Nanopartices Inhibiting Mfsd2a For Targeted Delivery Of Prodrugs To Brain Metastases

Objectives:The major facilitator super family domain-containing protein 2a(Mfsd2a)is overexpressed on brain micro vascular endothelial cells.Mfsd2a can inhibit the caveolin protein to reduce the efficiency of endocytosis by regulating the lipid composition on brain microvascular endothelial cells while inhibition of Mfsd2a can increase the efficiency of endocytosis.Tunicamycin(TM)is a reversible Mfsd2a inhibitor.We designed brain metastasis-specific intracellular hyaluronidase-1 cleavable hyaluronic-doxorubicin(HA-DOX/HD)to reduce the damage to normal cells caused by chemotherapy drugs.In conclusion,firstly,based on the starting material poly(lactic-co-glycolic acid)-poly(?-carbobenzoxy-L-lysine)(PLGA-PLL),nanoparticles(T@T-NPs)were constructed to inhibit Mfsd2a and promote chemotherapy drugs cross the blood-brain barrier by using Mfsd2a inhibitor tunicamycin(TM)loaded transcytosis-targeting peptide(TTP).Secondly,T@T-NPs were further anchored with CD44-specific hyaluronic acid(HA),yielding T@T-NPs-H to target brain metastatic tumor cells.Then,we loaded HA-DOX into T@T-NPs-H to get T@T-NPs-H/HD to selectively mediate apoptosis of cancer after prodrug activation and ameliorate toxicity to normal cells.Finally,after the successful construction of nanoparticles,we explored the efficiency and mechanism of brain accumulation and brain metastases targeting of nanoparticles in the further step,and evaluated the therapeutic effect of nanoparticles on brain metastases tumor bearing mice.Methods:(1)T@T-NPs-H/HD were prepared by double-emulsion solvent evaporation technique;dynamic light scattering and transmission electron microscopy were used to characterize the morphology,size,zeta potential and serum stability of the nanoparticles.Additionally,the in vitro drug release behavior of the T@T-NPs-H/HD were studied by dialysis method.(2)On brain microvascular endothelial cells,the influence of TM and TTP on nanoparticles uptake were investigated by flow cytometry and protein quantification.(3)In normal mice,the effect of TM and TTP on nanoparticles crossing blood-brain barrier were studied by IVIS image and%ID/g fluorescence quantification method respectively.(4)On brain metastatic tumor cells,the effect of HA on the uptake of nanoparticles was investigated by flow cytometry.(5)In brain metastasis tumor bearing mice,the impact of TM,TTP,and HA on nanoparticles brain metastases tumor targeting were examined by tissue sections.(6)The interaction between hyaluronidase-1 and HA-DOX was proved through DNA insertion experiments and enzymolysis experiments in vitro.Meanwhile,the cellular uptake and cytotoxicity of HA-DOX on brain metastatic tumor cells and astrocytes are compared through fluorescence microscopy and MTT.(7)Finally,pharmacodynamics of nanoparticles in brain metastases tumor bearing mice were investigated.Results:(1)The morphology of T@T-NPs-H/HD prepared in this study was irregular spherical;the particle size and zeta potential were 145.0±1.3 nm/-18.8± 0.6 mV;the particle size of T@T-NPs-H/HD was almost unchanged after 48 hours incubation with 10%FBS;HA-DOX in T@T-NPs-H/HD was controlled released(26.0%at 48 h)in PBS 7.4 with 0.5%SDS.(2)Compared with unmodified nanoparticles,the uptake of TTP-modified nanoparticles(T-NPs/DOX)was significantly enhanced on brain microvascular endothelial cells;free TM could further increase the uptake of T-NPs/DOX by inhibiting Mfsd2a;TM loaded nanoparticles(T@T-NPs)still had the effect mentioned above.(3)In normal mice,T@T-NPs significantly increased the brain accumulation of T-NPs/DOX when compared with free TM;T@T-NPs had self-promoting effect in passing blood-brain barrier.(4)On brain metastatic tumor cells,the uptake of HA-modified nanoparticles(T-NPs-H/DOX)was obviously stronger than that of unmodified HA nanoparticles(T-NPs/DOX).(5)In brain metastases tumor bearing mice,T@T-NPs-H+T@T-NPs-H could efficiently target brain metastases because of the combined effects of TTP,TM,and HA.(6)Compared with unpretreated HA-DOX,it was easier for HA-DOX with hyaluronidase-1 pretreatment to insert into DNA.Up to 26.1%HA-DOX was cleaved after 24 hours' hyaluronidase-1 treatment.Compared with astrocytes,the uptake and toxicity of HA-DOX were higher on brain metastatic tumor cells.(7)Compared with free drug,T@T-NPs-H/HD significantly prolonged the survival time of brain metastases tumor bearing mice.Conclusion:In this study,TM/HA-DOX co-loaded and TTP/HA co-modified nanoparticles were successfully constructed.We demonstrated that TM and TTP based T@T-NPs significantly cross the BBB;HA-DOX reduced damage to normal brain cells;prodrug HA-DOX loaded T@T-NPs-H/HD obviously prolonged the median survival of brain metastases tumor bearing mice.It could provide a reference for brain metastasis treatment.