascular endothelial growth factor (VEGF) siRNA to PEG through a disulfide linkage. The conjugate can spontaneously form polyelectrolyte complex (PEC) micelles with polycations such as PEI, PLL or KALA, a cationic fusogenic peptide (Fig. 5).¹¹⁷ PEG conjugation of VEGF siRNA greatly enhanced its stability against serum which was further improved by the formation of PEC micelles. The PEC micelles of PEI achieved enhanced siRNA transfection efficiency compared to the polyplexes formed by the intact siRNA and PEI, especially in the presence of serum.¹¹⁸ These PEC micelles demonstrated great potential as carriers for siRNA-based cancer therapy through local or systemic administration.¹¹⁹ Recently, Kataoka et al. prepared hybrid siRNA nanocomposites using PEG-SS-siRNA and calcium phosphate. These nanocomposites exhibited reduction-triggered siRNA release and effecient gene knockdown.¹²⁰

Fig. 5 Reduction-sensitive PEC micelles formed by siRNA-SS-PEG and polycations. Adapted from ref. 117.

The conjugates of siRNA or AON with other synthetic materials through a disulfide linkage, including poly(PEG acrylate),¹²¹ polystyrene microspheres,¹²² carbon nanotubes,^123,124 and cationic penetrating peptides¹²⁵ have also been reported.

2.4. Miscellaneous reduction-sensitive gene delivery systems

While it is commonly accepted that cytoplasm is a highly reductive environment, some studies demonstrated that there are protein disulfide isomerases on the exofacial surface of cell membrane, which may be involved in endosomal compartments after endocytosis^116,126 Cleavage of the disulfide bond may occur on the cell surface and/or within the endosomes. On the basis of this assumption, Kataoka et al. prepared a novel block catiomer, PEG-SS–P(Asp(DET)), in which the PEG and polycation segments were connected by a disulfide linkage. This catiomer can condense pDNA to form PIC micelles of ∼80 nm stabilized by the PEG palisades. The reductive PIC micelles showed a significantly improved pDNA transfection efficiency, 1–3 orders of magnitude higher than the micelles without the disulfide linkage. Detachment of the PEG palisades in endosomes was thought to facilitate the endosomal escape and thus efficient gene expression, which was supported by confocal laser scanning microscopy (CLSM) observations.¹²⁷ This type of PIC micelle is expected to have potential as low-toxicity and efficient vehicles for siRNA delivery. Park et al. reported a family of reduction-cleavable hyaluronic acid (HA) nanogels (200–500 nm) that were prepared by an inverse water-in-oil emulsion approach and can physically encapsulate siRNA. The nanogels containing GFP-siRNA did not show cytotoxicity but induced significant GFP gene silencing when co-delivered with GFP plasmid/lipofectamine to HCT-116 cells that have HA-specific CD44 receptors on their surface. A unique feature of these nanogels is the excellent biocompatibility because of the absence of polycations.¹²⁸ They also developed reduction-degradable PEG/DNA nanogels. In polar organic solvent such as DMSO, a thiol-terminated six-arm PEG interacted with DNA through hydrogen bonding to form complex nanoparticles which were reversibly stabilized by in situ crosslinking through disulfide linkages. These nanogels are stable in water but can release DNA under reduced condition, having potential for local gene delivery. However, surface coating with a positively charged KALA peptide is necessary for efficient cellular uptake and transfection.¹²⁹ Other reduction-sensitive gene delivery systems including biocleavable polyrotaxane, cationic polyaspartamide derivatives, chitosan/pDNA nanoparticles, terplex systems and so forth have also been reported.^52,130,131

In a recent article, reduction-cleavable poly-siRNA ranging from 50 bps to above 300 bps was prepared by oxidative polymerization of 5,5′-dithiol-modified siRNA (mono-siRNA). Compared to the mono-siRNA, this poly-siRNA formed the more compact and stable polyplexes with low M_w PEI (1.8 kDa), and showed more efficient intracellular siRNA delivery and higher RNAi activity. Importantly, this work provides an alternative approach for cationic polymer-based siRNA delivery systems.¹³²

3. Low pH-sensitive systems

There are numerous pH gradients in physiological and pathological processes. The extracellular pH values of some solid tumor tissues and inflammation sites are in general slightly lower than in blood or other normal tissues.^133,134 It is also well known that the lower pH values are found in endosomes (∼5.5–6.0) and lysosomes (4.5–5.5).³⁷ On the basis of these discoveries, various pH/acid-sensitive polymers have been developed as carriers for pDNA, ODN or siRNA delivery.

3.1. pH-dependent endosomolytic polymers

As mentioned above, one of the critical barriers for efficient gene delivery is the endosomal escape. Viruses and some pathogenic bacteria have pH-sensitive surface proteins that change conformation in mildly acidic environments such as in endosomes, and exhibit membrane-disruptive (fusogenic or endosomolytic) properties. Synthetic fusogenic peptides that mimic the sequences of these natural proteins have been confirmed to increase cytoplasmic gene delivery.¹³⁵ In the past two decades, synthetic pH-sensitive polymers as endosomolytic agents have attracted great interest due to their low or non immunogenicity, which is a concern of using fusogenic proteins or peptides. In general, these smart polymers have both hydrophobic parts and weakly acidic groups, which afford them pH-dependent endosomolytic properties. At physiological pH, the polymers have little endosomolytic activity but undergo a conformational change at endosomal pH and show membrane-disruptive properties. This endows the polymers with reduced toxicity to the other bio-membranes at neutral pH, but with the ability to facilitate endosomal escape.^136,137 The pH-sensitivity and membrane-disruptive activity can be manipulated by finely tuning the structures and compositions of the (co)polymers.^138,139 For example, a family of alkylamine derivatives of poly(styrene-alt-maleic anhydride) copolymers have been prepared. These polymers exhibited pH-dependent membrane-disruptive activity which can be tailored by changing the alkyl chain length of the side groups, the degree of modification, and the molecular weight of the copolymer backbone.¹⁴⁰ These membrane-disruptive polymers were proven to enhance the cytoplasmic delivery of pDNA or siRNA in the forms of hybrid lipoplexes or polyplexes.^138,141,142 Another type of commonly used endosomolytic polymer is polyamines that have a strong buffering capacity around the endosomal pH. Typical examples include PEI and imidazole-containing polymers, whose endosomolytic activity is thought to be due to the “proton-sponge” effect.^143,144 Also, the hydrophilic/hydrophobic balance and the pK_a are important parameters that control the pH-dependent membrane-disruptive activities of these amino-containing polymers.¹⁴⁵ Recently, pH-dependent endosomolytic polymers were applied to coat quantum dots which can be used as fluorescently traceable siRNA nanocarriers.^146,147

3.2. Low pH-sensitive reversible shielding/masking

In general, polyplexes or nanoparticles with positively charged surfaces are preferred for gene delivery in cell culture but undesired for in vivo uses via systemic administration. Although surface modification with neutral hydrophilic polymers such as PEG (termed as PEGylation) has proven to be useful for a prolonged circulation in blood, it often results in poor transfection efficacy due to inefficient cellular uptake and/or endosomal escape. In addition, some cationic fusogenic peptides and synthetic endosomolytic polycations are cytotoxic probably due to their strong interaction with plasma membranes. In order to solve these conflicting issues, pH-sensitive reversible shielding or masking strategies have been developed through dynamic covalent linkages or non-covalent interaction.^50,148,149 For example, Walker and colleagues reported a type of targeted polyplexes with endosomal pH-triggered deshielding properties, enabled by conjugating PEG to PLL via a hydrazone linkage or to PEI via an acetal linkage. The polyplexes with the acid-sensitive linkages showed much higher plasmid gene delivery efficiencies (1–2 orders of magnitude) than those with stable linkages, both in vitro and in vivo.^150,151 Lin et al. also prepared acid-labile block copolymers of poly(2-(dimethyl amino)ethyl methacrylate) (PDMAEMA) and PEG with a cyclic orthoester linkage by atom transfer radical polymerization. The acid-sensitive polyplexes formed from this polymer showed greatly enhanced transfection efficacy after being treated in the medium of endosomal pH (Fig. 6). A unique point of these copolymers is that the cationic chain length can be easily controlled by tuning the ratio of initiator to monomer.¹⁵² Recently, acid-triggered dePEGylation strategies based on covalent linkages were also applied by other scientists for pDNA/siRNA delivery^153,154 or polycationic micelles.¹⁵⁵

Fig. 6 Schematic illustration of the pH-sensitive PEG-shielded polyplexes.¹⁵²

Since the acid-sensitivity is not high enough to respond to such a small pH drop like those occurring in tumor tissues, the present PEGylated polyplexes with dynamically covalent linkages are designed mainly on the basis of the endo/lysosomal pH triggering. By contrast, Bae et al. reported a ternary gene delivery system that effectively targeted the acidic extracellular matrix of tumors. The polyplexes formed by PEI and pDNA were coated electrostatically with an ultra pH-sensitive diblock copolymer, poly(methacryloyl sulfadimethoxine)-b-PEG. These PEG-shielded nanoparticles showed a drastic change, in terms of cytotoxicity and transfection efficiency, when the pH values dropped from 7.4 (normal tissue) to 6.6 (tumor). Although this system is expected to be applicable for siRNA delivery to tumors, possible polyelectrolyte exchanges have to be considered in the coating process because of the smaller molecular weight of siRNA and its weaker interaction with polycations compared to those of plasmids.^156,157 Furthermore, they developed a biodegradable pH-sensitive diblock copolymer (poly(L-cystine bisamide-g-sulfadiazine)-b-PEG) which is thought to have potential as a coating material for positively charged polyplexes or other nanoparticles. In a recent review paper, various extracellular nanocarriers that are designed specifically for tumor targeted delivery systems have been demonstrated on the basis of mildly lower pH and overproduction of enzymes, such as matrix metalloproteinases in most solid tumor tissures.¹⁵⁸

To reduce cytotoxicity but retain the endosomolytic activity of some fusogenic peptides or membrane-disruptive polymers, Rozema and his colleagues demonstrated a novel concept of “endosomolysis by masking of a membrane-active agent (EMMA)” by reversibly modifying melittin using a maleic anhydride derivative.¹⁵⁹ Similar approaches have been applied for other pDNA or siRNA delivery systems.^160–162

3.3. Acid-labile polycations

Most acid-labile polycations used in this context are derivatives of PEI with different molecular weights. In a recent report, branched PEI with low and high M_ws were partially conjugated with acid-cleavable primary amine-bearing ketals. The purpose of the incorporation of ketal linkages is to reduce the stability of the polyplexes in endosomes and to facilitate the endosomal escape and release of the nucleic acids. Ketalization of high-M_w PEI significantly reduced cytotoxicity while enhancing transfection efficiency.¹⁶³ Further experiments revealed that the ketalization ratio, M_w and topology of PEI all had a great effect on the nucleic acid binding/condensation capability, pDNA transfection efficiency and RNAi activity, which were associated with the intracellular localization of the nucleic acid polyplexes.^71,164 Although the detailed intracellular mechanism is not completely elucidated, these results imply that finely tailored polymeric gene carriers with different structures are needed to selectively deliver pDNA and siRNA to the correct sites of action.

Crosslinking of low-M_w PEIs is another way to prepare acid-sensitive degradable polycations. Wagner et al. synthesized two types of acid-degradable polycations by crosslinking PEI (800 kDa) with linkers containing ketal and acetal linkages.⁸³ The polyplexes made of these acid-degradable polycations showed improved toxicity, both in vitro and in vivo, compared to those of the acid-stable polycations, demonstrating the important role of the acid-labile linkages. Acid-labile PEI derivatives with imine linkages showing lower cytotoxicity than PEI (25 kDa) were also prepared from PEI (1.8 kDa) with glutadialdehyde as a linker.⁸²

3.4. Low pH-sensitive siRNA/ODN–polymer conjugates

Low-pH sensitive conjugates of ODN and PEG were independently prepared by Nagasaki et al. using a β-thiopropionate linkage¹⁶⁵ and by Park et al. using a phosphoramidate linkage.¹⁶⁶ These conjugates can form PIC (or PEC) micelles with cationic polymers or peptides. In addition, lactose or folate as a ligand has been incorporated into the telechelic PEG end of the respective conjugate. The micelles formed from these ligand-containing conjugates and cationic polymers (PLL) or lipid (lipofectamine) showed an efficient antisense effect against plasmid gene expression in the cell lines with lactose- or folate-receptor.^167,168 siRNA was also conjugated to PEG which has a telechelic lactose group, via an acid-labile β-thiopropionate linkage. The PIC micelles formed by Lac-PEG-siRNA conjugates with PLL showed much more efficient RNAi activity compared to siRNA or PEG-siRNA alone in human hepatoma (HuH-7) cells. The presence of the acid-labile linkage and ligand, and the formation of PIC micelles were confirmed to be important for the efficient siRNA delivery.¹⁶⁹ When applied to multi-cellular HuH-7 spheroids, a system used as an in vitro three-dimensional model that mimics the in vivo biology of tumors, the acid-labile Lac-PEGylated PIC micelles showed an appreciable growth inhibition of the spheroids for up to 21 days.¹⁷⁰ Recently, Mahato and coworkers also reported a ³³P-labeled PEGylated conjugate of ODN with a galactose moiety at the telechelic end of PEG and an acid-labile linkage between PEG and the ODN, Gal-PEG-³³P-ODN.¹⁷¹ In contrast to Nagasaki's work, cationic polymers or lipids were not used in order to avoid the possible cytotoxicity. Following tail vein injection in rats, after 30 min, ∼60% of the injected conjugate, being much higher than ³³P-ODN itself, was accumulated in liver.

4. Thermoresponsive systems

In the past several decades, thermoresponsive synthetic and natural polymers have been extensively investigated due to their potential applications in various fields including tissue engineering and smart drug delivery systems. Regarding gene delivery, the first family of thermoresponsive cationic polymer gene carriers was prepared by Hennink et al. through copolymerization of DMAEMA and N-isopropylacrylamide (NIPAM).¹⁷² They found that the stability, cytotoxicity, and transfection efficiency of the polyplexes were significantly affected by the composition and molecular weight of the polymers. However, the effect of temperature on the cytotoxicity and transfection efficiency was not demonstrated. Okano and colleagues also reported a thermoresponsive copolymer which was prepared by copolymerization of DMAEMA, NIPAM and butyl methacrylate.¹⁷³ This copolymer was capable of condensing pDNA and the polyplex formation/dissociation was dependent on temperature. Cooling treatment at the temperature below the LCST of the copolymer during the transfection procedure greatly enhanced its transfection efficiency. Although detailed mechanism of this cooling-induced enhancement was not clear, this work demonstrated a new concept for controlling gene expression by temperature through a thermoresponsive polymer-based gene delivery system. In recent years, a number of other thermoresponsive cationic polymers were prepared and studied as gene carriers.⁵¹ Most of these polymers contain PNIPAM or its derivatives as the thermosensitive segment, and the cationic segment including PLL,¹⁷⁴ PEI,^51,175 chitosan or a derivative,^176,177 and polyarginine.¹⁷⁸ Park et al. reported another temperature-sensitive gene delivery system. They prepared hydrogel nanoparticles by crosslinking activated Pluronic^® with PEI, through a modified emulsification/solvent evaporation method. These nanoparticles showed a thermally reversible swelling/deswelling profile. When the temperature was decreased from 37 °C to 20 °C, the particle volumes drastically increased with 15–40 swelling ratios which could induce endosomal disruption.¹⁷⁹ When the nanoparticles were used for in vitro siRNA delivery, their RNAi activity was significantly increased by cold-shock treatment (Fig. 7).¹⁸⁰

Fig. 7 Schematic demonstration of thermosensitive nanogels for siRNA delivery. Adapted from ref. 180.

Thermoresponsive and YSA peptide-functionalized nanogels with a core–shell structure were prepared by precipitation polymerization of N-isopropylmethacrylamide (NIPMAM), and applied to effectively deliver siRNA into the cytosol of ovarian carcinoma cells in a receptor-mediated way. The nanogels have a high siRNA-loading capacity and can slowly release the active payload, which is particularly promising for systemic delivery of siRNA. Because the experiments of both siRNA loading and release were carried out at 37 °C which is lower than the phase transition temperature of PNIPMAM (∼43 °C), it is not clearly demonstrated why the siRNA-loaded nanogels release the water-soluble payload with a slow rate.¹⁸¹

Although some of the aforementioned thermoresponsive systems showed temperature-dependent transfection behavior, in all cases an increased plasmid expression or RNAi activity was found only by the cold-shock treatment, i.e., incubation at a lower temperature (< LCST) for several hours during the transfection. However, the cold-shock treatment is not easy to apply to practical in vivo uses. Recently, Wagner et al. reported that temperature-sensitive cationic copolymers could be used for a gene delivery system whose transfection efficiency was enhanced upon heating.¹⁸² The copolymers consist of a PEI block and a NIPAM-derived thermosensitive block with a cloud point of ca. 40 °C. These copolymers can condense pDNA to form polyplex nanoparticles of 150–200 nm with a neutral and thermoresponsive corona. When injected via the tail vein of A/J mice, significantly enhanced accumulation of the polyplexes and gene expression in the hyperthermia-treated tumor sites.¹⁸³ This work implies that other nucleic acids such as siRNA can also be site-specifically delivered by a combination of hyperthermia and thermoresponsive polymers. Furthermore, if magnetic nanoparticles are incorporated into these thermoresponsive gene delivery systems, hyperthermia can be achieved using an alternating current magnetic field, which may enable the delivery systems to function in the remote disease sites of human body.¹⁸⁴

5. Other stimuli-responsive gene delivery systems

Light is a convenient stimulus for spatially and temporally controlling drug delivery and gene expression (or knockdown by siRNA/ODN). The concept of photodynamic therapy (PDT) was proposed a century ago and gradually developed into a clinical modality for curing various diseases such as cancer and age-related macular degeneration.¹⁸⁵ Photochemical internalization (PCI), a novel technology as a branch of PDT, has been developed specifically to promote the release of macromolecular therapeutic agents such as toxins, pDNA in the form of a polyplex or lipoplex, and so forth, from endosomes into cytoplasm.¹⁸⁶ In recent years, PCI technology was also utilized for siRNA delivery.¹⁸⁷ The basic principle of PCI is the light-induced activation of a photosensitizer, which causes disruption of the endosomal or lysosomal membranes.

Another approach to manipulate DNA delivery is to use photolabile linkages. Kostiainen et al. reported an optically triggered DNA release system that is based on multivalent dendrons with photoremovable spermine groups attached to the periphery through an o-nitrobenzyl linkage. The dendrons were capable of binding DNA which can be released due to the disappearance of the multivalent electrostatic interaction upon light irradiation (∼365 nm). However, cell-related experiments were not performed in this work.¹⁸⁸ Similar photochemistry was also applied for the development of photo-labile gold and silica nanoparticles, of which the positively charged surface was converted to a negatively charged one upon near-UV irradiation (∼365 nm).^189,190 The gold nanoparticles with a positively charged surface were successfully used to deliver DNA into a cell nucleus in a light-controlled fashion. In principle, these cationic dendrimers or nanoparticles can also be used for light-sensitive siRNA delivery. Recently, gold nanoshell–siRNA conjugates which can be used for temporal and spatial control of RNAi have been developed. The TAT-lipid coating layer on the outside of the gold core promoted cellular internalization, while detachment from the gold surface and escape from endosomes of the free siRNA were triggered by pulsed near-infrared laser irradiation.¹⁹¹

Magnetic field and ultrasound have also been widely used for gene deliveries, including pDNA, ODN, and siRNA.^192–196 However, these intelligent delivery systems depend on the external physical stimulus, and in principle are not necessarily relevant to stimuli-responsive polymers, as such we do not discuss these systems in detail here. There are also several papers that report enzyme-responsive, polymer-based gene carriers for pDNA and ODN delivery.^197–199

6. Multi-functional polymers for siRNA delivery

Polymer-based in vivo siRNA delivery, in particular via systemic administration, is a complex process and there are a number of biological barriers, of which some are conflicting to overcome. Recently, bio-mimicking polymer-based siRNA vehicles that combine multiple functions targeting to different barriers have attracted great interest. Stayton et al. developed a new class of multi-functional drug/nucleic acid vehicles termed ‘encrypted polymers’ (Fig. 8A). These polymers consist of a hydrophobic, membrane-disruptive backbone onto which PEG chains are attached via acid-labile acetal linkages. Targeting ligands such as lactose are covalently conjugated to the telechelic end of some PEG chains. Therapeutic agents such as peptides or oligonucleotides can be covalently or electrostatically attached to the PEG chain ends. The membrane-disruptive activity of the encrypted polymers is weak due to the masking effect of the PEG chains, which may reduce toxicity. After receptor-mediated endocytosis, the polymer backbone is unmasked at the endosomal pH and becomes membrane-disruptive, allowing cytoplasmic delivery of the therapeutic agents.²⁰⁰ By a similar strategy, Rozema and colleagues reported a kind of dynamic polyconjugates for siRNA delivery to hepatocytes both in vitro and in vivo. In these polyconjugates, oligo PEG as a shielding agent and N-acetylgalactosamine as a hepatocyte targeting ligand were attached to an amphipathic poly(vinyl ether) (PBAVE) backbone through acid-labile maleamate linkages. The backbone has membrane-disruptive properties. siRNAs targeting ApoB or peroxisome proliferator-activated receptor α (Pparα) were conjugated to the end of the PBAVE backbone via a reducible disulfide linkage (Fig. 8B). Following a single intravenous injection of the siRNA-containing polyconjugates in mice, effective gene silencing of ApoB or Pparα was demonstrated with minimal toxicity and inflammatory response.¹⁶² The pH-sensitive masking concept was further applied to prepare other multi-functional cationic polymer-based siRNA vehicles. PEI or PLL were modified with PEG and DMMAn-protected melittin peptide. The functionalized polycations can effectively bind siRNA to form small polyplexes, and show much greater siRNA delivery efficiency in vitro than PEI or PLL.¹⁶¹ In order to develop stable polyplexes that do not dissociate in the extracellular environment and have potential for in vivo systemic delivery, siRNA was further conjugated onto the modified PLL backbone through a disulfide linkage. The extracellular stability of the siRNA-containing conjugates against polyelectrolyte exchange of heparin was significantly improved compared to the electrostatic polyplexes. Good in vitro biocompatibility and efficient gene silencing was also found. Unfortunately, when this multi-functional siRNA-conjugate was applied in vivo in mice through intravenous or intratumoral administration, high toxicity was observed. Aggregation of the conjugate due to incomplete PEG shielding and lack of targeting ligand may account for this in vivo toxicity.²⁰¹

Fig. 8 Multi-functional vehicles for deliveries of drug and nucleic acids.^162,200

Recently, a new class of polymerizable peptide-based surfactants containing a basic amino head group, two cysteine residues, and two lipophilic tails has been developed as siRNA carriers. A unique feature of these surfactants is their pH-sensitive membrane-disruptive properties. The surfactants can form compact nanoparticles of 160–260 nm with siRNA at appropriate N/P ratios through first complexation followed by in situ oxidative polymerization of dithiols.²⁰² Surface modification of the nanoparticles by PEG with a RGD or bombesin peptide as ligand at the telechelic chain end afforded a novel multi-functional siRNA delivery system, showing low toxicity and high in vitro and in vivo (systemic injection) RNAi efficiency.²⁰³ Agrawal et al. developed a new type of multi-functional siRNA vehicles called “dendriworms” composed of magnetofluorescent nanoworms on which the 4^th-generation PAMAM dendrimers were conjugated via a disulfide bond. These dendriworms can bind and protect siRNA through electrostatic interaction, and effectively deliver siRNA into cells in vitro or in vivo by slow intracranial infusion within the CNS tissues of mice. Their magnetofluorescent features make the dendriworms easy to track in cells or in vivo using different imaging techniques. However, at the present stage, the siRNA-loaded dendriworms are not suitable for systemic delivery, probably due to their positively charged surface. In addition, the authors did not demonstrate how the siRNA-loaded dendriworm enters cells: as a whole worm or in the form of an individual magnetic nanoparticle.²⁰⁴ Multi-functional magnetic nanoparticles composed of an RGD ligand, a fluorescent dye, PEG chains and therapeutic siRNA have also been reported, showing potential as siRNA vehicles for future in vivo cancer therapy.²⁰⁵

7. Conclusion and perspectives

Despite significant advances in the past two decades, gene therapy is still in the stage of clinical trials worldwide mainly due to the lack of safe and efficient delivery vehicles for therapeutic nucleic acids. Among the various attempts of driving gene therapy to real clinical application, polymer-based nucleic acid delivery systems, including the intelligent ones, play an important role, especially for the exciting RNAi-based gene therapy.

As mentioned in the previous sections, there are some conflicting demands for overcoming different extra- and intracellular barriers met by nucleic acid-loaded nanoparticles during delivery. The delivery systems, which response to only one stimulus and have less functionality, may not be efficient enough to achieve a satisfactory therapeutic effect in vivo. The development of virus-mimicking, multi-functional gene delivery systems is considered to be a practicable strategy in the future, in particular for intravenous administration. Ideal polymer-based, nucleic acid-loaded nanoparticles for potential in vivo applications should have several components that function at the appropriate stages during the delivery (Fig. 9).^22,34 The ligands on the particle surface can be used to recognize a specific cell/tissue, and facilitate cellular uptake through receptor-mediated endocytosis. A reversibly removable hydrophilic palisade, such as PEG chains, provides stealth protection of the nanoparticles during their circulation in the blood as well as travelling through the ECM, and may reduce toxicity. Upon some stimuli such as changes in pH and reduction potential, or light irradiation, the palisade can be removed, which might enhance the cellular uptake and/or endosomal escape. In the inner part of the nanoparticles, nucleic acids can be temporally loaded through electrostatic interaction, covalent conjugation, or physical encapsulation, which will protect the nucleic acid against enzymatic degradation. The inner part should be stable enough until delivery to the correct site (cytoplasm and/or nucleus) where it is disassembled to release the naked nucleic acid upon some kind of stimulus. In addition, it is preferred that the inner part contains endosomolytic components that help the endosomal escape. If cell penetrating peptide (CPP) is incorporated onto the surface of the inner part, cellular uptake may be further improved. Besides the therapeutic nucleic acids, in some cases, other drug and/or imaging reagents can also be incorporated into the nanoparticles, which may improve the therapeutic efficacy or help us to understand the delivery mechanism.^33,206 However, acquiring such an ideal and sophisticated nucleic acid delivery system is still far off. Many challenges remain to be overcome for future clinical approval of gene therapy, including polymer-based delivery systems.

Fig. 9 Ideal virus-mimicking multi-functional nanoparticles for nucleic acid delivery.

Although the experiences obtained from pDNA do provide useful information, different designs or structural modifications of the carriers are necessary for the development of polymer-based intelligent siRNA delivery systems. For example, siRNA and pDNA differ in terms of molecular weight, rigidity, as well as action site, the optimized structure of some polycations suitable for pDNA condensation and delivery may not be appropriate for siRNA. It is still necessary to do the basic research that helps to elucidate the structural effects of polymers, such as molecular weight, topology, cationic density and so forth, on their siRNA/pDNA packing/unpacking capability, the stability and physicochemical properties of the formed polyplex nanoparticles. In this regard, novel synthetic methodologies in organic and polymer chemistry such as “click” chemistry, combinatorial chemistry, controlled radical polymerization, and so forth, will facilitate the development of polymers with well-defined structures. It is generally considered that siRNA functions in cytoplasm while pDNA has to enter the nucleus for effective gene expression. In addition, it has been reported that free pDNA travels with difficulty within the cytoplasm.²⁰⁷ Thus, reduction-sensitive systems which can release the nucleic acid payload in cytoplasm may be preferred for siRNA delivery.

Issues of off-targeting effect and possible triggering of the innate immune system have to be carefully considered before siRNA-based gene therapy goes to clinical usage. Unmodified siRNAs are reported to activate the mammalian immune system²⁰⁸ and/or exhibit off-target gene regulation.^209,210 Although chemical modification of siRNA may avoid off-targeting effects²¹¹ and/or reduce its immunostimulatory activity,²¹² global gene expression and immune response are still significantly influenced by siRNA–lipoplexes or cationic liposomes.²¹³ Polymer-based siRNA vehicles that can release the naked siRNA in a controlled and programmable fashion may be useful to solve these problems. In addition, anti-PEG IgM was produced after intravenous injection of PEG-coated liposomes or siRNA–lipoplexes, which implies that more attention should be paid to other hydrophilic shielding polymers such as poly(N-vinyl pyrrolidone) and poly(oligo(ethylene glycol) methacrylate).²¹⁴

Considering the complex process during nucleic acid delivery, it is difficult to clearly clarify all the factors that play the key roles in successful gene therapy in the coming several years. In this regard, combinatorial or high-throughput approaches provide a very attractive strategy to drastically shorten the development cycles by rapidly screening various vehicles, from the synthesis of non-viral carriers with various chemical structures to the physiochemical and biological evaluation of these carriers or the corresponding nucleic acid-containing formulations.^22,215,216

Regarding in vivo applications, polymeric materials used with the nanoparticles have to be biocompatible. Not only acute (cyto)toxicity and inflammatory response, but also long-term pharmacological and toxicological effects of the materials have to be taken into account. Biodegradable and/or naturally occurring polymers are the preferable candidates for fabricating such intelligent, multi-functional nucleic acid delivery systems. In addition, non-cationic polymeric nanogels or nanoparticles with low toxicity may provide the alternatives as carriers, in particular for siRNA delivery.

Abbreviations used in this article

AON	antisense oligonucleotide
ApoB	apolipoprotein B
CHO-K1 cell	Chinese hamster ovary K1 cell
CLSM	confocal laser scanning microscope
CNS	central nervous system
CPP	cell penetrating peptide
DMAEMA	N,N-dimethylaminoethyl methacrylate
DMMAn	2,3-dimethylmaleicanhydride
dsRNA	double-stranded RNA
ECM	extracellular matrix
EGFP	enhanced green fluorescent protein
HA	hyaluronic acid
HCT116 cell	human colorectal carcinoma cell
HuH-7 cell	human hepatoma cell
IgM	Immunoglobulin M
KALA	a fusogenic polypeptide with the sequence of WEAK LAKA LAKA LAKH LAKA LAKA LKAC.
LbL	layer-by-layer
LCST	lower critical solution temperature
LHRH	Luteinizing Hormone-Releasing Hormone
NADPH	nicotinamide adenine dinucleotide phosphate
NIPAM	N-isopropylacrylamide
NIPMAM	N-isopropylmethacrylamide
NLS	nuclear localization sequence
ODN	oligodeoxynucleotide
PAA	poly(amido amine)
P(Asp)	poly(α,β−aspartic acid)
PBAVE	a random copolymer of butyl vinyl ether and aminoethyl vinyl ether
PCI	photochemical internalization
pDNA	plasmid DNA
PDT	photodynamic therapy
PEC	polyelectrolyte complex
PEG	poly(ethylene glycol)
PEI	poly(ethylenimine)
PIC	polyion complex
PLL	poly(L-lysine)
Pparα	peroxisome proliferator-activated receptor α
PPI	poly(propylenimine)
RNAi	RNA interference
RPC	reducible polycation
SCID	severe combined immunodeficiency
siRNA	small interfering RNA
TAT	a peptide with the sequence of GRKKRRQRRRG, being amino acid residues 47–57 of the transactivating transcriptional activator protein from HIV-1
VEGF	vascular endothelial growth factor

Acknowledgements

We thank the National Natural Science Foundation of China (20874001, 20474005 & 20534010) and Nitto Denko Technical Corp. for financial support.

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