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Polymers for gene delivery: Charged for success
Abstract
Several researchers are working on the syntheses of DNA-complexated cationic polymers with reduced charge density, high molecular weight and increased hydrophobicity. The researchers synthesized biodegradable polymers with low charge density, higher molecular weight and additional hydrophobic groups that were able to deliver plasmid DNA to target cells in vitro with higher efficiency and lower toxicity than those of commercially available transfection agents. The cationic groups of the synthesized polymers provided DNA-binding capacity, whereas the hydrophobic groups ensured further DNA condensation into polyplexes 90-220 nm in size having a slightly positive surface charge. Zhou and colleagues used various lactones and different lactone contents to synthesize a library of 12 terpolymers varying in cationic charge density and hydrophobicity. The researchers observed that polyplexes based on the terpolymers with higher lactone content and larger lactone group showed the highest transfection activity, supporting their hypothesis that hydrophobicity modulates gene transfer.
... Except for the polycationic gene carriers above mentioned, the researchers also designed and prepared some polycationic carriers with low charge density for gene transfer. These polycationic gene carriers possessed low cytotoxicity and satisfactory biocompatibility [133]. Zhou et al. synthesized a series of poly(amine-co-ester) terpolymers with low charge density for efficient gene delivery [133,134] (Fig. 5A). ...
... These polycationic gene carriers possessed low cytotoxicity and satisfactory biocompatibility [133]. Zhou et al. synthesized a series of poly(amine-co-ester) terpolymers with low charge density for efficient gene delivery [133,134] (Fig. 5A). These polymers of high molecular weight and low charge density exhibited efficient gene delivery, and the transfection efficiency was even better than commercial reagents PEI and lipofectamine 2000. ...
... siRNA, smallinterfering ribonucleic acid; pDNA, plasmid DNA. [133,134]. (B) Decationized polymer for redox-triggered gene delivery [135,136]. (C) Brush-like polycations with low charge density for gene delivery [137]. ...
Gene therapy, as a novel therapeutic strategy, is used to treat various diseases. Nevertheless, gene carriers are necessary for gene therapy. Among them, polycationic gene carriers are drawing increasing attention. At present, a variety of strategies have been employed to construct highly efficient polycationic gene carriers. This review focuses on the development of polycationic gene carriers in recent decades and is illustrated to construct efficient polycationic carriers through regulating physicochemical properties of carriers. Moreover, the interactions between polycations and nucleic acid or cell membrane, including electrostatic interaction, hydrogen bonding interaction, hydrophobic interaction, and metal coordination interaction, are discussed in detail for constructing highly efficient gene carriers. Furthermore, this review systematically expounds the relationship between the physicochemical properties of polycations and the transfection efficiency. Polycationic gene carriers have the bright prospects in the field of gene therapy.
... A high total positive charge on the polyplex can have various side effects, such as increased systemic cytotoxicity of the polyplex and random interaction between the polyplex and biological membranes, thus attenuating targeting ability and causing a loss of specificity. 23 In order to overcome these problems, a polypeptide, such as PEG, can be conjugated to the polyplex to reduce the overall charge ratio without affecting its gene complexation capability. 23 Alternatively, the high-charge problem can be overcome by the use of newly designed cationic polymers with hydroxyl or amide groups, such as poly(vinyl alcohol) dimethylaminoacetal (PVA3), that have been shown to have effective gene transfer, less toxicity, and increased DNA release in the cytoplasm. ...
... 23 In order to overcome these problems, a polypeptide, such as PEG, can be conjugated to the polyplex to reduce the overall charge ratio without affecting its gene complexation capability. 23 Alternatively, the high-charge problem can be overcome by the use of newly designed cationic polymers with hydroxyl or amide groups, such as poly(vinyl alcohol) dimethylaminoacetal (PVA3), that have been shown to have effective gene transfer, less toxicity, and increased DNA release in the cytoplasm. This effect can be explained by a decrease in the total positive charges of the complex or the formation of hydrogen bonds between the DNA and hydroxyl groups. ...
Prostate cancer is the second-most widespread cancer in men worldwide. Treatment choices are limited to prostatectomy, hormonal therapy, and radiotherapy, which commonly have deleterious side effects and vary in their efficacy, depending on the stage of the disease. Among novel experimental strategies, gene therapy holds great promise for the treatment of prostate cancer. However, its use is currently limited by the lack of delivery systems able to selectively deliver the therapeutic genes to the tumors after intravenous administration without major drawbacks. To remediate this problem, a wide range of nonviral delivery approaches have been developed to specifically deliver DNA-based therapeutic agents to their site of action. This review provides an overview of the various nonviral delivery strategies and gene therapy concepts used to deliver therapeutic DNA to prostate cancer cells, and focuses on recent therapeutic advances made so far.
... Therefore, to improve the stability of the vector/gene complexes, it is usually necessary to use an excess of cationic vectors. However, it will increase the charge density and nitrogen-to-phosphorus (N:P, N is the number of cationic groups in the polymer, and P is the number of phosphate groups in the nucleic acid) ratio of the complex, leading to severe cytotoxicity [11]. Therefore, there is an urgent need to prepare gene carriers that work well at low N:P ratios. ...
Introduction
Gene delivery vectors are a crucial determinant for gene therapeutic efficacy. Usually, it is necessary to use an excess of cationic vectors to achieve better transfection efficiency. However, it will cause severe cytotoxicity. In addition, cationic vectors are not resistant to serum, suffering from reduced transfection efficiency by forming large aggregates. Therefore, there is an urgent need to develop optimized gene delivery vectors. Recently, fluorination of vectors has been extensively applied to increase the gene delivery performance because of the unique properties of both hydrophobicity and lipophobicity, and chemical and biological inertness.
Areas covered
This review will discuss the fluorophilic effects that impact gene delivery efficiency, and chemical modification approaches for fluorination. Next, recent advances and applications of fluorinated polymeric and lipidic vectors in gene therapy and gene editing are summarized.
Expert opinion
Fluorinated vectors are a promising candidate for gene delivery. However, it still needs further studies to obtain pure and well-defined fluorinated polymers, guarantee the biosafety, and clarify the detailed mechanism. Apart from the improvements in gene delivery, exploiting other versatility of fluorinated vectors, such as oxygen-carrying ability, high affinity with fluorine-containing drugs, and imaging property upon introducing 19F, will further facilitate their applications in gene therapy.
... Employing high molecular weight polymers with a high charge density is one way around this problem, but as the polymer molecular weight increases, the cytotoxicity also increases, as a direct consequence of the surplus positive charges interacting with biomembranes and interfering with vital cellular functions. A major trade-off between the cytotoxicity and the gene delivery efficiency of cationic-polymer-based NPs must be dealt with [34]. In order to try to balance the cytotoxicity and transfection efficiency of polymers, polyethylenimine in particular, some researchers have tested various modifications. ...
Despite the development of many novel carriers for the delivery of various types of genetic material, the lack of a delivery system with high efficiency and low cytotoxicity is a major bottleneck. Herein, low molecular weight polyethylenimine (PEI1.8k) was functionalized with saponin residues using phenylboronic acid (PBA) as an ATP-responsive cross-linker, and a fluorinated side chain to construct PEI-PBA-SAP-F polycation as a highly efficient delivery vector. This vehicle could transfect small plasmid DNA ( 3 kb) with outstanding efficiency into various cells, including HEK293T, NIH3T3, A549, PC12, MCF7 and HT-29, as well as robust transfection of a large plasmid ( 9 kb) into HEK293T cells. The carrier could maintain transfection efficacy even at high concentration of serum and low doses of plasmid. The use of green fluorescent protein (GFP) knock-out analysis demonstrated transfection of different types of CRISPR/Cas9 complexes (Cas9/sgRNA ribonucleoproteins RNP, plasmid encoding Cas9 plus sgRNA targeting GFP, Cas9 expression plasmid plus in vitro-prepared sgRNA). The transfection of a large plasmid into local mouse brain tissue was also shown. In summary, we report an effective PEI-PBA-SAP-F gene carrier with the appropriate lipophilic/cationic balance for biomedical applications.
... Consistent with this finding, another study illustrated that the toxicity decreased with the increase of particle size . Using high molecular weight and increased hydrophobicity to compensate for low charge density may be a good strategy to balance performance and toxicity (Mastrobattista and Hennink 2011). ...
Cationic nanomaterials are defined as nanoscale structures smaller than 100 nm bearing positive charges. They have been investigated to apply to many aspects including clinical diagnosis, gene delivery, drug delivery, and tissue engineering for years. Recently, a novel concept has been made to use cationic nanomaterials as cell-free nucleic acid scavengers and inhibits the inflammatory responses in autoimmune diseases. Here, we highlighted different types of cationic materials which have the potential for autoimmune disease treatment and reviewed the strategy for autoimmune diseases therapy based on cationic nanoparticles. This review will also demonstrate the challenges and possible solutions that are encountered during the development of cationic materials-based therapeutics for autoimmune diseases.
... Nevertheless, cationic polymers and polyethyleneimine (PEI, 25 kDa) in particular, although they can deliver nucleotide sequences into the targeted cells with relatively high efficiency, have been reported to be highly toxic and unstable in serum. 18 As a way around this issue, it has been suggested that other vectors could be modified with PEI 25k to enhance their efficiency while reducing the toxicity of PEI 25k . To that end, Liu et al. 19 used photoluminescent carbon dots with properties including photoluminescence (PL) stability, wavelength emission tunability, high biocompatibility, low toxicity, facile synthesis methods, and their convenient monitoring ability. ...
... Internalization and utilization of therapeutic genes facilitated by so-called "vectors" into targeted cells in the process of gene delivery are essential and responsible for the success of gene therapy. Compared to viral vectors, polymeric vectors are safer alternatives for gene delivery owing to their advantages including ease of preparation, low cost, structural diversity, manipulable physiochemical properties, vector stability upon storage and reconstitution and lager payload capacity of gene cargo [1]. Although great efforts have been made in recent years to develop various polymeric vectors, very few of them are employed for gene therapy in Chaoyu Liu and Yuancai Xie have contributed equally. ...
Polymeric vectors are safer alternatives for gene delivery owing to their advantages as compared to viral vectors. To improve the stability and transfection efficiency of poly(lactic-co-glycolic acid) (PLGA)- and poly(ethylenimine) (PEI)-based vectors, poly(ethylene glycol) (PEG), folic acid (FA), arginylglycylaspartic acid (RGD) peptides and isoleucine-lysine-valine-alanine-valine (IKVAV) peptides were employed and PLGA–PEI–PEG–FA and PLGA–PEI–PEG–RGD copolymers were synthesized. PLGA–PEI–PEG–FA/DNA, PLGA–PEI–PEG–RGD/DNA and PLGA–PEI–PEG–RGD/IKVAV/DNA nanocomplexes (NCs) were formed through bulk mixing. The structure and properties, including morphology, particle size, surface charge and DNA encapsulation, of NCs were studied. Robust NCs with spherical shape, uniform size distribution and slightly positive charge were able to completely bind DNA above their respective N/P ratios. The critical N/P ratio for PLGA–PEI–PEG–FA/DNA, PLGA–PEI–PEG–RGD/DNA and PLGA–PEI–PEG–RGD/IKVAV/DNA NCs was identified to be 12:1, 8:1 and 10:1, respectively. The covalent modification of PEI through a combination of biodegradable PLGA, hydrophilic PEG and targeting motifs significantly decreased the cytotoxicity of PEI. The developed NCs showed both N/P ratio and cell type-dependent transfection efficiency. An increase in N/P ratio resulted in increased transfection efficiency, and much improved transfection efficiency of NCs was observed above their respective critical N/P ratios. This study provides a promising means to produce polymeric vectors for gene delivery.
... The structure-property relationship of cationic polymer in gene transfection and toxicity has been extensively investigated (41)(42)(43). ...
Severe sepsis represents a common, expensive, and deadly health care issue with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis would help develop new therapeutic strategies against severe sepsis. In this study, we identified the crucial role of cell-free DNA (cfDNA) in the regulation of the Toll-like receptor 9–mediated proinflammatory pathway in severe sepsis progression. Hypothesizing that removing cfDNA would be beneficial for sepsis treatment, we used polyethylenimine (PEI) and synthesized PEI-functionalized, biodegradable mesoporous silica nanoparticles with different charge densities as cfDNA scavengers. These nucleic acid–binding nanoparticles (NABNs) showed superior performance compared with their nucleic acid–binding polymer counterparts on inhibition of cfDNA-induced inflammation and subsequent multiple organ injury caused by severe sepsis. Furthermore, NABNs exhibited enhanced accumulation and retention in the inflamed cecum, along with a more desirable in vivo safety profile. Together, our results revealed a key contribution of cfDNA in severe sepsis and shed a light on the development of NABN-based therapeutics for sepsis therapy, which currently remains intractable.
... This has been canvassed as a breakthrough in molecular medicine. Unfortunately, its application in vivo is inhibited due to difficulties in transportation of negatively charged molecules like DNA, fragile and biomacromolecules into the nucleus without degradation [2][3][4]. Even after the delivery of these biomolecules to the patient, they are still vulnerable to degradation; and thus require protection to assure potency and maintain integrity [1,5]. ...
Here, human genomic DNA (hDNA) was encapsulated into supermacroporous alginate beads (SMPA) fabricated via microinjection and freeze-thawing cryogelation protocol. The scanning electron microscopy of the SMPA beads demonstrated smooth and centred burgled morphologies, while the encapsulated hDNA showed brilliant green fluorescence when visualised by fluorescence microscopy. The encapsulation efciency of 89.1 − 96.7% was achieved when the concentration of hDNA and alginate varied within 0.05 − 0.075% and 0.5 − 0.75 wt%, respectively in the presence of 0.1 M CaCl2 crosslinking agent. ~80% hDNA was released over an extended period of 80 h when SMPA was immersed in a 0.5 M Na2HPO solution diluted with 10 mM Tris buffer (pH 7). Results and trends here demonstrated that SMPA beads have great potential to be used as a biocompatible vehicle for transporting biomacromolecules
... This has been canvassed as a breakthrough in molecular medicine. Unfortunately, its application in vivo is inhibited due to difficulties in transportation of negatively charged molecules like DNA, fragile and biomacromolecules into the nucleus without degradation [2][3][4]. Even after the delivery of these biomolecules to the patient, they are still vulnerable to degradation; and thus require protection to assure potency and maintain integrity [1,5]. ...
Here, human genomic DNA (hDNA) was encapsulated into supermacroporous alginate beads (SMPA) fabricated via microinjection and freeze-thawing cryogelation protocol. The scanning electron microscopy of the SMPA beads demonstrated smooth and centred burgled morphologies, while the encapsulated hDNA showed brilliant green fluorescence when visualised by fluorescence microscopy. The encapsulation efficiency of 89.1 − 96.7% was achieved when the concentration of hDNA and alginate varied within 0.05 − 0.075% and 0.5 − 0.75 wt%, respectively in the presence of 0.1 M CaCl2 crosslinking agent. ~80% hDNA was released over an extended period of 80 h when SMPA was immersed in a 0.5 M Na2HPO solution diluted with 10 mM Tris buffer (pH 7). Results and trends here demonstrated that SMPA beads have great potential to be used as a biocompatible vehicle for transporting biomacromolecules.
... Both synthetic and natural polymers have been used for gene delivery, and several reviews are available in the literature [48][49][50][51][52]. However, these reviews were published approximately a decade ago, and hence, we prepared this review to outline earlier research in this area and to describe developments during the past decade, especially in the area of biodegradable polymers for gene delivery applications. ...
The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.
... During the past decade, alternatives to PEI have been developed for improved transfection efficiency while displaying lower toxicity [136]. Similar to PBAE carriers, poly(amido amines) (PAMAMs) carry cationic charge through the presence of various amine groups and degrade into oligomeric products in aqueous media. ...
Introduction: The delivery of nucleic acid therapeutics through non-viral carriers face multiple biological barriers that reduce their therapeutic efficiency. Despite great progress, there remains a significant technological gap that continues to limit clinical translation of these nanocarriers. A number of polymeric materials are being exploited to efficiently deliver nucleic acids and achieve therapeutic effects.
Areas covered: We discuss the recent advances in the polymeric materials for the delivery of nucleic acid therapeutics. We examine the use of common polymer architectures and highlight the challenges that exist for their development from bench side to clinic. We also provide an overview of the most notable improvements made to circumvent such challenges, including structural modification and stimuli-responsive approaches, for safe and effective nucleic acid delivery.
Expert opinion: It has become apparent that a universal carrier that follows “one-size” fits all model cannot be expected for delivery of all nucleic acid therapeutics. Carriers need to be designed to exhibit sensitivity and specificity towards individual targets diseases/indications, and relevant subcellular compartments, each of which possess their own unique challenges. The ability to devise synthetic methods that control the molecular architecture enables the future development that allow for the construction of “intelligent” designs.
... In particular, the emergent physics have attracted the interest of many scientists and continuous research upsurge, such as the cuprate superconductors, [1,2] multiferroics, [3][4][5][6] colossal magnetoresistance (CMR) effect, [7] and 2-dimensional electron gas. [8][9][10][11][12] Among these intriguing properties, the CMR effect has sparked a number of studies on the electromagnetic properties and the exploration of new phases. A milestone of the CMR effect is the discovery in doped perovskite-type manganites in 1993 by Helmholt et al. [13] The appearance of CMR effect can be qualitatively explained with Zener double exchange model, [14] the cooperative Jahn-Teller effect, [15] as well as electronic phase separations (EPS). ...
Light-matter interaction plays an important role in the non-equilibrium physics, especially in strongly correlated electron systems with complex phases. Photoinduced effect can cause the variation in the physical properties and produce some emergent phases. As a classical archetype, manganites have received much attention due to their colossal magnetoresistance (CMR) effect and the strong interaction of charge, spin, orbital, and lattice degrees of freedom. In this paper, we give an overview of photoinduced effect in manganites and their heterostructures. In particular, some materials, including ZnO, Si, BiFeO3 (BFO), titanate-based oxides, and 0.7Pb(Mg1/3Nb2/3)O3-0.3PbTiO3 (PMN-PT) have been integrated with manganites. Heterostructures composed of these materials display some exciting and intriguing properties. We do hope that this review offers a guiding idea and more meaningful physical phenomena will be discovered in active areas of solid state physics and materials science.
... One main aspect focused on the manipulation of the surface charge and hydrophobicity of the dendrimers. Charge density of dendrimers contributes not only to complex formation, cellular uptake, and endosomal escape but also cytotoxicity [24]. In order to reduce the inherent cytotoxicity of dendrimers, general approaches including partial acetylation or PEGylation have been carried out [22] and Liu et al. [23] to reduce the surface charge density. ...
Gene therapy requires successful delivery of therapeutic nucleic acids into target cells; thus, efficient and safe gene delivery carriers are crucial to its success. Although many polymeric materials have shown their potential as effective nucleic acid carriers, the inherent heterogeneity and polydispersity of these polymers hinder their application in clinical studies because of difficulties in their further precise modification, structure-activity relationship study, as well as consistent manufacturing. Therefore, precisely defined polymers, with potential for site-specific optimization according to structure-activity relationship information and highly controllable production, have been extensively investigated. In this review, we focus on the design and development of precisely defined polymers for efficient gene delivery, illustrated with examples including dendrimers, peptide-based polymers, and sequence-defined oligoaminoamide oligomers.
In the realm of cancer therapy, the spotlight is on nanoscale pharmaceutical delivery systems, especially polymer‐based nanoparticles, for their enhanced drug dissolution, extended presence in the bloodstream, and precision targeting achieved via surface engineering. Leveraging the amplified permeation and retention phenomenon, these systems concentrate therapeutic agents within tumor tissues. Nonetheless, the hurdles of systemic toxicity, biological barriers, and compatibility with living systems persist. Fluorinated polymers, distinguished by their chemical idiosyncrasies, are poised for extensive biomedical applications, notably in stabilizing drug metabolism, augmenting lipophilicity, and optimizing bioavailability. Material science heralds the advent of fluorinated polymers that, by integrating fluorine atoms, unveil a suite of drug delivery merits: the hydrophobic traits of fluorinated alkyl chains ward off lipid or protein disruption, the carbon–fluorine bond's stability extends the drug's lifecycle in the system, and a lower alkalinity coupled with a diminished ionic charge bolsters the drug's ability to traverse cellular membranes. This comprehensive review delves into the utilization of fluorinated polymers for oncological pharmacotherapy, elucidating their molecular architecture, synthetic pathways, and functional attributes, alongside an exploration of their empirical strengths and the quandaries they encounter in both experimental and clinical settings.
Osteosarcoma often occurs in children and adolescents with high invasiveness and high mortality. Polo‐like kinase 1 (PLK1) overexpressed in most tumors promotes cancer cell proliferation and transformation. PLK1 has been considered as a therapeutic target for osteosarcoma. RNAi‐based therapies have been employed to combat osteosarcoma through silencing PLK1 gene expression. However, the treatment results remain unsatisfactory due to the lack of a safe and efficient nonviral gene vector. To tackle this hurdle, biodegradable and CO2‐derivative cationic poly(vinylcyclohexene carbonates) (CPCHCs) has been used as gene vectors to perform a siPLK1 therapeutic strategy for osteosarcoma treatment. Of those CPCHCs, CPCHC60 has demonstrated the most excellent performance in gene transfection efficiency, endo‐lysosome escaping, biodegradability and biosafety. With the treatment of CPCHCs/siRNA nanoparticles, the expression level of PLK1 gene in osteosarcoma cells is significantly down‐regulated. Subsequently, cells were arrested in the G2/M phase are subsequently dead in the form of apoptosis, resulting in significant tumor regression both in vitro and in vivo. This study brings a new insight into the development of superior nonviral gene vectors for practical cancer treatment. Based on the results, the resulting nanoparticle‐based gene drug formation is considered to have a highly successful chance in further translational nanomedicine applications. This article is protected by copyright. All rights reserved
The construction of non-viral gene delivery faces two major challenges: cytotoxicity caused by high cationic charge units and easy degradation by lysosomes. Herein, highly water-dispersible polymeric carbon nitride (PCN) nanosheets were utilized as the core to construct a light-controlled non-cationic gene delivery system with sufficient lysosomal escape ability. In this system, these nanosheets exhibited efficient DNA condensation, outstanding biocompatibility, transfection tracking, light responsiveness and high transfection efficiency. Once PCN-DNA was taken up by the tumor cells, the accumulated ROS generated by photosensitizers (PSs) under light irradiation would destroy the structure of lysosomes, promote the escape of PCN-DNA and increase the efficiency of gene transfection. Simultaneously, the gene transfection process could be tracked in real time through fluorescence imaging technology, which was conducive to investigate the transfection mechanism. In vitro and in vivo experiments further confirmed that PCN nanosheets loaded with the P53 gene were beneficial to the regeneration of the P53 apoptotic pathway, increased tumor sensitivity to PSs, and further induced tumor cell apoptosis. In summary, the highly water-dispersible PCN nanosheets were applied to light-controlled self-escaping gene delivery for the first time, and tumor gene therapy was successfully realized.
Gene therapy is a promising method to treat acquired and inherited diseases by introducing exogenous genes into specific recipient cells. Polymeric micelles with different nanoscopic morphologies and properties hold great promise for gene delivery system. Conventional cationic polymers, poly(ethyleneimine)(PEI), poly(L-lysine)(PLL), poly(2-dimethylaminoethyl methacrylate)(PDMAEMA) and novel cationic polymers poly(2-oxazoline)s(POxs), have been incorporated into block copolymers and decorated with targeting moieties to enhance transfection efficiency. In order to minimize cytotoxicity, nonionic block copolymer micelles are utilized to load gene through hydrophilic and hydrophobic interactions or covalent conjugations, recently. From our perspective, properties(shape, size, and mechanical stiffness, etc.) of block copolymer micelles may significantly affect cytotoxicity, transfection efficiency, circulation time, and load capacity of gene vectors in vivo and in vitro. This review briefly sums up recent efforts in cationic and nonionic amphiphilic polymeric micelles for gene delivery.
Simple Summary
Immunotherapy, including adaptive and innate immunotherapy, exhibits promising future for the treatment of hepatocellular carcinoma. As a common tool for immunotherapy, the safe and efficient gene delivery turns to be especially important. Biocompatible polymers are a category of promising materials used in gene delivery, while there still lacks a comprehensive review article to discuss the updates on multiple disciplines covering biocompatible polymers, gene therapy, tumor immune microenvironment, and immunotherapy. This review is well-integrated with biocompatible polymers, nonviral gene therapy, and cancer immunotherapy. Our investigation will provide different perspective for the scientists focusing on the domains of biomaterials, gene therapy, and oncologists to move their research work forward.
Abstract
Hepatocellular carcinoma (HCC) is the third-largest cause of cancer death worldwide, while immunotherapy is rapidly being developed to fight HCC with great potential. Nucleic acid drugs are the most important modulators in HCC immunotherapy. To boost the efficacy of therapeutics and amplify the efficiency of genetic materials, biocompatible polymers are commonly used. However, under the strong need of a summary for current developments of biocompatible polymeric nucleic acid carriers for immunotherapy of HCC, there is rare review article specific to this topic to our best knowledge. In this article, we will discuss the current progress of immunotherapy for HCC, biocompatible cationic polymers (BCPs) as nucleic acid carriers used (or potential) to fight HCC, the roles of biocompatible polymeric carriers for nucleic acid delivery, and nucleic acid delivery by biocompatible polymers for immunotherapy. At the end, we will conclude the review and discuss future perspectives. This article discusses biocompatible polymeric nucleic acid carriers for immunotherapy of HCC from multidiscipline perspectives and provides a new insight in this domain. We believe this review will be interesting to polymer chemists, pharmacists, clinic doctors, and PhD students in related disciplines.
Gene therapy is a promising treatment for hereditary diseases, as well as acquired genetic diseases, including cancer. Facing the complicated physiological and pathological environment in vivo, developing efficient non-viral gene vectors is needed for their clinical application. Here, poly(N-isopropylacrylamide) (p(NIPAM)) nanogels are presented with either protonatable tertiary amine groups or permanently charged quaternized ammonium groups to achieve DNA complexation ability. In addition, a quaternary ammonium-functionalized nanogel was further provided with an aliphatic moiety using 1-bromododecane to add a membrane-interacting structure to ultimately facilitate intracellular release of the genetic material. The ability of the tertiary amine-, quaternized ammonium-, and aliphatic quaternized ammonium-functionalized p(NIPAM) nanogels (i.e., NGs, NGs-MI, and NGs-BDD, respectively) to mediate gene transfection was evaluated by fluorescence microscopy and flow cytometry. It is observed that NGs-BDD/pDNA complexes exhibit efficient gene loading, gene protection ability, and intracellular uptake similar to that of NGs-MI/pDNA complexes. However, only the NGs-BDD/pDNA complexes show a notable gene transfer efficiency, which can be ascribed to their ability to mediate DNA escape from endosomes. We conclude that NGs-BDD displays a cationic lipid-like behavior that facilitates endosomal escape by perturbing the endosomal/lysosomal membrane. These findings demonstrate that the presence of aliphatic chains within the nanogel is instrumental in accomplishing gene delivery, which provides a rationale for the further development of nanogel-based gene delivery systems.
Conjugated polymers (CPs) are capable of coordinating the electron coupling phenomenon to bestow powerful optoelectronic features. The light‐harvesting and light‐amplifying properties of CPs are extensively used in figuring out the biomedical issues with special emphasis on accurate diagnosis, effective treatment, and precise theranostics. This review summarizes the recent progress of CP materials in bioimaging, cancer therapeutics, and introduces the design strategies by rationally tuning the optical properties. The recent advances of CPs in bioimaging applications are first summarized and the challenges to clear the future directions of CPs in the respective area are discussed. In the following sections, the focus is on the burgeoning applications of CPs in phototherapy of the tumor, and illustrates the underlying photo‐transforming mechanism for further molecular designing. Besides, the recent progress in the CPs‐assistant drug therapy, mainly including drug delivery, gene therapeutic, the optical‐activated reversion of tumor resistance, and synergistic therapy has also been discussed elaborately. In the end, the potential challenges and future developments of CPs on cancer diagnosis and therapy are also illuminated for the improvement of optical functionalization and the promotion of clinical translation. This review summarizes the recent progress of conjugated polymer (CP) materials in bioimaging, cancer therapeutics, and introduces design strategies by rationally tuning the optical properties. Challenges and future developments of CPs on cancer diagnosis and therapy are also illuminated to boost next development of CPs design and biomedical applications, and improving optical functionalization for the promotion of clinical translation.
pH‐sensitive polyelectrolytes provide enormous opportunity for siRNA delivery. Especially, their tertiary amine structures can not only bind genes but also act as pH‐sensitive hydrophobic structure to control genes release. However, the influence of molecular structures on siRNA delivery still remains elusive, especially for the asymmetric alkyl substituents of the tertiary amine groups. Herein, a library of N‐methyl‐N‐alkyl aminoethyl methacrylate monomers (MsAM) with asymmetric alkyl substituents on the tertiary amine group is synthesized and used to prepare a series of tri‐block polycationic copolymers poly(aminoethyl methacrylate)‐block‐poly (N‐methyl‐N‐alkyl aminoethyl methacrylate)‐block‐poly(ethylene glycol methacrylate) (PAMA‐PMsMA‐PEG). And the properties of these polycations and their self‐assembled micelles are characterized, including molecular structure, proton buffering capacity, pH‐sensitivity, size, and zeta potential. With the length increase of one alkyl substituent, the proton buffering capacity of both monomers and polycations is demonstrated to be narrowed down. The siRNA delivery efficiency and cytotoxicity of these micelles are also evaluated on HepG2 cells. In particular, poly(aminoethyl methacrylate)‐block‐poly(N‐methyl‐N‐ethyl aminoethyl methacrylate)‐block‐poly(ethylene glycol methacrylate) (PAMA‐PMEMA‐PEG) elicited the best luciferase knockdown efficiency and low cytotoxicity. Besides, PAMA‐PMEMA‐PEG/siRRM2 also induced significant anti‐tumor activity in vitro. These results indicated PAMA‐PMEMA‐PEG has potential for further use in the design of gene vehicles with the improved efficiency of siRNA delivery.
Fluoropolymers have unique physicochemical properties such as hydrophobicity and lipophobicity, good chemical stability and bio-inertness, low surface energy and phase segregation. Owing to these properties, fluoropolymers have been widely used to prepare high performance materials. Especially, the use of fluoropolymers in biomedical applications has grown rapidly during the past decade. This critical review focuses on the recent advances of fluoropolymers in gene delivery, cytosolic protein delivery, drug delivery, magnetic resonance imaging, photodynamic therapy, anti-fouling and anti-bacterial applications, and tissue engineering. The mechanisms and features of fluoropolymers in these specific applications are discussed. Besides, we have reviewed the methods to synthesize water-soluble fluoropolymers for the applications and explained their supramolecular assembly behaviors in solutions. Finally, the opportunities and challenges of fluoropolymers in biomedical applications are discussed.
The use of genetic material by non-viral transfer systems is still in its initial stages, but there are high expectations for the development of targeted therapies. However, nucleic acids cannot enter cells without help, they must be well protected to prevent degradation and overcome a variety of biological barriers, the endosomal barrier being one of the greatest cellular challenges. Herein, the structure-property-relationship was investigated in detail, using well-defined polymers. Polyacrylamides were synthesized via RAFT polymerization resulting in a polymer library of (i) different cationic groups as aminoethyl acrylamide (AEAm), dimethylaminoethyl acrylamide (DMAEAm), dimethylaminopropyl acrylamide (DMAPAm) and guanidinopropyl acrylamide (GPAm); (ii) different degree of polymerization; and investigated (iii) in different cell culture settings. The influence of molar mass and cationic moiety on complex formation with pDNA, cytotoxicity and transfection efficiency of the polymers were investigated. The systematic approach identified a pH-independent guanidinium-containing homopolymer (PGPAm89) as the polymer with the highest transfection efficiency and superior endosomal release under optimal conditions. Since PGPAm89 is not further protonated inside endosomes, common escape theories appear unsuitable. Therefore, the interaction with bis(monoacryloylglycerol)phosphate, a lipid specific for endosomal vesicles, was investigated. Our research suggests that the interactions between amines and lipids may be more relevant than anticipated.
Novel amphiphilic poly(DMAEMA)-block-poly(NVP-co-BA-co-AEM) (BP83-1) forms stable micelles and BP83-1/pDNA complexes possessing controlled size, charge and enhanced aggregation degree. It was found that the formation of the micelles by BP83-1 is necessary for successful DNA binding and compaction. The polyamphiphile micelle aggregation degree defined their crucial effect on the compaction and morphology of polyplexes. Strong compaction of the DNA upon interaction with polymer at CMC value, positive charge, and high stability of the polyplex are key factors promoting the penetration of DNA through bio-surfaces that define the efficiency of gene delivery in mammalian cells.
Transfection is a process by which oligonucleotides (DNA or RNA) are delivered into living cells. This allows the synthesis of target proteins as well as their inhibition (gene silencing). However, oligonucleotides cannot cross the plasma membrane by themselves; therefore, efficient carriers are needed for successful gene delivery. Recombinant viruses are among the earliest described vectors. Unfortunately, they have severe drawbacks such as toxicity and immunogenicity. In this regard, the development of non-viral transfection vectors has attracted increasing interests, and has become an important field of research. In the first part of this review we start with a tutorial introduction into the biological backgrounds of gene transfection followed by the classical non-viral vectors (cationic organic carriers and inorganic nanoparticles). In the second part we highlight selected recent reports, which demonstrate that hybrid vectors that combine key features of classical carriers are a remarkable strategy to address the current challenges in gene delivery.
A reliable and sensitive detection of biogenic amines (BAs) is essential to ensure food safety and maintain public health. In this study, two naphthyl end‐capped terthiophene derivatives, namely 5‐(naphthalen‐1‐yl)‐2,2':5',2''‐terthiophene (NA‐3T) and 5,5''‐di(naphthalen‐1‐yl)‐2,2':5',2''‐terthiophene (NA‐3T‐NA), were employed to develop chemiresistive sensors for detecting gaseous BAs. In contrast to NA‐3T, the NA‐3T‐NA‐based sensor showed a higher sensitivity for trimethylamine (TMA) with experimental detection limit lower than 22 ppm, and also for aromatic BAs including dopamine, histamine, tryptamine, and tyramine. Besides, the recovery time for TMA was found to be shorter than 23 s. In addition, both sensors were successfully used for an in situ evaluation of meat freshness via monitoring the concentration of relevant volatile BAs. Difference in the sensing performances of the two chemiresistive sensors was tentatively ascribed to different packing structures of the derivatives and the adlayer structures of the films developed with the compounds.
Micelles have demonstrated excellent ability to deliver several different types of therapeutic agents, including chemotherapy drugs, proteins, siRNA, and DNA into tumor cells. Cationic micelles, self‐assemblies of amphiphilic cationic polymers, have exhibited tremendous promise in delivery of therapy genes and gene transfection. Present time, researches in the field have focused on achieving enhanced stability of the micellar assembly, prolonged circulation times and controlled release of the gene. This review focuses on the micelles as a nano‐sized carrier system for gene delivery, the system‐related modifications for the cytoplasm release, the stability and biocompatibility, as well as the clinic trials. As the development of synthetic chemistry and self‐assembly technology, the micelles’ structures and functionalities can be precisely controlled, and hence the synthetic micelles can not only efficiency condense DNA, but also facilitate DNA endocytosis, endosomal escape, DNA uptaking and nuclear transport, resulting in comparable gene transfection of virus.
Inefficient transfection of biocompatible low-molecular-weight (LMW) polycations, such as 1.8k polyethylenimine (PEI), is a major challenge for successful nucleic acid delivery. Current strategies to improve transfection efficiency are bottlenecked by maintaining the balance between efficient gene encapsulation and on-demand cargo release. Here, we developed a new class of Zn (II)-coordinated micelles, which showed tight siRNA binding and pH switchable release. The dipicolylamine modified PEI 1.8k (PD) and dopamine conjugated cholesterol (Chol-Dopa) assemble into coordinative micelles (Zn-PD/Chol-Dopa) via the coordination of DPA and Dopa through Zn (II) as a bridge. The high phosphate binding affinity of Zn-DPA enhanced the siRNA packaging. Moreover, the coordination effect weakened in the acidic environment of lyso/endosome, triggering the disassembly of micelles and siRNA release. These properties of the micelles resulted in strong siRNA transfection efficiencies in various cell lines. Our strategy of constructing coordinative micelles improves the transfection efficiency of LMW PEI and holds tremendous potential to develop the endogenous responsive gene delivery systems.
Improving siRNA delivery efficiency often encounters a dilemma with poor or decreased biocompatibility for polycationic micelles. To address the dilemma, this work focused on the structural exploration of hydrophobic core in amphiphilic polycationic micelles by preparing two amphiphilic polycations with block or random hydrophobic segments, poly(ethylene glycol)-block-poly(aminoethyl methacrylate)-block-poly(2-diamylamine ethyl methacrylate)-block-poly(2-diethylamine ethyl methacrylate (mPEG-PAMA-PD5A-PDEA, PADE) and poly(ethylene glycol)-block-poly(aminoethyl methacrylate)-block-poly(2-diamylamine ethyl methacrylate-co-2-diethylamine ethyl methacrylate) (mPEG-PAMA-P(D5A/DEA), PA(D/E)). The properties of the two copolymers and their self-assembly micelles were characterized including structure, morphology, size and zeta potential. Cytotoxicity, siRNA silencing efficiency and cellular uptake of PADE/siRNA and PA(D/E)/siRNA complexes were evaluated in HepG2 and MDA-MB-231 cells in vitro. The endosome escape and intracellular distribution of PADE/siRNA and PA(D/E)/siRNA in HepG2 cells were also observed by CLSM. Significantly, the results indicated that PA(D/E)/siRNA showed not only better gene silencing efficiency but also lower cytotoxicity, which may be attributed to the homogeneous morphology of the hydrophobic core of PA(D/E) micelles. Therefore, this work provided a new pathway to overcome the dilemma between siRNA delivery efficiency and biocompatibility for development of efficient polycation carriers.
Linear fluorinated polyamides with reversible cationic charges are feasibly prepared to be used as highly efficient gene vectors in HEK293 cell line. Due to the uniform polymer structure, the relationship between the physicochemical properties and transfection efficiency could be unambiguously investigated. The different efficiency in the application of gene delivery between the parent polyethylenimine (PEI) and the polyamides is directly associated with the differences in chemical and physical properties between secondary amines and fluorinated amides. We found that fluorination not only increases the cellular uptake of polymer/DNA polyplexes, but it also decreases cytotoxicity in terms of inducing lower concentrations of proinflammatory cytokine TNF‐α. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res B Part B, 2019.
Structure activity relationship (SAR) studies are very critical to design ideal gene vectors for gene delivery. However, It is difficult to obtain SAR information of low generation dendrimers due to lacking of easy structural modification ways. Here, we synthesized a novel family of rigid aromatic backbone-based low generation polyamidoamine (PAMAM) dendrimers. According to the number of primary amines, they were divided into two types: four-amine-containing PAMAM (DL1-DL5) and eight-amine-containing PAMAM (DL6-DL10). Due to the introduction of rigid aromatic backbone, low generation PAMAM was modified easier by different hydrophobic aliphatic chains. Several assays were used to study the interactions of PAMAM dendrimers with plasmid DNA, and the results revealed that they not only had good DNA binding ability but also could efficiently condense DNA into spherical shaped nanoparticles with suitable sizes and zeta potentials. The SAR studies indicated that the gene transfection efficiency of the synthesized materials depended on not only the structure of hydrophobic chains but also the number of primary amines. It was found that four-amine-containing PAMAM prepared from oleylamine (DL5) gave the best transfection efficiency, which was 3 times higher than that of lipofectamine 2000 in HEK293 cells. The cellular uptake mechanism mediated by DL5 was further investigated, the results indicated that DL5/DNA complexes entered the cells mainly via caveolae and clathrin-mediated endocytosis. In addition, these low generation PAMAM modified with single hydrophobic tail showed lower toxicity than Lipofectamine 2000 in MC3T3-E1, MG63, HeLa and HEK293 cells. These results reveal that such type of low generation polyamidoamine might be promising non-viral gene vectors, and also give us clues for the design of safe and high efficiency gene vectors.
Amines have been extensively involved in vector design thus far, however, their clinical translation has been impeded by several obstacles: cytotoxicity, polyplex serum instability and low efficacy in vivo. In pursuit of functional groups to substitute amines in vector design to address these disadvantages is of great significance. Herein, we report well-tailored noncationic copolymers that contain hydrophilic, hydrophobic, and zinc coordinative moieties through reversible addition–fragmentation chain transfer (RAFT) polymerization for efficient and safe gene delivery. These polymers are capable of condensing DNA, enabling the formation of uncharged polyplexes. Especially, the zinc coordinative ligand can simultaneously benefit strong DNA binding, robust cellular uptake, efficacious endosomal destabilization, low cytotoxicity, and avoidance of serum protein adsorption. The coordinative module holds great promise to substitute amines and inspires the development of next-generation gene vectors. More importantly, the coordinative copolymers illuminate the possibility and potential of noncationic gene delivery systems for clinical applications.
Development of biodegradable thermogels as intracameral injectable carriers for ocular delivery of antiglaucoma medications can provide a better treatment modality with low dosing frequency than eye drop formulations. For the first time, this study investigates the effect of deacetylation degree (DD) of the polysaccharide component in chitosan-g-poly(N-isopropylacrylamide) (CN) carriers on controlled release of pilocarpine in the management of glaucoma. Our results showed that increasing the chitosan DD from 60.7% to 98.5% leads to enhanced biodegradation resistance of carrier and prolonged release profile of the drug. Significant DNA damage and caspase-3 activation could be detected in lens epithelial cell cultures exposed to CN made from highly deacetylated polysaccharides, indicating apoptosis-related cytotoxicity due to relatively high positive charge density of the graft copolymers. Postoperative outcomes demonstrated that long-term therapeutic efficacy in glaucomatous rabbits is governed by intraocular pressure changes in response to intracamerally administered pilocarpine-loaded CN, strongly suggesting the usefulness of deacetylation in this injectable drug delivery carrier.
The assembly of low molecular weight polymers into highly efficient and non-toxic nanostructures has broad applicability in gene delivery. In this study, we reported the assembly of coumarin-anchored low generation dendrimers in aqueous solution via hydrophobic interactions. The synthesized material showed significantly improved DNA binding and gene efficacy, and minimal toxicity on the transfected cells. Moreover, the coumarin moieties in the assembled nanostructures endow the materials with light-responsive drug delivery behaviors. The coumarin substitutes in the assembled nanostructures were cross-linked with each other upon irradiation at 365 nm, and the cross-linked assemblies were degraded upon further irradiation at 254 nm. As a result, the drug-loaded nanoparticle showed a light-responsive drug release behavior and light-enhanced anticancer activity. The assembled nanoparticle also exhibited a complementary anticancer activity through the co-delivery of 5-fluorouracil and a therapeutic gene encoding tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study provided a facile strategy to develop light-responsive polymers for the co-delivery of therapeutic genes and anticancer drugs.
This review discusses recent studies on dendrimer- based nanoparticles in cancer chemotherapy and genetherapy. In order to achieve the high efficacy and low side effects of chemotherapy and genetherapy, it is essential to combine the unique features of dendrimers and the specific tumor microenvironment to target delivery and control release of therapeutic agents to tumor tissues and cells. Strategies to design the dendrimer-based delivery system in this review include non-modified dendrimers, dendrimer conjugates, assembled amphiphilic dendrimers, nanohybrid dendrimer carriers and dendrimers with inherent activity. In addition, specific functional groups on these dendrimers as stimuli-responsive system for targeting delivery and controlled release of therapeutic agents are discussed.
Dendrimers are a class of nano-sized synthetic polymer with a well-defined composition and regularly branched tree-like structure produced by stepwise growth. The uniform size, globular shape and tunable surface chemistry make dendrimers a versatile nanoscaffold to encapsulate or stabilize various inorganic (metal, metal oxide, semiconductor) nanoparticles. In the past decade, the research interests on dendrimer-inorganic hybrid nanoparticles have evolved from the development of interesting properties to the exploitation of advanced and useful functions. In particular, because gold nanoparticles with controlled morphology and optical properties have been demonstrated to be promising and versatile candidates for a diverse field of biomedical applications including sensing, in vitro and in vivo imaging, drug delivery, diagnostics and therapies, the dendrimer-gold nanoparticle hybrids with biocompatibility have recently been intensively investigated for promising biomedical applications due to their controllable structures and dimensions, as well as their desirable internal and/or external functionalities. In this review, we discuss the recent progress regarding the development of functional dendrimer-gold nanoparticle hybrids for biomedical applications. The strategies for the fabrication of various structures of dendrimer-gold nanoparticle hybrids will first be summarized, followed by their biomedical applications in the order of drug and gene delivery, photothermal therapy and combined therapies.
Anti-microRNA-155 (anti-miR-155), an oligonucleotide with a complimentary sequence to microRNA-155, holds great promise for lung cancer therapy, and thus some cationic materials have been used to deliver anti-miR-155 into lung tumors. Although the gene delivery capacity in vitro was favorable, the application in vivo was limited by rapid removal and significant cytotoxicity, which were mainly caused by the positive charge of the gene complexes. Therefore, it was necessary to develop a novel carrier to decrease the positive charge and increase the gene delivery capacity into the tumor site. In this paper, biodegradable poly(ester amine) (PEA) was used to condense anti-miR-155 into PEA/anti-miR-155 complexes, and natural anionic polysaccharide hyaluronic acid (HA) was modified with a lung tumor cell targeting peptide and then coated on the surface of gene complexes. The formed hyaluronic acid shielding, PEA/anti-miR-155/HA–peptide complexes were monodispersed, and the particle size and zeta potential were 362.7 nm and −10.17 mV, respectively. In addition, the PEA/anti-miR-155/HA–peptide complexes had good biocompatibility and stability in vitro, and the lung tumor growth inhibitions of PEA/anti-miR-155/HA–peptide in vitro and in vivo were also excellent. The PEA/anti-miR-155/HA–peptide complexes play an active role in tumor growth inhibition and could be useful for lung cancer therapy.
A new strategy for the construction of fluorinated cationic polymers for gene delivery was introduced. The fluorinated polymers were synthesized by crosslinking low molecular weight PEI with diols containing various lengths of perfluoroalkyl chain via epoxide ring-opening polymerization. Such study presents the first example for the polymeric gene vectors with fluorination on the polymer backbone but not side chain. These materials showed good DNA condensation and protection ability and could condense DNA into nanoparticles with proper sizes and zeta-potentials. The fluorine atoms might strengthen the interaction toward DNA, leading to more stable polyplexes. In vitro transfection results showed that the fluorinated polymers could mediate efficient gene delivery toward both 2D and 3D cell culture at low weight ratio, and the transfection efficiency was higher than PEI 25 kDa and the non-fluorinated counterpart. Several assays including DLS, TEM, luciferase reporter gene transfection and flow cytometry revealed that fluorination improved the serum resistance of these polymeric vectors, and more fluorine atoms might lead to better serum tolerance. These fluorinated materials exhibited very low cytotoxicity at transfection dosage. Cellular uptake study with uptake inhibitors indicated that macro-pinocytosis and microtubule-mediated endocytosis were the major endocytosis pathways for these polyplexes.
Small interfering RNA (siRNA) offers a highly selective and effective pharmaceutical for various life-threatening diseases, including cancers. The clinical translation of siRNA is, however, challenged by its short plasma life, poor cell uptake, and cumbersome intracellular trafficking. Here, cNGQGEQc peptide-functionalized reversibly crosslinked chimaeric polymersomes (cNGQ/RCCPs) is shown to mediate high-efficiency targeted delivery of Polo-like kinase1 specific siRNA (siPLK1) to orthotopic human lung cancer in nude mice. Strikingly, siRNA is completely and tightly loaded into the aqueous lumen of the polymersomes at an unprecedentedly low N/P ratio of 0.45. cNGQ/RCCPs loaded with firefly luciferase specific siRNA (siGL3) or siPLK1 are efficiently taken up by α3β1-integrin-overexpressing A549 lung cancer cells and quickly release the payloads to the cytoplasm, inducing highly potent and sequence-specific gene silencing in vitro. The in vivo studies using nude mice bearing orthotopic A549 human lung tumors reveal that siPLK1-loaded cNGQ/RCCPs boost long circulation, superb tumor accumulation and selectivity, effective suppression of tumor growth, and significantly improved survival time. These virus-mimicking chimaeric polymersomes provide a robust and potent platform for targeted cancer siRNA therapy.
Many synthetic polycationic vectors for non-viral gene delivery show high efficiency in vitro, but their usually excessive charge density makes them toxic for in vivo applications. Here we describe the synthesis of a series of high molecular weight terpolymers with low charge density, and show that they exhibit efficient gene delivery, some surpassing the efficiency of the commercial transfection reagents Polyethylenimine and Lipofectamine 2000. The terpolymers were synthesized via enzyme-catalyzed copolymerization of lactone with dialkyl diester and amino diol, and their hydrophobicity adjusted by varying the lactone content and by selecting a lactone comonomer of specific ring size. Targeted delivery of the pro-apoptotic TRAIL gene to tumour xenografts by one of the terpolymers results in significant inhibition of tumour growth, with minimal toxicity both in vitro and in vivo. Our findings suggest that the gene delivery ability of the terpolymers stems from their high molecular weight and increased hydrophobicity, which compensates for their low charge density.
Nanotechnology is a rapidly expanding multidisciplinary field in which highly sophisticated nanoscale devices are constructed from atoms, molecules or (macro)molecular assemblies. In the field of gene medicine, systems for delivering nucleic acids are being developed that incorporate virus-like functions in a single nanoparticle. Although their development is still in its infancy, it is expected that such artificial viruses will have a great impact on the advancements of gene therapeutics.
The water soluble terpolymer, poly(N-isopropylacrylamide (IPAAm)-co-2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-butylmethacrylate (BMA)) was synthesized, and its efficiency in in vitro gene transfection was evaluated. Copolymers with different compositions were synthesized by radical polymerization. For a series of copolymers containing 60 mol% of DMAEMA, the plasmid bands were retained within the gel loading slot, independent of polymer/plasmid weight ratios or BMA monomer content. In contrast, for a series of copolymers containing 20 mol% DMAEMA, plasmid bands of complexes were retarded with increasing weight ratios. For the copolymer with 10 mol% BMA content, the plasmid was completely retained within the gel loading slot. The transfection efficiency of polymer/plasmid complexes was evaluated in COS-1 cells using a pCMV-lacZ plasmid, encoding for β-galactosidase as a reporter gene. Transfection efficiency of a series of copolymers containing 20 mol% of DMAEMA varied with BMA content. The transfection efficiency of the copolymers with 0, 2, and 5 mol% of BMA was low. The transfection efficiency of the copolymers with 10 mol% of BMA was about 2-fold higher than that of the PDMAEMA control homopolymer. The transfected cells were observed at a very wide range of polymer/plasmid weight ratios. The transfection efficiency of all copolymers containing 60 mol% of DMAEMA was lower than that of the PDMAEMA homopolymer.
Screening of new gene delivery candidates regarding transfection efficiency and toxicity is usually performed by reading out transgene expression levels relative to a reference formulation after in vitro transfection. However, over the years and among different laboratories, this screening has been performed in a variety of cell lines, using a variety of conditions and read-out systems, and by comparison to a variety of reference formulations. This makes a direct comparison of results difficult, if not impossible. Reaching a consensus would enable placing new results into context of previous findings and estimate the overall contribution to the improvement of non-viral gene delivery. In this paper we illustrate the sensitivity of transfection outcomes on testing conditions chosen, and propose a screening protocol with the aim of standardization within the field.
Various nanoviral vectors exhibiting reduced transfection efficiency, biocompatibality, and potentially for large-scale production, are found to be suitable for gene delivery and therapy. Significant research work is focused on developing vector systems with attached receptor ligands to promote gene delivery to specific cells and tissues. Studies have shown that in arsonium and phosphonium phosphonolipid derivatives, in vitro transfection efficiency in Hela cells increases proportional to the number of methylene units between the phosphonate group and the cationic moiety. DNA condensed with low molecular weight lysine oligomers containing terminal cysteine residues cross-linked to form complexes with reducible disulfide linkages show significant gene transfer compared to commercially available lipid genes. The in vivo transfection efficiency using PDMAEMA shows that DNA complexes injected intraperitoneally is negatively affected by hyaluronic acid present in ascites.
To date, over 1340 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. In 1997 we set up a database to bring together global information on gene therapy clinical trials as comprehensively as possible. The data are compiled and regularly updated from official agency sources, published literature, conference presentations and posters and from information kindly provided by investigators or trial sponsors themselves.
This review updates our descriptive overview of the data in 2004 1, presenting our analysis of the clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of July 30 2007, we have stored entries on 1309 trials in 28 countries. We have analyzed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and which genes have been transferred. Details of the analyses presented, and our interactive, searchable database can be found on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www.wiley.co.uk/genmed/clinical. Copyright
