Functionalization Of Melanin-like Nanomaterials And Their Application In Tumor Thermo-Chemotherapy/Immunotherapy

BackgroundCancer is still a major threat to human health.Traditional cancer treatments,including surgery,chemotherapy,radiotherapy and immunotherapy,relieve the burden of cancer to a certain extent,but there are many problems,such as serious side effect,poor targeted ability,multidrug resistance and tumor recurrence.With the vigorous development of nano-biomedicine,more and more nanomedicines have been developed for tumor diagnosis and treatment.Nanomedicines have many advantages including targeted delivery,controlled released,stimulus response,functional integration,etc.These advantages make nanomedicines show great potential in reducing the toxicity of traditional drugs,improving targeting,and improving multidrug resistance.One of the important components of nanomedicine is nanomaterials.Small molecule drugs are loaded on nanomaterials by covalent/non-covalent modification or physical encapsulation to obtain nanomedicines.Through the functionalization of nanocarriers,nanomedicines can modify target molecules,in vivo behavior optimization molecules,therapeutic drugs or genes,imaging agents,stimuli-responsive modules,etc.to achieve the functional optimization of nanomedicine,reduce toxicity and enhance efficacy.Among the numerous inorganic and organic nanomaterials used to construct nanomedicines,melanin-like nanomaterials have received increasing attention due to their excellent physicochemical properties.Natural melanin can be divided into eumelanin,non-melanin,neuromelanin,heterologous melanin and prokaryotic melanin according to the source.The main components of biosynthetic eumelanin granules extracted from cuttlefish ink include5,6-dihydroxyindole(DHI)and 5,6-dihydroxyindole-2-Carboxyl(DHICA).The natural nanoparticles isolated from cuttlefish ink by differential centrifugation and polydopamine(PDA)nanoparticles synthesized in vitro are called melanin-like nanoparticles(MNPs)because they have similar physical and chemical properties to natural eumelanin.MNPs have attracted extensive attention in the fields of drug delivery,in vivo imaging,and tumor photothermal therapy due to their advantages of easy access,easy surface modification,outstanding optical properties,and good biocompatibility.As a non-invasive treatment method,photothermal therapy(PTT)has the advantages of less side effects and precise control.Nanomaterial-mediated tumor photothermal therapy has also gained increasing attention.However,the uneven distribution of photothermal conversion agents in tumor tissue causes deep tumor cells survival and eventually leads to incomplete ablation of tumor tissue.In addition,the inflammation accompanying photothermal ablation of tumor cells recruits a large number of TAMs,which increases the burden of tumor immunosuppression and further promotes tumor development.In recent years,melanin-like nanomaterials have been widely studied in the field of tumor photothermal therapy due to their excellent photothermal properties and biocompatibility.In this study,two different functionalized melanin-like nano-systems were mainly constructed to achieve high-efficiency tumor treatment:1)The functionalization of melanin-like nanoparticles and their application in tumor photothermal-chemotherapy;2)The functionalization of PDA melanin-like nanoparticles and their application in tumor photothermal-immunotherapy.This study focuses on the scientific problems in tumor photothermal therapy,and designs different functionalized melanin-like nano-systems to enhance the effect of tumor treatment.The functional design of this novel provides new design ideas to overcome the dilemma in tumor therapy.Part 1 The functionalization of melanin-like nanoparticles and their application in tumor photothermal-chemotherapyObjectivesIn this part of the study,aiming at the problem that the uneven distribution of photothermal conversion agents leads to the limited killing of photothermal effect on deep tumor cells,the size-changing melanin-like nanoparticles of cascade response to drug release were designed for combined tumor photothermal-chemotherapy treatment.The nano-system is loaded with doxorubicin by ultra-small melanin-like nanoparticles and then encapsulated with thermosensitive liposomes.It has the ability of drug release in the cascade response of near-infrared light(NIR)and tumor microenvironment,and also meets the contradictory requirements for nanoparticle size in vivo circulation and tumor tissue penetration.Firstly,large-sized NPs were enriched at the tumor site through EPR effect,and when irradiated with NIR,tumor cells were ablated and the thermosensitive liposome structure was destroyed.Small-sized NPs and DOX were further released into the tumor microenvironment and penetrate into deep tumor tisssues to kill tumor cells.This size-transferable nanosystem was desinged to enhance tumor therapeutics.Methods1.Preparation and characterization of melanin-like nano-systemWater-soluble ultra-small melanin-like nanoparticles were obtained by dissolving the melanin granule and then oxidatively self-polymerization.The melanin-like nanoparticles were loaded with drugs and co-extruded with thermosensitive lipid films to obtain the final nano-system MNP/DOX@TSL.Transmission electron microscope(TEM)was used to characterize the morphology of nanoparticles;dynamic light scattering(DLS)was used to characterize the hydrated particle size and Zeta potential of nanoparticles;UV-vis spectrophotometer was used to analyze UV-vis absorption spectrum;Fluorescence spectrophotometer was used to characterize the fluorescence emission spectrum;An infrared thermal imager was used to record the temperature change of the nanoparticles under NIR irradiation.2.In vitro and in vivo permeation ability of melanin-like nano-systemsThe permeability of nanoparticles in simulated extracellular matrix,3D tumor cell spheroids,and in vivo tumor tissues was investigated before and after irradiation with near-infrared light.The in vivo experiments were performed as follows:a 4T1tumor-bearing mouse model was constructed.When the tumor volume reached 100mm~3,MNP/DOX or MNP/DOX@TSL nanoparticles(10 mg/kg)were injected intravenously,respectively.12 h after injection,tumor sites were exposed to NIR irradiation for 5 min if needed.Tumors were collected,sectioned and stained,then were observed using a confocal microscope.3.In vivo metabolism and photothermal effects of melanin-like nanosystemHealthy Balb/c female mice(6-8 weeks old)were injected intravenously with PBS,free Cy5.5,MNP/Cy5.5 or MNP/Cy5.5@TSL NPs(10 mg/Kg).Blood samples were obtained through the tail-tip vein at different time points after intravenous administration,and the fluorescence intensity of the samples was analyzed using a fluorescence imaging system.4T1 tumor-bearing mice were used to study the biodistribution of MNP/Cy5.5@TSL NPs.When the tumor volume reached 100 mm~3,the mice were injected with different preparations through the tail vein and euthanized 12 h after the injection.Major organs(heart,liver,spleen,lung,kidney)and tumor tissues were collected separately,and ex vivo fluorescence images were obtained using a fluorescence imaging system and the relative fluorescence intensity of the samples was analyzed.To investigate in vivo photothermal effect of nanoparticles,tumor-bearing mice were injected with PBS,MNP@TSL and MNP/DOX@TSL(10 mg/Kg)via tail vein,respectively.After 12 h of administration,the tumor sites were exposed to 808 nm laser light.(2 W/cm~2,5 min).An infrared thermal imager was used to record local temperature changes and take pictures.4.In vivo antitumor effect evaluationWhen the tumor volume of tumor-bearing mice was about 100 mm~3,the mice were randomly divided into 5 groups:control group(PBS),MNP@TSL,MNP@TSL+NIR,MNP/DOX@TSL,and MNP/DOX@TSL+NIR.Mice were intravenously injected with nanoparticles on days 0,3,and 6,respectively.Mice in laser groups were exposed to 808 nm laser light(2 W/cm~2)for 5 min.The treatment experiment lasted for 16 days.During the whole experiment,a vernier caliper was used to monitor the change of tumor volume every two days.The tumor volume(V)was calculated according to the following formula:V=(length×width~2)/2,the body weight of the mice was monitored every two days,At the end of the experiment,tumors were collected,weighed and photographed.Results1.Preparation and characterization of melanin-like nano-system.TEM results showed that we synthesized melanin-like nanoparticles with good water dispersibility,and the size was about 3.7 nm.After drug loading and extrusion,MNP/DOX@TSL showed spherical shape with obvious black dot-like particles inside,with a size of about 83 nm,which is consistent with the DLS results.UV-vis absorption spectra showed that after drug loading,the nanoparticles appeared characteristic absorption peaks of DOX at 254 nm and 480 nm,indicating that the drug was loaded successfully.The fluorescence emission spectra showed similar results.2.In vitro and in vivo permeation ability of melanin-like nano-systemsThe results of in vitro experiments show that the penetration depth of MNP/DOX@TSL in the simulated extracellular matrix is limited,and the DOX fluorescence intensity is weak.After NIR irradiation,the fluorescence signal distribution and intensity of the nanoparticles were significantly improved,which were similar to those of MNP/DOX with small particle size,indicating that NIR irradiation destroyed the structural integrity of thermosensitive liposomes and the released small-sized drug-loaded particles have better penetration.The infiltration results in 3D spheroids showed that after NIR irradiation,the fluorescence signal distribution of nanoparticles in spheroids was closer to the center of spheroids,and the fluorescence intensity was stronger.In vivo tumor tissue section experiment results showed that the DOX fluorescence signal distribution in MNP/DOX@TSL group was mainly concentrated at the edge,while the DOX fluorescence signal penetration was deeper and the intensity was stronger after laser irradiation.These results demonstrate that this size-switchable nano-system can realize the deep penetration of drug in tumor tissue.3.In vivo metabolism and photothermal effects of melanin-like nano-system.The results of metabolism experiment in vivo showed that thermosensitive liposomes could significantly prolong the circulation time of melanin-like nanoparticles in vivo.In the MNP/Cy5.5 group,no DOX fluorescence signal could be detected in the blood 8 h after intravenous administration,while a significant fluorescence signal could still be detected in MNP/Cy5.5@TSL 12 h after the injection.The results of in vivo photothermal effect showed that after intravenous injection of MNP/DOX@TSL nanoparticles for 12 h,the local temperature of the tumor could be increased to 52°C after 5 min of laser irradiation,indicating that the nano-system has a good photothermal conversion effect in vivo.4.In vivo antitumor effect evaluation.From the experimental results,the MNP/DOX@TSL+NIR group has the best tumor growth inhibition effect,significantly delaying tumor growth,and the inhibition rate can reach 74%,which is much higher than that of photothermal therapy alone(16%)and chemotherapy alone(27%).There was no significant change in the body weight of mice compared with the control group,indicating that the nano-system did not cause serious systemic toxicity and had good biological safety.ConclusionsThe size-switchable melanin-like nano-system MNP/DOX@TSL with cascade response to drug release was successfully constructed to realize photothermal therapy combined with chemotherapy of deep tumor cells.The design can significantly improve the infiltration ability of nano-drugs in tumor tissues while realizing long circulation in vivo,and ultimately significantly inhibit tumor growth in tumor-bearing mice,achieving enhanced anti-tumor effect.Part 2 The functionalization of PDA melanin-like nanoparticles and their application in tumor photothermal-immunotherapyObjectiveLocal persistent inflammation caused by photothermal therapy increases the burden of immune suppression by recruiting a large number of tumor-associated macrophages(TAMs),which in turn helps the survival of remaining tumor cells,thereby hindering anti-tumor therapy.To address this problem,this part designed a multifunctional polydopamine(PDA)melanin-like black nanoparticles(NPs)to reshape the tumor immune microenvironment after PTT to achieve photothermal synergistic immune anti-tumor therapy.The final P/T@MM nanoparticles were obtained by co-extruding the PDA NPs loading TMP195 small molecules and the macrophage membrane through a liposome extruder.Macrophage membranes endow nanoparticles with long-circulation and inflammatory targeting capabilities,and TMP195 repolarizes the phenotype of TAMs from an immunosuppressive M2phenotype to an antitumor M1 phenotype by affecting the epigenetic signature of monocyte-macrophages,PDA as drug carrier and photothermal conversion agent.This triple multifunctional(including biomimetic inflammatory targeting,phenotypic repolarization of TAMs,and photothermal conversion)melanin-like nano-system can reshape the inflammatory immunosuppressive microenvironment after PTT and is expected to achieve excellent combined antitumor efficacy.Methods1.Synthesis and characterization of P/T@MM nanoparticles.Polydopamine nanoparticles were prepared by controlling the oxidative self-polymerization of dopamine,adsorbing TMP195 small molecules through non-covalent bonds,and then co-extruding the drug-loaded nanoparticles and macrophage membranes through a liposome extruder to obtain a macrophage membrane coating.Nanoparticles were characterized by TEM,DLS,UV-vis spectrophotometer for morphology,hydrated particle size and Zeta potential,and UV-vis absorption spectrum.2.Photothermal effect investigation of P/T@MM nanoparticles and repolarized TAMs phenotypes in vitro.The temperature of nanoparticles under laser irradiation at 808 nm was recorded with an infrared thermal imager.The relationship between the photothermal effect and the concentration of nanoparticles,laser power and laser irradiation time were investigated,and the photothermal conversion efficiency was calculated.The cytotoxicity of nanocarriers was investigated by incubating cells with different concentrations of nanocarriers.4T1 cells were incubated with 100μg/m L PDA and irradiated with 808 nm laser for 10 min.Cell viability was detected to evaluate the photothermal toxicity of the nano-system.RAW264.7 cells were co-incubated with IL-4 for 24 h to induce M2 phenotypic differentiation,and then incubated with different nanoparticles for 24 h.Flow cytometry was used to detect the proportion of M2 phenotypic macrophages,and ELISA was used to analyze the secretion levels of relevant inflammatory factors in cell culture supernatants.3.Local inflammatory effect of tumor after PTT and P/T@MM inflammatory targeting investigation.The evaluation of local tumor inflammatory effects after PTT was performed on4T1 tumor-bearing mice:when the tumor volume reached 100 mm~3,the tumor-bearing mice were injected with PBS or P@MM NPs(10 mg/kg)via tail vein,respectively.The tumor-bearing mice were then randomly divided into three groups:PBS,P@MM,and P@MM+NIR,15 mice in each group.Mice in the laser group were irradiated with 808 nm laser for 10 min(1.6 W/cm~2)at 12 h after intravenous injection.At predetermined time points(6,12,24,48,72 h),mice were euthanized and tumors were collected.The same mass of tumors was weighed and ground,and the levels of inflammatory factors in tissue homogenate were detected with a multi-factor detection kit.According to the method for preparing P/T@MM,the near-infrared fluorescent dye Cy5.5 was loaded into the carrier to obtain P/Cy5.5@MM NPs.When the tumor volume reached 100 mm~3,P@MM NPs(10 mg/kg)were injected intravenously,and12 h later,the tumor site was irradiated with 808 nm laser for 10 min.Mice were randomly divided into three groups and injected with free Cy5.5,P/Cy5.5@Lip and P/Cy5.5@MM NPs(1 mg Cy5.5/kg)at 24 h after laser irradiation,respectively.In vivo fluorescence signal of mice at 2,6,12,24,and 48 h were analysis and major organs(heart,liver,spleen,lung,and kidney)and tumor tissues at 6 h after intravenous injection were collected for ex vivo fluorescence analysis.4.In vivo antitumor effect of P/T@MM.4T1 tumor-bearing mice were randomly divided into 5 groups:PBS+NIR,P@MM+NIR,P/T@Lip+NIR,P/T@MM and P/T@MM+NIR.On the 5th day after tumor inoculation,when the tumor volume was close to 100 mm~3,the mice were administration for the first time,and the doses of TMP195 and PDA were 736μg/kg and 10 mg/kg,respectively,and they were administered again three days later.In the laser group,808 nm laser(1.6 W/cm~2)was used to irradiate the tumor site for 10 min12 h after administration.Tumor volume and mouse body weight were recorded every other day,and mouse blood and tumor tissue were collected at the end of treatment for further analysis.The collected tumor tissue was digested,ground and sieved to obtain a single cell suspension.Flow cytometry was used to analyze the infiltration of M1-type TAMs(F4/80~+CD80~+)and M2-type TAMs(F4/80~+CD206~+),cytotoxic T lymphocytes(CD3~+CD8~+),regulatory T cells(CD3~+CD4~+CD25~+),myeloid-derived suppressor cells(CD11b~+Gr1~+).Results1.Synthesis and characterization of P/T@MM nanoparticles.The results of TEM showed that the synthesized PDA was spherical and had good dispersion,and the morphology of the nanoparticles did not change significantly after loading TMP195.After the encapsulation of macrophage membrane,P/T@MM nanoparticles showed a coating with a thickness of 5-10 nm,indicating that the macrophage membrane was successfully covered.DLS results showed that the hydrated particle size of PDA and P/T@MM NPs were about 115 nm and 160 nm,respectively.The Zeta potential of P/T@MM was close to that of macrophage membrane vesicles at about-20 m V.UV-vis absorption spectra show that P/T@MM has broad absorption in the range of 550-900 nm.2.Photothermal effect investigation of P/T@MM nanoparticles and repolarized TAMs phenotypes in vitro.Cytotoxicity results showed that the cell viability was still higher than 80%when the concentration of nanocarriers was as high as 200μg/m L,indicating good cell safety.The cell viability of 4T1 cells incubated with 100μg/m L nanoparticles was reduced to less than 10%after laser irradiation,indicating that the nano-system had good photothermal toxicity.The photothermal effect of nano-systems is positively correlated with the carrier concentration,laser power and irradiation time in a certain range.Flow cytometry results showed that the proportion of M2-phenotypic macrophages decreased from 57.2%to 31.4%after incubation with P/T@MM NPs.ELISA results showed that the secretion of M1-phenotypic inflammatory factor IL6 increased in cell culture supernatant,while the secretion of M2-phenotypic inflammatory factor IL10 decreased.These results demonstrate that P/T@MM nanoparticles can significantly repolarize macrophage phenotypes from M2 to M1 in vitro.3.Local inflammatory effect of tumor after PTT and P/T@MM inflammatory targeting investigation.The detection results of local inflammatory factors in tumor showed that a variety of inflammatory factors remained at a high level within 6-72 hours after photothermal treatment.As can be seen from the analysis figures,the concentration of CCL-2 reached the peak at 24h and decreased within 24-48h but was still higher than that in control group.In addition,granulocyte-macrophage colony stimulating factor(GM-CSF)persistently increased at 6-72 hours after PTT.These results suggest that inflammatory effects after PTT lead to increased secretion of several inflammatory factors.Among them,CCL-2 and GM-CSF play an important role in the recruitment and concentration of TAMs.The results of the inflammatory targeting effect of P/T@MM showed that the macrophage membrane-coated nanoparticles had the most enrichment in tumor,and the fluorescence signal was strongest at 6 h.The tumor tissue at this time was collected for in vitro fluorescence imaging.The results showed that the fluorescence signal of the nanoparticles wrapped by the macrophage membrane was significantly higher than that of the other groups,indicating that the macrophage membrane coating can achieve inflammation-mediated tumor targeting in vivo.4.In vivo antitumor effect of P/T@MM.The results of tumor growth curve in mice showed that the tumor volume of the control group(PBS+NIR)increased rapidly during the 17 days of monitoring,while the tumor volume of the PTT group alone(P@MM+NIR)was inhibited in the early stage and increased significantly after the drug administration was stopped.In contrast,PTT combined with TAMs repolarization(P/T@MM+NIR)significantly inhibited the growth of tumor volume,and the tumor clearance rate even reached 60%,which was much higher than that of PTT alone(10%),indicating that PTT combined with TAMs repolarization strategy had the best anti-tumor effect.Further analysis of tumor infiltrating immune cells showed that at the end of treatment,the proportion of M2-type TAM in the photothermal treatment group was significantly higher than that in the control group,and the proportion of M2-type TAMs in the final treatment group was significantly lower and the proportion of M1/M2 TAMs was significantly higher.It was noteworthy that the infiltrated proportion of T cells in the final treatment group was significantly increased.The proportion of some immunosuppressive cells such as Regulatory T cells(Treg)and Myeloid-derived suppressor cells(MDSCs)were reduced.These results suggest that the P/T@MM nano-system can reshape the immunosuppressive microenvironment after PTT and facilitate cytotoxic T lymphocytes(CTLs)to kill tumor cells.ConclusionBionic melanin-like nanoparticles with targeted inflammation ability(P/T@MM)were successfully constructed to achieve combined photothermal-immunotherapy of tumors by reshaping the tumor immunosuppressive microenvironment.In vitro and in vivo experiments have shown that P/T@MM can significantly repolarize the phenotype of TAMs from immunosuppressive M2 type to anti-tumor M1 type,exhibiting a powerful synergistic antitumor effect in vivo.