Targeted gene delivery with trisaccharide-substituted chitosan oligomers in vitro and after lung administration in vivo.

The aim of this study was to improve the gene delivery efficacy of chitosan oligomer polyplexes by introducing a trisaccharide branch that targets cell-surface lectins. For this purpose, chitosan oligomers were substituted by a trisaccharide with the N-acetylglucosamine residue at the free end, and the ability of the trisaccharide-substituted chitosan oligomers (TCO) polyplexes to transfect various cell lines in vitro and lung tissue after in vivo administration to mice was investigated. Live-cell confocal microscopy showed improved cellular uptake in HEK 293 cells (11-fold, p<0.001) for the TCO polyplexes compared with the linear chitosan oligomers. Colloidal stability was also enhanced with the substituted form, which suggests that the trisaccharide branch stabilised the polyplexes by means of a steric stabilisation mechanism. Interestingly, gene expression levels in the human liver hepatocyte (HepG2) cells were 10-fold higher with the TCO polyplexes than those mediated by polyethyleneimine. A similar improvement was obtained in a human bronchial epithelial cell line (16HBE14o-). Transfection with the TCO was significantly inhibited (by 30-80%), for all the cell lines tested, in the presence of the free trisaccharide branch, confirming lectin-mediated uptake. Finally, in vivo studies showed that, 24 h after lung administration to mice, luciferase gene expression was 4-fold higher with the TCO than with the corresponding linear chitosan oligomers.     

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.     

Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine.

Low-molecular-weight polyethylenimine (LMW-PEI) was synthesized by the acid-catalyzed, ring-opening polymerization of aziridine and compared with commercially available high-molecular-weight PEI (HMW-PEI) of 25 kDa. Molecular weights were determined by size-exclusion chromatography in combination with multi-angle laser light scattering. The weight average molecular weight (M(w)) of synthesized LMW-PEI was determined as 5.4+/-0.5 kDa, whereas commercial HMW-PEI showed a M(w) of 48+/-2 kDa. DNA polyplexes of LMW-PEI and HMW-PEI were characterized with regard to DNA condensation (ethidium bromide fluorescence quenching), size (photon correlation spectroscopy) and surface charge (laser Doppler anemometry). Compared with HMW-PEI, DNA condensation of LMW-PEI was slightly impaired at lower N/P ratios. Complexes with plasmid DNA at a N/P ratio of 6.7 showed significantly increased hydrodynamic diameters (590+/-140 vs. 160+/-10 nm), while the zeta-potential measurements were similar (23+/-2 vs. 30+/-3 mV). The cytotoxicity of LMW-PEI in L929 fibroblasts was reduced by more than one order of magnitude compared with HMW-PEI, as shown by MTT assay. LMW-PEI exhibited increased transfection efficiency in six different cell lines. Reporter gene expression was found to be increased by a factor of 2.1-110. The pharmacokinetics and biodistribution of 125I-PEI in mice were similar for both molecular weights with an AUC of ca. 330+/-100% ID/ml min. Approximately half of the injected dose accumulated in the liver. LMW-PEI proved to be an efficient gene delivery system in a broad range of cell lines. Due to differences in polyplex structure, as well as its relatively low cytotoxicity, which makes the application of high N/P ratios possible, LMW-PEI appears to possess advantageous qualities with regard to transfection efficiency over PEI of higher molecular weight.     

3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins.

This study describes the development of 3PO, a nonviral, protein-based gene delivery vector which utilizes the highly evolved cell-binding, cell-entry and intracellular transport functions of the adenovirus serotype 5 (Ad5) capsid penton protein. A penton fusion protein containing a polylysine sequence was produced by recombinant methods and tested for gene delivery capability. As the protein itself is known to bind integrins through a conserved consensus motif, the penton inherently possesses the ability to bind and enter cells through receptor-mediated internalization. The ability to lyse the cellular endosome encapsulating internalized receptors is also attributed to the penton. The recombinant protein gains the additional function of DNA binding and transport with the appendage of a polylysine motif. This protein retains the ability to form pentamers and mediates delivery of a reporter gene to cultured cells. Interference by oligopeptides bearing the integrin binding motif suggests that delivery is mediated specifically through integrin receptor binding and internalization. The addition of protamine to penton-DNA complexes allows gene delivery in the presence of serum.     

Different synergistic roles of small polyethylenimine and Dosper in gene delivery.

Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency as we have reported earlier. Here, we have further investigated the potential mechanisms of this synergy. Complex morphology, complex sizes and DNA condensation were studied using transmission electron microscopy, light scattering methods and ethidium bromide exclusion, respectively. Cellular uptake, transfection efficiency, and effect of proton pump inhibitor bafilomycin A1 were examined in cell cultures. The cellular uptake of DNA was negligible with PEI2K-DNA complexes, whereas the uptake of the PEI2K-DNA-Dosper or the Dosper-DNA complexes was maximally about 40%. The number of transfected cells was two times higher with PEI2K-DNA-Dosper complexes than with Dosper-DNA complexes. The PEI2K-DNA-Dosper combination was slightly less sensitive to bafilomycin A1 than the PEI25K-DNA or Dosper-DNA complexes. There were no differences between PEI2K and PEI25K in DNA condensation. Dosper condensed DNA slightly more in PEI2K complexes. The PEI25K-DNA complexes were much smaller (<250 nm) than the PEI2K-DNA complexes (0.5-12 micro m) which were also rather polydisperse. It is suggested that two independent mechanisms would lead to synergistic transfection efficiency: (1) Dosper improves the cellular uptake of PEI2K-DNA complexes, and (2) PEI2K improves a transfer of the complexes from lysosomes to nucleus.     

Pyridylthiourea-grafted polyethylenimine offers an effective assistance to siRNA-mediated gene silencing in vitro and in vivo

Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10 nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100 nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4 days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments.     

Dextran-spermine polycation: an efficient nonviral vector for in vitro and in vivo gene transfection

Dextran-spermine cationic polysaccharide was prepared by means of reductive amination between oxidized dextran and the natural oligoamine spermine. The formed Schiff-base imine-based conjugate was reduced with borohydride to obtain the stable amine-based conjugate. The transfection efficiency of the synthetic dextran-spermine was assessed in vitro on HEK293 and NIH3T3 cell lines and found to be as high as the DOTAP/Chol 1/1 lipid-based transfection reagent. Modification of the dextran-spermine polycation with polyethylene glycol resulted in high transfection yield in serum-rich medium. Intramuscular injection in mice of dextran-spermine-pSV-LacZ complex induced high local gene expression compared to low expression of the naked DNA. Intravenous injection of a dispersion of the dextran-spermine-pSV-LacZ complex resulted with no expression in all examined organs. When the partially PEGylated dextran-spermine-pSV-LacZ complex was intravenously applied, a high gene expression was detected mainly in the liver. Preliminary targeting studies indicated that the PEGylated dextran-spermine-pSV-LacZ complex bound to galactose receptor of liver parenchymal cells rather than the mannose receptor of liver nonparenchymal cells. This work offers a new biodegradable polycation based on natural components, which is capable of transfecting cells and tissues in vitro and in vivo.     

A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo

Polyethylenimine (PEI), a polycation with high ionic charge density, has recently been used as a gene therapy delivery agent. We have defined the optimal conditions for PEI-based transfection of airway epithelial cells in vitro and in vivo and used these conditions to restore Cl(-) channel activity in a CF mouse model. Three forms of PEI, a linear 22 kDa (ExGen 500) form and branched 25 or 50 kDa forms were evaluated. All forms of PEI significantly increased luciferase reporter gene expression compared to the liposome DCChol/DOPE in a human bronchial epithelial cell line (16HBE) irrespective of the extent of cell confluency. With subconfluent cells, gene expression was around 1000-, 200- and 25-fold higher than liposomes using linear 22, 25 and 50 kDa PEI, respectively. The transfection efficiency was reduced in confluent and polarized epithelial cells but linear 22 kDa PEI showed the smallest decrease and gave 8000-fold better transfection in polarized cells compared to liposomes. A comparison of linear 22 or 25 kDa PEI with DCChol/DOPE for airway delivery in vivo via intranasal instillation was also performed. Linear 22 kDa PEI gave significantly better luciferase reporter gene expression of 350-fold in the lung, 180-fold in the nose and 85-fold in the trachea compared to liposome. In contrast, the 25 kDa form of PEI was no better than DCChol/DOPE. Repeat dosing with linear 22 kDa PEI failed to give reporter gene delivery comparable to the initial dose. To establish that PEI can be used to deliver a physiologically relevent gene in vivo, we used it to restore Cl(-) secretion by CFTR gene delivery in the airways of a CF mouse model.     

Chitosan delivery systems for the treatment of oral mucositis: in vitro and in vivo studies.

Oral mucositis is a frequent and potentially severe complication of radiation or chemotherapy for cancer. Associated with atrophy and ulceration of the oral mucosa is an increased risk of infection, and the most common pathogenic agent is Candida. Chitosan is an excellent candidate for the treatment of oral mucositis. Its bioadhesive and antimicrobial properties offer the palliative effects of an occlusive dressing and the potential for delivering drugs, including anti-candidal agents. The aim of this study was to develop an occlusive bioadhesive system for prophylaxis and/or treatment of oral mucositis. Gel and film formulations were prepared using chitosans at different molecular weights and in different solvents. Nystatin, which is considered as a prophylactic agent for oral mucositis was incorporated into the formulations. The in vitro release of nystatin from the formulations was decreased with the increasing molecular weight of chitosan. The effect of the formulations was investigated in vivo in hamsters with chemotherapy-induced mucositis. Mucositis scores in groups treated with nystatin incorporated into gel and suspension formulations were significantly lower (p < 0.05) than those treated with the chitosan gel alone. Survival of animals in the treated groups was higher than that in the control group. The retention time and distribution of the gels in the oral cavity were investigated in healthy volunteers. A faster distribution of nystatin in the oral cavity was obtained using the suspension compared to the gels, but the nystatin saliva level decreased rapidly as well. A drug concentration above the minimum inhibitory concentration (MIC) value for Candida albicans (0.14 microg/ml) was maintained for longer periods of time at the application site (90 min) than at the contralateral site (45 min) in the oral cavity.     

In vitro and in vivo evaluation of a matrix-in-cylinder system for sustained drug delivery.

A matrix-in-cylinder system for sustained drug delivery, consisting of a hot-melt extruded ethylcellulose (EC) pipe surrounding a drug containing HPMC-Gelucire 44/14 core, was evaluated in vitro and in vivo. In an aqueous medium, the HPMC-Gelucire core forms a gel plug, which releases the drug-through the open ends of the EC pipe--by means of erosion. The influence of hydrodynamic and mechanical stress and the effect of different 'physiologically relevant' dissolution media on the in vitro drug release were investigated. From these in vitro dissolution tests, it was concluded that the EC pipe has a protective effect on the drug containing HPMC-Gelucire core. It largely protects the core against hydrodynamics and mechanical stress. Furthermore, drug release from the matrix-in-cylinder system was only slightly affected by the composition of the dissolution medium. A randomised crossover in vivo study in dogs revealed that the matrix-in-cylinder system containing propranolol hydrochloride has an ideal sustained release profile with constant plasma levels maintained over 24 h. Moreover, administration of the matrix-in-cylinder system resulted in a 4-fold increase in propranolol bioavailability when compared with a commercial sustained release formulation (Inderal).     

Fibrin hydrogels for lentiviral gene delivery in vitro and in vivo

Gene delivery from hydrogels represents a versatile approach for localized expression of tissue inductive factors that can promote cellular processes that lead to regeneration. Lentiviral gene therapy vectors were entrapped within fibrin hydrogels, either alone or complexed with hydroxylapatite (HA) nanoparticles. The inclusion of HA into the hydrogel led to the formation of small aggregates distributed throughout the hydrogel, with no obvious alteration of the pore structure outside the aggregates. The presence of HA slowed hydrogel degradation by collagenase and plasmin relative to fibrin alone, and also decreased the rate of cell migration. Lentivirus had similar release from the fibrin hydrogels formed with or without HA. The altered hydrogel properties suggest an interaction between the nanoparticle and fibrin, which may displace the virus from the particle leading to similar release profiles. Transgene expression by cells migrating into the hydrogel in vitro was reduced in the presence of HA, consistent with the role of cell migration on transgene expression. In vivo, lentivirus loaded fibrin hydrogels promoted localized transgene expression that increased through day 9 and decreased through day 14. For the fibrin only hydrogels, expression continued to decline after day 14. However, hydrogels with HA maintained this transgene expression level for an additional 2 weeks before declining. Immunostaining identified transgene primarily outside the fibrin-HA gel at day 9; however, at day 21, transgene expression was observed primarily within the fibrin-HA gel. The localized delivery of lentivirus provides an opportunity to enhance the bioactivity of fibrin hydrogels for a wide range of applications in regenerative medicine.     

Targeted delivery of oligodeoxynucleotides to mouse lung endothelial cells in vitro and in vivo.

Pulmonary endothelium plays an important role in the maintenance of normal pulmonary physiology and its dysfunction is involved in a number of pulmonary diseases. Correction of endothelial dysfunction via antisense oligodeoxynucleotides (ODN) is dependent on the development of a delivery vehicle that can efficiently deliver the ODN to pulmonary endothelium with minimal toxicity. To this end, we have developed a novel lipidic vector that is highly efficient in targeted delivery of ODN to pulmonary endothelium. This is based on a method that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane) and an ethanol-containing buffer system for encapsulating large quantities of polyanionic ODN in lipid vesicles. An endothelium-specific antibody (273-34A) is incorporated into the lipid vesicles via a distearoylphosphatidylethanolamine-poly(ethylene glycol) spacer. The 273-34A antibody efficiently mediated delivery of ODN to mouse lung endothelial cells in vitro and in vivo. Furthermore, systemic administration of this formulation is associated with minimal hematological toxicities and induces little acute change in systemic and pulmonary hemodynamics. These results provide a basis for lipid-mediated delivery of ODN for the treatment of pulmonary diseases. They also suggest the utility of this approach as a research tool to characterize the function of genes in the pulmonary endothelium.     

Mesenchymal stem cells as a gene delivery system to create biological pacemaker cells in vitro

Pacemaker cells differ from common cardiomyocytes due to the presence of a spontaneous depolarization process during the diastolic phase of the cardiac cycle. This is due to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are responsible for providing an inward current. Genetically engineered mesenchymal stem cells (MSCs) were transfected with hHCN4 genes using lentiviral transfection, and their potential use as biological pacemaker cells was investigated. In addition to expressing an anticipated high level of the hHCN4 gene, MSCs transfected with hHCN4 genes also expressed characteristic hHCN4 protein, a cardiac pacemaker-like current and were capable of increasing the spontaneous beating rate of co-cultured cardiac myocytes. Control MSCs did not exert these effects. It is hypothesized that genetically engineered MSCs transfected with hHCN4 genes by lentiviral transfection can be modified to be cardiac pacemaker cells in vitro.     

Butirosin compared with gentamicin in vitro and in vivo

Butirosin (BTN) (P. W. K. Woo, G. L. Coffey, H. W. Dion, S. A. Fusari, and G. D. Senos, U. S. Patent 3,541,078, 1970) is a new aminoglycoside antibiotic notably active against opportunist bacterial species within Pseudomonas, Klebsiella, Enterobacter, Serratia, and Proteus. Numerous comparative tests were carried out with BTN and gentamicin (GTM) in vitro and in experimental infections in mice. BTN was more active in Mueller-Hinton broth than in agar, but its activity was lessened at acid pH or under anaerobiosis, as has been observed with other aminoglycosides. In standard agar diffusion tests, inhibition zones greater than 12 mm around 30-mug BTN disks generally denoted susceptibility, equivalent to minimal inhibitory concentrations [Formula: see text] 25 mug/ml. Cross-resistance between BTN and GTM occurred in a variable manner, with a number of bacterial strains strongly resistant to GTM being moderately susceptible to BTN. In mice, after a single subcutaneous injection, absorption of both antibiotics was rapid, with peak serum levels occurring in 15 min; this was followed by rapid elimination with estimated serum half-lives of about 20 min for each. After peroral administration of high doses in mice, there was no appreciable absorption of BTN. Several tests were carried out to compare BTN and GTM with respect to minimal inhibitory concentrations in vitro, acute subcutaneous median mouse protective doses, peak serum levels at such doses, and the therapeutic ratios derived from acute median protective and lethal doses. Although GTM usually proved to be more potent antibacterially on a weight basis, observations on BTN indicated a superior effectiveness in terms of therapeutic ratios.     

In vivo gene delivery via portal vein and bile duct to individual lobes of the rat liver using a polylysine-based nonviral DNA vector in combination with chloroquine.

The objective of this study was to evaluate a bifunctional synthetic peptide as a DNA vector for regional gene delivery to the rat liver by the portal vein and bile duct routes. The 31-amino-acid peptide (polylysine-molossin) comprises an amino-terminal chain of 16 lysines for electrostatic binding of DNA, and the 15 amino acid integrin-binding domain of the venom of the American pit viper, Crotalus molossus molossus. Initial in vitro evaluation demonstrated that polylysine-molossin/DNA complexes were much smaller (approximately 50-100nm versus 500-1300nm), more positively charged, and more stable in isotonic dextrose in comparisons with salt-containing solutions. However, polylysine-molossin/DNA complexes in any solution other than complete culture medium were ineffective for gene delivery in vitro. Vector localization studies demonstrated that both the portal vein and bile duct routes provided excellent access of polylysine-molossin/DNA complexes to the liver. However, complexes delivered by the portal vein were rapidly lost (<15 min) following re-establishment of the portal circulation, whereas complexes delivered by the bile duct persisted much longer. Polylysine-molossin/DNA complexes in various isotonic solutions were delivered to the right lateral lobes either by perfusion through a branch of the portal vein or by infusion into appropriate branches of the bile duct. Two or three hours before gene delivery, rats were given a single injection of chloroquine. We report that the polylysine-molossin vector is much more effective (>10-fold) when delivered by the bile duct route with all isotonic solutions evaluated, and that polylysine-molossin/DNA complexes in isotonic dextrose are much more effective (>10-fold) than complexes in salt-containing solutions.     

Structure-function relationships of gene delivery vectors in a limited polycation library.

A library of 13 polylysine-graft-imidazoleacetic acid conjugates was synthesized to examine the collective effects of polymer molecular weight, side chain substitution, and DNA:polymer ratio on cytotoxicity, transfection efficiency, and polycation-DNA interaction. In general, the relationships between the physicochemical characteristics and the gene transfer capabilities of these polycations appear nonlinear. The in vitro cytotoxicity of these polymers decreased, while total protein expression increased, with decreasing molecular weight and increasing imidazole content. Flow cytometry experiments indicated, however, that an increase in marker gene expression does not always correlate with the total number of cells transfected, even when similar polymer structures are used for transfection. The maximum level of luciferase gene expression was mediated by transfection with a low molecular weight, high imidazole content (9400 Mw, 95 mol% imidazole) polymer. The extent of DNA condensation, as determined by ethidium bromide fluorescence quenching, also decreased with decreasing polymer molecular weight and increasing imidazole content. Relative binding affinity between DNA and the polycations, measured via competitive binding in the presence of a synthetic polyanion, decreased with decreasing polymer molecular weight; however, the relative affinity also appeared to increase with increasing imidazole, suggesting that electrostatic contributions are not solely responsible for DNA-polycation binding interactions. This limited library and corresponding structure/function analysis forms the foundation upon which larger, more comprehensive polycationic libraries can be designed and evaluated to further understand how polycation transfection reagents function.     

Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery

The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.     

Aminated gelatin microspheres as a nasal delivery system for peptide drugs: evaluation of in vitro release and in vivo insulin absorption in rats.

Aminated gelatin microspheres (AGMS) was investigated as a nasal drug delivery system for peptide drugs. The in vitro drug release from microspheres was evaluated using a fluorescein-labeled insulin (RITC-insulin) and FITC-dextran with a molecular weight of 4.4 kDa (FD-4) as model drugs. RITC-insulin release from AGMS was significantly slower than from native gelatin microspheres (GMS), with a cumulate release of 18.4% and 32.4% within 30 min, and 56.9% and 75.1% within 8 h respectively. However, the release of FD-4 from both AGMS and GMS was quite rapid and no difference was observed for the two microspheres. The electrostatic interactions between the model drugs and the microspheres were supposed to be the main factor that controlled the release behavior. The absorption enhancing effect was estimated by measuring the changes of plasma glucose concentrations of healthy rats following intranasal administration of insulin-incorporated microspheres in both suspension and dry powder forms. AGMS could significantly increase the nasal absorption of insulin in rats when administered in a dry powder formulation, but no significant hypoglycemic effect was observed when given in suspensions. One of the mechanisms for the increased insulin absorption was attributed to the hydrogel nature of the microspheres that could absorb water from the nasal mucosa and thus resulted in a temporarily dehydration of the epithelium membrane and opening of the tight junctions. The positive charge of the AGMS has also evidently contributed to the absorption enhancing effect. In addition, the mucoadhesive properties of AGMS might also have played a role to the total effect. AGMS might be a new candidate carrier for the nasal delivery of peptide drugs.     

Recombinant Semliki Forest virus as a vector system for fast and selective in vivo gene delivery into balloon-injured rat aorta

Previously, we demonstrated that recombinant Semliki Forest virus (SFV) vector rapidly and selectively transfers genes into cultured vascular smooth muscle cells (VSMC), leaving endothelial cells (EC) unaffected. From this, we hypothesized that recombinant SFV in vivo only transfers genes into the media of balloon-injured but not intact vessel, that gene expression in VSMC is fast, and that the specificity of SFV for VSMC is caused by specific binding sites. To address these hypotheses, we studied the time course of in vivo SFV-LacZ and Ad-LacZ expression in balloon-injured rat aorta. In addition, the fusion characteristics of fluorescent pyrene-labeled SFV were explored in cultured VSMC and EC. In intact aorta, no LacZ expression was found in the intima or media at 24 h. In contrast, in denuded aorta, LacZ expression was detected in as early as 12 h after incubation. LacZ expression was predominantly present in the media. Ad-LacZ expression started after 12 h, but was predominantly present in the adventitia. Ad-LacZ expression in the media started after 72 h. In vitro transfection with SFV showed that fusion was higher and, moreover, saturable in VSMC as compared with EC, indicating the presence of specific SFV binding sites on VSMC, but not EC. From this we conclude that in vivo selectivity of SFV in balloon-injured vessels is based on the removal of the endothelium, which results in accessibility of VSMC in the media that carry specific binding sites for the SFV vector.