Gold Nanoparticles AuNPP-FA Inhibit Tumor Metastasis By Promoting Tumor Vascular Normalization

BackgroundChemotherapy always plays a very important role in tumor treatment.Traditional chemotherapeutic drugs are characterized by non-selective killing of active growing cells.To a certain extent,though chemotherapy has the ability to control the progress of tumors,it also brings various systemic toxicities and side effects simultaneously,limiting the clinical use of chemotherapeutic drugs.In addition,different from normal tissues blood vessels,tumor blood vessels have the characteristic of excessive permeability because of their loose vascular endothelial connections and insufficient pericytes coverage.Vascular leakage and the compression of blood vessels caused by abnormal proliferation of tumor promote tumor blood vessels to have serious blood hypoperfusion and further to create a tumor microenvironment of high interstitial fluid pressure.All of these characteristics of tumor blood vessels often lead to serious consequences as the chemotherapy drugs can’t be effectively delivered into the tumors or reach the effective treatment concentration,affecting the therapeutic effects absolutely and inducing the high invasiveness of tumors and multiple chemotherapy resistance as well.Based on the above facts,cancer nanomedicine has got an emerging development,which has been used in all aspects including cancer imaging,diagnosis,treatment and prevention.In this area of cancer nanomedicine,the research on gold nanoparticles(AuNPs)as drug delivery carrier is in full swing.AuNPs can effectively resist the high interstitial fluid pressure in the tumor microenvironment and accumulate in the tumor tissue.Meanwhile,AuNPs can be modified easily through surface functionalization and then owns many prominent properties,such as acid sensitivity,targeting specific tumors,prolonging its half-life and the time of metabolism in vivo so that it can effectively improve the anti-tumor efficacy.Therefore,more stably and accurately biochemical modification of AuNPs is the cornerstone to promote its wider application in antitumor therapy.Some studies have found that AuNPs itself has the inherent antineoplastic activity,and more studies mainly focus on the surface functional modification of drug-loaded AuNPs by improving its tumor tissues targeting and circulating bioavailability to enhance the efficacy of chemotherapeutic drugs.At present,the research on the regulation of tumor vascular normalization by AuNPs is still dubious.More concrete mechanisms of whether the new nanomaterials based on AuNPs can participate in altering tumor microenvironment to promote the normalization of tumor blood vessels and inhibit the tumor metastasis need to be further explained.In this study,an amphiphilic block copolymer Poly(ethylene glycol)-bpoly(diethylaminoethyl acrylate)(PEG-PDEAEA)is constructed by using PEG and DEAEA as componets.Then,AuNPs is bound with PEG-PDEAEA-FA after PEGPDEAEAis conjugated with FA through surface functional modification to form a new type gold nanoparticles AuNPP-FA.Finally,We use molecular biology experiments,in vivo and in vitro experiments to explore the anti-tumor properties of AuNPP-FA on various tumors and determine whether AuNPP-FA is involved in regulating vascular endothelial cells in TME to promote the normalization of tumor blood vessels,which provide a powerful preclinical study for establishing the effective application of AuNPP-FA in anti-tumor therapy.Hypothesis1.AuNPP-FA has good stability and tumor tissues targeting,and can be absorbed by tumor tissues preferentially.2.AuNPP-FA can effectively kill tumor cells without obvious toxic and side effects.3.AuNPP-FA may be involved in the modification of tumor microenvironment and inhibit tumor proliferation and metastasis.4.AuNPP-FA may affect the functions of vascular endothelial cells,promote the normalization of tumor blood vessels and inhibit tumor metastasis in the process of microenvironment modification.5.In tumor immunotherapy,anti-PD-L1 combined with chemotherapy and AuNPP-FA may improve the efficacy of tumor therapy.Methods and materials1.Preparation and characterization of AuNPP-FA.Firstly,the copolymer PEG-PDEAEA was synthesized by PEG and DEAEA through reversible addition-fragmentation chain transfer polymerization,and then FA or-cy3/cy7 with fluorescence was conjugated onto PEG-PDEAEA by amidation or condensation reaction.At last,AuNPs was bound with PEG-PDEAEA-FA or PEGPDEAEA polymers through sodium borohydride reduction method and ionic interactions to self-assembly form AuNPP,AuNPP-FA,AuNPP materials with fluorescent groups or chemotherapeutic drug groups.Then,nuclear magnetic resonance hydrogen spectroscopy(1H NMR)and fourier transform infrared spectrometer were used to verify the successful preparation of AuNPP-FA and other derived materials during the synthetic process.Transmission electron microscopy(TEM)and dynamic light scatterometer(DLS)were used to determine the surface morphology,size,zeta potential and stability of AuNPP-FA.CCK8 method was performed to detect the antitumor characteristics of the synthesized materials.Finally,TEM,immunofluorescence,flow cytometry,breast carcinoma in situ model and inductively coupled plasma mass spectrometry were used to identify the good targeting and biological distribution of AuNPP-FA in vivo.2.Effects of AuNPP-FA on biological functions of tumors in vitro and in vivo.Firstly,after tumor cells were stimulated by AuNPP-FA,the effects of AuNPP-FA on tumor proliferation and migration were detected by cloning formation assay,EdU cell proliferation assay and wound healing assay.Secondly,the subcutaneous tumor model of gastric cancer and breast carcinoma in situ model were successfully established and the effect of AuNPP-FA on tumor proliferation and metastasis in vivo was detected after intraperitoneal inj ection of quantitative amount of AuNPP-FA every day.The specific steps of AuNPP-FA acting on tumor metastasis in vivo were further discussed by using tail vein lung metastasis model and spontaneous tumor metastasis mouse model.Finally,the in vivo safety of AuNPP-FA was evaluated by HE staining,body weight,blood routine,blood biochemistry,urine and stool routine examination.3.Effects of AuNPP-FA on tumor angiogenesis and tumor vascular normalization.After the tumor-bearing mice were treated with AuNPP-FA by intraperitoneal injection,the connection of tumor vascular endothelial cells were detected by immunofluorescence stained with CD31 and scanning electron microscope(SEM).Immunofluorescence stained with α-SMA,VE-cadherin and CD31 was used to evaluate the pericyte coverage and the connection between endothelial vessels.The situations of tumor vascular perfusion,leakage and hypoxia of tumor tissues were detected by intravenous injection of fluorescein-labeled Lycopersicon esculentum(Tomato)lectin,FITC-conjugated dextran or pimonidazole.The infiltration of cytotoxic T lymphocytes and the expression of PD-L1 in tumor tissues were detected by immunofluorescence and histochemistry.Then,the therapeutic effect of anti-PD-L1 combined with AuNPP-FA was evaluated.After we got different tumor conditioned mediums stimulated by AuNPP-FA or not,the vascular endothelial cells(HUVECs)were cultured with these conditioned mediums,and then effects on the proliferation and migration of vascular endothelial cells,whether vascular endothelial cells stayed in a resting state and the markers of vascular normalization were detected.4.Preliminary study on the mechanism of tumor cell-vascular endothelial cell interaction induced by AuNPP-FA.Real-time PCR,Western blot and ELISA assays were used to detect the expression of different vascular growth/inhibitory factors in a variety of tumor cells stimulated by AuNPP-FA.The changes of signal pathways in vascular endothelial cells stimulated by conditioned mediums were detected by Western blot.After we identified that SEMA3A as a secretory protein had the most obvious change in expression among these common cytokines,the changes of signal pathways in vascular endothelial cells with or without SEMA3A’s receptor NRP-1 knocked down were detected by western blot after vascular endothelial cell were stimulated by the recombinant human protein SEMA3A.Results1.AuNPP-FA has good stability and targeting,and possesses antineoplastic activity.Poly(ethylene glycol)-b-poly(diethylaminoethyl acrylate)(PEG-PDEAEA)amphiphilic diblock copolymer was synthesized through two sequential steps of reversible addition-fragmentation chain transfer(RAFT)polymerizations.There were tiny characteristic peaks of folate in the range of 6.0～8.5 ppm,the C=O stretching of secondary amide,and C-N stretching and N-H bending in amide groups,which confirmed the successful conjugation of Folate through amidation onto PEG-PDEAEA to form PEG-PDEAEA-FA through 1H NMR and FTIR spectra.And then AuNPP-FA was self-assembly formed by coulombic interaction between PEG-PDEAEA-FA and AuNPs.In a similar way,we also synthesized MPA Au,PEG-SH Au and AuNPP,AuNPP-FA-cy3/cy7,AuNPP-Dox,AuNPP-Dox-FA and other derivative materials.Transmission electron microscopy(TEM)and dynamic light scatterometer(DLS)showed that AuNPP-FA was uniform monodisperse spherical gold nanoparticles,the diameter of which was about 1-6nm in PBS solution,the zeta potential fluctuation was small and the relative stability was high as it could exist stably in aqueous solution.Compared with AuNPP-Dox-FA,AuNPP-FA also showed a great effect on killing tumor cells by CCK8 assay,which aroused our great interest.So AuNPP-FA was selected as the main object in this study.After verifying the high expression of folic acid receptor in a variety of tumor cells,transmission electron microscopy,immunofluorescence and flow cytometry showed that the amount of AuNPP-FA uptake by tumor cells was significantly higher than that in the AuNPP group.The distribution of AuNPs in vivo was detected by breast carcinoma in situ model and the results showed that AuNPP-FA accumulated clearly in the tumor site and was significantly better than that in the AuNPP group.At the same time,with the passage of time,more material fluorescence signals could still be detected in the AuNPP-FA group.What’s more,after imaging the organs of the mice,the tumor tissue of the AuNPP-FA group had more material accumulation.In addition,the material fluorescence signals were the highest in the kidney.Finally,through ICP-MS technique,it was found that there were a large number of Au exclusion in urine,which confirmed that AuNPP-FA was the main metabolic pathway through renal metabolism.2.AuNPP-FA can effectively inhibit tumor proliferation and metastasis in vivo but only play an anti-proliferation role in vitro.Cloning formation assay,EdU cell proliferation assay and wound healing assay were performed to detect the effect of AuNPP-FA on the proliferation and migration of tumor cells in vitro.The results showed that AuNPP-FA could effectively inhibit the proliferation of tumor cells,but had no effect on tumor cells migration.With the use of the tumor-bearing mouse models,it was confirmed that AuNPP-FA could significantly inhibit tumor proliferation and metastasis in vivo.At the same time,HE staining,body weight,blood routine,blood biochemistry,urine and stool routine examination showed that the toxic and side effects of AuNPP-FA on the main organs in animals were weak with its strong safety and high biocompatibility.3.AuNPP-FA promotes tumor vascular normalization.After intraperitoneal injection of AuNPP-FA,CD31 was detected by immunofluorescence assay and blood vessels were observed by scanning electron microscope.The results showed that the number and density of tumor neovascularization in AuNPP-FA group was significantly decreased,and the intercellular adhesion and connection of vascular endothelial cells were enhanced.Then,the pericytes and interendothelial junction protein VE-cadherin of tumor vascular endothelial cells were detected by immunofluorescence,and we got the the consistent results as the number of pericytes covered by blood vessels increased,and the connection between vascular endothelial cells was significantly enhanced in AuNPPFA group,which marked the maturity,functionalization and normalization of blood vessels.In the aspect of vascular function,the immunofluorescence was performed to observe the tumor vessels after mice were intravenous injected with fluorescein-labeled Lycopersicon esculentum(Tomato)lectin,FITC-conjugated dextran or pimonidazole.The results showed that the vascular perfusion increased,leakage decreased and hypoxia was significantly improved in AuNPP-FA group.In the process of tumor vascular normalization,the infiltration of cytotoxic T lymphocytes increased.At the same time,anti-PD-L1 combined with paclitaxel and AuNPP-FA could significantly inhibit the proliferation and metastasis of tumor.In vitro,after HUVEC was stimulated by conditioned medium,it was found that compared with tumor conditioned medium alone,tumor supernatant stimulated by AuNPP-FA could effectively reduce the permeability and migration ability of HUVEC,and tightened adherens junctions between vascular endothelial cells.What’s more,the expression of Ki-67 decreased and the expression of IL33 increased significantly in vascular endothelial cells,which was a key sign for quiescent vascular endothelial cells.The expression of vascular normalization index PHD2 was also significantly inhibited in the stimulation group by western blot.These results showed that the tumor supernatant stimulated by AuNPPFA could effectively promote the tumor vascular endothelial cells to be in a resting state,leading to the normalization of tumor blood vessels.However,simple tumor supernatant without the stimulation of AuNPP-FA had no such effects on HUVEC,which proved obviously that after stimulated by AuNPP-FA,tumor cells would interact with vascular endothelial cells to mediate the quiescent state and tight junction in vascular endothelial cells,resulting in decreased permeability of HUVECs and promoted tumor vascular normalization,while AuNPP-FA did not act directly on vascular endothelial cells.4.AuNPP-FA stimulates SEMA3A secretion from cancer cells and inhibites the Smad2/3 signaling pathway in vascular endothelial cells.Real-time PCR,Western blot and ELISA assays were used to detect the expression of various vascular growth/inhibitory factors stimulated by AuNPP-FA.The results showed that the secretion of SEMA3A protein by tumor cells was significantly increased.Both conditioned medium and recombinant human SEMA3A protein could significantly inhibit Smad2/3 signaling pathway in HUVECs.When NRP-1,the cell membrane receptor of SEMA3A,was knocked down in vascular endothelial cells,the inhibition of Smad2/3 signaling pathway by CMs or SEMA3A were restored.The results showed that the secretion of SEMA3A were inceased by tumor cells with the stimulus of AuNPP-FA and then,SEMA3A combined with NRP-1 of vascular endothelial cells to inhibite the Smad2/3 signaling pathway in vascular endothelial cells,thus promoting the normalization of tumor blood vessels.Conclusions1.The successfully synthetic gold nanoparticles AuNPP-FA with good stability,can effectively target tumor cells to possesses antineoplastic activity.2.AuNPP-FA can inhibit the proliferation of tumor cells in vitro,but has no significant effect on the migration of tumor cells,while it can inhibit the proliferation and metastasis of tumor cells at the same time in vivo.3.AuNPP-FA can promote the normalization of tumor vascular morphology and functions,which is the main effect stage to inhibite tumor metastasis in vivo,rather than depending on the inhibition of tumor cell extravasation or metastatic niches proliferation.4.The application of AuNPP-FA in tumor-bearing mice is safe,and no obvious toxic and side effects of AuNPP-FA on important organs of mice.5.Cancer cells secrete SEMA3 A after treated with AuNPP-FA and then SEMA3A binds to NRP-1 of vascular endothelial cells,which inhibits the smad2/3 signaling pathway of vascular endothelial cells and promotes the normalization of tumor blood vessels.6.After promoting the normalization of tumor blood vessels,AuNPP-FA can increase the infiltration of CTLs and improve the therapeutic effect of PD-L1 inhibitor combined with chemotherapy drugs.