Polymeric Gene Delivery Research Articles

Brevinin-2R-linked polyethylenimine as a promising hybrid nano-gene-delivery vector.

Objective(s):Polyethylenimine (PEI) is one of the most widely used polymers in gene delivery. The aim of this study was to modify PEI by replacing some of its primary amines with Brevinin 2R (BR-2R) peptide in order to increase the efficiency of gene delivery.Materials and Methods:Polyethylenimine was modified by BR-2R peptide by two different approaches; A) conjugation methods including (І) using succinimidyl 3-(2-pyridyldithio) propionate (SPDP), (П) EDC/NHS protocol and (ПІ) EDC/NHS+6-bromohexanoic acid protocol, and B) physical interaction method. The modified polymers were characterized for their ability of plasmid condensation, number of primary amines, size and zeta potential. The transfection efficiency and cytotoxicity were evaluated on HEK293, L929, WEHI164 and Neuro2A cell lines by green fluorescent protein (GFP)-based plasmid (pGFP) reporter gene and viability assays, respectively. Apoptosis induction ability was also evaluated via PI/Annexin V assay. Results:Polyplex had size and zeta potential between 200-270 nm and +21.5- +28.4 mV, respectively. All vectors were able to condense plasmid DNA in C/P=4 (carrier-plasmid ratio). Transfection results on the Neuro2A cell line showed that the vector containing the BR-2R peptide, which was synthesized using EDC-NHS protocol had the best transfection efficiency. Conclusion:Our results showed that conjugation of Brevinin 2R as cell penetrating peptide to polyethyleneimine could enhance the transfection ability of the polymer.

BR2 cell penetrating peptide improved the transfection efficiency of modified polyethyleneimine

Polyethyleneimine (PEI) is one of the most widely used polymers in gene delivery. Cell penetrating peptides (CPPs) are a group of peptides that can transfer materials from the membrane to the cytoplasm and nucleus. The aim of this study was to modify PEI with three different CPPs as a cationic polymer in order to investigate and compare their efficiency in gene delivery. The CPP-modified polymers were evaluated in terms of plasmid condensation, buffering capacity and number of primary amines. The prepared nanoparticles were examined for size, zeta potential, transfection efficiency and cytotoxicity in LNCAP cells. The resulting nanoparticles had a particle size between 183 and 313 nm. Measuring the zeta potential of the vectors indicated that the charge of the nanoparticles is between 21 and 25 mV. Vectors can condense plasmid in carrier-plasmid ratio of 4. Transfection results on the LNCAP cell line showed that the vector containing the BR2 peptide had the best transfection activity. In the cytotoxicity test, it was observed that vector containing CyLoP1 in carrier-plasmid ratio of 4 had the highest cell cytotoxicity. In conclusion, conjugation of CPPs to PEI due to physicochemical changes of the polymer resulted in improved transfection activity.

Exploring the Functions of Polymers in Adenovirus-Mediated Gene Delivery: Evading Immune Response and Redirect Tropism

Adenoviruses (Ads) are promising viral carriers for gene therapy because of their unique attribution. However, clinical applications of Ad vectors are currently restricted by their immunogenicity and broad native tropism. To address these obstacles, a variety of non-immunogenic polymers are utilized to modify Ad vectors chemically or physically. In this review, we systemically discuss the functions of polymers in Ad-mediated gene delivery from two aspects, evading the host immune responses to Ads and redirecting Ad tropism. With polyethylene glycol (PEG) first in order, a variety of polymers have been developed to shield the surface of Ad vectors, and well accomplished to evade the host immune response, block CAR-dependant cellular uptake and reduce liver accumulation. Besides these functions, shielding Ad vectors with targeted polymers (including targeting ligands conjugated polymers and bio-responsive polymers) can also efficiently retarget Ad vectors to tumor tissues and reduce their distribution at non-targeted tissues. With its potentials to evade the immune response and retarget Ad vectors, modification with polymers has been generally regarded as a promising strategy to facilitate the clinical applications of Ad vectors for virotherapy.

Identifying key barriers in cationic polymer gene delivery to human T cells.

T cells have emerged as a therapeutically-relevant target for ex vivo gene delivery and editing. However, most commercially available reagents cannot transfect T cells and designing cationic polymers for non-viral gene delivery to T cells has resulted in moderate success. Here, we assess various barriers to successful gene transfer in the Jurkat human T cell line and primary human T cells. Using two polymers previously developed by our group, we show that uptake is one barrier to gene delivery in primary human T cells but is not predictive of successful gene delivery. We then probe intracellular pathways for barriers to gene transfer including endosomal acidification, autophagy, and immune sensing pathways. We find that endosomal acidification is slower and not as robust in human T cells compared to the model HeLa human cell line commonly used to evaluate cationic polymers for gene delivery. These studies inform the future design of cationic polymers for non-viral gene delivery to T cells, specifically, to rely on alternative endosomal release mechanisms rather than on pH-triggered release.

A biofunctionalized viral delivery patch for spatially defined transfection.

Gene therapy holds the significance of correcting genetic defects. However, difficulties in the in vivo delivery to the targeted tissues and systemic delivery remain the biggest challenges to be overcome. Here, a robust system of biofunctionalized polymeric layer-mediated lentiviral delivery was designed for the site-specific spatial and temporal control of viral gene delivery. Poly glycidyl methacrylate (pGMA) modification of a substrate via initiated chemical vapor deposition (iCVD) followed by polyethyleneimine (PEI) immobilization provided the adhesion site for the lentivirus. Furthermore, the polymeric patch based gene delivery system showed a high rate of gene transduction compared to bolus treatment. Furthermore, by using mask patterning, we were able to spatially pattern the lentivirus which allowed spatially defined transfection.

Peptide-grafted dextran vectors for efficient and high-loading gene delivery.

Among various polymeric gene delivery systems, peptide-based vectors demonstrate great potential owing to their unique structure and properties, including flexibility; however, there is insufficient molecular understanding of the role and properties of amino acids as building blocks in gene delivery. In this work, we constructed a series of histidine (H)-containing peptide-grafted dextran (D-RxHy) vectors via a simple two-step reaction of dextran with five RxHyC peptides: R7H3C, R5H3C, R5H5C, R3H5C, and R3H7C. The gel electrophoresis study unveiled the DNA-binding ability of H residues. While all D-RxHy vectors possess similarly low cytotoxicity, D-R3H7 exhibited the highest gene transfection efficiency. Interestingly, at the low nitrogen to phosphate (N/P) ratio of 2, D-R3H7 displayed a 6-8-fold higher luciferase expression compared to the gold standard branched PEI (25k). D-R3H7 and D-R5H5 demonstrated favorable cell uptake rates. A chloroquine-treated transfection assay confirmed the key effect of the high buffering capacity of H-rich D-R3H7 on its high gene transfection efficiency, especially at low N/P ratios. The present work unveiled that histidine is critical for both DNA condensation and the accurate control of endosomal escape. The tunable D-RxHy platform not only demonstrates promising potential for therapeutic purposes but can also be used as a tool to elucidate the molecular mechanism of polymer-based transfection.

A multifunctional polymeric gene delivery system for circumventing biological barriers.

Aiming to circumvent the pre-defined obstacles in the journey of gene transportation, we attempt to compile a number of functional components into a tandem tri-copolymeric material. Herein, a β-cyclodextrin-functionalized poly(glycerol methacrylate) (PG) segment and a quaternary amine-functionalized poly[(2-acryloyl)-ethyl-(p-boronic acid pinacol ester benzyl)diethylammonium bromide] (BP) segment are attached to complex DNA to formulate a nanoscaled delivery system based on electrostatic interactions. The formulated polyplex is strengthened by a hydrophobic poly[2-(5,5-dimethyl-1,3-dioxan-2-yloxy)ethyl acrylate] (PDM) segment, affording improved complex stability. To retrieve the polyplex from entrapment by acidic and digestive endo/lysosomes, light-stimulated ROS-producing 4,4'-(1,2-diphenylethene-1,2-diyl)bis(1,4-phenylene)diboronic acid (TPE) is installed in the cavities of cyclodextrin. Upon light irradiation, TPE is triggered to produce abundant ROS, not only committing disruption of the endo/lysosome membrane for polyplex escape from the entrapment but also inducing the transformation of the positively charged BP to become negatively charged. This charge conversion behavior, together with the transformation of PDM to be hydrophilic and responsive to an acidic endosome pH gradient (pH 5.0) is envisioned to induce the dissociation of the electrostatically-assembled polyplex, thereby facilitating the release of the DNA payload for the subsequent transcription machinery. This strategically-tailored and easily synthesized tandem tri-copolymer exhibits excellent gene expression activity and provides a facile response to endogenous and exogenous stimuli for active gene expression.

Enhancing the In Vitro and In Vivo Stabilities of Polymeric Nucleic Acid Delivery Nanosystems.

Gene therapy holds great promise for various medical and biomedical applications. Nonviral gene delivery systems formed by cationic polymer and nucleic acids (e.g., polyplexes) have been extensively investigated for targeted gene therapy; however, their in vitro and in vivo stability is affected by both their intrinsic properties such as chemical compositions (e.g., polymer molecular weight and structure, and N/P ratio) and a number of environmental factors (e.g., shear stress during circulation in the bloodstream, interaction with the serum proteins, and physiological ionic strength). In this review, we surveyed the effects of a number of important intrinsic and environmental factors on the stability of polymeric gene delivery systems, and discussed various strategies to enhance the stability of polymeric gene delivery systems, thereby enabling efficient gene delivery into target cells. Future opportunities and challenges of polymeric nucleic acid delivery nanosystems were also briefly discussed.

Manipulation of Transgene Expression in Fibroblast Cells by a Multifunctional Linear-Branched Hybrid Poly(β-Amino Ester) Synthesized through an Oligomer Combination Approach.

Delivery of functional genetic materials into fibroblast cells to manipulate the transgene expression is of great significance in skin gene therapy. Despite numerous polymeric gene delivery systems having been developed, highly safe and efficient fibroblast gene transfection has not yet been achieved. Here, through a new linear oligomer combination strategy, linear poly(β-amino ester) oligomers are connected by the branching units, forming a new type of poly(β-amino ester). This new multifunctional linear-branched hybrid poly(β-amino ester) (LBPAE) shows high-performance fibroblast gene transfection. In human primary dermal fibroblasts (HPDFs) and mouse embryo fibroblasts (3T3s), ultrahigh transgene expression is achieved by LBPAE: up to 3292-fold enhancement in Gaussia luciferase (Gluc) expression and nearly 100% of green fluorescence protein expression are detected. Concurrently, LBPAE is of high in vitro biocompatibility. In depth mechanistic studies reveal that versatile LBPAE can navigate multiple extra- and intracellular barriers involved in the fibroblast gene transfection. More importantly, LBPAE can effectively deliver minicircle DNA encoding COL7A1 gene (a large and functional gene construct) to substantially upregulate the expression of type VII collagen (C7) in HPDFs, demonstrating its great potential in the treatment of C7-deficiency related genodermatoses such as recessive dystrophic epidermolysis bullosa.

Amphoteric poly(amido amine)s with adjustable balance between transfection efficiency and cytotoxicity for gene delivery.

In order to balance transfection efficiency and cytotoxicity as well as screen the optimal polymers for gene delivery, a series of amphoteric copolymers (poly(CBA-AGM/GABA)s) composed of different ratios between agmatine (AGM) and γ-aminobutyric acid (GABA) monomers were synthesized. The AGM containing positively charged guanidinium groups was used to improve transfection efficiency, while the GABA containing negatively charged carboxyl groups was used to decrease cytotoxicity. It is hypothesized that the amphoteric poly(CBA-AGM/GABA)s synthesized at the optimal ratio of both components would well balance transfection efficiency and cytotoxicity. By comparing these polymers' essential features in gene delivery, the ideal ratio between AGM and GABA was optimized. AGM80, which contained 80% AGM and 20% GABA, showed favorable properties for gene delivery, including moderate DNA condensation capacity, high cellular uptake, strong nuclear localization ability, high transfection efficiency, and low cytotoxicity, indicating that this polymer is very promising as a potent and nontoxic gene carrier.

Low molecular weight PEI-based fluorinated polymers for efficient gene delivery

Fluorinated biomaterials have been reported to have promising features as non-viral gene carriers. In this study, a series of fluorinated polymeric gene carriers were synthesized via Michael addition from low molecular weight polyethyleneimine (PEI) and fluorobenzoic acids (FBAs)-based linking compounds with different numbers of fluorine atoms. The structure-activity relationship (SAR) of these materials was systematically investigated. SAR studies showed that fluorine could screen the positive charge of these polymers. However, this shielding effect of fluorine would endow fluorinated polymers with good balance between DNA condensation and release. In vitro transfection results suggested that these fluorinated polymers could mediate efficient gene delivery. Flow cytometry and confocal microscopy studies demonstrated that more efficient cell uptake could be achieved by fluorinated materials with more fluorine atoms. Cytotoxicity assays showed that these fluorinated materials exhibited very low cytotoxicity even at high mass ratios. This study demonstrates that FBA-based fluorinated biopolymers have the potential for practical application.

Rethinking the impact of the protonable amine density on cationic polymers for gene delivery: A comparative study of partially hydrolyzed poly(2-ethyl-2-oxazoline)s and linear poly(ethylene imine)s

To gain a more profound insight into the impact of the number and the density of protonable amines on the performance of polycations as non-viral vectors, a series of linear poly(ethylene imine)s (LPEIs) with different numbers of ethylene imine (EI) units was compared to partially hydrolyzed (21 to 86%, 20 kDa) poly(2-ethyl-2-oxazoline)s (PHPEtOxs) with a corresponding number of EI units but with varying densities. PHPEtOx polyplexes demonstrated lower transfection efficiencies than the corresponding LPEIs although having the same number of EI units as LPEI, exhibiting smaller or comparable polyplex diameters, similar zeta potentials, and similar or even preferred cyto- and hemocompatibility profiles. The lower efficiency was found to be related to a lower DNA binding capacity and less efficient protection of plasmid DNA against enzymatic degradation. The direct comparison of both types of polymers revealed that the density of charges within the polymer backbone seems to be more important than the total number of EI units. In conclusion, the reduction of the EI density to produce more biocompatible polyplexes must be critically examined, since the presence of high numbers of EI next to each other seems to have a dramatically higher impact on the transfection efficiency than on the in vitro toxicity.

Desarrollo de vectores génicos basados en polímeros sintéticos: PEI y PDMAEMA

En años recientes hubo un auge del uso de terapias génicas para el tratamiento de enfermedades de gran incidencia, como el cáncer. Generalmente, estas se basan en la liberación de material genético como plásmidos, en el núcleo celular, con lo cual se corrige una función o se induce la producción de proteínas deficientes a nivel fisiológico. Para llevar a cabo la terapia génica se requiere de vectores capaces de encapsular el material genético y garantizar su entrega en el núcleo celular. Los polímeros catiónicos sintéticos han llamado la atención como vectores, debido a su capacidad de condensar ácidos nucleicos para formar partículas que los protegen de la degradación enzimática y facilitan su captación celular.La polietilenimina y el polimetacrilato de N, N-dimetilaminoetilo son los polímeros catiónicos más eficaces para la administración génica. Sin embargo, estos requieren modificaciones químicas específicas para eliminar o disminuir algunas limitaciones tales como su alta citotoxicidad y baja biodegradabilidad. En este artículo se analizan algunas de estas modificaciones, enfocándose en avances recientes en el desarrollo de copolímeros anfifílicos como precursores de nanopartículas usadas como vectores génicos.

Remarkable Amplification of Polyethylenimine-Mediated Gene Delivery Using Cationic Poly(phenylene ethynylene)s as Photosensitizers.

Conjugated polymers can serve as good photosensitizers in biomedical applications. However, it remains unknown whether they are phototoxic to the supercoiled structure of DNA in improving gene delivery by the photochemical internalization (PCI) strategy, which complicates the application of conjugated polymers in gene delivery. In this work, we introduced a trace amount of cationic poly(phenylene ethynylene)s (cPPEs) into the polymeric shell of branched polyethylenimine (BPEI)/DNA complexes, studied the photosensitization of singlet oxygen by cPPEs, and confirmed that the supercoiled DNA is undamaged by the singlet oxygen generated by the photoexcitation of cPPEs. By taking advantage of the cPPE-mediated PCI effect, we report that the addition of the trace amount of cPPEs to the outer shell of the BPEI/DNA polyplexes could greatly amplify the transfection of gene green fluorescent protein on tumor cells with the efficiency from 14 to 86% without decreasing the cell viabilities, well solving the problem with a poor transfection capability of BPEI under low DNA-loading conditions. Our strategy to employ conjugated polymers as photosensitizing agents in gene delivery systems is simple, safe, efficient, and promising for broad applications in gene delivery areas.

Effects of interaction with gene carrier polyethyleneimines on conformation and enzymatic activity of pig heart lactate dehydrogenase

Polyethyleneimine (PEI) has long been considered as “golden standard” for polymeric gene delivery carrier, however also induces cytotoxicity. To make a further insight into the molecular basis of PEI cytotoxicity, fluorescence, absorption and circular dichroism spectroscopy were conducted to investigate the influence of PEI (average molecular weight 25,000 and 1800 Da) on the conformation of pig heart lactate dehydrogenase (LDH) and its catalytic efficiency. Zeta-potential measurement and isothermal titration calorimetry were used to reveal the interaction between PEI and LDH. PEI was found to bind onto the surface of LDH predominantly via hydrophobic interaction, inducing a more compact conformation and an increased surface hydrophobicity of the enzyme. The conformational change of LDH induced by PEI binding had little influence on the complex formation between LDH and reduced nicotinamide adenine dinucleotide (NADH, the co-enzyme). However, the nonspecific binding of PEI on the surface of LDH retarded the turnover of the enzyme. Meanwhile, the large quantity of amine groups on the polymer chain made PEI subject to form complexes with NADH and pyruvate (the substrate) via hydrogen bond and electrostatic interaction, which greatly reduced the binding efficient of LDH. The polymer size played an important role in PEI-LDH interaction. The smaller size of lower molecular weight PEI facilitated the close contact with LDH and consequential reduction of the turnover number of the enzyme. However, higher molecular weight PEI was more favorable for competitive binding with NADH and pyruvate and generally decreased the catalytic efficient of LDH.

Platinated graphene oxide: A nanoplatform for efficient gene-chemo combination cancer therapy

Cisplatin (CisPt) is one of the most effective antitumor drugs against a wide range of solid cancers, and recent studies have indicated that combination of CisPt and RNA interference (RNAi) agents would effectively enhance therapeutic index, while the development of simple yet robust dual-delivery systems still remains a challenge. Here, we demonstrated that platinated graphene oxide is an excellent platform to achieve such goal. Nano-Graphene oxide (NGO) was easily platinated by CisPt, and the resulting CisPt/NGO was characterized by several aspects. As a proof-of-concept, an antisense microRNA-21 (Anti-miR-21) was employed as a potential RNAi agent. While most previous work functionalized NGO with cationic polymers for gene delivery, we demonstrated that platinated NGO is a potent carrier to load Anti-miR-21 with improved capacity and adsorption stability. With Anti-miR-21 loading, the system displayed significantly enhanced cytotoxicity to cancer cells, suggesting a synergistic effect. Finally, the underlying mechanism of the improved efficacy was explored, which can be ascribed to the cell apoptosis induced by Anti-miR-21 for gene silencing. This work demonstrated platinated graphene oxide as an effective nanocarrier to co-deliver CisPt and gene therapy for the treatment of cancer.

Effects of the Physicochemical, Colloidal, and Biological Characteristics of Different Polymer Structures between α-Poly(l-lysine) and ε-Poly(l-lysine) on Polymeric Gene Delivery.

Though α-poly(l-lysine) (APL) has been well-studied in gene delivery, ε-poly(l-lysine) (EPL) with same repeating unit of l-lysine but different structure has been rarely investigated. This study compared various effects of their different structures in gene delivery processes. EPL showed less cytotoxicity and more proton buffering capacity for endosomal release than APL. Also, EPL/pDNA polyplexes represented higher nucleus preference than APL/pDNA polyplexes. However, EPL had weaker affinities with pDNA than APL, leading to formation of larger EPL/pDNA complexes with less compactness and successively faster decomplexation. The resultant difference of their pDNA binding affinity caused lower cellular uptake and lower transfection efficiency of EPL/pDNA complexes than APL/pDNA complexes. Thus, this study confirmed that various effects of gene delivery processes are changed by chemical structure of polymeric gene carriers. Especially, despite the low transfection efficiency of EPL-based polyplexes, the study found potentials of EPL in cytocompatibility, endosomal release, and nuclear import.

Conformational change of lysozyme on the interaction with gene carrier polyethyleneimine

Polyethyleneimine (PEI) has been considered as “golden standard” for polymeric gene delivery carriers, however also induces cytotoxicity. To reveal the molecular basis of PEI cytotoxicity, absorption, resonance Rayleigh scattering, fluorescence, circular dichroism and isothermal titration calorimetry were conducted to investigate the interaction between PEI (average molecular weight 25,000 Da) and lysozyme and its influence on the conformation of the enzyme. PEI is an amphiphilic polymer with strong hydrogen bonding capability and weak hydrophobicity due to dense amine groups and methyl groups. However, the hydropathic characteristics that hydrophobic methyl groups and hydrophilic amine groups distributed alternatively along the polymer chain made it difficult to penetrate into the hydrophobic core, but subject to binding onto the surface of lysozyme via hydrophobic interaction and hydrogen bond, leading to a more compact conformation and an increased surface hydrophobicity of the enzyme.

Complexation of plasmid DNA and poly(ethylene oxide)/poly(propylene oxide) polymers for safe gene delivery

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene therapy utilizes gene delivery to deliver genetic material with the goal of treating a disease or condition in the cell. Actual viral vectors may have side effects, while actual systems using metal nanoparticles for gene delivery are toxic. Therefore, we designed here a biocompatible tri-block copolymer PEO20–PPO69–PEO20 as a gene delivery vector [PEO: poly(ethylene oxide); PPO: poly(propylene oxide)]. We studied the conjugation of PEO20–PPO69–PEO20 and DNA using various techniques. Results of gel retardation assay along with zeta potential and dynamic light scattering provide evidence of DNA sequestration. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy show that the PO43− groups of plasmid DNA are primarily involved during nanoconjugate construction. The integrity and functionality of plasmid DNA within the cellular environment is further demonstrated by the expression of green fluorescent protein gene in Escherichia coli. Overall, our findings support the use of block copolymers as delivery systems for mammalian and plant cells.

Membrane structural change of dimyristoylphosphatidylcholine liposome on the interaction with polyethyleneimine.

Polyethyleneimine (PEI) has long been considered as "golden standard" for polymeric gene delivery carriers. To get a better understanding on the molecular basis of PEI cytotoxicity, dynamic light scattering, zeta-potential measurement, fluorescence emission, Fröster resonance energy transfer and anisotropy measurement were conducted to reveal the interaction between PEI and dimyristoylphosphatidylcholine (DMPC) liposome and the influence on the structural properties of the membrane. PEI was found to bind onto the surface of the liposome, inducing an aggregation of the vesicle and an increase in surface potential at low PEI concentration up to 0.05 mg mL-1. A further increase in PEI concentration made little change on the surface potential, however reduced the aggregation of the vesicle due to the repulsion between the adsorbed PEI chains. PEI binding slightly increased the fluidity of lipid in interface region and decreased its packing density, and thus resulted in an enhanced leakage of calcein through the membrane. The polymer size played an important role in PEI-DMPC liposome interaction. PEI of higher molecular weight was more favorable to interact with DMPC and more efficient to perturb the structural properties of the membrane.