Stimuli-Responsive Liposomes For Chemo-Photodynamic Combination Therapy Of Triple Negative Breast Cancer

Triple-negative breast cancer(TNBC)is a subtype of breast cancer with low or absence expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor 2 receptor(HER-2)protein.TNBC is the most agrressive subtype characterized by high metastasis and poor prognosis.Chemotherapy is currently the only systemic treatment for triple-negative breast cancer.However,Chemotherapy suffers from restricted therapeutic effects and severe systemic side effects due to the lack of tumor-targeting ability.Fortunately,combining chemotherapy with photodynamic therapy(PDT)via co-delivery nano-carriers provides a new strategy for the efficient and safe treatment of TNBC.Despite promising,the obstacles,such as poor tumor permeability,insufficient uptake and unresponsive drug release,limit the clinical application of these existing nano-carriers.Hence,the developement of nanosized drug delivery systems to achieve chemotherapy-PDT combined therapy for TNBC still faces severe challenges.In this work,two kinds of stimuli-responsive liposomes which both co-delivered with chemotherapeutic prodrugs and photosensitizers were exploited for the chemotherapy-PDT combination therapy of TNBC.Firstly,reactive oxygen species(ROS)-cleavable doxorubicin(DOX)prodrug(PPC-TKDOX)and PEG-modified photosensitizer(PPa-PEG)were synthesized.Then the ROSactivatable DOX prodrug-loaded liposome(RADL)were engineered by the self-assembly of PPC-TK-DOX,PPa-PEG,unsaturated phospholipid DOPC and cholesterol.RADL exhibited a uniform spherical morphology with average size of 115.3±1.2 nm and surface charge of-1.34±0.20 mV.RADL was of high drug loading(DL)capacity and encapsulation efficiency(EE)of 27.5%and 96.7%,respectively,and showed excellent stability in 10%fetal bovine serum(FBS).Upon 671 nm laser irradiation,RADL could induce ROS generation,not only to activate PPC-TK-DOX,but also oxidize DOPC for the ultrafast drug release.To evaluate the effect and mechanism of RADL on inhibiting tumor cell viability in vitro,cellular uptake,photodynamic activity,drug release,cytotoxicity and apoptosis analysis were performed in 4T1 TNBC cells.Results showed that RADL could be internalized by 4T1 cell effectively and further to generate ROS,release drug and inhibit cell viability upon the laser illumination.On this basis,the pharmacokinetic behavior of RADL in Sprague Dawley(SD)rats and the inhibiting effect of tumor growth and lung metastasis in orthotopic 4T1 models were investigated.The results showed that the elimination half-life(t1/2β)and the area under the drug-time curve(AUC0-t)of RADL were 5.7-fold and 112.3-fold higher than DOX,respectively.In addition,RADL could efficiently and targetedly accumulate in tumor site,then induce photodynamic and chemotherapeutic toxicity upon tumor localized laser irradiation.The synergistic effect of RADL results in 84.8%and 87.6%inhibition of tumor growth and lung metastasis,respectively.Moreover,RADL displayed great biosafety since no obvious systemic side effects were observed in vivo.In order to further increase the uptake of lipid nano-carriers by tumor cells,thereby enhance the accumulation and penetration of drugs in tumor tissues,PEG-modified photosensitizer was re-designed by inserting a matrix metalloproteinase-2(MMP-2)cleavable peptide between PPa and PEG for the synthesis of PPa-GPLGLAG-PEG.An enzyme,laser and temperature multiple stimuli-responsive liposome(ELTSL)was then developed by integrating enzyme-cleavable PPa-GPLGLAG-PEG and temperature-sensitive phospholipids DPPC and DSPC into one single nanoplatform.A hexadecyl chain-modified hydrophobic oxaliplatin(OXA)prodrug,HOC,was subsequently synthesized and encapsulated in the lipid bilayer of ELTSL to obtain ELTSL-HOC.ELTSL-HOC has a round appearance and a uniform distribution with the average size of 88.1 ± 1.7 nm.Zeta potential of ELTSL-HOC was determined to be-11.9±0.5 mV.The DL and EE of ELTSL-HOC is 23.1%and 97.5%,respectively.ELTSL-HOC showed excellent dispersibility and stability in 10%FBS.Furthermore,ELTSL-HOC could be heated to around 45℃ for rapid drug release upon 671 nm laser irradiation.The results of in vitro experiments showed that ELTSL could remove its PEG envelope under MMP-2 incubation,thereby significantly enhancing the uptake of 4T1 tumor cells and the penetration of multicellular spheroids(MCSs).Moreover,ELTSL-HOC could increase the cytotoxicity of chemotherapy drugs through its enhanced cellar uptake in a variety of TBNC cells,such as 4T1,MDA-MB-231 and MDA-MB-468.In vivo fluorescence imaging and photoacoustic imaging results indicated that ELTSL displayed greater tumor accumulation and penetration capacity than non-enzyme sensitive liposomes LTSL.The pharmacokinetic behaviors of ELTSL-HOC in SD rats were investigated and results showed that the t1/2β and AUC0-t of ELTSL-HOC was 5.7-and 20.4-fold higher than OXA,respectively.On this basis,the tissue distribution and anti-tumor effects of ELTSL-HOC were investigated in the orthotopic 4T1 model.The results showed that the Pt contents of ELTSL-HOC in tumor sites were 3.7-and 8.1-fold higher than that of OXA at 4 h and 24 h,respectively.The 4T1 tumor growth inhibition rate of ELTSL-HOC was as high as 97.3%,due to the synergistic chemotherapy-photodynamic therapeutic effect.In addition,there was no abnormal change of body weight and no obvious pathological lesion or inflammatory reaction in the main organs of ELTSL-HOC group,indicating that ELTSL-HOC displayed superior biosafety in vivo.In conclusion,stimuli-responsive liposomes based on co-delivery of chemotherapy prodrugs and photosensitizers could achieve highly effective and safe TNBC treatment through the combination of chemotherapy and PDT.Stimuli-responsive liposomes exhibit great prospect of medical application and provide a novel and reliable platform for the clinical conbination treatment of TNBC.