Effective intracellular delivery of bevacizumab via PEGylated polymeric nanoparticles targeting the CD44v6 receptor in colon cancer cells.

Colorectal cancer (CRC) is one of the most common and deadly cancers in the world, mainly due to its metastatic and metabolic ability. The CD44 receptor isoform containing exon 6 (CD44v6) is a transmembrane protein that plays an important role in the establishment of tumors and metastasis, which make this molecule a potential target for therapy and diagnosis of tumors. Aiming at a targeted therapy, the anti-VEGF monoclonal antibody (mAb) bevacizumab was loaded into poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) functionalized with an antibody fragment (Fab) specific for CD44v6-expressing human cancer cells. The sizes of NPs were in the range of 150-250 nm and they had a negative charge between -5 and -10 mV, with an association efficiency (AE) of bevacizumab of 86%. v6 Fab-PLGA-PEG NPs containing bevacizumab specifically bonded to the CD44v6 cell surface receptor and exhibited higher internalization into CD44v6+ epithelial cells than bare and (-) Fab-PLGA-PEG NPs. To understand the biological effect of NP targeting, the intracellular levels of bevacizumab and VEGF were evaluated after the incubation of targeted and untargeted NPs. The intracellular levels of bevacizumab were significantly higher in cells incubated with v6 Fab-PLGA-PEG NPs and these NPs resulted in a significant decrease in the intracellular VEGF compared to untargeted NPs and free bevacizumab. PLGA-PEG NPs, surface-functionalized with a v6-specific Fab, have the potential to intracellularly deliver bevacizumab into CD44v6 expressing cancer cells.

Nanotechnology-based siRNA delivery strategies for metastatic colorectal cancer therapy.

Colorectal cancer (CRC) is the second most common cause of cancer-related death in the world, mainly due to distant metastases events. The metastatic CRC (mCRC) stages are resistant to the therapeutic recommended. Therefore, it urges the development of more efficient strategies to impair the disease. Small interfering RNA (siRNA) is a well-known silencing tool with impact on targeted cancer therapy, even though in vivo challenges difficult its clinical application. Here, multiple solutions to overcome the extracellular and intracellular barriers encountered by intravenous delivery of siRNA are discussed. Moreover, it is emphasized the association of siRNA with nanoparticles to enable the long-term release and to counteract the toxicity issues of siRNA. Particular attention is paid on the potential of poly(lactic-co-glycolic acid) (PLGA) nanoparticles for systemic siRNA delivery in mCRC. Despite of being less used so far due to technological difficulties, multiple strategies to successfully encapsulate siRNA into PLGA nanocarriers are summarized.

Antibacterial activity of propolis extracts from the south of Portugal.

To examine the antibacterial activity of diverse extracts of propolis harvested at winter and spring from several locations of Algarve, Portugal, against Gram-negative and Gram-positive bacteria was the main goal of the present work. For such, the antibacterial activity was determined by agar diffusion. The results showed that all tested bacterial strains showed susceptibility to diluted propolis extracts and in a dose-dependent manner. Two propolis samples collected at springtime showed higher antibacterial activity, in comparison with samples harvested at wintertime. Ethanolic and methanolic extracts have a very similar activity (P<0.05). Helicobacter pylori strains J99 and 26695 were the most susceptible strains to the tested extracts (33.67±2.52 mm and 35.67±0.58mm, respectively). This study constitutes the first approach of the biological activities of Portuguese propolis from the Algarve region and evidences its potential use to combat bacterial infections, in particular against the gastric pathogen H. pylori.

Enhancement of chitosan-mediated gene delivery through combination with phiC31 integrase.

Gene transfer efficiency and expression stability are key factors to a successful gene therapy approach. In the present work we have developed a combined system for gene transfer that integrates well established non-viral polymeric vectors based on chitosan particles with the properties of phiC31-integrase that promotes a relatively non-immunogenic, site-specific integration, with sustained gene expression. Simultaneously, to overcome one of the major limitations in adeno-associated virus mediated gene transfer--the delivery of large genes--we have tested the capacity of our non-viral vectors to incorporate a large (8 Kb) transgene. Polyplexes were extensively characterized for their size, surface charge, morphology, pDNA complexation, transfection efficiency and transgene expression in vitro using HEK293 cells. Co-transfection with integrase was done by complexation in a single polyplex preparation or the use of two separate polyplex preparations. Transgene expression, GFP and CEP290 (1Kb and 8 Kb, respectively), was evaluated by fluorescence microscopy, flow cytometry and Western blot analysis. DNA complexation efficiency, particle size and morphology were consistent with gene delivery for all formulations. In contrast, transfection efficiency and transgene expression varied with polymer and polyplex size. Following delivery by chitosan polyplexes, high levels of GFP expression were still visible 16 weeks post-transfection and over-expression of the large transgene was detected at least 6 weeks post-transfection. Polyplexes incorporating phiC1 integrase demonstrate prolonged gene expression of both small (GFP, 1 Kb) and large genes (CEP290, 8Kb). This approach, using a combined strategy of polymers and integrase may overcome the size limitation found in commonly used adeno-associated virus mediated gene transfer techniques, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for gene delivery.

Cationic polyene phospholipids as DNA carriers for ocular gene therapy.

Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approach. The eye is a good target due to its small size, minimal diffusion of therapeutic agent to the systemic circulation, and low immune and inflammatory responses. Currently, most approaches are based on viral vectors, but efforts continue towards the synthesis and evaluation of new nonviral carriers to improve nucleic acid delivery. Our objective is to evaluate the efficiency of novel cationic retinoic and carotenoic glycol phospholipids, designated C20-18, C20-20, and C30-20, to deliver DNA to human retinal pigmented epithelium (RPE) cells. Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compounds and coformulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol). Addition of DNA to the liposomes formed lipoplexes, which were characterized for binding, size, biocompatibility, and transgene efficiency. Lipoplex formulations of suitable size and biocompatibility were assayed for DNA delivery, both qualitatively and quantitatively, using RPE cells and a GFP-encoding plasmid. The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable to the known lipid references 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice. The results demonstrate that cationic polyene phospholipids have potential as DNA carriers for ocular gene therapy.