Deborah Michel

Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

BackgroundGene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency.ResultsNanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression.ConclusionAmino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the unsubstituted parent gemini surfactant. Glycyl-lysine substitution in the gemini spacer improved buffering capacity and imparted a pH-dependent increase of particle size. This property conferred to the P/12-7NGK-12/L nanoparticles the ability to escape efficiently from clathrin-mediated endosomes. Balanced binding properties (protection and release) of the 12-7NGK-12 in the presence of polyanions could contribute to the facile release of the nanoparticles internalized via caveolae-mediated uptake. A more efficient endosomal escape of the P/12-7NGK-12/L nanoparticles lead to higher gene expression compared to the parent gemini surfactant.

Amino Acid-Substituted Gemini Surfactant-Based Nanoparticles as Safe and Versatile Gene Delivery Agents

Gene based therapy represents an important advance in the treatment of diseases that heretofore have had either no treatment or cure. To capitalize on the true potential of gene therapy, there is a need to develop better delivery systems that can protect these therapeutic biomolecules and deliver them safely to the target sites. Recently, we have designed and developed a series of novel amino acid-substituted gemini surfactants with the general chemical formula C(12)H(25) (CH(3))(2)N(+)-(CH(2))(3)-N(AA)-(CH(2))(3)-N(+) (CH(3))(2)-C(12)H(25) (AA= glycine, lysine, glycyl-lysine and, lysyl-lysine). These compounds were synthesized and tested in rabbit epithelial cells using a model plasmid and a helper lipid. Plasmid/gemini/lipid (P/G/L) nanoparticles formulated using these novel compounds achieved higher gene expression than the nanoparticles containing the parent unsubstituted compound. In this study, we evaluated the cytotoxicity of P/G/L nanoparticles and explored the relationship between transfection efficiency/toxicity and their physicochemical characteristics (such as size, binding properties, etc.). An overall low toxicity is observed for all complexes with no significant difference among substituted and unsubstituted compounds. An interesting result revealed by the dye exclusion assay suggests a more balanced protection of the DNA by the glycine and glycyl-lysine substituted compounds. Thus, the higher transfection efficiency is attributed to the greater biocompatibility and flexibility of the amino acid/peptide-substituted gemini surfactants and demonstrates the feasibility of using amino acid-substituted gemini surfactants as gene carriers for the treatment of diseases affecting epithelial tissue.

Design and evaluation of cyclodextrin-based delivery systems to incorporate poorly soluble curcumin analogs for the treatment of melanoma

Various analogs of curcumin show high in vitro cytotoxic activity and are potential candidates for treating a deadly skin disease, melanoma. Due to the low solubility of the drugs, a new delivery agent, namely a cationic gemini surfactant-conjugated β-cyclodextrin, was designed to incorporate novel drug candidates of the 1,5-diaryl-3-oxo-1,4-pentadienyl family. Based on physicochemical parameters, such as particle size and zeta potential, a schematic model for the potential interaction of the drug with the delivery agent was developed. The drug formulations were highly efficient in inhibiting the growth of melanoma cells, with IC(50) values significantly lower than melphalan, the drug currently used for the treatment of in-transit melanoma. CDgemini formulations showed excellent cellular selectivity, triggering apoptosis in the A375 cell line while showing no cytotoxicity to healthy human epidermal keratinocytes. The goal is to develop this novel nanoparticle approach into a non-invasive therapy for in-transit melanoma metastasis that lacks adequate treatment to date.

Lysine-functionalized nanodiamonds: synthesis, physiochemical characterization, and nucleic acid binding studies

Purpose: Detonation nanodiamonds (NDs) are carbon-based nanomaterials that, because of their size (4–5 nm), stable inert core, alterable surface chemistry, fluorescence, and biocompatibility, are emerging as bioimaging agents and promising tools for the delivery of biochemical molecules into cellular systems. However, diamond particles possess a strong propensity to aggregate in liquid formulation media, restricting their applicability in biomedical sciences. Here, the authors describe the covalent functionalization of NDs with lysine in an attempt to develop nanoparticles able to act as suitable nonviral vectors for transferring genetic materials across cellular membranes. Methods: NDs were oxidized and functionalized by binding lysine moieties attached to a three-carbon-length linker (1,3-diaminopropane) to their surfaces through amide bonds. Raman and Fourier transform infrared spectroscopy, zeta potential measurement, dynamic light scattering, atomic force microscopic imaging, and thermogravimetric analysis were used to characterize the lysine-functionalized NDs. Finally, the ability of the functionalized diamonds to bind plasmid DNA and small interfering RNA was investigated by gel electrophoresis assay and through size and zeta potential measurements. Results: NDs were successfully functionalized with the lysine linker, producing surface loading of 1.7 mmol g−1 of ND. These modified NDs formed highly stable aqueous dispersions with a zeta potential of 49 mV and particle size of approximately 20 nm. The functionalized NDs were found to be able to bind plasmid DNA and small interfering RNA by forming nanosized “diamoplexes”. Conclusion: The lysine-substituted ND particles generated in this study exhibit stable aqueous formulations and show potential for use as carriers for genetic materials.

HLA-DQB1*02 and DQB1*06:03P are associated with peanut allergy

Peanut allergy (PA) is a common and serious food allergy and its prevalence has increased in the past decade. Although there is strong evidence of inheritance, the genetic causes of this disease are not well understood. Previously, a large-scale genome-wide association study described an association between human leukocyte antigen (HLA)-DQB1 and asthma; the aim of this study was to evaluate the association between HLA-DQB1 and PA. Genotypic and allelic profiles were established for 311 Caucasian members of a well-described Canadian group of children with PA and 226 Caucasian controls. Firth’s logistic regression analyses showed associations between HLA-DQB1 alleles and PA for DQB1*02 (P=1.1 × 10−8, odds ratio (OR)=0.09 (CI=0.03–0.23)) and DQB1*06:03P alleles (P=2.1 × 10−2, OR=2.82 (CI=1.48–5.45)). This study of HLA in PA demonstrates specific association between two allelic groups of the HLA-DQB1 gene (DQB1*02 and DQB1*06:03P) and PA, highlighting its possible role in the development of this disease.

Novel 3,5-bis(arylidene)-4-piperidone dimers: Potent cytotoxins against colon cancer cells

Two novel series of dimeric 3,5-bis(arylidene)-4-piperidones 7 and 8 were prepared as cytotoxic agents. A specific objective of this study was the discovery of novel compounds displaying potent anti-proliferative activities against colon cancers. Most of the compounds demonstrate potent cytotoxicity against HCT116 and HT29 colon cancer cell lines in which the IC50 values range from low micromolar to nanomolar values. In general, the majority of the compounds showed greater cytotoxicity and some degree of selectivity toward HCT116 cells compared to HT29 cells. Compound 9 in which the amidic carbonyl groups were excised was substantially less potent than 8a in both cell lines suggested that the amide groups are important components of the molecules for exhibiting cytotoxicity. Virtually all the compounds were more potent than a reference drug 5-fluorouracil which is used in treating colon cancers as well as a related enone curcumin. QSAR studies were undertaken and some guidelines for amplification of the project have been formulated. Flow cytometry analysis of a representative potent compound 7f revealed that it induces apoptosis in HCT116 cells.

Development and validation of fast and simple flow injection analysis-tandem mass spectrometry (FIA-MS/MS) for the determination of metformin in dog serum.

A simple, fast and sensitive quantification method for the drug metformin in dog serum was developed using flow injection analysis (FIA)-tandem mass spectrometry (MS/MS). The method was fully validated according to industry standards. It is the first time that FIA-MS/MS for metformin was developed surpassing all existing methods in terms of time of analysis. The quantification method was dependent on the formation of [M+H](+) using electrospray ionization (ESI) and employing multiple reaction monitoring (MRM) using quadrupole-linear ion trap (4000 QTRAP(®)) instrument. A deuterated internal standard (IS) of metformin bearing six deuterium atoms was used to compensate for matrix effects and for variation in ion current within the ESI source. The ion transitions that were monitored were m/z 130.1→m/z 71.0 and m/z 130.1→m/z 60.1 for metformin and m/z 136.0→m/z 77.0 for the internal standard. A linear response (r=0.9966) was established for a range of concentrations of 5-2340 ng/mL. The inter- and intra-day variations were within the acceptable criteria for all quality control samples. The method was successfully applied for measurement of serum metformin concentration in dogs after intravenous injection.

Development of Lyophilized Gemini Surfactant-Based Gene Delivery Systems: Influence of Lyophilization on the Structure, Activity and Stability of the Lipoplexes

PURPOSE Cationic gemini surfactants have been studied as non-viral vectors for gene therapy. Clinical applications of cationic lipid/DNA lipoplexes are restricted by their instability in aqueous formulations. In this work, we investigated the influence of lyophilization on the essential physiochemical properties and in vitro transfection of gemini surfactant-lipoplexes. Additionally, we evaluated the feasibility of lyophilization as a technique for preparing lipoplexes with long term stability. METHODS A gemini surfactant [12-7NH-12] and plasmid DNA encoding for interferon-γ were used to prepare gemini surfactant/pDNA [P/G] lipoplexes. Helper lipid DOPE [L] was incorporated in all formulation producing a [P/G/L] system. Sucrose and trehalose were utilized as stabilizing agents. To evaluate the ability of lyophilization to improve the stability of gemini surfactant-based lipoplexes, four lyophilized formulations were stored at 25˚C for three months. The formulations were analyzed at different time-points for physiochemical properties and in vitro transfection. RESULTS The results showed that both sucrose and trehalose provided anticipated stabilizing effect. The transfection efficiency of the lipoplexes increased 2-3 fold compared to fresh formulations upon lyophilization. This effect can be attributed to the improvement of DNA compaction and changes in the lipoplex morphology due to the lyophilization/rehydration cycles. The physiochemical properties of the lyophilized formulations were maintained throughout the stability study. All lyophilized formulations showed a significant loss of gene transfection activity after three months of storage. Nevertheless, no significant losses of transfection efficiency were observed for three formulations after two months storage at 25 ˚C. CONCLUSION Lyophilization significantly improved the physical stability of gemini surfactant-based lipoplexes compared to liquid formulations. As well, lyophilization improved the transfection efficiency of the lipoplexes. The loss of transfection activity upon storage is most probably due to the conformational changes in the supramolecular structure of the lipoplexes as a function of time and temperature rather than to DNA degradation.

Permeability and conjugative metabolism of flaxseed lignans by Caco-2 human intestinal cells.

Reports in the literature associate the dietary intake of flaxseed lignans with a number of health benefits. The major lignan found in flaxseed, secoisolariciresinol diglucoside (1), undergoes metabolism principally to secoisolariciresinol (2), enterodiol (3), and enterolactone (4) in the human gastrointestinal tract. Systemically, lignans are present largely as phase II enzyme conjugates. To improve understanding of the oral absorption characteristics, a systematic evaluation of the intestinal permeation was conducted and the conjugative metabolism potential of these lignans using the polarized Caco-2 cell system was analyzed. For permeation studies, lignans (100 μM) were added to acceptor or donor compartments and samples were taken at 2 h. For metabolism studies, lignans (100 μM) were incubated in Caco-2 for a maximum of 48 h. Cell lysates and media were treated with β-glucuronidase/sulfatase, and lignan concentrations were determined using HPLC. Apical-to-basal permeability coefficients for 2-4 were 8.0 ± 0.4, 7.7 ± 0.2, and 13.7 ± 0.2 (×10(-6)) cm/s, respectively, whereas efflux ratios were 0.8-1.2, consistent with passive diffusion. The permeation of compound 1 was not detected. The extent of conjugation after 48 h was <3%, ∼95%, ∼90%, and >99% for 1-4, respectively. These data suggest 2-4, but not 1 undergo passive permeation and conjugative metabolism by Caco-2 cells.

Evidence for lymphatic transport of insulin by topically applied biphasic vesicles.

The cutaneous delivery pathway through the lymphatics of a novel transdermal lipid‐based delivery system (biphasic vesicles), which was previously shown to deliver sustained physiological levels of basal insulin in a pain‐free manner across the skin, was evaluated in a diabetic rat model. Transdermal patches (one per rat) containing insulin in biphasic vesicles (1–10 mg recombinant human insulin dose) were applied to the shaved abdominal skin of streptozotocin‐induced diabetic rats for 73 h. Blood glucose was monitored approximately every 2–10 h using a Lifescan glucose meter. Inguinal lymph node insulin levels were analysed by ELISA. Insulin in the lymph nodes increased in a dose‐ and time‐dependent manner. Maximal transdermal insulin concentrations in the lymph nodes were observed with both 140 IU (5 mg: 43.0 + 18.0 μIU mg−1 (mean + s.e.m., n = 4)) and 280 IU (10 mg: 48.0 + 19.6 μIU mg−1 (mean + s.e.m., n = 4)) doses of recombinant insulin at t = 73 h. The level of insulin in the lymph nodes after subcutaneous injection of 1 mg insulin at the peak blood glucose response was 35.8 μIU mg−1 (n = 2), before falling to 0.35 μIU mg−1 by t = 48 h (n = 2). The lymphatics is involved in the transdermal insulin delivery by biphasic vesicles. This is the first report on the lymphatic transport of a protein after non‐invasive topical application on the skin.