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Neurotensin-polyplex nanoparticles provide efficient gene transfection of nigral dopaminergic neurons when intracerebrally injected in preclinical trials of Parkinson's disease because they do not cross the blood–brain barrier (BBB). Therefore, this study aimed to open BBB with focused ultrasound (FUS) on the substantia nigra to attain systemic and intranasal transfections and evaluate its detrimental effect in rats. Systemically injected Evans Blue showed that a two-pulse FUS opened the nigral BBB. Accordingly, 35 μL of neurotensin-polyplex nanoparticles encompassing the green fluorescent protein plasmid (79.6 nm mean size and + 1.3 mV Zeta-potential) caused its expression in tyrosine hydroxylase(+) cells (dopaminergic neurons) of both substantiae nigrae upon delivery via internal carotid artery, retro-orbital venous sinus, or nasal mucosa 30 min after FUS. The intracarotid delivery yielded the highest transgene expression, followed by intranasal and venous administration. However, FUS caused neuroinflammation displayed by infiltrated lymphocytes (positive to cluster of differentiation 45), activated microglia (positive to ionized calcium-binding adaptor molecule 1), neurotoxic A1 astrocytes (positive to glial fibrillary acidic protein and complement component 3), and neurotrophic A2 astrocytes (positive to glial fibrillary acidic protein and S100 calcium-binding protein A10), that ended 15 days after FUS. Dopaminergic neurons and axonal projections decreased but recuperated basal values on day 15 after transfection, correlating with a decrease and recovery of locomotor behavior. In conclusion, FUS caused transient neuroinflammation and reversible neuronal affection but allowed systemic and intranasal transfection of dopaminergic neurons in both substantiae nigrae. Therefore, FUS could advance neurotensin-polyplex nanotechnology to clinical trials for Parkinson's disease. [ABSTRACT FROM AUTHOR]

Oligonucleotide therapeutics have great potential to target the currently undruggable genes and to generate entirely new therapeutic paradigms in multiple types of disease, thus having attracted much attention in recent years. However, their applications are greatly hindered by a lack of safe and efficient oligonucleotide-delivery vectors. Polyplex nanovesicles formed from oligonucleotides and the cationic block have shown exceptional features for the delivery of therapeutic oligonucleotides and other biopharmaceuticals. Nevertheless, these polyplex nanovesicles are deeply fraught with difficulty in tolerating physiological ionic strength. Inspired by the high binding ability between the dipicolylamine (DPA)/zinc (II) complex and the phosphodiester moieties of oligonucleotides, herein, we designed a coordinative cationic block to solve the intrinsic stability dilemma. Moreover, we found the stability of the resulted polyplex nanovesicles could be easily tuned by the content of coordinated zinc ions. In vitro cellular studies implied that the prepared zinc (II)-coordinative polyplex nanovesicles preferred to retain in the lysosomes upon internalization, making them ideal delivery candidates for the lysosome-targeting oligonucleotide therapeutics. [ABSTRACT FROM AUTHOR]

The clinical applications of cationic polymer-based non-viral gene carriers are usually hindered by the instability of polyplex or complex between these cationic polymers and anionic genes. In order to increase the stability, an amphiphilic cationic block copolymer PHB-b-PDMAEMA was designed to form cationic micelle, which comprised hydrophobic poly[(R)-3-hydroxybutyrate] (PHB) block and cationic poly(2-(dimethylamino)ethyl methacrylate) (DMAEMA) block. Interestingly, this cationic micelle after complex formation with plasmid deoxyribonucleic acid (pDNA) exhibited enhanced stability within 24 h and showed improved gene transfection performance than PDMAEMA only or non-viral transfection gold standard poly(ethylene imine) with molecular weight of 25 kDa (PEI-25k). In short, the design of cationic amphiphilic polymer, with micelle formation ability to enhance stability of polyplex, might greatly benefit the designs of non-viral cationic polymers as potential gene vectors. [ABSTRACT FROM AUTHOR]

In this work, zoledronic acid-loaded cationic methylcellulose polyplex nanoparticles were developed for enhanced gene delivery efficiency and breast cancer cell killing effect. Zoledronic acid (Zol) is one of bisphosphonate derivatives, which is used for osteoporosis treatment and also can induce apoptosis to breast cancer cells. First, cationic methylcellulose (MCPEI) was synthesized by reductive amination of polyethylenimine (PEI, 0.8 kDa) with periodate-oxidized methylcellulose (MC). Negatively charged Zol was introduced to MCPEI polyplexes via surface coating of polyplex (Zol(MCPEI/pDNA)) or loading into polyplexes (MCPEI/(pDNA + Zol)). The complexes with suitable size for efficient transfection could be formed via loading method. MCPEI/(pDNA + Zol) complexes showed higher stability and endosome buffering capacity than MCPEI polyplexes, probably due to imidazole group of Zol. Although the transfection efficiency of Zol(MCPEI/pDNA) complexes was similar with or less than that of MCPEI polyplexes, MCPEI/(pDNA + Zol) complexes showed much higher transfection efficiency than MCPEI polyplexes in HeLa cells. In the bafilomycin A1-treated transfection results, the transfection efficiency of MCPEI/(pDNA + Zol) complexes was significantly decreased. These results mean that the transfection of MCPEI/(pDNA + Zol) complexes would be mediated by endocytosis and loaded Zol could improve the transfection efficiency of MCPEI polyplexes via endosome buffering. In addition, breast cancer cell killing effect of MCPEI/(pDNA + Zol) was examined using pJDK-apoptin in MDA-MB-231 cells. MCPEI/(pJDK-apoptin + Zol) complexes showed the highest cancer cell killing effect due to the combinatorial effect of Zol and apoptin. It is concluded that zoledronic acid-loaded polyplexes could improve the transfection efficiency and MCPEI/(pDNA + Zol) complexes showed the potential for anticancer therapy. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop biocompatible polyplex nanoparticles with physicochemical properties suitable for the delivery of photosensitizer temoporfin. We prepared, characterized, and compared the two types of polyplex nanoformulations consisting of sodium alginate in combination with chitosan polymer or chitosan oligomer lactate. We obtained the polyplex system by multiple electrostatic interactions between cationic chitosan and anionic alginate and identified key process parameters. Particle size distribution, dispersity, and zeta potential were determined by dynamic light scattering (DLS), and the diameter and the morphology of the individual particles were visualized by a transmission electron microscopy (TEM). It was found that size distribution of the polyplex nanoparticles depends on the concentrations of chitosan and alginate stock solutions and the order and ratio of addition of stock solutions as well as on the pH of the resulting mixture. It appears that the nanoparticles are homogeneous, although micrographs indicate some (vague, indistinct) core-shell structure. The nanoparticles are stable at pH 7.4 (pH of blood plasma) and show only very little drug leak in experiment modeling conditions of blood pool transport to target tissues. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most malignant primary brain tumor and is very resistant to treatment; hence, it has a poor prognosis. Neurotensin receptor type 1 (NTSR1) plays a key role in cancer malignancy and has potential therapeutic applications. However, the presence and function of neurotensin (NTS) receptors in glioblastoma is not clearly established. RT-PCR assays showed that healthy (non-tumor) astroglial cells and C6 glioma cells express NTSR2 and its isoform (vNTSR2) rather than NTSR1. In glioma cells, NTS promotes the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2), an effect that was completely abolished by blocking the internalization of the NTS/NTSR complex. We demonstrated pharmacologically that the internalization is dependent on the activation of NTSR2 receptors and it was prevented by levocabastine, a NTSR2 receptor antagonist. The internalization of NTSR2 and vNTSR2 was further demonstrated by its ability to mediate gene transfer (transfection) via the NTS-polyplex system. Expression of reporter transgenes and of the pro-apoptotic soluble form of growth arrest specific 1 (tGAS1) was observed in glioma cells. A significant reduction on the viability of C6 cells was determined when tGAS1 was transfected into glioma cells. Conversely, astroglial cells could neither internalize NTS nor activate ERK 1/2 and could not be transfected by the NTS-polyplex. These results demonstrate that the internalization process of NTSR2 receptors is a key regulator necessary to trigger the activation of the ERK 1/2. Our data support a new internalization pathway in glioma C6 cells that involve NTSR2/vNTSR2, which can be used to selectively transfer therapeutic genes using the NTS-polyplex system. [ABSTRACT FROM AUTHOR]

Neurotensin (NT)-polyplex is a nonviral system for the targeted gene delivery to cells that express and internalize the high-affinity NT receptor (NTSR1). In hemiparkinsonian rats, we previously demonstrated the morphological and functional recovery from dopaminergic neurodegeneration using the NT-polyplex as a vehicle to transfect a neurotrophic gene. The main objective of this work was to demonstrate the feasibility of NT-polyplex to transfect reporter or therapeutic genes into neuroblastoma tumors through the blood stream or by intratumoral injection. N1E-115 cells known to express NTSR1 were allografted into athymic mice to generate the neuroblastoma tumor model. Both routes of administration allowed the NT-polyplex to reach and transfect tumoral cells. A low transgene expression was also detected in intestinal tract cells only after the injection into the blood stream. The transfection of the thymidine kinase (HSVTK) suicide gene followed by ganciclovir (GCV) treatment decreased the size and weight of neuroblastoma tumors by 30–50% and increased apoptosis compared to controls. This study shows the potential of the NT-polyplex as specific gene-transfer system for NTSR1 cancer cells.Cancer Gene Therapy (2009) 16, 573–584; doi:10.1038/cgt.2009.1; published online 30 January 2009 [ABSTRACT FROM AUTHOR]

The design of efficient systems for the targeted delivery of nucleic acids into cells is a rapidly developing area of polymer chemistry, molecular biology, and medicine. Complexes between DNA or RNA polyanions and various polycations, which are usually called polyplexes, hold promise as such delivery systems. Polyethylenimines (PEIs) and their derivatives are often used in research for the preparation of such complexes with plasmid DNA, oligonucleotides, and small RNA. Polyplex nanoparticles are employed for the delivery of genetic material into cells in culture and for the development of methods for the treatment of genetic and cancer diseases. The properties of polyplexes depend on the size, dispersity, and hydrophilicity of the used PEI or its derivatives and the ratio of polymers in the complex, which are responsible for the size, surface charge, and hydrophilicity of the resulting nanoparticles. The efficiency of polyplexes is determined by their ability to interact with components of biological systems on the surface and inside the cells, as well as with the blood vascular walls and the extracellular matrix during systemic in vivo use. [ABSTRACT FROM AUTHOR]

Purpose: To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection. Methods: An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (HDCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level. Results: The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential ( ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system. Conclusions: This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection. [ABSTRACT FROM AUTHOR]

Abstract Purpose  For systemic gene delivery to pancreatic tumor tissues, we prepared a three-layered polyplex micelle equipped with biocompatibility, efficient endosomal escape, and pDNA condensation functions from three components tandemly aligned; poly(ethylene glycol) (PEG), a poly(aspartamide) derivative with a 1,2-diaminoethane moiety (PAsp(DET)), and poly(l-lysine). Materials and Methods  The size and in vitro transfection efficacy of the polyplex micelles were determined by dynamic light scattering (DLS) and luciferase assay, respectively. The systemic gene delivery with the polyplex micelles was evaluated from enhanced green fluorescence protein (EGFP) expression in the tumor tissues. Results  The polyplex micelles were approximately 80 nm in size and had one order of magnitude higher in vitro transfection efficacy than that of a diblock copolymer as a control. With the aid of transforming growth factor (TGF)-β type I receptor (TβR-1) inhibitor, which enhances accumulation of macromolecular drugs in tumor tissues, the polyplex micelle from the triblock copolymer showed significant EGFP expression in the pancreatic tumor (BxPC3) tissues, mainly in the stromal regions including the vascular endothelial cells and fibroblasts. Conclusion  The three-layered polyplex micelles were confirmed to be an effective gene delivery system to subcutaneously implanted pancreatic tumor tissues through systemic administration. [ABSTRACT FROM AUTHOR]

Purpose: siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency. Methods: Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers. Results: Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process. Conclusions: MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies. [ABSTRACT FROM AUTHOR]

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency. [ABSTRACT FROM AUTHOR]

Gene therapy is expected to open a new strategy for the treatment of refractory vascular diseases, so the development of appropriate gene vectors for vascular lesions is needed. To realize this requirement with a non-viral approach, cyclo(RGDfK) peptide (cRGD) was introduced to block copolymer, poly(ethylene glycol)-block-polycation carrying ethylenediamine units (PEG-PAsp(DET)). cRGD recognizes αvβ3 and αvβ5 integrins, which are abundantly expressed in vascular lesions. cRGD-conjugated PEG-PAsp(DET) (cRGD-PEG-PAsp(DET)) formed polyplex micelles through complexation with plasmid DNA (pDNA) and the cRGD-PEG-PAsp(DET) micelles achieved significantly more efficient gene expression and cellular uptake as compared with PEG-PAsp(DET) micelles in endothelial cells and vascular smooth muscle cells. Intracellular tracking of pDNA showed that cRGD-PEG-PAsp(DET) micelles were internalized via caveolae-mediated endocytosis, which is associated with a pathway avoiding lysosomal degradation and that, PEG-PAsp(DET) micelles were transported to acidic endosomes and lysosomes via clathrin-mediated endocytosis. Further, in vivo evaluation in rat carotid artery with a neointimal lesion revealed that cRGD-PEG-PAsp(DET) micelles realized sustained gene expression, whereas PEG-PAsp(DET) micelles facilitated rapid, but transient gene expression. These findings suggest that introduction of cRGD to polyplex micelles might create novel and useful functions for gene transfer and contribute to the establishment of efficient gene therapy for vascular diseases. [ABSTRACT FROM AUTHOR]

Localized stimulation of angiogenesis is an attractive strategy to improve the repair of ischemic or injured tissues. Several microRNAs (miRNAs) such as miRNA-92a (miR-92a) have been reported to negatively regulate angiogenesis in ischemic disease. To exploit the clinical potential of miR-92a inhibitors, safe and efficient delivery needs to be established. Here, we used deoxycholic acid-modified polyethylenimine polymeric conjugates (PEI-DA) to deliver a locked nucleic acid (LNA)-based miR-92a inhibitor (LNA-92a) in vitro and in vivo. The positively charged PEI-DA conjugates condense the negatively charged inhibitors into nano-sized polyplexes (135 ± 7.2 nm) with a positive net charge (34.2 ± 10.6 mV). Similar to the 25 kDa-branched PEI (bPEI25) and Lipofectamine RNAiMAX, human umbilical vein endothelial cells (HUVECs) significantly internalized PEI-DA/LNA-92a polyplexes without any obvious cytotoxicity. Down-regulation of miR-92a following the polyplex-mediated delivery of LNA-92a led to a substantial increase in the integrin subunit alpha 5 (ITGA5), the sirtuin-1 (SIRT1) and Krüppel-like factors (KLF) KLF2/4 expression, formation of capillary-like structures by HUVECs, and migration rate of HUVECs in vitro. Furthermore, PEI-DA/LNA-92a resulted in significantly enhanced capillary density in a chicken chorioallantoic membrane (CAM) model. Localized angiogenesis was substantially induced in the subcutaneous tissues of mice by sustained release of PEI-DA/LNA-92a polyplexes from an in situ forming, biodegradable hydrogel based on clickable poly(ethylene glycol) (PEG) macromers. Our results indicate that PEI-DA conjugates efficiently deliver LNA-92a to improve angiogenesis. Localized delivery of RNA interference (RNAi)-based therapeutics via hydrogel-laden PEI-DA polyplex nanoparticles appears to be a safe and effective approach for different therapeutic targets. [ABSTRACT FROM AUTHOR]

Systemic and target-specific delivery of large genetic contents has been difficult to achieve. Although viruses effortlessly deliver kilobase-long genome into cells, its clinical use has been hindered by serious safety concerns and the mismatch between native tropisms and desired targets. Nonviral vectors, in contrast, are limited by low gene transfer efficiency and inherent cytotoxicity. Here we devised virus-mimetic polyplex particles (VMPs) based on electrostatic self-assembly among polyanionic peptide (PAP), cationic polymer polyethyleneimine (PEI) and nucleic acids. We fused PAP to the engineered ligand-binding domain of integrin αLβ2 to target intercellular adhesion molecule-1 (ICAM-1), an inducible marker of inflammation. Fully assembled VMPs packaged large genetic contents, bound specifically to target molecules, elicited receptor-mediated endocytosis and escaped endosomal pathway, resembling intracellular delivery processes of viruses. Unlike conventional PEI-mediated transfection, molecular interaction-dependent gene delivery of VMPs was unaffected by the presence of serum and achieved higher efficiency without toxicity. By targeting overexpressed ICAM-1, VMPs delivered genes specifically to inflamed endothelial cells and macrophages both in vitro and in vivo. Simplicity and versatility of the platform and inflammation-specific delivery may open up opportunities for multifaceted gene therapy that can be translated into the clinic and treat a broad range of debilitating immune and inflammatory diseases. [ABSTRACT FROM AUTHOR]

High-level targeted gene delivery has been demonstrated by molecular conjugates in vitro; however, in vivo delivery has been limited. The complexity of the resulting protein/DNA polyplex and a lack of understanding of its formation are persistent limitations. In this report, we show the effect of the DNA-binding agent poly-L-lysine (PLL), the ligand epidermal growth factor (EGF), and the coupling protein streptavidin on particle size, charge and gene delivery. Smaller (<80 nm) and more stable polyplexes were obtained with pll1116 than with shorter versions of PLL, especially in 0.15 M NaCl. Stability was increased by adding streptavidin to the polyplex; however, EGF increased particle size (>1000 nm) and decreased gene delivery when >300 EGF molecules per polyplex were used, indicating that a critical number of EGF molecules was needed for efficient gene delivery. The correct combination of these components resulted in the most efficient gene delivery in vitro and now provide for testing a more stable protein/DNA polyplex to aid in enhancing gene delivery in vivo. [ABSTRACT FROM AUTHOR]

We recently reported a novel coupling strategy involving salicylhydroxamic acid and phenyl(di)boronic acid molecules to attach the CNGRC peptide to PEI/DNA for CD13 targeting in tumors. Here, we doubly coupled Simian Virus (SV) 40 peptide-(nuclear localization signal)) and oligonucleotide-based (DNA nuclear targeting signal) nuclear signals to the same vector using peptide nucleic acid chemistry. This vector, CNGRC/PEG/PEI/DNA-βgal/NLS/DNTS, was predominantly localized in the cell nucleus, yielding about 200-fold higher βgal gene expression in vitro, more than 20-fold increase in tumor-specific gene delivery, and a robust βgal gene expression as demonstrated in stained tumor sections. For gene therapy purposes, we further engineered a similar targeting polyplex, CNGRC/PEG/PEI/DNA-p53/NLS/DNTS, with EBV-based episomal vector for sustained p53 gene expression. A distribution of vector DNA and apoptosis in p53-containing tumors was observed, yielding a significant tumor regression and 95% animal survival after 60 days. This multicomponent vector also co-targeted tumor and tumor-associated endothelial cells but not normal cells, and had more efficient therapeutic index than each vector administered as a single modality. The use of an efficient coupling strategy without compromising the vector's integrity for DNA condensation and endosomal escape; nuclear import; tumor-specific and persistent p53 gene expression clearly provides a basis for developing a single combinatorial approach for non-viral gene therapy.Gene Therapy (2006) 13, 1512–1523. doi:10.1038/sj.gt.3302773; published online 8 June 2006 [ABSTRACT FROM AUTHOR]

In the present study, we wanted to determine whether efficient gene delivery using an epidermal growth factor (EGF)/DNA polyplex could be accomplished in small cell lung cancer (SCLC) cell lines expressing low EGF receptor (EGFR) levels. EGFR expression levels and transduction efficiencies with polyplexes were examined in five SCLC cell lines and two controls. EGFR expression was examined by binding assays and demonstrated low EGFR levels ranging from 3.6 to 87.4 fmol/mg protein. The SCLC cell lines exhibited high sensitivity to adenovirus infection, which was an important determinant for transduction efficiency when adenovirus was used as an endosomolytic agent. The transduction efficiencies with EGF/DNA polyplexes ranged from 41% ± 3.5% to 73% ± 4.6% in the EGFR-positive SCLC cell lines. In the controls lacking EGFRs, only 5% ± 1.0% and 8% ± 1.8% of the cells were transduced. Furthermore, the transduction efficiency could be reduced from 50% ± 4.9% to 18% ± 1.1% when excess EGF was added to compete with the EGF/DNA polyplexes. In the present study, receptor-targeted gene delivery to SCLC cell lines has been demonstrated for the first time. Our results indicate that even low receptor expression levels in the target cells are sufficient for efficient and specific in vitro gene delivery with EGF/DNA polyplexes. [ABSTRACT FROM AUTHOR]

Many experimental cancer vaccines are exploring toll-like receptor agonists (TLRas) such as CpG, a DNA motif that agonizes toll-like receptor 9 (TLR9), to trigger immune responses that are potent and molecularly-specific. The ability to tune the immune response is especially important in the immunosuppressive microenvironments of tumors. Because TLR9 is located intracellularly, CpG must be internalized by immune cells for functionality. Polyplexes can be selfassembled through electrostatics using DNA (anionic) condensed by a positively charged carrier. These structures improve cell delivery and have been widely explored for gene therapy. In contrast, here we use cationic poly (β-amino esters) (PBAEs) to assemble polyplexes from CpG as an adjuvant to target and improve immune stimulation in cells and mouse models. Polyplexes were formed over a range of PBAE:CpG ratios, resulting in a library of complexes with increasingly positive charge and stronger binding as PBAE:CpG ratio increased. Although higher PBAE:CpG ratios exhibited improved CpG uptake, lower ratios of PBAE:CpG—which condensed CpG more weakly, activated DCs and tumorspecific T cells more effectively. In a mouse melanoma model, polyplexes with lower binding affinities improved survival more effectively compared with higher binding affinities. These data demonstrate that altering the polyplex interaction strength impacts accessibility of CpG to TLRs in immune cells. Thus, physiochemical properties, particularly the interplay between charge, uptake, and affinity, play a key role in determining the nature and efficacy of the immune response generated. This insight identifies new design considerations that must be balanced for engineering effective immunotherapies and vaccines. [ABSTRACT FROM AUTHOR]

Purpose: This work investigates the effects of hyaluronic acid (HA) conjugated onto branched poly(ethylenimine) (bPEI) and varying loading concentrations of these polymers complexed with DNA on their release from poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and the transfection of target cells. Methods: To examine the effect of alteration of the gene delivery polymer on the system, we observed the morphology, size, loading efficiency, polymer and DNA release, and the transfection efficiency for the microparticles formed with three internal phase loading concentrations during microparticle formation. Results: Addition of HA to this vector allowed for increased loading concentration within these systems and significantly altered release kinetics without changing the morphology of the particles. The incorporation of HA onto the bPEI backbone significantly increased the transfection efficiency of the complexes released from the corresponding microparticle formulation. Conclusions: The results show that the modification of bPEI with HA and the concentration of loaded polymer/DNA complexes can significantly alter the entrapment and release profiles from PLGA microparticles. This is significant in that it offers insight into the effects of modification of gene delivery vectors on a controlled release system designed to achieve a sustained therapeutic response. [ABSTRACT FROM AUTHOR]

In this study we investigated the spectral properties, including electric absorption, circular and linear dichroism (CD and LD), and fluorescence emission, of DNA in a DNA-branched polyethyleneimine (BPEI) polyplex at various polymer molecular weights ( M ) and BPEI-amine-to-DNA-phosphate ratios (N/P ratios). All BPEIs exhibited a common N/P dependence in their absorption and CD spectra. At N/P ratios < 1.0, we observed some hyperchromism in the absorption spectrum, red-shifts in CD bands, and decreases in LD intensity and fluorescence intensity of intercalated ethidium. At intermediate N/P ratios, complete collapse of all spectra occurred. As the N/P ratio increased further, the polyplex dissolved in water. From its characteristic CD spectrum obtained under these conditions, we conclude that the DNA exists in a B-like form. The fluorescence and LD intensities never recovered-even at high N/P ratios- which indicates that the dissolved polyplex possesses positive charges and the DNA in the polyplex is condensed despite its B-form CD spectrum. The N/P range in which the absorption and CD signals collapsed was wider when the BPEIs Mw decreased. In the case where the BPEIs M was 0.8 k, recovery of the absorption and CD spectral properties at a high N/P ratio was never achieved, which suggests that the molecular weight of the polymer plays an important role in its dissolution at a high N/P ratio. [ABSTRACT FROM AUTHOR]

Based on the fact that aberrant overexpression of some growth factor receptors was observed in a variety of human cancer cells, a novel nonviral gene delivery system GE7, which contains a 16-amino-acid ligand for identifying EGF receptor was constructed for tumor-targeted gene therapy. Intravenous administration of GE7 system revealed that it has the ability to target β-galactosidase (β-gal) reporter gene into murine hepatoma (Hepa) cells. Owing to the limited antitumor effects elicited by a single-gene transfer, recent efforts to treat malignancy using combined gene therapy have been accomplished with varying degrees of success. In this study, the human cyclin-dependent kinase inhibitor gene p21[SUPWAF-1] and the murine cytokine gene granulocyte-macrophage colony-stimulating factor (GM-CSF) were used simultaneously for in vivo gene therapy through systemic injection of the EGF R targeted GE7/DNA complex into murine hepatoma-bearing mice. The results demonstrated that combined administration of p21[SUPWAF-1] and GM-CSF could remarkably inhibit the growth of subcutaneously transplanted hepatoma Hepa cells, and significantly increase the survival rate of tumor-bearing mice. The activities of natural killer (NK) cells and specific cytotoxic T lymphocytes (CTL) were clearly enhanced after combined gene therapy. In vitro experiments showed that p21[SUPWAF-1] gene transfer exhibited a suppressive function on the growth of Hepa cells and the expression of H-2K[SUPb] and B7-1 molecules on Hepa cells increased significantly after combined genes delivery. All these results suggested that the GE7 system was able to target therapeutic genes efficiently to cancer cells, which showed high EGF R expression. The cotransfer of p21[SUPWAF-1] and GM-CSF genes apparently inhibited the growth of tumors through (a) the arrest of tumor cell growth and (b) the enhancement of systemic antitumor immunity. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the influence of ceil cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation- or lipid-based transfection systems was 30- to more than 500-fold higher when transfection was performed during S or G2 phase compared with cells in G1 phase which have the lowest expression levels. In contrast, this effect was not observed with recombinant adenovirus which varied only four-fold. Our results indicate that mitotic activity enhances transfection not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown. Furthermore, low transfection success into G1 cells indicates that DNA complexes deposited in G1 cells are probably not retained long enough to take advantage of mitosis effects or that passage of transfected cells through S phase is inhibitory. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cellular spheroids have attracted much attention as a transplantation procedure because the increased cell-to-cell interaction in spheroids enhances cell survival and its functions after the transplantation into the body. Furthermore, the potency of spheroidal cells may be further improved by introducing transgenes to augment cellular functions as well as enhance the paracrine effects by secreting key proteins involved in the essential cellular signaling cascades. In this study, we organized a new platform for genetically-modified cell transplantation by combining a microfabricated culture system for 3D spheroid formation with a newly developed non-viral transfection system, polyplex nanomicelle. After transfection of Gaussia luciferase using the nanomicelle, the prolonged luciferase expression was obtained for more than a month with continuous albumin secretion from the hepatocyte spheroids to the level comparable with control spheroids receiving no transfection. In contrast, by the transfection using FuGENE HD, a commercially available lipid-based reagent, the luciferase expression was obtained, yet the albumin secretion was significantly decreased with disintegration of the spheroid architecture. To assess the feasibility of the hepatocyte spheroids for in vivo transplantation, the spheroids were recovered by the use of micropatterned culture plate functionalized with thermosensitive polymer and dispersed into Matrigel Matrix. The luciferase expression as well as albumin secretion was maintained for more than a month from the spheroids in the Matrix. Thus, the combination of spheroid cell culture on micropatterned plates with gene introduction using polyplex nanomicelle is a promising platform for genetically-modified cell transplantation to achieve sustained transgene expression with maintaining innate cell functions. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop chitosan-coated and polyplex-loaded liposomes (PLLs) containing DNA vaccine for Peyer's patch targeting. Plain liposomes carrying plasmid pRc/CMV-HBs were prepared by the reverse-phase evaporation method. Chitosan coating was carried out by incubation of the liposomal suspensions with chitosan solution. Main lipid components of liposomes were phosphatidylcholine/cholesterol. Sodium deoxycholate and dicetyl phosphate were used as negative charge inducers. The zeta potentials of plain liposomes were strongly affected by the pH of the medium. Coating with chitosan variably increased the surface charges of the liposomes. To increase the zeta potential and stability of the liposome, chitosan was also used as a DNA condensing agent to form a polyplex. The PLLs were coated with chitosan solution. In vivo study of PLLs was carried out in comparison with chitosan-coated liposomes using plasmid encoding green fluorescence protein as a reporter. A single dose of plasmid equal to 100 μg was intragastrically inoculated into BALB/c mice. The expression of green fluorescence protein (GFP) was detected after 24 h using a confocal laser scanning microscope. The signal of GFP was obtained from positively charged chitosan-coated liposomes but found only at the upper part of duodenum. With chitosan-coated PLL540, the signal of GFP was found throughout the intestine. Chitosan-coated PLL demonstrated a higher potential to deliver the DNA to the distal intestine than the chitosan-coated liposomes due to the increase in permanent positive surface charges and the decreased enzymatic degradation. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to investigate the influence of molecular weights on the chemical, biophysical, and biological properties of bioreducible oligoethylenimine conjugates. The cationic conjugates were synthesized by polyaddition between branched oligoethylenimine 800 Da (OEI) and the disulfide bond containing N, N′-cystamine bisacrylamide (CBA) linker. A correlation between the copolymer molecular weights and the polyplex transfection properties was found. The OEI–CBA copolymers differing in molecular weights (from 8.6 to 37 kDa) showed good plasmid DNA binding ability resulting in compact 90- to 150-nm-sized polyplexes. Colloidal stability of the polyplexes was lost in reductive environment. A low concentration of dithiothreitol of 5 µM was sufficient to render polyplexes unstable in size. Reducing conditions at physiological salt concentration triggered polyplex dissociation. The bioreducible polymers displayed much lower cytotoxicity (IC50 ∼ 100 μg/mL in cell culture) than branched polyethylenimine 25 kDa (BPEI) and linear polyethylenimine 22 kDa (LPEI). Reporter gene transfection experiments were done with CHO-K1 and B16-F10 cells. The largest (37 kDa) copolymer HC-6-8 demonstrated highest transfection levels among all the bioreducible copolymers, which was comparable with LPEI and much more effective than BPEI. [ABSTRACT FROM AUTHOR]

To improve transfection of plasmid DNA as well as serum protein stability of polyionic complex nanoparticles of branched polyethyleneimine (bPEI), poly(ethylene glycol) (PEG)-stabilized nanoparticles were made from L-histidine substituted bPEI (PEI-Histidine) synthesized by Fmoc chemistry. The polymer was characterized by TNBS assay, H NMR, GFC, potentiometric titration and elemental analysis of carbon and nitrogen, DNA condensation, and the stability against extracellular matrix (heparin sulfate) was investigated by dye exclusion and agarose gel retardation assays. The nanoparticles were characterized by dynamic light scattering-zeta potential analyzer. Cytotoxicity and expression of enhanced green fluorescent protein (EGFP) were determined in hepatocellular carcinoma by MTT assay and fluorescent techniques. PEI-Histidine showed a reduced p K without any significant loss of total primary amines. Plasmid DNA was condensed almost thoroughly with PEGylated polymers, either bPEI or PEI-Histidine, at lower critical N/P ratios. PEGylated PEI-Histidine showed the better resistance to heparin induced displacement and the lower cytotoxicity when it was compared to bPEI. Interestingly unlike bPEI, smaller and less positively charged nanoparticles were obtained from PEGylated PEI-Histidine at N/P ratio=2 that resulted in about 4 folds higher EGFP expression than bPEI without any significant cytotoxicity. These properties are consistent with the higher serum protein resistance and buffer capacity of PEGylated PEI-Histidine at endosomal acidic pH. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

We report the development of a small interfering RNA (siRNA) delivery vector based on cationic perfluorocarbon nanoemulsions. We have prepared perfluorodecalin (PFD) emulsions with a positive surface charge provided by a fluorinated poly(ethylenimine) (F-PEI). The fluorinated emulsion (F-PEI@PFD) reduced cytotoxicity of F-PEI and demonstrated effective binding with siRNAs to form nanosized emulsion polyplexes. The prepared emulsion polyplexes enhanced cellular uptake and improved endosomal escape of the siRNA. In addition to increased reporter gene silencing in multiple cancer cell lines, when compared with control F-PEI and PEI polyplexes, the siRNA emulsion polyplexes showed an excellent resistance to serum deactivation and maintained high activity, even in high-serum conditions. The F-PEI@PFD emulsion polyplexes carrying an siRNA to silence the expression of Bcl2 gene induced apoptosis and inhibited tumor growth in a melanoma mouse model in vivo and showed potential for in vivo ultrasound imaging. This study demonstrates the potential of F-PEI@PFD emulsions as a multifunctional theranostic nanoplatform for safe siRNA delivery, with integrated ultrasound imaging functionality. [ABSTRACT FROM AUTHOR]

Two uptake mechanisms were identified for PEGylated DNA polyplex biodistribution to the liver. At a low polyplex dose, a rapid-uptake mechanism dominates, resulting in 60% capture by liver in 5 min, due to a saturable receptor-mediated process. Rapid-uptake led to the fast metabolism of polyplexes by liver (t1/2=2.1 h), correlating with a 1-μg pGL3 polyplex dose losing full transfection competency after 4 h in the liver. Dose escalation of either polyplex or poly(ethylene glycol) (PEG) peptide led to the saturation of rapid-uptake and revealed a delayed-uptake mechanism for polyplexes by liver. Delayed-uptake was characterized by the slower liver accumulation of 40% of the polyplex dose over 40 min, followed by slow metabolism (t1/2=15 h) and an extended time (12 h) for a 1-μg pGL3 polyplex dose, remaining fully transfection competent in the liver. The delayed-uptake mechanism is consistent with polyplexes crossing liver fenestrated endothelial cells to reach steady state in the space of Disse. The results describe how to control polyplex biodistribution to liver to avoid rapid-uptake and metabolism, in favor of delayed-uptake, to preserve polyplex transfection competency in the liver for up to 12 h. [ABSTRACT FROM AUTHOR]

Cytotoxicity, speedy degradation, and limited cellular absorption are the foremost features influencing the successful delivery of RNAs. Chitosan (Cs) is a polymer that offers an advantage due to its bio-compatibility and biodegradable nature, making it an ideal polycationic vector for delivering siRNA. In this study, chitosan has been modified with arginine in order to increase its encapsulation of siRNA and improve cellular absorption. It was discovered that arginine and guanidino moieties could transport through membranes of cells and play an important part in membrane permeability. FTIR and 13C NMR were used to characterize the complex. These chitosan-arginine (CsAr) siRNA complexes are further encapsulated in anionic DPPC/cholesterol liposomes to combine the effects of liposome-chitosan-arginine complexes called lipopolyplexes (LCAr). Formed LCAr were investigated for their lipid/CsAr-siRNA ratios, size, zeta-potential, heparin, and serum RNase stability by agarose gel retardation, and cell uptake efficiency compared to their "parent" polyplexes. Results revealed complete lipidation of CsAr-siRNA polyplexes at lipid mass ratio 10 resulting in lipopolyplexes in the 120 to 230nm range. Polyplex entrapped ~70% of siRNA, whereas lipidation increases siRNA encapsulation to ~95%. Developed LCAr showed ~4 times less hemolytic potential as compared to the parent polyplexes at the highest siRNA dose. The CsAr-siRNA and its lipid-coated form showed enhanced cellular association as compared to the marketed Lipofectamine 2000 proving its effectiveness in siRNA delivery. CsAr-liposome conjugation is simple and safe, and serves as a robust carrier for gene transport in physiological situations without compromising transfection efficacy. [ABSTRACT FROM AUTHOR]

One of the major obstacles in current cancer gene therapy is the lack of a gene delivery system with high efficiency and targetability. In this paper, a nonviral gene delivery system GE7, which was designed to target EGF receptor (EGF R) overexpressed on the surface of cancer cells through an EGF R-binding oligopeptide (GE7), was used for in viva gene therapy in a murine subcutaneous hepatoma model. It was demonstrated that the GE7 system could target the reporter gene β-gal to EGF R-expressing hepatoma cells with high efficiency after in vitro transfection and in viva peritumoral injection. To improve the therapeutic effect elicited by single gene transfer, human cyclin-dependent kinase inhibitor gene p21[sup WAF-1] and murine cytokine gene GM-CSF were used simultaneously in peritumoral injection of the GE7/DNA polyplex. The results showed that combined gene transfer of p21[sup WAF-1] and GM-CSF could inhibit the growth of pre-established tumor more effectively and prolong the survival time of hepatoma-bearing mice more significantly than the transfer of a single gene. Apoptosis in the tumor tissues were found when injected with the p21[sup WAF-1] DNA polyplex. Prominent inflammatory infiltration was observed in the tumor tissue transfected with the GM-CSF DNA polyplex. Our data demonstrate that the GE7 system-mediated, EGF R-targeted cotransfer of p21[sup WAF-1] and GM-CSF genes exhibit more potent antitumor effect by inducing tumor cell apoptosis and inflammatory responses. [ABSTRACT FROM AUTHOR]

Despite recent advances in the treatment of human colon cancer, the chemotherapeutic efficacy against colon cancer is still unsatisfactory. The complexity in colorectal cancer treatment leads to new research in combination therapy to overcome multidrug resistance in cancer and increase apoptosis. The objective of the present research work was to develop polyplexes for co-delivery of plasmid DNA with retinoic acid against colorectal cancer cell line (HCT-15). Plain polyplexes were prepared using chitosan and hyaluronic acid solution (0.1% w/v), whereas retinoic acid polyplexes were prepared using ethanol: water (1:9 v/v) system. The particle size was observed in the order of chitosan solution > blank polyplex > retinoic acid-loaded polyplex. Encapsulation efficiency of retinoic acid was found to be 81.51 ± 4.33% for retinoic acid-loaded polyplex formulation. The drug release was observed to be in a controlled pattern with 72.23 ± 1.32% release of retenoic acid from polyplex formulation. Cell line studies of the formulation displayed better cell inhibition and low cytotoxicity for the retinoic acid-loaded polyplexes in comparison to pure retinoic acid, thus demonstrating better potential action against colorectal cancer cell line HCT-15. Retinoic acid-loaded polyplexes indicated higher potential for the delivery of the active whereas the cell line studies displayed the efficacy of the formulation against colorectal cancer cell line HCT-15. [ABSTRACT FROM AUTHOR]

We have investigated the spectral properties of DNA, including its electric absorption, circular and linear dichroism (CD and LD), and fluorescence emission, in the DNA-linear polyethyleneimine (LPEI) and DNA-branched polyethyleneimine (BPEI) complexes at various polymer concentrations. The spectral properties of both complexes are similar. We observed a relatively moderate change in the absorption and CD spectra at low amine/DNA phosphate (N/P) ratios (< 0.5), followed by a drastic collapse within the N/P range from 0.8 and 1.0. The absorption and CD spectra recovered as the N/P ratio increased to ca. 1.2. In contrast, the LD and emission of ethidium intercalated between the DNA bases decreased almost linearly at N/P ratios between 0.0 and 1.0. These spectra never recovered at higher N/P ratios. We believe that the moderate changes in the spectrum at low N/P ratios occurred because of electrostatic interactions between DNA and BPEI, while the collapsed spectra at N/P ratios between 0.5 and 1.5 occurred because of condensation/aggregation of the DNA. Considering the structure of the polymers, we suggest that the secondary amino group of LPEI and all three amino groups of BPEI are equally involved in DNA condensation. [ABSTRACT FROM AUTHOR]

A new era of medical technology in cancer treatment is a directly specific modification of gene expression in tumor cells by nucleic acid delivery. Currently, the main challenge to achieving this goal is to find a non-toxic, safe, and effective strategy for gene transfer to cancer cells. Synthetic composites based on cationic polymers have historically been favored in bioengineering due to their ability to mimic bimolecular structures. Among them, polyethylenimines (PEIs) with superior properties such as a wide range of molecular weight and a flexible structure may propel the development of functional combinations in the biomedical and biomaterial fields. Here, in this review, we will focus on the recent progressions in the formulation optimization of PEI-based polyplex in gene delivery to treat cancer. Also, the effect of PEI's intrinsic characteristics such as structure, molecular weight, and positive charges which influence the gene delivery efficiency will be discussed. [ABSTRACT FROM AUTHOR]

Gene therapy, a promising treatment for vascular disease, requires appropriate gene vectors with high gene transfer efficiency, good biocompatibility and low cytotoxicity. To satisfy these requirements from the approach of nonviral vectors, a novel block copolymer, poly(ethylene glycol) (PEG)-block-polycation, carrying ethylenediamine units in the side chain (PEG-b-P[Asp(DET)]) was prepared. PEG-b-P[Asp(DET)] formed a polyplex micelle through polyion complex formation with plasmid DNA (pDNA). The PEG-b-P[Asp(DET)] polyplex micelle showed efficient gene expression with low cytotoxicity against vascular smooth muscle cells in vitro. It also showed reduced interactions with blood components, offering its feasibility of gene delivery via the vessel lumen. To evaluate in vivo gene transfer efficiency for vascular lesions, PEG-b-P[Asp(DET)] micelle was instilled into rabbit carotid artery with neointima by an intravascular method, and expression of the reporter gene in vascular lesions was assessed. Polyplexes from homopolymer P[Asp(DET)] and branched polyethyleneimine (BPEI) were used as controls. Ultimately, only the polyplex micelle showed appreciable gene transfer into vascular lesions without any vessel occlusion by thrombus, which was in strong contrast to BPEI and P[Asp(DET)] polyplexes which frequently showed occlusion with thrombus. These findings suggest that the PEG-b-P[Asp(DET)] polyplex micelle may have promising potential as a nonviral vector for the treatment of vascular diseases.Gene Therapy (2007) 14, 1029–1038. doi:10.1038/sj.gt.3302945; published online 26 April 2007 [ABSTRACT FROM AUTHOR]

Purpose: Since the discovery of RNAi and its therapeutic potential, carrier systems have been developed to deliver small RNAs (particularly siRNA) for modulation of gene expression at the post-transcriptional level. An important factor determining the fate and usability of these systems in vivo is interaction with blood components, blood cells, and the immune system. In this study, a lipid-based and a polymer-based carrier system containing siRNA have been investigated in vitro in terms of their hemocompatibility. Methods: The nanocomplexes studied were Angiplex, a targeted lipid-based system, and pHPMA-MPPM polyplex, a formulation based on a cationic polymer. siVEGFR-2 was encapsulated in both carriers and activation of platelets, coagulation, and complement cascade as well as induction of platelet aggregation were evaluated in vitro. Results: Both systems had been shown before to cause significant silencing in vitro. Our findings indicated that pHPMA-MPPM polyplex triggered high platelet activation and aggregation although it did not stimulate coagulation substantially. Angiplex, on the other hand, provoked insignificant activation and aggregation of platelets and activated coagulation minimally. Complement system activation by Angiplex was in general low but stronger than pHPMA-MPPM polyplex. Conclusions: Taken together, these in vitro assays may help the selection of suitable carriers for systemic delivery of siRNA in early preclinical investigations and reduce the use of laboratory animals significantly. [ABSTRACT FROM AUTHOR]

In this study, a small library of prednisolone and budesonide conjugated polyethylenimine (PEI) derivatives was prepared. Since nuclear entry is a major barrier for plasmid DNA (pDNA) delivery, it is hypothesized that glucocorticoid-conjugated PEIs that employ their specific receptors to enter the cell nucleus may increase the gene transfer ability of PEI. In this study, prednisolone and budesonide were conjugated onto low and high molecular weight PEI (1.8 and 25 kDa) with different degrees of substitution via different linkers. The glucocorticoid-polyethyleneimine (GC–PEI)/pDNA polyplexes were prepared, and their biophysical characteristics, plasmid delivery, and cell-induced toxicity were evaluated. The polyplexes demonstrated a substantial ability to condense plasmid DNA and the protection of nucleic acids against enzymatic degradation. The polyplexes’ size was in the range of 50–250 nm with zeta potentials between 10 and 30 mV. Interestingly, prednisolone-conjugated low molecular weight PEI could enhance the level of desired protein expression by up to 4 folds with no toxicity compared to the parent unmodified polymer. The results of this study show a promising approach to preparing gene carrier vehicles based on low molecular weight PEI with low toxicity. [ABSTRACT FROM AUTHOR]

In this study, a suicide gene therapy approach was optimized by a non-viral polyplex system based on pEGFP-N1 vector harboring purine nucleoside phosphorylase gene conducted by vascular endothelial growth factor promoter for an in vitro breast cancer model (4T1 cell line). The VEGF promoter and purine nucleoside phosphorylase gene were cloned into the vector from the source of 4T1 and E. coli genomic DNA, respectively. A gene construct was developed by replacing VEGF promoter instead of CMV promoter in pEGFP-N1vector. PNP gene was integrated in to the multiple cloning site of the obtained vector. On the other hand, a construct from pEGFP-N1 harboring PNP gene under the control of the original CMV promoter was developed. The transfection method using cationic polymer was optimized based on N/P ratio, cell cytotoxicity, polyplex size, zeta potential and the green fluorescent protein (GFP) expression by fluorescent microscopy and flowcytometry. Also, the effect of hypoxia condition induced by 0.5 mM H2O2 on the promoter efficiency was investigated. The results showed that the performed gene delivery system is capable of the gene transfection to more than 30% of the cancer cells with both VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1 plasmids. The hypoxia condition did not show a significant effect on the VEGF promoter. But, it revealed that bystander effect can improve the efficacy of this system and reduce drug IC50 to 2 and fourfold for plasmids VEGF-PNP-pEGFP-N1 and PNP-pEGFP-N1, respectively. These results showed that the bystander effect could almost compensate the low efficiency of non-viral gene delivery systems. We suggest that the tumor-specific gene expression system mediated by the VEGF promoter can be especially useful in the present model of breast cancer gene therapy. [ABSTRACT FROM AUTHOR]

Polyethyleneimine (PEI) has been extensively investigated as an efficient carrier for nucleic acid delivery. Yet, it suffers from a high toxicity profile that hinders clinical translation. Fluorination has proven to be a valid approach to reduce the cytotoxicity of PEI and improve the in vitro siRNA delivery potency. Hydrophobicity and lipophobicity can be controllably introduced into the side chains of PEI. However, the effect of fluorination on siRNA delivery in vivo, particularly the biodistribution of siRNA polyplex nanoparticles with fluorinated PEIs, has not been extensively explored. Here, we introduce two series of fluorinated PEIs via amidation with ethyl trifluoroacetate and perfluorobutyryl chloride. Fluorination substantially improved the performance of PEI for siRNA delivery by reducing the cytotoxicity to MDA-MB-231 cells. Importantly, fluorinated PEI enabled the major accumulation of siRNA polyplex nanoparticles in the liver while non-fluorinated PEI delivered siRNA nanoparticles mainly to the lungs after intravenous administration to mice. It is envisioned that fluorination may be an important general strategy for lowering toxicity of cationic polymers, and that the fluorination-induced alteration of biodistribution may be applicable for improved delivery to different organs. [ABSTRACT FROM AUTHOR]

High molecular weight polyethylenimine (HMW PEI; branched 25 kDa PEI) has been widely investigated for gene delivery due to its high transfection efficiency. However, the toxicity and lack of targeting to specific cells have limited its clinical application. In the present investigation, L-3, 4‐dihydroxyphenylalanine (L-DOPA) was conjugated on HMW PEI in order to target L-type amino acid transporter 1 (LAT-1) and modulate positive charge density on the surface of polymer/plasmid complexes (polyplexes). The results of biophysical characterization revealed that the PEI conjugates are able to form nanoparticles ≤ 180 nm with the zeta potential ranging from + 9.5–12.4 mV. These polyplexes could condense plasmid DNA and protect it against nuclease digestion at the carrier to plasmid ratios higher than 4. L-DOPA conjugated PEI derivatives were complexed with a plasmid encoding human interleukin-12 (hIL-12). Targeted polyplexes showed up to 2.5 fold higher transfection efficiency in 4T1 murine mammary cancer cell line, which expresses LAT-1, than 25 kDa PEI polyplexes prepared in the same manner. The cytotoxicity of these polyplexes was also substantially lower than the unmodified parent HMW PEI. These results support the use of L-3, 4‐dihydroxyphenylalanine derivatives of PEI in any attempt to develop a LAT-1 targeted gene carrier. [ABSTRACT FROM AUTHOR]

Purpose: Use of RNA interference as novel therapeutic strategy is hampered by inefficient delivery of its mediator, siRNA, to target cells. Cationic polymers have been thoroughly investigated for this purpose but often display unfavorable characteristics for systemic administration, such as interactions with serum and/or toxicity. Methods: We report the synthesis of a new PEGylated polymer based on biodegradable poly(amido amine)s with disulfide linkages in the backbone. Various amounts of PEGylated polymers were mixed with their unPEGylated counterparts prior to polyplex formation to alter PEG content in the final complex. Results: PEGylation effectively decreased polyplex surface charge, salt- or serum-induced aggregation and interaction with erythrocytes. Increasing amount of PEG in formulation also reduced its stability against heparin displacement, cellular uptake and subsequent silencing efficiency. Yet, for polyplexes with high PEG content, significant gene silencing efficacy was found, which was combined with almost no toxicity. Conclusions: PEGylated poly(amido amine)s are promising carriers for systemic siRNA delivery in vivo. [ABSTRACT FROM AUTHOR]

Purpose: A reversibly-PEGylated diblock copolymer, poly(aspartate-hydrazide-poly(ethylene glycol))- block-poly(aspartate-diaminoethane) (p[Asp(Hyd-PEG)]- b-p[Asp(DET)]) was reported here for enhanced gene transfection and colloidal stability. The diblock copolymer possessed a unique architecture based on a poly(aspartamide) backbone. The first block, p[Asp(Hyd)], was used for multi-PEG conjugations, and the second block, p[Asp(DET)], was used for DNA condensation and endosomal escape. Methods: p[Asp(Hyd-PEG)]- b-p[Asp(DET)] was synthesized and characterized by H-NMR. Polyplexes were formed by mixing the synthesized polymers and pDNA. The polyplex size, ζ-potential, and in vitro transfection efficiency were determined by dynamic light scattering, ζ-potential measurements, and luciferase assays, respectively. pH-dependent release of PEG from the polymer was monitored by cationic-exchange chromatography. Results: The polyplexes were 70-90 nm in size, and the surface charge was effectively shielded by a PEG layer. The transfection efficiency of the reversibly PEGylated polyplexes was confirmed to be comparable to that of the non-PEGylated counterparts and 1,000 times higher than that of the irreversibly PEGylated polyplexes. PEG release was demonstrated to be pH-sensitive. Fifty percent of the PEG was released within 30 min at pH 5, while the polymer incubated at pH 7.4 could still maintain 50% of PEG after 8 h. Conclusion: The reversibly PEGylated polyplexes were shown to maintain polyplex stability without compromising transfection efficiency. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)–induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents. [ABSTRACT FROM AUTHOR]

Purpose: 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 (R), and the corresponding mechanism by O-methyl-free N,N,N-trimethyl chitosans (TMCs) as gene carriers. Methods: Four TMCs with various chain lengths were synthesized and used to evaluate the R. To investigate the details of R, a number of factors such as cytotoxicity, cellular uptake efficiency, cellular uptake pathway and intracellular trafficking, were evaluated. Results: 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 R rather than polycation-DNA binding affinity, polyplex particle size in water, zeta potential, serum, cytotoxicity, and cellular uptake efficiency. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Polyethylene glycol (PEG)ylated polyacridine peptides bind to plasmid DNA with high affinity to form unique polyplexes that possess a long circulatory half-life and are hydrodynamically (HD)-stimulated to produce efficient gene expression in the liver of mice. We previously demonstrated that acridine-modified lysine (Acr) in (Acr-Lys)6-Cys-PEG5kDa stabilizes a 1-μg pGL3 dose for up to 1 h in the circulation, resulting in HD-stimulated (saline only) gene expression in the liver, equivalent in magnitude to direct-HD dosing of 1 μg of pGL3. In this study, we report that increasing the spacing of Acr with either four or five Lys residues markedly increases the stability of PEGylated polyacridine peptide polyplexes in the circulation allowing maximal HD-stimulated expression for up to 5 h post DNA administration. Co-administration of a decoy dose of 9 μg of non-expressing DNA polyplex with 1 μg of pGL3 polyplex further extended the HD-stimulated expression to 9 h. This structure-activity relationship study defines the PEGylated polyacridine peptide requirements for maintaining fully transfection competent plasmid DNA in the circulation for 5 h and provides an understanding as to why polyplexes or lipoplexes prepared with polyethylenimine, chitosan or Lipofectamine are inactive within 5 min following intravenous dosing. [ABSTRACT FROM AUTHOR]

Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems. [ABSTRACT FROM AUTHOR]

Liposome-encapsulated polyplex system represents a promising delivery system for oligonucleotide-based therapeutics such as siRNA and asODN. Here, we report a novel method to prepare liposome-encapsulated cationic polymer/oligonucleotide polyplexes based on the reverse-phase evaporation following organic extraction of the polyplexes. The polyplexes of polyethylenimine and oligonucleotide were first formed in aqueous buffer at an N/P ratio of 6. The overall positively charged polyplexes were then mixed with the anionic phospholipids in overall organic media. The overall organic environment and electrostatic interaction between anionic phospholipids and positively charged polyplexes resulted in inverted micelle-like particles with the polyplexes in the core. After phase separation, the hydrophobic particles were recovered in organic phase. Reverse-phase evaporation of the organic solvent in the presence of hydrophilic polymer-grafted lipids resulted in a stable aqueous dispersion of hydrophilic lipid-coated particles with the polyplex in the core. Transmission electron microscopy visualization revealed spherical structures with heavily stained polyplex cores surrounded by lightly stained lipid coats. The lipid-coated polyplex particles showed colloidal stability, complete protection of the loaded oligonucleotide molecules from enzymatic degradation, and high loading efficiency of more than 80%. Thus, this technique represents an alternative method to prepare lipid-coated polyplex particles as a delivery system of oligonucleotide therapeutics. [ABSTRACT FROM AUTHOR]