<p>Characterization and Cytotoxicity of Polyprenol Lipid and Vitamin E-TPGS Hybrid Nanoparticles for Betulinic Acid and Low-Substituted Hydroxyl Fullerenol in MHCC97H and L02 Cells</p>
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This study demonstrated an innovative formulation including the polyprenol (GBP) lipid and vitamin E-TPGS hybrid nanoparticles (NPs) which was aimed to control the transfer of betulinic acid (BA) and low-substituted hydroxyl fullerenol (C60(OH)n). Additionally, it developed BA-C60(OH)n-GBP-TPGS-NPs delivery system and researched the anti-hepatocellular carcinoma (HCC) effects.The NPs were prepared by nanoprecipitation with ultrasonic-assisted emulsification (UAE) method. It was characterized by scanning electronic microscopy (SEM), transmission electron microscopy (TEM), FTIR spectrum, size distribution and zeta potential. Physical and chemical properties were evaluated through measurement of drug release, stability studies, drug loading efficiency (DE) and encapsulation efficiency (EE). Biological activities were evaluated through measurement of MTT assay, lactate dehydrogenase leakage assay (LDH), cell proliferation assays, cell apoptosis analysis, comet assay, wound healing assay, cell invasion and Western blot analysis.The NPs exhibited clear distribution characteristics, improved solubility and stability. BA and C60(OH)n for the NPs displayed a biphasic release pattern with sustained drug release properties. The mixture of C60(OH)n with different hydroxyl groups may have a certain effect on the stability of the NPs system itself. The NPs could effectively inhibit MHCC97H cell proliferation, migration and invasion in vitro. Combined use of C60(OH)n and BA in GBP lipids may improve the inhibit effect of C60(OH)n or BA against HCC cells and reduce cytotoxicity and genotoxicity of C60(OH)n for normal cells. We concluded that one of the important mechanisms of BA-C60(OH)n-GBP-TPGS-NPs inhibiting MHCC97H cells is achieved by up-regulating the expression of Caspase-3, Caspase-8 and Caspase-9.Keywords:
Betulinic acid
Zeta potential
MTT assay
Improvement of MTT assay by 2-chloroadenosine in activation test and cytotoxicity test of lymphocyte
Objective:To sutdy the effect of 2 chloroadenosine(2 ClA),which is specifically cytotoxic to macrophages,on MTT assay in activation test and cytotoxicity test of lymphocyte.Methods:Using cell culture technique,mouse splenic lymphocytes and peritoneal macrophages were cultured.Lymphocyte activation and specific cytotoxicity to tumor cells and toxicity of 2 ClA to macrophages were measured by MTT assay in the presence or absence of 2 ClA.Results:2 ClA had a strong cytotoxic effect on macrophages.When the activation test and cytotoxicity test of lymphocyte were measured by MTT assay,the optical density values of 2 ClA group was lower than that of control group,and statistic analysis showed P0 05.Conclusion:With the specific cytotoxicity to macrophages,2 ClA can exclude the influence of macrophages to MTT assay in activation test and cytotoxicity test of lymphocyte,and more reliable results will be obtained.
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Objective To compare three methods(MTT assay,living cells count and morphologic observation) for evaluation on cytotoxicity of medical products.Methods MTT assay,living cells count and morphologic observation were used to evaluate the cytotoxicity of nineteen pieces of medical products by cytotoxicity graduation and judgement of qualification,the results of these methods were analysed for degree of coincidence and comparison of advantages and disadvantages among these methods.Results The rate of coincidence of cytotoxicity graduation between MTT assay and living cells count was 73.7%(14/19) and that of qualification was 94.7%(18/19),the sample No.15 was tested four times with three methods,and the results show the difference between MTT assay and living cells-count were great,while the results of morphologic observation method were concordant with that of living cells count.Conclusion MTT assay is more suitable for evaluation of cytotoxicity of medical products for it is easy to operate and its errors are small,but living cells-count method and morphologic observation are needed to make a comprehensive judgement to unqualified medical products.
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Betulinic acid is found in the several foods and plants, such as sour jujube fruits, papaya, persimmon, betulinic acid exhibits low toxicity and a wide range of anticancer activities. Ferritinophagy and ferroptosis are related to tumor formation and therapeutic effects, both of which are controlled by iron metabolism and inextricably linked. In this study, we discovered that betulinic acid could suppress proliferation and migration of hepatoma cells, raised ROS level and inhibited antioxidation level in cells. Ferrostatin-1 was used to determine the death way of hepatoma cells induced by betulinic acid. Betulinic acid could cause ferritinophagy-related phenomenon in vivo and in vitro, promote ferritinophagy-protiens expressions, induce ferritinophagy to inhibit tumours finally. In vivo studies revealed the low toxicity of betulinic acid in mice, the dose of human was about 10 times that of mice, the using dose of human could also be achievable. These findings illustrate the potential of betulinic acid for treatment of cancer.
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Betulinic acid, a pentacyclic triterpene, is widely distributed throughout the tropics. It possesses several biological properties such as anticancer, anti-inflammatory, antiviral, antiseptic, antimalarial, spermicidal, antimicrobial, antileshmanial, antihelmentic and antifeedent activities. However, betulinic acid was highly regarded for its anticancer and anti-HIV activities. Anticancer role of betulinic acid appeared by inducing apoptosis in cells irrespective of their p53 status. Due to high order safety in betulinic acid, a number of structural modifications carried out to improve its potency and efficacy. The C-1, C-2, C-3, C-4, C-20 and C-28 positions are the diversity centers in betulinic acid, and the derivatives resulted on various structural modifications at these positions screened for their anticancer activity. This review presents the structure activity relationship carried out on C-1, C-2, C-3, C-4, C-20, C-28, A-ring, D-ring and E-ring modified betulinic acid derivatives. We have compiled the most active betulinic acid derivatives along with their activity profile in each series. Structure activity relationship studies revealed that C-28 carboxylic acid was essential for the cytotoxicity. The halo substituent at C-2 position in betulinic acid enhanced the cytotoxicity. Though the relation of the cytotoxicity with the nature of substituents at C-3 position could not be generalized but the ester functionality appeared to be a better substituent for enhancing the cytotoxicity. An interesting observation is that the three rings skeleton (A, B and C rings) had played an important role in eliciting anticancer activity, which could be a new molecular skeleton to design new anticancer drugs.
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Background : Silver nanoparticles are of interest to be used as antimicrobial agents in medical care, wound dressings and cosmetics. Despite the fact that AgNPs are among the most commercialized nano materials owing to their specific antimicrobial properties, there is limited information about their risk assessment and possible hazards to human health and environment. Thus, this study was carried out to evaluate the cytotoxicity and morphological changes of different concentrations of two samples of commercialized silver nanoparticles (A and B) in CHO-K1 cells using MTT assay. Methods: The cytotoxicity effect of silver nanoparticles AgNP-A (19.6 nm in diameter) and AgNP-B (15 nm in diameter) on CHO-K1 was evaluated in the range of 0.005-500 µg/ml after 24 hours of treatment using MTT assay. The morphological changes of treated cells were examined by light microscopy. Results: Based on the results of cytotoxicity by MTT assay, reduced viability of cells and cytotoxicity were observed. The 50% inhibitory concentration (IC50) of silver nanoparticles was recorded at 100 and 10 µg/ml for AgNP-A and AgNP-B, respectively. Moreover, microscopic observations indicated clear morphological changes of treated cells. Conclusion: Concentration and physicochemical properties of silver nanoparticles like size, shape, surface area, coating and zeta potential play a role in cytotoxicity and morphological changes of treated cells.
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Aim:To improve the anti-tumor effects and to screen candidate for anti-tumor agent,30-hydroxy derivatives 6a-e and 7a were obtained from the modification of 23-hydroxy betulinic acid.Methods:After protection of the hydroxyl at C-3 and C-23 and carboxyl at C-28 of 23-hydroxy betulinic acid,the oxidation of compounds 5a-e was processed with m-chloroperoxybenzoic acid in chloroform by refluxing for 6 h.Meanwhile,the anti-tumor activity of the target compounds was tested by MTT.Results:The structures of the target compounds have been confirmed by the analytical and spectral analyses.The results showed that the anti-tumor activity of the target compounds was much potent than that of 23-hydroxy betulinic acid.Conclusion:The derivative of 23-hydroxy betulinic acid(6e) is a promising lead compound as the anti-tumor agent worth further investigation.
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Betulinic acid, a natural product, is an anti-HIV and antitumor agent with novel structure and new mechanism of action. Not only it is found abundantly in the nature, but also its semi-synthesis approach has been successful. Betulinic acid is a perfect lead-compound. A lot of betulinic acid derivatives have been synthesized. The structure-activity relationships of Betulimic acid, The mechanisms and the effects of YK-FH312, RPR103611 and IC9564 derivated from Betulinic acid were reviewed in the paper.
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In this research work, a series of eighteen novel coumarinyl substituted thiazolidin-2,4-dione analogs (4a-4r) have been designed by molecular hybridization approach, synthesized and their structures were established on the basis of FTIR, 1H NMR, 13C NMR and elemental (CHN) analysis. These title compounds were screened for their cytotoxicity using MTT assay methodology against five different mammalian cancer cell lines viz. hormone dependant breast adenocarcinoma (MCF7), cervical carcinoma (HeLa), colorectal carcinoma (HT29), lung cancer (A549) and prostate adeno carcinoma (PC3). The cytotoxicity screening studies revealed that MCF-7, HeLa and A549 cancer cell lines were sensitive to all the tested compounds. Though the compounds showed varying degrees of cytotoxicity in the tested cell lines, most significant effect was observed for compounds 4i (1.06, 2.4 and 3.06 µM) and 4o (0.95, 3.2 and 2.38 μM) against MCF7, HeLa and A549 cell lines respectively. In conclusion, the anticancer results of these promising leads strongly encouraged us for additional lead optimization with the aim of developing more potential anticancer agents.
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Betulinic acid, a pentacyclic triterpene, is distributed in a variety of plants, such as birch, eucalyptus and plane trees. It shows a wide spectrum of biological and pharmacological properties, such as anti-inflammatory, antibacterial, antiviral, antidiabetic, antimalarial, anti-HIV and antitumor effects. Among them, the antitumor activity of betulinic acid has been extensively studied. However, obtaining betulinic acid from natural resources can no longer meet the needs of medicine and nutrition, so methods such as chemical synthesis and microbial biotransformation have also been used to prepare betulinic acid. At the same time, with the development of synthetic biology and genetic engineering, and the elucidation of the biosynthetic pathways of terpenoid, the biosynthesis of betulinic acid has also been extensively researched. This article reviews the preparation of betulinic acid and its pharmacological activities, in order to provide a reference for the research and utilization of betulinic acid.
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MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.
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