Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo.

We have developed polyelectrolyte gene delivery vectors that display good extracellular stability and are activated intracellularly to permit transgene expression. The strategy comprises covalent crosslinking of primary amines in poly-L-lysine/DNA complexes with a crosslinking agent that can later be cleaved by reduction. Crosslinked complexes maintained the same size and surface charge but showed increased stability against polyelectrolyte exchange with poly-L-aspartic acid. Surface modification with polyethyleneglycol improved solubility and masked their positive surface charge. Crosslinked complexes showed 10-fold increased plasma circulation following intravenous administration to Balb/c mice. In the absence of chloroquine, the levels of transgene expression in B16F10 murine melanoma cells were similar for crosslinked and non-crosslinked complexes, however, chloroquine selectively potentiated transgene expression by the non-crosslinked complexes. Cellular uptake of the complexes was the same, irrespective of crosslinking. Following microinjection into the cytoplasm of Xenopus oocytes, or the cytoplasm or nucleus of Rat-1 fibroblasts, crosslinked complexes mediated the same transgene expression as non-crosslinked complexes, indicating crosslinked complexes are rapidly reduced and activated intracellularly. We therefore hypothesize that the lower in vitro transfection activity of crosslinked complexes in the presence of chloroquine is due to reduced transfer from endosome to cytoplasm, mainly due to increased stability against destabilization by chloroquine. The extended systemic circulation together with triggered intracellular activation makes these complexes a promising system for targeted gene delivery in vivo.     

Impact of polyplex micelles installed with cyclic RGD peptide as ligand on gene delivery to vascular lesions

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 α(v)β(3) and α(v)β(5) 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.     

A reduction-responsive drug delivery with improved stability: disulfide crosslinked micelles of small amiphiphilic molecules

Micelles self-assembled from small amphiphilic molecules are unstable in biological fluids, and thus are poor drug carriers. In contrast, amphiphilic polymer micelles can encapsulate hydrophobic drugs in their core to greatly enhance their aqueous solubility and extend their retention time in blood circulation owing to their hydrophilic shell. However, the major disadvantages of conventional polymer micelles are the heterogeneity of the amphiphilic polymer structure and premature drug leakage. Thus, herein, to address these shortcomings, disulfide crosslinked micelles composed of a small amphiphilic molecule, di-lipoyl-glycerophosphorylcholine (di-LA-PC), were developed as redox-responsive drug carriers. Specifically, di-LA-PC was synthesized and self-assembled to form crosslinked micelles under catalysis by dithiothreitol. The disulfide crosslinked micelles maintained high stability in a simulated physiological environment, but rapidly disassembled under reductive conditions. Furthermore, paclitaxel (PTX), as a model drug, was encapsulated in the core of the crosslinked micelles with a high loading content of 8.13%. The in vitro release studies indicated that over 80% of PTX was released from the micelles in the reductive environment, whereas less than 20% PTX was released without reduction in the 68 h test. Benefiting from their nanoscale characteristics, the PTX-loaded micelles showed efficient cellular internalization and effectively induced the death of cancer cells, as revealed in the MTT, apoptosis and cell cycle tests. Moreover, pharmacokinetic studies demonstrated that the crosslinked micelles prolonged the circulation of the incorporated PTX in the bloodstream and increased its accumulation in the tumor tissue via the EPR effect. Finally, the PTX-loaded micelles displayed prominent in vivo anti-tumor activity in a 4T1 xenograft tumor model. In summary, the di-LA-PC crosslinked micelle platform possesses excellent stability, high loading capacity and reduction-responsive release profile, which may have applications in the delivery of PTX and other anti-cancer drugs.

Reduction-responsive crosslinked di-LA-PC micelles from amphiphilic bis-LA-PC conjugate for PTX loading and GSH-triggered release of PTX.     

Polymer micelles with cross-linked ionic cores for delivery of anticancer drugs.

This work reports the design of polymer micelles with cross-linked ionic cores that display high stability. Block ionomer complexes of poly(ethylene oxide)-b-poly(methacrylic acid) copolymer and divalent metal cations were utilized as micellar templates for the synthesis of the cross-linked micelles. Such micelles represent hydrophilic nanospheres of core-shell morphology. The core comprises a network of the cross-linked polyanions, which is surrounded by the shell of hydrophilic PEO chains. The ionic character of the core provided for pH-dependent swelling/collapse behavior of the nanogels. Cisplatin, a potent chemotherapeutic agent, was incorporated into the ionic core of the micelles with remarkably high efficiency (22% w/w). The drug-loaded micelles were stable in aqueous dispersions exhibiting no aggregation or precipitation for a prolonged period of time. Slow release of platinum complexes was observed in sustained manner from the cisplatin-loaded cross-linked micelles in physiological saline. In vitro studies using human A2780 ovarian carcinoma cells demonstrated that the cross-linked micelles rapidly internalized and delivered cisplatin into cells. These results indicated that polymer micelles with cross-linked ionic cores are promising for further fundamental material studies and practical applications as drug delivery carriers.     

Ethanol enhanced in vivo gene delivery with non-ionic polymeric micelles inhalation.

Modifications of both carriers and host barriers have been investigated for efficient inhalation gene delivery to lung. Here we used a biocompatible, non-ionic poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) polymeric micelles (PM) as a carrier and combined it with ethanol to enhance membrane penetration of delivered DNA. The inhalation delivery with six 100 microg doses of pCMV-Lac Z with PM co-formulated with 10%-40% ethanol to nude mice in 2 days at 8 h interval was performed. The beta-galatosidase (beta-Gal) activity was assessed using chlorophenol red-beta-d galactopyranoside (CPRG) and X-gal staining for quantitative and qualitative analysis in tissues. The results showed that beta-Gal activity was significantly increased by 38% in lung around bronchioles when inhalation with PM and 10% ethanol was given. The 10% ethanol also increased the intracellular apparent permeability by 42% in stomach and by 141% in intestine at 48 h after the first dosage of delivery. Also delivery of DNA encoding a functional human cystic fibrosis transmembrane protein (CFTR) using the same inhalation delivery method co-formulated with 10% ethanol, an increased expression of CFTR in lung was detected by immunostaining. We concluded that 10% ethanol co-formulated with the PM system could enhance inhaled gene delivery to airway and gastrointestinal (GI) tract.     

PEGylated polyethylenimine for in vivo local gene delivery based on lipiodolized emulsion system.

Polyethylenimine (PEI) is one of the most efficient vectors for non-viral gene delivery, whereas its poor transfection activity, compared to viral vectors, and cytotoxicity need to be improved for in vivo applications. In this study, we prepared two PEI conjugates with 6 and 10 wt.% of poly(ethylene glycol) (PEG) grafts (referred to PEI-PEG-6 and PEI-PEG-10, respectively) in order to investigate the effects of PEGylation on cytotoxicity and transfection activity in vitro. In addition, their suitability as vectors for local gene delivery in vivo was assessed by injecting lipiodolized emulsions containing polymer/DNA complexes into the femoral artery of Sprague-Dawley (SD) rats, occluded by a surgical suture to block inflow of the blood to the leg. Both PEGylated PEIs showed significantly lower cytotoxicity and higher transfection activity in COS-1 cells than PEI taken as a control; in particular, PEI-PEG-10 produced the most promising results. The stable water-in-oil emulsion, composed of aqueous domains containing the complexes and lipiodol as an oil phase, was formed in the presence of a hydrogenated castor oil. From in vivo experiments, it was found that all the complexes, dispersed in the lipiodolized emulsion, delivered effectively gene to muscle, surrounding the injection site, rather than other organs such as liver, spleen, kidney, heart and lung. The in vivo transfection activity of PEI-PEG-10 was 3-folds higher in muscle than that of PEI. Based on these results, it can be concluded that PEGylated PEIs (based on the lipiodolized emulsion system) hold a promising potential for local gene delivery in vivo.     

PEG shielded polymeric double-layered micelles for gene delivery.

A combination of A-B and B-C block copolymers was used to encapsulate DNA inside pEG coated particles, where A is a cationic block (poly(dimethylaminoethyl methacrylate), pDMAEMA) for DNA binding and condensation, B is a hydrophobic block (poly(butylmethacrylate), pBMA) and C is a polyethylene glycol (pEG) block. The AB and BC block copolymers were synthesized by transition metal mediated radical polymerization. The AB block copolymer had a fixed pBMA molecular weight of 3800 g/mol and a varying pDMAEMA molecular weight (from 22 to 65 kg/mol), the BC block copolymer had a fixed composition (pBMA 9000 g/mol; pEG 2000 g/mol). Plasmid DNA containing particles were made via a detergent dialysis method. By this method, particles of approximately 120 nm, as determined by dynamic light scattering (DLS), with a near neutral charge were formed, independent of the DMAEMA block size. DLS measurements and gel electrophoresis indicated that the particles were very stable in cell culture medium at 37 degrees C and resistant to anionic exchange by poly-l-aspartic acid. The particles were able to transfect COS-7 and OVCAR-3 cells with minor toxicity if incubated for 1 or 4 h; incubation for 24 h resulted in an increased toxicity. This paper shows that small polyplexes with near neutral charge can be obtained via a convenient detergent dialysis method using pDMAEMA-b-pBMA and pBMA-b-pEG. These particles may be interesting for in vivo experiments where particles with high positive charges have adverse interactions with blood components.     

Drug delivery in polymeric micelles: from in vitro to in vivo.

A new drug delivery modality was developed based on drug encapsulation in polymeric micelles followed by a controlled release at the tumor site triggered by ultrasound focused on the tumor. Ultrasound not only released drug from micelles but also enhanced the local uptake of both free and encapsulated drug by tumor cells, thus providing effective drug targeting. The significant success of in vitro studies of this new drug delivery technique warranted extending studies to animal experiments. Here the results of the in vitro studies of the above technique are summarized and the first in vivo experiments using colon cancer model in rats are reported. The in vivo results showed that application of low-frequency ultrasound (20 and 70 kHz) significantly reduced the tumor size when compared with non-insonated controls; this result indicated in vivo drug targeting to tumors by ultrasound.     

PEGylated dendrimer-entrapped gold nanoparticles with low immunogenicity for targeted gene delivery

The high efficiency and specificity of gene therapy are mainly ascribed to the excellent transfection ability of the gene carrier. Non-viral polymer vectors have attracted extensive attention because of their low cytotoxicity and outstanding genetic loading capacity compared with viral vectors. For safe and efficient transfection of nuclear acids, here we report a novel gene delivery system, dendrimer-entrapped gold nanoparticles modified with a folate-conjugated poly (ethylene glycol) (Au DENPs-PEG-FA), possessing superior gene transfection efficiency than that of partially hydrophilic methoxy poly(ethylene glycol) (mPEG)-modified dendrimer-entrapped gold nanoparticles (Au DENPs-mPEG). The prepared Au DENPs-PEG-FA were well characterized, and our data revealed that the vector showed good cytocompatibility. Additionally, the quantification of inflammatory cytokines detected by qRT-PCR showed that the vectors displayed low innate immune response. The efficiency of nucleic acid (encoding enhanced green fluorescent protein (EGFP) and luciferase (Luc) reporter) transfection evaluated via flow cytometry and confocal microscopic imaging suggested that the Au DENPs-PEG-FA were able to transfect nucleic acid into HeLa cells with enhanced transfection efficiency. Furthermore, the existence of FA rendered the Au DENPs with excellent targeting performance via FA receptor-ligand binding interaction. The designed Au DENPs-PEG-FA with low immunogenicity and enhanced gene transfection efficiency may hold a great promise to be a superior vector for gene therapy.

The designed Au DENPs-PEG-FA compacts pDNA into cells to enhance gene transfection efficiency.     

Polyethylenimines for in vivo gene delivery

Branched and linear polyethylenimines (PEIs) are proving to be efficient, non-toxic and versatile agents for in vivo gene delivery by a number of routes. A major factor in the successful use of PEIs seems to be the small size of PEI/DNA complexes which can be achieved under controlled conditions of formulation, mainly by using PEIs of low molecular weights. This review considers the in vivo use of PEI, from formulation to delivery and analysis of gene expression. PEI delivery is already used for the analysis of numerous physiological processes. It is hoped that scrutiny of the mechanisms involved with PEI-based gene delivery at different levels of the transfection process and in different in vivo contexts will aid the transition towards its use in therapeutic situations.     

Poly(cationic lipid)-mediated in vivo gene delivery to mouse liver

We have previously demonstrated that liposomes generated from poly(cationic lipid) (PCL) and cholesterol (Chol) have low cytotoxicity, are serum resistant, and display a transfection efficiency in vitro similar to commercially available cationic liposomes. Our in vivo experiments demonstrated that PCL-Chol liposomes bound much less avidly to serum proteins than did liposomes composed of 1,2-bis(dioleoyloxy)-3-(trimethylamonio)propane (DOTAP)-Chol or DOTAP-L-alpha dioleoyl phosphatidylethanolamine (DOPE). Injection of the lipoplexes (PCL-Chol+DNA) through the portal vein after partial hepatectomy (PH) led to much higher reporter gene expression (luciferase) in the liver than did naked DNA injection. Marked green fluorescent protein expression was visualized in almost all hepatocytes in the liver of mice receiving lipoplex injection, even in the absence of PH. Subcutaneous injection of thyroid hormone triiodothyromine (T(3)) significantly promoted hepatocyte regeneration and markedly enhanced PCL-Chol-mediated gene transfer in mouse liver when the lipoplex was administrated through either portal or tail vein. With T(3) pretreatment, PCL-Chol exerted a better gene transfer efficacy in mouse liver than DOTAP-Chol or DOTAP-DOPE. Two injections of lipoplexes through an indwelling catheter in the portal vein extended the transgene expression at a high level when T(3) injection was repeated. In conclusion, our findings demonstrate that the polymerized cationic liposomes are very stable in the blood and are effective agents for in vivo gene delivery, and that thyroid hormone administration offers a non-invasive approach to enhance liposome-mediated liver gene delivery.     

Dexamethasone-loaded peptide micelles for delivery of the heme oxygenase-1 gene to ischemic brain

The R3V6 peptides, which are composed of a 3-arginine block and a 6-valine block, formed self-assembled micelles in aqueous solution. Dye quenching assays showed that a hydrophobic fluorescent dye, 5-dodecanoylaminofluorescein (DAF), interacted with and was loaded into the hydrophobic core of the micelles. In this study, dexamethasone-loaded R3V6 peptide micelles (R3V6-Dexa) were evaluated as a gene carrier. R3V6-Dexa had higher gene delivery efficiency in human embryonic kidney 293 cells compared to those of the R3V6 peptides and poly-L-lysine (PLL). Dexamethasone might stabilize the micelle structure of the R3V6 peptides by forming strong hydrophobic cores and enhanced the transfection efficiency. Furthermore, R3V6-Dexa reduced the expression of an inflammatory cytokine, interleukin-6 (IL-6), more efficiently in lipopolysaccharide (LPS)-induced Raw264.7 cells than did dexamethasone, suggesting that R3V6-Dexa is also a useful carrier for dexamethasone delivery. A focal brain ischemia-reperfusion model was produced by middle cerebral artery occlusion (MCAO). A heme oxygenase-1 (HO-1) expression plasmid DNA, pSV-HO-1, was delivered into the brain using R3V6-Dexa as a carrier. The pSV-HO-1/R3V6-Dexa complex was injected into the brain 1hr prior to MCAO. Twenty-four hours later, the HO-1 expression of the pSV-HO-1/R3V6-Dexa injection group was higher than those of the MCAO control, pβ-Luc/R3V6-Dexa, and pSV-HO-1/PEI25k injection groups. In addition, the infarct size was reduced due to the delivery of pSV-HO-1/R3V6-Dexa complex. Therefore, R3V6-Dexa may be a useful carrier for HO-1 gene delivery and stroke gene therapy.     

Novel vectors for in vivo gene delivery to vascular tissue

Although some success has been achieved with gene delivery in animal models of vascular disorders, the results from some clinical trials have been less promising, possibly due, in part, to the use of suboptimal vectors for in vivo gene transfer. Non-viral vectors have a very low transfection efficiency so are largely unsuitable for most in vivo applications, and the relatively broad tropism of many of the commonly used viral vectors can limit efficient gene delivery specifically to target vascular tissues. However, characterisation of novel virus serotypes and advances in techniques that enable vectors to be targeted to the required tissue have led to progress in the development of novel vectors that could be utilised for gene delivery for vascular disorders.     

Phosphated crosslinked guar for colon-specific drug delivery. II. In vitro and in vivo evaluation in the rat.

Targeting of drugs to the colon, following oral administration, can be accomplished by the use of modified, biodegradable polysaccharides as vehicles. In a previous study, a crosslinked low swelling guar gum (GG) hydrogel was synthesized by reacting it with trisodium trimetaphosphate (STMP). In the present study the functioning of GG crosslinked products (GGP) as possible colon-specific drug carriers was analyzed by studying (a) the release kinetics of pre-loaded hydrocortisone from GGP hydrogels into buffer solutions with, or without GG degrading enzymes (alpha-galactosidase and beta-mannanase) and (b) direct measurements of the polymers' degradation in the cecum of conscious rats. The effect of GG diet on alpha-galactosidase and beta-mannanase activity in the cecum of the rat and GGP degradation was also measured. It was found that the product GGP-0.1 (loosely crosslinked with 0.1 equivalents of STMP) was able to prevent the release of 80% of its hydrocortisone load for at least 6 h in PBS, pH=6.4. When a mixture of alpha-galactosidase and beta-mannanase was added to the buffer solution, an enhanced hydrocortisone release was observed. In-vivo degradation studies in the rat cecum showed that despite the chemical modification of GG, it retained its enzyme-degrading properties in a crosslinker concentration-dependent manner. Eight days of GG diet prior to the study increased alpha-galactosidase activity in the cecum of the rat three-fold, compared to its activity without the diet. However, this increase in the enzyme activity was unable to improve the degradation of the different GGP products. The overall alpha-galactosidase activity in the rat cecum was found to be extracellular, while the activity of beta-mannanase was found to be bacterial cell-wall associated. It is concluded that because CG crosslinked with STMP can be biodegraded enzymatically and is able to retard the release of a low water-soluble drug, this polymer could potentially be used as a vehicle for colon-specific drug delivery.     

Self-assembling complexes for in vivo gene delivery

Non-viral DNA-containing particles represent a potentially attractive alternative to viruses for in vivo gene therapy applications. However, the inability to form small particles that remain stable and non-aggregating in vivo has limited their usefulness to date. The relatively simple self-assembling complexes that function so efficiently for in vitro gene delivery fail to provide similar gene delivery capabilities in vivo. Now for the first time, significant advances are being made into defining many of the in vivo barriers preventing stable particle formation. This improved understanding has been manifested into a rational design approach to DNA particle formation. This review highlights many of the recent studies in which rational design was employed in an effort to form polymer/DNA particles (polyplexes) that resist in vivo aggregation and inactivation.     

Comparative in vivo approaches for selective adenovirus-mediated gene delivery to the placenta

Gene delivery to the placenta is one potential way of specifically modifying placental biological processes and fetal development. The aim of this study was to determine the most efficient and least invasive route of placental adenovirus delivery. The feasibility of adenovirus-mediated gene transfer to the rat placenta was addressed by maternal intravenous or direct intraplacental injection of adenoviral vectors expressing the glucose transporter GLUT3, a noncirculating integral membrane protein. Both routes led to transgene expression in the placenta. However, direct intraplacental delivery on day 14 of gestation yielded a higher transduction efficiency than maternal intravenous administration, and markedly reduced transgene expression in maternal liver. Most importantly, the amount of the GLUT3 transgene and the adenovirus itself in fetal tissues was only 1 to 3% of that found in the placenta. These results indicate that the nature of the transgene and the route of adenovirus administration are key parameters in selective placental somatic gene transfer. This novel strategy may prove useful for modifying a placental function without altering the fetal genome.