Study On The Targeted Delivery Of GLA Loaded Nanoparticles To Breast Cancer Cells And Breast Cancer Stem Cells In Vitro

Breast cancer(BC)is the most common malignant tumors of chinese women,Every year chinese primary BC and the number of deaths accounted for 12.2% and 9.6% of the world.Among them,10-14% of breast cancer patients were diagnosed with triple negative breast cancer(TNBC),this type of BC with high mitotic rate,lymphocytes are highly invasive,high nuclear grade and larger tumor size,also associated with early diagnosis have lymph node metastasis and about 15% of patients will appear brain metastases.In the development,drug resistance and relapse,tumor stem cells(CSCs)are the main influencing factors.In view of the above problems,we present new idea by using good biocompatible carrier(nanoliposomes,human serum albumin nanoparticles)by loading traditional Chinese medicine which can anti tumors.,The entirely tumor killing effect and effect of decreasing of toxic and side effect would obtained through passive target effect to make drug enriched in breast cancer cells and breast tumor stem cells.Glaucocalyxin A(GLA)is a diterpenoid compounds with a variety of biological activity,according to reports,GLA have inhibitory effect on lots of tumors.In this research,GLA loaded nanoliposomes and human serum albumin nanoparticles were prepared to target delivery GLA to breast tumor cells and breast tumor stem cells.UV wavelength scanning method was used to determine the GLA and High Performance Liquid phase Chromatography(HPLC)was successfully used to establish detection method for GLA detectation in vitro,GLA in the concentration range from 7.82 to 1000 g·ml-1 shows good correlation with peak area(R2 = 0.99992),and linear regression equation is A= 30910 C + 86556.This method has a good specificity to GLA determination,in and between day precision and recovery rate of this method are all satisfied with the requirements of GLA detectation in vitro.The cell culture experiments such as passage,cryopreservation and thawing were processed.MCF-7 tumorsphere(MCF-7-TS)and sum149 PT tumorsphere(sum149PT-TS)were screened as breast cancer stem cells by culturing MCF-7 and sum149 PT cells in serum-free medium.Both breast cancer cell and breast cancer stem cells were identified by CD24-/CD44+ as surface marker.The optimal prescription and preparing technics of GLA nanoliposomes(GLA-LNs)were screened with single factor investigation method and orthogonal test design method.The entrapment efficiency(EE)and drug loading(DL)of GLA-LNs are determined by HPLC method,and they are 79.19±1.733% and 3.43 ±0.250%,respectively.The particle size,pd I and zeta potential are characterized with Malvern particle size analyzer,and they are 82.58±1.25 nm,0.19±0.009 and-11.86± 1.48 m V,respectively.The results of morphology show that GLA-LNs are round in shape and have uniform size.Through the comparative study with free GLA and GLA-LNs release behavior,we find that GLA-LNs have better sustained release effect and more stability.The results of confocal microscope observation show that the C6-LNs uptake of MCF-7 cells and MCF-7-TS are time dependent within 4 hours,and they have high uptake effect with C6 Nanoliposomes(C6-LNs)comparing with free C6,The cell cytotoxicity were measured by CCK-8 Kit method on MCF-7,sum149 PT cell lines and MCF-7-TS,the results show that GLA-LNs has high cell killing effect compared with free drug,and the cell killing effects of GLA-LNs and GLA is time dependent,and blank nanoliposomes have merely cell toxicity.Flow Cytometry Test Technology was used to determine the apoptosis effects on MCF-7,sum149 PT cell lines and MCF-7-TS,the result show that after drug treatment,the apoptosis effects of GLA-LNs are better than GLA;The microspheres formation rate experiments of MCF-7 cell lines and MCF-7-TS show GLA-LNs has better effect to reduce the cell differentiation and cell proliferation of stem cells.GLA human serum albumin nanoparticles(GLA-HSA-NPs)were prepared with high encapsulation efficiency by our laboratory patent successfully.By using Malvern particle size analyzer,the particle size,pd I and Zeta potential are detected as 158.33±31.37 nm,0.37±0.90,and-29.2±5.01 m V,respectively;The shape of GLA-HSA-NPs are observed by TEM,the results show that GLA-HSA-NPs are round in shape and have uniform size.The EE is measured by HPLC method and the result is 89.59±1.77%.Through the comparative studies with free GLA and GLA-HSA-NP release behavior,we find that GLA-HSA-NP has better sustained release effect in 120 h,and the cumulative released quantity in 24 h,48h and 96 h are 51.01%,66.93% and 77.45%,respectively.The uptake effects on MCF-7 cells and MCF-7-TS of C6 human serum albumin nanoparticles(C6-HSA-NPs)are observed by confocal,and C6-HSA-NPs have strong uptake effect comparing with control group.By using CCK-8 Kit method,we observe GLA-HSA-NPs have strong cytotoxicity in MCF-7 cell lines and MCF-7-TS.It is confirmed that GLA-HSA-NPs have cell apoptosis induced effect on MCF-7 cell lines and MCF-7-TS.By using microspheres formation rate experiments of MCF-7 cell lines and MCF-7-TS,we conclude that GLA-HSA-NPs have stem cell killing effect.In summary,GLA loaded nanoliposomes and human serum albumin nanoparticles can avoid the drug disadvantages such as low solubility,and they can improve the stability of drug,at the same time GLA loaded nanoliposomes and human serum albumin nanoparticles can be target delivered drug to tumours and enriched in tumor cells and tumor stem cells,which provides a new strategy for the treatment of breast cancer.