Biodistribution characteristics of amino acid dendrimers and their PEGylated derivatives after intravenous administration.

In this study, we synthesized dendritic poly(L-lysine)s (DPKs), dendritic poly(L-ornithine)s (DPOs), which are constructed as novel amino acid dendrimers, and PEGylated KG6 (the sixth generation of DPKs), and evaluated the physicochemical properties and biodistribution characteristics of these dendrimers. The particle size of DPKs and DPOs was well controlled in the nanometer range. The zeta-potential of these dendrimers was slightly positive and this gradually increased in association with their generation. After intravenous administration to mice, all tested dendrimers cleared rapidly from blood flow and mainly accumulated in the liver and kidney. The hepatic and renal accumulation changed in a generation-dependent manner. In contrast, no significant distributional differences between same generation of DPK and DPO were observed, although the constituent amino acids, particle size, and zeta-potential were different. However, PEGylation of KG6 caused great changes in particle size, zeta-potential, blood retention and organ distribution in vivo, indicating that the PEGylation is applicable strategy to improve biodistribution characteristics of dendrimeric molecules. The information provided by this study will be helpful for the development of future drug delivery systems using amino acid dendrimers as safe drug carriers.     

Nonionic amphiphilic block copolymers promote gene transfer to the lung

Various pulmonary disorders, including cystic fibrosis, are potentially amenable to a treatment modality in which a therapeutic gene is directly delivered to the lung. Current gene delivery systems, either viral or nonviral, need further improvement in terms of efficiency and safety. We reported that nonionic amphiphilic block copolymers hold promise as nonviral gene delivery systems for transfection of muscular tissues. To evaluate the efficiency of these vectors in the lung, intratracheal instillation or aerosolization of reporter genes complexed with Lutrol or PE6400 was performed. Lutrol-DNA and, to a lesser extent, PE6400-DNA complexes promoted efficient gene transfection into mouse airways in a dose-dependent manner. This improvement over naked DNA was observed irrespective of the reporter gene. Lutrol enabled us to deliver significantly higher DNA amounts than current nonviral vectors, with even greater increases in gene expression and without the formation of colloidally unstable complexes. Time course studies showed that Lutrol-DNA complexes permitted prolonged gene expression for up to 5 days whereas with poly(ethylenimine) (PEI)-DNA polyplexes, expression peaked on days 1-2 postinstillation, was strongly reduced by day 5, and reached background levels on day 7. Aerosolized delivery of Lutrol-DNA complexes, a less invasive approach to deliver genes to the lung, gave 5- to 15-fold higher reporter gene expression compared with PEI-DNA polyplexes administered via the same delivery route. After intratracheal instillation of Lutrol-DNA complexes, histochemical staining for beta-galactosidase expression showed the presence of large blue areas. Histopathological analysis showed that Lutrol alone did not elicit inflammation, and that the inflammatory response after intratracheal instillation of Lutrol-DNA complexes was reversible and was observed only with the highest amounts of DNA. We also found that Lutrol can efficiently deliver genes to the airways of cystic fibrosis mice. Thus, we conclude that Lutrol is a highly promising vector for gene delivery to the lung.     

Characterisation and tumour targeting of PEGylated polylysine dendrimers bearing doxorubicin via a pH labile linker

Polylysine dendrimers have potential as biodegradable vectors for the delivery of cytotoxic drugs to solid tumours. Here, the cytotoxicity, drug release and tumour targeting properties of Generation 5 PEGylated polylysine dendrimers comprising an outer generation of l-lysine or succinimyldipropyldiamine (SPN) and containing doxorubicin (DOX) linked through an acid labile 4-(hydrazinosulfonyl) benzoic acid (HSBA) linker have been characterised. Less than 10% of the DOX load was released from LYS or SPN dendrimers in pH 7.4 buffer over 3 days. In contrast approximately 100% release was evident at pH 5. The DOX-conjugated dendrimers also retained similar cytotoxic properties to free DOX in in vitro cell culture studies (presumably as a result of in situ liberation of free DOX). The clearance patterns of the DOX conjugated SPN and all-lysine dendrimers were similar to the equivalent non-DOX conjugated systems, however the SPN dendrimers showed reduced metabolic lability and increased uptake into RES organs when compared to the equivalent all-lysine dendrimers. In vivo assessment of the DOX-conjugated, PEGylated polylysine dendrimers (both SPN and LYS constructs) in rats bearing Walker 256 tumours revealed higher uptake into tumour tissue when compared with control tissue such as muscle (~ 8 fold) and heart (~ 3 fold). The data suggest that polylysine dendrimers containing DOX conjugated via an acid labile HSBA linker may provide a mechanism to target the delivery of DOX to tumours.     

Tumor-specific gene delivery mediated by a novel peptide-polyethylenimine-DNA polyplex targeting aminopeptidase N/CD13

We have developed a novel polyethylenimine (PEI)-DNA vector formulation that is capable of efficient tumor-specific delivery after intravenous administration to nude mice. To further increase the specificity of delivery, we have attached the peptide CNGRC to the vector, which is specific for aminopeptidase N (CD13). The strategy for coupling this peptide to PEI was based on a novel method involving the strong affinity between phenyl(di)boronic acid (PDBA) and salicylhydroxamic acid (SHA) as well as a polyethylene glycol (PEG) linker to reduce steric hindrance between the vector and the peptide. In vitro assessment of targeting by the CNGRC/PEG/PEI/DNA vector carrying a beta-galactosidase (beta-Gal)-expressing plasmid showed as much as a 5-fold increase in transduction, relative to the untargeted PEG/PEI/DNA-betagal vector, of CD13-positive lung cancer, fibrosarcoma, bladder cancer, and human umbilical vein endothelial cells. Competition with free peptide resulted in up to a 90% reduction in delivery, indicating that gene delivery was specific for CD13-positive cells. Intravenous administration of the CNGRC/PEG/PEI/DNA-betagal vector to nude mice bearing subcutaneous tumors resulted in as much as a 12-fold increase in beta-Gal expression in tumors as compared with expression in either lungs or tumors from animals treated with the original PEI/DNA-betagal vector. In vivo transduction analysis using the CNGRC/PEG/PEI/DNA vector to target the intravenous delivery of a yellow fluorescence protein (YFP)-expressing plasmid to subcutaneous H1299 tumors confirmed delivery of YFP to both tumor cells and tumor endothelial cells. The use of this peptide to further increase tumor-specific delivery mediated by our novel PEI/DNA vector now provides a basis for developing tumor-targeted gene therapies for use in the clinical treatment of cancer.     

Molecular modeling and in vivo imaging can identify successful flexible triazine dendrimer-based siRNA delivery systems

This study aimed to identify suitable siRNA delivery systems based on flexible generation 2-4 triazine dendrimers by correlating physico-chemical and biological in vitro and in vivo properties of the complexes with thermodynamic parameters calculated using molecular modeling. The siRNA binding properties of the dendrimers and PEI 25 kDa were simulated, binding and stability were measured in SYBR Gold assays, and hydrodynamic diameters, zeta potentials, and cytotoxicity were quantified. These parameters were compared with cellular uptake of the complexes and their ability to mediate RNAi. Radiolabeled complexes were administered intravenously, and pharmacokinetic profiles and biodistribution of these polyplexes were assessed both invasively and non-invasively. All flexible triazine dendrimers formed thermodynamically more stable complexes than PEI. While PEI and the generation 4 dendrimer interacted more superficially with siRNA, generation 2 and 3 virtually coalesced with siRNA, forming a tightly intertwined structure. These dendriplexes were therefore more efficiently charge-neutralized than PEI complexes, reducing agglomeration. This behavior was confirmed by results of hydrodynamic diameters (72.0 nm-153.5 nm) and zeta potentials (4.9 mV-21.8 mV in 10 mM HEPES) of the dendriplexes in comparison to PEI complexes (312.8 nm-480.0 nm and 13.7 mV-17.4 mV in 10 mM HEPES). All dendrimers, even generation 3 and 4, were less toxic than PEI. All dendriplexes were efficiently endocytosed and showed significant and specific luciferase knockdown in HeLa/Luc cells. Scintillation counting confirmed that the generation 2 triazine complexes showed more than twofold prolonged circulation times as a result of their good thermodynamic stability. Conversely, generation 3 complexes dissociated in vivo, and generation 4 complexes were captured by the reticulo-endothelial system due to their increased surface charge. Molecular modeling proves very valuable for rationalizing experimental parameters based on the dendrimers' structural properties. Non-invasive molecular imaging predicted the in vivo fate of the complexes. Therefore, both techniques effectively promote the rapid development of safe and efficient siRNA formulations that are stable in vivo.     

In vivo tumor transfection mediated by polyplexes based on biodegradable poly(DMAEA)-phosphazene.

In recent years, increasing interest is being paid to the design of transfectants based on non-toxic and biodegradable polymers for gene therapy purposes. We recently reported on a novel, biodegradable polymer, poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) for use in non-viral gene delivery. In this study, the biodistribution and in vivo transfection efficiency of polyplexes composed of plasmid DNA and p(DMAEA)-ppz were investigated after intravenous administration in tumor bearing mice. Data were compared with those of polyplexes based on the non-biodegradable polyethylenimine (PEI 22kDa). Both polyplex systems were rapidly cleared from the circulation (<7% ID, at 60 min after administration) and showed considerable disposition in the liver and the lung, all in line with earlier work on cationic polyplex systems. The lung disposition is attributed to aggregates formed by interaction of the polyplexes with blood constituents. Redistribution of the polyplexes from the lung was observed for both polyplex formulations. Importantly, both polyplex systems showed a substantial tumor accumulation of 5% and 8% ID/g for p(DMAEA)-ppz and PEI22 polyplexes, respectively, at 240 min after administration. The tumor disposition of the p(DMAEA)-ppz and PEI22 polyplexes was associated with considerable expression levels of the reporter gene. In contrast to PEI22 polyplexes, p(DMAEA)-ppz polyplexes did not display substantial gene expression in the lung or other organs (organ gene expression<1/100 of tumor gene expression). The observed preferential tumor gene expression mediated by the p(DMAEA)-ppz polyplexes enables the application of this polymer to deliver therapeutic genes to tumors.     

Non-toxic phototriggered gene transfection by PAMAM-porphyrin conjugates

Development of controllable and non-toxic gene transfection systems is a core issue in gene therapy. Photochemical internalization, an innovative strategy in cytosolic release, provides us with an opportunity to develop a light-inducible gene delivery system. In this study, a novel photochemical internalization (PCI)-mediated gene delivery system was synthesized by surface modification of polyamidoamine (PAMAM) dendrimers via 5,10,15-tri(4-acetamidophenyl)-20-mono(4-carboxyl-phenyl)porphyrin (TAMCPP) conjugated to the generation 4 PAMAM dendrimer (G4). This water-soluble PAMAM-TAMCPP conjugate was characterized for cell viability, phototoxicity, DNA complexation, and in vitro transfection activity. The results show that TAMCPP conjugation did not increase the cytotoxicity of the PAMAM dendrimer below 20 μM, but significantly induced cell death after suitable irradiation. Under almost non-toxic G4-TAMCPP-mediated PCI treatment, the expression of green fluorescent protein determined by flow cytometry could be markedly enhanced in HeLa cells. Therefore, the G4-TAMCPP conjugate had an inducible and effective gene transfection activity, and showed considerable potential as a bimodal biomaterial for PCI-mediated gene therapy.     

Luciferin detection after intranasal vector delivery is improved by intranasal rather than intraperitoneal luciferin administration

In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which to continually measure expression of this marker gene after gene transduction. Its substrate, luciferin, is typically injected into the peritoneal cavity before bioimaging. Here we demonstrate that, compared with intraperitoneal injection, intranasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed after administration of viral vectors based on adenovirus type 5, adeno-associated virus type 8, and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after nonviral polyethylenimine (PEI)-DNA delivery. Detection increased relative to the concentration of luciferin; however, a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intraperitoneal injection, intranasal instillation yields about a 10-fold increase in sensitivity with an approximate 30-fold reduction in luciferin usage when bioimaging in the nasal and pulmonary airways of mice.     

Phase I study of liposome-DNA complexes encoding the interleukin-2 gene in dogs with osteosarcoma lung metastases

Systemic gene delivery using cationic liposome-DNA complexes (LDCs) has been shown to elicit potent antitumor activity in mice with tumor metastases to the lungs. However, intravenous gene delivery for treatment of established cancer has not been evaluated previously in a spontaneous, large animal model. We therefore evaluated the safety, toxicity, and efficacy of intravenous gene delivery, using LDCs in dogs with established tumor metastases. Twenty dogs with chemotherapy-resistant osteosarcoma metastases to the lungs received a series of intravenous infusions of cationic liposomes and plasmid DNA encoding the canine interleukin-2 (IL-2) cDNA. Effects of intravenous gene delivery on immune activation, clinical and hematologic parameters, tumor responses, and survival times were assessed. We found that slow intravenous administration of IL-2 LDCs resulted in detectable IL-2 transgene expression in lung tissues of dogs. Repeated intravenous infusions of LDCs were well tolerated by dogs with lung tumor metastases and elicited systemic immune activation, as reflected by fever, leukogram changes, monocyte activation, and increased natural killer cell activity. Three of 20 dogs experienced partial or complete regression of lung metastases after infusion of IL-2 LDCs. Overall survival times were significantly increased in treated dogs compared with historical control animals with the same stage of disease. We conclude that repeated intravenous infusion of LDCs in cancerbearing dogs is safe and well tolerated at low doses and may be capable of eliciting antitumor activity in some animals with advanced tumor metastases.     

Nanoparticles Modified With Tumor-targeting scFv Deliver siRNA and miRNA for Cancer Therapy

Targeted delivery of RNA-based therapeutics for cancer therapy remains a challenge. We have developed a LPH (liposome-polycation-hyaluronic acid) nanoparticle formulation modified with tumor-targeting single-chain antibody fragment (scFv) for systemic delivery of small interfering RNA (siRNA) and microRNA (miRNA) into experimental lung metastasis of murine B16F10 melanoma. The siRNAs delivered by the scFv targeted nanoparticles efficiently downregulated the target genes (c-Myc/MDM2/VEGF) in the lung metastasis. Two daily intravenous injections of the combined siRNAs in the GC4-targeted nanoparticles significantly reduced the tumor load in the lung. miRNA-34a (miR-34a) induced apoptosis, inhibited survivin expression, and downregulated MAPK pathway in B16F10 cells. miR-34a delivered by the GC4-targeted nanoparticles significantly downregulated the survivin expression in the metastatic tumor and reduced tumor load in the lung. When miR-34a and siRNAs were co-formulated in GC4-targeted nanoparticles, an enhanced anticancer effect was observed.     

Highly compacted DNA nanoparticles with low MW PEG coatings: In vitro, ex vivo and in vivo evaluation

Highly compacted DNA nanoparticles, composed of single molecules of plasmid DNA compacted with block copolymers of poly-l-lysine and 10 kDa polyethylene glycol (CK₃₀PEG_10k), mediate effective gene delivery to the brain, eyes and lungs in vivo. Nevertheless, we found that CK₃₀PEG_10k DNA nanoparticles are immobilized by mucoadhesive interactions in sputum that lines the lung airways of patients with cystic fibrosis (CF), which would presumably preclude the efficient delivery of cargo DNA to the underlying epithelium. We previously found that nanoparticles can rapidly penetrate human mucus secretions if they are densely coated with low MW PEG (2-5 kDa), whereas nanoparticles with 10 kDa PEG coatings were immobilized. We thus sought to reduce mucoadhesion of DNA nanoparticles by producing CK₃₀PEG DNA nanoparticles with low MW PEG coatings. We examined the morphology, colloidal stability, nuclease resistance, diffusion in human sputum and in vivo gene transfer of CK₃₀PEG DNA nanoparticles prepared using various PEG MWs. CK₃₀PEG_10k and CK₃₀PEG_5k formulations did not aggregate in saline, provided partial protection against DNase I digestion and exhibited the highest gene transfer to lung airways following inhalation in BALB/c mice. However, all DNA nanoparticle formulations were immobilized in freshly expectorated human CF sputum, likely due to inadequate PEG surface coverage.     

Dendrimers: relationship between structure and biocompatibility in vitro, and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo.

Dendrimers are highly branched macromolecules of low polydispersity that provide many exciting opportunities for design of novel drug-carriers, gene delivery systems and imaging agents. They hold promise in tissue targeting applications, controlled drug release and moreover, their interesting nanoscopic architecture might allow easier passage across biological barriers by transcytosis. However, from the vast array of structures currently emerging from synthetic chemistry it is essential to design molecules that have real potential for in vivo biological use. Here, polyamidoamine (PAMAM, Starburst), poly(propyleneimine) with either diaminobutane or diaminoethane as core, and poly(ethylene oxide) (PEO) grafted carbosilane (CSi-PEO) dendrimers were used to study systematically the effect of dendrimer generation and surface functionality on biological properties in vitro. Generally, dendrimers bearing -NH(2) termini displayed concentration- and in the case of PAMAM dendrimers generation-dependent haemolysis, and changes in red cell morphology were observed after 1 h even at low concentrations (10 microg/ml). At concentrations below 1 mg/ml CSi-PEO dendrimers and those dendrimers with carboxylate (COONa) terminal groups were neither haemolytic nor cytotoxic towards a panel of cell lines in vitro. In general, cationic dendrimers were cytotoxic (72 h incubation), displaying IC(50) values=50-300 microg/ml dependent on dendrimer-type, cell-type and generation. Preliminary studies with polyether dendrimers prepared by the convergent route showed that dendrimers with carboxylate and malonate surfaces were not haemolytic at 1 h, but after 24 h, unlike anionic PAMAM dendrimers they were lytic. Cationic 125I-labelled PAMAM dendrimers (gen 3 and 4) administered intravenously (i.v.) to Wistar rats ( approximately 10 microg/ml) were cleared rapidly from the circulation (<2% recovered dose in blood at 1 h). Anionic PAMAM dendrimers (gen 2.5, 3.5 and 5.5) showed longer circulation times ( approximately 20-40% recovered dose in blood at 1 h) with generation-dependent clearance rates; lower generations circulated longer. For both anionic and cationic species blood levels at 1 h correlated with the extent of liver capture observed (30-90% recovered dose at 1 h). 125I-Labelled PAMAM dendrimers injected intraperitoneally were transferred to the bloodstream within an hour and their subsequent biodistribution mirrored that seen following i.v. injection. Inherent toxicity would suggest it unlikely that higher generation cationic dendrimers will be suitable for parenteral administration, especially if they are to be used at a high dose. In addition it is clear that dendrimer structure must also be carefully tailored to avoid rapid hepatic uptake if targeting elsewhere (e.g. tumour targeting) is a primary objective.     

Aerosolized antibiotics for treating hospital-acquired and ventilator-associated pneumonia

Hospital-acquired pneumonia is a common complication that continues to have a poor cure rate in some patients with intravenous therapy alone. Aerosolized antibiotics are theoretically attractive in an attempt to optimize lung concentrations of antibiotics. Limited data suggest that aerosolized aminoglycosides or colistin in addition to intravenous therapy results in good response rates in patients with multidrug-resistant organisms or nonresponding pneumonia. Adverse events can occur, especially with colistin. When used, care should be taken to properly compound and administer aerosolized antibiotics to ensure tolerability and good drug delivery.     

Aerosolized antibiotics for treating hospital-acquired and ventilator-associated pneumonia

Hospital-acquired pneumonia is a common complication that continues to have a poor cure rate in some patients with intravenous therapy alone. Aerosolized antibiotics are theoretically attractive in an attempt to optimize lung concentrations of antibiotics. Limited data suggest that aerosolized aminoglycosides or colistin in addition to intravenous therapy results in good response rates in patients with multidrug-resistant organisms or nonresponding pneumonia. Adverse events can occur, especially with colistin. When used, care should be taken to properly compound and administer aerosolized antibiotics to ensure tolerability and good drug delivery.     

Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA

Matrix systems based on biocompatible and biodegradable polymers like the United States Food and Drug Administration (FDA)-approved polymer poly(DL-lactide-co-glycolide acid) (PLGA) are promising for the delivery of small interfering RNA (siRNA) due to favorable safety profiles, sustained release properties and improved colloidal stability, as compared to polyplexes. The purpose of this study was to design a dry powder formulation based on cationic lipid-modified PLGA nanoparticles intended for treatment of severe lung diseases by pulmonary delivery of siRNA. The cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) was incorporated into the PLGA matrix to potentiate the gene silencing efficiency. The gene knock-down level in vitro was positively correlated to the weight ratio of DOTAP in the particles, and 73% silencing was achieved in the presence of 10% (v/v) serum at 25% (w/w) DOTAP. Optimal properties were found for nanoparticles modified with 15% (w/w) DOTAP, which reduced the gene expression with 54%. This formulation was spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. The spray-drying process did not affect the physicochemical properties of the readily re-dispersible nanoparticles, and most importantly, the in vitro gene silencing activity was preserved during spray-drying. The siRNA content in the powder was similar to the theoretical loading and the siRNA was intact, suggesting that the siRNA is preserved during the spray-drying process. Finally, X-ray powder diffraction analysis demonstrated that mannitol remained in a crystalline state upon spray-drying with PLGA nanoparticles suggesting that the sugar excipient might exert its stabilizing effect by sterical inhibition of the interactions between adjacent nanoparticles. This study demonstrates that spray-drying is an excellent technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue.     

Alternative formulations,delivery methods,and administration options for psychotropic medications in elderly patients with behavioral and psychological symptoms of dementia

Objective: The purpose of this paper was to review alternative formulations, delivery methods, and administration options for psychotropic medications in elderly patients with behavioral and psychological symptoms of dementia (BPSD).Methods: A MEDLINE search was conducted initially in December 2008 and was updated in September 2009, including the search terms pharmacologic treatment and dementia, behavioral and psychological symptoms of dementia, alternative psychotropic medication formulations, alternative dosing methods of medication, drug delivery options, antidepressants and dementia, anxiolytics and dementia, antipsychotics and dementia, mood stabilizers and dementia, cognitive enhancers and dementia, medications and enteral feeding tubes, and hiding medication. Studies were limited to English-language articles dated from 1950 to 2009. Additional relevant articles were obtained by reviewing the references in the initial articles. Drug Facts and Comparisons 4.0 Online, Lexi-Comp Online, and Lexi-Drugs Online were used to obtain additional information. Targeted patients were elderly individuals with BPSD who were considered difficult to treat because they were unable to swallow, were refusing medications, or were not able to eat or drink per physician order.Results: In addition to the standard capsule or tablet given orally, a variety of formulations and delivery methods for psychotropic medications are available. Options include short- and long-acting intramuscular, intravenous, liquid, orally disintegrating, transdermal patch, sublingual, and rectal forms. Additionally, all formulations can be further altered in substance, delivery, or both. For example, tablets may be crushed and capsules opened; this changes their formulation and allows the option of mixing with food or liquids to be taken by mouth or through a tube. Caution must be used, however; in certain cases, alteration of the original form or the intended delivery method is contraindicated. In addition, many alternative administration options are not formally approved for use in the manner in which they are commonly applied and are therefore used with little or no information on tolerability and effectiveness. Ethical and legal issues include patient consent and off-label use.Conclusions: Overall, few studies have examined the use and efficacy of alternative psychotropic formulations and delivery methods in elderly patients with BPSD, and none have specifically addressed drug-alteration and alternative-administration issues. There is no evidence to compare alternative delivery forms (eg, tablet or capsule) of a given medication in terms of efficacy or tolerability. Still, alternative methods may be the only option for treatment of some patients. Practitioners must be familiar with the range of formulations and delivery options available so that they can optimize their patients' medication regimens. More data are needed on the use of alternative formulations, delivery methods, and administration options and their limitations in this population.     

Organic-inorganic hybrid-nanocarrier of siRNA constructing through the self-assembly of calcium phosphate and PEG-based block aniomer.

The development of siRNA delivery systems is a major key for practical RNA therapy that holds promise for the treatment of life-threatening human diseases, yet there still exists significant difficulties in their construction because of the various requirements including high transfection efficacy, tolerability in the biological medium, and low toxicity. Here we report the novel preparation route of organic-inorganic hybrid-nanocarriers entrapping siRNA based on the self-assembly of the block aniomer, poly(ethylene glycol)-block-poly(methacrylic acid), with calcium phosphate crystals. The nanocarriers have diameters in the range of several hundreds of nanometers and revealed excellent colloidal stability due to the steric stabilization effect of the PEG palisade. The biological activity of siRNA loaded in nanocarriers was assessed using 293 cells stably expressing luciferase gene, showing the remarkably high gene silencing-efficacy without the use of any adjuvant molecules such as chroloquin. Further advantage of the system is the serum tolerability, which is of a critical issue in in vivo application.     

Inhibition of pulmonary metastasis in mice by all-trans retinoic acid incorporated in cationic liposomes.

The purpose of this study was to investigate whether all-trans retinoic acid (ATRA), an active metabolite of retinal, incorporated in cationic liposomes composed of 1,2 dioleoyl-3-trimethylammonium propane (DOTAP)/cholesterol could inhibit established metastatic lung tumors by delivery to the pulmonary tumor site after intravenous injection. After intravenous injection in mice, the highest lung accumulation of [(3)H]ATRA was observed by the DOTAP/cholesterol liposomes formulation, while other formulations including [(3)H]ATRA dissolved in serum or [(3)H]ATRA incorporated in distearoyl-l-phosphatidylcholine (DSPC)/cholesterol liposomes produced little accumulation in the lung. In mice used as a model of lung cancer metastasis, ATRA incorporated in DOTAP/cholesterol liposomes, injected intravenously, reduced the number of tumor nodules compared with free ATRA or ATRA incorporated in DSPC/cholesterol liposomes. These results suggest that ATRA incorporated in cationic liposomes would be an effective strategy for differentiation therapy of lung cancer metastasis.     

Cationic polymers for gene delivery: designs for overcoming barriers to systemic administration

Cationic, polymer-based delivery systems have faced limitations in the systemic delivery of therapeutic gene drugs due to difficulties in formulation, in vivo stabilization, toxicity and low transfection efficiencies. Strategies for overcoming some of these barriers have utilized knowledge gained from the fields of colloidal stabilization and protein trafficking. This review highlights recent efforts in polycation preparations that include the development of new polymers for gene delivery, the modification of traditional polycations with hydrophilic polymers for salt and serum stability and the addition of bioactive functionalities to polymers for enhanced intracellular trafficking. These studies have resulted in polymer/DNA composites with increased stability and delivery efficiencies.     

Susceptibility of 40 Haemophilus ducreyi strains to 34 antimicrobial products

A study was performed to examine compounds that might improve the selectivity of the primary isolation medium for Haemophilus ducreyi. The susceptibility of 40 H. ducreyi strains to 34 antimicrobial agents, including 10 antibiotics, 3 quaternary ammonium compounds, 3 phenolic derivatives, 3 acridines, and 15 heavy metal compounds, was investigated by using an agar plate dilution technique. Results were compared with the susceptibilities of other gram-negative rods which may be contaminants on isolation media. The minimal inhibitory concentration range for colistin (16 to 128 micrograms/ml) indicated that this antibiotic might be of use as a selective agent. H. ducreyi was susceptible to spectinomycin (minimal inhibitory concentration range, 16 to 64 micrograms/ml), thiamphenicol (0.12 to 1 microgram/ml), chloramphenicol (0.12 to 0.5 micrograms/ml), and streptomycin (4 to 32 micrograms/ml) and moderately susceptible to kanamycin (2 to 8 micrograms/ml). For the heavy metal compounds, a high susceptibility was seen with copper(II) chloride (2 to 8 micrograms/ml, corresponding to a concentration of 0.75 to 3 micrograms of Cu2+ ions per ml), sodium selenite (1 to 4 micrograms/ml, or 0.45 to 1.83 micrograms of Se- ions per ml), and phenylmercury acetate (0.12 to 0.5 micrograms/ml). The minimal inhibitory concentrations of quaternary ammonium compounds, acridines, and phenolic derivatives were between 1 and 32 micrograms/ml, 8 and 32 micrograms/ml, and 8 and 250 micrograms/ml, respectively.