Polycations for Gene Delivery: Dilemmas and Solutions - American

Subscriber access provided by University of Sunderland

Review

Polycations for Gene Delivery: Dilemmas and Solutions Jie Chen, Kui Wang, Jiayan Wu, Huayu Tian, and Xuesi Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00688 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Polycations for Gene Delivery: Dilemmas and Solutions Jie Chen,†,‡,# Kui Wang,†,‡ Jiayan Wu,†,‡ Huayu Tian,*,†,‡,# and Xuesi Chen†,‡,# †Key

Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Changchun 130022, P. R. China ‡University #Jilin

of Science and Technology of China, Hefei 230026, P. R. China

Biomedical Polymers Engineering Laboratory, Changchun 130022, P. R. China

ABSTRACT: Gene therapy has been a promising strategy for treating numerous gene-associated human diseases by altering specific gene expressions in pathological cells. Application of non-viral gene delivery is hindered by various dilemmas encountered in systemic gene therapy. Therefore, solutions must be established to address the unique requirements of gene-based treatment of diseases. This review will particularly highlight the dilemmas in polycations-based gene therapy by systemic treatment. Several promising strategies, which are expected to overcome these challenges, will be briefly reviewed. This review will also explore the development of polycations-based gene delivery systems for clinical applications. INTRODUCTION Gene therapy has emerged as a promising strategy for specific treatment of human genetic diseases.1-3 Gene therapy introduces therapeutic genes into pathological cells by altering expressions of targeted endogenous genes to prevent the progression or to cure related diseases.4 Gene therapy has become one of the most potential biotechnological applications for treating genetic disorder-induced diseases, which generally cannot be treated by conventional methods.5-7 Gene-based strategy is expected to cure these disease fundamentally.8 However, naked genes are extremely difficult to be internalized by targeted cells because of their nuclease susceptibility in vivo, rapid clearance by phagocytic cells, and rare uptake by pathological cells. All these factors further restrict clinical application of gene therapy.9 These limitations highlight the crucial role of gene carriers in gene therapy.10 Although considerable progress has been achieved in this field, safety and effectiveness of gene carriers remain as major challenges.11 Commonly applied viral vectors are restricted by

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

their cargo loading and safety concerns.12 Over the past two decades, non-viral carriers have been proposed as safer alternatives for gene therapy.8,

13-15

Recently, nanoparticle-based

preliminary clinical trials have achieved promising effects for gene therapy.1, 16, 17 Nonetheless, wide scale application of gene therapeutics still faces many unavoidable challenges related to disease prevention and treatment. Dilemmas encountered in polycations-based non-viral gene therapy,18 include surface potentials, particle sizes, molecular weights, cytotoxicity, amphiphilicity, and stability of nanoparticles. Many strategies have been proposed to overcome these challenges encountered by existing gene carriers.16,

19, 20

In recent years, many clinical

trials, registered at http://www.clinicaltrials.gov, have been performed or are ongoing by making use of polycations-based gene delivery systems. However, to date, none of gene therapeutics based on polycations has so far been approved for marketing by the US Food and Drug Administration (FDA).1 The success of clinical applications mainly depends on development of safe, controllable, and efficient polycations-based gene delivery systems. In this review, we will particularly highlight the dilemmas encountered specifically in polycations-based gene therapy rather than cover all the aspects of this field. Then, we will review a number of conceivable solutions that have been extensively applied in recent years. Finally, the prospect of developing polycations-based carriers for gene therapy in the future will be summarized and assessed. VARIOUS TYPES OF DILEMMAS AND THEIR SOLUTIONS Clinical trials have reported that insufficient gene transfection efficiency remains as the most significant limitation of non-viral carrier-based gene therapy. Successful gene therapy depends on the complex process between physiological fluids and the extracellular space during gene transfection (Figure 1).16,

18, 21

Plasmid DNA in mice features a half-life of approximately

10 min following intravenous injection.22 Thus, a suitable gene carrier is necessary for packaging DNA into nanoparticles to improve circulation time and transfection efficiency. However, the complicated microenvironment in vivo requires gene carriers with appropriate physical and chemical properties.23 Several "dilemmas" must be balanced in overcoming obstacles of gene therapy. These factors include surface charges, particle sizes, molecular weight, cytotoxicity, amphiphilicity, bioconjugation, and stability.


Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Barriers of non-viral carrier-based in vivo gene therapy. Adapted with permission from ref 16. Copyright 2014, Nature Publishing Group. Charge Transport Dilemma Surface charge density of carrier/DNA complexes plays an important role not only in circulation in the physiological environment, but also in intracellular endocytosis.9 On the one hand, gene delivery systems with cationic components have attracted considerable attention because of their supernal water solubility and doughty capacity to package pDNA through electrostatic interactions.24 Compared with other types of vehicles, cationic polymers are



generally more effective because they can attach to anionic cell surfaces in large amounts, thus facilitating endocytosis in vacuolar compartments and promoting intracellular release of genes.25, 26

On the other hand, these abundant positively charged polymers may strongly adsorb the

negatively charged serum proteins to form large aggregations and induce vigorous immune responses.11 Therefore, cationic polymer-based gene delivery systems generally fail to achieve satisfactory responses in cancer therapy via systemic administration due to the nonspecific interactions with the negative components of blood and phagocytosis by non-target cells, leading to a rapid clearance from blood circulation.27 On basis of the above analysis, charge transport dilemma has been recognized as one of the most significant challenges in systemic gene therapy. In general, an excellent gene delivery system should possess a strong positive charge density when complexing with pDNA, and a weak charge density when circulating in the blood, and it should be capable of recovering positive charge density when encountering target cells. To address the "dilemma" in charge transport, polyethylene glycol modification (PEGylation) of cationic polymers can stabilize gene delivery systems, minimize nonspecific interactions, and prolong the circulation time, thereby increasing aggregation in tumor sites through enhanced permeation and retention (EPR) effect.28,

29

Shielded positive charges with polyanions30-32 of

cationic polymers is another effective strategy for prolonging circulation time of gene delivery systems to tumor sites via the EPR effect. Although introduction of PEG and polyanions has achieved significant progress in recent years, some obstacles should also be resolved. PEGylation or polyanions can significantly reduce intracellular uptake and endosomal escape of gene delivery systems after their accumulation in tumor tissues, thereby markedly diminishing gene transfection and antitumor efficiency.33 To overcome this challenge, Wang et al. developed a ternary gene delivery system with a tumor acidity-responsive PEGylated anionic polymer on the surface of positively charged polymer/siRNA complexes via electrostatic interactions (Figure 2).34 This ternary gene delivery system is stable at neutral or alkaline pH values and can separate the surface PEG layer at slightly acidic tumor extracellular microenvironments because of the existence of amide bonds, which facilitate delivery of siRNA to tumor cells after their accumulation at tumor tissues, thus promoting silencing efficiencies and enhancing inhibition of tumor growth.


Page 4 of 25

Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Schematic illustration of the acidity-responsive capacity and enhanced tumoral cell uptake of sheddable ternary nanoparticles. Adaped with permission from ref 34. Copyright 2012, American Chemical Society. An ideal gene delivery system should be capable of prolonging circulation time in the blood, improving transport efficiency to tumor sites, and promoting intracellular uptake of target cells.35 We previously designed a series of charge-reversal copolymers, which can be triggered by extracellular acidic microenvironments to enhance cellular internalization.9,

11,

36-38

Polyethylenimine-poly(ʟ-lysine)-poly(ʟ-glutamic acid) (PELG), a zwitterionic copolypeptide with rapid pH-responsive capacity, was prepared as the shielding system. PELG was negatively charged at a physical pH value and could be utilized as a shielding system to improve circulation and EPR effect of positively charged gene delivery systems in vivo. When the ternary polyplexes contacted with the acidic microenvironment of tumor tissues, they become positively charged by undergoing rapid charge conversion, thereby promoting intracellular uptake, enhancing transfection efficiency, and achieving an excellent tumor treatment effect.39 Different from amide bond cleavage charge-conversional systems, PELG-based system can achieve rapid charge conversion and be precisely controlled as amide bond cleavage occurs in a wide pH range.36 In addition, we further developed a facile strategy for constructing an ultrasensitive pH-triggered charge/size dual-rebound gene delivery system for tumor treatment (Figure 3).11 An anti-angiogenesis gene was complexed by polyethylenimine (PEI) and poly-ʟ-glutamate, and



then tightened in situ by aldehyde-modified PEG. The generated Schiff base bonds were stable under physiological conditions but were rapidly cleavable in the acidic microenvironment of tumors. Compared with the former strategy, this gene delivery system possesses more favorable properties as follows: (1) simpler preparation technique without chemical bench; (2) rapid peeling off of PEG shielding by acidic pH in a tumor microenvironment; (3) PEG cross-linking shielding not only shield the positive charges, but also tightening complex nanoparticles; and (4) dual charge/size rebounding mechanism. Accordingly, long circulation time and high tumor accumulation were achieved by the charge/size dual-rebound gene delivery system. Excellent antitumor therapeutic efficacy was also achieved, as evidenced by marked inhibition of tumor volume without affecting body weight, and without abnormal histomorphology and pathology of the main organs.11 Therefore, this strategy exhibits remarkable potential for tumor therapy in the future.


Page 6 of 25

Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Schematic illustration of the ultrasensitive pH triggered charge/size dual-rebound gene delivery system. Adapted with permission from ref 11. Copyright 2017, American Chemical Society. Size Dilemma Previous studies have illustrated that particle size of carrier/DNA complex critically affects encapsulation efficiency, stability, clearance, biodistribution, penetration, endocytosis, endosomal escape, and therapeutic efficacy against tumors in in vivo gene therapy.40,

41

In

general, large size gene delivery systems can be rapidly cleared from in vivo circulation, whereas small size ones exhibit inefficient endocytosis by tumor cells, leading to low gene transfection efficiency and antitumor effect.42 Size dilemma is another critical issue in non-viral gene therapy. Wang et al. demonstrated that 90 nm can be the optimal size of nanoparticles for gene delivery systems for macromolecular gene drugs.41 Researchers investigated the optimal size of cationic mixed micellar nanoparticles, which possess identical physiochemical properties except for size, for gene-based cancer therapy. Various sizes of nanocarriers for siRNA delivery were evaluated by the rational design. The previous study comprehensively evaluated the effects of nanoparticle size on circulation, retention, internalization, and antitumor effects. Results indicated that increasing size of nanocarriers not only enhanced the cellular uptake of siRNA, but also improved gene silencing efficiency and promoted cell apoptosis. Therefore, the size of nanocarriers plays a critical role in siRNA delivery by determining their biological properties and antitumor efficiency. Excellent gene delivery systems should utilize nanocarriers with a suitable size when circulating in blood and large size ones when encountering tumor cells. Guan et al. designed a pH-responsive size increasing strategy by detaching PEG shielding for the gene delivery system in tumor therapy (Figure 4).37 In this study, a PEI/DNA complex was in situ-shielded by the reaction of aldehyde-modified PEG with amino groups from PEI. The compressed size of the complex improved stabilization and prolonged the circulation time in vivo, thereby enhancing the EPR effect. PEG-shielded nanoparticles were stable under physiological conditions during transport. Once accumulated in the slightly acidic tumor environment with extracellular pH



value, cleavage of Schiff base bond was rapidly triggered, resulting in an increased size of the complex.

Figure 4. Schematic illustration of the pH-responsive size increasing gene delivery system. Adapted with permission from ref 37. Copyright 2017, American Chemical Society. Molecular Weight Dilemma The molecular weight of polycations-based gene delivery system plays an important role in the final encapsulation efficiency and releasing.43 Generally, the low molecular weight formed complexes presented higher sizes than those of the high molecular weight.44 The molecular weight has also shown influence in toxicity. Polycations with higher molecular weight can increase transfection efficiency, as well as more toxic.45 PEI with its molecular weight ranging from 5 to 25 kDa can effectively condense nucleic acid and form stable gene transfection complexes.46 Although oligoethylenimine (OEI) displays lower cytotoxic effect, it demonstrates poor DNA-binding ability because of forming big aggregate (0.5 to 2 μm) and reduces gene transfection efficiency compared with PEI25k (high molecular weight, 25 kDa). For PEI25k, it shows delightful efficiency for gene expression, while its application has been severely restricted by the significant cytotoxicity induced by nondegradable methylene backbones and high cationic charged density.47 Related with this dilemma, many strategies have been


Page 8 of 25

Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


performed to improve the feasibility of polycations-based gene delivery system. Chen et al. designed and synthesized a series of cationic copolymers by grafting different kinds of OEI onto a determinate and biodegradable multi-armed poly(ʟ-glutamic acid) backbone (MP-g-OEI) (Figure 5).48 The MP-g-OEI copolymers showed lower cytotoxicity and higher transfection efficiency than PEI25k both in the presence and absence of serum in different cell lines. The cytotoxicity of MP-g-OEI copolymers was further illustrated with their molecular weight and charge density. This strategy demonstrates considerable potentiality as non-viral carriers for gene therapy.

Figure 5. Schematic illustration of MP-g-OEI copolymers adapted with permission from ref 48. Copyright 2009, John Wiley & Sons, Ltd. Disulfide cross-linked biodegradable cationic polymers showed great potential as gene carriers for cancer therapy due to the reduction-sensitive property.49-51 Liu et al. developed a hyperbranched disulfide cross-linked PEI by ring-opening reaction of propylene sulfide. The efficiency of hyperbranched disulfide cross-linked PEI (lPEI-SS) was investigated as the siRNA



Page 10 of 25

vector for antitumor therapy on 4T1 tumor model. Significant anti-proliferation effect on 4T1 cell line was observed due to the effective induction of apoptosis of tumor cells. In addition, the tumor growth and metastasis of 4T1 tumor model were effectively inhibited by lPEI-SS/siRNA polyplexes.49 Jiang and co-workers prepared reduction degradable disulfide-containing azobenzene-terminated poly(2-(dimethylamino)ethyl methacrylate)s and supramolecular host– guest self-assembly systems with poly(cyclodextrin) (PCD/Az-ss-BPDMs), which was evaluated

as

a

non-viral

gene

delivery

system.

The

transfection

efficiency

of

PCD/Az-ss-BPDM/DNA polyplexes achieved almost 10 times higher amount than that of PEI25k. Whereas the cytotoxicity of the polyplexes was much lower than that of PEI, which was mainly due to the degradation of redox responsive supramolecular host–guest systems.50 Cytotoxicity Dilemma Cytotoxicity is one of the most major concerns for cationic polymer mediated gene delivery. The cationic components can interact with the negatively charged proteins and proteoglycans on cell surfaces, thereby inducing cytomembrane destabilization and necrosis.52 The cytotoxicity of gene delivery systems is derived from the density of positive charges on their surfaces.53 Usually, the density of positive charges increases with their improved molecular weight, thus resulting in much higher toxicity. However, with the increased cytotoxicity of cationic gene delivery systems, enhanced transfection efficiency emerges. For all these reasons, cytotoxicity dilemma is urgent to be solved or avoided in the process of gene therapy. Shuai and co-workers synthesized a ternary copolymer, by introducing disulfide linkages to graft lPEI to the block polymer of poly(ʟ-lysine) (PLL) and PEG for siRNA therapy.54 The disulfide linkage and PLL block of the carrier rendered the favorable biodegradability and low cationic toxicity. Conjugation of Herceptin antibody as a targeting ligand for Her2/neu receptor of Skov-3 cells significantly improved the silencing activity of the nanocomplex. The distinct features of easy degradability, low cytotoxicity, and strong gene silencing efficiency make the copolymer a promising candidate for gene therapy. Several other strategies have also been attempted to reduce the toxicity of cationic polymer-mediated gene delivery, including introduction of PEG segments,55 cross-linking via reducible bonds,49, 56 conjugation with ligands,57 and introduction of hydrophobic or hydrophilic segments.58-62


Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Amphiphilicity Dilemma It is commonly believed that amphiphilic polycation derivatives can improve gene delivery efficiency due to their similar structures with the lipophilic cell membrane, thus increasing the cellular uptake efficiency of complex particles.63, 64 The hydrophobic segments are suggested to conjugate with polycations to obtain amphiphilic copolymers, which may influence some major processes of gene therapy. (1) Hydrophobic interactions can contribute to complex formation between amphiphilic carriers and genes because that the hydrophobic moieties may provide a cooperative binding with genes.65 (2) Amphiphilic carriers are able to promote the charge inversion of carrier/DNA complex, which facilitating contact with the negatively charged cell membrane and permitting the subsequent gene transfection.66 (3) Introduction of hydrophobic segments can enhance the interactions between plasma and phospholipid bilayer membranes. In addition, the enhanced adsorption can facilitate the subsequent endocytosis.63 (4) Conjugation of polycations with hydrophobic segments effectively achieves the alleviation of serum inhibition due to the stable complexes formation in serum.67 (5) Hydrophobization can facilitate gene dissociation from cationic carriers because of the weakened bonding efficiency between amphiphilic carriers and DNA.68 However, the degree of hydrophobicity and chemical structure in the amphiphilic gene delivery systems is the most critical point in many aspects of gene therapy. Thomas et al. carried out systematic modifications of commercial PEIs with hydrophobic segments to enhance PEI-mediated transfection by alerting the proton sponge capacity, hydrophobic-hydrophilic balance and lipphilicity.69 Anderson's group synthesized and optimized a library of amphiphilic carriers for gene delivery to different types of cells. Different length of alkyl chains modified dendrimers were performed to evaluate for their ability to deliver genes to target cells.70-72 Yang and colleagues prepared a series of amphiphilic gene carriers by modifying PEI1.8k with different hydrophobic functional groups to investigate how molecular structure, hydrophobicity, and conjugation degree in the conjugated side groups influence transfection efficiency.73 This study demonstrated that hydrophobic segments modified PEI1.8k polymer can effectively condense pDNA into cationic nanoparticles of about 100 nm, whereas PEI1.8k showed much larger particle sizes of 2 µm. Hydrophobic modification also increased gene transfection efficiency. Importantly, the modification degree greatly influenced gene transfection, and complete substitution of primary amine groups could lead to



Page 12 of 25

significantly lower transfection efficiency. These findings provided us insights to modification of cationic carriers for the development in the field of gene therapy. Bioconjugation Dilemma Ineffective transfection efficiency remains a critical issue in non-viral gene delivery.74,

75

Improving delivery and target efficiencies of nanocarriers is a key strategy for augmenting the effectiveness of their application. Conjugation of targeting bioligands to gene carriers has demonstrated remarkable potential in treating various genetic diseases through active targeted payload delivery.18 High-affinity interactions between bioligands and receptors on the target cells, can improve cellular uptake and accumulation of therapeutic genes.30 Anderson et al. first conducted an in-human clinical trial of systemic administration of transferrin-targeted non-viral gene delivery systems to a 4-year-old girl with severe combined immunodeficiency disease.5 Since then, numerous theoretical studies on bioconjugated nanoparticle-based gene delivery systems have progressed into clinical trials.1 Most of these systems consisted of target ligands that bind to the receptors of pathological cells, PEG that improve steric stabilization, and cationic units that bind to nucleic acids. Despite the advances in clinical studies and many successful outcomes of experimental studies, no universal procedure has been established for targeting ligands.76,

77

A common dilemma is that bioconjugate ligands can accelerate their

clearance from the circulation and further hinder their target tissue penetration, although they can promote cell internalization once they arrive at the target site.18, 78 Bioconjugation dilemma can be primarily solved by improving anti-fouling capacity to protect metabolism during circulation and to promote effective targeting to specific sites. PEGylation can effectively decrease non-specific phagocytosis and prolong circulation time at the expense of compromising target-specific internalization.79 Wagner et al. synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with PEGylated epidermal growth factor (EGF) peptides for targeted gene delivery. The targeted strategy demonstrated more than 10-fold gene transfection efficiency compared with PEGylated complexes without EGF.80 Cleavable PEG-conjugated nanocarriers can potentially overcome this dilemma by preventing rapid clearance and inefficient targeted delivery.81, 82 Torchilin et al.83 constructed a multifunctional immunoliposomal nanocarrier that contains TAT peptides, long pH-sensitive


Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PEG-PE segments, and cancer cell-specific mAb 2C5. TAT peptides were conjugated with a short PEG1k chain and shielded by long pH-sensitive PEG-PE segments, whereas mAb 2C5 was attached to a long PEG3.4k chain. Under normal conditions, TAT peptides were "hidden" by the long PEG chains. When exposed to a low pH environment, degradation of hydrazone bonds prompted the removal of long PEG chains, thereby exposing the TAT conjugates. This strategy showed promising potential in overcoming bioconjugation dilemma to realize efficient gene therapy. Stability Dilemma Excellent non-viral carriers for clinical gene therapy should be stable in storage and degradable in in vivo applications. Gene carriers must package nucleic acids into stable nanoparticles in storage, but must be degradable under specific microenvironment, including pH, redox, and bio-enzymes in the body.84, 85 To improve storage stability, polypeptide-based gene delivery systems have attracted significant attention owing to their inherent bioactivity, biocompatibility, and biodegradability. Peptides play prominent roles in normal metabolic processes, including biological identification based on peptide sequences, antigen–antibody reactions, and signal transduction.30,

86, 87

Researchers have recently developed various other

biodegradable gene delivery systems, such as poly(lactic-co-glycolic acid),88 carbothydrate,89 polyphosphoesters,25 and polyamidoamine.90 Transmission stability also restricts transfection efficiency of many non-viral carriers. An ideal delivery system should be stable in the extracellular environment to protect nucleic acids from enzymatic degradation and must be capable of controlling the release of nucleic acids once inside the cells. In other words, integrity of carrier/DNA complex should be maintained during circulation, then, carrier/DNA complex must be de-packaged appropriately to release DNA to achieve its bioavailability to therapeutic sites and to realize effective gene therapy.91 Environmentally responsive non-viral gene delivery systems, triggered by abnormal disease pathology or unique microenvironments in cells, can potentially overcome this dilemma. Tumor microenvironments have been extensively studied because of their specific markers, such as high temperature, low pH, hypoxia, and overexpression of some enzymes.24 Radical differences between extracellular and intracellular microenvironments may be the key to resolving this issue.



Microenvironmental sensitive gene delivery systems have recently been explored for treating various diseases.21, 92, 93 A notable redox gradient exists between the extracellular environment and intracellular cytosol due to the intrinsic biological signal of cytoplasmic glutathione (GSH), which has been extensively explored for enhanced cancer therapy.94 GSH levels in the blood measure approximately 2 µM, whereas that of the intracellular environment ranges from 2 mM to 10 mM.95 Mckenzie et al. developed a class of peptide-based delivery agents by inserting multiple cysteine residues into synthetic peptides (Figure 6).96 Low molecular-weight peptides could oxidize spontaneously after binding to pDNA to form interpeptide disulfide bonds. The stability of cross-linked peptide/DNA complexes significantly improved with increasing number of cysteines incorporated into the peptides. Formation of disulfide linkages prompted compression in particle size and prevented their dissociation in concentrated sodium chloride compared with control peptide/DNA complexes. Maximal transfection efficiency was achieved depending on the number of incorporated cysteine residues, which were involved in the intracellular release of DNA triggered by disulfide bond reduction rather than increasing DNA uptake by cells.

Figure 6. Formation of peptide-based cross-linked peptide/DNA condensates. Adapted with permission from ref 96. Copyright 2000, American Society for Biochemistry and Molecular Biology. This cross-linked strategy can effectively improve transmission stability in vitro of non-viral gene delivery systems. However, transport barriers in vivo should be further improved. Guan et al. developed a pH-responsive detachable PEG shielding strategy that can overcome the obstacle


Page 14 of 25

Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in circulation. Carriers/DNA complexes can be further compressed after shielding with PEG. The shielded nanocomplexes were stable during transport. When accumulated at tumor tissues, the acidic tumor microenvironment triggered the cleavage of Schiff base bonds and detached the shielding systems, thereby exposing binary cationic complexes and improving gene therapy efficiency. The detailed strategies were illustrated in the previous summarization (Figure 3 and Figure 4)11,

37.

Overall, environmental sensitive cross-linked non-viral gene delivery systems

possess significant potential in enhancing gene therapy through a new mechanism of action. Dilemma in Co-delivery systems Compared with monotherapy, the combinatorial delivery of genes and drugs through polymeric carriers will be a potentially effective strategy for favoring synergistic therapeutic effects of both agents to maximize therapeutic efficiency.97 This approach is expected to be effective for combating cancer and other diseases in future. Co-delivery systems can simultaneously encapsulate therapeutic genes and drugs in a stable manner and transport them to the desired cellular compartments to facilitate their controlled release.98,

99

Therefore,

combination therapy of appropriate genes and drugs can effectively overcome multidrug resistance of cancers, inhibit the anti-apoptotic process, and achieve synergistic therapeutic effects. However, co-delivery systems face certain key dilemmas, including stability, loading capacity, and release kinetics.100 On the one hand, co-delivery systems should possess the capacity to load therapeutic genes and drugs simultaneously within a single platform. Thus, carriers should possess the chemical functionalities to combine with genes via electrostatic interactions and encapsulate small molecule drugs via covalent or non-covalent interactions.101 On the other hand, to achieve maximum therapeutic effect, the release of genes or drugs from the co-delivery systems should be controlled strictly based on their functional mechanism. Duration of translation of exogenous DNA or gene silencing by siRNA lasts for 24-72 h after cell endocytosis, whereas drugs can work immediately upon release, implying that genes and drugs should be sequentially released from co-delivery systems.100, 101 Shuai et al. synthesized a diblock copolymer comprising hydrophobic poly(ε-caprolactone) (PCL) and hydrophilic linear PEI to assemble biodegradable nanoparticles. PEGylation of the nanoparticles was performed through hierarchical assembly strategy by electrostatic coating of



folate-encoded PEGylated polyglutamic acid to PEI-PCL/siRNA complexes to achieve EPR effect and receptor-mediated endocytosis.102 The results showed that co-delivery of B-cell lymphoma-2 (BCL-2) siRNA and doxorubicin (DOX) could significantly inhibited tumor growth and significantly prolonged survival of C6 glioma-bearing rats. Zhang et al. prepared a triblock copolymer micelle based on N-succinyl chitosan–poly-ʟ-lysine–palmitic acid for co-delivery of siRNA-P-glycoprotein (P-gp) and DOX. DOX was encapsulated in the core by hydrophobic interaction, whereas siRNA was bonded in the interface layer via electrostatic interactions (Figure 7).103 This strategy can overcome the membrane transport protein (P-gp)-related pump resistance, which is the mechanism of cancer multidrug resistance. P-gp can efflux drugs from cells and significantly decrease drug concentration and efficiency. Although, significant achievements have been attained, additional systematic studies should be conducted to ensure that each therapeutic agent can act sufficiently at its target site.

Figure 7. Schematic illustration of the gene/drug co-delivery system for synergistic tumor therapy. Adapt with permission from ref 103. Copyright 2016, Macmillan Publishers Limited, part of Springer Nature. Synergistic treatment of an adjuvant together with DNA vaccine will be a potential effective strategy for immunotherapy. In order to achieve an ideal immune response, DNA vaccines should be delivered to the cytoplasm of the target cells to induce effective gene expression, thereby enabling antigen presentation and T-cell recognition. Although several early


Page 16 of 25

Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


researches have confirmed the potential of nucleic acid vaccines, many practical problems still need to be addressed. Among them, the limited gene expression of DNA vaccines extremely restricts their further application.104 Recently, cationic polymers have been extensively applied as a robust adjuvant of nucleotide vaccine. These polymers exhibit the special capacity of improving the efficiency of DNA vaccines by enhancing maturation rates of antigen-presenting cells (APCs), increasing proliferation of effector T cells, and enlarging the production of antigen specific antibodies and cytokines.105 In regard to the advantages of cationic polymer adjuvants, some optimal properties have been proposed: (1) compared with naked DNA, cationic polymer/DNA complexes can improve the endocytosis process by APCs; (2) multiple antigens are synthesized in APCs and other bystander cells (myocytes or fibroblasts) due to the effective transfection of polymer/DNA complexes; (3) many biological properties of the cationic polymers have attracted worldwide attention, including controllable spatial structure, easy modification, and other aspects of biological activities.106 Nevertheless, the application of polycations-based gene vaccines is restricted not only by the complicated microenvironment in vivo, but also by the safety issues of polycations for their toxicity.105 To solve this dilemma, polycations with the property of biodegradability have potential values, such as disulfide cross-linked low molecular weight cationic polymers and polypeptides. Furthermore, conjugation of target ligands (such as mannose) to polymers for the receptors of APCs can also improve the efficiency of DNA vaccines. Several other strategies also have great potential to reduce the toxicity of cationic polymer-mediated gene delivery, including introduction of PEG segments, cross-linking via reducible bonds, and introduction of hydrophobic or hydrophilic segments.85, 107 Conclusions and Future Perspectives In summary, a comprehensive understanding of the "dilemmas" in gene therapy can guide the design of more intelligent carriers (Table 1). With rapid developments in nanotechnology and biological sciences, the comprehensive route of gene delivery systems entering target cells and realization of their functions will be further clarified. Multifunctional delivery systems that feature controllable on and off switch for various functions at the appropriate time and space can overcome physiological barriers and carry DNA to the targeted regions. More importantly,



Page 18 of 25

security of the non-viral gene delivery systems should be reconsidered thoroughly before clinical applications. Table 1 Representative polycations for overcoming the dilemmas of gene delivery Dilemmas Surface charge

Particle size Molecular weight/ Cytotoxicity

Amphiphilicity

Bioconjugation

Stability

Co-delivery systems

Polycations

Solutions

Reference

OEAL/PEI

pH-sensitive shielding

9

PEG/PLG/PEI PLG/PEI PEG/ssPEI800 PEG-PAsp(MEA)-PEI Albumin/rPAMG mPEG-b-PCL/PCL-b-PPEEA PEG/PEI Star-(PEG-b-PEI)

pH-sensitive shielding Shielding system pH-sensitive shielding Redox and pH dual-sensitive Redox-sensitive Different particles sizes pH-sensitive shielding Multi-arm-block low MW PEIs

11 31, 32 34 19 23 41 37 47

MP-g-OEI lPEI-SS

Multi-arm-grafted low MW PEIs Disulfide cross-linked low MW PEIs Redox and light dual-sensitive Redox-sensitive Hydrophobic modification Hydrophobic modification Hydrophobic modification Hydrophobic modification

48 49

PEGylated PEGylated PEGylated and targeting Targeting PEGylated and targeting Peptide corona and stabilized interface Redox and pH dual-sensitive TPP modified pH-sensitive Targeting and amphiphilic micelles Amphiphilic micelles

25 26 29 30 5 90

PCD/Az-ss-BPDM PGED-NG Fluorinated carriers PEI-PBLG Alkyl chains modified dendrimers Different hydrophobic moieties modified PEI1.8k mPEG-b-PCL-b-PPEEA mPEG-b-PCL-b-PLL PEG/Tf-PEI RGD-HA/PEI-PBLG Tf-PEG-AD/CDP Supramolecular hybrid dendrimers PEG-PAsp(MEA)-PEI iRGD-PMDM D-PCE/BCL-2/FA NSC-PLL-PA

AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Phone: +86-431-85262539


50 56 14 59

71 64, 73

19 93 102 103

Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ORCID Jie Chen: 0000-0003-1945-0047 Huayu Tian: 0000-0002-2482-3744 Xuesi Chen: 0000-0003-3542-9256 ACKNOWLEDGEMENTS This work was supported by the National Key Research and Development Program of China (Grant No. 2016YFC1100701), National Natural Science Foundation of China (Grant Nos. 51873208, 51803210, 51520105004, 51390484 and 21474104), Jilin province science and technology development program (Grant Nos. 20160204032GX and 20180414027GH). REFERENCES (1) (2) (3) (4)

(5)

(6) (7) (8) (9)

(10) (11) (12) (13)

Chen, J., Guo, Z. P., Tian, H. Y., and Chen, X. S. (2016) Production and clinical development of nanoparticles for gene delivery. Mol Ther-Meth Clin D 3, 16023. Scholz, C., and Wagner, E. (2012) Therapeutic plasmid DNA versus siRNA delivery: Common and different tasks for synthetic carriers. J Control Release 161, 554-565. Mulligan, R. C. (1993) The Basic Science of Gene-Therapy. Science 260, 926-932. Chen, J., Jiao, Z. X., Lin, L., Guo, Z. P., Xu, C. N., Li, Y. H., Tian, H. Y., and Chen, X. S. (2015) Polylysine-modified polyethylenimines as siRNA carriers for effective tumor treatment. Chinese J Polym Sci 33, 830-837. Davis, M. E., Zuckerman, J. E., Choi, C. H. J., Seligson, D., Tolcher, A., Alabi, C. A., Yen, Y., Heidel, J. D., and Ribas, A. (2010) Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464, 1067-U140. Sun, W. J., and Gu, Z. (2017) Tailoring non-viral delivery vehicles for transporting genome-editing tools. Sci China Mater 60, 511-515. Anderson, W. F. (1984) Prospects for Human-Gene Therapy. Science 226, 401-409. Li, S. D., and Huang, L. (2006) Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Ther 13, 1313-1319. Chen, J., Dong, X., Feng, T. S., Lin, L., Guo, Z. P., Xia, J. L., Tian, H. Y., and Chen, X. S. (2015) Charge-conversional zwitterionic copolymer as pH-sensitive shielding system for effective tumor treatment. Acta Biomater 26, 45-53. Xu, J., Ganesh, S., and Amiji, M. (2012) Non-condensing polymeric nanoparticles for targeted gene and siRNA delivery. Int J Pharmaceut 427, 21-34. Guan, X. W., Guo, Z. P., Lin, L., Chen, J., Tian, H. Y., and Chen, X. S. (2016) Ultrasensitive pH Triggered Charge/Size Dual-Rebound Gene Delivery System. Nano Lett 16, 6823-6831. Sun, J. Y., Anand-Jawa, V., Chatterjee, S., and Wong, K. K. (2003) Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther 10, 964-976. Zhao, B. W., Zhou, Z. X., and Shen, Y. Q. (2016) Effects of chirality on gene delivery efficiency of polylysine. Chinese J Polym Sci 34, 94-103.



(14) (15) (16) (17)

(18) (19)

(20) (21) (22)

(23) (24) (25)

(26)

(27)

(28) (29)

(30) (31) (32)

(33)

Cheng, Y. Y. (2017) Fluorinated Polymers in Gene Delivery. Acta Polym Sin, 1234-1245. Zhang, Y., Liu, L., Wang, T. H., Tian, H. Y., and Chen, X. S. (2017) Synthesis of pH-sensitive Nanogels and Their Application in siRNA Delivery. Acta Polym Sin, 1150-1158. Yin, H., Kanasty, R. L., Eltoukhy, A. A., Vegas, A. J., Dorkin, J. R., and Anderson, D. G. (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet 15, 541-555. Fang, H. P., Guo, Z. P., Lin, L., Chen, J., Sun, P. J., Wu, J. Y., Xu, C. N., Tian, H. Y., and Chen, X. S. (2018) Molecular Strings Significantly Improved the Gene Transfection Efficiency of Polycations. J Am Chem Soc 140, 11992-12000. Zou, H. J., Wang, Z. J., and Feng, M. (2015) Nanocarriers with tunable surface properties to unblock bottlenecks in systemic drug and gene delivery. J Control Release 214, 121-133. Li, J. G., Yu, X. S., Wang, Y., Yuan, Y. Y., Xiao, H., Cheng, D., and Shuai, X. T. (2014) A Reduction and pH Dual-Sensitive Polymeric Vector for Long-Circulating and Tumor-Targeted siRNA Delivery. Adv Mater 26, 8217-8224. Xu, M. M., Liu, R. J., and Yan, Q. (2018) Biological Stimuli-responsive Polymer Systems: Design, Construction and Controlled Self-assembly. Chinese J Polym Sci 36, 347-365. Li, Y., Gao, J., Zhang, C., Cao, Z., Cheng, D., Liu, J., and Shuai, X. (2017) Stimuli-Responsive Polymeric Nanocarriers for Efficient Gene Delivery. Topics in current chemistry 375, 27. Kawabata, K., Takakura, Y., and Hashida, M. (1995) The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharmaceutical research 12, 825-30. Chen, Y., Li, J., and Oupicky, D. (2018) Conjugate Polyplexes with Anti-Invasive Properties and Improved siRNA Delivery In Vivo. Bioconjugate chemistry 29, 296-305. Tian, H. Y., Chen, J., and Chen, X. S. (2013) Nanoparticles for Gene Delivery. Small 9, 2034-2044. Sun, T. M., Du, J. Z., Yan, L. F., Mao, H. Q., and Wang, J. (2008) Self-assembled biodegradable micellar nanoparticles of amphiphilic and cationic block copolymer for siRNA delivery. Biomaterials 29, 4348-4355. Qi, R. G., Liu, S., Chen, J., Xiao, H. H., Yan, L. S., Huang, Y. B., and Jing, X. B. (2012) Biodegradable copolymers with identical cationic segments and their performance in siRNA delivery. J Control Release 159, 251-260. Dash, P. R., Read, M. L., Barrett, L. B., Wolfert, M., and Seymour, L. W. (1999) Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery. Gene Ther 6, 643-650. Sun, Q. H., Zhou, Z. X., Qiu, N. S., and Shen, Y. Q. (2017) Rational Design of Cancer Nanomedicine: Nanoproperty Integration and Synchronization. Adv Mater 29. Ogris, M., Brunner, S., Schuller, S., Kircheis, R., and Wagner, E. (1999) PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 6, 595-605. Tian, H. Y., Lin, L., Chen, J., Chen, X. S., Park, T. G., and Maruyama, A. (2011) RGD targeting hyaluronic acid coating system for PEI-PBLG polycation gene carriers. J Control Release 155, 47-53. Xia, J. L., Chen, J., Tian, H. Y., and Chen, X. S. (2010) Synthesis and characterization of a pH-sensitive shielding system for polycation gene carriers. Sci China Chem 53, 502-507. Xia, J. L., Tian, H. Y., Chen, J., Guo, Z. P., Lin, L., Yang, H. Y., and Feng, Z. C. (2016) Polyglutamic acid based polyanionic shielding system for polycationic gene carriers. Chinese J Polym Sci 34, 316-323. Hatakeyama, H., Akita, H., and Harashima, H. (2013) The Polyethyleneglycol Dilemma: Advantage


Page 20 of 25

Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34) (35) (36)

(37)

(38)

(39) (40)

(41)

(42)

(43) (44)

(45) (46) (47)

(48)

(49)

(50)

and Disadvantage of PEGylation of Liposomes for Systemic Genes and Nucleic Acids Delivery to Tumors. Biol Pharm Bull 36, 892-899. Yang, X. Z., Du, J. Z., Dou, S., Mao, C. Q., Long, H. Y., and Wang, J. (2012) Sheddable Ternary Nanoparticles for Tumor Acidity-Targeted siRNA Delivery. Acs Nano 6, 771-781. Tian, H. Y., Tang, Z. H., Zhuang, X. L., Chen, X. S., and Jing, X. B. (2012) Biodegradable synthetic polymers: Preparation, functionalization and biomedical application. Prog Polym Sci 37, 237-280. Tian, H. Y., Guo, Z. P., Lin, L., Jiao, Z. X., Chen, J., Gao, S. Q., Zhu, X. J., and Chen, X. S. (2014) pH-responsive zwitterionic copolypeptides as charge conversional shielding system for gene carriers. J Control Release 174, 117-125. Guan, X., Guo, Z., Wang, T., Lin, L., Chen, J., Tian, H., and Chen, X. (2017) A pH-Responsive Detachable PEG Shielding Strategy for Gene Delivery System in Cancer Therapy. Biomacromolecules 18, 1342-1349. Guan, X., Li, Y., Jiao, Z., Lin, L., Chen, J., Guo, Z., Tian, H., and Chen, X. (2015) Codelivery of antitumor drug and gene by a pH-sensitive charge-conversion system. ACS applied materials & interfaces 7, 3207-15. Chen, J., Li, X. Z., Tian, H. Y., Zhu, X. J., and Chen, X. S. (2015) Application of Zwitterionic Polymers in the Treatment of Malignant Tumors. Chem J Chinese U 36, 2148-+. Zhao, P. F., Zheng, M. B., Yue, C. X., Luo, Z. Y., Gong, P., Gao, G. H., Sheng, Z. H., Zheng, C. F., and Cai, L. T. (2014) Improving drug accumulation and photothermal efficacy in tumor depending on size of ICG loaded lipid-polymer nanoparticles. Biomaterials 35, 6037-6046. Liang, S., Yang, X. Z., Du, X. J., Wang, H. X., Li, H. J., Liu, W. W., Yao, Y. D., Zhu, Y. H., Ma, Y. C., Wang, J. et al. (2015) Optimizing the Size of Micellar Nanoparticles for Efficient siRNA Delivery. Adv Funct Mater 25, 4778-4787. Guo, Z. P., Chen, J., Lin, L., Guan, X. W., Sun, P. J., Chen, M. W., Tian, H. Y., and Chen, X. S. (2017) pH triggered size tncreasing gene carrier for efficient tumor accumulation and excellent antitumor effect. ACS applied materials & interfaces 9, 15297-15306. Diez, S., and de Ilarduya, C. T. (2006) Versatility of biodegradable poly(D,L-lactic-co-glycolic acid) microspheres for plasmid DNA delivery. Eur J Pharm Biopharm 63, 188-197. vandeWetering, P., Cherng, J. Y., Talsma, H., and Hennink, W. E. (1997) Relation between transfection efficiency and cytotoxicity of poly(2-(dimethylamino)ethyl methacrylate)/plasmid complexes. J Control Release 49, 59-69. Godbey, W. T., Wu, K. K., and Mikos, A. G. (1999) Size matters: Molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle. J Biomed Mater Res 45, 268-275. Neu, M., Fischer, D., and Kissel, T. (2005) Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. J Gene Med 7, 992-1009. Petersen, H., Kunath, K., Martin, A. L., Stolnik, S., Roberts, C. J., Davies, M. C., and Kissel, T. (2002) Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines. Biomacromolecules 3, 926-936. Chen, L., Tian, H. Y., Chen, J., Chen, X. S., Huang, Y. B., and Jing, X. B. (2010) Multi-armed poly(L-glutamic acid)-graft-oligoethylenimine copolymers as efficient nonviral gene delivery vectors. J Gene Med 12, 64-76. Liu, S., Huang, W., Jin, M. J., Fan, B., Xia, G. M., and Gao, Z. G. (2016) Inhibition of murine breast cancer growth and metastasis by survivin-targeted siRNA using disulfide cross-linked linear PEI. Eur J Pharm Sci 82, 171-182. Jiang, Q. M., Zhang, Y. T., Zhuo, R. X., and Jiang, X. L. (2016) A light and reduction dual sensitive



(51) (52)

(53)

(54)

(55) (56)

(57)

(58)

(59)

(60)

(61)

(62)

(63) (64)

(65)

(66)

supramolecular self-assembly gene delivery system based on poly(cyclodextrin) and disulfide-containing azobenzene-terminated branched polycations. J Mater Chem B 4, 7731-7740. Guo, Z. P., Tian, H. Y., Xia, J. L., Chen, J., Lin, L., and Chen, X. S. (2010) Bioreducible crosslinked low molecular weight branched PEI-PBLG as an efficient gene carrier. Sci China Chem 53, 2490-2496. Chen, J., Tian, H. Y., Dong, X., Guo, Z. P., Jiao, Z. X., Li, F. F., Kano, A., Maruyama, A., and Chen, X. S. (2013) Effective Tumor Treatment by VEGF siRNA Complexed with Hydrophobic Poly(Amino Acid)-Modified Polyethylenimine. Macromol Biosci 13, 1438-1446. Chen, J., Liang, H., Lin, L., Guo, Z., Sun, P., Chen, M., Tian, H., Deng, M., and Chen, X. (2016) Gold-Nanorods-Based Gene Carriers with the Capability of Photoacoustic Imaging and Photothermal Therapy. ACS applied materials & interfaces 8, 31558-31566. Li, J. G., Cheng, D., Yin, T. H., Chen, W. C., Lin, Y. J., Chen, J. F., Li, R. T., and Shuai, X. T. (2014) Copolymer of poly(ethylene glycol) and poly((L)-lysine) grafting polyethylenimine through a reducible disulfide linkage for siRNA delivery. Nanoscale 6, 1732-1740. Moghimi, S. M., Hunter, A. C., and Murray, J. C. (2001) Long-circulating and target-specific nanoparticles: Theory to practice. Pharmacol Rev 53, 283-318. Li, R. Q., Wu, W., Song, H. Q., Ren, Y. L., Yang, M., Li, J. S., and Xu, F. J. (2016) Well-defined reducible cationic nanogels based on functionalized low-molecular-weight PGMA for effective pDNA and siRNA delivery. Acta Biomater 41, 282-292. Luo, Y. F., Zhai, X. Y., Ma, C. N., Sun, P., Fu, Z. P., Liu, W. G., and Xu, J. (2012) An inhalable beta(2)-adrenoceptor ligand-directed guanidinylated chitosan carrier for targeted delivery of siRNA to lung. J Control Release 162, 28-36. Li, R. Q., Wu, Y. N., Zhi, Y., Yang, X. C., Li, Y. L., Xua, F. J., and Du, J. (2016) PGMA-Based Star-Like Polycations with Plentiful Hydroxyl Groups Act as Highly Efficient miRNA Delivery Nanovectors for Effective Applications in Heart Diseases. Adv Mater 28, 7204-+. Chen, J., Tian, H. Y., Guo, Z. P., Xia, J. L., Kano, A., Maruyama, A., Jing, X. B., and Chen, X. S. (2009) A Highly Efficient siRNA Carrier of PBLG Modified Hyperbranched PEI. Macromol Biosci 9, 1247-1253. Chen, J., Tian, H. Y., Guo, Z. P., Lin, L., Dong, X., Zhu, X. J., and Chen, X. S. (2012) Hyperbranched PEI Grafted by Hydrophilic Amino Acid Segment Poly[N-(2-hydroxyethyl)-L-glutamine] as an Efficient Nonviral Gene Carrier. J Appl Polym Sci 123, 2257-2265. Chen, J., Guo, Z. P., Lin, L., Hu, Y. Y., Tian, H. Y., Chen, M. W., and Chen, X. S. (2017) Combination therapy of pDNA and siRNA by versatile carriers composed of poly(L-serine) modified polyethylenimines. Mater Chem Front 1, 937-946. Gao, S. Q., Tian, H. Y., Xing, Z. K., Zhang, D. W., Guo, Y., Guo, Z. P., Zhu, X. J., and Chen, X. S. (2016) A non-viral suicide gene delivery system traversing the blood brain barrier for non-invasive glioma targeting treatment. J Control Release 243, 357-369. Liu, Z. H., Zhang, Z. Y., Zhou, C. R., and Jiao, Y. P. (2010) Hydrophobic modifications of cationic polymers for gene delivery. Prog Polym Sci 35, 1144-1162. Ong, Z. Y., Yang, C., Cheng, W., Voo, Z. X., Chin, W., Hedrick, J. L., and Yang, Y. Y. (2017) Biodegradable cationic poly(carbonates): Effect of varying side chain hydrophobicity on key aspects of gene transfection. Acta Biomater 54, 201-211. Alshamsan, A., Haddadi, A., Incani, V., Samuel, J., Lavasanifar, A., and Uludag, H. (2009) Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethylenimine. Molecular pharmaceutics 6, 121-33. Kuhn, P. S., Levin, Y., and Barbosa, M. C. (1999) Charge inversion in DNA-amphiphile complexes:


Page 22 of 25

Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(67)

(68) (69) (70)

(71)

(72) (73)

(74)

(75)

(76) (77)

(78) (79)

(80)

(81)

(82)

possible application to gene therapy. Physica A 274, 8-18. Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents. Gene Ther 7, 126-138. Gabrielson, N. P., and Pack, D. W. (2006) Acetylation of polyethylenimine enhances gene delivery via weakened polymer/DNA interactions. Biomacromolecules 7, 2427-2435. Thomas, M., and Klibanov, A. M. (2002) Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells. P Natl Acad Sci USA 99, 14640-14645. Cho, S. W., Goldberg, M., Son, S. M., Xu, Q. B., Yang, F., Mei, Y., Bogatyrev, S., Langer, R., and Anderson, D. G. (2009) Lipid-Like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells. Adv Funct Mater 19, 3112-3118. Khan, O. F., Zaia, E. W., Yin, H., Bogorad, R. L., Pelet, J. M., Webber, M. J., Zhuang, I., Dahlman, J. E., Langer, R., and Anderson, D. G. (2014) Ionizable Amphiphilic Dendrimer-Based Nanomaterials with Alkyl-Chain-Substituted Amines for Tunable siRNA Delivery to the Liver Endothelium In Vivo. Angew Chem Int Edit 53, 14397-14401. Nguyen, D. N., Green, J. J., Chan, J. M., Longer, R., and Anderson, D. G. (2009) Polymeric Materials for Gene Delivery and DNA Vaccination. Adv Mater 21, 847-867. Teo, P. Y., Yang, C., Hedrick, J. L., Engler, A. C., Coady, D. J., Ghaem-Maghami, S., George, A. J. T., and Yang, Y. Y. (2013) Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection. Biomaterials 34, 7971-7979. Long, Z., Wu, Y. P., Gao, H. Y., Li, Y. F., He, R. R., and Liu, M. (2018) Functionalization of Halloysite Nanotubes via Grafting of Dendrimer for Efficient Intracellular Delivery of siRNA. Bioconjugate chemistry. Zha, Z., Hu, Y., Mukerabigwi, J. F., Chen, W., Wang, Y., He, C., and Ge, Z. (2018) Thiolactone Chemistry-Based Combinatorial Methodology to Construct Multifunctional Polymers for Efficacious Gene Delivery. Bioconjugate chemistry 29, 23-28. Wang, X. Y., Shao, N. M., Zhang, Q., and Cheng, Y. Y. (2014) Mitochondrial targeting dendrimer allows efficient and safe gene delivery. J Mater Chem B 2, 2546-2553. Tan, J., Wang, H. Y., Xu, F., Chen, Y. Z., Zhang, M., Peng, H. G., Sun, X., Shen, Y. Q., and Huang, Y. Z. (2017) Poly-gamma-glutamic acid-based GGT-targeting and surface camouflage strategy for improving cervical cancer gene therapy. J Mater Chem B 5, 1315-1327. Noble, G. T., Stefanick, J. F., Ashley, J. D., Kiziltepe, T., and Bilgicer, B. (2014) Ligand-targeted liposome design: challenges and fundamental considerations. Trends Biotechnol 32, 32-45. Merdan, T., Callahan, J., Peterson, H., Bakowsky, U., Kopeckova, P., Kissel, T., and Kopecek, J. (2003) Pegylated polyethylenimine-Fab ' antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells. Bioconjugate chemistry 14, 989-996. Blessing, T., Kursa, M., Holzhauser, R., Kircheis, R., and Wagner, E. (2001) Different strategies for formation of PEGylated EGF-conjugated PEI/DNA complexes for targeted gene delivery. Bioconjugate chemistry 12, 529-537. Song, N., Ding, M., Pan, Z., Li, J., Zhou, L., Tan, H., and Fu, Q. (2013) Construction of targeting-clickable and tumor-cleavable polyurethane nanomicelles for multifunctional intracellular drug delivery. Biomacromolecules 14, 4407-19. Zheng, J., Wan, Y., Elhissi, A., Zhang, Z. R., and Sun, X. (2014) Targeted Paclitaxel Delivery to Tumors Using Cleavable PEG-Conjugated Solid Lipid Nanoparticles. Pharmaceutical research 31, 2220-2233.



(83)

Koren, E., Apte, A., Jani, A., and Torchilin, V. P. (2012) Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity. J Control Release 160, 264-273. (84) Grigsby, C. L., and Leong, K. W. (2010) Balancing protection and release of DNA: tools to address a bottleneck of non-viral gene delivery. J R Soc Interface 7, S67-S82. (85) Chen, J., Guan, X., Hu, Y., Tian, H., and Chen, X. (2017) Peptide-Based and Polypeptide-Based Gene Delivery Systems. Topics in current chemistry 375, 32. (86) Geysen, H. M. (1985) Antigen-Antibody Interactions at the Molecular-Level - Adventures in Peptide-Synthesis. Immunol Today 6, 364-369. (87) Kieffer, B. L. (1995) Recent advances in molecular recognition and signal transduction of active peptides: Receptors for opioid peptides. Cell Mol Neurobiol 15, 615-635. (88) Yuan, X. D., Shah, B. A., Kotadia, N. K., Li, J. A., Gu, H., and Wu, Z. Q. (2010) The Development and Mechanism Studies of Cationic Chitosan-Modified Biodegradable PLGA Nanoparticles for Efficient siRNA Drug Delivery. Pharmaceutical research 27, 1285-1295. (89) Williford, J. M., Wu, J., Ren, Y., Archang, M. M., Leong, K. W., and Mao, H. Q. (2014) Recent Advances in Nanoparticle-Mediated siRNA Delivery. Annu Rev Biomed Eng 16, 347-370. (90) Xu, X. H., Jian, Y. T., Li, Y. K., Zhang, X., Tu, Z. X., and Gu, Z. W. (2014) Bio-Inspired Supramolecular Hybrid Dendrimers Self-Assembled from Low-Generation Peptide Dendrons for Highly Efficient Gene Delivery and Biological Tracking. Acs Nano 8, 9255-9264. (91) Wang, T., Upponi, J. R., and Torchilin, V. P. (2012) Design of multifunctional non-viral gene vectors to overcome physiological barriers: Dilemmas and strategies. Int J Pharmaceut 427, 3-20. (92) Wu, J. T., Zhang, J., Deng, C., Meng, F. H., Cheng, R., and Zhong, Z. Y. (2017) Robust, Responsive, and Targeted PLGA Anticancer Nanomedicines by Combination of Reductively Cleavable Surfactant and Covalent Hyaluronic Acid Coating. ACS applied materials & interfaces 9, 3985-3994. (93) Zhou, G. Y., Xu, Y. M., Chen, M. W., Cheng, D., and Shuai, X. T. (2016) Tumor-penetrating peptide modified and pH-sensitive polyplexes for tumor targeted siRNA delivery. Polym Chem-Uk 7, 3857-3863. (94) Chen, W., Zhong, P., Meng, F. H., Cheng, R., Deng, C., Feijen, J., and Zhong, Z. Y. (2013) Redox and pH-responsive degradable micelles for dually activated intracellular anticancer drug release. J Control Release 169, 171-179. (95) Hassan, S. S. M., and Rechnitz, G. A. (1982) Determination of Glutathione and Glutathione-Reductase with a Silver Sulfide Membrane-Electrode. Anal Chem 54, 1972-1976. (96) McKenzie, D. L., Kwok, K. Y., and Rice, K. G. (2000) A potent new class of reductively activated peptide gene delivery agents. J Biol Chem 275, 9970-9977. (97) Chen, J., Lin, L., Guo, Z. P., Xu, C. N., Tian, H. Y., Park, K., and Chen, X. S. (2015) Synergistic treatment of cancer stem cells by combinations of antioncogenes and doxorubicin. J Drug Deliv Sci Tec 30, 417-423. (98) Chen, W. C., Yuan, Y. Y., Cheng, D., Chen, J. F., Wang, L., and Shuai, X. T. (2014) Co-Delivery of Doxorubicin and siRNA with Reduction and pH Dually Sensitive Nanocarrier for Synergistic Cancer Therapy. Small 10, 2678-2687. (99) Xu, C. N., Wang, P., Zhang, J. P., Tian, H. Y., Park, K., and Chen, X. S. (2015) Pulmonary Codelivery of Doxorubicin and siRNA by pH-Sensitive Nanoparticles for Therapy of Metastatic Lung Cancer. Small 11, 4321-4333. (100) Yang, Z., Gao, D., Cao, Z., Zhang, C., Cheng, D., Liu, J., and Shuai, X. T. (2015) Drug and gene co-delivery systems for cancer treatment. Biomater Sci-Uk 3, 1035-1049.


Page 24 of 25

Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(101) Khan, M., Ong, Z. Y., Wiradharma, N., Attia, A. B. E., and Yang, Y. Y. (2012) Advanced Materials for Co-Delivery of Drugs and Genes in Cancer Therapy. Adv Healthc Mater 1, 373-392. (102) Cheng, D., Cao, N., Chen, J. F., Yu, X. S., and Shuai, X. T. (2012) Multifunctional nanocarrier mediated co-delivery of doxorubicin and siRNA for synergistic enhancement of glioma apoptosis in rat. Biomaterials 33, 1170-1179. (103) Zhang, C. G., Zhu, W. J., Liu, Y., Yuan, Z. Q., Yang, S. D., Chen, W. L., Li, J. Z., Zhou, X. F., Liu, C., and Zhang, X. N. (2016) Novel polymer micelle mediated co-delivery of doxorubicin and P-glycoprotein siRNA for reversal of multidrug resistance and synergistic tumor therapy. Sci Rep-Uk 6. (104) Li, L., and Petrovsky, N. (2016) Molecular mechanisms for enhanced DNA vaccine immunogenicity. Expert review of vaccines 15, 313-29. (105) Shen, C., Li, J., Zhang, Y., Li, Y. C., Shen, G. X., Zhu, J. T., and Tao, J. (2017) Polyethylenimine-Based Micro/Nanoparticles as Vaccine Adjuvants. International Journal of Nanomedicine 12, 5443-5460. (106) Gao, M., Zhu, X., Wu, L., and Qiu, L. (2016) Cationic Polyphosphazene Vesicles for Cancer Immunotherapy by Efficient in Vivo Cytokine IL-12 Plasmid Delivery. Biomacromolecules 17, 2199-209. (107) Zhao, X., Li, X., Zhao, Y., Cheng, Y., Yang, Y., Fang, Z., Xie, Y., Liu, Y., Chen, Y., Ouyang, Y. et al. (2017) Immune Activities of Polycationic Vectors for Gene Delivery. Frontiers in pharmacology 8, 510.

TOC Polycations for Gene Delivery: Dilemmas and Solutions


Polycations for Gene Delivery: Dilemmas and Solutions - American

Recommend Documents