| Breast cancer is a malignant tumor arising from breast epithelial tissue.It is one of the most common malignant tumors that harm women's health in recent years.For the treatment of breast cancer,the use of surgical treatment and chemotherapy,or a variety of ways combined treatment.In the process of chemotherapeutic drug development,most drugs are difficult to be absorbed in the body due to their poor water solubility and low bioavailability.Therefore,nanotechnology has been used to improve the chemical and pharmacokinetic properties of drugs,thereby improving the efficacy of drug therapy.The insoluble drugs were prepared as nanosuspensions to improve the stability of the drugs in vivo and increase the circulation time of the drugs in the blood so as to achieve continuous drug release in vivo.The solubility,pharmacokinetics,biodistribution and pharmacodynamics may be affected by the size of nanocrystals.The size of intravenous nanocrystals is generally less than 1 ?m.However,the particle size is too small and the dissolution rate of the drug is too fast.The drug behaves like a solution in vivo,leading to the excessive clearance rate in vivo.If the particle size is too large,it is considered not suitable for the further development of nanomaterials.It does not have the advantages of nanomaterials,and the dissolution rate is too slow,it quickly accumulates in the reticuloendothelial system,and it is easy to be cleared by the mononuclear macrophage system.At present,there are many studies on the size of nanocrystals for drug absorption,distribution and drug efficacy in vivo,while there are few reports on the comprehensive and systematic study of nanosuspensions particle size on drug fate in vivo.Betulinic acid(BA)was selected as the model drug,and BA was a pentacyclic triterpenoid compound,which is a natural and safe anti-tumor drug and has a good therapeutic effect on a variety of cancers such as breast cancer,melanoma and ovarian cancer.But due to its poor solubility in water and blood circulation time is short,severely restricted the clinical application,therefore,the preparation of betulinic acid nanosuspensions with different particle sizes(BA/NSs)agent to improve the delivery efficiency in order to maximize of BA antitumor efficacy,different particle size of BA/NSs combined Taxol(?)achieves the expected effect of the treatment of breast cancer.Taxol(?)is a first-line drug for the treatment of breast cancer and has a good effect on the treatment of breast cancer.However,the addition of Criophore EL to the preparation is prone to allergic reactions and hepatorenal toxicity,which severely limits patient compliance with Taxol(?).BA/NSs combination with Taxol(?)can not only improve the accumulation of drugs in tumor cells,but also reduce the hepatorenal toxicity caused by allergic reactions caused by Taxol(?),thus achieving the synergistic and detoxification effect on breast cancer.In this study,the efficient green preparation method?high-pressure homogenization method was used to prepare BA/NSs with different particle sizes by changing the homogenization pressure and the number of homogenization cycles,using P188 and lecithin as the co-solvent.The particle sizes of BA/NS(160 nm),BA/NS(400 nm),and BA/NS(700 nm)were 161.2±1.9 nm,406.5±17.1 nm and 722.9 ± 73.7 nm,respectively.PDI was less than 0.3,Zeta potential was more than-20 mV,and RDI after lyophilization was less than 110%,indicating that there was no significant difference in particle size before and after lyophilization,and PDI and potential did not significant change.The stability of dilution and placement was good,and the nanosuspensions sizes were observed to be evenly distributed under transmission electron microscopy,all of which presented a long-rod shape and no adhesion.DSC and XRD results showed that the freeze-dried BA/NSs powder preparations with different particle sizes were all in amorphous state,and FTIR test results showed that there was no chemical reaction between the BA drugs and the auxiliary materials,which further indicated that the preparations were relatively stable.In vitro release experimental results showed that compared with the free BA,BA/NSs prepared with different particle sizes could significantly improve the release rate of BA,among which the highest BA release rate was found in BA/NS(160 nm),and the highest cumulative drug release rate was found in BA/NSs,and the BA release rate decreased as the particle size increased.In vitro cytological studies investigated the effects of BA/NSs with different particle sizes combined with Taxol(?)on the toxicity and apoptosis of mouse breast cancer 4T1 cells,and further studied the intracellular uptake of BA/NSs with different particle sizes and the uptakemechanism.The cytotoxic effect of BA/NSs combined with Taxol(?)with different particle sizes was stronger than that of free BA,and the uptake of BA by 4T1 cells increased with the decrease of BA/NSs particle sizes.BA/NS(160 nm)entered the cell through the clathrinid-mediated,and BA/NS(400 nm)was absorbed by the cell through two pathways caveolin-mediated and micropinocytosis.The BA/NS(700 nm)with the maximum particle size entered the cell through micropinocytosis.BA/NSs with different particle sizes increased the uptake of BA by 4T1 cells in different mechanisms to improve the anti-tumor effect.Pharmacokinetic experiment was carried out to investigate the pharmacokinetic characteristics of free BA and different particle sizes BA/NSs in rats.The half-life(T1/2)of BA/NS(160 nm),BA/NS(400 nm)and BA/NS(700 nm)were significantly prolonged,and AUC0-? were increased by 1.59-,1.20-and 1.19-fold,respectively.After the preparation of BA/NSs,the circulation time of the BA in rats can be prolonged,and the smaller the particle size,the higher the drug accumulation and the slower the drug clearance rate,and the significantly improved bioavailability in vivo.The tumor model of in situ breast cancer mice was established,and the drug distribution of BA/NSs with different particle sizes in vivo showed that compared with free BA,the BA content in tumor tissues at BA/NS(160 nm),BA/NS(400 nm)and BA/NS(700 nm)was 4.42-,2.62-and 2.02-fold higher,respectively.Different particle size of BA/NSs can improve the drug into tumor tissue,and with the decrease of the particle size distribution in the tumor cumulants.In addition,BA/NSs with different particle sizes were less accumulation in liver and kidney tissues than other groups,which could improve the hepatorenal toxicity caused by Taxol(?).The experimental results of tissue distribution fully demonstrated that the nanosuspensions could enter tumor cells in different ways by reducing the particle size,and BA/NSs with different particle size could increase the drug accumulation in tumor tissues.The mouse model of 4T1 in situ breast cancer was established to study the pharmacodynamics of free BA drugs,Taxol(?)and different preparations in vivo.The results showed that the tumor inhibition effect of free BA and Taxol(?)in vivo was relatively weak,while the tumor inhibition effect order of the combined treatment of different preparations in vivo was BA/NS(160 nm)+Taxol(?)>BA/NS(400 nm)+Taxol(?)>BA/NS(700 nm)+ Taxol(?).The results were consistent with the results of cytological evaluation in vitro.Immunohistochemical study results show that BA/NSs and Taxol(?)combination,could inhibit the Bcl-2 factor expression,enhance the expression of Bax factor,inducing tumor cell apoptosis,at the same time,activate the NF-?B signaling pathways,cut is associated with tumor cell migration and invasion matrix metalloproteinases(MMP-2 and MMP-9)expression and inhibiting tumor cell migration.The safety evaluation in vitro showed that the Taxol(?)group was prone to induce acute liver and kidney inflammation and had a serious toxic effect.However,BA/NSs combined with Taxol(?)showed no obvious damage to the main tissues(heart,liver,spleen,lung and kidney),no adverse reactions,no impact on liver and kidney functions,without hepatorenal toxicity,the combined treatment was highly safe. |
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