Fundamental Study Of GSH-responsive Micelles Based Photodynamic-chemo Combined Therapy Against Breast Cancer

Cancer has been a major public health issue.Breast cancer is the most frequently occurring cancer in women.The traditional therapy routes against breast cancer are surgery and chemotherapy.However,surgery has the shortcomings of large wound and high recurrence.Although chemotherapy is generally considered to be one of the most efficient approaches for cancer therapy,its poor selectivity does harm to normal cells' metabolism.DOX is an effective chemotherapeutic drug used for the treatment of solid tumors.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 drug-resistance.Photodynamic therapy(PDT)has been utilized as a new strategy to improve the treatment of tumor.Photosensitizers accumulated in tumor tissues are activated by specific light to generate therapeutic reactive oxygen species(ROS),which results in a sequence of photochemical and photobiology processes with macromolecules to induce cell apoptosis or necrosis.It shows advantages of effectiveness,less side effect,no drug-resistance and repeatable treatment.PDT has been a clinically approved therapeutic approach against tumor by FDA since 1997.The limited light penetration,photosensitizers with poor water solubility and low tumor accumulation hampered its clinical application.Combination therapy is routinely used for the treatment of cancer and improves the therapeutic effect.PDT is used in cancer therapy combining with chemotherapy to inhibit tumor recurrence and metastasis after surgery.Furthermore,there are no overlapping toxicities between PDT and chemotherapy.However,PDT combined with chemotherapy is limited due to poor water-solubility of photosensitizers and side effects of chemotherapeutic drugs.In recent years,nanoparticles have been employed as drug delivery system(DDS)to improve anti-cancer agents' accumulation in tumor and decrease drug toxicity.Stimuli-responsive N-(2-hydroxypropyl)methacrylamide(HPMA)has attracted extensive attention because of its desire water solubility and biocompatibility.Drug in HPMA polymer is delivered to the tumor site via enhanced permeability and retention(EPR)effect and released quickly to reach an effective concentration.Some HPMA polymeric prodrug systems have been entered into clinical trials.To overcome these challenges,a GSH responsive HPMA polymeric prodrug conjugating DOX was developed.It was aimed to improve the water solubility of DOX and photosensitizer-chlorin e6(Ce6),promote drug accumulation in the tumor site and enhance the anti-breast cancer effect via targeted micelle and combined treatment.The amphiphilic block polymer p HPMA-SS-DOX with GSH-responsibility was synthesized by reversible addition-fragmentation chain transfer(RAFT)polymerization and characterized by size exclusion chromatograph(GPC).The drug loading of chemically bonded DOX was measured by ultraviolet visible spectrophotometer(UV-Vis).DOX/Ce6 co-loaded micelle(CLM)was prepared via thin-film hydration method.The particle size,polymer dispersity index(PDI),zeta-potential,morphology,critical micelle concentration(CMC),encapsulation efficacy,drug loading and stability were characterized by Zetasizer Nano ZS,transmission electron microscope(TEM),fluorescence spectrophotometer(FL)and UV-Vis,respectively.The release profile of CLM was studied by dialysis bag diffusion method.The singlet oxygen(1O2)generated from CLM was determined using anthracene-9,10-dipropionic acid disodium salt(ADPA)as 1O2 quencher.The cell experiments were conducted on 4T1 breast cancer cells which overexpressed GSH.The cellular uptake of CLM was performed by confocal fluorescence imaging.Various endocytosis inhibitors were used to investigate the micelles internalization mechanisms.The intracellular ROS generation was monitored by staining the cells with the dichlorofluorescin diacetate(DCFH-DA).The dark toxicity and phototoxicity of CLM were determined by cell counting kit-8(CCK-8)assay.Flow cytometry was used to test the apoptotic and necrotic cell population quantitatively.The biodistribution of micelles was measured at different time points in 4T1 breast tumor bearing mice.In vivo targeted PDT-chemotherapy anti-breast cancer efficacy was evaluated according to tumor volume growth,tumor growth inhibition(TGI)and body weight.The cardiotoxicity and tumor cell necrosis induced by CLM were carried out using hematoxylin-rosin(HE)staining.The results demonstrated that the GSH responsive HPMA polymeric prodrug conjugating DOX was successfully synthesized and drug loading of DOX was 5.0 %.It formed micelle through self-assembly in aqueous solution.The particle size of CLM was 122.0 ? 8.0 nm.PDI was 0.149 ? 0.03.Its morphology was well-distributed spherical.Zeta potential was-7.54 ? 0.87 mV.CMC was measured to be 0.014 mg/m L.The encapsulation efficacy and drug loading of physically encapsulated Ce6 were 76.2 ? 5.2 % and 4.82 ? 0.4 %,respectively.CLM showed decent stability in PBS under 4? within 7 days.The releasing behavior of DOX and Ce6 from CLM displayed GSH-responsive property.CLM was able to effectively generate 1O2 after laser irradiation.Cellular uptake studies suggested more efficient uptake of micelle was attributed to the clathrin-mediated endocytosis.As expected,CLM generated remarkable reactive oxygen species(ROS)in 4T1 cells under irradiation.The IC50 of CLM was 60 ?g/m L in the dark.Cell viability of 4T1 cells treated with CLM and irradiated for 0 min and 10 min(0.3 W/cm2)were 81.1 ? 10.7 % and 25.1 ? 1.3 %,respectively.The ratio of apoptosis and necrosis cell population induced by CLM was 55.2 ? 5.3 %,which was much higher than that of chemotherapy alone(23.1 ? 2.1 %).The results of micelle biodistribution in mice bearing breast cancer by intravenous injection revealed that micelle enhanced drug accumulation in tumor site and prolonged action time.It reached a maximum at 36 h post-injection.PDT combination with chemotherapy based on CLM showed an excellent effect on inhibiting tumor growth.HE staining exhibited that there was no obvious damage observed in heart treated with CLM.The tumor tissue damage in CLM group was the most severe among the groups.In conclusion,a GSH responsive HPMA polymeric prodrug DOX has been synthesized successfully.It formed micelle by self-assembly process in aqueous solution under lower concentration and improved water solubility of poorly water soluble drugs.Drug-loaded micelles were spherical-shaped with well-distributed small size and high encapsulation efficacy.DOX and Ce6 released from micelles were GSH-dependent.The polymeric micelle improved drug accumulation in tumor via EPR effect.PDT combining with chemotherapy showed an outstanding anti-tumor effect both in vitro and in vivo.The polymeric micelles applying for targeting drug delivery system will be a safe and effective approach to anti-breast cancer.