| BackgroundCancer is still a serious disease in human lives. In the past decade, nano-drug delivery system (NDDS) has generated great interests for targeted anticancer drug delivery because they possess several advantages. Such as its permeability, targeting, controlled drug release and increase drug solubility advantages in cancer therapy. However, most studies only deliver drugs to cancer cells cytoplasm, which have achieved their ultimate goal. But most anti-cancer drugs, such as etoposide (VP-16) or cisplatin, destroy DNA topoisomerase in nucleus and induce apoptosis. These drugs as p-gp substrate with free in cell cytoplasm, and these drugs are likely to be discharged outside of MDR cells, thereby reducing the anti-cancer effect. In addition, these drugs may also be weakened by the degradation or isolated by lysosomes in the cytoplasm, therefore lead to (MDR).The intranuclear NDDS delivery drugs into the nucleus have been paid more and more attention in recent years. The extracellular pH value (pHe) of normal tissues and blood is constant at pH 7.4; however, the pHe of most solid tumors ranges from 6.0 to 7.0 due to the increase in glycolytic metabolism and the decrease of blood perfusion within the tumors. Moreover, intracellular pH values (pHi) also vary, being acidic in endosomes (pH from 5.0-6.0) and lysosomes (pH from 4.0-5.0) and alkaline (pH 7.4) in the nucleus (pHn).Based on the pH gradients within cellular compartments, an effective approach to improve the efficacy of cancer therapy is to develop delivery systems that utilize pH-sensitive mixed micellesAimwe designed and prepared a novel NDDS, CS/PAA/VP-16@ TPGS/PLGA NPs, which contained a number of drug loading smaller nanoparticles chitosan/ polylactic acid copolymer nanoparticles(CS/PAA/VP-16 NPs) loaded at tocopheryl polyethylene glycol succinate (TPGS) emulsified lactic acid-glycolic acid (TPGS/ PLGA NPs) large nanoparticles, herein, VP-16 as a model drug. We have made physical and chemical characterization, verified pH sensitive multi-stage release characteristics, and studied the cytotoxicity, endocytosis and intracellular migration properties of nanoparticles and analyzed mechanisms of reversal multidrug resistance.Methods1. Physicochemical characterization:CS/PAA NPs were prepared by the dropping method. CS/PAA/VP-16@TPGS/PLGA NPs were prepared by a coaxial electro-dropping method with double emulsion (W/O/W) principle, so that CS/ PAA/VP-16 NPs loaded into the TPGS/PLGA NPs large nanoparticles. Particle size and surface potential analysis, scanning electron microscopy determination of CS/ PAA NPs, CS/PAA/VP-16 NPs, CS/PAA@TPGS/PLGA NPs, CS/PAA/VP-16 @TPGS/PLGA NPs particle size, Zeta potential and surface morphology; We verified CS/PAA@ TPGS/PLGA NPs special structure by transmission electron microscopy and confocal laser scanning electron microscope. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) to detect the physical and chemical structure of nanoparticles; Encapsulation efficiency, drug loading and pH-sensitive release characteristics in vitro were measured using a UV-Vis spectrophotometer.2. Cytotoxicity in vitro:The cytotoxicity of VP-16, CS/PAA/VP-16 NPs, CS/ PAA/VP-16@ TPGS/PLGA NPs or CS/PAA NPs and CS/PAA@ TPGS/PLGA NPs on A549/DDP were measured using the WST-1 kit. Then, we determined CS/PAA NPs and CS/PAA@ TPGS/PLGA NPs using theAnnexin V-FITC/PI apoptosis detection kit and flow cytometry.3. Cellular uptake and migration:Preparation of green fluorescent membrane probe (Dio) labeled TPGS/PLGA NPs and rhodamine-123 (Rh-123) labeled CS/PAA NPs that formatting of CS/PAA/Rh-123@TPGS/PLGA/Dio NPs. We compares the migratory process of CS/PAA/Rh-123@TPGS/PLGA/Dio NPs with free Rh-123 from uptake to nucleus distribution in MDR cell using a confocal microscope.4. Mechanisms of overcoming MDR:We prepared Dio labeled/PAA@ TPGS/ PLGA/Dio NPs and Rh-123 labeled CS/PAA/Rh-123@ TPGS/PLGA NPs, then lysosomes were labeled by red fluorescent probe or green fluorescent probes. We verified degradation of the larger TPGS/PLGA NPs and the relationship of CS/ PAANPs with lysosomes and nucleus by CLSM. We observed the ultrastructure of A549/DDP cells treated with CS/PAA@TPGS/PLGA NPs for various times by TEM. The autophagy effect of CS/PAA@TPGS/PLGA NPs on A549/DDP cells is detected by MDC.Results1. We prepared CS/PAA NPs, CS/PAA/VP-16 NPs have spherical particle size of 30 nm, and CS/PAA@TPGS/PLGA NPs, CS/PAA/VP-16@TPGS/PLGA NPs have particle size of about 300nm. The entire surface charge drug delivery system of S @ LNPs is negative, the encapsulation efficiency was 91.7%, drug loading was 1.35%. The results of Transmission electron microscopy and confocal laser showed that CS/PAA NPs loaded at TPGS/PLGA NPs. The results of XRD, FTIR further proof thesuccessful synthesis of S @ L NPs and theState of drug being innanoparticles. The release characteristics of S @ L NPs revealed that the larger TPGS/PLGA NPs were undermined and released CS/PAA NPs in an acidic environment, however S@L NPs keep stable in alkaline environment. Further studies showed that CS/PAA NPs display minimal drug leakage in an acidic environment but show enhanced drug release in an alkaline environment. All theseresults proved the pH sensitive release characteristics of S @ LNPs.2. WST-1 test results indicate that the cytotoxicity of CS/PAA@TPGS/PLGA NPs loaded with VP-16 was stronger than the cytotoxicity of the free drug. The toxicity of the material shown that CS/PAA@TPGS/PLGA NPs at concentrations of 2.0 mg/ml significantly enhanced cytotoxicity of A549/DDP cells. While CS/PAA@TPGS/PLGA NPs at the 1.0 mg/ml have a low toxicity, which is consist with the results of apoptosis experiments.3. The results of cellular uptake and migration indicate that Rh-123 loaded in the CS/PAA/Rh-123@TPGS/PLGA/Dio NPs are able to pass cell membranes into the cytoplasm, followed by released the smaller CS/PAA/Rh-123 NPs. The CS/PAA/Rh-123 NPs were capable of entering the nucleus and release Rh-123 into the nucleus. At the free Rh-123 group, there was no red fluorescence appeared in the nucleus.4. The study of mechanism shown that CS/PAA/VP-16@TPGS/PLGA NPs were internalized into MDR cells via endocytosis. Following this, the outer layerof TPGS/PLGA NPs were destroyed in the acidic environment of lysosomes, and released CS/PAA NPs. CS/PAA NPs are able to escape the lysosome into the nucleus through nuclear pores, while the fragments of TPGS/PLGA NPs were retained in the cytoplasm or lysosomes, triggering autophagy, caused by cytotoxicity.ConclusionWe prepared a novel NDDS, designated S@L NPs has the pH-sensitive release characteristics. The S@L NPs delivered the drug directly into the nucleus, thereby reversing multidrug resistance of cancer cells and reduce the side effects. |
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