Biodegradable, cationic methacrylamide-based polymers for gene delivery to ovarian cancer cells in mice

A series of cationic, methacrylamide polymers was tested for use as a biodegradable gene carrier in ovarian cancer. Tumor transfection activity of polyplexes consisting of a reporter gene and different methacrylamide polymers was assessed, after intraperitoneal injection in mice bearing an ovarian cancer xenograft. In this model, polyplexes based on poly(HPMA-DMAE) showed transfection activity similar to polyplexes based on the nondegradable and rather toxic polyethylenimine (PEI22). The tumor transfection activity of the pHPMA-DMAE polyplexes was remarkable considering their poor transfection activity in in vitro assays. Polyplexes based on pHPMA-DMAE were devoid of any cytotoxicity and mediated highest transfection activity at the highest N/P ratio investigated. Tumor cell gene expression after a single administration of these polyplexes rapidly declined within time, at a similar rate to that observed after injection with polyplexes based on PEI22. Incubation of the polyplexes with hyaluronic acid (HA), a polyanion accumulating in the ascitic fluid of ovarian cancer bearing mice, changed the physical characteristics of the pHPMA-DMAE and PEI22 polyplexes. The transfection activity of PEI22-based polyplexes, but not that of pHPMA-DMAE based polyplexes, was strongly impaired by HA. Differences in HA sensitivity might have contributed to the in vivo gene expression activities of pHPMA-DMAE- and PEI22-based polyplexes. pHPMA-DMAE-based polyplexes have potential for use in ovarian cancer therapy due to their considerable transfection activity, their low cytotoxicity, and their HA resistance.     

Photocytotoxicity of mTHPC (Temoporfin) Loaded Polymeric Micelles Mediated by Lipase Catalyzed Degradation

PURPOSE: To study the in vitro photocytotoxicity and cellular uptake of biodegradable polymeric micelles loaded with the photosensitizer mTHPC, including the effect of lipase-catalyzed micelle degradation. METHODS: Micelles of mPEG750-b-oligo(epsilon-caprolactone)5 (mPEG750-b-OCL5) with a hydroxyl (OH), benzoyl (Bz) or naphthoyl (Np) end group were formed and loaded with mTHPC by the film hydration method. The cellular uptake of the loaded micelles, and their photocytotoxicity on human neck squamous carcinoma cells in the absence and presence of lipase were compared with free and liposomal mTHPC (Fospeg). RESULTS: Micelles composed of mPEG750-b-OCL5 with benzoyl and naphtoyl end groups had the highest loading capacity up to 30% (w/w), likely due to pi-pi interactions between the aromatic end group and the photosensitizer. MTHPC-loaded benzoylated micelles (0.5 mg/mL polymer) did not display photocytotoxicity or any mTHPC-uptake by the cells, in contrast to free and liposomal mTHPC. After dilution of the micelles below the critical aggregation concentration (CAC), or after micelle degradation by lipase, photocytotoxicity and cellular uptake of mTHPC were restored. CONCLUSION: The high loading capacity of the micelles, the high stability of mTHPC-loaded micelles above the CAC, and the lipase-induced release of the photosensitizer makes these micelles very promising carriers for photodynamic therapy in vivo.     

Well-defined and potent liposomal meningococcal B vaccines adjuvated with LPS derivatives

Potent liposomal PorA formulations containing various lipopolysaccharide (LPS) derivatives were developed. The following adjuvants were compared: the commonly used aluminum phosphate (AlPO(4)), and three LPS like adjuvants: monophosphoryl lipid A (MPL), lipopolysaccharide (galE LPS) and the less toxic LPS mutant lpxL1. The immunogenicity in mice was evaluated and compared with that against an outer membrane vesicle (OMV) vaccine. The IgG isotype distribution and bactericidal activity were determined. Furthermore, PorA specific proliferation of lymph node cells after immunization and restimulation in vitro was studied with selected formulations. Both AlPO(4) and MPL were unable to improve the functional immunogenicity (i.e. bactericidal response) of liposomal PorA. Besides, when these adjuvants were used, the percentage of responders in the groups did not reach 100%. This was also observed with non adjuvated PorA-liposomes or OMV. Of the adjuvants studied, only galE LPS and lpxL1 LPS were capable of increasing the immunogenicity and avoid non responsiveness against PorA-liposomes. Importantly, the adjuvant activity of lpxL1 LPS was accompanied by an improved PorA specific proliferation of lymph node cells and a concomitant increase in IL-2 production. In conclusion and considering its lower toxicity, lpxL1 LPS adjuvated liposomes are superior to other formulations tested.     

A step-by-step approach to study the influence of N-acetylation on the adjuvanticity of N,N,N-trimethyl chitosan (TMC) in an intranasal nanoparticulate influenza virus vaccine

Recently we reported that reacetylation of N,N,N-trimethyl chitosan (TMC) reduced the adjuvant effect of TMC in mice after intranasal (i.n.) administration of whole inactivated influenza virus (WIV) vaccine. The aim of the present study was to elucidate the mechanism of this lack of adjuvanticity. Reacetylated TMC (TMC-RA, degree of acetylation 54%) was compared with TMC (degree of acetylation 17%) at six potentially critical steps in the induction of an immune response after i.n. administration in mice. TMC-RA was degraded in a nasal wash to a slightly larger extent than TMC. The local i.n. distribution and nasal clearance of WIV were similar for both TMC types. Fluorescently labeled WIV was taken up more efficiently by Calu-3 cells when formulated with TMC-RA compared to TMC and both TMCs significantly reduced transport of WIV over a Calu-3 monolayer. Murine bone-marrow derived dendritic cell activation was similar for plain WIV, and WIV formulated with TMC-RA or TMC. The inferior adjuvant effect in mice of TMC-RA over that of TMC might be caused by a slightly lower stability of TMC-RA-WIV in the nasal cavity, rather than by any of the other factors studied in this paper.     

非穿透性小梁切除术联合人羊膜植入临床观察

目的：观察非穿透性小梁切除术联合人羊膜植入的临床效果。方法：对18例（30眼）中、晚期开角型青光眼进行非穿透性小梁切除术联合人羊膜植入，术后观察视力、视野、眼压及滤过泡形成情况。结果：术后随访3－9月，视力、视野稳定，眼压≤20mmHg(1mmHg=0.133kPa)，滤过泡形成良好，并发症少。结论：非穿透性小梁切除术 联合人羊膜植入治疗开角型青光眼，能有效降低眼压，形成功能性滤过泡和并发症少等优点，是治疗开角型青光眼较理想的方法。     

Renal targeting of kinase inhibitors

Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tubular cells and reduced unwanted systemic effects. In our approach kinase inhibitors are linked to the renal carrier lysozyme using a platinum-based linker that binds drugs via a coordinative linkage. Many kinase inhibitors contain aromatic nitrogen atoms able to bind to this linker without the need of prior derivatization. The resulting drug-lysozyme conjugates are rapidly filtered in the glomerulus into the tubular lumen and subsequently reabsorbed via the endocytic pathway for clearance of low-molecular weight proteins. An important property of the formed conjugates is their in vivo stability and the sustained drug release profile within target cells. This review summarizes the state-of-the-art of drug targeting to the kidney. Furthermore, we will highlight recent results obtained with kinase inhibitor-lysozyme conjugates targeted to different kinases, i.e. the transforming growth factor (TGF)-beta-receptor kinase, p38 MAPkinase and Rho-associated kinase. Both in vitro and in vivo results demonstrated their efficient tubular uptake and beneficial therapeutic effects, superior to treatment with free kinase inhibitors. These proof-of-concept studies clearly indicate the feasibility of drug targeting for improving the renal specificity of kinase inhibitors.     

A versatile family of degradable non-viral gene carriers based on hyperbranched poly(ester amine)s.

A variety of degradable hyperbranched poly(ester amine)s containing primary, secondary and tertiary amino groups, were synthesized and evaluated as non-viral gene carriers. The polymers were obtained in high yields through a Michael-type conjugate addition of diacrylate monomers with trifunctional amine monomers. Analysis of degradation products using liquid chromatography-mass spectroscopy (LC-MS) demonstrated that all poly(ester amine)s had a hyperbranched structure with a degree of branching of approximately 0.30. These poly(ester amine)s were readily water-soluble and degradable under physiological conditions (pH 7.4, 37 degrees C), in which more than 10% ester bonds were hydrolyzed within 4 h. Moreover, these hyperbranched poly(ester amine)s showed high buffering capacities between pH 5.1 and 7.4. Three out of nine synthesized polymers, i.e. p(HDDA-AEP), p(HDDA-AMP), and p(BDDA-AMP), were shown to effectively condense plasmid DNA into small-sized (approximately 94-135 nm) and positively charged complexes. Polymer/DNA complexes ('polyplexes') based on these three polymers, and larger complexes of p(BDDA-AEP) (approximately 497 nm) were able to transfect COS-7 cells in vitro. Importantly, the transfection activity of polyplexes was preserved in the presence of serum proteins. The highest transfection level was observed for p(HDDA-AEP) polyplex which had a transfection efficiency higher than or comparable to that polyplexes of polyethylenimine (PEI) and poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA). Furthermore, these poly(ester amine)s revealed no or low cytotoxicity. These results demonstrated that hyperbranched poly(ester amine)s can be applied as safe and efficient gene delivery polymers.