"Trinitarian" Echelon Responsive Nano-drug Delivery System Inhibit PI3K/Akt Pathway-mediated Pulmonary Metastasis Of Breast Cancer

Breast cancer ranks first in the incidence of female malignancies in China,with an annual mortality rate of more than 70,000 making it the first killer of women’s health.Tumor metastasis is one of the leading causes of clinical failure.Co-delivery of a phosphoinositide3-kinase/serine/threonine protein kinase(PI3K/Akt)inhibitor and a chemotherapeutic agent in responsive nanocarriers is a promising strategy to inhibit of tumor growth and metastasis,however,the design and construction of a stimuli-responsive nanomaterial that is safe,stable and able to break through various biological barriers are still challengingThe drug delivery system needs to overcome three barriers:(ⅰ)Avoid clearance in the blood circulation;(ⅱ)Accumulate in tumor cells;and(ⅲ)Escaping from the endosomes.Based on these three barriers,we designed a serial of nancarries,which named "Trinitarian" echelon response nanocarrier.In order to increase tumor cell accumulation,an Akt inhibitor(quercetin,Qu)was co-delivered with a chemotherapeutic agent(docetaxel,DTX)by using hyaluronic acid(HA)-modified nanoparticles(NPs)as vectors.The dual DTX/Qu-loaded HA modified PLGA-PEI NPs(PP-HA/NPs)were prepared through a modified emulsion solvent evaporation technique.The particle size and Zeta potential of PP-HA/NPs were 209.8±10.8 nm and+(12.0±1.07)mv,respectively.Wound healing assay,cell migration assay and invasion assay revealed that Qu co-delivery and HA modification elicited synergistic inhibitory effects on cell motility.The inhibition rates of migration and invasion were 95.6%and 99.3%,respectively PP-HA/NPs could down regulate the expression of p-Akt and matrix metalloproteinase-9(MMP-9).With HA modification,PP-HA/NPs could be efficiently uptaken by 4T1 breast cancer cells and could further induce cytotoxicity,decrease colony formation and promote cell apoptosis.Biodistribution assay demonstrated PP-HA/NPs also enhanced drug accumulation in the tumor and lungs and predicted that PP-HA/NPs could be employed as an effective therapy for primary tumor and pulmonary metastasisAccording to the design of "Trinitarian" echelon response nanocarrier,we used enzyme sensitivity,charge reversal and endosome escape technology to inhibit breast cancer metastasis.The peptide of GFLGF was used as a linker to connect PLGA and PEI,and the PEI was then modified with 2,3-dimethylmaleic anhydride(DA).The material of PLGA-peptide-PEI-DA was successfully synthesized.GDC0941(GDC),an inhibitor of PI3K,was co-delivered with DTX in PLGA-peptide-PEI-DA(PPP-DA/NPs).The particle size and Zeta potential were 135.6±2.8 nm and-(27.1±0.8)mV,respectively.The material of PPP-DA has proton-buffering capability,and the charge of PPP-DA/NPs could gradually reverse with the decrease of pH Moreover,the drug accumulation was increased in the presence of cathepsin B(Cath B).The celluar uptake indicated that PPP-DA/NPs could be efficiently uptaken by 4T1 breast cancer cells in lower pH.More inmportantly,PPP-DA/NPs could inhibit cell growth and induce apoptosis.Wound healing assay,cell migration assay and invasion assay showed that the inhibition rates were 53.4%,96.1%and 97.4%,respectively.These data revealed the effective inhibitory effects of PPP-DA/NPs on cell motility.The inhibition of tumor growth and metastasis may attribute to the down-regulate the expression of p-Akt,MMP-9,caspase-9,caspase-3 and Bcl-2.Compared with the saline group,the TIR of PPP-DA/NPs on 4T1 tumor bearing mice was 68.4%,and the pulmonary metastasis inhibition rate reached 95.3%.The third drug delivery system was constructed as follows.Graphene oxide(GO)was used as the carrier,folic acid(FA)and heparin were then modified on the GO with PEI as the liker,therefore the vector of GO-PEI-FA/Heparin(GPFH)was successfully synthesized.Doxorubicin(DOX)was loaded on GPFH(DOX@GPFH)with high drug loading efficiency of 125.1%,and the particle size and Zeta potential were 232.8±9.0 nm and-(42.8±1.6)mV,respectively.Then DOX@GPFH was mixed with GDC(DOX:GDC=1:5,m/m),labeled as(DOX@GPFH)+GDC.The celluar assays showed that(DOX@GPFH)+GDC could increase cellular uptake,induce apoptosis and down-regulated the expression of p-Akt and MMP-9.Furthermore,the wound healing assay,cell migration assay and invasion assay showed that the inhibition rates were 61.2%,81.0%and 76.8%,respectively.Compared with the saline group,the TIR of(DOX@GPFH)+GDC on 4T1 tumor bearing mice was 77.0%,and the pulmonary metastasis inhibition rate reached 73.7%.In conclusion,based on the concept of "rinitarian",the stimuli-responsive co-delivery nanocarriers could be an effective approach,which provides an important theoretical basis for the development of delivery system for the metastasis of breast cancer.