Polymeric Gene Carriers Research Articles

Activated tyrosine kinase c‐Src‐responsive artificial gene carrier: in vitro and in vivo evaluation

AbstractThe proto‐oncogene c‐Src is a protein‐tyrosine kinase that is associated with increased risk of acute and chronic kidney diseases, cardiovascular diseases, neurological diseases, and cancers. The purpose of this study was to develop a gene delivery carrier responding to activated c‐Src and to evaluate its efficacy in vitro and in vivo. The polymeric gene carrier consists of a neutral main chain bearing a peptide substrate of c‐Src or negative control peptide. Complexes of the polymer containing c‐Src‐targeting peptides and DNA were phosphorylated in the presence of c‐Src, resulting in release of the DNA. Green fluorescent protein (GFP) expression was observed after microinjection of complexes of polymer containing c‐Src‐targeting peptides and GFP‐encoding DNA into A431 cells at the nitrogen/phosphate (N/P) ratio of 1.0. GFP was not expressed in cells microinjected with negative control polymer/DNA complex. Direct injection of complexes of polymer containing c‐Src‐targeting peptides and luciferase‐encoding DNA at N/P ratios of 1.0 and 2.0 into tumor‐bearing mice resulted in luciferase expression in A431 tumors but not in normal skin tissues. No luciferase expression was observed in A431 tumors or skin tissues after injection of control polymer/DNA complex. These results indicate that our gene delivery carrier enables activated c‐Src‐specific gene expression.

Synthesis and characterization of oligo aminoglycosides and polyethylenimine conjugates as polymeric gene carriers

Synthesis and characterization of oligo aminoglycosides and polyethylenimine conjugates as polymeric gene carriers

Synthetic Helical Polypeptide as a Gene Transfection Enhancer.

The relatively low transfection efficiency limits further application of polymeric gene carriers. It is imperative to exploit a universal and simple strategy to enhance the gene transfection efficiency of polymeric gene carriers. Herein, we prepared a cationic polypeptide poly(γ-aminoethylthiopropyl-l-glutamate) (PALG-MEA, termed PM) with a stable α-helical conformation, which can significantly improve the gene transfection efficiency of cationic polymers. PM can be integrated into polymeric gene delivery systems noncovalently through electrostatic interactions. With the assistance of PM, polymeric gene delivery systems exhibited excellent cellular uptake and endosomal escape, thereby enhancing transfection efficiency. The transfection enhancement effect of PM was applicable to a variety of cationic polymers such as polyethylenimine (PEI), poly-l-lysine (PLL), and polyamidoamine (PAMAM). The ternary gene delivery system PM/pshVEGF/PEI exhibited an excellent antitumor effect against the B16F10 tumor model. Moreover, we demonstrated that PM could also enhance the delivery of gene editing systems (sgRNA-Cas9 plasmids). This work provides a facile and effective strategy for constructing polymeric gene delivery systems with a high transfection efficiency.

Interleukin-12 Plasmid DNA Delivery by N-[(2-Hydroxy-3-trimethylammonium)propyl]chitosan-Based Nanoparticles.

Cationic polysaccharides are capable of forming polyplexes with nucleic acids and are considered promising polymeric gene carriers. The objective of this study was to evaluate the transfection efficiency and cytotoxicity of N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan salt (HTCS), a quaternary ammonium derivative of chitosan (CS), which benefits from non-ionizable positive charges. In this work, HTCS with a full quaternization of amino groups and a molar mass of 130,000 g·mol−1 was synthesized to use for delivery of a plasmid encoding the interleukin-12 (IL-12) gene. Thus, a polyplex based on HTCS and the IL-12 plasmid was prepared and then was characterized in terms of particle size, zeta potential, plasmid condensation ability, and protection of the plasmid against enzymatic degradation. We showed that HTCS was able to condense the IL-12 plasmid by the formation of polyplexes in the range of 74.5 ± 0.75 nm. The level of hIL-12 production following the transfection of the cells with HTCS polyplexes at a C/P ratio of 8:1 was around 4.8- and 2.2-fold higher than with CS and polyethylenimine polyplexes, respectively. These findings highlight the role of HTCS in the formation of polyplexes for the efficient delivery of plasmid DNA.

Cationic Heteropolymers with Various Functional Groups as Efficient and Biocompatible Nonviral Gene Vectors.

With the rise and development of gene therapy, it is of great significance to develop highly efficient and biocompatible polymeric gene carriers. In this work, a series of heteropolymers from ring-opening polymerization of diepoxide compounds and various functional primary amines were synthesized. The feed dosage of amines was adjusted to obtain the polymers with different functional group contents, and the structure-activity relationships of these polymers as nonviral gene vectors were examined in detail. Results revealed that, although the amine with the fluorinated chain seemed to be less reactive in the polymerization, the relative content of each component in the target product was consistent with the feed dosage. Compared to the "golden standard" polyethylenimine (PEI) 25 kDa, these heteropolymers showed much lower cytotoxicity and higher gene transfection efficiency, especially in serum-containing medium, and up to 78 times of efficiency than PEI was obtained. Meanwhile, they exhibited much better serum resistance than PEI, compared with the transfection efficiency in serum-free experiments, and even higher efficiency could be achieved with serum in HeLa cells. Mechanism study results suggest that the content of fluorinated chain and histamine might distinctly influence their transfection. The fluorinated chains could enhance the serum tolerance and cellular uptake efficiency (with serum), while the imidazole group in the histamine chain would improve the endosome/lysosome escape.

Polyplex transfection from intracerebroventricular delivery is not significantly affected by traumatic brain injury

Traumatic brain injury (TBI) is largely non-preventable and often kills or permanently disables its victims. Because current treatments for TBI merely ameliorate secondary effects of the initial injury like swelling and hemorrhaging, strategies for the induction of neuronal regeneration are desperately needed. Recent discoveries regarding the TBI-responsive migratory behavior and differentiation potential of neural progenitor cells (NPCs) found in the subventricular zone (SVZ) have prompted strategies targeting gene therapies to these cells to enhance neurogenesis after TBI. We have previously shown that plasmid polyplexes can non-virally transfect SVZ NPCs when directly injected in the lateral ventricles of uninjured mice. We describe the first reported intracerebroventricular transfections mediated by polymeric gene carriers in a murine TBI model and investigate the anatomical parameters that dictate transfection through this route of administration. Using both luciferase and GFP plasmid transfections, we show that the time delay between injury and polyplex injection directly impacts the magnitude of transfection efficiency, but that overall trends in the location of transfection are not affected by injury. Confocal microscopy of quantum dot-labeled plasmid uptake in vivo reveals association between our polymers and negatively charged NG2 chondroitin sulfate proteoglycans of the SVZ extracellular matrix. We further validate that glycosaminoglycans but not sulfate groups are required for polyplex uptake and transfection in vitro. These studies demonstrate that non-viral gene delivery is impacted by proteoglycan interactions and suggest the need for improved polyplex targeting materials that penetrate brain extracellular matrix to increase transfection efficiency in vivo.

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.

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.

A synergistic polyphosphoester-based co-delivery system of the anticancer drug doxorubicin and the tumor suppressor gene p53 for lung cancer therapy.

Lung cancer is one of the most frequently occurring cancers worldwide and its pathological complexity necessitates combination therapies of various therapeutic elements such as anti-cancer drugs and genes to achieve synergistic treatment. In this study, we designed a co-delivery carrier of the anti-cancer drug doxorubicin (DOX) and the tumor suppressor gene p53 for lung cancer treatment. First, a copolymer precursor (mPEG-b-PBYP) was prepared via ring-opening polymerization of 2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane (BYP). Subsequently, on the basis of the precursor, a pH-sensitive prodrug (abbreviated as mPEG-b-PBYP-hyd-DOX) and a polycation gene vector (abbreviated as mPEG-b-PBYP-g-DAE) were separately prepared via CuAAC and thiol-yne "click" chemistry, wherein DAE represents 2-dimethylaminoethanethiol hydrochloride. After that, the prodrug and the gene vector copolymers were mixed in an aqueous solution in order to self-assemble into hybrid micelles, which could then condense the p53 gene and finally form DOX prodrug/p53 co-loaded nanoparticles. The average particle size and morphologies of the hybrid micelles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The gel retardation assay showed that the p53 gene could be well immobilized and maintained stably under the electronegative conditions, similar to those in the blood circulation. A cytotoxicity assay showed the obvious antitumor effect of the hybrid micelle/p53 gene nanoparticles on A549 and H1299 cells when compared to drug or gene therapy applied alone, respectively. Furthermore, the results from the live cell imaging system revealed that the hybrid micelle/p53 gene nanoparticles could effectively deliver and release DOX and the p53 genes into A549 cells. All of the results showed that the hybrid micelles containing the DOX prodrug and p53 genes could be widely used in the treatment of lung cancer.

Enhanced Systemic Anti-Angiogenic siVEGF Delivery Using PEGylated Oligo-d-arginine.

Angiogenesis mainly mediated by upregulation of vascular endothelial growth factor (VEGF) provides a hallmark of rapidly proliferating tumor cells and an essential component of the tumor growth and microenvironment, making it a targetable process for antitumor therapy. RNA interference (RNAi) provides a very effective tool for developing antitumor therapies; however, its application to date has been hampered due to the lack of efficient small interfering RNA (siRNA) delivery systems in vivo. Here, we report a polymeric gene carrier system based on PEGylation of a cationic cysteine-ended 9-mer arginine oligopeptide (CR9C), which provides effective siRNA systemic delivery and specifically suppresses VEGF (siVEGF). The PEG500-CR9C/siVEGF oligopeptoplex provided improved blood circulation, enhanced protection from serum proteases, reduced uptake in the liver and kidneys, enhanced tumor targeting, and down-regulated intratumoral VEGF level, which comprehensively resulted in improved antitumor efficacy without significant toxicity in vivo. PEG500-CR9C has a great potential for safe and efficient siRNA delivery with diverse applications.

Rational design of acid-labile branched polycation with superior gene transfection capacity

There is much need in advancing rational design of synthetic cationic polymers as gene carriers. Inspired by the molecular blueprint of the famed branched polyethylenimine (bPEI), here we describe a new series of acid-labile branched poly(ortho ester amino alcohol)s (bPOEAAs) that embodies multiple design principles about polymer degradability, chain branching, and type of cationic amino groups. The bPOEAAs were facilely synthesized via expoxide-amine reaction of monomers with various ratios, allowing a range of branching degrees and amine compositions to be explored. A polymer (bPOEAA2) with an intermediate degree of branching and approximately equal fraction of secondary and tertiary amines was identified to be optimal in forming stable polyplexes with plasmid DNA. Rapid release of DNA from the polymer was achieved by accelerated backbone degradation at endosomal acidic pH. Together, these unique features of bPOEAA2 enabled markedly improved transfection efficiency in cells in physiological medium and much lower cytotoxicity than bPEI. Insights gained from the rational design and screening of bPOEAAs may contribute to deeper understanding of structure-activity relationship of polymeric gene carriers, which may ultimately lead to safer and more effective gene therapies.

Cationic Polythiophenes as Gene Delivery Enhancer.

There is urgent demand of easily available and highly effective method to improve transgene performance of polymeric gene carriers at low consumption of delivery materials. We developed biocompatible multicomponent nanocomposites in which small quantities of cationic polythiophenes were engineered into the outer shell of polypeptide/DNA polyplexes without covalent linkages. We revealed the introduction of polythiophenes in small quantities led to multiple outcomes including modulation of polyplex size and zeta potential, increase in polyplex stability, promotion of endolysosome membrane disruption, light-induced generation of reactive oxygen species (ROS), and significant enhancement of gene delivery to tumor cells. The factors such as structural architectures, molecular weights, photosensitizing capability, and percentage composition of polythiophenes were investigated.

A smart gene delivery platform: Cationic oligomer

Low transfection efficiency and high cytotoxicity of polymeric gene carriers have hampered the application of numerous polycations for gene therapy. To overcome this barrier, a cationic glycoconjugate of kanamycin and di(ethylene glycol) diacrylate was prepared via a facile approach. Nuclear magnetic resonance, Fourier transform infrared spectroscopy, and size exclusion chromatography were employed to investigate the resulting materials. Agarose gel electrophoresis, atomic force microscopy, and circular dichroism spectroscopy were used to record the interaction of the cationic oligomer and plasmid DNA. Finally, the cytotoxicity and transfection efficiency were evaluated by using COS-7 cells. The results indicated that cationic oligomers had been obtained and plasmid DNA was condensed into nanocomplexes, with a high transfection efficiency of the oligomer and a low toxicity in COS-7 cell line. It provided a novel perspective to develop gene carrier, with better safety and greater transfection efficiency, compared to traditional high molecular weight polymers.

Recent progress in cationic polymeric gene carriers for cancer therapy

In recent years, various carriers for gene delivery have been developed for biomedical applications. Among all kinds of gene carriers, cationic polymeric carriers for delivery therapeutic gene as non-viral carriers have received growing interests due to their improved high transfection efficiency with the relative safety. In particular, the advancement of novel polymeric gene carriers has gained much progress in the development of effective anticancer therapy. Herein, this review focused on the development of cationic polymeric carriers for cancer therapy, including polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimers, polylysine (PLL), chitosan and modified cationic polymers. And recent progresses in the development of novel polymeric carriers for gene delivery, such as targeted gene carriers, responsive gene carriers and multifunctional gene carriers, were summarized. Finally, the future perspectives in the development of novel polymeric carriers for delivery gene were presented.

Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

In this report, we exploited the potential of PEI-PLL to deliver VEGF gene for the potential therapeutic treatment of PD by using both cell culture and animal models of PD. To the best of our knowledge, this is the first report describing the use of novel polymeric gene carriers for the delivery of VEGF gene to DA neurons with improved transfection efficiency. Finally, the study will lead to a significant advancement in the field of non-viral PD gene therapy treatment.

Layer‐by‐layer inorganic/polymeric nanoparticles for kinetically controlled multigene delivery

Nonviral gene delivery methods represent a potential safe and effective approach for treating myriad diseases. For many gene therapy applications, delivering multiple exogenous genes and controlling the time profile that these genes are expressed would be advantageous. Polymeric nonviral gene carriers are versatile and can be readily tailored for particular therapeutic applications, have the ability to carry multiple large genes within each particle, and can be more easily manufactured than viruses used for gene delivery. A layer-by-layer (LbL) theranostic-enabling nanoparticle was developed to incorporate two plasmid types which have differing expression time profiles. Temporally controlling the expression of exogenous DNA enables superior control over the microenvironment and could lead to better control over differentiation pathways and cell fate. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 707-713, 2016.

Novel glycol chitosan-based polymeric gene carrier synthesized by a Michael addition reaction with low molecular weight polyethylenimine

A glycol chitosan-based polymer that spontaneously assembles with plasmid DNA into nanorods was evaluated as a non-viral vector for gene delivery. Glycol chitosan–methyl acrylate-polyethylenimine (GMP) was synthesized by grafting polyethylenimine onto glycol chitosan via amidation after Michael addition using methyl acrylate. Gel retardation and PicoGreen assay experiments showed complete complex formation with plasmid DNA. GMP/pDNA complexes were characterized using biophysical techniques and were found to be positively charged rod-shape structures with widths in the nanometer scale and lengths in the micrometer scale. Transfection efficiency and cytotoxicity of GMP polymer was evaluated in human epithelial ovary carcinoma (HeLa) cells, human embryonic kidney 293 (HEK293) cells, and human hepatocellular liver carcinoma (HepG2) cells, in comparison to high molecular weight polyethylenimine, a commonly used transfection reagent. Intracellular polymer uptake was compared and confirmed by confocal microscopy. The results demonstrate that GMP, a hybrid polymer of glycol chitosan grafted with branched polyethylenimine, may serve as a promising vehicle for efficient gene delivery.

Tumor accumulation of protein kinase-responsive gene carrier/DNA polyplex stabilized by alkanethiol for intravenous injection

We synthesized polymeric gene carriers consisting of poly-L-lysine (PLL) main chain modified both with substrate peptide for protein kinase Cα (PKCα) and alkanethiol (pentadecanethiol). Due to the grafted substrate peptide, the polyplex prepared from these carriers is expected to show gene expression triggered by the phosphorylation of the peptide by intracellular PKCα. The modified alkanethiol on the main chain stabilized the polyplex both via disulfide crosslinking and hydrophobic interaction. The polyplex found to show gene expression in vitro when the alkanethiol content in the main chain was enough low (4-mol%-modification of PLL’s ε-amine group) to minimize cytotoxic effect. Even though the content of alkanethiol is low, the polyplex had significant stability in a model serum solution and showed longer blood circulation in vivo. The polyplex clearly accumulated in tumor after intravenous injection.

Complexation of bioreducible cationic polymers with gold nanoparticles for improving stability in serum and application on nonviral gene delivery.

Widespread applications of conventional polymeric gene carriers are greatly hampered by their inefficient transfection performance under serum-containing environment. Aiming to overcome this limitation, we propose a bioreducible polyethylenimine-gold nanocomplex system (SSPEI-Au NC), which can be prepared by a simple layer-by-layer (LBL) assembly procedure. SSPEI-Au NC contains sequentially deposited layers of bioreducible polyethylenimine (SSPEI) and poly(γ-glutamic acid) (γ-PGA) for efficient binding and delivery of plasmid DNA (pDNA). SSPEI-Au NC was characterized for various physicochemical properties, including: UV-vis spectra, TEM imaging, hydrodynamic size, and pDNA binding ability. The SSPEI-Au NC were efficiently uptaken by mammalian cells as observed using dark-field microscopy. Comparing to nondegradable PEI25k, the bioreducible SSPEI-Au NC exhibited superior transfection capability under serum-containing condition while causing lower cytotoxicity on mammalian cell lines. The effect of serum on SSPEI-Au NC dispersity was studied using UV-vis spectrometry and the results suggest that serum-assisted colloidal stability of SSPEI-Au NC contributed to its serum-resistant 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.