Co-delivery of curcumin and STAT3 siRNA using deformable cationic liposomes to treat skin cancer

Skin cancer is one of the most widely prevalent cancer types with over expression of multiple oncogenic signaling molecules including STAT3. Curcumin is a natural compound with effective anti-cancer properties. The objective of this work was to investigate the liposomal co-delivery of curcumin and STAT3 siRNA by non-invasive topical iontophoretic application to treat skin cancer. Curcumin was encapsulated in cationic liposomes and then complexed with STAT3 siRNA. The liposomal nanocomplex was characterized for particle size, zeta-potential, drug release and stability. Human epidermoid (A431) cancer cells were used to study the cell uptake, growth inhibition and apoptosis induction of curcumin-loaded liposome–siRNA complex. Topical iontophoresis was applied to study the skin penetration of nanocomplex in excised porcine skin model. Results showed that curcumin-loaded liposome–siRNA complex was rapidly taken up by cells preferentially through clathrin-mediated endocytosis pathway. The co-delivery of curcumin and STAT3 siRNA using liposomes resulted in significantly (p?<?.05) greater cancer cell growth inhibition and apoptosis events compared with neat curcumin and free STAT3 siRNA treatment. Furthermore, topical iontophoresis application enhanced skin penetration of nanocomplex to penetrate viable epidermis. In conclusion, cationic liposomal system can be developed for non-invasive iontophoretic co-delivery of curcumin and siRNA to treat skin cancer.     

The cationic liposomal adjuvants CAF01 and CAF09 formulated with the major outer membrane protein elicit robust protection in mice against a Chlamydia muridarum respiratory challenge

Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 10⁴ inclusion forming units (IFU) of C. muridarum. Following the challenge the mice were weighed daily. At 10 days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had significant C. muridarum-specific antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted robust cell mediated immune responses with high production of IFN-γ and IL17 and low levels of IL-4. Following the challenge the four groups of mice lost significantly less body weight than the MEM-immunized group. Lungs of mice vaccinated with CAF01, or CAF09, and nMOMP were significantly lighter than those from mice immunized using rMOMP. The number of IFU recovered from the lungs of mice vaccinated with CAF01, or CAF09, and nMOMP was similar to the number of IFU recovered from mice immunized with live EB. Mice that received rMOMP had significantly higher numbers of IFU than other groups. In conclusion, CAF01 and CAF09 elicited very robust protective humoral and cellular immune responses and were equally effective at adjuntavizing the C. muridarum MOMP. Mice vaccinated with nMOMP were significantly better protected than those immunized with rMOMP, indicative of the importance of the structural conformation of this antigen in protection.     

大鼠β-防御素-2基因气道转染增强肺抗感染防御功能的研究

β-防御素体外试验显示具广谱抗菌活性，本研究应用转基因方法评估其体内抗菌作用。构建大鼠β-防御素-2(rBD2)重组质粒pBK-CMV-rBD2和pCDNA-3．1-Myc-His( )-rBD2，通过脂质体包裹的方法将重组质粒经气管滴入，检测其在气管和肺组织表达情况，并应用肺组织匀浆上清菌落计数法检察对绿脓杆菌的肺清除率。结果显示在基因转染大鼠的气管和肺组织内均检测到rBD2-His融合蛋白基因mRNA和蛋白的表达，说明脂质体包裹的重组p防御素-2pBK-CMV-rBD2质粒可有效转染气道上皮组织。肺细菌清除率实验显示构建重组质粒pBK-CMV-rBD2气道转染与对照相比可显著提高肺组织对绿脓杆菌的清除率(n(8，p(0．01)．本研究表明β-防御素基因气道转染可提高呼吸道天然抗感染防御功能，可能在呼吸道感染的防治实践中具有潜在应用价值。     

Peptide-mediated Gene Transfer of Cationic Lipid/Plasmid DNA Complexes to Endothelial Cells

The purpose of this research is to develop ligand-targeted plasmid based gene delivery systems for gene transfer to tumor endothelium. Cell adhesion assays were used to test the peptide inhibition of human endothelial cell adsorption to vitronectin-treated tissue culture plates. A series of RGD containing peptides were tested in linear form and with one and two disulfide bonds. The linear and two disulfide bond peptides yielded similar IC₅₀ (≈1 × 10^-7 M). Substitution of two methionines for cysteines yielded a single disulfide bond that increased the IC₅₀ by 10-fold. The single and double disulfide peptides were derivatized to N-succinyl-dioleoylphopsphatidylethanolamine and incorporated into 100 nm liposomes radiolabeled with ³H-cholesterylhexadecylether. Liposome uptake by human umbilical vein endothelial cells was tested as a function of lipopeptide surface density. Increase in membrane surface density from 5 to 20 mol% increased human umbilical derived endothelial cell (HUVEC) uptake of the liposomes for both the single and double disulfide peptides. Liposome uptake by HUVECs was 3-fold greater for the double disulfide compared to the single disulfide. The single and double disulfide lipopeptides were then tested for gene transfer to HUVECs using DOTMA:Cholesterol cationic liposomes. The polyplexes were formed by rapidly mixing plasmid DNA with DOTMA:CHOL liposomes at a 3:1 charge ratio in 2% ethanol, 10% lactose. The ethanol was removed by lyophilization and upon rehydration, the lipoplexes had a mean diameter of approximately 100 nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20 mol% increased gene transfer by 10-fold. This increase in transfection could be reduced to that obtained in the absence of lipopeptide by co-incubating the HUVECs with a 100-fold excess of the single disulfide RGD peptide, thus demonstrating lipopeptide mediated gene transfer to endothelial cells.     

Hydrosoluble polymers for muscular gene delivery

Striated muscle tissue is an attractive target for gene delivery as it can be easily reached and can express exogenous proteins. However, administration of naked DNA results in low transfection levels, and the design and development of safe and efficient gene delivery systems are thus required.This review is focusing on the characteristics of the striated muscle tissue with regards to features possibly affecting gene transfer, as well as the different soluble polymers that have been evaluated as gene carriers. The described formulations are ranging from polymers displaying a high density of positive charges to non-ionic molecules. Nevertheless, polymers exhibiting few or no positive charges appear to our opinion as the most promising approach to achieve both safe and efficient transfection of the striated muscles.     

Lipid-based emulsion system as non-viral gene carriers

The genetic materials for systemic administration meet a number of huddles before they reach the nucleus of the target cells, such as enzymatic degradation in the bloodstream, extravascularization around the target tissue, endocytosis by the target cells, and endosomal escape of the genes. Therefore, there have been tremendous needs of effective gene carriers that can deliver the genetic materials to the target site. Of numerous approaches, recent studies have demonstrated that the lipid-based emulsion systems have the high potential as non-viral gene carriers: 1 lipid emulsions are biocompatible because their major constituents are composed of the non-toxic oils and amphiphilic lipids; 2 the cationic lipid emulsions can form nano-sized complexes with negatively charged DNAs, through which the genetic materials can be protected from the enzymatic degradation in the body fluids; 3 The emulsion/DNA complexes are shown to be stable in the bloodstream since their surfaces are rarely recognized by the immune-related cells and serum proteins; and 4 the surfaces of the emulsion complexes are readily modified by varying the lipid composition. In this review, highlighted are the recent advances in the emulsion-based gene carriers.     

The antibacterial activity of photodynamic agents against multidrug resistant bacteria causing wound infection

Antimicrobial photodynamic inactivation (aPDI) of multidrug-resistant (MDR) wound pathogens was evaluated with cationic porphyrin derivatives (CPDs). MDR bacterial strains including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae were used. The CPDs named PM, PE, PN, and PL were synthesized as a photosensitizer (PS). A diode laser with a wavelength of 655 nm was used as a light source. aPDI of the combinations formed with different energy densities (50, 100, and 150 J/cm²) and PS concentrations (ranging from 3.125 to 600 µM) were evaluated on each bacterial strain. Dark toxicity, cytotoxicity, and phototoxicity were determined on fibroblast cells. In the aPDI groups, survival reductions of up to 5.80 log₁₀ for E. coli, 5.90 log₁₀ for P. aeruginosa, 6.11 log₁₀ for K. pneumoniae, and 6.78 log₁₀ for A. baumannii were obtained. The cytotoxic effect of PL and PM on fibroblast cells was very limited. PN was the type of CPD with the highest dark toxicity on fibroblast cells. In terms of providing broad-spectrum aPDI without or with very limited cytotoxic effect, the best result was observed in aPDI application with PL. The other CPDs need some modifications to show bacterial selectivity for use at 50 µM and above.     

Cardiovascular toxicity assessment of poly (ethylene imine)- based cationic polymers on zebrafish model

Poly(ethylene imine)s (PEIs) have gained enormous attention in designing novel drug delivery systems for cancer treatment. High molecular weight of PEIs such as PEI 25 kD are promising for their drug carrying capacity. However, increased molecular weight is associated with toxicity. Currently, the toxicity evaluation of PEIs is mainly focused on the culture cell models, with very few studies investigating the risk assessment in vivo. Herein, the systemic evaluation of branched PEI 25 kD and PEI-CyD (PC) composed of low molecular PEI (Mw 600) and β-cyclodextrin (β-CyD) is performed in zebrafish model and endothelial cells. Our finding indicate that exposure of PC and PEI 25 kD can induce high mortality rate, shorten hatching time, promote malformations and cell apoptosis of zebrafish embryos in a dose-dependent manner. Most significantly, the cationic polymer PC and PEI 25 kD can decrease heart rate of zebrafish embryos and down-regulate the expression of heart development-related genes, which demonstrate their cardiovasculature toxicity. In this case, we further investigate the effect of PC on endothelial cells. Indeed, PC can induce endothelial cells dysfunction, including oxidative stress and apoptosis which are involved in cardiovascular diseases. These fundamental studies provide valuable insights into the biocompatible evaluation of PEI based drug carriers.     

Minimizing liver uptake of cationic Tc radiotracers with ether and crown ether functional groups

Ischemia-related diseases, particularly coronary artery disease (CAD), account for the majority of deaths worldwide. Myocardial ischemia is a serious condition and the delay in reperfusion of ischemic tissues can be life-threatening. This is particular true in the aged population. Rapid and accurate early detection of myocardial ischemia is highly desirable so that various therapeutic regiments can be given before irreversible myocardial damage occurs. Myocardial perfusion imaging with radiotracers is an integral component in evaluations of patients with known or suspected CAD. (99m)Tc-Sestamibi and (99m)Tc-Tetrofosmin are commercial radiopharmaceuticals currently available for myocardial perfusion imaging. Despite their widespread clinical applications, both (99m)Tc-Sestamibi and (99m)Tc-Tetrofosmin do not meet the requirements of an ideal perfusion imaging agent, largely due to their high liver uptake. The intense liver uptake makes it difficult to interpret the heart activity in the inferior and left ventricular wall. Photon scattering from the high liver radioactivity accumulation remains a significant challenge for diagnosis of heart diseases. This review will summarize the most recent research efforts to minimize the liver uptake of cationic (99m)Tc radiotracers by using ether and crown ether-containing chelators. Fast liver clearance will shorten the duration of imaging protocols (< 30 min post-injection), and allow for early acquisition of heart images with high quality. Improvement of heart/liver ratio may permit better detection of the presence and extent of coronary artery disease. Identification of such a new radiotracer that allows for the improved noninvasive assessment of myocardial perfusion would be of considerable benefit in treatment of patients with suspected CAD.     

Comparison of in vivo fate and immunogenicity of hepatitis B surface antigen incorporated in cationic and neutral liposomes

To compare cationic liposomes (CatL) and neutral liposomes (NeuL), as a vaccine carrier, the in vivo fate and immunogenicity of hepatitis B surface antigen (HBsAg), incorporated in CatL and NeuL, were investigated. CatL, composed of phosphatidyl choline (PC) and stearyl amine (SA) with a molar ratio of 9:1, showed a 2.5-fold higher incorporating efficiency of HBsAg than NeuL composed of PC alone. Most of HBsAg incorporated in both liposomes existed in an antibody-available form on the outer surface of liposomes. After intramuscular injection to rats, HBsAg in CatL resided at the injection site for a longer period than that in NeuL with terminal half lives of 52.5 and 42.9h, respectively. However, HBsAg in NeuL was more efficiently taken up by the lymphatic organs and spleen than that in CatL. Furthermore, the group treated with HBsAg in NeuL showed earlier sero-conversion with higher anti-HBsAg titre than the group treated with HBsAg in CatL. Sero-conversion rates (SCRs) in both CatL- and NeuL-treated animals were 100% after every injection carried out, except the primary injection of CatL. These results demonstrate that CatL can enhance the retention of incorporated antigen at the injection site, compared with NeuL. However, the production of antibody by HBsAg in NeuL is more effective than that by HBsAg in CatL, probably due tothe higher lymphatic targeting ability of NeuL. Thus, NeuL appears to be a better carrier for HBsAg than CatL.