| Leptomeningeal carcinomatosis(LMC)is a serious complication in the late stage of tumors,referring to invasion of the leptomeninges and subarachnoid space by malignant tumors metastasis which are common in breast cancer,lung cancer and melanoma.The prognosis of patients with LMC is poor with a high mortality rate,and median survival is only 4-6 weeks without treatment.Even the application of systemic or local chemotherapy,radiotherapy,molecularly targeted therapy,immunotherapy,etc.,have merely been found to extend the median survival period of patients from 1-3 months to 3-11 months.Intrathecal injection of methotrexate(MTX)is considered the most frequently used drug in clinical treatment of LMC.Our research team has gained plenty of clinical data and experience in the long-term diagnosis and treatment of LMC,with deep understanding of its efficacy and related side effects.However,due to the heterogeneity of patients,intrathecal injection treatment plan,and the existence of confounding factors such as combination of other treatments,the effect of intrathecal MTX may be completely different in various studies accompanied by white matter demyelination,bone marrow suppression,explosive cerebral edema and other side effects.Since nanoparticles with their excellent optical,electrical properties hold significant potential to broaden the diagnosis and treatment options for diseases,nanomedicine has shown unique advantages in the treatment of malignant tumors,Therefore,the development of novel nanomedicine suitable for LMC treatment and systematically exploring their efficacy and safety are highly scientific with prospects of clinical application.In recent years,dynamic therapy based on reactive oxygen species(ROS)(e.g.,hydrogen peroxide(H2O2),singlet oxygen(1O2),and hydroxyl radicals,·OH))within tumor cells capable of inducing cell death by nonspecific oxidative damage on cellular life-sustaining biomolecules(e.g.,proteins,DNA,and lipids)has attracted extensive attention of researchers duo to its controllability and high efficiency.Metal organic frameworks(MOFs),which are crystalline porous materials formed by the self-assembly of organic ligands and metal ions or metal clusters through coordination bonds,have become increasingly popular for constructing nanosystems for biomedical applications,benefiting from their unique properties(adjustable structure,high specific surface area,porosity,etc.).In particular,the porous structures and abundant functional groups of MOF nanoparticles endow them with easy integration of functional moieties,such as drugs,photo-/sono-sensitizers,nanocatalysts,targeting molecules,for varied tumor therapies.In terms of above perspectives,we selected LMC patients treated with intrathecal MTX and control group for case-control study to explore clinical efficacy and safety of intrathecal MTX by observing the changes of cerebrospinal fluid(CSF)indexes,clinical response,and survival period.In addition,we herein fabricated a targeted Zirconium(Zr)-MOF-based nanosystem(MOF@MP-RGD)as a dynamic therapy platform through an"one-pot synthetic approach".In such nanosystem,bifunctional TCPP(Fe)was employed not only as single-site Fenton-type catalyst for chemodynamic therapy(CDT)which could catalyze the conversion of endogenous H2O2 into·OH in the mildly acidic and H2O2-overexpressed tumor microenvironment(TME),but also as a porphyrin-type sonosensitizer for sonodynamic therapy(SDT)which could induce change of O2 to 1O2 upon ultrasound(US)irradiation.We further selected human breast cancer cells MDA-MB-231 cells and an orthotopic LMC mouse model with MOF@MP-RGD nanosystems intrathecally injected to explore their role and mechanism in inhibiting tumor cell proliferation both in vivo and in vitro,and provide a theoretical basis for the treatment of LMC with novel nanomedicine via intrathecal administration.Part one The efficacy and safety analysis of intrathecal methotrexate in the treatment of leptomeningeal carcinomatosisObjective:To explore clinical efficacy and safety of intrathecal MTX by observing the changes of CSF indexes,clinical response and survival time,and further analyze the predictive factors of good prognosis and side effects.Methods:According to the LMC diagnostic guideline of the European Association of Neuro-Oncology-European Society for Medical Oncology(EANO-ESMO)in 2017,35 LMC patients were enrolled with 29 patients treated with intrathecal MTX and 6 patients as the control group.The past history,present history,treatment process,laboratory blood and CSF related tests,and Magnetic Resonance Imaging(MRI)were collected,and the treatment response and related side effects were evaluated and graded,to analyze the clinical characteristics of LMC patients and the therapeutic efficacy and safety of intrathecal MTX.Cox proportional-hazards model and binary logistic regression were used to explore the predictors of good prognosis and side effects of intrathecal MTX.Results:1.The most common primary tumor in 35 patients with LMC was lung cancer(23 cases),followed by 5 cases of breast cancer,3 cases of gastric cancer,and 1 case of glioma,melanoma,pancreatic cancer,and myeloid sarcoma,respectively.Epidermal Growth Factor Receptor(EGFR)mutation happened in 78.3%patients with lung cancer.11 cases had only meningeal metastasis,13 cases had brain parenchyma and meningeal metastasis,and extraneural metastatic sites(bone,liver,lymph node,lumbar spine,thoracic cavity,abdominal cavity and other organ metastasis)were seen in 17 cases.2.Lumbar puncture was performed in all 35 patients before treatment.Thereinto,18(51.4%)patients had increased intracranial pressure(ICP)(223.6±117.7 mmH2O),19(54.3%)patients had decreased glucose content(2.2±1.3 mmol/L),and 22(62.9%)patients had increased protein content(1.0±1.0 g/L).3.29 patients received standard intrathecal MTX treatment,of which 3 patients had complete remission,19 had partial remission,5 patients were stable and 2 patients progressed.Six patients did not receive intrathecal injection,of which 4 had stable disease and 2 had progressive disease.4.25 patients in intrathecal MTX group underwent lumbar puncture post-treatment.12 patients(48.%)had increased ICP(221.4±120.4 mmH2O)before therapy,and 5 patients(20.%)had increased ICP(151.5 mmH2O ±93.6 mmH2O,P=0.003)post-treatment.14 patients(56.%)had decreased glucose(2.2±1.3 mmol/L)before therapy,and 8 patients(32.%)had decreased glucose(3.0±1.2 mmol/L,P=0.009)post-treatment.15 patients(60.%)had increased protein(0.9±0.9 g/L)before therapy,and 10 patients(40.%)had increased protein(0.7±0.8 g/L,P=0.020)post-treatment.5.The median survival time in the intrathecal chemotherapy group was 6.1 months,and 1.9 months in the non-intrathecal chemotherapy group(P=0.007).The median survival was 17.9 months in the targeted therapy group and 3.6 months in the non-targeted therapy group(P=0.015).6.Grade 1 myelosuppression occurred in 33.3%of patients,and bone metastasis was a risk factor for myelosuppression(P=0.032).White matter demyelination happened in 27.3%of patients,and the duration of disease ≥1 year was the risk factor of white matter demyelination(P=0.034).7.Intrathecal injection(P=0.023)and targeted therapy(P=0.011)were were related to good prognosis.Conclusions:1.Lung cancer was the most common primary cancer in LMC with a high EGFR mutation rate,followed by breast cancer and gastric cancer.Increased ICP,decreased glucose,increased protein,brain parenchymal and other organs metastasis frequently happened in patients with LMC.2.Intrathecal injection of MTX could significantly improve the CSF indexes and prolong the median survival of patients to 6.1 months.Targeted therapy extended median survival to 17.9 months.3.Systemic toxicity such as myelosuppression and white matter demyelination often occurred during intrathecal MTX.Myelosuppression was associated with bone metastasis,and white matter demyelination was associated with disease duration ≥1 year.4.Intrathecal MTX and targeted therapy were related to good prognosis.Part two Construction of metalloporphyrin-loaded targeted Zr-based Metal Organic Frameworks and in vitro antitumor studyObjective:To fabricate a novel targeted Zr-based MOF nanosystem(MOF@MP-RGD)loaded with TCPP(Fe),and utilizing MDA-MB-231 cells to explore the targeting ability of the nanoparticles and mechanism of synergistic inhibition of tumor cell through CDT and SDT.Methods:TCPP(Fe)loaded Zr-based MOF nanoparticles(MOF@MP)were constructed by a "one-pot synthetic approach",and further modified with RGD peptide as an active targeting ligand through the classic EDC-NHS coupling(denoted as MOF@MP-RGD).3,3’,5,5’-tetramethylbenzidine(TMB)and 1,3-diphenylisobenzofuran(DPBF)were used as detection agents to monitor the generation of·OH and 1O2.The cell internalization of MOF@MP-RGD nanosystems was evaluated in MDA-MB-231 cells,with human dermal fibroblasts(HDFs)as the control,by a fluorescent inverted microscope.2’,7’-Dichlorodihydrofluorescein diacetate(DCFH-DA)was utilized to detect the MOF@MP-RGD nanosystems-mediated ROS production within tumor cells in simulated tumor microenvironment(TME)or/and irradiated by ultrasound(US).Cytotoxicities of MOF@MP-RGD nanosystems on MDA-MB-231 via CDT and SDT were measured by CCK-8 assay and Calcein-AM/PI staining.Cytotoxicities of MTX on MDA-MB-231 cells and HDFs were also measured by CCK-8 assay.Results:1.The MOF@MP-RGD nanosystems exhibited the smooth octahedral geometry with a size of<200 nm and good dispersion.The loading of TCPP(Fe)was 24.1 wt%,and the targeted RGD peptides were successfully modified on the surface of nanoparticles through covalent bonds.The MOF@MP-RGD nanosystems showed excellent stability in different physiological environments(DMEM,CSF,and 0.%NaCl).2.The Fenton reaction catalytic performance of MOF@MP-RGD nanosystem exhibited properties of concentration-dependence and acidic H2O2 environmental responsiveness.The MOF@MP-RGD nanosystems were able to catalyze the conversion of O2 to 1O2 under US irradiation.3.The MOF@MP-RGD nanosystems displayed concentration-dependent cytotoxicity to MDA-MB-231 cells,but no cytotoxicity to HDFs.When MDA-MB-231 cells and HDFs were co-cultured with MTX for 48 h,the cell viabilities were 60%and 53.8%,respectively.4.Obvious stronger nanoparticle fluorescence was observed in MDA-MB-231 tumor cells treated with MOF@MP-RGD nanosystems,compared with MOF@MP and in HDFs.5.It was found that the green fluorescence in the cells simultaneously treated with MOF@MP-RGD nanosystems and US irradiation under acidic H2O2 environment was the strongest.The cell viability of MOF@MP-RGD nanosystems was 59.7%in single simulated TME,and the viabilities of the MOF@MP-RGD nanosystems after different US duration(60 s,90 s,and 120 s)were decreased to 70.7%,56.2%,and 49.6%,respectively.Only 19.8%cell survived under US irradiation in acidic H2O2 environment superior to the clinically-used chemotherapeutic MTX.Conclusions:1.MOF@MP nanoparticle loaded with high-efficiency therapeutic agent TCPP(Fe)was successfully synthesized by a " one-pot synthetic approach",then modified with RGD,finally forming the targeted nanosystem(MOF@MP-RGD).MOF@MP-RGD nanosystems proved high TCPP(Fe)loading efficiency,excellent stability,good dispersibility and a particle size<200 nm.2.MOF@MP-RGD nanosystems could catalyze the conversion of H2O2 to·OH in simulated TME and induce the change of O2 to 1O2 under US irradiation,which demonstrated excellent TME-responsive CDT and US-controlled SDT.3.In vitro,MOF@MP-RGD nanosystems could be selectively taken up by tumor cells.4.In vitro,MOF@MP-RGD nanosystems could effectively and synergistically increase intracellular ROS levels through tumor-specific CDT and SDT,causing oxidative stress and significantly inhibiting tumor cell proliferation without cytotoxicity to normal cells meaning high biosafety.MTX had no selective and relatively weak cytotoxicities on MDA-MB-231 cells and HDFs.Part three In vivo efficacy and biosafety of intrathecal administration of targeted Zr-based Metal Organic Frameworks in the therapy of leptomeningeal carcinomatosisObjective:To confirm the metabolic pathway,anti-tumor effect and biosafety of intrathecal injection of MOF@MP-RGD nano systems in orthotopic LMC mice,and to provide theoretical basis for new nanomedicine in the remedy of LMC.Methods:The LMC mouse model was established by intrathecal injection of 1 × 105 MDA-MB-231-luc cell into Balb/c male mice.The contents of nanoparticles in brain tissues,main organs and excretions post-injection of nanosystems were analyzed by In Vivo Imaging System(IVIS)and inductively coupled plasma optical emission spectrometer(ICP-OES)to observe its metabolism in vivo.MOF@MP-RGD nanosystems were intrathecally injected into the LMC mice and US irradiation was executed,which were repeated 3 times on day 1,5,8 after grouping.The tumor volumes were measured by IVIS once a week;the weight and survival period of the mice were recorded;tumor tissue and main organs were collected for Hematoxylin-Eosin staining(HE staining)and Ki-67 staining,to systematically evaluate the anti-tumor effect of such nanosystem.Healthy ICR mice were intrathecally injected with MOF@MP-RGD nanosystems twice a week.After one month,blood samples were obtained for blood panel analysis and blood biochemistry test,and the main organs were collected for HE staining,to observe the biosafety of intrathecal injection of MOF@MP-RGD.Results:1.Fluorescence qualification and elemental quantification showed MOF@MP-RGD nanosystems could selectively target LMC tumor tissue after intrathecal injection,showing the highest brain enrichment(26.8%ID/g)and small amount of Zr element(1.5%ID/g)in the liver at the 24th hour.Within 72 hours of intrathecal injection,29.0%and 5.1%of the nanoparticles were excreted in feces and urine,respectively.2.MOF@MP-RGD nanosystems and US irradiation significantly inhibited tumor growth(P=0.008)and prolonged survival(P=0.003)of LMC mice.Whereas the effect of MTX in suppressing tumor growth and lengthening the survival period were limited without statistical difference.3.The intrathecal injection of MOF@MP-RGD nanosystems had good biocompatibility without damage to the blood system,major organs,and body weight increase of mice,while the MTX group showed a significant inhibition of weight augment(P=0.010).Conclusions:1.This study confirmed that after intrathecal injection of the MOF@MP-RGD nanosystems in an orthotopic LMC mouse model,it was rapidly and efficiently enriched in the tumor tissue,metabolized by the liver,and excreted mainly through feces,avoiding long-term retention and potential toxicity in vivo which are beneficial for clinical translation.2.MOF@MP-RGD nanosystems effectively alleviated the LMC progression and lengthened the survival period without serious systemic toxicity.In comparison,MTX,as one of the most commonly utilized chemotherapy for the clinical remedy of LMC,presented restricted effect followed by unsatisfactory adverse event.3.This work demonstrated the therapeutic effectiveness of applying intrathecal administration way to accomplish MOF@MP-RGD nanosystems delivery against LMC,offering a new but promising strategy for clinical LMC treatment with high biosafety. |
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