M2pep Peptide And PTPRZ1 Antibody Comodified Micelles Targeting Tumor Microenvironment Drug Delivery For Glioma Treatment

Glioblastoma（GBM）is the most malignant primary brain tumor accounting for about 50%of glioma and showing invasive growth.Although the development of surgical resection is combined with radiotherapy and chemotherapy,the survival rate is still very low.In addition,the existence of the Blood Brain Barrier（BBB）prevents most small molecules and almost all macromolecular drugs reaching the brain.Therefore,how to across the BBB and reach the brain is the key to intracranial drug delivery.The cell types in tumors will affect the treatment for glioma.For example,microglia/macrophages will affect the growth and invasion of glioma.Among them,the TAMs correlated with glioma malignant grade and poor prognosis.M1 TAMs play an immune surveillance role in inhibiting tumor growth,and M2 TAMs promote tumor growth.Glioma stem cells（GSCs）in the microenvironment of glioma are considered to be tumor-initiating"seed"cells.The surface-specific receptor PTPRZ1 of GSCs can interact with the pleiotrophin（PTN）highly expressed in M2 TAMs.The interaction of PTN-PTPRZ1 signal can promote glioma growth.What’s more,GSCs secrete the periostin（POSTN）to recruit peripheral blood macrophages through the POSTN-α_vβ₃signal pathway.Therefore,targeting M2 TAMs and GSCs can not only inhibit the recruitment of macrophages,but also block the PTN-PTPRZ1 signal axis,then inhibit the proliferation and invasion of glioma.In recent years,nanocarriers have been widely used for BBB drug delivery.The monosialoganglioside（GM1）can be used to promote nerve damage repair and regeneration and have a good BBB permeability.In this paper,we used DSPE-PEG linker conjugated with the M2pep polypeptide and the PTPRZ1 antibody,the dual-targeting GM1 micelles（DT-GM1）named a"hybrid micelle"can target glioma microenvironment.Evaluation the DT-GM1 micelle targeting ability for M2macrophages and GSCs in vivo and vitro.And evaluating the anti-tumor effect of DT-GM1/DOX on orthotopic glioma model in vivo using doxorubicin（DOX）as antineoplastic model drugs.The main research contents and results as follows:（1）Synthesis and characterization of DSPE-PEG-M2pep:The free sulfhydryl group at the C terminal of M2pep reacts with the maleimide of DSPE-PEG-Mal to form a thioether bond.The results of time-of-flight mass spectrometry showed that the molecular ion peak of M2pep C（2403.64）,DSPE-PEG-Mal（1400-3000,mainly concentrated in 2114.69）,DSPE-PEG-M2pep（mainly concentrated in 4520.81）,respectively.The proton nuclear magnetic resonance spectrum results showed that after the M2pep C sulfhydryl group modified by DSPE-PEG-Mal,the Mal peak of DSPE-PEG-M2pep was disappeared,indicating that M2pep C has been successfully conjugated with the DSPE-PEG-Mal,and the conjugated compound DSPE-PEG-M2pep was obtained.（2）Synthesis and characterization of DSPE-PEG-PTPRZ1:The PTPRZ1 antibody（free amino groups）conjugated with the DSPE-PEG-NHS（NHS active ester）through the amide condensation reaction.The SDS-PAGE and immunofluorescence results indicated that the successful conjugation of the PTPRZ1（Ig G）antibody with the DSPE-PEG-NHS.The reducing SDS-PAGE results demonstrated that the DSPE-PEG-PTPRZ1have higher molecular weight than unconjugated antibodies.Fluorescence microscopy recognized that Cy3-labeled secondary antibodies bound to Ig G-GM1 micelles with high fluorescence intensity compared with GM1 micelles and free Ig G.（3）Preparation and characterization of DT-GM1/DOX:The drug loading and the encapsulation efficiency of DT-GM1/DOX is 2.66±0.11%and 74.62±3.12%,respectively.When the DOX concentration was 3 mg/m L,the particle size of the micelle was 12.14±0.10 nm,and the surface charge was-15.9±0.6 m V.The TEM results showed that the micelles exhibited compact and spherical morphology.（4）The cellular uptake and the transport pathways of DT-GM1:The cellular uptake of DT-GM1 micelles by LN229 was significantly stronger than b End.3 cells,and the PTPRZ1 receptor is highly expressed on LN229 cells,indicating the modification of the PTPRZ1 antibody promoted the absorption of micelles by LN229 cells.After pre-incubation the b End.3 cells with inhibitors（chloroquine,colchicine,filipin,chlorpromazine and brefeldin A）,the results showed that both of the GM1 and DT-GM1micelles were affected by brefeldin A which the absorption of the b End.3 cells decreased by 40.50%and 32.96%,respectively,indicating that GM1 micelles are mainly mediated by clathrin-medicated pathway.After GM1 micelles modified by M2pep and PTPRZ1 antibody,the transport pathways were changed slightly which promoted the cell absorption by microtubule pathway,caveolin pathway and clathrin pathway.（5）Evaluate the DT-GM1 micelles across BBB in vitro:Both of the GM1 and DT-GM1 micelles could penetrate the b End.3 monolayer cell layer and absorbed by LN229cells.The penetration of micelles increased and the cell uptake of LN229 cells were also increased with the extension of time.The nanoparticle across the BBB reached the strongest at 12 h,and the DT-GM1 micelles penetrated the upper chamber b End.3 cells which the uptake by LN229 cells were more than GM1 micelles.（6）Evaluation of M2pep-GM1 micelles targeting M2 macrophages in vitro:The bone marrow-derived macrophages（BMDMs）were extracted and verified by flow cytometry.The expression of F4/80⁺CD11b⁺was 88.86%.The Q-PCR results showed that both the expression of i NOS for M1 macrophages and Arg-1 for M2 macrophages were increased.The immunofluorescence showed the expression of CD86（M1macrophages）and CD206（M2 macrophages）was increased,respectively.The co-localization of M2pep with M2 macrophages was more than the Sc M2pep.The binding of M2pep to M1 macrophages was less,but higher than the scrambled peptide Sc M2pep to M1 macrophages.After incubation the GM1,M2pep-GM1 and Sc M2pep-GM1/Di D micelles with M2 macrophages,we found that the M2 macrophages uptake by M2pep-GM1 micelles were higher than the GM1 and Sc M2pep-GM1 micelles.（7）Evaluation of PTPRZ1-GM1 micelles targeting GSCs in vitro:The LN229tumorspheres model was constructed in vitro.The LN229 cells after induction by B27,EGF and b FGF,the immunofluorescence of LN229 tumorspheres showed that the expression of CD133,SOX2 and PTPRZ1 were increased.The CSLM and the semi-quantification of fluorescence intensity results showed that compared with Ig G-GM1and GM1 micelles,the PTPRZ1-GM1 micelles showed better targeting and penetrating ability to LN229 tumorspheres,whereas the uptake of N229 cells have no significant difference.Compared with Ig G-GM1 micelles,the PTPRZ1-GM1 micelles penetrated more than Ig G-GM1 micelles in tumorspheres with higher fluorescence intensity at 60,80 and 100μm.The semi-quantification of fluorescence intensity showed that when the the PTPRZ1 receptors blocking by PTPRZ1 antibodies,the penetration of PTPRZ1-GM1 micelles for the LN229 tumorsphereswas significantly decreased.（8）Evaluation of the DT-GM1 micelles across BBB and target glioma microenvironment in vivo:In vivo distribution experiments of LN229-Luc bearing mice showed that the GM1/Di R micelles could penetrate the BBB faster and easier than free Di R.The accumulation of DT-GM1 micelles in the brain were more than other micelles（GM1/Di R,M2pep-GM1/Di R,PTPRZ1-GM1/Di R）.The fluorescence intensity of brain site gradually increased with time and reached the peak at 24 h.The drug accumulated on the right brain consistent with the location of tumor injection,indicating that the drug was mainly accumulated near the tumor site.The immunofluorescence frozen sections in different groups showed that M2pep-GM1/Di R micelles were enriched in the area of higher CD206 expression,and PTPRZ1-GM1/Di R micelles accumulated near CD133-positive tumor cells.The DT-GM1 micelles were accumulated more near the area with the higher expression of CD206 and CD133 indicated that DT-GM1 micelles could target M2 macrophages and GSCs,ultimately target glioma tumor microenvironment.（9）In vivo pharmacodynamic evaluation of DT-GM1/DOX:The LN229-Luc glioma bearing-mice after administration the saline,free DOX,GM1/DOX and DT-GM1/DOX,the median survival was 33.5,36,40 and 46 days,respectively.The DT-GM1/DOX micelles significantly inhibited tumor proliferation comparing with GM1/DOX micelles which the bioluminescence intensity of Luc was reduced and the median survival was prolonged for 6 days.After treatment with different group,the proportion of M2 TAMs,M1 TAMs and GSCs in each treatment group was evaluated by flow cytometry.The proportions of M2 TAMs（CD206⁺F4/80⁺CD45⁺CD11b⁺Ly6G^-）in the saline group,free DOX,GM1/DOX and DT-GM1/DOX groups were 31.66%,18.20%,14.73%and 8.28%,respectively.DT-GM1/DOX not only reduced the proportion of M2 TAMs but also increased the proportion of M1 TAMs,enhancing the targeted killing effect.Furthermore,the proportion of GSCs（CD133⁺CD15⁺）in the saline group decreased from 3.74%to 2.12%for DT-GM1/DOX group.After H&E staining in each group found that the tumor area in the DT-GM1/DOX group was the smallest,and there was no difference in the organs（heart,liver,spleen,lung and kidney）in each group.The results of immunohistochemistry（IHC）showed that the expression of Ki67 of DT-GM1/DOX was much lower than other administration groups,and the expression of GFAP was the highest.The IHC results showed that DT-GM1/DOX micelles improved the curative effect without increasing the cytotoxicity of organs,and reduced the tumor proliferation and the malignant of glioma.