Preparation Of Betulinic Acid-targeted Liposomes And Mechanism Of Anti-colorectal Canccer

Colon cancer?CRC?has attracted widespread attention as a multiple malignant tumor.The current treatment of CRC mainly relies on chemotherapy drugs with large side effects.Betulinic acid?BA?is a natural compound extracted from medicinal plants and has a significant inhibitory effect on CRC.However,the poor water solubility prevents its clinical application.Encapsulating BA in a nano drug-loading system is beneficial to improve its solubility and bioavailability.With the specific performance of the load system,it is expected to achieve targeted release of BA in the colon.This article reviews the occurrence,development and treatment of CRC,introduces the research progress of BA and nano-preparation technology,and provides a theoretical basis for the research of BA nano-formulation and its anti-CRC research.The specific research content and results of this article are as follows:?1?First,a method for accurate detection of BA content based on HPLC was established,and a series of methodological investigations was conducted to prove the accuracy and reliability of the method.The linear range is 1⁵00?g·mL^-1,The limit of detection is 0.042?g·mL^-1 and the limit of quantification is 0.18?g·mL^-1.The types of BA nano-formulations were screened from three different nano-dosage forms,Including nanoparticles?BA-NPS?,self-microemulsions?BA-SMEDDS?and liposomes?BA-LP?.The experiments found that BA-LP has the smallest particle size,the most uniform distribution,higher encapsulation rate and slower release among the three forms,so liposomes were selected as the dosage form for subsequent research.?2?The liposome BA-LP was prepared by the thin film dispersion method,and the pH targeting effect is achieved by adding Eudragit S100.The preparation conditions were optimized through single factor experiments and orthogonal experiments.The best pH-BA-LP preparation prescription was:soybean lecithin fixed at 30 mg,45 ^oC of water bath,and the dosage of BA,cholesterol,Eudragit S100,sodium cholate and IPM was 2 mg,6mg,4 mg,20 mg and 60?L,respectively.The pH-BA-LP prepared under the optimized condition has a good appearance,with an average particle size of about 90 nm.The PDI is 0.182 and the Zeta potential value is-32±5.1 mV.And the encapsulation rate was90.66%.In vitro release studies have shown that the cumulative release amount of pH-BA-LP in pH 7.4 medium is higher than that in pH 6.8 and pH 1.2 medium,and the release rate reaches 91.86%at 72 h,which is much higher than the corresponding release rate of free BA.It is proved that pH-BA-LP improves the solubility of BA in water and has a pH-targeted release effect.Stability evaluation found that pH-BA-LP can be stably stored at 4 ^oC for more than 15 days,but will rapidly deteriorate and lose stability at 60 ^oC.The preparation of pH-BA-LP into precursor liposomes by lyophilization can effectively enhance its stability.The optimized optimal lyophilization conditions are:2.5%trehalose as lyoprotectant,pre-freezing time is 8 h.Proliposomes can be stably stored at 25 ^oC for more than 30 days,and the preparation of proliposomes will not impair the pH-targeted release ability of pH-BA-LP.?3?The results of in vitro cell experiments showed that the IC₅₀ of pH-BA-LP to five CRC cells SW620,HCT116,HT29,SW1116 and CT26 and normal colon cells HCoEpiC were 56.11,43.23,59.49,56.63,66.35 and 89.11?M,respectively.It shows that the effect of the drug on CRC cells is stronger than that of normal colon cells,and the inhibitory effect of pH-BA-LP on each kinds of CRC cell is stronger than free BA.The scratch experiment found that pH-BA-LP can effectively reduce the migration rate of colon cancer cells.Western blot experiments found that BA and pH-BA-LP significantly inhibited the expression of Akt and p-Akt,and up-regulated the expression of Raptor and VDAC1 in a dose-dependent manner.Low concentration?25?M?of pH-BA-LP can up-regulate the expression levels of TLR-4 and TLR-9,but at high concentration?50¹00?M?the expression of TLR-4 and TLR-9 decreases rapidly.Examination of NFAT1 and NFAT4 protein found that the drug also showed the result of first enhancing and then inhibiting protein expression.?4?Animal experiments show that pH-BA-LP can effectively inhibit tumor growth in mice,and the tumor inhibition rate reaches 59.6%at high dose?50 mg/kg?,which is higher than free BA?49.4%?at the same dose.The organ coefficient of heart,liver,lung and kidney of mice in pH-BA-LP group had no significant difference compared with the blank control group,indicating that pH-BA-LP had higher biological safety,and the increased organ coefficient of spleen might be caused by immune enhancement.HE staining and TUNEL staining experiments found that the pH-BA-LP administration group had a higher degree of apoptosis than free BA.Flow cytometry was used to detect the proliferation and subpopulation changes of immune cells in tumor,peripheral blood and spleen of tumor-bearing mice.It was found that pH-BA-LP can increase NK cells and CD3+cells in various tissues,and CD8 in CD3 was also increased,which proves that pH-BA-LP can play an anti-cancer effect by enhancing the autoimmunity level of tumor-bearing mice.Immunohistochemistry showed that the positive infiltration rate of CD8and CD68 in the pH-BA-LP group was increased compared with the blank control,and CD163 was relatively decreased,which proved that pH-BA-LP can regulate the immune system of tumor-bearing mice.