Preparation and Optimization of MiR-375 Nano-Vector Using Two Novel Chitosan-Coated Nano-Structured Lipid Carriers as Gene Therapy for Hepatocellular Carcinoma.

Currently, there is still a lack of effective carriers with minimal side effects to deliver therapeutic miRNA. Thus, it is crucial to optimize novel drug delivery systems. MiR-375 has proven superior therapeutic potency in Hepatocellular carcinoma (HCC). The purpose of this study was to fabricate 2 novel and smart nano-carriers for the transportation efficiency of miR-375 in HCC cells and enhance its anti-tumor effects. We established the miR-375 construct through the pEGP- miR expression vector. Two nano-carriers of solid/liquid lipids and chitosan (CS) were strategically selected, prepared by high-speed homogenization, and optimized by varying nano-formulation factors. Thus, the two best nano-formulations were designated as F1 (0.5% CS) and F2 (1.5% CS) and were evaluated for miR-375 conjugation efficiency by gel electrophoresis and nanodrop assessment. Then, physio-chemical characteristics and stability tests for the miR-375 nano-plexes were all studied. Next, its efficiencies as replacement therapy in HepG2 cells have been assessed by fluorescence microscopy, flow cytometry, and cytotoxicity assay. The obtained data showed that two cationic nanostructured solid/liquid lipid carriers (NSLCs); F1 and F2 typically had the best physio-chemical parameters and long-term stability. Moreover, both F1 and F2 could form nano-plexes with the anionic miR-375 construct at weight ratios 250/1 and 50/1 via electrostatic interactions. In addition, these nano-plexes exhibited physical stability after three months and protected miR-375 from degradation in the presence of 50% fetal bovine serum (FBS). Furthermore, both nano-plexes could simultaneously deliver miR-375 into HepG2 cells and they ensure miR re-expression even in the presence of 50% FBS compared to free miR-375 (p-value < 0.001). Moreover, both F1 and F2 alone significantly exhibited minimal cytotoxicity in treated cells. In contrast, the nano-plexes significantly inhibited cell growth compared to free miR-375 or doxorubicin (DOX), respectively. More importantly, F2/miR-375 nano-plex exhibited more anti-proliferative activity in treated cells although its IC50 value was 55 times lower than DOX (p-value < 0.001). Collectively, our findings clearly emphasized the multifunctionality of the two CS-coated NSLCs in terms of their enhanced biocompatibility, biostability, conjugation, and transfection efficiency of therapeutic miR-375. Therefore, the NSLCs/miR-375 nano-plexes could serve as a novel and promising therapeutic strategy for HCC.

Cytotoxic and Apoptotic Effects of Luffa Cylindrica Leaves Extract against Acute Lymphoblastic Leukemic Stem Cells.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is an aggressive malignancy defined by accumulation of lymphoblasts in the bone marrow. Leukemic stem cells (LSCs) are the major cause of the recurrence and metastasis of ALL. This study aimed to develop an effective anti-cancer agent targeting these LSCs. Luffa Cylindrica (L.C.) leaves extract was selected to evaluate its effect on ALL via eradicating the LSCs as it contains many active anti-cancer flavonoids. METHODS: Thirty-two bone marrow samples of ALL patients were used in this study. LSCs population was identified in the selected samples. Cell viability was measured by MTT assay and flow cytometry. Cell cycle, apoptosis, proliferation marker; ki-67 and colony forming assay were further analyzed. RESULTS: This study revealed the expression of CD34+/CD38+ cells in addition to CD34+/CD38- population and the extract was effective against the two LSCs populations. MTT assay showed that treated leukemic cells exhibited significant reduction in the viable cells in a dose dependent manner with IC50 of 3 µg/µl which was then confirmed by flow cytometry. Cell cycle analysis results showed significant reduction in the percentage of cells treated with L.C. extract in both the S and G0/G1 phases, with concomitant increase in the G2/M phase. Also, L.C. extract could effectively induce apoptosis, inhibit proliferation and suppress colonogenecity of leukemic cells. CONCLUSION: This study validated the medicinal potential of L.C. leaves extract as a promising anti-leukemic agent targeting both LSCs and blasts in ALL patients, which may be explained by the synergy found between its potent flavonoids especially apigenin, luteolin and kaempferol.

Therapeutic role of a synthesized calcium phosphate nanocomposite material on hepatocarcinogenesis in rats.

Nanotechnology research is booming worldwide, and the general belief is that medical and biological applications will form the greatest sector of expansion over the next decade. With this in mind, this study was designed to evaluate the therapeutic effects of a synthesized tricalcium phosphate nanocomposite material (nano-TCP) on hepatocarcinoma in a rat model, as initiated with diethylnitrosamine (DEN) and promoted with phenobarbital (PB). Hepatocarcinoma was induced with intraperitoneal injections of DEN (50 mg·(kg body mass)(-1)) 3 times a week for 2 weeks. Three weeks after the last dose of DEN, the rats received PB (0.05 %, w/v) in their drinking water for a further 6 weeks. Nano-TCP (100 mg·(kg body mass)(-1)) was administered intraperitoneally 3 times per week to rats with HCC. At the end of the experimental period, liver samples were collected from all animals for biochemical and histopathological analysis. The degree of DNA fragmentation was analyzed, in addition to immune status, by measuring the levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2). The activities of the most important free-radical scavengers of the antioxidant defense system as well as malondialdehyde (MDA) content and liver enzymes were measured. The levels of hepatic heat shock protein-70 (HSP-70), caspase-3, and metalloproteinase-9 were also measured as markers for inflammation and apoptosis. Histopathological examination of liver tissue was performed. The results revealed the potent efficacy of nano-TCP in repairing the fragmented DNA and ameliorating most of the investigated parameters by significant elevation in the levels of hepatic alanine aminotransferase (ALT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. On the other hand, there was a significant decrease in hepatic gamma-glutamyl transpeptidase (γ-GT), MDA, IL-2, IFN-γ, TNF-α, matrix metalloproteinase-9 (MMP-9), HSP-70, and caspase-3 levels upon treatment. The findings form histopathological examination of the liver tissues agreed with the biochemical results and confirmed the difference between the control and treatment groups. In conclusion, nano-TCP succeeded in treating hepatocarcinoma efficiently, and presents a new hope for patients to get safe, fast, and effective treatment.