RNAi-Mediated Knockdown of Cottontail Rabbit Papillomavirus Oncogenes Using Low-Toxicity Lipopolyplexes as a Paradigm to Treat Papillomavirus-Associated Cancers.

The cottontail rabbit papillomavirus (CRPV)-associated VX2 carcinoma of the New Zealand White rabbit serves as a model system for human papillomavirus (HPV)-associated head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to evaluate the tumor-inhibiting effect of RNAi-mediated knockdown of the CRPV oncogenes, E6 and E7, using siRNA-loaded lipopolyplexes (LPPs). VX2-carcinoma-derived cells were cultured for up to 150 passages. In addition, CRPV E6 and E7 oncogenes were transiently expressed in COS-7 cells. Efficiency and safety of LPPs were evaluated in both VX2 cells and the COS-7 cell line. Both of these in vitro CRPV systems were validated and characterized by fluorescence microscopy, Western blot, and RT-qPCR. Efficient knockdown of CRPV E6 and E7 was achieved in VX2 cells and COS-7 cells pretransfected with CRPV E6 and E7 expression vectors. Knockdown of CRPV oncogenes in VX2 cells resulted in reduced viability, migration, and proliferation and led to a G0/G1 block in the cell cycle. CRPV E6 and E7 siRNA-loaded LPPs could represent promising therapeutic agents serving as a paradigm for the treatment of papillomavirus-positive cancers and could be of value for the treatment of CRPV-associated diseases in the rabbit such as papillomas and cancers of the skin.

Photodynamic and antiangiogenic activities of parietin liposomes in triple negative breast cancer.

Parietin (PTN) is an anthraquinone with promising efficacy in the inhibition of cancer cell proliferation and tumor growth. Due to its hydrophobicity, PTN is sparingly soluble under physiological conditions and has a low bioavailability. Hence, we presented PTN in liposomes to overcome these drawbacks. The prepared liposomes were characterized and their stability was also assessed in serum. Singlet oxygen quantum yield of PTN loaded liposomes was indirectly quantified using uric acid. The intracellular uptake of liposomes was studied by CLSM which indicated the perinuclear localization of PTN liposomes. Cellular viability assay and live/dead staining demonstrated both light and dose-dependent phototoxicity of PTN on the human breast cancer cell line. The mechanism of cellular uptake was investigated using different pathway inhibitors and the results showed that clathrin-mediated endocytosis is predominant. The colocalization experiment indicated that PTN is localized in both mitochondria and lysosomes. These findings together with flow cytometry analysis elucidated that apoptosis is the main mechanism underlying cell death post-PDT. Finally, the antiangiogenic effect of PTN liposomes was further evaluated in the chorioallantoic membrane (CAM) model and the results indicated that PDT induced vascular response was confined to the irradiated area leaving the non-irradiated unscathed.

Cucumber-Derived Exosome-like Vesicles and PlantCrystals for Improved Dermal Drug Delivery.

(1) Background: Extracellular vesicles (EVs) are considered to be efficient nanocarriers for improved drug delivery and can be derived from mammalian or plant cells. Cucumber-derived EVs are not yet described in the literature. Therefore, the aim of this study was to produce and characterize cucumber-derived EVs and to investigate their suitability to improve the dermal penetration efficacy of a lipophilic active ingredient (AI) surrogate. (2) Methods: The EVs were obtained by classical EVs isolation methods and by high pressure homogenization (HPH). They were characterized regarding their physico-chemical and biopharmaceutical properties. (3) Results: Utilization of classical isolation and purification methods for EVs resulted in cucumber-derived EVs. Their dermal penetration efficacy for the AI surrogate was 2-fold higher when compared to a classical formulation and enabled a pronounced transdermal penetration into the viable dermis. HPH resulted in submicron sized particles composed of a mixture of disrupted plant cells. A successful isolation of pure EVs from this mixture was not possible with classical EVs isolation methods. The presence of EVs was, therefore, proven indirectly. For this, the lipophilic drug surrogate was admixed to the cucumber juice either prior to or after HPH. Admixing of the drug surrogate to the cucumber prior to the HPH resulted in a 1.5-fold increase in the dermal penetration efficacy, whereas the addition of the AI surrogate to the cucumber after HPH was not able to improve the penetration efficacy. (4) Conclusions: Results, therefore, indicate that HPH causes the formation of EVs in which AI can be incorporated. The formation of plant EVs by HPH was also indicated by zeta potential analysis.

Surface tailored zein as a novel delivery system for hypericin: Application in photodynamic therapy.

Zein is an FDA-approved maize protein featured by its manipulative surface and the possibility of fabrication into nanomaterials. Although extensive research has been carried out in zein-based technology, limited work is available for the application of zein in the field of cancer photodynamic therapy (PDT). In this work, we report zein as a carrier for the natural photosensitizer hypericin in the PDT of hepatocellular carcinoma in vitro. Zein was modified through chemical PEGylation to form PEGylated zein micelles that were compared with two zein nanoparticle formulations physically stabilized by either the lecithin/pluronic mixture or sodium caseinate. FT-IR, 1HNMR and HP-SEC MALS approaches were employed to confirm the chemical PEGylation of zein. Our developed zein nanoparticles and micelles were further characterized by photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). The obtained results showed relatively smaller sizes and higher encapsulation of hypericin in the micellar zein than the nanoparticle-based formulations. Phototoxicity on hepatocellular carcinoma (HepG2 cells) manifested a dose-dependent toxicity pattern of all designed zein formulations. However, superior cytotoxicity was prominent for the hypericin-based micelles, which was influenced by the higher cellular uptake profile. Consequently, the treated HepG2 cells manifested a higher level of intracellular generated ROS and disruption of mitochondrial membrane potential, which induced apoptotic cell death. Comparatively, the designed hypericin formulations indicated lower phototoxicity profile in murine fibroblast L929 cells reflecting their safety on normal cells. Our investigations suggested that the surface-modified zein could be employed to enhance the delivery of the hydrophobic hypericin in PDT and pave the way for future in vivo and clinical applications in cancer treatment.

Lipoparticles for Synergistic Chemo-Photodynamic Therapy to Ovarian Carcinoma Cells: In vitro and in vivo Assessments.

PURPOSE: Lipoparticles are the core-shell type lipid-polymer hybrid systems comprising polymeric nanoparticle core enveloped by single or multiple pegylated lipid layers (shell), thereby melding the biomimetic properties of long-circulating vesicles as well as the mechanical advantages of the nanoparticles. The present study was aimed at the development of such an integrated system, combining the photodynamic and chemotherapeutic approaches for the treatment of multidrug-resistant cancers. METHODS: For this rationale, two different sized Pirarubicin (THP) loaded poly lactic-co-glycolic acid (PLGA) nanoparticles were prepared by emulsion solvent evaporation technique, whereas liposomes containing Temoporfin (mTHPC) were prepared by lipid film hydration method. Physicochemical and morphological characterizations were done using dynamic light scattering, laser doppler anemometry, atomic force microscopy, and transmission electron microscopy. The quantitative assessment of cell damage was determined using MTT and reactive oxygen species (ROS) assay. The biocompatibility of the nanoformulations was evaluated with serum stability testing, haemocompatibility as well as acute in vivo toxicity using female albino (BALB/c) mice. RESULTS AND CONCLUSION: The mean hydrodynamic diameter of the formulations was found between 108.80 ± 2.10 to 405.70 ± 10.00 nm with the zeta (ζ) potential ranging from -12.70 ± 1.20 to 5.90 ± 1.10 mV. Based on the physicochemical evaluations, the selected THP nanoparticles were coated with mTHPC liposomes to produce lipid-coated nanoparticles (LCNPs). A significant (p< 0.001) cytotoxicity synergism was evident in LCNPs when irradiated at 652 nm, using an LED device. No incidence of genotoxicity was observed as seen with the comet assay. The LCNPs decreased the generalized in vivo toxicity as compared to the free drugs and was evident from the serum biochemical profile, visceral body index, liver function tests as well as renal function tests. The histopathological examinations of the vital organs revealed no significant evidence of toxicity suggesting the safety and efficacy of our lipid-polymer hybrid system.

Potent Cytotoxicity of Four Cameroonian Plant Extracts on Different Cancer Cell Lines.

In this study, the potential cytotoxicity of four plant extracts originated from Cameroon: Xylopia aethiopica (XA), Imperata cylindrica (IC), Echinops giganteus (EG) and Dorstenia psilurus (DP) were examined in vitro. We tested the anti-proliferative activity of the methanolic extracts of these compounds using MTT assay on seven different human cancer cell lines: HeLa, MDA-MB-231, A549, HepG2, U-87, SK-OV-3 and HL60. Induction of cell death was assessed by cell cycle analysis, apoptosis was determined by Annexin V-FITC binding and caspase 3/7 activity. As well, changes in mitochondrial membrane potential (MMP) and cell migration were tested. The genetic toxicity, using the alkaline comet assay, was evaluated. The studied extracts inhibited the cell proliferation of all tested cancer cell lines with concentration dependent effect over time. All of these extracts mainly induced apoptosis of HeLa cells by the accumulation of hypodiploid cells in the sub-G0/G1 phase and increasing the activity of caspase 3/7, as well they showed potential MMP disturbance and expressed a marked inhibitory effect on cell migration. Assessment of probable genetic toxicity by these extracts revealed no or minimum incidence of genetic toxicity. Therefore, the studied plant extracts are exhibiting potent anticancer activity based upon marked induction of tumor-cell death.

Sensitivity of Papilloma Virus-Associated Cell Lines to Photodynamic Therapy with Curcumin-Loaded Liposomes.

Photodynamic therapy (PDT) is a minimally invasive therapeutic approach used in the treatment of various medical conditions and cancerous diseases, involving light, a photosensitizing substance, and oxygen. Curcumin, a naturally occurring compound, carries antitumor activities and potentially could be exploited as a photosensitizer in PDT. Only little is known about liposomal-encapsulated curcumin that could help in increasing the efficacy, stability, and bioavailability of this compound. This study investigates the in vitro effects of curcumin-loaded liposomes in combination with PDT. Three papilloma virus-associated cell lines were treated with curcumin-loaded liposomes corresponding to a curcumin concentration of 0-100 µmol/L for 4 h followed by illumination at 457 nm (blue) for 45, 136, and 227 s at a fluence of 220.2 W/m2 (100 mA) corresponding to 1, 3 and 5 J·cm-2. After 24 h, the biological outcome of the treatment was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), SYTO9/PI (propidium iodide), Annexin V-FITC (fluorescein isothiocyanate)/PI, clonogenic survival, and scratch (wound closure) assays. Photoactivation of curcumin-loaded liposomes led to a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death. The results point to the combination of curcumin-loaded liposomes with PDT as a potentially useful tool for the treatment of papillomavirus-associated malignancies.

Downregulation of MDR 1 gene contributes to tyrosine kinase inhibitor induce apoptosis and reduction in tumor metastasis: A gravity to space investigation.

P-glycoprotein (P-gp) associated multidrug resistance (MDR) represents a major failure in cancer treatment. The overexpression of P-gp is responsible for ATP-dependent efflux of drugs that decrease their intracellular accumulation. An effective downregulation of MDR1 gene using small interfering RNA (siRNA) is one of the safe and effective tools to overcome the P-gp triggered MDR. Therefore, the development of an efficient and non-toxic carrier system for siRNA delivery is a fundamental challenge for effective cancer treatment. Polyamidoamine (PAMAM) dendrimer has been used for efficient delivery of siRNA (dendriplexes) to the tumor cells but the associated toxicity problems render its use in biological applications. A non-covalent lipid modification (lipodendriplexes) is supposed to offer a promising strategy to overcome the demerits linked to the naked dendriplexes system. In the current study, we deliver siRNA, designed against MDR1 gene (si-MDR1), in colorectal carcinoma cells (Caco-2), having overexpression of P-gp, to check the role of MDR1 gene in tumor progression and multidrug resistance using two dimensional (2D) and three dimensional (3D) environment. Imatinib mesylate (IM), a P-gp substrate, was used as model drug. Our results revealed that the effective knockdown by lipodendriplexes system can significantly reduce the tumor cell migration in 2D (p < 0.001) and 3D (p < 0.001) cell cultures as compared to unmodified dendriplexes and si-Control groups. It was also observed that lipodendriplexes aided downregulation of MDR1 gene effectively, re-sensitized the Caco-2 cells for IM uptake and showed a significantly (p < 0.001) higher apoptosis. Our findings imply that our lipodendriplexes system has a great potential for siRNA delivery, however, further in vivo application using a suitable targeted system can play a major role for better cancer therapeutics.

Development and formulation of metformin (Antidiabetic) effervescent Granules: to increase patient compliance and its stability study.

Convenience of administration and patient compliance are gaining importance in the formulation of dosage forms. Many patients, like elderly people and person with dysphagia find difficulty to swallow the tablets and thus do not comply with prescriptions. So the present study was conducted to develop and formulate metformin effervescent granules. The stability study was carried out for 24 weeks (168 days) at temperatures of 4°C, Room temperature, 40°C & 60°C and at the end, the %age of drug remaining in the formulation was determined. The results showed that the formulation of metformin effervescent granules were remained best stable at 4°C in refrigerator, as the %age of drug remaining is not decreased more than 5% and the formulation stored at room temperature, was also found to be very close to the standard at the end of 24 weeks. It is concluded from the study that granules may be another dosage form to use as antidiabetic pharmaceutical product.