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Aims: To develop an appropriate liposomal formulation for gene delivery against primary effusion lymphoma (PEL), a herpesvirus HHV8-associated B-cell lymphoma., Materials & Methods: Cationic, cationic PEGylated and cationic PEGylated anti-CD138 liposomes (ILp) linking a monoclonal antibody expressed on PEL cells were prepared by a thin layer evaporation method, followed by extrusion technique. The formulations were mixed with a model oligonucleotide to form the lipoplexes and tested on a BCBL-1 cell (a PEL cell line). The transfection efficiency was evaluated by flow cytometry and confocal laser scanning microscopy analysis., Results: Based on antigen-antibody interaction, ILp mediated a specific gene delivery as shown by a significant increase in the transfection rate and a localized internalization of the oligonucleotide, in comparison with cationic liposomes and cationic PEGylated liposomes., Conclusion: ILp could be proposed as effective carriers for oligonucleotide transfer in BCBL-1 cells. In vitro experimental results encourage us to further test the in vivo therapeutic potential of ILp for specific delivery of antitumoral agents.

Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical-chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers., (Copyright 2010 Elsevier B.V. All rights reserved.)

This study aims to evaluate the effects of the two most widely used glycosaminoglycans (dermatan sulphate and heparin) on both the structural and biological properties of collagen-based modified membranes (COL/GAGs membranes) designed for tissue engineering. The molecular weight of dermatan sulphate and heparins was correlated with the membrane feasibility and the cell (fibroblasts and keratinocytes) ability to adhere and proliferate on the COL/GAG membranes. Microstructure and physico-chemical properties of COL/GAGs membranes were examined using scanning electron microscopy and differential scanning calorimetry; the free amino group content and the swelling properties were also detected. The morphology, proliferation and growth behaviour of keratinocytes and fibroblasts were investigated using microscopical approach and in vitro colorimetric assay. Both fibroblasts and keratinocytes are able to grow and proliferate on COL/dermatan sulphate membranes. Fibroblasts revealed significantly higher proliferation on the membranes prepared with heparin if compared to the proliferation on the membrane without heparin (COL membrane). Particularly, a combination of the membranes formulated adding high molecular weight dermatan sulphate and high molecular weight heparin could be suitable to be used as biomaterials for epidermal substitute.

The pharmaceutical treatment of central nervous system (CNS) disorders is the second largest area of therapy, following cardiovascular diseases. Nowadays, noninvasive drug delivery systems for CNS are actively studied. The development of these new delivery systems started with the discovery that properly surface-engineered colloidal vectors, and in particular liposomes and polymeric nanoparticles, with a diameter approximately 200nm, were shown to be able to cross the blood-brain barrier (BBB) without apparent damage, and to deliver drugs or genetic materials into the brain. However, even if this ability was confirmed by confocal microscopy and measured by biodistribution experiments or by means of the pharmacological effect exerted by the embedded drugs, a clear understanding of the main characteristics of the colloidal systems that are important for BBB crossing is still lacking. It is also shown that the presence of the drug is able to modify the surface of these systems, with unpredictable results on the colloidal systems biodistribution; thus, the results obtained in the absence of the loaded drug have to be taken cautiously. Moreover, since the loaded drug is only a fraction of the colloidal system that is administered, the presence of the carrier in the body and into CNS, especially in the case of long-term therapies, might cause adverse effects not yet fully understood. Thus, even if promising results have been obtained, and some colloidal systems loaded with a drug are the US Food and Drug Administration (FDA) approved for human use (but not for brain targeting), a long way of research has to be done in order to use these drug delivery systems for the treatment of CNS pathologies., (Copyright 2009 Elsevier B.V. All rights reserved.)

Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.

The relatively hydrophilic bile acid, ursodeoxycholic acid (UDCA), was used as an additive to DOTAP cationic liposomes to evaluate the effect on the cellular uptake of an oligonucleotide. Nuclear magnetic resonance studies were applied to estimate the relative amount of incorporated UDCA into the lipidic bilayers. DOTAP or DOTAP-UDCA vesicles (MixVes; DOTAP/UDCA molar ratios 1:0.25, 1:0.5, 1:1, and 1:2) formed complexes with 5'-fluorescein conjugated 29-mer phosphorothioate oligonucleotides (PS-ODNs) and studied using gel electrophoresis. In addition, the complexes were tested after transfection to assess the cellular uptake and the localization of the oligo in a HaCaT cell line by the use of cytofluorimetric and confocal microscopic analysis. DOTAP lipid formulated in the presence of a defined amount of UDCA forms more stable, flexible, and active MixVes. In particular, the MixVes at 1:0.25 and 1:0.5 molar ratios increase and modify the cellular uptake of PS-ODNs if compared with DOTAP liposomes 3 hours after the transfection studies. Moreover, the in vitro data suggest that these new formulations are not toxic.

The aim of this work was to develop a novel strategy for the formulation of biodegradable PLA microspheres as delivery systems for proteins or peptides. The strategy is based on the exploitation of the gel-sol transition of the thermoreversible Pluronic F127 gel. The gel allows the formation of the particles without be co-entrapped in the matrix. The microspheres prepared using the novel technique (TG-Ms, or thermoreversible gel-method microspheres) were characterized in vitro (as concerns the size, the morphology, the protein encapsulation, the release and the protein distribution in the polymer matrix), in comparison with microspheres prepared using the classical double emulsion/solvent evaporation method (w/o/w-Ms). Two types of bovine serum albumin (BSA), with different water solubility, were used as model proteins. TG-Ms exhibited small size (7-50 m) and high protein content (8.6%, w/w) regardless of the BSA water solubility, in contrast with w/o/w-Ms, which revealed a size range of 100-130 microm and a protein content related to the BSA water solubility. TG-Ms, in spite of their smaller size respect of the w/o/w-Ms, displayed a reduced initial burst effect and a higher rate in the second release phase that resulted in a quasi-constant profile. The release behavior of the TG-Ms may be attributable to both the localization of the protein in the particle core, as shown by the confocal laser scanning microscopy analysis on labeled-BSA loaded microspheres, and the few pores in the matrix, as shown by the scanning electron microscopy. A working hypothesis about the mechanism of the particle formation was also discussed.

Vegetable cells are unusual findings in Papanicolaou-stained cervical smears; these structures could be wrongly mistaken for abnormal human cells, worm eggs, or spores by a cytologist encountering the possibility of meeting those elements in cytological analysis. Five cervicovaginal smears showing similar vegetable cells have been detected over a 3-yr period (2002-2004) in the course of a population screening program for cancer of the uterine cervix in Modena (Italy) involving 32,500 women. According to the clinical histories of the patients, the vaginal pharmaceutical drugs or appliances used were of different types: vaginal lavages, pessaries, and vaginal creams. Following a careful investigation, the only substance that can lead to vegetal elements has been identified as polysaccharide galactomannan, which is one of the excipient present in the drugs used. The authors have identified the origin of these contaminants and the means of pollution, using cytological and pharmaceutical investigation.

The direct evaluation of the heterogeneity of the particle population of nanometric drug delivery systems as liposomes is difficult to achieve owing to the dimension and the carrier characteristics. The influence of the lipidic ratio and composition on the physical stability of liposomes during their storage was investigated using atomic force microscopy (AFM) and photon correlation spectroscopy (PCS). Liposomes were made by a mixture of different lipids and obtained using distinct methods of preparation. AFM images, acquired immediately after the deposition of the sample on mica surface, clearly showed the spherical shape of the lipidic vesicles. In all the 7 months of the experiment, the average sizes of the different liposomes evaluated using the two techniques were comparable. According to PCS analysis, AFM images confirmed that almost all the diversified vesicular systems tended to form aggregates during their storage; this loss of stability was strengthened by the increase of polydispersity index value. The different behaviours observed were to ascribe to the lipidic composition more than the methods of liposome preparation. In conclusion, AFM technique owing to the relative simplicity cold be useful for the technological control of size distribution profile according to the preparative factors and moreover to the batch-to-batch reproducibility.