Investigation Of Doxorubicin Liposome Modified With ASP8 And Folate Targeting To Bone Metastasis From Breast Cancer

BackgroundBreast cancer,a common malignancy in women,often metastasizes to bone tissue after onset,which can lead to poor clinical outcomes.Due to the specific composition of bone cel LS,uptake of chemotherapeutic drugs often occurs in non-target sites and subsequently instigates similar degree of cytotoxicity on both cancer and normal cel LS.Despite the considerable effort scholars have invested to address and bring about an in-depth understanding,they are still unable to improve the status quo.Therefore,hindering the invasiveness of cancer cel LS,effectively controlling the metastasis of cancer cel LS and improving the diagnosis and treatment of cancer metastases are the major directions researchers must focus on for improving cancer therapy in the future.Clinical treatment of breast cancer bone metastases mainly focuses on surgical resection,endocrine therapy,chemotherapy and radiotherapy.However,due to the particular pathophysiological characteristics of bones,after the spread of cancer cel LS to bones,treatment options are limited and not ideally effective,which is reflected by a poor clinical prognosis.Moreover,because bone metastases are usually characterized by multiple nodules,it is difficult to eliminate bone metastases by complete removal of thesenodules via local radiotherapy or surgical resection.For chemotherapy,due to the low blood circulation in the bones,systemic medications do not produce enough drug concentration at the bone site.In order to maintain effective chemotherapeutic drug concentrations and increase the therapeutic effect of drugs on skeletal metastatic lesions,it is usually necessary to increase the drug dose or increase the number of administrations,both of which may lead to severe systemic toxicity.Therefore,designing a drug delivery system with high selectivity and high efficiency to better deliver antitumor drugs to target cancer cel LS in bone tissue in order to achieve better therapeutic effects has become a clinical strategy to solve this problem.In this study,a dual co-modified liposome delivery system(A/F-LS)was designed using aspartic acid(ASP8)and folic acid(FR),in which ASP8 can target bone tissue while folic acid is able to further target tumor cel LS in bone tissue.This dual co-modification drug carrier can effectively deliver liposome-encapsulated chemotherapeutic drugs to tumor cel LS in bone tissue while protecting normal cel LS from damage.In vivo experiments were performed to characterize and optimize the density of A/F-LS,study its pharmacokinetics,observe its effects on animal pain and survival and evaluate its cytotoxicity in animal.In vitro experiments were equally performed to observe its effects on the biological behavior of breast cancer MDA-MB-231 cel LS.By comprehensively assessing the effect of A/F-LS on targeted treatment of bone metastases,the results of this study laid the experimental basis for clinical treatment of bone metastases in breast cancer.Objectives1.Prepare dual co-modified doxorubicin liposomes,characterize and optimize their densities to improve the targeting of DOX-A/F-LS selective transport.2.Establish a mouse model of breast cancer bone metastasis by injecting MDA-MB-231 cel LS into tibiae,study the in vivo the pharmacokinetics,toxicity and efficacy of DOX-A/F-LS in anima LS.3.Evaluate the effect of DOX-A/F-LS on the proliferation,migration and invasion of breast cancer MDA-MB-231 cel LS in vitroMethods1.Preparation and characterization of A/F-LS: DSPE-PEG2000-ASP8 and DSPE-PEG2000-folic acid were synthesized and used to prepare A/F-LS.The prepared A/F-LS was characterized by measuring the size of various liposomes with a laser particle analyzer.The intracellular and extracellular drug release of various liposomal preparations were measured by dialysis.The binding efficiency of SP8 and HA at different concentrations in liposome was studied to determine the optimal combination ratio of ASP8 to HA.2.Efficacy and toxicity of A/F-LS in vivo: A mouse model of breast cancer bone metastasis was established by iliac bone injection of MDA-MB-231 cel LS,followed by administration of an equivalent dose of 5 mg doxorubicin/kg body weight by tail vein injection.Anima LS were divided into 5 groups,namely the control,free doxorubicin,doxorubicin A-LS,doxorubicin F-LS and doxorubicin A/F-LS groups.The free doxorubicin,doxorubicin A-LS,doxorubicin F-LS and doxorubicin A/F-LS preparations were injected separately to study the pharmacokinetics.In vivo imaging of mice was performed and animal pain behavior evaluated.Toxicological studies were performed by histological and hemogram analysis to evaluate the safety of various liposomal preparations.3.In vitro targeted therapy with A/F-LS: The inhibitory effect of liposomal doxorubicin on the growth of MDA-MB-231 cel LS was detected in vitro by MTT assay.The scratch test and Transwell invasion assay were used to compare the in vitro migratory and invasive ability of breast cancer cel LS,respectively.Western blotting was used to detect the expression of proteins involved in tumor-associated signaling pathways.Results1.The observed mass-to-charge ratios of synthesized DSPE-PEG2000-ASP8 and DSPE-PEG2000-folic acid were approximately 3864.154 and 3239.984,respectively.The particle sizes of various liposomal preparations namely N-LS,A-LS,F-LS,and A/F-LS measured using the laser particle size analyzer showed that the diameter was approximately between 95.82±0.13 nm and 96.61±0.11 nm,indicating that the sizes of N-LS,A-LS,F-LS,and A/F-LS were not significantly affected by ASP8 or folic acidmodification.The binding efficiency of different concentrations of ASP8 and HA in liposomes was experimentally studied and the results showed the ability of ASP8-modified liposomes(A-LS)to bind to HA.With the increase of ASP8/lipid molar ratio in liposomal preparations,the binding amount to HA was a LSo significantly increased.The optimal densities of DSPE-PEG2000-ASP8 and DSPE-PEG2000-folate in the liposomes were 15% and 10%(molar ratio),respectively.2.Breast cancer bone metastasis mouse model was injected with free doxorubicin,doxorubicin A-LS,doxorubicin F-LS and doxorubicin A/F-LS via tail vein injection with the equivalent dose of 5 mg doxorubicin/kg body weight and blood samples were taken from the jugular vein at 0.083,0.25,0.5,1,2,3,4,6,8,12 and 24 hours after injection.The results indicated that the liposomal surface binds a sufficient amount of ASP8 and folic acid without compromising the circulatory characteristics of PEG.Data obtained from in vivo imaging indicated that the A/F-LS system is likely to achieve specific targeting of metastatic tumor cel LS in bone by drugs delivery and transport in vivo.For evaluation of pain behavior,the pain-related behaviors of mice were detected on days 4,6,8,10,12,14,16,18,20,22 and 24 after inoculation of tumor cel LS.The results indicated that A/F-LS treatment significantly reduced the frequency of animal retraction(3.25 ± 0.63)and reduced the animal pain leve LS.From histological and hematological examination indicators,the A/F-LS group showed only mild hepatotoxicity,and red blood cell(RBC)and white blood cell(WBC)leve LS did not significantly decrease.A/F-LS greatly reduced the cytotoxicity of DOX and was relatively safe at the dose tested.The weight loss values of mice in the A/F-LS group were lower compared to the free DOX group throughout the experiment,indicating that DOX-A/F-LS bone-targeted delivery can reduce non-specific cell uptake by enhancing endocytosis of tumor cel LS.These results indicated that DOX-A/F-LS exhibits strong antitumor activity.2.The In vitro scratch assay showed that different concentrations of DOX-A/F-LS effectively inhibited the migration ability of MDA-MB-231 cel LS(P <0.05).The migration rate was 50.36±1.1% in DOX-A/F-LS(10u M)group,which was significantlydecreased compared with the blank control group(79.89±1.60%;P<0.05).This indicated that DOX-A/F-LS has a significant inhibitory effect on the migration of breast cancer MDA-MB-231 cel LS.In Transwell assay,compared with the control group,the results showed that A/F-LS effectively hindered the invasive ability of MDA-MB-231 cel LS,and the inhibitory effect was dose-dependent.Western blotting was used to detect the expression of p-ERK1/2,p-Akt and p-p38 protein.The results showed that DOX-A/F-LS treatment significantly inhibited the phosphorylation of ERK1/2 and AKT but did notaffect the expression leve LS of p38,suggesting that AKT and ERK1/2 pathways are involved in the inhibitory effect of doxorubicin liposomes on the proliferation and migration of MDA-MB-231 cel LS.The cytotoxicity of DOX-containing liposome preparations against MDA-MB-231 cel LS was detected by MTT assay.The results showed that the cytotoxicity of folic acid-modified liposomes(DOX-F-LS and-DOX-A/F-LS)were significantly increased(p < 0.05).The IC50 values of DOX-F-LS and DOX-A/F-LS were 17.8 l?g/m L and 15.3?g/m L,respectively.The folic acid-free liposomes DOX-A-LS and DOX-N-LSIC50 values were 100.5 ?g/m L and 94.9 ?g/m L,respectively.The results showed that folic acid-modified liposomes significantly inhibited the proliferation of MDA-MB-231 cel LS overexpressing the folic acid receptor on the cell surface,and the liposomal preparations facilitated the further transport of the drug to the bone compared to free DOX and subsequently enhanced the therapeutic effect on bone metastases.Conclusion1.The particle size of DOX-A/F-LS is between 95.82±0.13 nm and 96.61±0.11 nm.The optimal density of ASP8 and folic acid on liposome are 15% and 10% respectively(molar ratio).DOX-A/F-LS can effectively improve the active targeting of drug delivery and may reduce the EPR effect of intrinsic transcendental dependence2.DOX-A/F-LS is an ideal pharmacokinetic model for breast cancer bone metastasis.It is suitable for targeted drug delivery to tumor cel LS through systemic administration with concomitant reduction of the toxicity on normal cel LS.3.DOX-A/F-LS can effectively regulate the cell cycle of breast cancer in the process of breast cancer metastasis to the bone metastasis,significantly inhibit the function of cell metastasis and invasion,effectively exert anti-tumor effect and reduce normal cell damage.