Analysis of factors affecting stability of high concentration VHH-Fc fusion protein / 中国生物制品学杂志

@#Objective To explore the factors affecting the stability of high concentration variable domain of heavy-chain antibody-Fc(VHH-Fc) fusion protein.Methods Three groups of forced degradation experiments,shaking,light and 40℃ high temperature were set up.Differential scanning fluorimetry,dynamic light scattering(DLS) and ultra performance liquid chromatography-mass spectrometry(UPLC-MS) were used to detect the effects of the three forced degradation conditions on the conformational stability,colloidal stability,average hydrodynamic diameter and post-translational modifications of high concentration VHH-Fc fusion protein.Results Under the light condition,the onset temperature of unfolding(T_(onset)),melting temperature(T_m) and aggregation onset temperature(T_(agg)) of high concentration VHH-Fc fusion protein decreased the most,and the oxidation ratio of Met160 and Met266 increased significantly.Under the condition of shaking,the variation of the diffusion interaction parameter(k_D) and the average hydrodynamic diameter was the largest.Conclusion Light can significantly reduce the conformational stability of high concentration VHH-Fc fusion protein and induce methionine oxidation.Shaking has the most significant effect on its colloidal stability and promotes aggregation.

Silver nanoparticles-resistance of HeLa cell associated with its unusually high concentration of α-ketoglutarate and glutathione / 生物工程学报

Silver nanoparticles (AgNPs) is known as one of the most valuable metal nanoparticles in antibacterial and anticancer application. AgNPs-resistant bacteria has been documented, but it is unclear whether cancer cells can also escape the anti-cancer effect of AgNPs. In this study, we aimed to investigate this phenomenon and its underlying mechanism. The antibacterial activity and cytotoxicity of AgNPs were measured in the presence of HeLa cell metabolites. The status of AgNPs in the system associated with metabolites were characterized by UV-Vis, Zetasizer Nano ZS, and transmission electron microscopy. Non-targeted metabolomics was used to reveal the metabolites components that bind with AgNPs. HeLa cells were injected intraperitoneally to establish the tumor-bearing mice model, and the stability of AgNPs in mice serum was analyzed. The results manifested that HeLa cell metabolites inhibited the anticancer and antibacterial effects of AgNPs in a dose-dependent manner by causing AgNPs aggregation. Effective metabolites that inhibited the biological activity of AgNPs were stable in 100 ℃, insoluble in chloroform, containing sulfur elements, and had a molecular weight less than 1 kDa in molecular weight. There were 115 compounds bound with AgNPs. In vitro experiments showed that AgNPs aggregation occurred only when the concentration of α-ketoglutarate (AKG) and glutathione (GSH) together reached a certain threshold. Interestingly, the concentration of AKG and GSH in HeLa cellular metabolites was 10 and 6 times higher than that in normal cervical epithelial cells, respectively, which explained why the threshold was reached. Furthermore, the stability of AgNPs in the serum of tumor-bearing mice decreased by 20% (P < 0.05) compared with the healthy mice. In conclusion, our study demonstrates that HeLa cells escaped the anti-cancer effect of AgNPs through the synergistic effect of AKG and GSH, suggesting the need to develop strategies to overcome this limitation.

Tumor-targeting intravenous lipid emulsion of paclitaxel:Characteristics,stability,toxicity,and toxicokinetics / 药物分析学报

Lipid nanoemulsions are promising nanodrug delivery carriers that can improve the efficacy and safety of paclitaxel(PTX).However,no intravenous lipid emulsion of PTX has been approved for clinical treatment,and systemic safety profiles have not yet been reported.Here we outline the development of a PTX-loaded tumor-targeting intravenous lipid emulsion(PTX Emul)and describe its characteristics,colloidal stability,and systemic safety profiles in terms of acute toxicity,long-term toxicity,and tox-icokinetics.We also compare PTX Emul with conventional PTX injection.Results showed that PTX Emul exhibited an ideal average particle size(approximately 160 nm)with narrow size distribution and robust colloidal stability under different conditions.Hypersensitivity reaction and hemolysis tests revealed that PTX Emul did not induce hypersensitivity reactions and had no hemolytic potential.In addition,where the alleviated systemic toxicity of PTX Emul may be attributed to the altered toxicokinetic characteristics in beagle dogs,including the decreased AUC and increased plasma clearance and volume of distribution,PTX Emul alleviated acute and long-term toxicity as evidenced by the enhanced the median lethal dose and approximate lethal dose,moderate body weight change,decreased bone marrow suppression and organ toxicity compared with those under PTX injection at the same dose.A fundamental understanding of the systemic safety profiles,high tumor-targeting efficiency,and superior antitumor activity in vivo of PTX Emul can provide powerful evidence of its therapeutic potential as a future treatment for breast cancer.

Encapsulación de un filtro solar (avobenzona) en liposomas / Encapsulation of a sunscreen (avobenzone) in liposomes

El objetivo de este trabajo fue comprobar la optimización de la encapsulación de avobenzona en liposomas, y evaluar si constituye una barrera física de protección contra la fotodegradación de avobenzona en presencia de octilmetoxicinnamato. Se aplicó un diseño experimental para optimizar los procesos de encapsulación. Los resultados obtenidos mostraron un aumento significativo en la eficiencia de encapsulación al encontrar una relación óptima del agente encapsulante con el agente a encapsular y las interacciones apropiadas entre los factores evaluados. Los valores obtenidos en la eficiencia de encapsulación están alrededor de un 90,00 por ciento y el tamaño logrado fue de 9,156 mm. La fotoestabilidad de la avobenzona en presencia del filtro solar UVB, octilmetoxicinnamato, mejoró al estar encapsulado en liposomas con un porcentaje de degradación del 22,07 por ciento contra un 32,96 por ciento de la avobenzona sin encapsular, y la estabilización coloidal de la dispersión de liposomas mejoró con la utilización de carbopol 940 al 1,00 por ciento. En conclusión, la encapsulación de avobenzona en liposomas al usar isolecitina se logra con alta eficiencia, y se comproba que la degradación de la avobenzona promovida por la luz disminuye al estar encapsulada, aun en presencia de octilmetoxicinnamato.

This study was aimed at confirming the optimization of Avobenzone encapsulation in liposomes, and at evaluating whether this is a physical barrier to protect AVO from photodegradation in presence of octylmetoxycinnamate or not. An experimental design served to optimize the processes of encapsulation. The results showed a significant increase in the encapsulation efficiency since optimal relationship between the encapsulating agent and the agent to be encapsulated, as well as adequate interactions among the studied factors were found. The values of encapsulation efficiency were roughly 90.00 percent and the particle size obtained was 9.156 mm. The Avobenzone photostability in presence of UVB filter octylmetoxycinnamate improved when being encapsulated in liposomes, with a degradation percentage of 22.07 percent against 32.96 percent of the non-encapsulated, and the colloidal stabilization of liposomal dispersion improved with the use of 1.00 percent Carbopol 940. It can be concluded that the encapsulation of avobenzone in liposomes using isolecitine is highly efficient, and it is confirmed that Avobenzone photodegradation decreases when it is encapsulated, regardless of octylmetoxycinnamate.