TPGS And Folate-PEG-DSPE Stabilized Resveratrol Nanosuspensions With Long-cycling Potential Target Delivery And Reversal Of Multidrug Resistance

Resveratrol?RSV?is a active ingredient of traditional Chinese medicine,which is discovered from polygonum cuspidatum and possess good anti-oxidation and liver protection.RSV possesses more benefits for hunman health,such as anti-cell membrane lipid peroxidation,scavenging oxygen free radicals and producing protective effect on cardiovascular.Besides,RSV has more broad prospects in the clinical application,which has a significant inhibitory effect on breast cancer,lung cancer,skin cancer,colon cancer and other tumor cell proliferation.However,the poor water soluble and unstable to light and heat of RSV limits its applying.Therefore,this project intends to use nanocrystal technology to construct a high-stability and delivery-oriented RSV delivery system which not only enhances the drug solubility,but also achieves the target drug delivery and reverses the growth of tumor cells drug resistance potential.In this study,we used RSV as a model drug and distearoylphosphatidyl ethanolamine-polyethylene glycol(DSPE-PEG₂₀₀₀-NH₂)grafted onto folate?FA?to obtain polymer?folate-PEG-DSPE?as a stabilizer,combined with polyethylene glycol vitamin E succinate?TPGS?to prepare RSV nanocrystals by anti-solvent precipitation method,and constructed long-cycle and active targeting reverse drug-resistant nanocrystal system.The main contents of this study include:pre-prescription study of resveratrol,screening and preparation of prescription resveratrol nano-crystallization,physical and chemical properties of resveratrol nano-crystallization,evaluation of RSV nanocrystals in vitro and in vivo.The maximum absorption wavelength of resveratrol was determined to be 306nm based on UV-Vis spectra.Analytical methodological study results showed that the linear relationship in the range of 1.0⁶.0?g·ml^-1 was good.The results of intraday precision,daytime precision and recovery measurements showed that the relative standard deviation?RSD?was less than 2%,which was in line with the methodological requirements.RSV in different media solubility and oil-water partition coefficients were determined,the results showed that RSV was in the poor water solubility(about 28.75?g·ml^-1),and the log P is about 2.3 in positive octanol-water which indicated that the fat solubility is better.Due the lower solubility of RSV limits its application,this study uses nanocrystal technology to increase solubility and bioavailability.TPGS and folate-PEG-DSPE modified RSV nanocrystals were prepared by anti-solvent precipitation method.The results of fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy showed that folate was successfully grafted on DSPE-PEG,and finally synthesized folate-PEG-DSPE.The effects of organic solvent,stabilizer,ratio of TPGS to folate-PEG-DSPE,ratio of organic to antisolvent,the rate of anti-solvent addition in organic phase,ultrasonic time and crystallization temperature on the particle size were investigated by single factor method.The effect of drug concentration and the concentrations of TPGS and folate-PEG-DSPE on the response-value of particle size,Zeta potential and polydispersity index?PDI?were investigated by establishing a mathematical model using central composite design-response surface model?CCD-RSM?.Under the optimal process prescription,the morphology of RSV nanocrystals was observed by transmission electron microscope?TEM?and atomic force microscope?AFM?,and the particle size,particle size distribution and Zeta potential were measured by dynamic light scattering?DLS?.The experimental results showed that the RSV nanoparticles had a regular spherical shape with good dispersibility and the mean particle size of 210.32±4.86 nm,PDI of 0.106 and Zeta potential of-31.23±2.11mV.Stability results showed that RSV nanocrystals were stable within 20 days at room temperature and unstable at 4°C.The freeze-drying process of nanocrystals was investigated and screened out the best cryoprotectants based on morphology,appearance,particle size and redispersibility,and finally determined the optimal freeze-drying process.Scanning electron microscopy?SEM?results showed that the RSV nanocrystals were flaky and spherical in shape with uniform particle size distribution.The results of differential scanning calorimetry?DSC?and X-ray powder diffraction?XRPD?showed that the crystal form of RSV were changed and formed an amorphous state during the preparation of RSV nanocrystals by anti-solvent precipitation method.Solubility results showed that the saturated solubility of the freeze-dried powder was 945.87?g·ml^-1,which was about 33 times higher than the bulk drug.When the freeze-dried powder placed at room temperature for three months,the particle size increased slightly,and appearance and re-dispersibility remained good,stability improved significantly.In vitro MTT experimental results showed that TPGS and folate-PEG-DSPE were no significant toxicity on human non-small cell lung cancer A549 cells,which proved the safety of the stabilizer.Compared with the RSV solution group,RSV nanocrystals could significantly inhibit the growth of A549 cells,and the inhibitory effect is stronger.The antitumor results in vivo showed that the tumor volume of the RSV nanocrystal injection group was significantly smaller than that of the RSV solution injection group?10 mg/kg??P<0.05?,and the tumor suppression effect was obvious.It was proved that the multi-effect stabilizer formulated nanocrystals could significantly enhance the tumor inhibitory effect of RSV.In summary,RSV nanocrystals showed narrow particle size distribution which was consisted with the morphological analysis of TEM,and RSV nanocrystals showed higher stability and solubility was significantly improved.The designed stabilizers showed higher safety effect.Besides,the RSV nanocrystals showed good anti-tumor effect and targeting effect both in vivo and in vitro,meanwhile reducing the toxic and side effects,which can improve the curative effect of poorly-soluble drugs and actively target to reverse the multi-drug resistance of tumors.Of course,this study provides a new idea,which has a larger application development prospects.