DNA Condensation Ability Research Articles

Temperature/GSH Dual Responsive Fluorinated Mesoporous Silica‐Coated Au NRs for Serum‐Resistant Gene Delivery**

AbstractMesoporous silica nanoparticles (MSN) are emerging as one of the most appealing candidates for gene carriers. Nevertheless, one major obstacle for their clinical application is the lack of efficient controlled release and gene delivery. In this study, a mesoporous silica‐modified gold nanorods (Au NRs) controlled release platform was synthesized. Then a thermosensitive copolymer was grafted to the surface of release platform, to impart the switching response of carrier under the photothermal of Au NRs. Fluorination were modified on the surface of MSN through a disulfide bond to achieve glutathione (GSH) response release and improve serum‐resistant ability. This gene carrier, with notable DNA condensation ability and negligible cytotoxicity, could be easily internalized into cells, enhanced green fluorescent protein expression in the presence of serum under laser irradiation. This work inspired us to solve the problem of traditional controllable sustained release and realize the performance of intracellular switch response to gene sustained release.

Self-Assembled Nanoparticle-Forming Derivatives of Dextrin-Conjugated Polyethylenimine Containing Urethane Bonds for Enhanced Delivery of Interleukin-12 Plasmid

Background and Objective: In the present investigation, low molecular weight polyethylenimine (LMW PEI, 1.8 kDa PEI) was conjugated to dextrin via urethane units and tested to transfer plasmid encoding interleukin-12 (IL-12) plasmid. Although high molecular weight PEI (HMW PEI, 25 kDa PEI) has shown substantial transfection efficiency, its wide application has been hampered due to considerable cytotoxicity. Therefore, LMW PEI with low toxic effects was used as the core of our gene transfer construct. Methods: LMW PEI was conjugated to dextrin via urethane units to improve its biophysical characteristics as well as cytotoxic effects. The conjugates were characterized in terms of buffering capacity, plasmid DNA condensation ability, particle size, and zeta potential as well as protection against enzymatic degradation. In Vitro experiments were carried out to evaluate the ability of these LMW PEI conjugates to transfer plasmid encoding human interleukin-12 (hIL- 12) to the cells. The MTT assay was performed to measure the cell-induced toxicity of the conjugates. Results: The results of our study demonstrated that the PEI derivatives with higher amounts of amine content (i.e. higher conjugation degrees) have considerable buffering capacity and plasmid condensation ability. These conjugates could condense plasmid DNA at Carrier to Plasmid ratios (C/P) ≥2 and form polyplexes at the size range of 120-165 nm while their zeta potential was around 5.5-8.5 mV. The results of transfection efficiency demonstrated that the level of IL- 12 production increased by 2-3 folds compared with unmodified LMW PEI while the level of cytotoxicity was not higher than 20%. Conclusion: The strategy used in this study shows a promising way to prepare gene carriers with high transfection efficiency and low toxicity.

Recombinant VAR2CSA malaria protein-targeted exosome-mediated DACT2 gene therapy for effective glioma treatment

A good non-viral vector is the key to the success of gene delivery and therapy; hence, modified exosomes may overcome the physiological barrier in the delivery in vivo. This study aims to design a novel non-viral vector and verify its gene delivery function in vitro and in vivo for dapper homolog 2 (DACT2) genes. In this study, amphipathicity cationic exosomes with receptor targeted function and DACT2 gene loading function were constructed by exosomes, PEG, glycidyl hexadecyl dimethylammonium chloride, and recombinant VAR2CSA malaria protein (RVP) antibody. Characterizations of RVP antibody and PEG modified cationic lipid exosome (RVP-CL) nanocomplexes were evaluated by dynamic light scattering, atomic force microscopy, FTIR, and so on. The DNA condensation ability and stability were assessed by agarose gel electrophoresis. Cellular uptake efficiency and cytotoxicity in glioma cells were investigated. Furthermore, the tumor suppressive effect was evaluated in vitro and in vivo. The results indicated that RVP-CL had a uniform size of 100–200 nm and positive zeta potential. With high uptake efficiency, RVP-CL can rapidly target, recognize, and enter the glioma cell (KNS-42 and U118 mg) and release the gene. Moreover, RVP-CL/DACT2 can effectively inhibit the growth of glioma cells in vitro and in vivo. The modified cationic exosomes may offer a promising strategy for gene delivery in the treatment of RVP-positive gliomas or other tumors.

DNA Condensation Triggered by the Synergistic Self-Assembly of Tetraphenylethylene-Viologen Aggregates and CT-DNA.

Development of small organic chromophores as DNA condensing agents, which explore supramolecular interactions and absorbance or fluorescence-based tracking of condensation and gene delivery processes, is in the initial stages. Herein, we report the synthesis and electrostatic/groove binding interaction–directed synergistic self-assembly of the aggregates of two viologen-functionalized tetraphenylethylene (TPE-V) molecules with CT-DNA and subsequent concentration-dependent DNA condensation process. TPE-V molecules differ in their chemical structure according to the number of viologen units. Photophysical and morphological studies have revealed the interaction of the aggregates of TPE-V in Tris buffer with CT-DNA, which transforms the fibrous network structure of CT-DNA to partially condensed beads-on-a-string-like arrangement with TPE-V aggregates as beads via electrostatic and groove binding interactions. Upon further increasing the concentration of TPE-V, the “beads-on-a-string”-type assembly of TPE-V/CT-DNA complex changes to completely condensed compact structures with 40–50 nm in diameter through the effective charge neutralization process. Enhancement in the melting temperature of CT-DNA, quenching of the fluorescence emission of ethidium bromide/CT-DNA complex, and the formation of induced CD signal in the presence of TPE-V molecules support the observed morphological changes and thereby verify the DNA condensation abilities of TPE-V molecules. Decrease in the hydrodynamic size, increase in the zeta potential value with the addition of TPE-V molecules to CT-DNA, failure of TPE-V/cucurbit(8)uril complex to condense CT-DNA, and the enhanced DNA condensation ability of TPE-V2 with two viologen units compared to TPE-V1 with a single viologen unit confirm the importance of positively charged viologen units in the DNA condensation process. Initial cytotoxicity analysis on A549 cancer and WI-38 normal cells revealed that these DNA condensing agents are non-toxic in nature and hence could be utilized in further cellular delivery studies.

Multifunctional Neomycin-Triazine-Based Cationic Lipids for Gene Delivery with Antibacterial Properties.

Cationic lipids (CLs) have gained significant attention among nonviral gene delivery vectors due to their ease of synthesis and functionalization with multivalent moieties. In particular, there is an increasing request for multifunctional CLs having gene delivery capacity and antibacterial activity. Herein, we describe the design and synthesis of a novel class of aminoglycoside (AG)-based multifunctional vectors with high transfection efficiency and noticeable antibacterial properties. Specifically, cationic amphiphiles were built on a triazine scaffold, allowing for an easy derivatization with up to three potentially different substituents, such as neomycin (Neo) that serves as the polar head and one or two lipophilic tails, namely stearyl (ST) and oleyl (OL) alkyl chains and cholesteryl (Chol) tail. With the aim to shed more light on the effect of different types and numbers of lipophilic moieties on the ability of CLs to condense and transfect cells, the performance of Neo–triazine-based derivatives as gene delivery vectors was evaluated and compared. The ability of Neo–triazine-based derivatives to act as antimicrobial agents was evaluated as well. Neo–triazine-based CLs invariably exhibited excellent DNA condensation ability, even at a low charge ratio (CR, +/−). Besides, each derivative showed very good transfection performance at its optimal CR on two different cell lines, along with negligible cytotoxicity. CLs bearing symmetric two-tailed OL proved to be the most effective in transfection. Interestingly, Neo–triazine-based derivatives, used as either free lipids or lipoplexes, exhibited strong antibacterial activity against Gram-negative bacteria, especially in the case of CLs bearing one or two aliphatic chains. Altogether, these results highlight the potential of Neo–triazine-based derivatives as effective multifunctional nonviral gene delivery vectors.

Redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles for controllable gene delivery and magnetic resonance imaging

AbstractRecently, theranostic candidates that provide a combination of gene delivery and image diagnosis have attracted much interest in medical research. However, there are still many challenges for their clinical applications, such as uncontrollable gene delivery, high cytotoxicity, low transfection efficiency and reduced image contrast. Herein, redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles (IONs@rPEI) were prepared for both efficient gene delivery and magnetic resonance (MR) imaging. Firstly, crosslinked rPEI was synthesized by Michael addition reaction with N,N‐bis(acryloyl)cystamine, dopamine and low‐molecular‐weight branched PEI. The rPEI was then coated onto IONs by ligand exchange reaction forming IONs@rPEI. The physicochemical properties of the IONs@rPEI, such as chemical structure, size, zeta potential and DNA condensation ability, were investigated. In addition, a rapid degradation of the as‐prepared nanoparticles was observed, which was triggered by reducing glutathione via destruction of disulfide linkages suggesting a potential controllable DNA release in tumor cells. In MR imaging detection, the IONs@rPEI had a high T2 relaxivity of 81 L mmol−1 s−1 indicating a potential usage as MR imaging contrast reagent. In cell assay, the IONs@rPEI exhibited low cytotoxicity and good transfection efficiency. In conclusion, the as‐prepared crosslinked IONs@rPEI can be used as a promising technology platform for gene therapy and MR imaging in theranostics. © 2019 Society of Chemical Industry

Cancer gene therapy mediated by RALA/plasmid DNA vectors: Nitrogen to phosphate groups ratio (N/P) as a tool for tunable transfection efficiency and apoptosis

Cancer gene therapy based on p53 tumor suppressor gene supplementation emerges as one of the most challenging and promising strategies. The development of a suitable gene delivery system is imperative to ensure the feasibility and viability of cancer gene therapy in a clinical setting. The conception of delivery systems based on cell- penetrating peptides may deeply contribute for the evolution of therapy efficacy. In this context, the present work explores the p53 encoding plasmid DNA (pDNA) condensation ability of RALA peptide to produce a suitable intracellular delivery platform. These carriers, formed at several nitrogen to phosphate groups (N/P) ratio, were characterized in terms of morphology, size, surface charges, loading and complexation capacity and the fine structure has been analyzed by Fourier-transformed infrared (FTIR) spectroscopy. Confocal microscopy studies confirmed intracellular localization of nanoparticles, resulting in enhanced sustained pDNA uptake. Moreover, in vitro transfection of HeLa cells mediated by RALA/pDNA vectors allows for gene release and p53 protein expression. From these progresses, apoptosis in cancer cells has been investigated. It was found that N/P ratio strongly tailors gene transfection efficiency and, thus, it can be fine-tuned for desired degree of both protein expression and apoptosis. The great asset of the proposed system relies precisely on the use of N/P ratio as a tailoring parameter that can not only modulate vector´s properties but also the extent of pDNA delivery, protein expression and, consequently, the efficacy of p53 mediated cancer therapy.

A co-delivery platform based on plasmid DNA peptide-surfactant complexes: formation, characterization and release behavior.

The development of delivery systems based on cell penetrating peptides represents an incredible asset and may deeply contribute for the evolution of therapies efficacy. In this context, we explore the plasmid DNA (pDNA) condensation ability of TAT peptide to produce a suitable intracellular delivery platform. The nanoparticles were formed at various ratios of nitrogen to phosphate groups (N/P) and the variation of polyplexes properties with this parameter was studied. Beyond the large size exhibited by these carriers, their low pDNA immobilization profile instigates the need for an additional compacting agent. To maximize the performance of this peptide delivery system, a series of alkyl trimethyl ammonium bromide surfactants (CnTAB) were employed to further condense pDNA. In general, not only this strategy promotes the formation of lower sized vectors, but also greatly enhances particle characteristics such as surface charge and pDNA encapsulation. The magnitude of this effect is intimately dependent on surfactant chain length. Furthermore, the known cytotoxicity of cationic surfactants has been dramatically reduced by their incorporation into TAT/pDNA complexes. The release kinetics can be tailored and optimized to promote the controlled/sustained release of pDNA. Following this, the surfactant alkyl chain length and the N/P ratio are important controlling parameters. In addition, doxorubicin and paclitaxel can be efficiently loaded and encapsulated into peptide/pDNA/surfactant carriers. The presented platform reveals a great potential for therapeutic payloads loading and controlled release open advanced and new approaches in the design/formulation of innovative biomedical systems towards clinical translation.

Design, Synthesis, and In Vitro Evaluation of Low Molecular Weight Protamine (LMWP)-Based Amphiphilic Conjugates as Gene Delivery Carriers.

Development of efficient non-viral carriers is one of the major challenges of gene delivery. In the current study, we designed, synthesized, and evaluated the in vitro gene delivery efficiency of novel amphiphilic constructs composed of cholesterol and low molecular weight protamine (LMWP: VSRRRRRRGGRRRR) peptide. Vectors having both hydrophobic and hydrophilic moieties were evaluated in terms of particle size and charge, DNA condensation ability, cytotoxicity, and gene transfection efficiency. The prepared vectors spontaneity self-assembled into the liposome-like particles with a high local positive density. The nano-vehicle A (H5-LMWP-Cholestrol) and nano-vehicle B (LMWP-Cholesterol) could form micelles at concentrations above 50μg/mL and 65μg/mL, respectively. The gel retardation assay showed that nano-vehicles A and B could condense pDNA more efficiently than the corresponding unconjugated peptides. The mean of size and zeta potential of complexed nano-vehicle A at N/P ratios of 5, 15, and 30 were 151nm and 23mv, and those of nano-vehicle B were 224nm and 19mv, respectively. In terms of transfection efficiency, the designed nano-vehicles showed almost two-fold higher gene expression level compared to PEI 25kDa at optimal N/P ratios, and also exhibited negligible cytotoxicity on a model cancer cell, Neuro 2a. The findings of the present study revealed that these cationic micelles can be promising candidates as non-viral gene delivery vehicles.

Reaching for the Stars in the Brain: Polymer-Mediated Gene Delivery to Human Astrocytes

Astrocytes, the “star-shaped” glial cells, are appealing gene-delivery targets to treat neurological diseases due to their diverse roles in brain homeostasis and disease. Cationic polymers have successfully delivered genes to mammalian cells and hence present a viable, non-immunogenic alternative to widely used viral vectors. In this study, we investigated the gene delivery potential of a series of arginine- and polyethylene glycol-modified, siloxane-based polyethylenimine analogs in primary cultured human neural cells (neurons and astrocytes) and in mice. Plasmid DNAs encoding luciferase reporter were used to measure gene expression. We hypothesized that polyplexes with arginine would help in cellular transport of the DNA, including across the blood-brain barrier; polyethylene glycol will stabilize polyethylenimine and reduce its toxicity while maintaining its DNA-condensing ability. Polyplexes were non-toxic to human neural cells and red blood cells. Cellular uptake of polyplexes and sustained gene expression were seen in human astrocytes as well as in mouse brains post-intravenous-injections. The polyplexes also delivered and expressed genes driven by astrocyte-restricted glial fibrillary acidic protein promoters, which are weaker than viral promoters. To our knowledge, the presented work validates a biocompatible and effective polymer-facilitated gene-delivery system for both human brain cells and mice for the first time.

Facile synthesis of semi-library of low charge density cationic polyesters from poly(alkylene maleate)s for efficient local gene delivery

Cationic polymers are one of the main non-viral vectors for gene therapy, but their applications are hindered by the toxicity and inefficient transfection, particularly in the presence of serum or other biological fluids. While rational design based on the current understanding of gene delivery process has produced various cationic polymers with improved overall transfection, high-throughput parallel synthesis of libraries of cationic polymers seems a more effective strategy to screen out efficacious polymers. Herein, we demonstrate a novel platform for parallel synthesis of low cationic charge-density polyesters for efficient gene delivery. Unsaturated polyester poly(alkylene maleate) (PAM) readily underwent Michael-addition reactions with various mercaptamines to produce polyester backbones with pendant amine groups, poly(alkylene maleate mercaptamine)s (PAMAs). Variations of the alkylenes in the backbone and the mercaptamines on the side chain produced PAMAs with tunable hydrophobicity and DNA-condensation ability, the key parameters dominating transfection efficiency of the resulting polymer/DNA complexes (polyplexes). A semi-library of such PAMAs was exampled from 7 alkylenes and 18 mercaptamines, from which a lead PAMA, G-1, synthesized from poly(1,4-phenylene bis(methylene) maleate) and N,N-dimethylcysteamine, showed remarkable transfection efficiency even in the presence of serum, owing to its efficient lysosome-circumventing cellular uptake. Furthermore, G-1 polyplexes efficiently delivered the suicide gene pTRAIL to intraperitoneal tumors and elicited effective anticancer activity.

Mussel-inspired polydopamine-polyethylenimine conjugated nanoparticles as efficient gene delivery vectors for mammalian cells

Efficient delivery of DNA to cells is the primary concern to address the objective of gene therapy. Many attempts have been made to develop polymeric carriers for gene delivery. To have an efficient carrier, it is vital to understand the properties of the vector for better stability, transfection efficiency and minimal toxicity. Branched polyethylenimine (bPEI) has been considered as the ‘gold standard’ for gene delivery but suffers a major drawback of exhibiting high cytotoxicity. Here, we have attempted to develop a mussel-derived polymer, polydopamine (PDA), conjugated polyethylenimine nanoparticles in such a way that the toxic nature of bPEI is suppressed by the conversion of free primary amine groups to secondary and tertiary amines. Keeping the amount of PDA fixed, varying amounts of bPEIs of different molecular weights (25, 10 and 1.8kDa) were conjugated via Michael addition and/or Schiff base. A trend in hydrodynamic size of the conjugated nanoparticles was observed in the range from 160 to 300nm and zeta potential from +12–30mV in the projected three series, viz., (i) PDA1-25bPEI0.5, PDA1-25bPEI1, PDA1-25bPEI2; (ii) PDA1-10bPEI0.5, PDA1-10bPEI1, PDA1-10bPEI2; and (iii) PDA1-1.8bPEI0.5, PDA1-1.8bPEI1, PDA1-1.8bPEI2. A visible trend in the DNA condensation ability and buffering capacity was also noticed. Further, cell cytotoxicity assays revealed that pDNA complexes of PDA-bPEI nanoparticles were non-toxic to mammalian cells and these complexes exhibited several folds higher transfection efficiency than the complexes of native bPEIs as demonstrated by fluorescence measurements and flow cytometry. Altogether, the results advocate the promising potential of these conjugated nanoparticles for future in vivo applications.

Efficient megalin targeted delivery to renal proximal tubular cells mediated by modified-polymyxin B-polyethylenimine based nano-gene-carriers

Non-viral vectors have attracted great interest, as they are simple to prepare, easy to modify and relatively safe, compared to viral vectors. Kidney-targeted gene delivery systems depict a promising technology to improve drug efficacy in renal diseases treatments. In order to develop a novel kidney-targeted gene delivery system, we synthesized polyamine-PEI conjugates using polymyxin B as ligand and investigated their potential targeting efficiency. After grafting either PEI25 kDa or PEI10 kDa with polymyxin B through amidation reaction, the modified-polymyxin-PEI/DNA-nanoplexes were produced via electrostatic attraction between the cationic polymers and EGFP plasmid. The properties of modified polymers including size, surface charge density, DNA condensation ability, buffering capacity and cytotoxicity were evaluated. Results revealed that the average size of -modified-polymyxin- PEI25kDa was about 143–180nm and modified-polymyxin-PEI10kDa 115–194nm. The zeta potentials were in the range of 16.4±1.87 to 23.43±1.25mV and 11.3±1.4 to 19.3±2.1mV for conjugates based on PEI25 and PEI10 respectively. The AFM images revealed that the complexes were spherical and nano-sized at C/P=4. The buffering capacity of both PEI 10 and 25kDa increased as the percentage of polymyxin B grafting increased. In vitro study demonstrated that modified-polymyxin-PEI conjugates could remarkably improve the gene transfection efficiency to kidney cells. The transfection efficiency of the polyplexes was dependent on the weight ratio of ligand in the formulation (~12 and 8 fold increase for PEI25 and PEI10kDa, respectively) and was significantly higher than that of unmodified PEIs/DNA nanoparticles. These results suggest that modified-polymyxin-PEI /DNA nanoparticles can effectively target megalin-expressing kidney cells and show improved transfection efficiency and low cytotoxicity in In vitro and In vivo studies. Animal studies confirmed the in vivo study. Thus, these conjugates can be considered as a safe and efficient non-viral therapeutic therapy vector for kidney diseases.

Megalin-targeted enhanced transfection efficiency in cultured human HK-2 renal tubular proximal cells using aminoglycoside-carboxyalkyl- polyethylenimine -containing nanoplexes

Non-viral vectors are of interest as therapeutic gene delivery agents in gene therapy, because they are simple to prepare, easy to modify and have definable safety profiles compared to viral vectors. The potential of gene therapy in the treatment of renal diseases is limited by a lack of effective kidney-targeted gene delivery systems. Aminoglycoside antibiotics gentamicin and neomycin were connected by amide linkages to carboxyl groups on carboxyalkylated-PEI25 (25kDa PEI) or carboxyalkylated-PEI10 (10kDa PEI). Aminoglycoside-carboxyalkylated-PEI conjugates were characterized with respect to size, surface charge density, DNA condensation ability, and buffering capacity. Polyplexes prepared by electrostatic interaction between aminoglycoside-carboxyalkylated-PEIs and enhanced green fluorescent protein-expressing (EGFP) plasmid DNA had appropriate nano-scale size (143–173nm). Their targeting potential was investigated in cultured HK-2 immortalized human cortex/proximal tubule kidney epithelial cells, which expresses megalin, a scavenger receptor that mediates endocytosis of a diverse group of ligands, including aminoglycoside antibiotics. Aminoglycoside-carboxyalkylated-PEIs significantly increased EGFP gene transfection efficiency in HK-2 cells by ∼13-fold for aminoglycoside-carboxyalkylated-PEI25 and ∼7-fold increase for aminoglycoside-carboxyalkylated-PEI10 relative to the corresponding PEIs without aminoglycosides. The transfection efficiency of polyplexes was dependent on the weight ratio of aminoglycoside-containing ligand in the carrier. In the presence of a range of concentrations of human serum albumin, which competes for megalin binding, aminoglycoside-carboxyalkylated-PEI-mediated transfection was reduced to background levels. These results suggest that aminoglycoside-carboxyalkylated-PEI polyplexes can target megalin-expressing kidney-derived cells in vitro resulting in improved transfection efficiency with low cytotoxicity.

The effect of cell penetrating peptides on transfection activity and cytotoxicity of polyallylamine.

Introduction: Cationic polymers have the potential to be modified to achieve an ideal gene vector lacking viral vector defects. The aim of the present study was to improve polyallylamine (PAA) transfection efficiency and to reduce cytotoxicity by incorporating of cell-penetrating peptides (CPPs). Methods: To prepare the peptide-based polyplexes, PAA (15 kDa) was modified with 2 peptides (TAT and CyLoP-1) by covering the 0.5% and 1% of amines. Buffer capacity and DNA condensation ability of modified polymer, particle size and zeta potential of nanoparticles, cell viability, and transfection activity of vectors were evaluated. Results: In low carrier to plasmid (C/P) weight ratios such as 0.5 and 1, the unmodified polymer was more capable to condense the DNA compared to the synthesized vectors. In C/P ratio of 2, the plasmid was fully condensed in all vectors. The size of polyplexes ranged from 195 to 240 nm. The zeta potential was almost as the same as PAA and varied from 25 to 27 mV. All polyplexes increased the buffer capacity compared to PAA. The transfection efficiency was improved compared to unmodified polymer especially in the vectors modified with 1% of TAT or CyLoP-1 peptides in C/P ratio of 2. The cytotoxicity of prepared vectors was less than PAA. In most ratios, the cytotoxicity of the CyLoP-1 modified samples was less than the TAT modified ones. Conclusion: Modification of PAA with CPPs improved the transfection activity of vector.

Cationic Liposomes Modified with Polyallylamine as a Gene Carrier: Preparation, Characterization and Transfection Efficiency Evaluation.

Purpose: Cationic polymers and cationic liposomes have shown to be effective non-viral gene delivery vectors. In this study, we tried to improve the transfection efficiency by employing the advantages of both. Methods: For this purpose, modified polyallylamines (PAAs) were synthesized. These modifications were done through the reaction of PAA (15 KDa) with acrylate and 6-bromoalkanoic acid derivatives. Liposomes comprising of these cationic polymers and cationic lipid were prepared and extruded through polycarbonate filters to obtain desired size. Liposome-DNA nanocomplexes were prepared in three carrier to plasmid (C/P) ratios. Size, zeta potential and DNA condensation ability of each complex were characterized separately and finally transfection efficiency and cytotoxicity of prepared vectors were evaluated in Neuro2A cell line. Results: The results showed that mean particle size of all these nanocomplexes was lower than 266 nm with surface charge of 22.0 to 33.9 mV. Almost the same condensation pattern was observed in all vectors and complete condensation was occurred at C/P ratio of 1.5. The lipoplexes containing modified PAA 15 kDa with 10% hexyl acrylate showed the highest transfection efficacy and lowest cytotoxicity in C/P ratio of 0.5. Conclusion: In some cases nanocomplexes consisting of cationic liposome and modified PAA showed better transfection activity and lower cytotoxicity compared to PAA.

Tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding IL-12 gene

Targeted delivery by polymer-based nanoparticles has been considered as an efficient approach to transfer genetic materials into cells. Considering the over expression of integrin αVβ3 receptor on tumor cells and the presence of the binding site for tetraiodothyroacetic acid (tetrac) on integrin receptor, we hypothesized that the conjugation of tetrac to polyethylenimine (PEI) might be an effective strategy for pDNA delivery into the cells over-expressing integrins on their surfaces. In order to test the hypothesis, tetrac conjugated PEI/plasmid DNA complexes were prepared and their ability in the delivery of plasmid encoding IL-12 gene was investigated. Moreover, the conjugates were characterized with respect to plasmid DNA condensation ability, particle size and zeta potential as well as cell-induced toxicity and plasmid protection against DNase degradation. The results demonstrated that tetrac conjugated derivatives of PEI were able to condense the plasmid and protect it against enzyme degradation. The results of dynamic light scattering (DLS) and atomic force microscopy (AFM) revealed that the formed nanoparticles were in the size range of 85–125nm. The highest level of IL-12 gene expression was achieved by terac-conjugated PEIs at the carrier to plasmid ratio of 8 where they could increase the level of gene expression up to 4 fold in the cell lines over-expressing integrin αVβ3 receptor whereas no increase in the level of IL-12 expression in the cell lines lacking integrin receptors was observed. Also, the results of the competitive inhibition of the receptors demonstrated the specificity of transfection for the cells over expressing αvβ3 receptor. On the other hand, tetrac conjugation of PEI significantly reduced the polymer-induced apoptotic effects. The results obtained in this investigation suggest the potential of tetrac as a small molecule mimicking the binding properties of integrin binding peptides (e.g., RGD) for targeted gene delivery.

A Facile Strategy to Prepare Hyperbranched Hydroxyl-Rich Polycations for Effective Gene Therapy.

For effective gene therapy, nonviral gene carriers with low toxicity and high transfection efficiency are of much importance. In this work, we developed a facile strategy to prepare hyperbranched hydroxyl-rich polycations (denoted by TE) by the one-pot method involving ring-opening reactions between two commonly used reagents, ethylenediamine (ED) with two amino groups and 1,3,5-triglycidyl isocyanurate (TGIC) with three epoxy groups. The hyperbranched TEs with different molecular weights were investigated on their DNA condensation ability, protein absorption property, biocompatibility, transfection efficiency, and in vivo cancer therapy and toxicity. TE exhibited low cytotoxicity and protein absorption property due to the plentiful hydroxyl groups. The optimal transfection efficiency of TE was significantly higher than that of the gold standard polycationic gene carrier branched polyethylenimine (PEI, 25 kDa). Furthermore, TE was applied for in vivo tumor inhibition by the delivery of antioncogene p53, which showed good antitumor efficiency with low adverse effects. The present work provides a new concept for the facile preparation of hyperbranched hydroxyl-rich polycationic carriers with good transfection performances.

Acid-labile poly(ortho ester amino alcohols) by ring-opening polymerization for controlled DNA release and improved serum tolerance

A new type of linear copolymers, poly(ortho ester amino alcohols) (POEAAs), has been synthesized by ring-opening polymerization between diglycidyl ethers and amine monomer with ortho ether bonds. POEAAs have uniform distribution of tertiary amine main-chains along with abundant hydroxyl groups and acid-cleavable tertiary amine side-chains. POEAA1 and 2 containing different diglycidyl ether moieties were prepared to regulate their hydrophilicity and DNA condensation ability. POEAAs could efficiently condense plasmid DNA into nanoparticles, which have much lower cytotoxicity than branched PEI (25 KDa). The breakage of tertiary amines in side chains of POEAAs caused by hydrolysis of ortho ester bonds at mildly acid-pHs could obviously facilitate the release of DNA from the polyplexes. More significantly, the effective transfection on 293T cells mediated by POEAAs was not negatively affected by 10% serum concentration because of their improved serum tolerance.

Gene delivery to neuroblastoma cells by poly (l-lysine)-grafted low molecular weight polyethylenimine copolymers

Polyethylenimine (PEI) and poly (l-lysine) (PLL) are among the most investigated non-viral gene carriers. However, both polymers contain deficiencies that restrict their applications. In the present study, we synthesized PLL-alkyl-PEI conjugates via 6-carbon alkyl linker and investigated their possible advantages in gene delivery. Four PLL copolymers were synthesized with different molecular weights and ratios of PEI. The physiochemical properties of synthesized conjugates such as size, zeta potential, DNA condensation ability, buffering capacity and cytotoxicity were investigated. Renilla luciferase assay was employed to evaluate the gene transfection efficiency of pDNA-polymer to Neuro2A cell line. DNA condensation and particle size measurements showed that new PLL-PEI conjugates could form polyplexes in nano-scale size in the range of 99–122 nm and were able to condense DNA at low concentration. While cytotoxicity reduced in some groups, the transfection efficiency increased about 2.8 and 4 fold as compared to the unmodified PEI 1.8 kDa and 10 kDa, respectively. The results of the present study showed that the chemical modifications of PEI with PLL could significantly improve transfection efficiency and PLP10-10% shows the most promise as a new gene carrier.