Major degradable polycations as carriers for DNA and siRNA.

Journal of Controlled Release 193 (2014) 74–89

Contents lists available at ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Review

Major degradable polycations as carriers for DNA and siRNA Mohammad Ariful Islam a,b,c,1, Tae‐Eun Park b,1, Bijay Singh b, Sushila Maharjan b, Jannatul Firdous b,c, Myung-Haing Cho d, Sang-Kee Kang e, Cheol-Heui Yun b,c, Yun‐Jaie Choi b, Chong-Su Cho b,⁎ a

Brigham and Women's Hospital & Harvard Medical School, Boston, MA 02115, USA Department of Agricultural Biotechnology & Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, South Korea Center for Food and Bioconvergence & World Class University Biomodulation Major, Seoul National University, Seoul 151-742, South Korea d Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea e Institute of Green-Bio Science and Technology, Seoul National University, Pyeongchanggun, Gangwondo 232-916, South Korea b c

a r t i c l e

i n f o

Article history: Received 25 February 2014 Accepted 27 May 2014 Available online 3 June 2014 Keywords: Degradable polymer Polyethylenimine Polyamidoamine Cyclodextrin DNA siRNA

a b s t r a c t Non-viral gene delivery systems are one of the most potential alternatives to viral vectors because of their less immunogenicity, less toxicity and easy productivity in spite of their low capacity of gene transfection using DNA or silencing using siRNA compared to that of viral vectors. Among non-viral systems, the polycationic derivatives are the most popular gene carriers since they can effectively condense nucleic acids to transfer into the cells, especially the polyethylenimine (PEI) which has been used as a golden standard polymer owing to its high buffering ability for endosomal escape of gene to be expressed. However, PEI has severe problems for its toxicity due to the high positive charge density and non-degradability although the toxicity of PEI depends on its molecular weight (MW) and structure. Therefore, a considerable attention has been paid on synthesis of degradable PEI derivatives using low MW one because low MW PEI is much less toxic than high MW PEI. Other degradable polycationic gene carriers such as polyamidoamines (PAA) and cyclodextrin (CD)-based polycations are also in a significant interest because of their high transfection efficiency with low toxicity. This review in detail explains the recent developments on these three major degradable polycations as promising carriers for deoxyribonucleic acid (DNA) and small interfering RNA (siRNA). © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . Degradable PEI derivatives and their linkages . . . 2.1. Ester linkage . . . . . . . . . . . . . . 2.1.1. Linear PEI . . . . . . . . . . . . 2.1.2. Branched PEI . . . . . . . . . . 2.1.3. Grafted PEI . . . . . . . . . . . 2.2. Disulfide linkages . . . . . . . . . . . . 2.2.1. Main chain . . . . . . . . . . . 2.2.2. Side chain . . . . . . . . . . . 2.3. Imine linkage . . . . . . . . . . . . . . 2.4. Carbamate linkage . . . . . . . . . . . . 2.5. Amide linkage . . . . . . . . . . . . . . 2.6. Ketal linkage . . . . . . . . . . . . . . Degradable PAA derivatives and their characteristics 3.1. Charge density . . . . . . . . . . . . . . 3.2. Density of disulfide linkages . . . . . . . 3.3. Architectural features . . . . . . . . . .

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⁎ Corresponding author. Tel.: +82 2 880 4868; fax: +82 2 875 2494. E-mail address: [email protected] (C.-S. Cho). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jconrel.2014.05.055 0168-3659/© 2014 Elsevier B.V. All rights reserved.

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3.4. Cell penetrating peptide (CPP)-based PAA . . . . 3.5. PEGylated PAA . . . . . . . . . . . . . . . . 3.6. Boronic acid-modified PAA . . . . . . . . . . . 3.7. Specific ligand-conjugated PAA . . . . . . . . . 4. Cyclodextrin (CD)-based polycations and their properties 4.1. Stability . . . . . . . . . . . . . . . . . . . 4.2. Transfection activity and cellular uptake . . . . . 4.3. Endosomal escape capacity . . . . . . . . . . . 4.4. Cell specificity . . . . . . . . . . . . . . . . . 4.5. Dual delivery of biologics . . . . . . . . . . . . 5. Concluding remarks and future perspectives . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Gene therapy is one of the promising approaches in treating a variety of inherited and acquired diseases but the therapeutic potential of gene therapy has been limited by the lack of efficient gene delivery system [1]. The majority of the gene therapy researches have been relied on viral vectors due to high efficiency of gene transfer in vivo. However, the viral vectors raise safety concern due to severe off-target immunogenicity, inflammatory response and toxicity [2]. Therefore, non-viral gene delivery systems have been extensively explored as alternative gene carriers owing to their less immunogenicity and less toxicity than viral vectors. However, most of the non-viral vectors are less efficient for gene transfer, especially in clinical trials. Among non-viral vectors, cationic polymers have been increasingly applied due to several advantages such as their versatility, easy selection of appropriate gene size, introduction of specific targeting ligands and easy production. Among cationic polymers, PEI has been used as a ‘state-of-the-art’ polymer in vitro and in vivo due to its high buffering ability for endosomal escape of the delivered gene [3]. However, PEI has several disadvantages such as non-degradability, high cytotoxicity and aggregation of polyplexes during blood circulation in vivo. Moreover, the cytotoxicity of the PEI depends on the MW, structure and concentration of the PEI [4]. Elucidation of relationship between physicochemical properties such as degradability, charge density, solubility, MW, crystallinity, hydrophobicity, rigidity and pKa value of the cationic polymers and function of gene is very important for the application of these gene carriers in clinical trials. Among them, degradability of the polymeric carriers is the most critical factor because it reduces cytotoxicity by degrading the polymers into small MW molecules which are easily eliminated by in vivo excretion pathway. Moreover, it increases the transfection efficiency of DNA or gene silencing capacity of siRNA by unpacking the polymer/DNA and/or polymer/siRNA complexes, respectively, in order to release the genes inside the cells [5]. Therefore, many studies have been focused on the preparation of degradable PEIs (also ligand-conjugated degradable PEIs) which consisted of low MW PEIs and crosslinkers through degradable linkages as shown in Scheme 1. It is also worthy to mention that few PEI derivatives such as jetPEI [6] and one linear PEI [7] are recently being in clinical trials which offer their promising practical application in the future. The jetPEI is also widely used as a commercial transfection agent as a positive control because of their high transfection efficiency with less toxic effect [6]. On the other hand, the linear PEI which is under phase I clinical trial has been studied in advanced and/or metastatic pancreatic cancer patients. The trial has been intended to administer increasing doses of the PEI-complexed DNA encoding two genes such as somatostatin receptor subtype 2 and deoxycitidine kinase with uridylmonophosphate kinase. The result of the study showed complementary therapeutic effects, although the work has not been completed and the final results are yet to be reported.

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Other major degradable polycations such as PAAs [8] and CD-based polymers [9] have also been considered potential candidates for efficient delivery of DNA and siRNA. Various properties such as charge density, density of degradable linkages, architectural features, PEGylation and conjugation of specific ligands or cell penetrating peptides can significantly affect the efficacy of PAAs for nucleic acid delivery. On the other hand, several factors such as stability, cellular uptake and transfection activity, endosomal escape capacity, cell specificity and dual delivery capacity are required when designing effective CD-based polymers. In this review, we have elaborately described the above three polycationic polymers and their degradable derivatives for efficient delivery of nucleic acids such as DNA and siRNA. Firstly, the degradable PEI derivatives prepared by using several degradable linkages are described with their design and synthesis methods in detail. The degradable PEIs are classified and described based on linkages into ester, disulfide, imine, carbamate, amide and ketal linkages. Secondly, various degradable PAA derivatives and their characteristics are explained based on various structural features of the linkages and their potential modification to improve specificity, transfection ability or circulation time. Thirdly, the usefulness of degradable CD-based polymers and their structural as well as functional properties are elaborately focused. Finally, this review offers several suggestions as a comprehensive future direction on this promising field of gene therapy in order to translate the bench experiments toward the bed-side clinical applications.

2. Degradable PEI derivatives and their linkages 2.1. Ester linkage The degradable PEIs with ester linkages can be classified into linear, branched and grafted according to the structural differences.

Scheme 1. A schematic representation of designing ligand-conjugated degradable PEIs.

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2.1.1. Linear PEI The degradable linear PEIs with ester linkage can be simply prepared by Michael addition reaction between linear low MW PEI and diacrylate compound as cross-linker. The ester linkages are easily degraded through hydrolysis mechanism. In general, the degradable linear PEIs have high transfection efficiency (or gene silencing ability) with low cytotoxicity due to degradation of the ester linkages and pH buffering capacity. The degradable linear PEI with ester linkage was firstly prepared by the reaction of 1,3-butanediol diacrylate as a cross-linking agent with low MW PEI (800 Da) [10]. Half-life, the amount of time required to get half molecular weight from initial molecular weight of gene carrier obtained by gel permeation chromatography, is a convenient index which represents the degradation rates of polymers. The half-life of the polymer was 4 h due to rapid hydrolysis of the ester linkages at physiological condition. Gene transfection efficiency of the degradable linear PEI in MDA-MB-231 cells was 9-fold higher than PEI 25 kDa with much reduced cellular toxicity [10]. Another degradable linear PEIs have been synthesized by Michael addition reaction between linear low MW PEI (423 Da) and poly(ethylene glycol) (PEG) diacrylate with three different MW (285, 575 and 700 Da) as shown in Fig. 1 [11]. The MW of copolymer ranged from 7980 to 12,860 Da depending on the reaction conditions. The half-life of the copolymer (MW of PEG: 575 Da) was 8 h, suggesting a rapid degradation at physiological condition primarily due to its hydrophilic property. Interestingly, the MW of PEG significantly affected the transfection efficiency in various cells due to the shielding effect of the PEG part in the copolymer, where the shielding effect of PEG increased with the increase of MW of PEG. They also compared transfection efficiency of the gene carrier between aerosol administration and intravenous (IV) (a systemic delivery) in mice [12]. Interestingly, aerosol route showed much higher transfection efficiency in lungs than IV due to easy access to pulmonary tissues by aerosol delivered first-pass effect. Recently, Islam et al. prepared poly(amino ester) (PAE) based on linear low MW PEI (MW: 423 Da) and sorbitol dimethacrylate having hyperosmotic property as cross-linker as shown in Fig. 2 [13]. The polymer/DNA complexes showed excellent stability in the presence of serum and after lyophilization compared to that of PEI/DNA complexes due to the presence of many hydroxyl groups in the polymer backbone, which was also responsible for low surface charge of the polyplexes even at higher N/P ratios. Accordingly, the polymer showed higher transfection efficiency than PEI 25 kDa due to synergistic effect of osmotic active property by polysorbitol chain and buffering capacity by PEI backbone. The same carrier was used to achieve therapeutic RNA interference in vitro mediated by osteopontin (OPN) siRNA [14] as the OPN plays an important role in tumor angiogenesis [15]. These studies demonstrated that the carrier accelerated the DNA and siRNA delivery better than that of PEI 25 kDa due to caveolin-1 and cyclooxygenase-2mediated caveolae-dependent endocytosis by osmotic active property of the polysorbitol gene carrier.

2.1.2. Branched PEI The degradable branched PEIs prepared by branched low MW PEI and diacrylate have merits over the linear ones because they are effective for condensation and protection of gene due to their high amine density. The degradability of the branched PEIs is low compared to linear type due to less water accessibility in their branched structure for hydrolysis of ester linkages [16]. Forrest et al. [10] prepared degradable branched PEIs by reaction of 1,6-hexanediol diacrylate as a cross-linker with the primary and secondary amine groups in low MW PEI (800 Da). The synthesized degradable PEI had a high MW of 14 kDa due to highly branched product. The carrier expressed 2- to 16-fold higher transfection efficiency in MDAMB-231 cells than that of PEI 25 kDa. Degradable branched PEI was also prepared by reaction of hexanediol diacrylate with low MW PEI (800 Da) through polylibrary technique [17]. The carrier showed higher transfection efficiency in Neuro2a and BI6F10 cells than that of linear PEI (22 kDa) with hemocompatibility. However with long term reaction, ester linkages are converted into amide bonds by inter- and intra-molecular ester aminolysis. This study further showed that an elevated reaction temperature such as 60 or 80 °C provided mixed ester and amide linkages which provided a slow degradation of the polymer [18]. Kim et al. prepared degradable branched PEI based on low MW poloxamer (2500 Da) diacrylate and low MW PEI (600, 1200 and 1800 Da) as shown in Fig. 3 [19]. The obtained polymer had high MW of 83 kDa with a characteristic of slow degradation owing to the highly branched product. The polymer showed much higher transfection efficiency with a reduced cytotoxicity than that of PEI 25 kDa. Arote et al. prepared degradable branched PEIs by Michael addition reaction between low MW branched PEIs (600, 1200 and 1800 Da) with hydrophobic polycaprolactone (PCL) diacrylate (Fig. 4) [20]. The half-life of the polymer was 4.5 to 5 days, which is longer than that of degradable

Fig. 1. Reaction scheme for the formation of degradable PEI-alt-PEG copolymers [11].

Fig. 3. Scheme of degradable, hyperbranched poly(ester amine)s (PEAs) based on poloxamer diacrylate and low molecular weight branched PEI [19].

Fig. 2. Schematic illustration on the design of polysorbitol or polymannitol gene transporter based on LMW PEI and sorbitol or mannitol backbone as a form of diacrylate or dimethacrylate cross-linking chain. Different parts of the transporters show different functional properties [13].


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Fig. 4. Scheme of degradable poly(ester amine) based on polycaprolactone and polyethylenimine by Michael addition [20].

linear PEI-alt-PEG due to the hydrophobic property of PCL. The transfection efficiency of the polymer was 15–20 fold higher than that of PEI 25 kDa. Due to slow degradation of the hydrophobic PCL, the cytotoxicity of the polymer should be further examined with repeated injection in vivo. The same group synthesized another derivative of degradable branched PEI based on glycerol dimethacrylate with hyperosmotic property as cross-linker and low MW branched PEI (1200 Da) as shown in Fig. 5 [21]. The half-life of the polymer was 9–10 days, probably due to the polymeric hyperbranched structure. Impressively, the polymer elucidated much higher transfection efficiency than PEI 25 kDa, indicating the synergistic effect of buffering capacity by PEI and hyperosmotic property by polyglycerol with enhanced in vivo gene transfection efficiency after aerosol administration. Similar tendency of high transfection efficiency was obtained by the degradable branched PEI based on glycerol triacrylate and low MW PEI (1200 Da) [22]. Yu et al. prepared degradable branched PEIs based on PEG dimethacrylate (550 Da) and low MW (600, 1200 and 1800 Da) PEIs [23]. The half-life of the polymer was longer at pH 5.6 than that at pH 7.4. Moreover, the polymer exhibited comparable transfection

Fig. 5. Scheme of poly(ester amine)s based on glycerol dimethacrylate and low molecular weight PEI. Dimethacrylate linker reacts with primary and secondary amines of polyethylenimine resulting in formation of ester bonds [21].

Fig. 6. Scheme of polymannitol-base gene transporter based on by Michael addition resulting in the formation of degradable ester bonds [25].

efficiency with PEI 25 kDa in 293T, HeLa and HepG2 cells where the transfection efficiency of the polymer increased with an increase of MW of starting PEIs due to the increased buffering capacity. Similarly, Luu et al. [24] synthesized degradable branched PEIs based on osmotic sorbitol diacrylate and low MW PEI (1200 Da). Previously, they also prepared degradable branched PEIs using sorbitol dimethacrylate. The cytotoxicity of the polymer prepared by sorbitol diacrylate was less than that of the polymer prepared by sorbitol dimethacrylate, suggesting that the hydrophobic methylated group in the polymer increased the cellular toxicity. The polysorbitol-mediated gene carrier exhibited higher transfection efficiency than PEI 25 kDa through selective caveolae endocytosis pathway by hyperosmotic polysorbitol backbone despite the existence of the PEI part in the copolymer.

Fig. 7. Scheme of polyxylitol-based gene carrier synthesized by Michael addition reaction resulting in the formation of degradable ester bonds [26].

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Park et al. prepared degradable branched PEIs based on osmotic mannitol diacrylate and low MW PEI as shown in Fig. 6 [25]. Similarly, the polymannitol-based gene transporter showed higher transfection efficiency with lower cytotoxicity than those of PEI 25 kDa owing to the hyperosmotic property by polymannitol backbone and ‘proton sponge effect’ by PEI part in the polymer. Interestingly, the caveolae-mediated endocytosis pathway of the polyplexes prevented lysosomal fusion and avoided gene degradation. Recently, Lee et al. prepared degradable PEIs based on osmotic xylitol diacrylate and low MW PEI as shown in Fig. 7 [26]. The polyxylitol-based gene transporter showed higher transfection efficiency with lower cytotoxicity than those of PEI 25 kDa due to superior osmotic effect compared to PEI inside endosomes and facilitation of enhanced endosomal osmolysis. The higher osmotic properties of polyxylitol gene carrier enabled the gene to avoid lysosomal degradation and transfer more genes into cytosol and nucleus. 2.1.3. Grafted PEI Zhang et al. [28] grafted low MW PEI (800 Da) onto PEG dithiothreitol using 1,1′-carbonyldiimidazole as the linker where the polymer has two degradable linkages, ester linkage as the main chain and carbamate linkage as the side chain. It should be noted that the polymer showed higher transfection efficiency and no inhibitory effect on the transfection efficiency in the presence of serum. The characteristics of degradable PEIs with ester linkages are summarized in Table 1. 2.2. Disulfide linkages Disulfide linkage is easily degraded in the presence of reducing enzymes such as glutathione or glutathione reductase [29]. While the

disulfide bonds are relatively stable in the extracellular condition, they can be rapidly cleaved in the intracellular environment because the concentration of glutathione (0.5 to 10 mM) is very high in the cytoplasm. The degradable PEIs with disulfide linkages can be classified into PEIs with disulfide bond in the main chain and side chain according to the location of disulfide linkages in the polymer.

2.2.1. Main chain The first degradable PEIs with disulfide linkages were prepared by cross-linking low MW PEI (800 Da) with dithiobis(succinimidylpropionate) (DSP) and dimethyl 3,3′-dithiobispropionimidate (DTBP) [30]. The introduced disulfide bonds of the polymers were degraded by glutathione, an intracellular reducing agent. The transfection efficiency of the degradable PEI was dependent on the nature of the cross-linking agent, the extent of conjugation and the N/P ratio. Degradable PEIs based on linear low MW PEI (423 Da) and cystamine bisacrylamide (CBA) as a crosslinking agent showed about 5-fold higher transfection efficiency than that of PEI 25 kDa with a lower cellular toxicity in brain endothelial cells due to the cleavage of the disulfide linkages in the polymer [31]. Bauhuber et al. [32] synthesized a library of PEG–PEI copolymer with disulfide bond between PEG block and PEI block to study the changes in gene delivery function in relation to polymer structure. It was found that a higher content (more than 50%) of PEG in the copolymer provided a greater effect on the physicochemical properties of the polyplexes that significantly decreased cellular uptake followed by reduced transfection efficiency. Recently, Lei et al. [33] prepared reductively reversible and hydrolytically degradable PEIs based on RGD-coupled PEG pyridyldithio propionate and PEI-SH to get disulfide and amide linkages in the main chain. The polymer showed higher transfection efficiency in U87 cells

Table 1 Representative examples of degradable PEIs having ester linkages (modified from Ref [129]). Type

MW of starting PEI (Da)

Cross-linker

Used cells

Characteristics

References

Linear

800

1,3-butanediol diacrylate

MDA-MB-231

[10]

423

PEG diacrylate

HepG2, MG63

423

Sorbitol dimethacrylate

A549, HeLa, H322

423

Sorbitol dimethacrylate

A549

Branched 800

1,6-Hexane diacrylate

MDA-MB-231

800

1,6-Hexane diacrylate

N2a, BI6F10

600, 1200, 1800 600, 1200, 1800

Poloxamer diacrylate Polycaprolactone diacrylate

A549, 293 T, HepG2 293T, HepG2, HeLa

1200

Glycerol dimethacrylate

HeLa, HepG2, 293T

600, 1200, 1800

PEG dimethacrylate

293T, HeLa HepG2

1200

Sorbitol diacrylate

A549, 293T, HeLa

1200

Mannitol diacrylate

A549. HeLa, N2a

1200

Xylitol diacrylate

A549, HeLa, C2C12

1200

Mannitol dimethacrylate

A549, HeLa, HepG2

800

PEG dithiothreitol

HeLa, MCF-7, COS-7, HepG2

– Rapid hydrolysis (half-life: 4 h) – Higher (up to 16-fold) gene transfection efficiency – MW of PEG is critical – Half-life, the amount of time required to get half MW from initial MW of gene carrier, is ~ 8 h – Aerosol administration is more efficient than IV one – No aggregation of polyplexes after lyophilization due to many hydroxyl groups – Accelerated gene transfection by osmotic active property – Accelerated gene silencing – Involvement of caveolin-1 and COX-2 expression for regulating cellular uptake mechanism – High MW of 14 kDa, reaction with primary and secondary amines of PEI – Longer reaction time and high temperature provided mixed ester and amide linkages – High MW of 83 kDa, a slight serum dependency on transfection – Half-life is 4.5 to 5 days – 15–20-fold higher gene transfection efficiency due to hydrophobic crosslinking agent – Half-life is 9 to 10 days – Synergistic effect of buffering capacity by PEI and hyperosmotic property by polyglycerol – Half-life is pH-dependent – Longer half-life than PEG diacrylate – Reduced cytotoxicity than sorbitol dimethacrylate – Caveolae endocytosis pathway – Stimulation of caveolae endocytosis pathway – Prevention of delivered gene from lysosomal fusion by regulation of cellular uptake – Avoidance of gene degradation in lysosome – Enhanced osmolysis of endosome cooperatively with the proton sponge effect – Stimulation of COX-2 induction which was responsible for the enhancement intracellular uptake by activating caveolae-mediated endocytosis. – Two degradable linkages – No serum dependency on transfection efficiency

Grafted

[11,12]

[13]

[14]

[10] [17,18] [19] [20]

[21]

[19] [23] [25]

[26] [27]

[28]


(a human primary glioblastomacellline) with reduced cytotoxicity than PEI 25 kDa and enhanced gene expression in the brain of intracranial U87 glioblastoma-bearing nude mice through IV injection via the tail vein due to the glutathione-sensitive disulfide linkage and RGD sequence. 2.2.2. Side chain Synthesis of degradable PEIs with disulfide linkages in the side chain of the polymer based on low MW (2600, 3100 and 4600 Da) PEI and N,N ′-bis(terbutoxycarbonyl) cysteine or 3,3′-dithiodipropionicacid di(N-succinimidyl ester) as cross-linkers has been reported [34]. The polymers showed high transfection efficiency with low toxicity when compared to commercial transfection reagent. The results further showed that the combination of high branching density and reductively cleavable bonds in the polymer is a very important parameter for efficient gene silencing of siRNA [35]. Peng et al. [36] synthesized degradable PEIs containing disulfide bonds through cross-linking of thiolated PEIs after the reaction of methylthiirane with low MW PEI (800 Da). The polymer showed gene transfer activity, comparable to PEI 25 kDa, with low toxicity and no effect on the transfection efficiency in the presence of serum. In another study, they found that the polymer with either very low or very high thiolation degrees had poor transfection efficiency where the polymer synthesized from PEI (800 Da) provided the best transfection activity compared to the polymer prepared from PEI (1800 Da and 25 kDa) [37]. Liu et al. [38] prepared degradable PEIs by click chemistry through the reaction of azide-terminated low MW PEI (1800 Da) with a disulfidecontaining dialkyne as a cross-linker. The degradable polymer showed higher transfection efficiency with lower toxicity than those of PEI 25 kDa in both the presence and absence of serum. They also prepared brush-type degradable PEIs which have the advantages of biodegradability and ideal configuration for protecting genes to improve transfection efficiency and cell viability [39]. Recently, Zhang et al. [40] prepared degradable PEIs based on Pluronic® diacrylate and LMW PEI crosslinked with disulfide bond to get dual-degradable disulfide and ester linkages. The polymer showed higher transfection efficiency in BGC-823 and 293T cell lines with lower cytotoxicity than PEI 25 kDa. The consequences are due to the formation of stable polyplexes by ester bonds in the extracellular environment and the cleavage of disulfide bonds in the intracellular environment to release complexed DNA. The characteristics of degradable PEIs containing disulfide linkage are summarized in Table 2. 2.3. Imine linkage The imine linkage in the polymeric gene carrier can be introduced by nucleophilic addition reaction of aldehyde or ketone with amine. The imine linkage can be cleaved into corresponding amine and carbonyl compound in water and rapidly hydrolyzed at the acidic condition. Besides, the imine is reduced to an amine by the reducing agent. Many researchers reported degradable PEIs containing imine linkages based on low MW PEI and chitosan because the limitation of low transfection efficiency of chitosan can be overcome by the introduction of PEI due to its buffering capacity. Low MW PEI (1800 Da) was grafted into periodate-oxidized chitosan through imine linkages by Jiang et al. [44] as shown in Fig. 8. The chitosan-graft-PEI demonstrated about 1000-fold higher transfection efficiency in three different cell lines with lower cytotoxicity than chitosan itself due to the buffering ability of the incorporated PEI in the carrier. They introduced galactose moiety into the chitosan-graft-PEI for hepatocyte targeting as shown in Fig. 9. The polymer showed higher transfection efficiency in HepG2 cells and liver cells of mouse after intraperitoneal injection than the chitosan-graft-PEI due to the galactose specific ligand to receptors of the hepatocytes [45]. They also introduced PEG as a spacer into the galactosylated chitosan-graft-PEI to get stability of polyplexes during blood circulation in vivo [46]. The polymer showed higher transfection efficiency in liver cells of mouse than PEI 25 kDa after IV administration although the transfection efficiency of the

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polymer in vitro was lower than that of PEI 25 kDa due to the hydrophilic property of PEG in the polymer. Furthermore, they introduced mannose moiety into the chitosan-graft-PEI to get receptor-mediated endocytosis for targeting antigen-presenting cells as shown in Fig. 10 [47]. The polymer showed higher transfection efficiency in RAW264.7 cells with lower cytotoxicity than chitosan-graft-PEI due to the specific mannose ligand to receptors of macrophage cells. Similarly, they coupled folic acid into chitosan-graft-PEI to deliver Akt1 shRNA in vitro and in vivo as shown in Fig. 11 [48]. The polymer showed higher gene silencing in A549 cells than the chitosan-graft-PEI and greater lung tumor suppression in a urethane-induced lung cancer model mouse after aerosol delivery. Recently, the polymer was used to deliver PDCD4 gene in vitro and in vivo after the introduction of PEG as spacer [49]. The polymer showed high transfection efficiency in folate receptor-overexpressing cancer cells and reduced tumor numbers and tumor sizes in H-ras 12V liver cancer mouse than PEI 25 kDa after IV injection. Ping et al. [50] synthesized chitosan-graft-[PEI-β-cyclodextrin (CD)] via reductive amination between oxidized chitosan and low MW PEI-conjugated β-CD to deliver DNA or siRNA. Although the supramolecular PEGylation of the polymer through self-assembly of adamantyl-modified PEG with β-CD moieties decreased transfection ability in HEK 293 and L929 cells, the polymer showed higher gene silencing in both cells with lower cytotoxicity than PEI 25 kDa. Recently, Liu et al. prepared degradable PEI by crosslinking low MW PEI (2 kDa) with Pluronic® through carbamate linkages and further conjugated RGD as the tumor-targeting peptide in conjunction with Tat as cell-penetrating peptide to get tumor cell specificity and to increase cellular uptake of genes [51]. The polymer showed targeting specificity to αvβ3 receptor of HeLa cells and B16 cells, and higher transfection efficiency in two cell lines with lower cytotoxicity than PEI 25 kDa. They also grafted low MW PEI (20 kDa) into N-octyl-Nquaternary methyl chitosan through carbamate linkages [52]. The polymer protected the DNA by DNase I and the polyplexes were resistant to dissociation induced by 50% BSA. The polymer also showed higher transfection efficiency than PEI 25 kDa in vitro and in vivo with lower cytotoxicity even at high doses. Kim et al. reported degradable PEI containing imine linkage by reaction of low MW PEI (1800 Da) with glutaraldehyde [53]. The half-life of the copolymer was 1.1 h at physiological pH, suggesting a rapid degradation due to imine linkage. This polymer showed high transfection capacity with low cytotoxicity. Recently, Jiang et al. grafted low MW PEI (800 and 2000 Da) with periodated dextran through imine reaction [50]. The polymer showed significantly higher transfection than PEI 25 kDa in the presence of serum when the incubation time of polyplexes increased up to 48 h. However, the polymer showed moderately lower transfection ability without serum for 4 h [54]. 2.4. Carbamate linkage The carbamate linkage is relatively stable because the carbamic acid is often chemically interconverted to the carbamate ester such as urethane. Degradable PEI was prepared by a reaction of low MW PEI (600, 1200 and 1800 Da) with PEG succinimidyl succinate via carbamate linkage [55]. The polymer showed 3-fold higher transfection capability than starting PEI with high cell viability of over 80%, although the transfection efficiency of the polymer was lower than PEI 25 kDa because of the decreased cellular uptake of the polyplexes by the hydrophilic nature of PEG. Degradable PEI was also prepared by a reaction of low MW PEI (800 Da) with 1,4-butanediol bis(chloroformate) through carbamate linkage [56]. The synthesized polymer showed remarkably higher transfection efficiency than PEI 25 kDa despite of the low MW of the obtained polymer (2800 Da). Liu et al. synthesized poly(ester-co-urethane)-grafted-PEI via aminolysis of poly(ester-co-urethane) by low MW PEI (800 Da) where the polymer has two degradable linkages such as ester bond in the main chain and carbamate bond in the side chain [57]. The polymer

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Table 2 Representative examples of degradable PEIs having disulfide linkages (modified from Ref [129]). Type

MW of initial PEI (Da)

Crosslinker

Cells

Characteristics

References

Main chain

800

DSP and DTBP

CHO

[30]

423 1500, 5000, 10,800 25,000

CBA Methoxy PEG thioacetate PEG-pyridyldithiopropionate

bEnd.3 CHO U87

800, 25,000 2600, 3100, 4600

CBA N,N′-bis(terbutoxycarbonyl) cysteine and 3,3′-dithiodipropionic acid di(N-succinimidyl ester) Thiolated PEI

HeLa BC HEK

– The first synthesis of the polymer – Cleavage of the polymer by glutathione – About 5-fold higher transfection efficiency – Study on polymer structure–gene function relationship – Incorporation of RGD sequence for cancer cell targeting – Enhanced gene expression in the brains of cancer bearing mice – Enhanced transfection efficiency by addition of a intermediate Pluronic® – Very high transfection efficiency of 70% – Elucidate the importance in combination of branching density and disulfide bond for efficient siRNA delivery – Simple cross-linking – Elucidate the importance in disulfide density and MW of starting PEI on transfection – Click chemistry, disulfide and carbamate linkages – Click chemistry, brushed type PEI

Side chain

800, 1800, 25,000

1800 1800 800

25,000 1800

293T, HeLa, COS7, CHO

Disulfide containing dialkyne Disulfide and imidazole containing dialkyne Poloxamer diacrylate

293T, HeLa 293T

3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester 3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester

HEK

293T, BGC-823

COS-1 HepG2 293T C2C12

exhibited higher transfection efficiency with lower cytotoxicity than those of PEI 25 kDa due to the short half-life of 14 h at pH 7.4. Zhao et al. [58] compared gene expression in vitro and in vivo between acidlabile degradable PEI containing imine linkages prepared by a reaction of low MW PEI (2000 Da) and glutaraldehyde and degradable PEI containing biscarbamate linkages prepared after the reaction with low MW PEI (2000 Da) and activated triethylene glycol. The results indicated that the degradable PEI with carbamate linkages exhibited higher transfection efficiency with lower toxicity in vitro and in vivo than that of degradable PEI with imine ones due to the degradability of carbamate linkages at neutral pH. Another degradable PEI was prepared by a reaction of low MW PEI (800 Da) with ethylene bis(chloroformate) through biscarbamate linkages [59] and showed higher transfection activity with lower cytotoxicity than PEI 25 kDa.

2.5. Amide linkage The amide linkage is more stable to hydrolytic degradation than ester bond at physiological condition but it is susceptible to degradation by enzymes. Xiong et al. [60] synthesized poly(aspartate-graft-PEI) by grafting of low MW PEI (800 Da) to the polyaspartate obtained by ring opening polymerization of β-benzyl-L-aspartate-N-carboxyanhydride through amide linkage. The polymer did not show tissue damage at day 5 post-administration in mouse, whereas PEI 25 kDa showed apoptosis and necrosis in the kidney and spleen. However, the polymer induced inflammation, apoptosis and necrosis in the spleen and liver of rodent at day 1. The polymer exhibited higher transfection efficiency and lower toxicity than PEI 25 kDa. Similarly, Wen et al. [61] prepared PEG-block-polyglutamine-graft-PEI through aminolysis of PEG-blockpoly(γ-benzyl L-glutamate) by using low MW (423 Da) PEI. The polymer was degraded by papain enzyme solution. Moreover, the polymer exhibited markedly higher transfection efficiency in four different cell lines (HepG2, HeLa, 293T and Bel9402) with lower cellular toxicity than those of PEI 25 kDa. Furthermore, the transfection was not affected by serum due to the shielding effect of PEG in the block copolymer. Yu et al. [62] prepared a core–shell typed gene carrier based on poly(L-succinimide) (PSI)-graft-PEI by reaction of PSI obtained by polycondensation of L-aspartic acid with low MW PEI (423 Da) via amide

– Two degradable bonds having disulfide and ester – Control of degradable bonds in the extracellular and intracellular environment – Elucidation of the importance of disulfide density for balance between transfection efficiency and toxicity. – Very high transfection efficiency of chitosan-SS-PEI.

[31] [32] [33] [41] [34,35]

[36,37]

[38] [39] [40]

[42] [43]

linkages. Because the shell region partially shielded the positive charges of PEI and reduced serum aggregation during the blood circulation, the polymer exhibited significantly higher transfection with lower toxicity than PEI 25 kDa. Similarly, they grafted low MW branched PEI (600 and 1200 Da) to the PSI via amide linkages to observe the effect of MW of starting PEI on the transfection efficiency [63]. The transfection ability decreased as the MW of the starting PEI increased owing to the increased charge density and toxicity of PEI. Namgung et al. synthesized degradable star-shaped copolymer based on 3- or 6-arm PEG succinimidyl succinate and low MW PEI (2500 Da) via amide linkages [64]. The polymer obtained from the 6-arm PEG showed higher transfection capacity than that of the 3-arm PEG due to higher charge density and more prevention of aggregation of the polyplexes made by the 6-arm PEG than those of PEI 25 kDa. 2.6. Ketal linkage The ketal linkage is rapidly degraded at mild acidic pH of 5 that results in the release of DNA from the acidic endosome and lysosome into the cytoplasm [65]. Shim et al. prepared degradable PEI by partial conjugation of low MW PEI (800 Da) using amine-bearing ketal. The polymer showed improved transfection with low cytotoxicity because the ketalization of the PEI facilitated dissociation of the polymer complexes upon hydrolysis of ketal linkages and release of DNA. They also optimized the ketalization of low MW PEI to enhance the transfection efficiency [66]. For instance, ketalization up to 70% increased transfection capacity whereas ketalization more than 70% dramatically decreased the transfection activity of the synthesized polymeric gene carrier [67]. 3. Degradable PAA derivatives and their characteristics PAAs are a family of peptidomimetic polymers containing tertiary amino and amido groups regularly arranged along their polymer chain [68]. They can be prepared by Michael addition reaction of primary or secondary amines with acrylamide derivatives in mild condition [64]. In this section, only bioreducible PAAs will be covered because they have been extensively studied for gene delivery systems due to the rapid cleavage of the disulfide bonds inside the cells by reductive


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Fig. 8. Scheme of chitosan-graft-PEI (CHI-g-PEI) copolymer by an imine reaction between periodate-oxidized chitosan and an amine group of PEI. (IO − 4 : an anion periodate; N2: nitrogenous condition; PEI: polyethylenimine) [44].

Fig. 10. Scheme of mannosylated chitosan-graft-polyethylenimine (Man-CHI-g-PEI) as a gene carrier for RAW264.7 cell targeting. (IO− 4 : an anion periodate; N2: nitrogenous condition; NaBH4: sodium borohydride; PEI: polyethylenimine) [47].

enzymes. Especially, here we discuss the effects of several important factors of the PAAs on their gene delivery properties.

3.1. Charge density

Fig. 9. Scheme of galactosylated chitosan (GC)-graft-PEI (GC-g-PEI) as a gene carrier for hepatocyte targeting. (NHS:N-hydroxysuccinimide; EDC: ethyl(dimethylaminopropyl) carbodiimide; IO− 4 : an anion periodate; PEI: polyethylenimine) [45].

Generally, the high cationic charge density in the polymeric carriers easily condenses DNA to form polyplexes. Although these positively charged polyplexes confer high cellular uptake, the high cationic density also results in severe toxicity. In order to investigate the effects of charge density of polymers on the transfection efficiency, Piest et al. [69] has prepared PAAs by the reaction of CBA and 1,4-diaminobutane (DAB) by varying the degree of acetylation and benzoylation of amino butyl

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optimization of cationic charge density and hydrophobicity/hydrophilicity balance are the important parameters to enhance the transfection efficiency. They also optimized the charge density of PAAs prepared by the reaction of CBA with 4-amino-1-butanol and ethylene diamine (EDA) or triethylenetetramine (TETA) for the efficient siRNA delivery [70]. It was found that at least 20–30 mol% of EDA or TETA in the copolymers is required to make stable polyplexes that showed excellent gene silencing in H1299 cells with lower cytotoxicity. At the same time, the increase in cationic charge in the copolymers showed increase in cytotoxicity and haemolytic activity, indicating that positive charge density in gene carriers need to be controlled to obtain maximum gene silencing by siRNA. 3.2. Density of disulfide linkages

Fig. 11. Scheme of the folate-chitosan (FC)-graft-PEI copolymer (FC-g-PEI). The copolymer was synthesized by an imine reaction between periodate-oxidized folate and an amine group of low molecular weight polyethylenimine. (NHS:N-hydroxysuccinimide; DCC: dicyclohexylcarbodiimide; IO− 4 : an anion periodate; N2: nitrogenous condition; NaBH4: sodium borohydride; PEI: polyethylenimine) [48].

The effect of density of disulfide linkages on gene delivery properties of the bioreducible PAAs has received much attention in chemistry and biology because the density of disulfide bonds in PAAs is closely related with intracellular vector unpacking and gene delivery. Lin et al. [71] prepared a series of bioreducible PAAs based on CBA and 1-(2-aminoethyl) piperazine (AEP) or N,N′-hexamethylenebisacrylamide (HMBA)/CBA and AEP to contain various amounts of disulfide linkages in the main chain. The results indicated that poly(HMBA/CBA-AEP) copolymers improved the transfection efficiency in COS-7 cells with lower cytotoxicity as compared to poly(HMBA-AEP) homopolymer without disulfide linkages. Further, high transfection efficiency and cell viability were obtained for the poly(HMBA 80/CBA 20-AEP) with 20 mol% disulfide content. But, the increase in disulfide content in the copolymer to 42 mol% and 63 mol% showed only marginal effect on the transfection efficiency, indicating that the presence of appropriate amount of disulfide linkages in the PAAs have a favorable effect on cell viability and transfection efficiency. Chen et al. [72] tuned the disulfide content in the hyperbranched PAAs based on HMBA/CBA and N,N-dimethylaminodipropylene-triamine (DMDPTA) to check the effect of disulfide content on the molecular weight of the polycation degradation products, ease of polyplexes disassembly, cell viability and transfection efficiency. The results indicated that higher disulfide content having 50, 74 and 100 mol% in the copolymers showed about 10-fold higher transfection efficiency in B16F110 mouse melanoma cells than PEI 25 kDa and higher cell viability was obtained for the higher disulfide content having 74 and 100 mol%. They also investigated the effect of disulfide content in PAAs based on poly(HMBA/CBAAEP) copolymer on the gene transfection using DNA of different molecular weight [73]. Interestingly, increase in the disulfide content in the PAAs increased DNA transfection but had no effect on the antisense oligonucleotides. The presence of disulfide bonds in PAAs had no significant effect on the rate of intracellular DNA clearance, suggesting that enhanced intracellular disassembly of the bioreducible polyplexes is not a major factor to the improvement in transfection efficiency while the molecular weight of the DNA affects the activity of the bioreducible polyplexes. Furthermore, they compared cytotoxicity of PAAs based on CBA and DMDPTA between intracellular glutathione and exofacial plasma membrane thiol to elucidate the mechanism of the decreased toxicity of bioreducible PAAs [74]. It was found that the toxicity of PAAs decreased in a linear fashion with an increase in disulfide content. The toxicity was low in cells that contain higher concentration of glutathione whereas plasma membrane protein thiols increased toxicity of PAAs, suggesting that the decrease of toxicity of PAAs is directly related to the intracellular glutathione concentration. 3.3. Architectural features

side chains in the PAAs. It was found that benzoylation of PAAs reduced charge density and cytotoxicity. Besides, the benzoylated PAAs showed much higher transfection efficiency than the acetylated ones in COS-7 cells. The consequence is attributed to the increased stabilization of polyplexes by the hydrophobic benzoyl groups, suggesting that the

Due to the structure–function relationships of bioreducible PAAs, it can be expected that PAAs with different topographical structures will have different gene delivery properties. Martello et al. [75] prepared linear PAAs based on CBA, N,N′-dimethylethylenediamine (DMEDA)


and 4-aminobutanol (ABOL) or 2-aminoethanol (ETA), and branched PAAs based on cystamine, N,N′-bisacryloylpiperazine and ABOL (or ETA) to study the effects of architecture of the PAAs on their gene delivery properties. Both linear and branched PAAs showed remarkably low cytotoxicity without much difference of cytotoxicity between linear and branched PAAs. Moreover, hyperbranched PAAs showed higher gene efficiency in COS-7 cells than linear ones even in the presence of serum due to the increased buffering capacity conferred by the high density of secondary and tertiary amines in the polymers. Zhang et al. [76] prepared different branched bioreducible PAAs based on CBA and 1-(2-aminoethyl)piperazine (AEPZ) using mixed solvents to compare gene delivery properties. It was found that low-branched PAA more effectively condenses DNA into positively charged nanoparticles than linear PAA and high-branched PAA. Similarly, low-branched PAA showed the highest transfection efficiency with lower cytotoxicity among the three PAAs due to the endocytosis rate without change in endocytosis pathways. Recently, Ping et al. [77] prepared bioreducible hyperbranched PAAs with tertiary amino cores and hydroxyl terminal groups by reaction of CBA, AEPZ and 3-amino-1,2-propandiol to investigate the effect of tertiary amine groups in the polymer backbone on the gene transfection. It was observed that PAAs with amino terminal groups had the highest efficiency in HEK 293, COS-7, MCF-7 and HepG2 cell lines with lower cytotoxicity. However, the transfection efficiency of PAAs with hydroxyl terminal groups was cell-type dependent, suggesting that the terminal structure in the PAAs is one of parameters to affect gene transfection. 3.4. Cell penetrating peptide (CPP)-based PAA It is well-documented that CPPs or protein transduction domains play an important role in intracellular transportation of cargo molecules in receptor- and temperature-independent manner [78]. Kim et al. [79] grafted arginine (R) as a main component of the CPPs into PAA based on CBA and diaminohexane (DAH) to enhance efficacy of VEGF gene silencing for cancer gene therapy. The siRNA/poly(CBA-DAH-R) complexes increased membrane permeability with arginine modification and VEGF siRNA/poly(CBA-DAH-R) complexes more inhibited VEGF expression than VEGF siRNA/PEI in various human cancer cell lines due to enhanced intracellular delivery and effective localization to the cytoplasm of the VEGF siRNA by the grafted arginine. They also incorporated poly(CBA-DAH-R) into the PAA dendrimer (PAM) to overcome the limitation of low molecular weight of poly(CBA-DAH-R) [80]. The PAM-poly(CBA-DAH-R) showed superior condensing ability for DNA, enhanced cellular uptake and greater transfection efficiency than poly(CBA-DAH-R) due to the formation of more compact nanosized polyplexes by the increased high molecular weight of the carrier. Furthermore, they prepared bioreducible PAAs based on CBA, argininegrafted DAH and 1-(3-amino-propyl) imidazole (API) to seek cell penetrating and endosome buffering functionality [81]. It was found that decrease in arginine portions with an increase in API in the copolymers reduced cytotoxicities of the polymers and transfection efficiencies also due to the decreased cellular uptakes of polyplexes, suggesting the predominance of arginine portions over API as endosome buffering moieties for efficient gene delivery. Guanidine as a component of CPPs is also known to function for the efficient translocation of non-permeant molecules [82]. Therefore, Kim et al. [83] introduced guanidine into bioreducible PAA based on CBA and DAH to enhance cellular uptake and nuclear localization ability. The guanidine-grafted PAA showed about 8-fold higher transfection efficiency in mammalian cell line with lower cytotoxicity due to the high cellular uptake efficiency (96.1%) and strong nuclear localization ability by the introduced guanidine group in the carriers. Recently, they also examined the possibility of guanidine-grafted PAA for gene therapy of erectile dysfunction (ED) in mice model of vasculogenic ED induced by a high cholesterol diet [84]. The polymer showed 7-fold higher transfection efficiency in A7r5 rat

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vascular smooth muscle cells than PEI 25 kDa and higher gene expression in the corpus cavernosum tissue of hypercholesterolaemic mice. 3.5. PEGylated PAA PEGylated cationic polymers have been proven to overcome extracellular barriers of the gene therapy because PEGylated polymers/gene complexes improved colloidal stability in systemic circulation, reduced interaction with blood components and cell surfaces which resulted in prolonged circulation with lower cytotoxicity. However, PEGylated polyplexes also exhibited decreased cellular uptake, diminished endosomal escape properties and hence low efficiency of the delivered gene [5]. Brumbach et al. [85] studied feasibility of altering and optimizing PEG content in mixtures of PAAs based on CBA and TETA, and their corresponding PEGylated counterparts to identify optimal gene carrier candidate. It was found that an increase of PEG content in the formulation adversely affected polyplexes formation whereas optimum mixtures of PAA and PEGylated PAA improved better physiochemical properties of the gene carrier and higher gene expression than PEI 25 kDa and PAA in the presence of serum. Similarly, they also mixed PAAs based on CBA, ABOL and EDA and their corresponding PEGylated counterparts to check the effect of PEGylation on the gene delivery properties [86]. These results indicated that PEGylation decreased polyplexes surface charge and increased their stability against salt and serum but decreased resistance against heparin displacement. Although gene silencing efficiency in H1299 cells was decreased with an increase in PEG content, polyplexes with PEG contents of 30 and 45 wt.% showed significant silencing efficiency without cytotoxicity. 3.6. Boronic acid-modified PAA Boronic acids as the organic derivatives of boric acid are known for their reversible ester bond formation with cis-1,2 and 1,3-diols in the alcohols. Therefore, phenylboronic acid containing polymers have been used for recognition of L-DOPA [87], glucose responsive delivery of insulin [88] and targeting to tumor cells through the formation of reversible covalent binding between the diol-function of the sialic acid and boronate functions of the polymers [89]. For example, Piest et al. [90] grafted para-carboxyphenylboronic acid (4CPBA) or ortho-aminomethylphenylboronic acid (2AMPBA) with PAAs based on CBA and DAB to evaluate effects of boronic acid on gene delivery properties. It showed that phenylboronic acid moieties improved polyplexes formation with DNA and the transfection efficiency of 4-CPBAPAA/DNA complexes was similar to PEI (Exgen) in COS-7 cells both in the absence and presence of serum. However, phenylboronic acid-carrying PAAs showed increased cytotoxicity due to the increased cell membrane disruptive interaction. Recently, they also incorporated 2AMPBA into PAAs based on CBA and ABOL to confirm the interaction of phenylboronic acid with the glycocalyx of the cells and the interaction of phenylboronic acid with siRNA [91]. It was found that boronic acid moieties as side groups in the polymer chain strongly enhanced the stability of the polyplexes without aggregation for 6 days at 37 °C due to the intermolecular Lewis acid–base interactions between the phenylboronic groups and alcohol and amine groups in the polymer. Further, phenylboronic acidcarrying PAAs gave a 35% knockdown of luciferase expression in H1299 cells while addition of 0.9% sorbitol to the transfection medium strongly enhanced the knockdown efficiency to 59% due to boronic ester formation with the added sorbitol and reduced binding interactions with the glycocalyx of the cells. 3.7. Specific ligand-conjugated PAA Target-specific gene delivery is another important strategy to overcome the extracellular barriers in non-viral gene delivery as specific enhancement of cellular uptake occurs through receptor-mediated

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endocytosis. Nam et al. [92] introduced primary cardiomyocyte (PCM) specific peptide screened by phage display into PAAs based on CBA and DAH for specific delivery of Fas siRNA because the inhibition of Fas inhibits cardiomyocyte apoptosis without immune stimulation [93]. It was found that the cellular binding and uptake of the PCM-PAA/DNA complexes in H9C2 rat cardiomyocytes were inhibited by the addition of free PCM peptide while Fas siRNA/PCM-PAA complexes showed significant Fas gene silencing in rat cardiomyocytes under hypoxic conditions with an inhibition of cardiomyocytes apoptosis, a clear indication of receptor-mediated endocytosis mechanism. They also conjugated RGD peptide into PAAs based on CBA and DAH to deliver oncolytic adenovirus (AD) shRNA for overcoming limitations of transduction targeting and immune privilege of AD because RGD peptide is a specific ligand for cell-surface integrins on tumor endothelium [94]. The results indicated that cytopathic effects of oncolytic AD coated with RGD-PAA were much more enhanced than that of AD itself in cancer cells selectively and HT 1080 cells treated with AD shRNA/RGD-PAA showed strong induction of apoptosis and suppression of IL-8 and VEGF expression, indicating the receptor-mediated endocytosis mechanism. Furthermore, they conjugated PCM and Tat peptides into siRNA via cleavable disulfide linkages to deliver siRNA specifically to cardiomyocytes with a high gene silencing by PAA based on CBA and DAH [95]. It was found that the peptide-modified siRNA/PAA complexes enhanced the cellular uptake and gene-silencing capacity of the siRNA in cardiomyocytes without significant cytotoxicity and the combined siRNA/PAA complexes were delivered to the mouse heart at significantly high levels compared to the unmodified siRNA via systemic administration, suggesting the synergistic gene silencing effect of cell targeting peptide and CPP. 4. Cyclodextrin (CD)-based polycations and their properties Recently, CD-based polymers have attracted great attention for gene therapy because CD-based polymer system is the first representative of polymeric gene carriers that entered clinical trials for siRNA delivery less than a decade after its introduction. In this section, several strategies such as stability, cellular uptake, endosomal escape, cell specificity and dual delivery will be discussed to overcome extracellular and intracellular barriers of CD-based polymers as gene carriers. However, CDbased polyrotaxanes, CD-based dendrimers and monodisperse CD derivatives will not be covered here because recent comprehensive reviews on CD-based drug delivery are available [96–98].

supramolecular PEGylation of polyplexes with siRNA demonstrated higher silencing efficiency in HEK293 and L929 cells comparable to commercial DharmaFECT. Likewise, amino-β-CD was assembled with adamantane–poly(vinyl alcohol) (PVA)–PEG or cholesterol–PVA–PEG to deliver DNA [104] or siRNA [105] respectively. The self-assembled amino-β-CD/adamantane– PVA–PEG/DNA complexes showed higher transfection efficiency in HeLa cells with lower cytotoxicity than PEI 25 kDa due to the stabilized nanosized particles by the hydrophilic PVA [104]. On the other hand, the self-assembled amino-β-CD/cholesterol–PVA–PEG/siRNA complexes showed almost similar gene silencing efficiency in CHO cells with 3–4 orders of less toxicity compared to PEI 25 kDa [105]. 4.2. Transfection activity and cellular uptake An efficient gene delivery system must be able to overcome at least two major barriers; an extracellular barrier to enhance cellular uptake of the polyplexes and an intracellular barrier to promote the efficient escape of DNA from early endosomes after cellular uptake. In this regard, Choi et al. [106] developed a sunflower-shaped β-CD-conjugated poly(ε-lysine) (β-CDPL) polyplexes that promotes the removal of cholesterol from the cell membrane, facilitating the transfer of DNA into cells. β-CDPL polyplexes showed higher transfection efficiency in NIH3T3 cells with lower cytotoxicity than linear PEI or poly(ε-lysine) due to the synergistic effect of high cellular uptake and proton sponge ability of β-CDPL. A new generation of hybrid polysaccharide nanocarriers composed of chitosan and anionic β-CD such as sulfobutylether-β-CD and carboxymethyl-β-CD were prepared by ionotropic gelation to deliver DNA in human mucus-producing cell line Calu-3 [107]. The hybrid nanoparticles showed higher transfection efficiency in the Calu-3 cells with lower cytotoxicity than naked DNA due to the enhanced cellular uptake. The influence of β-CD on the cellular uptake and transfection efficiency was also studied using β-CD-modified polyplexes with uptake inhibitors [108]. Interestingly, β-CD-modified PEI significantly promoted the route of caveolae-mediated endocytosis in the HEK293T cells with high cellular uptake and high transfection efficiency whereas PEI polyplexes were internalized by both clathrin-mediated and caveolaemediated endocytosis. It should be noted that the cellular uptake and transfection efficiency of polyplexes depend on the type of the used cells. 4.3. Endosomal escape capacity

4.1. Stability The delivery system must retain its physicochemical properties during blood circulation in the body because negatively charged proteins compete with gene binding and induce aggregation of polyplexes in the systemic circulation. Several hydrophilic polymers such as PEG [99], polyvinylpyrrolidone [100], and dextran [101] have been used to increase the circulation of the polyplexes by masking the surface charge and by preventing protein opsonization. Pun et al. [102] studied the effect of PEGylation of β-CD-containing polycations (β-CDP), obtained by preDNA-complexation PEGylation and post-DNA-complexation PEGylation, on the stability of the polyplexes at physiological salt concentration. It was found that both PEGylated β-CDP polyplexes were stable at the physiological conditions whereas unmodified polyplexes rapidly aggregated and precipitated. They also investigated effect of added adamantinePEG on the stability of the CD-modified PEI polyplexes in vitro and in vivo [103]. The PEGylated CD-PEI-based polyplexes were stable at physiological salt concentrations without affecting gene expression in the PC3 cells compared to linear PEI and systemic delivery of gene into mice by the PEGylated carrier showed accumulation of the gene in the liver without toxicity. Similarly, self-assembly of adamantyl-modified PEG with the β-CD moieties of chitosan-graft-(PEI-β-CD) allowed the supramolecular PEGylation which significantly improved the stability of the polyplexes at physiological conditions [50]. Remarkably, the

In order to improve the buffering capacity and to promote the endosomal escape capability of the polyplexes, imidazole was introduced into β-CDP [109]. Imidazole, the functional unit of histidine amino acid, has a proton-sponge property and enhances release of gene into the cytoplasm [110]. From a live, single cell-based assay, imidazole-introduced CDP buffered the endocytic vesicles while PLL and CDP did not. The buffering activity is assumed to confer enhanced ability to escape the endocytic pathway that is correlated with an increase in transfection efficiency. However, it is unclear if the increased transfection efficiency of imidazole-introduced CDP is an outcome of enhanced endosomal escape, as imidazole-introduced CDP generates larger amounts of unpackaged intracellular DNA than CDP. These consequences raise the possibilities of multiple effects of CDP by simple modifications [111]. Another example of modification of β-CD is the conjugation of PEI with tosylated β-CD [112]. PEI has an excellent buffering capacity in an endosomal environment and facilitates endosomal escape to the cytoplasm [3]. The PEI-conjugated CD mediated nearly 4-fold higher transfection efficiency in HEK 293 cells with lower cytotoxicity than PEI 25 kDa due to the buffering capacity of the conjugated PEI and biocompatibility of CD moiety. Similarly, Yang et al. [113] conjugated low molecular weight PEI (MW of 423 and 600) onto 1,1′-carbonyldiimididazole-activated αCD to deliver DNA in HEK 293 and COS 7 cells. The oligoethylenimine-


conjugated α-CD star polymers showed higher transfection efficiency in the used cells with lower cytotoxicity than PEI 25 kDa and the polymers with longer and branched OEI arms showed higher transfection efficiency. However, they did not show relationship between buffering capacity and transfection efficiency.

4.4. Cell specificity Addition of targeting ligands to the gene carriers allows for receptormediated delivery. Therefore, galactosylated-based particles are used for selective targeting to hepatocytes via the asialoglycoprotein receptor. As a consequence, Pun et al. [104] introduced galactose as a specific ligand into adamantane-peptide-PEG which forms polyplexes with β-CDP due to the host/guest interaction of CD/adamantane. The galactosylated βCDP polyplexes transfected hepatoma cells with 10-fold higher efficiency than that of glucosylated β-CDP ones. Davis and his coworkers introduced transferrin (TF) as a targeting ligand into adamantane-PEG to target malignant cells that overexpress TF receptor [114]. The TF-containing CDP polyplexes transfected K562 leukemia cells with higher efficiency than only CDP polyplexes. Furthermore, they took the advantages of these polyplexes for siRNA delivery [115–119]. The TF-containing CDP/ siRNA polyplexes dramatically inhibited tumor growth in a murine model of metastatic Ewing's sarcoma with no innate immune responses and no abnormalities in liver and kidney functions via IV administration [115]. They also delivered ribonucleotide reductase subunit M2 (RRM2) siRNA in vitro and in vivo by TF-containing CDP for DNA replicationdependent diseases such as cancer [116]. The TF-containing CDP/siRNA showed significant knockdown of RRM2 protein in cultured cancer cells and significant tumor growth inhibition in murine model mice. Likewise, three consecutive daily doses of TF-containing CDP/siRNA 2.5 mg/kg in tumor-bearing mice via IV administration showed tumor growth inhibition [113]. Due to these effects, the delivery system termed as RONDEL™ was applied for the human Phase I clinical trial to patients having solid cancer refractory [118]. The TF-containing CDP/siRNA polyplexes were detected in post-treatment tumor biopsy sections and significant reduction in both the specific mRNA and protein was obtained. Furthermore, they delivered RRM2 siRNA in non-small cell lung cancer (NSCLC) and neck squamous cell carcinoma (HNSCC) cell lines and in a mouse xenograft model of HNSCC by the same carrier [119]. The polyplexes inhibited cell growth in both lung cancer cell lines and systemic delivery of the polyplexes via IV route significantly reduced tumor progression in a mouse model. Recently, Zhao et al. [120] conjugated folic acid (FA) into star-shaped γ-CD–OEI with disulfide linker for targeting to specific tumor cells overexpressing folate receptors (FRs). The FA–γ-CD–OEI delivered DNA to specific KB cells expressing FRs and showed higher gene expression in the KB cells due to facilitation of continuous FR-mediated endocytosis by recycling of FRs onto cellular membranes. Similarly, Li et al. [121] introduced FA into low molecular weight PEI cross-linked with 2hydroxy-propyl (HP)–β-CD to deliver VEGF siRNA in vitro and in vivo. The FA–HP–β-CD–PEI/siRNA complexes showed higher gene silencing efficiency (90%) in FA receptor-enriched HeLa cells with reduction of VEGF protein expression in the presence of 20% serum and marked inhibition of tumor growth in tumor-bearing mouse via tail vein injection. Oligopeptides were coupled to HP–β-CD–PEI [122] or β-CD–PEI [123] for targeting to human epidermal growth factor receptor 2 (HER 2) overexpressing in breast and ovary cancers, and for targeting to epidermal growth factor receptor (EFGR)-positive liver cancers. Compared to HP–β-CD–PEI, HP–β-CD–PEI-peptide showed strong targeting specificity to HER 2 receptor and significantly enhanced the anti-tumor effect on tumor-bearing mice by delivering interferon-α (IFN-α) gene in mice model via subcutaneous injection [122]. Similarly, β-CD-PEI-peptide showed higher acetylcholinesterase gene expression in EGFR-positive liver cancer cells and effectively inhibited tumor growth in tumorbearing mice via intraperitoneal injection [123].

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4.5. Dual delivery of biologics Recently, there has been an increasing interest in the development of multifunctional polymeric carriers by combining drug delivery and gene therapy to achieve a synergistic effect of drug and gene therapies [124]. Especially, CD-containing polymeric gene carriers have advantages to deliver hydrophobic drugs through inclusion complexes between hydrophobic moieties of the drugs and pending CD moieties. It is now well-documented that the host/guest interaction between hydrophobic benzyl groups and the pending CD moieties contributes to the nano-assembly of PEI-grafted β-CD and poly(β-benzyl-L-aspartate) (PBLA) [125]. The β-CD–PEI/PBLA assemblies co-delivered dexamethasone as a hydrophobic drug and DNA in an osteoblast cell line. It was found that a slight increase in transfection efficiency was obtained for the co-delivery system compared to the counterpart without DMS due to the increased nuclear translocation by the DMS. A well-known PLL dendron was conjugated with per-6-azido-β-CD to form β-CD–PLLD by click chemistry to co-deliver methotrexate as an anticancer drug and DNA in human breast cancer MCF-7 cells [126]. The nanocomplexes of β-CD–PLLD exhibited high gene transfection efficiency with anticancer activity demonstrating combinatorial delivery of DNA and anticancer drugs without a complicated micellization process. Recently, they co-delivered docetaxel (DOC) as the anticancer drug and MMP-9 siRNA as the therapeutic gene by the same carrier for the cancer therapy [127]. The β-CD–PLLD/DOC/siRNA complexes induced a more significant apoptosis in HNE-1 cells than DOC or MMP-9 siRNA only. To develop a synergistic co-delivery system for anticancer drug paclitaxel (PTX) and p53 gene for potential cancer therapy, supramolecular self-assembled inclusion complex was prepared from PTX, γ-CD and OEI arms with FA [128]. The inclusion complex, γ-CD–OEI–SS–FA/PTX enhanced the gene transfection even at low N/P ratios in the FR-positive KB cells and induced significant cell apoptosis, indicating the synergistic effects of co-delivery of PTX and p53 gene may be promising for cancer therapeutic application. 5. Concluding remarks and future perspectives Polymeric diversity is the most significant blessings for the material scientists to design effective non-viral gene vectors as alternative to viral carriers where the cationic polymers have received considerable attention in vitro and in vivo for efficient DNA or siRNA delivery. As efficient cationic gene carriers, PEI is the ‘state-of-the-art’ polymer for its high transfection efficiency due to its endosomal escape capacity; however, its high toxicity and non-degradable nature have blocked their promising use. Therefore, modification of PEI using various biodegradable cross-linkers to make them degradable and less toxic is a very useful strategy for their wide clinical application. At present, few of PEIbased gene carriers are underway of clinical trials which show their prosing applications in the future. It is worthy to mention that we have been extensively investigating for the last almost one decade to develop and screen a wide variety of PEI derivative as gene delivery vehicle. In this review, we have critically analyzed several of our previous gene carrier systems based on cationic polymer, especially, PEI derivatives. Moreover, we have elaborately explained various other gene carriers such as PAA and CD-based polycationic polymers. The degradable PEIs were classified into six categories such as ester, disulfide, imine, carbamate, amide and ketal linkages according to the degradable linkages in the PEI derivatives. The degradable PEIs having ester linkages were classified into linear, branched and grafted according to the structural differences after reactions. Generally, linear PEIs degrade more rapidly than branched and grafted PEIs through hydrolysis mechanism. The degradable PEIs having disulfide linkages were classified into degradable PEIs with disulfide bond in main chain and side chain according to the location of disulfide linkage in accordance with the location of disulfide linkage in the PEI derivatives. The degradable PEIs having disulfide linkages degrade in the intracellular reductive

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Table 3 Major degradable polycationic gene carriers and their therapeutic intervention against diseases. Types

Gene carriers

DNA or RNA

Target diseases (disease model)

Characters

References

Degradable PEI derivatives

RGD–PEG–SS–PEI

Luciferase gene

Intracranial glioblastoma (xenograft model)

[33]

FC-g-PEI

Akt1 shRNA

Lung cancer (urethane-induced lung cancer mouse model)

Degradable PAA derivatives

Guanidinylated poly(CBA-DAH)

Luciferase gene

Vasculogenic erectile dysfunction (hypercholesterolaemic mouse)

CD-based polymers (CDP)

TF-containing CDP

EWS-FLI1 siRNA

Ewing's sarcoma (metastatic Ewing's sarcoma mouse model) Head and neck tumor (xenograft model)

– Cancer targeting by RGD ligand – Redox-responsive disulfide linkage – Cancer targeting by folate ligand – Suppression of oncogene – Imine linkage – Cell penetrating ability by guanidine groups – Disulfide linkage – Cancer targeting by transferrin – Suppression of Ewing's family tumors – Cancer targeting by transferrin – Suppression of malignant progression – Cancer targeting by folic acid – Tumor growth suppression

RRM2 siRNA

FA–HP–β-CD–PEI

MC-10–HP–γ-CD–PEI

YC21–β-CD–PEI

Vascular endothelial growth factor protein siRNA IFN-α

Cancer (xenograft model)

Acetylcholinesterase gene

Liver cancer (xenograft model)

Breast and ovary cancer (xenograft model)

environment through reductively cleavable bonds in the PEI derivatives. However, this kind of PEI derivative is very stable in the extracellular environment, because cross-linking of low MW PEIs with functional cross-linking agents enhanced gene delivery efficiency with lower cytotoxicity due to the degradability of synthesized PEI and functionality of the cross-linking agents. The other cationic gene carriers such as PAA and CD also showed tremendous potential to increase gene transfer efficacy with lower level of toxicity. Although, enormous research works have been conducted on developing these cationic gene carriers, however more extensive investigations are urgently required to validate their promising clinical use in the future. In conclusion, therefore we propose several key suggestions for the future studies on this field as follows which would be important to consider: (1) a close evaluation on the structure–function relationship should be required for future direction of developing degradable cationic non-viral gene carriers; (2) investigation on the relationship between the physicochemical properties of gene carriers and gene functions can be another interesting field of study; (3) more elaborated in vivo experiments should be conducted and it is required to fully understand the in vivo behavior of the gene carriers because most of the gene carriers still represent their poor in vivo translatability, although they show nice in vitro results. The degradable polymeric gene carriers whose in vivo therapeutic effects were examined are listed in Table 3; (4) it would be very interesting to find out how gene carriers control their release profile based on the environment and timing. A successful observation on their controlled release manner at different intracellular compartment can open up revolutionary diversity to design efficient gene carriers as of interest; (5) until to date, the development of non-viral gene carriers are mainly focused on the improvement of gene transfer efficacy and reduction of their toxic effect. Although there are many successful results that have been reported, their regulation and effectivity on the body's immunological systems as well as cellular and molecular signaling mechanism have been mostly untouched and even not understood properly. A detailed investigation on these areas in the future can give the ultimate benefits to open up the door for their more potential use; and (6) finally; it is worth mentioning that this is the prime time to come up with the interdisciplinary approaches to work with non-viral gene vectors involving material scientists, chemists, engineers, molecular biologists, immunologists and clinicians altogether to understand

– Cancer targeting by MC-10 peptide which binds to human epidermal growth factor receptor 2 – Suppression of tumor growth and enhancement of survival time – Cancer targeting by YC21 peptide which binds to EGFR – Tumor growth suppression by regulating cell proliferation

[48]

[84]

[115] [119]

[117]

[122]

[123]

this very promising field of biomedical research more deeply for the real translation of these laboratory bench works to the bed-side clinical application.

Acknowledgments This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government through the Ministry of Science, ICT and Future Planning (MSIP) (2014R1A1A2007163).

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Polyamidoamine Dendrimer Conjugates with Cyclodextrins as Novel Carriers for DNA, shRNA and siRNA.

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Dendrimers as carriers for siRNA delivery and gene silencing: a review.

Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications.

Potential Use of Polyamidoamine Dendrimer Conjugates with Cyclodextrins as Novel Carriers for siRNA.

Cationic polyene phospholipids as DNA carriers for ocular gene therapy.

Degradable Cross-Linked Nanoassemblies as Drug Carriers for Heat Shock Protein 90 Inhibitor 17-N-Allylamino-17-demethoxy-geldanamycin.

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Cocoon-like self-degradable DNA nanoclew for anticancer drug delivery.

Layer-by-layer films with bioreducible and nonbioreducible polycations for sequential DNA release.

Multifunctional selenium nanoparticles as carriers of HSP70 siRNA to induce apoptosis of HepG2 cells.

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes.

A flexible microneedle array as low-voltage electroporation electrodes for in vivo DNA and siRNA delivery.

Chemotherapy Carriers for Triple Negative Breast Cancer.

Topical delivery of siRNA into skin using SPACE-peptide carriers.

A general strategy to prepare different types of polysaccharide-graft-poly(aspartic acid) as degradable gene carriers.

Nano-Sized Sunflower Polycations As Effective Gene Transfer Vehicles.

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Polyacylurethanes as Novel Degradable Cell Carrier Materials for Tissue Engineering.

Native chemical ligation for conversion of sequence-defined oligomers into targeted pDNA and siRNA carriers.

Magnesium alloys as a biomaterial for degradable craniofacial screws.

Local Delivery of PHD2 siRNA from ROS-Degradable Scaffolds to Promote Diabetic Wound Healing.

Polyspermine imine, a pH responsive polycationic siRNA carrier degradable to endogenous metabolites.