A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study.

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120⁻290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.

Plasmid-Templated Control of DNA-Cyclodextrin Nanoparticle Morphology through Molecular Vector Design for Effective Gene Delivery.

Engineering self-assembled superstructures through complexation of plasmid DNA (pDNA) and single-isomer nanometric size macromolecules (molecular nanoparticles) is a promising strategy for gene delivery. Notably, the functionality and overall architecture of the vector can be precisely molded at the atomic level by chemical tailoring, thereby enabling unprecedented opportunities for structure/self-assembling/pDNA delivery relationship studies. Beyond this notion, by judiciously preorganizing the functional elements in cyclodextrin (CD)-based molecular nanoparticles through covalent dimerization, here we demonstrate that the morphology of the resulting nanocomplexes (CDplexes) can be tuned, from spherical to ellipsoidal, rod-type, or worm-like nanoparticles, which makes it possible to gain understanding of their shape-dependent transfection properties. The experimental findings are in agreement with a shift from chelate to cross-linking interactions on going from primary-face- to secondary-face-linked CD dimers, the pDNA partner acting as an active payload and as a template. Most interestingly, the transfection efficiency in different cells was shown to be differently impacted by modifications of the CDplex morphology, which has led to the identification of an optimal prototype for tissue-selective DNA delivery to the spleen in vivo.

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors.

The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%.

Nanoparticulated Polymeric Systems for Gene Delivery.

Gene therapy, currently, represents one of the new therapies that have emerged for the treatment of different diseases. Between the different approaches that can be chosen concerning this therapy, gene delivery with non-viral systems has focused the attention during the past decades, because of the reduced toxicity compared to the viral systems. Non-viral vectors are formed by a group of different molecules with a wide variety of sources and features. Among them, polymeric systems have been extensively studied due to the ability to form nanoparticles in the presence of nucleic acids, protect the included nucleic acid and more importantly, improve the entrance of the desired nucleic acid fragment into the cell and hopefully achieve a therapeutic effect. In this review, some of the most used polymeric systems are commented with the main characteristics that can influence the activity of each of them, such as the molecular weight, N/P ratio (positive charges of cationic polymer/negative charges of pDNA), stability or the molecular structure.

Host-Guest-Mediated DNA Templation of Polycationic Supramolecules for Hierarchical Nanocondensation and the Delivery of Gene Material.

Only a few examples of monodisperse molecular entities that can compact exogenous nucleic acids into nanocomplexes, protect the cargo from the biological environment, facilitate cell internalization, and promote safe transfection have been reported up to date. Although these species open new venues for fundamental studies on the structural requirements that govern the intervening processes and their application in nonviral gene-vector design, the synthesis of these moieties generally requires a relatively sophisticated chemistry, which hampers further development in gene therapy. Herein, we report an original strategy for the reversible complexation and delivery of DNA based on the supramolecular preorganization of a ß-cyclodextrin-scaffolded polycationic cluster facilitated by bisadamantane guests. The resulting gemini-type, dual-cluster supramolecules can then undergo DNA-templated self-assembly at neutral pH value by bridging parallel DNA oligonucleotide fragments. This hierarchical DNA condensation mechanism affords transfectious nanoparticles with buffering capabilities, thus facilitating endosomal escape following cell internalization. Protonation also destabilizes the supramolecular dimers and consequently the whole supramolecular edifice, thus assisting DNA release. Our advanced hypotheses are supported by isothermal titration calorimetry, NMR and circular dichroism spectroscopic analysis, gel electrophoresis, dynamic light scattering, TEM, molecular mechanics, molecular dynamics, and transfection studies conducted in vitro and in vivo.

Differential role of gene hypermethylation in adenocarcinomas, squamous cell carcinomas and cervical intraepithelial lesions of the uterine cervix.

Cervical cancer is the third most common cancer in women worldwide. The hypermethylation of P16, TSLC-1 and TSP-1 genes was analyzed in squamous cell carcinomas (SCC), cervical intraepithelial lesions (CIN) and adenocarcinomas (ADC) of the uterine cervix (total 181 lesions). Additionally human papillomavirus (HPV) type, EPB41L3, RASSF1 and RASSF2 hypermethylation were tested in ADC and the results were compared with those obtained previously by our group in SCC. P16, TSLC-1 and TSP-1 hypermethylation was more frequent in SCCs than in CINs. These percentages and the corresponding ones for EPB41L3, RASSF1 and RASSF2 genes were also higher in SCCs than in ADCs, except for P16. The presence of HPV in ADCs was lower than reported previously in SCC and CIN. Patients with RASSF1A hypermethylation showed significantly longer disease-free survival (P = 0.015) and overall survival periods (P = 0.009) in ADC patients. To our knowledge, this is the first description of the EPB41L3 and RASSF2 hypermethylation in ADCs. These results suggest that the involvement of DNA hypermethylation in cervical cancer varies depending on the histological type, which might contribute to explaining the different prognosis of patients with these types of tumors.

Evaluation of improved PAMAM-G5 conjugates for gene delivery targeted to the transferrin receptor.

The transfection activity of non-viral vectors is highly dependent on the delivery capacity of the carriers. Therefore, the aim of this work was to evaluate the activity of a new PAMAM dendrimer-Transferrin conjugate (P-Tf) with improved gene delivery activity to cancer cells. The formulations containing the novel P-Tf were able to bind pDNA and protect it from the activity of DNAse I enzyme. Moreover, it formed nanoparticles with positive surface charge, although the presence of Tf led to a decrease of the zeta potential to almost electroneutral values. This new vector, formulated at N/P 6, exhibited excellent transfection efficacy in HeLa, HepG2 and CT26 cell lines, whereas in Neuro2A no improvement was achieved. Compared to control complexes with branched polyethylenimine (bPEI), targeted dendriplexes (complexes formed by cationic polymeric dendrimers and DNA) were more efficient in HepG2 and HeLa cells. Cellular viability was always kept over 80% in these cell lines with higher values than bPEI control polyplexes. The uptake via receptor-mediated endocytosis was ensured by a competition assay, by adding an excess of free Tf, which led to a decrease in the transfection activity of targeted dendriplexes.

Efficient targeted gene delivery by a novel PAMAM/DNA dendriplex coated with hyaluronic acid.

AIM: To design and develop a novel target-specific DNA-delivery system using hyaluronic acid (HA)-polyamidoamine (PAMAM) conjugates (P-HA). MATERIALS & METHODS: The coupling of HA to the PAMAM dendrimer was analyzed by (1)H-NMR and elemental analysis (CHN). Their properties were characterized in terms of size and zeta-potential and evaluated for in vitro and in vivo transfection efficiency. RESULTS: The designed covalent HA-dendriplexes enhanced gene transfection of pCMV-Luc reporter gene in overexpressing CD44-receptor cancer cells. They were also more efficient in transfecting MDA-MB231 cells than conventional PEI-polyplexes. The cytotoxicity of the covalent HA-dendriplexes was lower than when using conventional polyethylenimine-polyplexes. In vivo studies showed that these targeted complexes were also efficient for delivering pCMVLuc in different organs of healthy mice, as well as in tumors of C57BL/6 animals. CONCLUSIONS: The HA-dendriplexes developed in this work may offer an advantageous alternative to conventional cationic polymer-based formulations for DNA delivery into cancer cells in an efficient and safe manner.

Differential gene hypermethylation in genital lichen sclerosus and cancer: a comparative study.

AIMS: Lichen sclerosus (LS) is a chronic inflammatory disease of the genital skin of unknown aetiology. The role of LS in penile squamous cell carcinogenesis is not well characterized. HPV has been implicated in both, as have epigenetic changes. The presence of HPV and hypermethylation of the MGMT, p16, RASSF1, RASSF2, TSLC1 and TSP1 genes were studied in penile LS; MGMT, RASSF2 and TSLC1 hypermethylation in penile cancer and TSLC1 hypermethylation in vulvar LS and cancer extends previous results reported by our group. METHODS AND RESULTS: Thirty-seven HPV genotypes and hypermethylation were evaluated by PCR/reverse-line-blot and methylation-specific PCR respectively, in 27 preputial LS, 24 penile SCC, 30 vulvar SCC, 21 vulvar LS and 22 normal skin cases. HPV66 was present in 3.7% of penile LS cases, and p16 and RASSF2 hypermethylation were more frequent in penile cancer than in penile LS. p16, RASSF1, RASSF2 and TSP1 hypermethylation were similar in penile and vulvar LS. CONCLUSIONS: Gene hypermethylation is a common event in penile LS, and occurs approximately as frequently as in vulvar LS. Certain genes can be hypermethylated as an early or late event in LS or cancer, respectively. This suggests a possible sequential role for these alterations in the transition from benign to malignant lesions.

RASSF2 hypermethylation is present and related to shorter survival in squamous cervical cancer.

Ras association (RalGDS/AF-6) domain family member 2 (RASSF2) is a gene involved in the progression of several human cancers, including breast, colorectal and lung cancer. The aims of this study were to determine the hypermethylation of the gene in squamous cervical cancer and precursor lesions, along with that of RASSF1 and the recently described EPB41L3, and to analyze the potential prognostic role of these genes. Methylation-specific PCR and bisulfite sequencing were used to analyze the methylation status of RASSF2 and EPB41L3 gene in 60 squamous cervical cancer, 76 cervical intraepithelial neoplasias grade III, 16 grade II, 14 grade I and 13 cases of normal tissue adjacent to cervical intraepithelial neoplasia. RASSF2 expression was evaluated by immunohistochemistry and the re-expression of RASSF2 and EPB41L3 was analyzed by quantitative reverse-transcription PCR in HeLa, SiHa, C33A and A431 cell lines treated with 5-aza-2'-deoxycytidine and/or trichostatin. RASSF1 hypermethylation and human papillomavirus type were also analyzed in all the cases by methylation-specific PCR and reverse line blot, respectively. RASSF2 hypermethylation was predominant in squamous cervical cancer (60.9%) compared with cervical intraepithelial neoplasias (4.2%) and was associated with a lower level of RASSF2 expression and vascular invasion in squamous cervical cancer. EPB41L3 and RASSF1 hypermethylations were also more frequent in cancer than in precursor lesions. Patients with RASSF2 hypermethylation had shorter survival time, independent of tumor stage (hazard ratio: 6.0; 95% confidence interval: 1.5-24.5). Finally, the expressions of RASSF2 and EPB41L3 were restored in several cell lines treated with 5-aza-2'-deoxycytidine. Taken together, our results suggest that RASSF2 potentially functions as a new tumor-suppressor gene that is inactivated through hypermethylation in cervical cancer and is related to the bad prognosis of these patients.