| Camptotheca acuminata Decne is a plant belonging to the blueberry family and the genus Camptotheca,which is a tall deciduous perennial tree unique to China.Camptothecin(CPT)and 10-Hydroxycamptothecin(10-HCPT/HCPT)are indole alkaloids contained in Camptotheca acuminata.As natural ingredients with highly effective anti-cancer effects,CPT and HCPT have a broad spectrum of anti-cancer effects and are clinically used to treat primary liver cancer,gastric cancer,lung cancer,rectal cancer,bladder cancer,etc.CPT and HCPT are difficult to dissolve in water and have low bioavailability,resulting in instability of drug molecules during the internal circulation process,time-consuming and laborious extraction processes,lengthy operating techniques,and the need for a large amount of solvents.The final target molecules are thermally decomposed at continuous high temperatures,which poses significant difficulties for the large-scale production of CPT and HCPT.In this study,Camptotheca acuminata seeds,which are easy to store,were selected as the extraction raw material to establish a green and efficient extraction method that combines modern extraction technology with green solvents,has simple operation,high efficiency,and low energy consumption.On this basis,a bionic drug delivery system with“homing effect”was prepared.The in vitro and in vivo anticancer activity of the drug was evaluated,and the specific research results are as follows:(1)Ultrahigh pressure(UHP)combined with sodium lauryl sulfonate(SLS)aqueous micelle media was used to simultaneously extract two insoluble alkaloids,CPT and HCPT,from Camptotheca acuminata seeds.The optimal extraction process for CPT and HCPT was obtained by single factor and response surface methodology.The concentration of SLS was 60mg/m L,the material liquid ratio was 1:25,the pressure holding time was 3.9 min,and the extraction pressure was 110 MPa.The extraction rates of CPT and HCPT were 82.68±1.2%and 89.02±1.2%,respectively.The purity of CPT and HCPT was 94.51±0.22%and 96.76±0.29%respectively after decolorization,solid-liquid extraction,column chromatography,and reverse solvent recrystallization purification.Research shows that this extraction method has good stability and high repeatability.(2)Studies on the energy consumption,extraction rate,and extraction mechanism of two insoluble alkaloids by traditional extraction methods such as ultra-high pressure extraction,Soxhlet extraction,ethanol homogenization,and microwave extraction.The results show that UHPE has the characteristics of high extraction efficiency and low energy consumption.The extraction rate of ultra high pressure extraction(UHPE)is comparable to that of Soxhlet extraction,ethanol homogenate extraction,microwave extraction,and ultrasonic extraction,but the energy consumption is the lowest,only 4.68×10~3 J/mg(Calculate based on the total amount of CPT and HCPT extracted)and significantly reduced extraction time.The unit energy consumption for Soxhlet extraction and ethanol homogenization extraction were 3.29×10~6 J/mg and 3.24×10~6 J/mg,respectively,and the unit energy consumption for microwave extraction and ultrasonic extraction were 1.09×10~6 J/mg and 1.07×10~6 J/mg,respectively.Studies on density functional theory(DFT)and mass transfer kinetics have verified the mechanism of ultra-high pressure binding micelle media extraction.SLS combines with CPT and HCPT through intermolecular hydrogen bonds and electrostatic forces to form supramolecules and fold them into micelles,effectively extracting CPT and HCPT from camptotheca acuminata seeds.The combination of forces increases the solubility of the target component in the mixed system.Extracting the target active ingredient from plant tissue consists of two simultaneous processes,rapid washing and slow diffusion.High pressure and efficient fragmentation of the substructure of camptotheca acuminata seed cells,and rapid release of the active ingredient from the endosperm.(3)Hydroxycamptothecin(HCPT)and bletilla striata polysaccharide(BSP)were chemically coupled with succinic anhydride to prepare a hybrid biomimetic shell of liposome tumor cell membrane coated with nanoparticles of prodrug micelles,and biomimetic drug delivery system(T-Lipo-HCPT-BSP)was finally obtained.The successful synthesis of HCPT and BSP was confirmed by FTIR,~1HNMR,UV,XRD,DSC,and TG characterization.The critical micelle concentrations of the obtained samples were 0.004(6:1),0.008(5:1),0.063(4:1),0.080(3:1),0.360(2:1),0.507(1:1),0.725(0.5:1),and 1.212 mg/m L(0.1:1),respectively.Based on the optimization of drug loading and yield,HCPT:BSP=3:1 was ultimately selected as the optimal synthesis ratio.Characterization by DLS,Zeta potential,and SEM confirmed that the prepared prodrug micelle nanoparticles had a uniform particle size,good morphology,and an average particle size of 210±2.3 nm.At the same time,the liposome tumor cell membrane hybrid biomimetic shell was coated on the exterior of the prodrug micelles.The successful coating of the biomimetic shell was demonstrated by TEM and SDS PAGE characterization by selecting the prodrug micelles with high drug loading,high yield,and a critical micelle concentration of 0.0804 mg/m L(3:1).(4)The stability and hemolysis experiments of T-Lipo-HCPT-BSP in PBS buffer under physiological conditions have demonstrated the safety of intravenous injection of T-Lipo-HCPT-BSP.The in vitro release and cell uptake experiments further demonstrated that T-Lipo-HCPT-BSP can be released and accumulated under tumor environmental conditions,and enter cells in different ways to inhibit tumor cell angiogenesis,thereby inhibiting tumor growth.In the PBS buffer solution under physiological conditions,the nanoparticles did not exhibit significant aggregation,the particle size tended to stabilize,and there was no hemolysis,which demonstrated that T-Lipo-HCPT-BSP could be administered intravenously.HCPT-BSP and T-Lipo-HCPT-BSP can be ingested through cellular endocytosis pathways mediated by reticulin,caveolin,and energy consumption,thereby entering intestinal epithelial cells.Due to the increase in particle size,some particles of T-Lipo-HCPT-BSP can also enter the cells through macrocytosis.The chicken embryo chorioallantoic membrane(CAM)experiment found that BSP,HCPT,HCPT-BSP,and T-Lipo-HCPT-BSP all had an inhibitory effect on angiogenesis,and the effect of synergistic administration was greater than that of single administration.In the T-Lipo-HCPT-BSP group,blood vessels generally became thinner,vascular morphology was abnormal,and complete blood vessels were not formed,demonstrating the potential of the synergistic effect of HCPT and BSP in effectively inhibiting tumor growth.T-Lipo-HCPT-BSP releases different amounts of drugs in different p H buffers,with the cumulative release of drugs reaching 84.67%at a maximum of 120 h in an acidic environment simulating the tumor site.The cumulative release of drugs from T-Lipo-HCPT-BSP is higher than that from the HCPT-BSP group,which is also confirmed by the Caco-2 endocytosis experiment.The average fluorescence intensity detected in HCPT-BSP and T-Lipo-HCPT-BSP is much higher than that of free HCPT at 1 h and 4 h,The prodrug T-Lipo-HCPT-BSP,which is coated with a biomimetic cell membrane,is more easily absorbed by cells and exhibits a high time dependence.This is mainly due to the good apparent permeability coefficient of T-Lipo-HCPT-BSP.Compared with free HCPT,the apparent permeability coefficients of HCPT-BSP and T-Lipo-HCPT-BSP are 4.15 and 8.61 times higher than those of the original drug,respectively.(5)To investigate the antitumor effects of HCPT-BSP and T-Lipo-HCPT-BSP in vivo and in vitro.The ability of T-Lipo-HCPT-BSP to exert cytotoxicity,promote cell apoptosis,inhibit cell cycle,prevent cell migration,and target tumor sites to inhibit tumor growth in tumor bearing mice has been verified through in vitro and in vivo mouse experiments.The HCPT-BSP and T-Lipo-HCPT-BSP groups showed higher cytotoxicity to LLC cells and Caco-2 cells in vitro than the original HCPT group.Moreover,due to the homologous cell membrane of the coated LLC cells,T-Lipo-HCPT-BSP exhibited significantly greater cytotoxicity to LLC cells than to Caco-2 cells.The increased cytotoxicity also further demonstrates that LLC cells have a better uptake of T-Lipo-HCPT-BSP,enabling effective accumulation of drugs in cells.In addition,HCPT is a cycle specific drug.Cell cycle experiments show that the high-dose T-Lipo-HCPT-BSP treatment group can block LLC cells at 43.01±2.1%in the S phase and10.27±2.6%in the G2/M phase,thereby inducing apoptosis.T-Lipo-HCPT-BSP has a significant inhibitory effect on the migration ability of LLC cells.Compared with the blank control group(calculated at 100%cell migration rate),the migration rate of T-Lipo-HCPT-BSP cells of 100μg/m L is only 9.26%.In addition,the ability of T-Lipo-HCPT-BSP to induce apoptosis was further demonstrated in the flow cytometry investigation of cell apoptosis.After36 h of co incubation with the drug,the late apoptosis rate of the T-Lipo-HCPT-BSP treatment group was 95.68%higher than that of the HCPT original drug(45.03%).The above cell experimental results have also been reflected in the near infrared fluorescence distribution of tumor bearing mice and the therapeutic experiments of tumor bearing mice,proving that T-Lipo-BSP-HCPT can better target tumor sites,overcome the multidrug resistance(MDR)of tumor drugs during chemotherapy,and then distribute and accumulate in tumor sites.After 21days of treatment,compared with the blank saline group,the tumor inhibition rate of the T-Lipo-HCPT-BSP/H group in tumor bearing mice was as high as 89.9%,and the therapeutic effect was better than that of the HCPT technical drug group and the HCPT-BSP group.The results of H&E section staining experiment showed that the high-dose group had no significant toxicity to the visceral tissues of tumor bearing mice while achieving high antitumor activity,and had the characteristics of safety and low toxicity.Based on the above experimental results,T-Lipo-HCPT-BSP,which can play a synergistic role in drug delivery,as a drug delivery system with“homing effect”,has a high application prospect and clinical value. |
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