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Abstract: This work was designed to investigate the effect of the degree of substitutions of caproic acid on plasmid DNA (pDNA) binding, cellular uptake, biocompatibility, and transfection efficiency of caproic acid grafted chitosan (CGC). The CGC with three substitution degrees (CGC-5, CGC-15, and CGC-25) were synthesized by coupling caproic acid with chitosan. Chemical characterization of graft polymers was performed using FTIR, NMR, and elemental analysis. The CGC polymers showed good pDNA condensing capacity and efficient protection of pDNA from DNase I. The nanosized CGC/pDNA polyplexes exhibited well-defined spherical shapes and stability in serum. Isothermal titration calorimetry demonstrated reduction in CGC–pDNA binding constant with increase in the degree of caproic acid substitution. Caproic acid substitution resulted in 2–7-fold higher cellular uptake in HEK 293 cells mainly via the clathrin-mediated pathway without affecting biocompatibility. In vitro transfection study suggested a dependence of transfection efficiency on the variability of caproic acid substitution. The CGC-15 polymer exhibited 31-fold and 1.33-fold higher gene expression compared to chitosan and the marketed non-viral vector FuGENE®HD, respectively. These finding suggests that the CGC-15 graft polymer is a promising non-viral gene delivery vector due to its superior transfection efficiency and biocompatibility. [Copyright &y& Elsevier]

Abstract: Polycations have been explored as non-viral carriers for effective delivery of small interfering RNA (siRNA). Modifying polycations such as polyethylenimine (PEI) with lipid substitution was found to improve the siRNA delivery efficiency of polycationic carriers. However, the role of such lipid modification is not clear and remains to be probed at the atomistic level. In this work, we elucidate the role of lipid modification through a series of all-atom molecular dynamics simulations on siRNA complexation mediated by a native PEI and four analogous obtained by different lipid modifications. The lipid modification does not affect PEI''s capability of neutralizing the siRNA charge, neither does it affect the polyion bridging which plays an important role in siRNA complexation. Significant linkages among the lipid modified PEIs via association of lipid side-groups are observed and this results in more stable and compact PEI/siRNA polyplexes. The lipid associations between short lipids form and break frequently while the lipid associations between long lipids are more stable. For PEIs modified with short lipids, increasing the lipid substitution level results in more compact and stable siRNA structure. For PEIs modified with long lipids, increasing the lipid substitution does not change the amount of PEI linkage via lipid association, and has a reverse effect on compacting siRNA structure due to increased steric hindrance brought by the lipid association on individual PEIs. The simulation results generally correlate well with experimental data and suggest a framework of designing and systematic evaluation of polycation-based siRNA carriers using molecular dynamics simulations. [Copyright &y& Elsevier]

Abstract: To achieve successful delivery of siRNA therapeutics, cytocleavable cationic polyrotaxanes (PRXs) composed of N,N-dimethylaminoethyl (DMAE) group-modified α-cyclodextrins (CDs) that were threaded onto a poly(ethylene glycol) (PEG) axis and capped with a bulky stopper using cytocleavable disulfide linkages (DMAE-PRX) were utilized as an siRNA carrier. DMAE-PRXs with various numbers of threading CDs and modified DMAE groups were synthesized, and the physicochemical properties, cellular internalization, and gene silencing activity of DMAE-PRX/siRNA were investigated to elucidate the relationship between its supramolecular structure and its function. When the numbers of modified DMAE groups were increased, the DMAE-PRXs formed closely associated polyplexes with siRNA and increased their polyanion exchange resistance. Additionally, the DMAE-PRXs with 52 threading CDs (52CD-PRXs) showed greater binding capabilities with siRNA and greater resistance to polyanion competition than 31CD-PRXs, indicating that the highly CD-threaded PRX structure in the 52CD-PRXs is superior in forming stable polyplexes with siRNA. Indeed, 52CD-PRX/siRNA showed greater intracellular uptake of siRNA than 31CD-PRX/siRNA with comparable numbers of DMAE groups. 52CD-PRX/siRNA successfully induced gene silencing of a targeted luciferase expressed in human cervical carcinoma without marked cytotoxicity and non-specific gene silencing. Although the gene silencing activities of DMAE-PRX/siRNA were comparable to those of linear poly(ethylenimine) (L-PEI), L-PEI showed cytotoxicity and non-specific gene silencing. Additionally, DMAE-PRXs with cytocleavable capabilities were found to enhance gene silencing, in comparison with non-cleavable DMAE-PRX. Thus, the cytocleavable cationic PRXs are suggested to be attractive supermolecules for the delivery of therapeutic siRNAs. [Copyright &y& Elsevier]

Abstract: Cationic lipid- and polymer-based nanodevices are considered appropriate alternatives for virus-based particles for delivery of nucleic acids, including genes and siRNA, into eukaryotic cells. Because of colloidal stability concerns and toxicity issues the potential in vivo application of these so-called non-viral systems, in particular cationic lipids, was met with considerable skepticism. However, in recent years, the development of novel ionizable cationic lipid formulations in conjunction with sophisticated procedures to carefully control the size of the nanoparticles has rapidly advanced options for a successful therapeutic application. Thus it would appear that cationic lipids have taken a prominent step ahead in their potential use as nanocarriers for siRNA delivery in gene silencing of target genes in a variety of diseases. Verification and improvement of delivery efficiency as well as screening of targeting ligands justify further work in revealing underlying mechanisms that are instrumental in efficient crossing of cellular barriers by cationic lipid-based nanocarriers. In this regard, triggering entry into specific pathways or modulating trafficking along such pathways, either by targeting of nanoparticles or by affecting specific cellular signaling pathways, may represent promising tools. Such options may involve, for example, facilitating nanoparticle transport across endothelial cells by transcytotic mechanisms, or improving delivery efficiency by affecting nanoparticle trafficking that avoids lysosomal delivery. Here, recent progress in the field of lipid-based nanocarriers is discussed, with a focus on mechanisms underlying their interactions with cells in vitro. Where appropriate, we will include mechanisms for polymer-based systems in our discussion. [Copyright &y& Elsevier]

Abstract: The stability of DNA-polyplexes and intracellular DNA release are important features of gene delivery systems. To study these features, we have evaluated reducible cysteine-flanked linear lysine and arginine-rich peptides, modified with histidine residues. The reducible disulfide bonds in cysteine flanked peptides and histidine residues should augment DNA release from the peptide–DNA complexes upon disintegration of the reducible bonds. Template polymerization and oxidative polycondensation were applied to obtain peptide oligomers used for DNA-polyplex preparation. The peptides and DNA–peptide complexes were investigated with physical, chemical and transfection measurements. Physicochemical and transfection properties of DNA-polyplexes depended on the amino acid sequence of the peptidic polymers and type of the polymerization. MALDI-TOF analysis of oxidatively polycondensed products revealed several forms of peptide oligomers corresponding to 5–8 amino acid monomers. DNA–peptide particles based on template-polymerized complexes were more resistant to relaxation by negatively charged heparan sulfate than polyplexes formed with oxidatively condensed peptides. Complexes of DNA with the polycations prepared by oxidative polycondensation exhibited a 100–1000-fold higher level of gene expression compared to DNA/template-polymerized peptide complexes. The most efficient transgene expression was shown with arginine-rich polyplexes. Transfection efficacy of the arginine-rich polyplexes was even 10-fold better than that of DNA/PEI complexes. On average, polyplexes based on cysteine-flanked peptide oligomers showed lower cytotoxicity than non-reducible high molecular weight polylysine/DNA particles. We conclude that reducible peptide oligomers provide efficient DNA transfection and have the potential as vehicles for gene delivery. [Copyright &y& Elsevier]

Abstract: In this study, poly(ethylene glycol) (PEG)-block-polycation/siRNA complexes (PEGylated polyplexes) were wrapped with a hydrated silica, termed "silica nanogelling", in order to enhance their stability and functionality. Silica nanogelling was achieved by polycondensation of soluble silicates onto the surface of PEGylated polyplexes comprising a disulfide cross-linked core. Formation of silica nanogel layer on the PEGylated cross-linked polyplexes was confirmed by particle size increase, surface charge reduction, and elemental analysis of transmission electron micrographs. Silica nanogelling substantially improved polyplex stability against counter polyanion-induced dissociation under non-reductive condition, without compromising the reductive environment-responsive siRNA release triggered by disulfide cleavage. Silica nanogelling significantly enhanced the sequence-specific gene silencing activity of the polyplexes in HeLa cells without associated cytotoxicity, probably due lower endosomal entrapment (or lysosomal degradation) of delivered siRNA. The lower endosomal entrapment of the silica nanogel system could be explained by an accelerated endosomal escape triggered by deprotonated silanol groups in the silica (the proton sponge hypothesis) and/or a modulated intracellular trafficking, possibly via macropinocytosis, as evidenced by the cellular uptake inhibition assay. Henceforth, silica nanogelling of PEGylated siRNA polyplexes is a promising strategy for preparation of stable and functional siRNA delivery vehicles. [Copyright &y& Elsevier]

Abstract: Design and synthesis of efficient drug delivery systems are of vital importance for medicine and healthcare. Materials innovation and nanotechnology have synergistically fueled the advancement of drug delivery. Innovation in material chemistry allows the generation of biodegradable, biocompatible, environment-responsive, and targeted delivery systems. Nanotechnology enables control over size, shape and multi-functionality of particulate drug delivery systems. In this review, we focus on the materials innovation and processing of drug delivery systems and how these advances have shaped the past and may influence the future of drug delivery. [Copyright &y& Elsevier]

Abstract: Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water‐soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest. [Copyright &y& Elsevier]

Abstract: In this study, the gene delivery properties of new hyperbranched poly(amido amine)s (PAAs) with disulfide linkages in the main chain were investigated in comparison with their linear analogs. Eight different bioreducible PAAs were prepared by Michael addition of N,N′-bisacryloylpiperazine (BP) with cystamine (CYST) or N,N′-dimethylcystamine (DMC) and of N,N′-cystaminebisacrylamide (CBA) with N,N′-ethylenediamine (EDA) or N,N′-dimethylethylenediamine (DMEDA). In order to study the effect of terminal groups on the transfection efficiency, each polymer was terminated with 4-aminobutanol (ABOL) or with 2-aminoethanol (ETA). The hyperbranched and the linear PAAs generally formed polyplexes with plasmid DNA with sizes around 200nm and positive zeta potentials ranging from +10 to +22mV at polymer/DNA weight ratios equal or higher than 3/1. Remarkably low or no cytotoxicity was observed for both hyperbranched and linear PAAs. Hyperbranched CBA-containing PAAs showed higher gene expression in DNA transfection tests with COS-7 cells than their linear analogs and up to two times higher than linear PEI that was used as the reference polymer. Transfection efficiencies of the branched PAAs were generally enhanced by the presence of serum, which is a promising property for future in vivo studies with these hyperbranched PAAs. In this study the ease of synthetic modification of both linear and hyperbranched poly(amido amide)s and the versatility of hyperbranched PAAs in regulating DNA transfection and cytotoxicity are demonstrated. The results show the large possibilities for this class of polymers to provide polymeric vectors with controllable properties for gene therapy applications. [Copyright &y& Elsevier]

Abstract: RNA-based therapeutics has emerged as a novel and powerful approach for targeting a broad range of human diseases. Currently, a number of RNA-based drugs are under clinical investigation. The development of such drugs, however, has been slow and encountered multiple challenges. The clinical progress of such therapeutics strongly depends on whether a delivery vehicle efficiently and safely directs the drug into the target cells. Among the variety of non-viral vectors, dendritic carriers are particularly attractive due to their unique molecular architectures, globular shape, and multivalent groups on their surface. Lipid-based vectors were among the earliest strategies used for gene transfection and they are the most studied carriers for siRNA delivery. However, so far only a few of such systems have been studied in vivo. This review focuses on the most widely studied dendritic as well as lipid-based carriers for gene/siRNA delivery. [Copyright &y& Elsevier]

Abstract: Small interfering RNA (siRNA) that silence genes by the process of RNA interference offers a new therapeutic modality for disease treatment. Polycation-based nanoparticles termed polyplexes have been developed to maximise extracellular and intracellular siRNA delivery, a key requirement for enabling the clinical translation of RNAi-based drugs. Medical applications are dependent on safety; therefore, detailed investigation into potential toxicity to the cell or organism is required. This review addresses potential adverse effects arising from cellular and tissue interactions, immune stimulation and altered gene expression that can be associated with the assembled polyplex or the polycation and siRNA component parts. A greater understanding of the cellular mechanisms involved allows design-based solutions for rationale development of safe, effective and clinically relevant polyplex-based RNAi drugs. [Copyright &y& Elsevier]

Abstract: Polyplex particles formed with plasmid DNA (pDNA) and Pluronic P85-block-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (P85-b-P[Asp(DET)]) demonstrated highly effective transfection ability compared to PEG-based block cationomer, PEG-b-P[Asp(DET)]. Ternary polyplexes comprising PEG-b-P[Asp(DET)], poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-b-P[Asp(DET)] (P(EPE)-b-P[Asp(DET)]) used as an analog of P85-b-P[Asp(DET)], and pDNA were prepared in this work aiming at maintaining adequate transfection efficiency while solving the stability issues of the P85-b-P[Asp(DET)] polyplexes. Furthermore, a bioreducible P(EPE)-SS-P[Asp(DET)] possessing a redox potential-sensitive disulfide linkage between the P(EPE) polymer and the cationic block was used as a substitute for P(EPE)-b-P[Asp(DET)] during ternary complex formation to investigate whether the transfection ability of the ternary polyplex system could be enhanced by triggered release of P(EPE) polymers from the polyplexes. The ternary complexes showed significant improvement in terms of stability against salt-induced aggregation compared to binary complexes, although the gene delivery ability dropped with the amount of PEG-b-P[Asp(DET)] used for complexation. By manipulating the difference in redox potential between the extracellular and intracellular environments, the reducible ternary complexes achieved higher transfection compared to the non-reducible polyplexes; moreover, the reducible polyplexes exhibited comparable stability to the non-reducible ones. These results suggest that reducible ternary complexes could provide satisfactory transfection efficiency without comprising the colloidal stability of the particles. [Copyright &y& Elsevier]

Abstract: Increasing knowledge about molecular mechanisms of endogenous RNA interference (RNAi) and small interfering RNAs (siRNAs) has been incorporated into innovative nucleic acid medicines for treatment of diseases such as cancers. Although RNAi and siRNA have the potential to become powerful therapeutic drugs, their delivery to the target site represents a major challenge. The design and creation of nanocarriers for the safe and efficient delivery of siRNA towards their potential applications site is one of the challenging and rapidly growing areas of research since they have to overcome the commonly encountered biological barriers. In this review, we discuss the recent nanotechnological strategies for siRNA delivery by using different carriers such as liposomes, dendrimers and carbon nanotubes. [Copyright &y& Elsevier]

RNAi-mediated gene silencing has great potential for treating various diseases, including cancer, by delivering a specific short interfering RNA (siRNA) to knock down pathogenic mRNAs and suppress protein translation. Although many researchers are dedicated to devising polymer-based vehicles for exogenous in vitro siRNA transfection, few synthetic vehicles are feasible in vivo. Recent studies have presented copolymer-based vectors that are minimally immunogenic and facilitate highly efficient internalizing of exogenous siRNA, compared with homopolymer-based vectors. Cationic segments, organelle-escape units, and degradable fragments are essential to a copolymer-based vehicle for siRNA delivery. The majority of these cationic segments are derived from polyamines, including polylysine, polyarginine, chitosan, polyethylenimines and polyamidoamine dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Degradable fragments of copolymers must be derived from various polyelectrolytes to release the siRNA once the complexes enter the cytoplasm. This review describes recent progress in copolymer-mediated siRNA delivery, including various building blocks for biocompatible copolymers for efficient in vitro siRNA delivery, and a useful basis for addressing the challenges of in vivo siRNA delivery. [ABSTRACT FROM AUTHOR]

Abstract: Sequence defined oligo (ethane amino) amides produced by solid-phase supported synthesis using different building blocks and molecular shapes were tested for structure–activity relationships in siRNA delivery. Efficient reporter gene knockdown was obtained in a variety of cell lines using either branched three-armed structures, or lipid-modified structures with i-shape, T-shape, U-shape configuration. For the majority of structures (apart from U-shapes), the presence of 2 or 3 cysteines was strictly required for polyplex stabilization and silencing activity. Although all four building blocks contain the ethylenediamine proton sponge motif, only oligomers assembled with the tetraethylenepentamine based amino acids (Stp, Gtp, Ptp) but not with the triethylenetetramine based amino acid (Gtt) were able to mediate efficient gene silencing. For the lipopolymeric structures, out of the tested saturated (from C4 to C18) and unsaturated (C18) fatty acid moieties, two proximate oleic acids or linolic acids provided the oligomers with the best gene silencing activity and also pH specific lytic activity at pH 5.5, presumably facilitating endosomal escape of the polyplexes. Evaluation of oligomer chain length revealed a minimal number of at least two oligo (ethane amino) building blocks per oligomer arm as necessary for the vast majority of structures, but only marginal changes were found with higher numbers (structures with up to 60 ethane amino nitrogens were evaluated). Two promising carriers (T-shape 49, i-shape 229) were also evaluated for EG5 siRNA delivery. This resulted in tumor cell cycle arrest, and appearance of mitotic monoastral spindles both in vitro and in vivo upon systemic delivery. Repeated intratumoral treatment with EG5 siRNA polyplexes significantly reduced Neuro2A-eGFPLuc tumor growth in a siRNA-specific manner. [Copyright &y& Elsevier]

To improve the understanding of the physicochemical behavior of polyplexes (DNA-polycation complexes) and to avoid the complexity of blood, the formation and stability of polyelectrolyte complexes of degradable polycations and polyanions, we previously studied under pH, temperature, and ionic strength typical of human blood. In this study, the investigation is extended to polycationic macromolecules added into mixtures of polyanions selected to mimic polyanionic species present in blood. The poly(l-lysine) polycation was added to binary mixtures of degradable polyanions with different charge densities and hydrophobicity. The polyanions were poly(l-lysine citramide imide), poly(l-lysine citramide), and poly(l-lysine citramide) partially esterified with heptyl groups. We found selectivity and fractionation in the molar mass, which depended on the structural characteristics of the polyanions. The affinity of polycationic poly(l-lysine) macromolecules to polyanions increased in the following order: poly(l-lysine citramide imide) < poly(l-lysine citramide) < hydrophobized poly(l-lysine citramide). These data complements the previous information with respect to the possibility of polyplex destabilization and/or the interactions of polycationic macromolecules in excess with the polyanionic species present in blood, depending on the physicochemical characteristics of the polyplex, the excess polycation, and blood elements. [ABSTRACT FROM PUBLISHER]

In order to prepare gene carriers responsive to an endosomal pH, amino groups of linear poly(allylamine) (PAA) or poly( L-lysine) (PLL) were coupled with a-carboxyl groups of α-amino acids (Gly, His, Lys, Arg, and Orn). Acid-base titration indicated that Lys-, Arg-, and Orn-pendant polymers had both strongly basic groups and endosomal pH-responsive α-amino groups. These polymers, like PAA and PLL, formed stable complexes with DNA. Lys-, Arg-, and Orn-pendant polymers were effective transfection reagents independent of the backbone polymers. The pH-responsive α-amino groups enhanced transfection activity as shown by the observation that acetylation of the α-amino group resulted in a considerable loss in transfection activity. These results strongly suggested a lysosomotropic activity of the α-amino groups. Among the α-amino acid-pendant polymers tested, the Orn-pendant polymer exhibited the highest transfection activity/toxicity index. Since PLL with α-amino acid-pendants is composed of naturally occurring amino acids, it is expected to be biodegradable, and these reagents have promise as gene carriers. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: Direct targeting of dendritic cells is an ideal goal for DNA vaccine delivery in order to stimulate both arms of the immune system. However, dendritic cells are often difficult to transfect using nonviral polyplexes. Here we show that transfecting bystander cells such as fibroblasts with PEI/DNA complexes leads to efficient cross-presentation of a model antigen by dendritic cells and subsequent activation of antigen-specific CD8+ T cells. Maturation of dendritic cells is also stimulated after co-culture with transfected fibroblasts. Such outcomes depend on a proper balance between transfection efficiency and polyplex-induced cytotoxicity in the fibroblasts. In fact, substantial cytotoxicity is desirable and even necessary for cross-presentation and cross-priming of T cells. This study illustrates a new pathway of polymer-based DNA vaccine delivery via bystander cells without direct targeting of antigen-presenting cells and highlights the importance of exploiting polymer-induced cytotoxicity for the benefit of immune activation. [Copyright &y& Elsevier]

Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems. [ABSTRACT FROM AUTHOR]

Abstract: Polyplexes formed from cationic polymer/pDNA have been known to be vulnerable to external ionic strength. To improve polyplex stability against ionic strength, we attempted the chemical conjugation of the hydrophobic deoxycholate (DC) moiety to the polyamidoamine-diethylenetriamine (PAM-DET) dendrimer. Dynamic light scattering studies showed that the tolerance of the resulting PAM-DET-DC against ionic strength is higher than that of PAM-DET. In addition, we confirmed that the stability of polyplex has a strong relationship with the degree of conjugation of the DC moiety to the PAM-DET dendrimer and the charge ratio of PAM-DET-DC. Furthermore, the transfection efficiency of the PAM-DET-DC polyplex is higher than that of PAM-DET but its cytotoxicity remains the same. Therefore, the chemical conjugation of DC is a safe and effective method for increasing the stability of supramolecules formed from electrostatic interaction. [Copyright &y& Elsevier]

Abstract: Cellular entry of nanoparticles for drug- and gene delivery relies on various endocytic pathways, including clathrin- and caveolae-mediated endocytosis. To improve delivery, i.e., the therapeutic and/or cell biological impact, current efforts are aimed at avoiding processing of the carriers along the degradative clathrin-mediated pathway towards lysosomes, and promoting that along the caveolae-mediated pathway. Here, we demonstrate the effective internalization of branched polyethylenimine polymers (BPEI), complexed with nucleic acids, by HeLa cells along both pathways. However, transfection efficiency or nuclear ODN delivery primarily occurs via the caveolae-mediated pathway, along which delivery into lysosomes is avoided. Interestingly, inhibition of intracellular protein kinase A (PKA) activity modulates the intracellular trafficking of both poly- and lipoplexes along the clathrin-mediated pathway by impeding trafficking into the late endosomal/lysosomal compartments, thus avoiding degradation. In case of BPEI polyplexes this promotes their transfection efficiency by 2–3 fold. Evidence excludes early endosomes as a major site for BPEI-mediated release/delivery. Rather, we identify a novel compartment, tentatively characterized as a transferrin−/rab9−/LAMP1− compartment, to which cargo within the clathrin-mediated pathway of endocytosis is rerouted upon inhibition of PKA, and which may act as an alternative and effective site of cargo release in gene delivery. Our findings offer new opportunities for improving gene delivery by non-viral based nanoparticles. [Copyright &y& Elsevier]

Abstract: In the present study we compare LPEI and BPEI characteristics related to DNA condensation and their role as free polycation chains in gene transfection. Using radioactive 32P labeled DNA, we investigated the effect of free PEI chains on the cellular uptake of polyplexes. Our investigations show different properties of BPEI and LPEI polyplexes in condensation and de-condensation processes as well as in cellular uptake, which was tightly correlated with transfection efficiency. In agreement with earlier reports we find all DNA to be condensed at N/P = 3. Further added PEI chains remain free in solution. We found that both the cellular uptake and gene transfection of BPEI polyplexes is much more efficient than LPEI polyplexes at a low N/P ratio of 3 (i.e., without free PEI chains). When N/P is high (10, with 7 portions of free PEI), the LPEI and BPEI polyplexes have similar transfection efficiency even though the cellular uptake of the LPEI polyplexes is significantly lower. In addition, we found that addition of free short or long PEI chains (2.5 and 25 kDa) leads to a comparable gene transfection efficiency. [Copyright &y& Elsevier]

Abstract: The effects of the presence of two different types of phenylboronic acids as side groups in disulfide-containing poly(amido amine)s (SS-PAA) were investigated in the application of these polymers as gene delivery vectors. To this purpose, a para-carboxyphenylboronic acid was grafted on a SS-PAA with pending aminobutyl side chains, resulting in p(DAB–4CPBA) and an ortho-aminomethylphenylboronic acid was incorporated through copolymerization, resulting in p(DAB–2AMPBA). Both polymers have 30% of phenylboronic acid side chains and 70% of residual aminobutyl side chains and were compared with the non-boronated benzoylated analogue p(DAB–Bz) of similar Mw. It was found that the presence of phenylboronic acid moieties improved polyplex formation with plasmid DNA since smaller and more monodisperse polyplexes were formed as compared to their non-boronated counterparts. The transfection efficiency of polyplexes of p(DAB–4CPBA) was approximately similar to that of p(DAB–Bz) and commercial PEI (Exgen), both in the absence and the presence of serum, indicating that p(DAB–4CPBA) and p(DAB–Bz) are potent gene delivery vectors. However, the polymers with phenylboronic acid functionalities showed increased cytotoxicity, which is stronger for the ortho-aminophenylboronic acid containing polyplexes of p(DAB–2AMPBA) than for the p(DAB–4CPBA) analog. The cytotoxic effect may be caused by increased membrane disruptive interaction as was indicated by the increased hemolytic activity observed for these polymers. [Copyright &y& Elsevier]

Abstract: Polycations are one of the most frequently used classes of materials for non-viral gene transfer in vivo. Several studies have demonstrated a sensitive relationship between polymer structure and delivery activity. In this work, we used reverse addition-fragmentation chain transfer (RAFT) polymerization to build a panel of N-(2-hydroxypropyl)methacrylamide (HPMA)-oligolysine copolymers with varying peptide length and polymer molecular weight. The panel was screened for optimal DNA-binding, colloidal stability in salt, high transfection efficiency, and low cytotoxicity. Increasing polyplex stability in PBS correlated with increasing polymer molecular weight and decreasing peptide length. Copolymers containing K5 and K10 oligocations transfected cultured cells with significantly higher efficiencies than copolymers of K15. Four HPMA-oligolysine copolymers were identified that met the desired criteria. Polyplexes formed with these copolymers demonstrated both salt stability and transfection efficiencies on-par with poly(ethylenimine) PEI in cultured cells. [Copyright &y& Elsevier]

Abstract: The nano-polyplexes based gene delivery using the combination of water-soluble chitosan and polyethyleneimine (PEI) previously showed synergistic effect on gene transfection in a variety of human cell lines. This system simply facilitates gene therapy via a non-viral gene delivery system. Here, we have started to apply this system for therapeutic gene delivery into the osteoblastic-like cell line, MG-63. The transfection efficiency and cytotoxicity in MG-63 were investigated, intracellular uptake of nano-polyplexe was confirmed through confocal laser scanning microscope. Finally, bone morphogenesis protein (BMP) gene delivery was performed using our developed transfection system. The expression level of BMP-2 therapeutic gene was measured via reverse transcriptase polymerase chain reaction (RT-PCR). Comparing with commercial available system, lipofectamine, the expression of BMP were prolonged. The chitosan-PEI blend polyplex studied here showed the advantages in the aspects of high gene transfection with low cytoxicity into MG-63 cells. This study proposes the non-viral vector as a promising approach for bone regeneration though gene therapy. [Copyright &y& Elsevier]

Abstract: Recent advances in efficient microRNA (miRNA) delivery techniques using brain-targeted nanoparticles offer critical information for understanding the functional role of miRNAs in vivo, and for supporting targeted gene therapy in terms of treating miRNA-associated neurological diseases. Here, we report the rabies virus glycoprotein (RVG)-labeled non-toxic SSPEI nanomaterials capable of neuron-specific miR-124a delivery to neuron in vivo. The RVG-labeled BPEI-SS (RVG-SSPEI) nanocarrier showed less toxicity in acetylcholine receptor-positive Neuro2a cells, and electrostatic interaction of RVG-SSPEI with miR-124a exhibited optimal transfection efficacy. The RVG-SSPEI polymer specifically targeted Neuro2a using cy5.5-miR-124a mixed with RVG-SSPEI. The functional action of miR-124a oligomers released from polyplexes in the cytoplasmic region was evaluated by a reporter vector containing a miR-124a -binding sequence, and showed a significantly reduced reporter signal in a dose-dependent manner. Cy5.5-miR-124a/RVG-SSPEI- injected into mice via tail veins displayed the enhanced accumulation of miR-124a in the isolated brain. Hindrance of the efficient penetration of neuronal cells by size limitation of the miR-124a/RVG-SSPEI improved with the help of mannitol through blood-brain barrier disruption. These findings indicated that the RVG peptide combined with mannitol infusion using SSPEI polymer for neuron-specific targeting in vivo is sufficient to deliver neurogenic microRNA into the brain. [Copyright &y& Elsevier]

Abstract: PEGylated cationic polymers have been extensively studied for substituting virus as gene delivery vehicles. These polymers can produce water-soluble polyionic complexes (polyplexes) with plasmid DNA (pDNA) and show enhanced stability compared to non-PEGylated polyplexes. However, PEGylation always diminishes the transfection efficiency of polyplexes probably due to poor cellular internalization of the particles and difficulty in releasing the pDNA cargo from the complexes intracellularly for gene expression. As non-ionic surfactants, Pluronic block copolymers have been shown to interact with plasma membrane and promote cellular uptake of various small molecules and biomacromolecules. To evaluate whether Pluronic could improve the transfection efficiency of polyplexes, Pluronic P85- and PEG-based cationomers comprising poly{N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide (P[Asp(DET)]) cationic blocks were synthesized and tested for their transfection ability. In this study, it was demonstrated that although the stability of the PEG-based polyplexes was better than that of the P85-based polyplexes based cationic polymers, the P85-based polyplex could achieve significantly higher transfection than the PEG counterparts. The improvement of gene delivering ability was shown to be correlated with the enhanced cellular internalization of the P85-based polyplexes. [Copyright &y& Elsevier]

Purpose: As the next step in the development of an intra-articular controlled release system to treat painful temporomandibular joint (TMJ) inflammation, we developed several biodegradable poly(DL-lactic-co-glycolic acid) (PLGA)-based microparticle (MP) formulations encapsulating a model anti-inflammatory small interfering RNA (siRNA) together with branched poly(ethylenimine) (PEI) as a transfecting agent. The effect of siRNA loading and N:P ratio on the release kinetics of siRNA-PEI polyplexes was determined, and the size and N:P ratio of the polyplexes released over time was characterized. Methods: Polyplex-loaded PLGA MPs were prepared using an established double emulsion technique. Increasing the pH of the release samples enabled siRNA-PEI dissociation and subsequent measurement of the release of each component over 28 days. Polyplex diameter was measured for all release samples and compared to freshly prepared siRNA-PEI under simulated physiologic conditions. Results: Systematic variation of siRNA loading and N:P ratio resulted in distinct siRNA and PEI release profiles. Polyplex diameter remained constant despite large variations in the relative amounts of siRNA and PEI. Excess PEI was sequestered through complexation with 500-1,000 nm diameter PLGA MP-derived particles, including small MPs and PLGA degradation products. Conclusions: These PLGA MP formulations show exciting potential as the first intra-articular TMJ controlled release system. [ABSTRACT FROM AUTHOR]

Abstract: Incorporating ligands into nano-scale carriers for specific delivery of therapeutic nucleic acids to tumor sites is a promising approach in anti-cancer strategies. Current artificial vector systems however still suffer from efficient and specific delivery, compared to their natural counterparts and addressed receptor types rarely are exclusively expressed on target cells. In this study synthetic dual receptor targeted polyplexes were developed, mimicking biphasic cell entry characteristics of natural viruses to increase efficiency and specificity by a dual-receptor internalization mechanism. For engineering the synthetic dual targeted vector system, the transferrin targeting peptide B6 was evaluated for the first time in the context of PEGylated PEI based polyplexes. As a second ligand, arginine–glycine–aspartic acid (RGD) containing peptide was incorporated for simultaneous integrin targeting. Cellular association, cellular uptake, transfection efficiency and accordant competition experiments displayed specificity of both ligands for each targeted receptor in two prostate cancer cell lines. A clear synergy of dual targeting over the combination of single-targeted polyplexes was found, suggesting that the dual targeting strategy is one step towards safe vectors for therapeutic approaches. [Copyright &y& Elsevier]

Abstract: Surface modification using poly(ethylene glycol) (PEG) is a widely used strategy to improve the biocompatibility of cationic polymer-based nonviral gene vectors (polyplexes). A novel method based on intravital real-time confocal laser scanning microscopy (IVRTCLSM) was applied to quantify the dynamic states of polyplexes in the bloodstream, thereby demonstrating the efficacy of PEGylation to prevent their agglomeration. Blood flow in the earlobe blood vessels of experimental animals was monitored in a noninvasive manner to directly observe polyplexes in the circulation. Polyplexes formed distinct aggregates immediately after intravenous injection, followed by interaction with platelets. To quantify aggregate formation and platelet interaction, the coefficient of variation and Pearson''s correlation coefficient were adopted. In contrast, polyplex micelles prepared through self-assembly of plasmid DNA with PEG-based block catiomers had dense PEG palisades, revealing no formation of aggregates without visible interaction with platelets during circulation. This is the first report of in situ monitoring and quantification of the availability of PEGylation to prevent polyplexes from agglomeration over time in the blood circulation. This shows the high utility of IVRTCLSM in drug and gene delivery research. [Copyright &y& Elsevier]

Purpose: To develop a novel polyethylenimine (PEI)-based polymeric carrier for tumor-targeted delivery of cytotoxic double-stranded RNA polyinosinic:polycytidylic acid, poly(I:C). The novel carrier should be chemically less complex but at least as effective as a previously developed tetra-conjugate containing epidermal growth factor (EGF) as targeting ligand, polyethylene glycol (PEG) as shielding spacer, 25 kDa branched PEI as RNA binding and endosomal buffering agent, and melittin as endosomal escape agent. Methods: Novel conjugates were designed employing a simplified synthetic strategy based on 22 kDa linear polyethylenimine (LPEI), PEG spacers, and recombinant EGF. The efficacy of various conjugates (different PEG spacers, with and without targeting EGF) in poly(I:C)-mediated cell killing was evaluated in vitro using two human U87MG glioma cell lines. The most effective polyplex was tested for in vivo activity in A431 tumor xenografts. Results: Targeting conjugate LPEI-PEG2 kDa-EGF was found as most effective in poly(I:C)-triggered killing of tumor cells in vitro. The efficacy correlated with glioma cell EGFR levels. Repeated intravenous administration of poly(I:C) polypexes strongly retarded growth of A431 human tumor xenograft in mice. Conclusions: The optimized LPEI-PEG2 kDa-EGF conjugate displays reduced chemical complexity and efficient poly(I:C)-mediated killing of EGFR overexpressing tumors in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Chitosan (CHI), a linear polysaccharide, has been intensively studied as a nonviral gene delivery vector. The low physiological solubility of CHI has limited its gene transfection efficiency. Here we report the synthesis of different substitution degrees of succinated chitosans (CHI-succ) to increase water solubility. According to the proton nuclear magnetic resonance spectra, the degree of deacetylation of hydrolyzed CHI was roughly 88% and the degrees of succinylation in three CHI-succ polymers were approximately 5, 10, and 20%. Various weight ratios of CHI/DNA and CHI-succ/DNA polyplexes were prepared for gel electrophoresis retardation, particle size, zeta potential, and morphology studies. The results suggest that the plasmid DNA is readily entrapped at a CHI-succ/DNA weight ratio of 20; the sizes and zeta potentials were between 110 and 140 nm and ±1–5 mV, and the polyplexes exhibited low cytotoxicity against HEK 293T cells. CHI-succ with 5 and 10% degrees of substitution showed improved transfection efficiency as compared with nascent CHI. From the Clinical Editor: Chitosan, a cationic polysacchride with gene therapy potential, has inherently poor water solubility, which is improved by partial succinylation according to this report. The new DNA/Chitosan polyplexes exhibit improved safety against HEK 293T cells. [Copyright &y& Elsevier]

Abstract: The polymer OEI-HD-1, a degradable 25kDa carrier for siRNA transfer based on β-propionamide-crosslinked oligoethylenimine, was functionalized to further improve biocompatibility and/or endosomolytic characteristics of the polymer. For this purpose OEI-HD-1 was either modified with succinic acid to reduce cationic charge density; alternatively OEI-HD-1 was grafted with 1molar equivalent of 5kDa polyethyleneglycol (PEG), or with 1 equivalent of PEG plus 8molar equivalents of dimethylmaleic anhydride (DMMAn) – modified melittin peptide to enhance endosomal escape. Polymers were characterized in their capability of siRNA complex formation, cytotoxicity, and luciferase reporter gene silencing activity. Succinylation of OEI-HD-1 at 12% of nitrogens strongly reduced siRNA binding and silencing activity. All other OEI-HD-1 derivates displayed improved in vitro biocompatibility and siRNA mediated luciferase gene knockdown in Neuro2A/eGFPLuc cells. Succinylation at 7% of nitrogens strongly reduced cytotoxicity of the polymer and extended the functional window between gene silencing and cytotoxicity. Incorporation of DMMAn-Mel and PEG into OEI-HD-1 generated a pH-responsive lytic polymer conjugate with the highest potency in siRNA mediated gene knockdown and largest functional activity/biocompatibility window. [Copyright &y& Elsevier]

Abstract: Nucleic acid based therapies have so far mainly been focused on plasmid DNA (pDNA), small interfering RNA (siRNA), antisense and immunostimulatory oligonucleotides. Messenger RNA (mRNA) was the subject of only a few studies. The objective of this investigation was the preparation of new composite polyplexes with mRNA consisting of poly(ethylene imine) (PEI) and poly(ethylene imine)–poly(ethylene glycol)-copolymers (PEI–PEG) as blends. These complexes were designed to increase the stability of mRNA, to improve transfection efficiency and to reduce cytotoxicity. Hydrodynamic diameters of the polyplexes were measured by dynamic light scattering, polyplex stability was analyzed by gel retardation assay and transfection efficiency of luciferase (Luc) encoding mRNA was evaluated under in vitro conditions. Most of the polyplexes generated showed small particle sizes <200nm and positive zeta-potentials of +20mV to +30mV. Stable complexes were formed even at low nitrogen to phosphate ratios. Polyplexes with mRNA Luc and blends of low molecular weight PEI(5kDa) and PEI(25kDa)–PEG(20kDa)1-block-copolymer showed protein expression as high as polyplexes with PEI(25kDa). Moreover, luciferase expression was significantly higher than that obtained with one of the components alone. These results suggest that delivery systems for pulmonary application of mRNA merit further investigation under in vitro and in vivo conditions. [ABSTRACT FROM AUTHOR]

Abstract: High cationic charge densities in polymeric vectors result in tight DNA condensation, leading to small highly positively charged polyplexes which show generally high cellular uptake in vitro. However, high cationic charge densities also introduce membrane-disruptive properties to the polymers, thereby frequently causing high cytotoxities. We previously developed poly(amido amine)s with repetitive disulfide linkages in the main chain (SS-PAAs) that are significantly less toxic than PEI, due to fast intracellular degradation of these polymers by bioreductive cleavage of the disulfide bonds. In this study we have investigated the effects of variation in charge density and hydrophobicity on the gene delivery properties of these polymers by varying the degree of acetylation and benzoylation in SS-PAAs with aminobutyl side chains. It was found that introduction of hydrophobic benzoyl groups results in higher transfection efficiencies, both in the absence and presence of serum. [ABSTRACT FROM AUTHOR]

Abstract: Efficient intracellular delivery of siRNA is a significant hurdle to its therapeutic success. For biological studies on the efficiency of carrier-mediated uptake of siRNA, quantitative determination of the amount of internalized siRNA is required. In this study, when the apparent uptake of fluorescently labeled siRNA, formulated in different lipo- and polyplexes, was examined using different techniques, major differences were observed. Additional experiments showed that these differences could be explained by quenching phenomena that were dependent on interactions between siRNA and carrier and their intracellular environment. Differences in fluorescent signal of complexed siRNA due to quenching could be overcome by measuring the fluorescent signal after lysing the transfected cells in lysis buffer that contained 2% SDS to dissociate siRNA from the complexes. This method offers a simple approach for quantifying cellular uptake of siRNA, which might help in the development of more efficient delivery systems. [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]

To develop novel gene delivery carriers, aminated polysorbate 80 (P80-NH) was synthesized with strong positively charged properties through the introduction of three amine groups. The resulting P80-NH and DNA polyplex exhibited superb condensation abilities due to the high densities of positively charged amines groups. Size and surface charge of polyplex were shown to be well suited for cellular internalization. In addition, the P80-NH/DNA polyplex demonstrated acceptable transfection efficiency in HeLa cells and was nontoxic relative to the conventional 25-kDa polyethyleneimine system. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [ABSTRACT FROM AUTHOR]

Abstract: Poly(l-lysine), (PLL), and poly(dl-amino serinate), (PSA), are respectively enzymatically and hydrolytically degradable polycations. This work was aimed at investigating their degradability when they are complexed with polyanions, namely poly(acrylic acid) and poly(l-lysine citramide), taken as simple models of DNA in polyplexes. Comparison was made with degradation characteristics of the same polycations in solution in the absence of polyanion on the basis of size exclusion chromatography and capillary zone electrophoresis. Complexed PLL remained enzymatically degradable by trypsin, an endopeptidase, but was no longer degradable by aminopeptidase, an exopeptidase. Trypsin yielded a mixture of trilysin and tetralysin. Complexed PSA remained hydrolytically degradable in aqueous media. The hydrolysis of PSA led to dl-serine. However, traces of anionic species were also detected that were identified as residues of constituting repeating units issued from the N-benzyloxycarbonyl polyaminoserinate precursor (PSAZ). [ABSTRACT FROM AUTHOR]

Abstract: Applications of siRNA for cancer therapy have been spotlighted in recent years, but the rational design of efficient siRNA delivery carriers is still controversial, especially because of possible toxicity of the carrier components. Previously, a cationic polyaspartamide derivative, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), was reported to exert high transfection efficacy for plasmid DNA with negligible cytotoxicity. However, its direct application for siRNA delivery was fairly limited due to the unstable polymer/siRNA complex formation. In this study, to overcome such instability, stearic acid as a hydrophobic moiety was conjugated to the side chain of PAsp(DET) with various substitution degrees. The stearoyl introduction contributed not only to siRNA complex formation with higher association numbers but also to complex stabilization. The obtained stearoyl PAsp(DET)/siRNA complex significantly accomplished more efficient endogenous gene (BCL-2 and VEGF) knockdown in vitro against the human pancreatic adenocarcinoma (Panc-1) cells than did the unmodified PAsp(DET) complex and commercially available reagents, probably due to the facilitated cellular internalization. This finding suggests that the hydrophobic PAsp(DET)-mediated siRNA delivery is a promising platform for in vivo siRNA delivery. [Copyright &y& Elsevier]

Abstract: Silica-coating of positively charged polyplexes was demonstrated through silicic acid condensation to improve the polyplexes for enhanced complex stability and transfection efficiency. Silicic acid was efficiently condensed by polycations to form a silica network in the polyplex through electrostatic interaction and hydrogen bonding. The silica-coated (SC) polyplexes had an anionic surface charge of −20 mV and were 10–20 nm larger in size compared to the non-silica-coated control (+33.4 mV, 106 nm). Silica-coating significantly improved the polyplex stability against both dissociations by counter polyanion exchange and aggregation by salt. The silica network was dissolved to form silicic acid by removing free silicic acid based on the equilibrium, SiO2 + 2H2O ⇄ Si(OH)4. Indeed, dialysis of the SC polyplex solution against excess silica-free buffer permitted plasmid DNA release from the silica-coated polyplex, indicating the reversible nature of the silica-layer. The SC polyplex achieved significantly higher transfection efficiency without serious cytotoxicity compared to the polyplex without silica-coating. Detailed examinations of transfection using SC polyplexes revealed that the enhanced transfection efficiency was because of facilitated endosomal escape, possibly due to the protonation of the silica in acidic endosomal compartments. These findings demonstrate the utility of the silica-coating technique for polyplex-mediated gene delivery. [Copyright &y& Elsevier]

Abstract: Gene delivery using cationic polymers has attracted much attention due to their potential advantages, such as large DNA loading capacity, ease of large-scale production, and reduced immunogenicity. We recently reported that polyplexes from poly[N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide] (P[Asp(DET)]), having an efficient endosomal escape due to pH-selective membrane destabilization, showed high transfection efficiency with minimal toxicity. Pharmacogenomic analysis demonstrated that P[Asp(DET)] also provided long-term security after transfection. We hypothesized that the biodegradability of P[Asp(DET)] played a significant role in achieving effective transfection. Gel permeation chromatography (GPC) and electrospray ionization mass spectrometry (ESI-MS) measurements of P[Asp(DET)] revealed their ability to undergo rapid degradation. In contrast, a derivative polycation, N-substituted polyglutamide (P[Glu(DET)]), showed no degradability, indicating that the degradation of P[Asp(DET)] was induced by a specific self-catalytic reaction between the PAsp backbone and the side-chain amide nitrogen. Degradation products of P[Asp(DET)] caused no cytotoxicity, even at high concentrations in the culture medium. Repeated transfection by administering the polyplexes for every 24h showed that biodegradable P[Asp(DET)] provided a continuous increase in transgene expression, while non-degradable P[Glu(DET)] showed a decrease in transgene expression after 48h, coupled with fluctuations in expression profiles of endogenous genes. In vivo intraperitoneal injection of P[Asp(DET)] induced minimal inflammatory cytokine induction to a level comparable to that of normal saline. These results indicate that the biodegradability of P[Asp(DET)] played a key role in achieving safe and sustained transgene expression, by minimizing cumulative toxicity caused by polycations remaining in cells or in the body. [Copyright &y& Elsevier]

Abstract: Non-viral gene delivery is emerging as a realistic alternative to the use of viral vectors with the potential to have a significant impact on clinical therapies. The documented dangers of using the efficient recombinant viruses as carriers have led many to explore the possible advantages of using polymer-based non-viral vectors. To date there is no gene delivery vehicle that contains all the desirable characteristics but they do exist individually in a variety of non-viral carriers, e.g. degradable, low toxicity, cell specific, relatively efficient and capable of delivering multiple genes. Polymers may not be as effective as the viral vehicles; however, the continued focus and growth of knowledge in this field has already resulted in improved delivery. Over the past 10 years, significant progress has been made through the design of specific polymers for this application. Another interesting development in this field is the influx of research on combination approaches to non-viral gene delivery. Scaffolds made of both natural and synthetic materials are being utilized to aid in sustained delivery of the polymer vectors. While the non-viral gene therapy field is currently receiving a large degree of dedicated research there is now the realistic potential of a clinically relevant output. This review presents a summary of combinatorial delivery systems of non-viral polyplexes delivered via tissue engineered scaffolds. For polyplexes to move into the clinical arena, it is important that we uncover and understand the technical hurdles that need to be overcome so that the efficacy of this promising technology can be established. [Copyright &y& Elsevier]

Gene therapy is becoming a promising therapeutic modality for the treatment of genetic and acquired disorders. Nonviral approaches as alternative gene transfer vehicles to the popular viral vectors have received significant attention because of their favorable properties, including lack of immunogenicity, low toxicity, and potential for tissue specificity. Such approaches have been tested in preclinical studies and human clinical trials over the last decade. Although therapeutic benefit has been demonstrated in animal models, gene delivery efficiency of the nonviral approaches remains to be a key obstacle for clinical applications. This review focuses on existing and emerging concepts of chemical and physical methods for delivery of therapeutic nucleic acid molecules in vivo. The emphasis is placed on discussion about problems associated with current nonviral methods and recent efforts toward refinement of nonviral approaches. [ABSTRACT FROM AUTHOR]

Abstract: With increasing knowledge on the molecular mechanisms of endogenous RNA interference, small interfering RNAs (siRNAs) have been emerging as innovative nucleic acid medicines for treatment of incurable diseases such as cancers. Although several siRNA candidates for the treatment of ocular and respiratory diseases are undergoing clinical trials, there are challenges inherent in the further development of siRNAs for anti-cancer therapeutics, because systemic administration will be required in most cases. In addition to nonspecific off-target and immune stimulation problems, appropriate delivery remains a major hurdle. The technologies developed for delivery of nucleic acid medicines such as plasmid DNA and antisense oligonucleotides have paved the way to rapid progress for in vivo delivery of siRNAs. Here, we review various in vivo delivery strategies including chemical modification, conjugation, lipid-based techniques, polymer-based nanosystems, and physical methods. Moreover, the current progress in siRNA delivery systems for gynecologic, liver, lung, and prostate cancers is discussed. [Copyright &y& Elsevier]

Third generation cationic dendritic polymeric polypropyleneimine (PPI) was modified by Pluronic P123 and investigated for gene delivery. The cytotoxicity of P123-PPI was evaluated by the MTT assay and shown to be much lower than that of PPI alone. P123-PPI and PPI can both condense plasmid DNA into nanoparticles with a size of approximately 100 nm and a zeta potential of about 15 mV at the N/P ratio 20:1. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 0.4 U/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 75 μg/mL sodium heparin. The transfection efficiency of SPC-A1 cells using P123-PPI/DNA nanoparticles was much higher than the transfection utilizing PPI/DNA nanoparticles. The addition of free P123 during the preparation of P123-PPI/DNA nanoparticles could significantly enhance the transfection efficiency in the presence of 10% fetal bovine serum. Therefore, P123-PPI/DNA complex nanoparticles may be a safe, efficient and promising cationic conjugate for gene delivery. [ABSTRACT FROM AUTHOR]

This report investigates the comparative in vitro controlled release and transfection efficiencies of pDNA‐lipofectamine complex (lipoplex) and pDNA‐poly(ethylene imine) complex (polyplex), from a biodegradable polycaprolactone (PCL) film. The effect of molecular weight of gelatin used as a porogen on in vitro release and transfection efficiency was also studied. A sustained release profile was obtained for naked pDNA and lipoplex from polymeric films for a month, while the release of polyplexes (PEI/DNA) is simply a burst at day 5, with little or no release thereafter. The release of polyplexes from PCL films is retarded due to interaction between the polyplexes and the polymer. A high burst release was seen for naked pDNA which was suppressed in the presence of gelatin. The extent of suppression of the burst effect by gelatin increased with its molecular weight. For complexed pDNA (lipoplex), the release was slow, but could be accelerated using gelatin; again the acceleration in release is dependant on the molecular weight of the gelatin used. The addition of gelatin as a porogen has no effect on the release of polyplexes from PCL films. The bioactivity of released plasmid DNA and complexes was studied by in vitro transfection using COS‐7 cells. Transfection was observed from released lipoplexes samples till day 9 from PCL film with lower MW gelatin and till day 18 in the case of PCL films with higher MW gelatin. The results also showed that the bioactivity of released lipoplexes was superior to that of the naked pDNA. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [ABSTRACT FROM AUTHOR]

A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC. [ABSTRACT FROM AUTHOR]