Screening potential P-glycoprotein inhibitors by combination of a detergent-free membrane protein extraction with surface plasmon resonance biosensor

P-glycoprotein (P-gp) highly expressed in cancer cells can lead to multidrug resistance (MDR) and the combination of anti-cancer drugs with P-gp inhibitor has been a promising strategy to reverse MDR in cancer treatment. In this study, we established a label-free and detergent-free system combining surface plasmon resonance (SPR) biosensor with styrene maleic acid (SMA) polymer membrane proteins (MPs) stabilization technology to screen potential P-gp inhibitors. First, P-gp was extracted from MCF-7/ADR cells using SMA polymer to form SMA liposomes (SMALPs). Following that, SMALPs were immobilized on an SPR biosensor chip to establish a P-gp inhibitor screening system, and the affinity between P-gp and small molecule ligand was determined. The methodological investigation proved that the screening system had good specificity and stability. Nine P-gp ligands were screened out from 50 natural products, and their affinity constants with P-gp were also determined. The in vitro cell verification experiments demonstrated that tetrandrine, fangchinoline, praeruptorin B, neobaicalein, and icariin could significantly increase the sensitivity of MCF-7/ADR cells to Adriamycin (Adr). Moreover, tetrandrine, praeruptorin B, and neobaicalein could reverse MDR in MCF-7/ADR cells by inhibiting the function of P-gp. This is the first time that SMALPs-based stabilization strategy was applied to SPR analysis system. SMA polymer can retain P-gp in the environment of natural lipid bilayer and thus maintain the correct conformation and physiological functions of P-gp. The developed system can quickly and accurately screen small molecule ligands of complex MPs and obtain affinity between complex MPs and small molecule ligands without protein purification.

Evaluation of the effects of two novel irrigants on intraradicular dentine erosion, debris and smear layer removal

OBJECTIVES: To evaluate the effects of copolymer of acrylic acid and maleic acid (Poly[AA-co-MA]) and calcium hypochlorite (Ca(OCl)2) on root canal dentin using scanning electron microscope (SEM). MATERIALS AND METHODS: Twenty-four single-rooted teeth were instrumented and the apical and coronal thirds of each root were removed, leaving the 5 mm middle thirds, which were then separated into two pieces longitudinally. The specimens were randomly divided into six groups and subjected to each irrigant for 5 min as follows: G1, Ca(OCl)2; G2, Poly(AA-co-MA); G3, Ca(OCl)2 + Poly(AA-co-MA); G4, sodium hypochlorite (NaOCl); G5, ethylenediaminetetraacetic acid (EDTA); G6, NaOCl+EDTA. The specimens were prepared for SEM evaluation. Smear layer, debris and erosion scores were recorded by two blinded examiners. One image from G3 was analyzed with energy dispersive spectroscopy (EDS) on suspicion of precipitate formation. Data were analyzed using the Kruskal-Wallis and Dunn tests. RESULTS: G1 and G4 showed the presence of debris and smear layer and they were statistically different from G2, G3, G5 and G6 where debris and smear layer were totally removed (p < 0.05). In G1 and G4, erosion evaluation could not be done because of debris and smear layer. G2, G3 and G5 showed no erosion, and there was no significant difference between them. G6 showed severe erosion and was statistically different from G2, G3 and G5 (p < 0.05). EDS microanalysis showed the presence of Na, P, and Ca elements on the surface. CONCLUSIONS: Poly(AA-co-MA) is effective in removing the smear layer and debris without causing erosion either alone or with Ca(OCl)2.

The preliminary research of PSM on the inhibition of HIV-1 / 重庆医学

Objective To investigate the inhibiton effect of 4‐styrenesulfonic acid‐co‐maleic acid(PSM ) in HIV‐1 .Methods The inhibition effect of different doses of PSM on HIV‐1 in susceptible cells GHOST (3) X4/Hi5 was observed by Luciferase ,and so did the inhibitory effect of PSM on JR‐FL、HXB2、CNE6 ,CNE30 ,CNE50 ,CNE55 .The cellular toxicity of PSM on the VK2/E6E7 was also evaluated by CCK8 kit .The transcript level of tight junction proteins (ZO‐1 ,E‐cadherin and Occludin) of HEC‐1‐A were analyzed by qRT‐PCR .And then observed the effect of PSM on expression of genitourinary epithelial cells HEC‐1‐A ,so we could e‐valuated the effect of integrity of local mucosal indirectly .Results The results showed that PSM exhibited potent antiviral activity against a broad spectrum of HIV‐1 major isolates with different genotypes and biotypes (EC50 value of JR‐FL ,HXB2 ,CNE6 , CNE30 ,CNE50 ,CNE55 were 5 .78 ,0 .77 ,1 .85 ,3 .15 ,1 .70 ,2 .27 μg/mL respectively) .Meanwhile ,it had less cytotoxicity on VK2/E6E7 .qRT‐PCR showed that no obvious restrain effect on expression of ZO‐1 was observed and PSM increased the level of tran‐scription of E‐cadherin and Occludin .Conclusion PSM may be a potential agent for the prevention of HIV‐1 infection .

Potential drug delivery system: study of the association of a model nitroimidazole drug with aggregates of amphiphilic polymers on aqueous solution

This study evaluated the association of N-hexyl-2-methyl-4-nitroimidazol, a model drug, to aggregates formed by anionic polyelectrolytes on aqueous solution. The alternating copolymers of maleic anhydride and N-vinyl-2-pyrrolidone were synthesized and then modified by reaction of the anhydride groups with aliphatic amines and alcohols of varying length of the alkyl chain. The partition of the model drug between water and the hydrophobic microdomains provided by the copolymers was studied using the pseudo-phase model to determinate the distribution coefficient K S, and the standard free energy of transfer ∆µ°t. The results indicate that all copolymers assessed are potential pharmaceutical reservoirs of the model drug. Nevertheless, the solubility of N-hexyl-2-methyl-4-nitroimidazol on the polymeric solutions is independent from the length of the alkyl chain of the copolymer.

Realizou-se estudo sobre a associação da N-hexil-2-metil-4-nitroimidazol, fármaco modelo, aos agregados formados por polieletrólitos aniônicos em solução aquosa. Os copolímeros alternados de anidrido maléico e N-vinil-2-pirrolidona foram sintetizados e, em seguida, modificados pela reação dos grupos de anidrido com aminas e álcoois alifáticos de duração variável da cadeia alquílica. A partição do fármaco modelo entre a água e os microdomínios hidrofóbicos fornecido pelos copolímeros foi estudada usando o modelo de pseudo-fase, a fim de determinar a distribuição do coeficiente K S e a energia livre padrão de transferência ∆µ°t. Os resultados indicam que todos os copolímeros avaliados são potenciais reservatórios farmacêuticos do fármaco. No entanto, a solubilidade do N-hexil-2-metil-4-nitroimidazol sobre as soluções poliméricas é independente do comprimento da cadeia alquílica do copolímero.