Study Of Doxorubicin Conjugated Enzyme Responsive Micelles Loading Nifuroxazide Against Metastasis Breast Cancer In Lung

Cancer has been a major public health issue.Breast cancer is the most frequently occurring cancer in women,and more than 90%of patients died from tumor metastasis.Lung,liver and brain are mainly metastatic organs,and the rate of lung metastasis can be as high as 60%.The commonly treatment against breast cancer and its metastases are surgery and chemotherapy.However,surgery has the shortcomings of large wound and high recurrence.Chemotherapy is a systemic treatment,but it has side effects and poor prognosis,which can not inhibit tumor metastasis effectively.Doxorubicin?DOX?is a kind of amphoteric anthracycline antibiotic with high anti-cancer activity,which is one of the first line drug for chemotherapeutic.The major mechanism of DOX is its ability to insert into DNA to inhibit DNA synthesis.However,application of free DOX is limited due to its short half-life,cardiotoxicity and other side effects,such as it can inhibit hematopoietic function of bone marrow.Nifuroxazide?NFX?is a nitrofuran drug,and they are broad-spectrum antibiotics.Recent results have shown that this kind of drugs are inhibitors of the signal transduction and activators of transcription?STAT-3?,because they can inhibit the STAT-3 pathway to down-regulate the expression of matrix metalloproteinase,and this process can suppresse tumor metastasis effect.However,nifuroxazide has poor solubility,and it is mainly dissolved in mixed solvent for intraperitoneal injection for murine test.As a vector for drug delivery system,polymeric micelles can increase the solubility and stability,and also can reduce toxicity.The important one is it can passive target by improving Enhanced permeability and retention effect?EPR?.Although there has unique advantages of micelle drug carrier system to deliver antitumor drug to the target.However,the metastasis have small volume,large quantity and highly dispersed in the invaded organs,thus the nanoscale drug-carriers are difficult to distribute to the metastasis tumors by passive targeting.Therefore,drug delivery based on nano drug targeting tumor metastasis is also facing a severe challenge.As we known,there are large differences between the tumor microenvironment and normal tissues,and the expression of protease is very high in tumor.Recently,reseachers studied a new type of stimuli sensitive nano drug delivery system that cathepsin B sensitive drug delivery system.The drug loaded in this system can be delivered to the tumor,and the drug is released at the tumor,so it can enhance therapeutic effect,and also reduce the damage for normal tissue.This study is foucused on a cathepsin B responsive produrg loaded with nifuroxazide.The drug delivery system is amied to inhibit breast cancer lung metastasis.This paper has carried out the related research work from the following three parts.The first part is the synthesis and self-assembly of the enzyme sensitive polymer prodrug.The Diblock polyDOX-OEG was synthesized via a one-step reversible addition-fragmentation chain transfer reaction?RAFT?.The content of DOX in polymer was determined by UV spectrophotometry.The molecular weight of the polymeric prodrug was determined by size exclusion chromatography?SEC?.The thin-film hydration method was used to prepare DOX/NFX co-loaded micelles?CLM?;the determination of critical nanopartical concentration?CMC?of polymer by pyrene fluorescence probe;according to the entrapment efficiency and Loading capacity of NFX to insure the feed ratio;The morphology,particle size,zeta potential and stability of drug-loaded nanoparticles were determined by transmission electron microscopy?TEM?and Dynamic light scattering?DLS?;The enzymatic sensitivity and in vitro release of CLM were investigated by dialysis method.Conclusions:The prodrug POEGMA-GFLG-DOX was synthesized and the mass fraction of DOX in the polymer was 9.2%?w/w?;the CMC of polymer was 0.025 mg/ml;the encapsulation efficiency and loading capacity of NFX were 65.4%and 11.6%;the micelle shape was approximately spherical and the average diameter was 131.33±6.0 nm.;the size of micelle was stable in 36h;the release of CLM was depended the enzyme,the cumulative release rate reached more than 90%in 48 h.The results showed that we synthesized the polymer with dependence of cathepsin B,and it could release DOX and NFX in tumor sites,improve the efficacy and bioavailability of drugs,and reduce the damage to normal tissues.The second part is the in vitro study of drug-loaded micelles.4T1 cell viability was determinted by CCK-8 method;Flow cytometry and confocal microscopy were used to investigate cell uptake and apoptosis of CLM;The effect of CLM on cell healing by wound-healing migration assay;The migration and invasion for cell was used boyden chamber?Transwell?migration and invasion assay.Conclusions:The IC₅₀ of free drug DOX,NFX and CLM micelles were 0.48 g/mL,1.74 g/mL,8.93 g/mL;4T1 cells could uptake the CLM,and the drug mainly distributed in the cytoplasm;CLM could significantly induce apoptosis of 4T1 cells;CLM could inhibit 4T1 cell growth,migration and invasion.The results showed that CLM could effectively kill cancer cells and inhibit the metastasis of breast cancer cells.The third part is the pharmacodynamic evaluation of the drug loaded micelles in vivo.Using 4T1 cells to construct the murine orthotopic breast cancer spontaneous lung metastasis model and breast cancer lung metastasis model estabilished by intravenous injecting cancer cells,and investigated anti-cancer efficancy of CLM;conducted a histopathological?H&E?study on heart,liver,spleen,lung and kidney;the immunohistochemistry?Ki67,CD31,MMP-9,MMP-2?were examed on tumor and lung sections;TUNEL staining was investigated apoptotic cells on tumor;double staining of lung cord sections with anti-Gr-1 and anti-CD11b antibodies;The targeting of ability of CLM to tumor tissue and the distribution in major organs were observed by using in vivo small animal imaging.Conclusions:The isolated organ imaging showed that CLM has the largest fluorescence intensity at 36 h in tumor,and also distributed in lung;CLM in two kinds of animal tumor models significantly inhibited tumor growth and metastasis,especially lung;slice results showed that the CLM group's positive cell numbe of Ki67,CD31,MMP-9,MMP-2 were decreased;The CLM group was not observed MDSCs;TUNEL results showed that CLM group showed the most aptotic population;H&E showed that CLM group was not observed cardiotoxicity.The results showed that the drug delivery system not only has anti-tumor effect,but also could effectively inhibit lung metastasis of breast cancer by combinational administration of NFX.In summary,the enzyme responsive polymer prodrug with DOX was synthesized,and then encapsulated nifuroxazide as drug delivery system aiming to anti-metastatic breast cancer.The polymeric carrier improved the solubility of nifuroxazide,and could be able to release of DOX the site of action.Polymeric micelles enhanced the drug distribution in the tumor by EPR effect,which showed the drug delivery system has good effect for anti-cancer growth and anti-metastasis in breast cancer.Otherwise,it has good safety,and expected to design as a safe and effective drug delivery system against breast cancer metastasis.