Stable Polyplexes Research Articles

Gene delivery efficiency and intracellular trafficking of novel poly(allylamine) derivatives

Non-viral gene carriers for safe and efficient gene transfection have become of particular interest among researchers of different disciplines ranging from physical chemistry to biotechnology. Recently polymeric vectors have been extensively studied as potentially new gene transfer agents. Until now most of the research efforts were made to optimize the gene-to-polymer weight ratio of polyplexes for safe and efficient gene transfection. In this work, we report on the development of novel poly(allylamine) derivatives with different balance of the primary, secondary, tertiary, and quaternary amino groups. All derivatives were able to complex pDNA into polyplexes at low gene-to-polymer weight ratios i.e., 1:1 or 1:2. Moreover, the examined polyplexes were less cytotoxic and showed better transfection efficiency when compared to linear poly(ethyleneimine). These results indicate that the presence of quaternary ammonium groups is important in the formation of stable polyplexes. Polymers with all types of amino groups showed large potential for gene delivery. Furthermore, polyplexes with such derivatives were well internalized by cells and ended up into acidic late endosomes.

Synergistic effect of pendant hydroxypropyl and pyrrolidine moieties randomly distributed along polymethacrylamide backbones on in vitro DNA-transfection

In this work, the cationic monomer N-ethyl pyrrolidine methacrylamide (EPA) was copolymerized with the neutral monomer N-hydroxypropyl methacrylamide (HPMA) at different molar ratios obtaining linear random copolymers that were characterized and evaluated in vitro as non-viral gene carriers using murine Swiss 3T3 fibroblasts. The copolymers with excess or equimolar amount of EPA were able to complex DNA forming stable polyplexes with an average size between 50 and 200nm, while the copolymers with an excess of HPMA do not. Cell viability was shown to depend on the EPA/HPMA molar ratio, exhibiting the equimolar copolymer poly (EPA-co-HPMA) 50:50 (EPA50) a full cytocompatibility, similar to the HPMA-rich systems. This copolymer EPA50 has also shown significantly higher transfection levels than the systems with other compositions and the positive controls poly l-lysine (PLL) and poly EPA (pEPA). This statistical equimolar copolymer EPA50 has unique properties related to its composition and microstructure, which allows it to complex DNA, showing an excellent biocompatibility and high transfection efficiency.

Modulated protonation of side chain aminoethylene repeats in N-substituted polyaspartamides promotes mRNA transfection.

Fine-tuning of chemical structures of polycation-based carriers (polyplexes) is an attractive strategy for safe and efficient mRNA transfaction. Here, mRNA polyplexes comprising N-substituted polyaspartamides with varied numbers of side chain aminoethylene repeats were constructed, and their transfection ability against human hepatoma cells was examined. Transfection efficacy clearly correlated with the number of aminoethylene repeats: polyplexes with odd number repeats (PA-Os) produced sustained increases in mRNA expression compared with those with even number repeats (PA-Es). This predominant efficacy of PA-Os over PA-Es was contradictory to our previous findings for pDNA polyplexes prepared from the same N-substituted polyaspartamides, that is, PA-Es revealed superior transfection efficacy of pDNA than PA-Os. Intracellular FRET analysis using flow cytometry and polyplex tracking under confocal laser scanning microscopy revealed that overall transfection efficacy was determined through the balance between endosomal escaping capability and stability of translocated mRNA in cytoplasm. PA-Es efficiently transported mRNA into the cytoplasm. However, their poor cytoplasmic stability led to facile degradation of mRNA, resulting in a less durable pattern of transfection. Alternatively, PA-Os with limited capability of endosomal escape eventually protect mRNA in the cytoplasm to induce sustainable mRNA expression. Higher cytoplasmic stability of pDNA compared to mRNA may shift the limiting step in transfection from cytoplasmic stability to endosomal escape capacity, thereby giving an opposite odd-even effect in transfection efficacy. Endosomal escaping capability and nuclease stability of polyplexes are correlated with the modulated protonation behavior in aminoethylene repeats responding to pH, appealing the substantial importance of chemistry to design polycation structures for promoted mRNA transfection.

Intercalating quaternary nicotinamide-based poly(amido amine)s for gene delivery

In the development of potent polymeric gene carriers for gene therapy, a good interaction between the polymer and the nucleotide is indispensable to form small and stable polyplexes. Polymers with relatively high cationic charge density are frequently used to provide these interactions, but high cationic charge is usually associated with severe cytotoxicity. In this study an alternative, nucleotide specific binding interaction based on intercalation was investigated to improve polymer/pDNA complex formation. For this purpose bioreducible poly(amido amine) copolymers (p(CBA-ABOL/Nic)) were synthesized with different degrees of intercalating quaternary nicotinamide (Nic) groups and amide-substituted derivatives in their side chains. The quaternary nicotinamide group was chosen as intercalating moiety because this group is part of the naturally occurring NAD+ coenzyme and is therefore expected to be non-toxic and non-carcinogenic. The presence of the quaternary nicotinamide moieties in the poly(amido amine) copolymers showed to effectively promote self-assembled polyplex formation already at low polymer/DNA ratios and results in decreased polyplex size and increased stability of the polyplexes. Furthermore, in contrast to the primary amine functionalized analogs the quaternary nicotinamide polymers showed to be non-hemolytic, indicating their compatibility with cell membranes. Polymers with 25% of Nic in the side chains induced GFP expressions of about 4–5 times that of linear PEI, which is comparable with p(CBA-ABOL), the parent PAA without Nic, but at a two- to fourfold lower required polymer dose. N-phenylation of the nicotinamide functionality even further reduces the required polymer dose to form stable polyplexes, which is a major improvement for these kinds of cationic polymers.

Polycationic star polymers with hyperbranched cores for gene delivery

Core-shell type stars synthesized via atom transfer radical polymerization were used for the delivery of nucleic acids. The interior of the stars consisted of hyperbranched poly(arylene oxindole), while the arms were composed of poly(N,N-dimethylaminoethyl methacrylate). The length of the star arms varied in degree of polymerization (DP) from 14 to 98. The hydrodynamic radius of the structures measured in water indicated the presence of small aggregates, while isolated stars ranging in size from 14 to 29 nm were seen in organic solvent. The phase transition temperatures of the stars in water, measured in basic conditions, were shifted to lower values with increasing DP of the arms. Stable polyplexes of stars with plasmid DNA were formed. Their size varied from 300 nm to 400 nm, depending upon the DP of arms. The zeta potential of the polyplexes was positive, which facilitated their cellular uptake. The DP of the arms influenced the transfection efficiency of HT-1080 cells, demonstrating that stars are promising candidates for synthetic gene vectors.

Polymeric Plerixafor: effect of PEGylation on CXCR4 antagonism, cancer cell invasion, and DNA transfection.

To determine the effect of PEG modification on pharmacologic and gene delivery properties of polymeric CXCR4 antagonist based on Plerixafor. Polymeric Plerixafor (PAMD) was synthesized from Plerixafor (AMD3100) and grafted with different amounts of PEG (2kDa). CXCR4 antagonism of the synthesized polymers was determined using receptor redistribution assay. Inhibition of cancer cell invasion by the polyplexes of the synthesized polymers was assessed using Boyden-chamber method. Transfection activity of DNA polyplexes formed with the synthesized polymers was evaluated in U2OS osteosarcoma and B16F10 melanoma cells. Our results demonstrate that modification of PAMD with PEG decreased toxicity of the polymers, while preserving their CXCR4 antagonism. Polyplexes prepared with PEG-PAMD inhibited invasion of cancer cells to an extent similar to the commercial CXCR4 antagonist Plerixafor. Negative effect of PEG on transfection activity of PEG-PAMD polyplexes could be overcome by using polyplexes formulated with a mixture of PAMD and PEG-PAMD. Modification of PAMD with PEG is a viable strategy to preserve the desirable CXCR4 antagonism and ability to inhibit cancer cell invasion of PAMD, while improving safety and colloidal stability of the PAMD polyplexes.

The potential and advances in RNAi therapy: chemical and structural modifications of siRNA molecules and use of biocompatible nanocarriers.

Small interfering RNA (siRNA) has attracted great attention as a potential new drug due to its highly sequence-specific gene silencing ability and generality in therapeutic target. However, the medical applications of siRNA have been severely hindered by the lack of an optimal systemic delivery methodology. This poor delivery performance of siRNA is mainly caused by its inherent physicochemical properties including short and stiff structure, low charge density and vulnerability to nuclease cleavage. Thus, the successful development of efficient systemic delivery platform for siRNA is a fundamental requirement necessary to bring siRNA-based drugs to the market. Herein, we describe some siRNA delivery methods based on the chemical and structural modifications of delivery materials and siRNA itself to carry siRNA therapeutics safely to the targeted place without adverse effects. This review particularly explains the latest progress of chemically and structurally modified siRNA polymer (poly-siRNA)-based delivery systems. The stable and compact siRNA polyplexes, which are formed by poly-siRNA and different types of biocompatible materials, can enhance serum stability and target delivery efficiency in vitro and in vivo. In addition, this review provides specific information on poly-siRNA delivery systems from basics to therapeutic applications in different animal disease models.

Genetic diseases, immunology, viruses, and gene therapy.

Human Gene TherapyVol. 25, No. 4 Pioneer PerspectiveGenetic Diseases, Immunology, Viruses, and Gene TherapyJames M. WilsonJames M. WilsonSearch for more papers by this authorPublished Online:22 Apr 2014https://doi.org/10.1089/hum.2014.2511AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byOne-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA29 July 2021 | Journal of the American Chemical Society, Vol. 143, No. 31Progress and setbacks: The two faces of technology emergenceResearch Policy, Vol. 49, No. 1Gene Therapy for Neurologic Disease: A Neurosurgical ReviewWorld Neurosurgery, Vol. 121Sugar-based systemsTrehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery22 December 2015 | ACS Biomaterials Science & Engineering, Vol. 2, No. 1Gene-Modified Cells for Stem Cell Transplantation and Cancer Therapy16 July 2014 | Human Gene Therapy, Vol. 25, No. 7Magnetic mesoporous silica nanoparticles for CpG delivery to enhance cytokine induction via toll-like receptor 91 January 2014 | RSC Adv., Vol. 4, No. 86 Volume 25Issue 4Apr 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:James M. Wilson.Genetic Diseases, Immunology, Viruses, and Gene Therapy.Human Gene Therapy.Apr 2014.257-261.http://doi.org/10.1089/hum.2014.2511Published in Volume: 25 Issue 4: April 22, 2014PDF download

A Molecular Dynamics Simulation Study on the Effect of Endogenous Molecules on SiRNA Polyplexes

Small interfering RNA (siRNA) based RNA interference (RNAi) is an important therapeutic strategy in treatment of cancer and hereditary diseases. RNAi is composed of several important stages, the first being siRNA delivery into cells. Polymeric carriers are used in delivery to aggregate siRNA molecules into nano-scale polyplexes. Upon delivery of polyplexes into cytosol, siRNA has to be released to trigger RNAi. However, mechanism of siRNA release from polyplexes is not clear; how endogenous molecules may affect the stability of polyplexes in cytosol is yet to be explored. In this study, we performed a series of molecular dynamics (MD) simulations to study the behavior of siRNA polyplexes in the presence of microRNA (miRNA), the most abundant nucleic acid inside cells. Polyethylenimine (PEI), known as a ‘gold-standard’ among polymeric delivery vehicles, was used as the carrier. miRNAs were introduced into pre-formed siRNA-PEI polyplexes. Quantitative analysis of the MD trajectories was carried out to characterize the change in the structure, compactness and charge distribution of the polyplex upon miRNA addition. PEI affinity toward siRNA and miRNA was determined and compared through individual simulations on siRNA-PEI and miRNA-PEI complexation. Data obtained from the MD simulations was compared with gel electrophoresis assays of siRNA-PEI-miRNA mixtures as well as siRNA-PEI-heparin mixtures; the latter served as a control due to the known capability of heparin to cause siRNA release. Our results revealed the ability of miRNAs to bind to pre-formed siRNA polyplexes through electrostatic interactions with PEI nitrogens. Therefore, upon the addition of miRNA, the size of the polyplex increased and its charge became negative. In agreement with the simulations, in gel electrophoresis, at the same concentrations, heparin was able to release siRNA while polyplexes maintained their integrity in the presence of miRNA.

Highly efficient gene transfection by a hyperosmotic polymannitol based gene tranporter through regulation of caveolae and COX-2 induced endocytosis.

The regulation of cellular uptake to cross the cell membrane is one of the key strategies of importance for efficient gene transfection of non-viral vectors. Hyperosmotic activity of polyplexes may facilitate crossing of the membrane barrier by elevating the osmolarity of the extracellular matrix. In this study, we demonstrated that a polymannitol based gene transporter (PMGT) utilizes the hyperosmoticity contributed by the polymannitol backbone leading to accelerated cellular uptake and enhanced gene transfection. Mannitol dimethacrylate (MDM) monomer was synthesized by esterification of mannitol and methacryloyl chloride. The prepared MDM was then cross-linked with low molecular weight (LMW) branched polyethyleneimine (bPEI) by Michael addition reaction to produce PMGT. PMGT provided polyplex stability in serum, low cytotoxicity, and degradability due to the ester linkages present in the polymannitol backbone. Elevated transfection activity and efficiency, both in vitro and in vivo, were achieved by modulating the mode of cellular uptake due to the effect of the hyperosmotic properties of PMGT. Cyclooxygenase-2 (COX-2) inhibition by SC58236 revealed the up-regulation of this osmoprotectant molecule against the hyperosmotic activity of polymannitol, inducing rapid endocytosis of PMGT in order to re-balance the hyperosmotic environment. Various inhibition studies of endocytosis showed caveolae-mediated endocytosis to be the main route of cellular internalization to account for the enhanced transgene expression.

Simultaneous RGB Emitting Au Nanoclusters in Chitosan Nanoparticles for Anticancer Gene Theranostics

Advanced theranostic materials hold promise for targeted delivery of drugs, with the ability to follow the transport as well as its consequences. This should, ideally, be possible with minimum invasive surgery and having no or minimum cytotoxicity of the materials. It requires development of newer materials whose physical properties would allow for easy probe, which could carry the therapeutic molecules, which will be stable under physiological conditions, and at the same time would be able to permeate barriers to the target. We report the development of a composite consisting of highly fluorescent Au nanoclusters and the biopolymer chitosan, which could easily be converted into nanoparticles and would form a stable polyplex with suicide gene for induction of apoptosis in cervical cancer cells. The simultaneous red, green, and blue fluorescence from the nanoclusters provided convenient optical imaging and flow cytometry probes, without having to use additional dyes. Moreover, the colloidal nanocluster-polymer composite could be converted into solid film and be stored with the retention of optical properties. The pH tunable optical properties in the medium were also intact in the films that quickly dissolved in water with retention of properties.

Assessment of Cholesterol-Derived Ionic Copolymers as Potential Vectors for Gene Delivery

A library of cholesterol-derived ionic copolymers were previously synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization as 'smart' gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA)) and cationic poly(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P(DMAEMA-co-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P(DMAEMA-co-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P(MAA-co-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P(MAA-co-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P(DMAEMA-co-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P(MAA-co-CMA)-DNA) and 20:1 (Q-P(DMAEMA-co-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P(DMAEMA-co-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P(DMAEMA-co-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P(DMAEMA-co-CMA)-siRNA particles are extremely promising candidates for in vivo applications of siRNA therapy.

Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds

Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine–lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively 15N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1–H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5–1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.

Insight into Polycation Chain Length Affecting Transfection Efficiency by O-Methyl-Free N,N,N-Trimethyl Chitosans as Gene Carriers

The structure-function relationship and mechanism of polycations as gene carriers have attracted considerable research interest in recent years. The present study was to investigate the relationship between polycation chain length and transfection efficiency (RCL-TE), and the corresponding mechanism by O-methyl-free N,N,N-trimethyl chitosans (TMCs) as gene carriers. Four TMCs with various chain lengths were synthesized and used to evaluate the RCL-TE. To investigate the details of RCL-TE, a number of factors such as cytotoxicity, cellular uptake efficiency, cellular uptake pathway and intracellular trafficking, were evaluated. In comparison to short chain length TMCs (S-TMCs), long chain length ones (L-TMCs) mediated higher gene expression. The polyplexes formed by L-TMCs and pDNA showed higher stability. The cellular uptake pathway and intracellular trafficking of these TMC/pDNA polyplexes were different. These above factors are probably the key ones in RCL-TE rather than polycation-DNA binding affinity, polyplex particle size in water, zeta potential, serum, cytotoxicity, and cellular uptake efficiency. For rational design of chitosan-based polycations as gene carriers, polycations with relative long chain lengths are more favorable and more attention should be paid to polyplex stability, function of uncomplexed polycation chains, cellular uptake pathway, and intracellular trafficking.

Poly(trehalose): sugar-coated nanocomplexes promote stabilization and effective polyplex-mediated siRNA delivery.

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or "poly(trehalose)" and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.

Design and formulation of trimethylated chitosan-graft-poly(ɛ-caprolactone) nanoparticles used for gene delivery

The ideal gene polyplexes should have a subtle balance between polyplex stability to protect DNA against nucleases, and polyplex instability to permit DNA dissociation inside cells. In this research, low molecular weight trimethylated chitosan was chemically modified with poly(ɛ-caprolactone). Owing to the amphiphilic character, trimethylated chitosan-graft-poly(ɛ-caprolactone) (TMC-g-PCL) formed nanoparticles in aqueous media. TMC-g-PCL nanoparticles could effectively condense pDNA into polyplexes about 200nm in size. The TMC-g-PCL/DNA polyplexes were stable in physiological salt condition and showed high uptake efficiency probably due to the increasing cell membrane-carrier interaction as a result of hydrophobic modification. However, the high degree of quaternization influenced the buffer capacity of TMC-g-PCL and led to a reduction in the release from the lysosomes. By adding chloroquine to exclude the limitation of lysosome escape, the transfection efficiency of TMC-g-PCL/DNA polyplexes was similar to that of PEI/DNA polyplexes. This study demonstrated the potential of TMC-g-PCL/DNA nanoparticles as an efficient carrier for gene delivery.

HA‐Coated Chitosan Nanoparticles for CD44‐Mediated Nucleic Acid Delivery

CD44, the main receptor of hyaluronic acid (HA), is overexpressed in several pathological conditions and therefore can be seen as an interesting target for therapeutic intervention. Here, an approach using HA-coated chitosan (CS)-triphosphate (TPP) nanoparticles is investigated, using CS with different molecular weight (25 and 684 kDa), which influences HA presentation, and enzymatic and pH stability. In a study of nuclease stability, post-digestion of nanoparticles with chitosanase reveals that pDNA is at least partially degraded by DNAse; this may suggest that literature results overestimate the polyplex stability against nucleases. Using cells with a significantly different CD44 expression (RAW 264.7 macrophages-high levels; K562 leukemia cells-low levels; Kelly neuroblastoma cells-absent), the selectivity of CD44-mediated transfection is proven. Further, using luciferase pDNA and then later anti-luc siRNA, low MW CS-based nanoparticles show the best results despite a lower internalization efficiency; this effect is ascribed to a more efficient endosomal disruption and nucleic acid de-complexation.

Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness

Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.

Parallel High-Throughput Screening of Polymer Vectors for Nonviral Gene Delivery: Evaluation of Structure–Property Relationships of Transfection

In recent years, "high-throughput" (HT) has turned into a keyword in polymer research. In this study, we present a novel HT workflow for the investigation of cationic polymers for gene delivery applications. For this purpose, various poly(ethylene imine)s (PEI) were used as representative vectors and investigated via HT-assays in a 96-well plate format, starting from polyplex preparation up to the examination of the transfection process. In detail, automated polyplex preparation, complex size determination, DNA binding affinity, polyplex stability, cytotoxicity, and transfection efficiency were performed in the well plate format. With standard techniques, investigation of the biological properties of polymers is quite time-consuming, so only a limited number of materials and conditions (such as pH, buffer composition, and concentration) can be examined. The approach described here allows many different polymers and parameters to be tested for transfection properties and cytotoxicity, giving faster insights into structure-activity relationships for biological activity.

A Comparison of Thiolated and Disulfide-Crosslinked Polyethylenimine for Nonviral Gene Delivery

Branched polyethylenimine (25 kDa) is thiolated and compared with redox-sensitive crosslinked derivatives. Both polymers thiol contents are assessed; the thiolated polymers have 390-2300 mmol SH groups/mol, whereas the crosslinked polymers have lower thiol contents. Cytotoxicity assays show that both modified polymers give lower hemolysis than unmodified PEI. Increased thiol content increases gene transfer efficiency but also elevates cytotoxicity. Crosslinking improves plasmid DNA condensation and enhances transfection efficiency, but extensive crosslinking overstabilizes the polyplexes and decreases transfection, emphasizing the need to balance polyplex stabilization and unpacking. Thus, at low levels of crosslinking, 25 kDa PEI can be an efficient redox-sensitive carrier system.