Preparation And In Vitro Biological Effects Of HPMCP / SN-38 @ PLGA / TPGS Nanoparticles

BackgroundSN-38(7-ethyl-10-hydroxy-camptothecin) is an active metabolite of irinotecan, which is pertained to the anti-cancer drug camptothecin (CPT) family. Irinotecan can not maintain stability in the liver and tumor. In vivo, only a small fraction of irinotecan, about 2%-8%, can be decomposed to SN-38. Active metabolite SN-38 is a topoisomerase I inhibitor, this inhibitory effect on topoisom erase I leads to the processes of DNA replication and translation. The toxicity of SN-38 is about 1000 times of irinotecan. Due to itself extremely hydrophobic, insoluble and low biological utilization degree that was largely limits the immediate clinical application. Therefore, the development of a drug delivery system to overcome the problems of SN-38. will be very significant and have a good prospect.In the past few decades nano drug delivery system has been widely studied. Using biocompatible, biodegradable materials to prepare nano drug delivery system can significantly improve drug water solubility and stability; reduce the toxic effects of drugs on the body. Compared with the original drug, nano drug delivery system exhibit bigger particle size, increase the accumulation of it, which can promote the uptake of the drug and improve the treatment concentration of drug in the tumor tissue. In addition, the nano drug delivery system also has the role of sustaining the release of drug in tumor site, and ensures a sustained and stable concentration of drug therapy. It is a very effective method to enhance the effect of most anticancer drugs which are difficult to dissolve.Experimental purposesIn this study, a double emulsion solvent evaporation method was used to prepare a shell and core structure bilayer HPMCP@PLGA/TPGS NPs, with an alkaline environment sensitive core and an acidic environment sensitive shell,which is used to carry the model drug SN-38.Drug loading nanoparticles are able to deliver the drug to a target location, within the cell nucleus, by means of multi-level release.In the experiment, the physical and chemical properties of the synthesized core-shell structure nanoparticles were characterized, and in vitro cell experiments were carried out to observe the uptake behavior of nanoparticles. We also conducted experiment to explore the anticancer effect of HPMCP/SN-38@PLGA/TPGS NPs.MethodsBilayer nano particles with a core-shell structure were prepared by solvent evaporation method. Using Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and dynamic light scattering instrument to detect the nanoparticles. The physical and chemical structure, surface morphology and particle size of the nanoparticles were detected. The drug loading, encapsulation efficiency and in vitro release behavior of drug loaded nanoparticles were detected by UV spectrophotometry.The effects of HPMCP NPs,HPMCP@PLGA/TPGS NPs, original drug SN-38, HPMCP/SN-38@PLGA/TPGS NPs on A549 cell survival rate were tested by WST-1 kit for detection of material safety and the cytotoxicity of drug nanoparticles. Using AnnexinV-FITC/PI kit to detect apoptosis to explore whether the cell death were caused by apoptosis, the aim is to further confirm the safety of the nanoparticles.Using two kinds of different fluorescence labeled core-shell bilayer nanoparticles, the behavior of the nanoparticles in the cell was observed by confocal microscopy.Result1.The double-layer nanoparticles which are applied to deliver SN-38 in our experiment,shown to be spherical with smooth surface, kernel nano particle size is about 40nm and the size is uniform without agglomeration.The size of double-layer nanoparticles is about 240 nm,particle size distribution is uniform without agglomeration. X-ray powder diffractometry (XRD) and fourier transform infrared (FTIR) techniques showed that the nanoparticles were successfully loaded and the drug showed a non crystalline state in the nanoparticles.The encapsulation efficiency of the inner core nanoparticles is 90.97%, and the drug loading rate is 4.95%. The encapsulation efficiency of bilayer nanoparticles is 86.91%, and the drug loading rate is 1.32%. In vitro drug release experiment showed that the inner core particles have a better release behavior in a slightly alkaline environment, however in acid environment seldom release. The bilayer nanoparticle has different release behavior as the change of pH.2.WST-1 assay results showed that the materials themselves have a low toxicity to cancer cell,carrying SN-38 bilayer nanoparticles and Compare with the original drug SN-38,the toxicity of HPMCP/SN-28@PLGA/TPGS NPs toA549 cells was significantly enhanced.Flow cytometry suggested nanoparticles can promote apoptosis of A549 cell when the concentration is higher.3.The behavior of bilayer nanoparticles inside the cell Was observed by confocal microscopy,after the bilayer nanoparticles was uptaken by A549 cell the outer shell nanoparticles were gradual degrade and release the inner core nanoparticles. Then the inner core nanoparticles migrate into nucleus, the whole process involved multi-release behavior.Conclusion:Double nano particles prepared by this experiment can directly deliver drug to the target location in the cell nucleus, enhance the solubility of SN-38 and reduce the drug efflux and degradation, improve its effective concentration, thereby reducing the toxic effects on the body and enhance the anti-tumor effect of drugs. This shows that this drug delivery strategy in nuclear targeting drug delivery is meaningful.