The Anti-tumor Research Of Albumin-bound Paclitaxel Nanoparticle By Kupffer Cell Uptake Saturation Mechanism

In the last decade,it has been found that the median delivery efficiency of nanoparticles to solid tumors remained at 0.7%.This low value seriously affects the transformation and application of nanomedicine in clinical practice.Most nanoparticles are trapped in Mononuclear phagocyte system（MPS）,85%of which is Kupffer cell.In 2020,Warren C.W.Chan’s team at the University of Toronto,Canada,published a study on the delivery mechanism of nanoparticles in Nature Materials（IF=38.6）.They found that there were kinetic limits and saturation thresholds in the course of Kupffer cell uptake nanoparticles.Beyond this saturation threshold,the liver clearance of the nano-drug was decreased nonlinearly,the circulation time of nano-drugs was prolonged,the efficiency of nano-drugs delivery was increased,and the accumulation of nano-drugs in the tumor was increased in the tumor-bearing mice.At the same time,a new concept of nano-quantitative,particle concentration,that is,the number of nanoparticles per milliliter of nano-solution is proposed.Therefore,the uptake of Kupffer cell is temporarily saturated by blank nanoparticles without drug loaded,which can increase the delivery efficiency of drug-loaded nanoparticles to tumors and enhance their anti-tumor effects.Based on the newly discovered mechanism of Kupffer cell saturation,in our study,we investigated the saturation thresholds of poly-lactic-co-glycolic acid（PLGA）blank nanoparticles and bovine serum albumin（BSA）blank nanoparticles in RAW264.7 cells in vitro,respectively.The lowest injection dose and saturation time of blank nanoparticles by which the state of liver reach saturation in tumor-bearing mice were studied.In addition,the phenomenon of macrophage and liver uptake saturation were characterized.At last,it was evaluated systematically that the therapeutic efficacy and safety of blank nanoparticles combined with albumin-bound paclitaxel（ABP）against breast cancer via using the method of factorial analysis of variance.This research is mainly divided into three chapters.The first chapter is the construction and characterization of blank nanoparticles.In this chapter,the formulation of blank nanoparticles was optimized by single factor analysis;the blank PLGA and BSA nanoparticles were successfully prepared by the optimal formulation;the particle size,Zeta potential,morphology and the particle concentration of the blank nanoparticles were characterized.PLGA blank nanoparticles were prepared by emulsion-solvent evaporation method with the particle size of（78.9±0.67）nm and the potential of（-37.1±1.63）m V,and BSA blank nanoparticles were prepared by solvent removal-heat curing method,the particle size was（132.2±0.85）nm and the potential was（-20.4±0.50）m V.In order to achieve liver saturation adequately,BSA blank nanoparticles are similar in size to ABP and can meet the experimental requirement of imitating the uptake of ABP by Kupffer cell.In morphology,the two kinds of blank nanoparticles were spherical in shape,and the diameter detected by transmission electron microscopy（TEM）was consistent with the diameter detected by hydration.Finally,the particle concentration of blank nanoparticles was detected by nanoparticle tracking analysis（NTA）technique.The particle concentration of PLGA and BSA were 3.2×10¹² and 2.4×10¹² particles/m L,respectively.In the second chapter,the saturation threshold of macrophage uptake nanoparticles in vitro and in tumor-bearing mice was studied in vitro and in vivo.In vitro,the uptake saturated threshold of blank nanoparticles by RAW264.7 cells was detected.The uptake saturation of blank nanoparticles was characterized by the fluorescence intensity of macrophage uptake Nile-red labeled blank nanoparticles,so that a standard curve of fluorescence intensity-particle number was established.A series of concentration gradient Nile-Red labeled blank nanoparticles were given to macrophages to obtain the saturated fluorescence intensity taken up by macrophages.Fluorescence intensity thresholds and standard curves were used to obtain that the number of PLGA and BSA blank nanoparticles taken up by a RAW264.7 cell was 88,000 and 23,000,respectively.Furthermore,CCK-8 method was used to detect the viability of RAW264.7 cells.It was found that blank nanoparticles of different concentrations had no toxic or inhibitory effect on the viability of RAW264.7 cells,but promoted cell proliferation.Finally,saturation phenomena of RAW264.7 cells was further confirmed via using Fluorescence microscope and confocal laser scanning microscopy（CLSM）.In vivo,Di R was used to trace the blank nanoparticles.The distribution of the blank nanoparticles in the tumor-bearing mice,the threshold of liver uptake fluorescence intensity and the peak time were observed.The results showed that the liver of tumor-bearing mice reached saturation state after injection of 4.8×10¹¹PLGA blank nanoparticles for 5 minutes and 25 minutes after injection of 3×10¹¹BSA blank nanoparticles,respectively.In the third chapter,we studied the anti-breast cancer effect and safety of liver-saturated dose of blank nanoparticles combined with ABP.This chapter is divided into two parts:anti-tumor therapy research and safety analysis.We designed a 2×3 factorial analysis of variance study based on different levels of cross combinations of ABP factor and blank nanoparticle factor.Firstly,a 4T1 mouse model of breast cancer in situ was established.Subsequently,the growth curve of tumor volume,the survival curve of mice,the results of tumor weight,and the results of tumor pathology showed that the liver-saturated dose of blank nanoparticles combined with ABP had a stronger inhibitory effect on tumor.The results of pathological analysis of the major organs of the mice,the changes in body weight of the mice and biochemical analysis of the plasma showed that the blank nanoparticles had a good biocompatibility at the saturated dose in vivo.Compared with the ABP alone group,the combination of liver-saturated dose of blank nanoparticles with ABP reduced the damage of liver and kidney function.Therefore,the liver-saturated dosage of blank nanoparticles is safe and has a protective effect on liver and kidney function.In summary,PLGA and BSA blank nanoparticles were successfully prepared under the experimental conditions.In RAW264.7 cells and tumor-bearing mice,the threshold dose and saturation time of macrophage uptake of blank nanoparticles were obtained.The experimental results showed that the threshold dose of blank nanoparticles was safe.Blank nanoparticles combined with ABP have stronger tumor inhibition and alleviated the damage of ABP on liver and kidney function comparing with ABP alone.The results showed that tumor-bearing mice and RAW264.7 cells could be saturated by blank nanoparticles.When liver uptake was saturated,the delivery efficiency of ABP to tumor was increased and the anti-tumor effect was enhanced.At the same time,the liver-saturated dose of blank nanoparticles did not damage the main organs and liver and kidney function of the tumor-bearing mice,but alleviated the liver and kidney injury during the use of ABP.This study provides a new design idea for improving the efficiency of nano-drug delivery and a new research method for studying the Kupffer cell saturation threshold.