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BACKGROUND: The global cellular landscape of the tumor microenvironment (TME) combining primary and metastatic liver tumors has not been comprehensively characterized. METHODS: Based on the scRNA-seq and spatial transcriptomic data of non-tumor liver tissues (NTs), primary liver tumors (PTs) and metastatic liver tumors (MTs), we performed the tissue preference, trajectory reconstruction, transcription factor activity inference, cell-cell interaction and cellular deconvolution analyses to construct a comprehensive cellular landscape of liver tumors. RESULTS: Our analyses depicted the heterogeneous cellular ecosystems in NTs, PTs and MTs. The activated memory B cells and effector T cells were shown to gradually shift to inhibitory B cells, regulatory or exhausted T cells in liver tumors, especially in MTs. Among them, we characterized a unique group of TCF7+ CD8+ memory T cells specifically enriched in MTs that could differentiate into exhausted T cells likely driven by the p38 MAPK signaling. With regard to myeloid cells, the liver-resident macrophages and inflammatory monocyte/macrophages were markedly replaced by tumor-associated macrophages (TAMs), with TREM2+ and UBE2C+ TAMs enriched in PTs, while SPP1+ and WDR45B+ TAMs in MTs. We further showed that the newly identified WDR45B+ TAMs exhibit an M2-like polarization and are associated with adverse prognosis in patients with liver metastases. Additionally, we addressed that endothelial cells display higher immune tolerance and angiogenesis capacity, and provided evidence for the source of the mesenchymal transformation of fibroblasts in tumors. Finally, the malignant hepatocytes and fibroblasts were prioritized as the pivotal cell populations in shaping the microenvironments of PTs and MTs, respectively. Notably, validation analyses by using spatial or bulk transcriptomic data in clinical cohorts concordantly emphasized the clinical significance of these findings. CONCLUSIONS: This study defines the ontological and functional heterogeneities in cellular ecosystems of primary and metastatic liver tumors, providing a foundation for future investigation of the underlying cellular mechanisms.
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Células Endoteliais , Neoplasias Hepáticas , Humanos , Ecossistema , Neoplasias Hepáticas/genética , Perfilação da Expressão Gênica , Microambiente TumoralRESUMO
The cyclic GMP-AMP synthase-stimulator interferon gene (cGAS-STING) pathway is an emerging therapeutic target for the prophylaxis and therapy of a variety of diseases, ranging from cancer, infectious diseases, to autoimmune disorders. As a cytosolic double stranded DNA (dsDNA) sensor, cGAS can bind with relatively long dsDNA, resulting in conformational change and activation of cGAS. Activated cGAS catalyzes the conversion of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) into cGAMP, a cyclic dinucleotide (CDN). CDNs, including 2'3'-cGAMP, stimulate adapter protein STING on the endoplasmic membrane, triggering interferon regulatory factor 3 (IRF3) phosphorylation and nuclear factor kappa B (NF-κB) activation. This results in antitumor and antiviral type I interferon (IFN-I) responses. Moreover, cGAS-STING overactivation and the resulting IFN-I responses have been associated with a number of inflammatory and autoimmune diseases. This makes cGAS-STING appealing immunomodulatory targets for the prophylaxis and therapy of various related diseases. However, drug development of CDNs and CDN derivatives is challenged by their limited biostability, difficult formulation, poor pharmacokinetics, and inefficient tissue accumulation and cytosolic delivery. Though recent synthetic small molecular CDN- or non-CDN-based STING agonists have been reported with promising preclinical therapeutic efficacy, their therapeutic efficacy and safety remain to be fully evaluated preclinically and clinically. Therefore, it is highly desirable and clinically significant to advance drug development for cGAS-STING activation by innovative approaches, such as drug delivery systems and drug development for pharmacological immunomodulation of cGAS. In this Account, we summarize our recent research in the engineering and delivery of immunostimulatory or immunoregulatory modulators for cGAS and STING for the immunotherapy of cancer and autoimmune diseases. To improve the delivery efficiency of CDNs, we developed ionizable and pH-responsive polymeric nanocarriers to load STING agonists, aiming to improve the cellular uptake and facilitate the endosomal escape to induce efficient STING activation. We also codelivered STING agonists with complementary immunostimulatants in nanoparticle-in-hydrogel composites to synergetically elicit potent innate and adaptive antitumor responses that eradicate local and distant large tumors. Further, taking advantage of the simplicity of manufacturing and the established nucleic acid delivery system, we developed oligonucleotide-based cGAS agonists as immunostimulant immunotherapeutics as well as adjuvants for peptide antigens for cancer immunotherapy. To suppress the overly strong proinflammatory responses associated with cGAS-STING overactivation in some of the autoimmune disorders, we devised nanomedicine-in-hydrogel (NiH) that codelivers a cGAS inhibitor and cell-free DNA (cfDNA)-scavenging cationic nanoparticles (cNPs) for systemic immunosuppression in rheumatoid arthritis (RA) therapy. Lastly, we discussed current drug development by targeting cGAS-STING for cancer, infectious diseases, and autoimmune diseases, as well as the potential opportunities for utilizing cGAS-STING pathway for versatile applications in disease treatment.
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Doenças Autoimunes , Doenças Transmissíveis , Interferon Tipo I , Neoplasias , Humanos , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Interferon Tipo I/metabolismo , DNA/metabolismo , Neoplasias/terapia , Imunoterapia , Fatores Imunológicos , Adjuvantes Imunológicos , HidrogéisRESUMO
Cancer has been one of the major healthcare burdens, which demands innovative therapeutic strategies to improve the treatment outcomes. Combination therapy hold great potential to leverage multiple synergistic pathways to improve cancer treatment. Cancer cells often exhibit an increased generation of reactive oxygen species (ROS) and antioxidant species compared with normal cells, and the levels of these species can be further elevated by common therapeutic modalities such as photodynamic therapy (PDT) or chemotherapy. Taking advantage that cancer cells are vulnerable to further oxidative stress, we aim to design a drug delivery system by simultaneously increasing the cellular ROS level, reducing antioxidative capacity, and inducing anticancer chemotherapy in cancer cells. Here, we designed a star-shape polymer, PEG(-b-PCL-Ce6)-b-PBEMA, based on the Passerini three-component reaction, which can both enhance ROS generation during PDT and decrease the GSH level in cancer cells. The polycaprolactone conjugated with photosensitizer Ce6 served as hydrophobic segments to promote micelle formation, and Ce6 was used for PDT. The H2O2-labile group of arylboronic esters pendent on the third segment was designed for H2O2-induced quinone methide (QM) release for GSH depletion. We thoroughly investigated the spectral properties of blank micelle during its assembling process, ROS generation, and H2O2-induced QM release in vitro. Moreover, this polymeric micelle could successfully load hydrophobic anticancer drug Doxorubicin (DOX) and efficiently deliver DOX into cancer cells. The triple combination of ROS generation, GSH elimination, and chemotherapy dramatically improved antitumor efficiency relative to each of them alone in vitro and in vivo.
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Traumatic brain injury (TBI) is one of the major causes of death and disability worldwide. Novel and effective therapy is needed to prevent the secondary spread of damage beyond the initial injury. The aim of this study was to investigate whether berberine has a neuroprotective effect on secondary injury post-TBI, and to explore its potential mechanism in this protection. The mice were randomly divided into Sham-saline, TBI-saline and TBI-Berberine (50 mg/kg). TBI was induced by Feeney's weight-drop technique. Saline or berberine was administered via oral gavage starting 1 h post-TBI and continuously for 21 days. Motor coordination, spatial learning and memory were assessed using beam-walking test and Morris water maze test, respectively. Brain sections were processed for lesion volume assessment, and expression of neuronal nuclei (NeuN), cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), 8-hydroxy-2-deoxyguanosine (8-OHdG), ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) were detected via immunohistochemistry and immunofluorescence. There were statistically significant improvement in motor coordination, spatial learning and memory in the TBI-Berberine group, compared to the TBI-saline group. Treatment with berberine significantly reduced cortical lesion volume, neuronal loss, COX-2, iNOS and 8-OHdG expression in both the cortical lesion border zone (LBZ) and ipsilateral hippocampal CA1 region (CA1), compared to TBI-saline. Berberine treatment also significantly decreased Iba1- and GFAP-positive cell number in both the cortical LBZ and ipsilateral CA1, relative to saline controls. These results indicated that berberine exerted neuroprotective effects on secondary injury in mice with TBI probably through anti-oxidative and anti-inflammatory properties.
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Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Berberina/farmacologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
As the implications of reactive oxygen species (ROS) are elucidated in many diseases, ROS-responsive nanoparticles are attracting great interest from researchers. In this work, a ROS sensitive thioketal (TK) moiety with a π-conjugated structure was introduced into biodegradable methoxy poly(ethylene glycol)-thioketal-poly(ε-caprolactone)mPEG-TK-PCL micelles as a linker, which was designed to speed up the drug release and thus enhance the therapeutic efficacy. The micelle showed a high drug loading content of 12.8% and excellent stability under physiological conditions because of the evocation of π-π stacking and hydrophobic interactions with the anticancer drug doxorubicin (DOX). The polymeric micelle presented a better drug carrier capacity and higher in vitro anticancer efficacy towards cancer cells. The in vivo study showed that DOX-loaded mPEG-TK-PCL micelles displayed lower toxicity towards normal cells and remarkably enhanced antitumor efficacy. This research provides a way to design potential drug carriers for efficient cancer chemotherapy.
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Acetais/química , Portadores de Fármacos/química , Micelas , Polímeros/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Cetonas/química , CamundongosRESUMO
Activating cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) holds great potential for cancer immunotherapy by eliciting type-I interferon (IFN-I) responses. Yet, current approaches to cGAS-STING activation rely on STING agonists, which suffer from difficult formulation, poor pharmacokinetics, and marginal clinical therapeutic efficacy. Here, we report nature-inspired oligonucleotide, Svg3, as a cGAS agonist for cGAS-STING activation in tumor combination immunotherapy. The hairpin-shaped Svg3 strongly binds to cGAS and enhances phase separation to form Svg3-cGAS liquid-like droplets. This results in cGAS-specific immunoactivation and robust IFN-I responses. Remarkably, Svg3 outperforms several state-of-the-art STING agonists in murine and human cells/tissues. Nanoparticle-delivered Svg3 reduces tumor immunosuppression and potentiates immune checkpoint blockade therapeutic efficacy of multiple syngeneic tumor models in wild-type mice, but in neither cGas-/- nor Sting-/- mice. Overall, these results demonstrate the great potential of Svg3 as a cGAS agonistic oligonucleotide for cancer combination immunotherapy.
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Ionizable lipid nanocarriers have made historical contribution to COVID-19 mRNA vaccines. Here, we report ionizable polymeric nanoparticles that co-deliver bi-adjuvant and neoantigen peptides for cancer immunotherapy in combination with immune checkpoint blockade (ICB). Current cancer ICB benefits only a small subset of patients, largely due to a lack of pre-existing target cells and checkpoint targets for ICB, tumor antigenic heterogeneity, and tumor immunosuppression. Therapeutic vaccines hold the potential to enhance ICB therapeutic efficacy by expanding antitumor cell repertoires, upregulating immune checkpoint levels and hence sensitizing ICB, and reducing tumor immunosuppression. Chemically defined peptide vaccines are attractive, but their current therapeutic efficacy has been limited due to 1) poor vaccine delivery to immunomodulatory lymph nodes (LNs) and antigen (Ag)-presenting cells (APCs), 2) poor immunostimulant adjuvant efficacy with restricted target cell subsets in humans, 3) limited adjuvant/Ag codelivery to enhance Ag immunogenicity, and 4) limited ability to overcome tumor antigenic heterogeneity. Here, we developed nanovaccines (NVs) using pH-responsive polymeric micellular nanoparticles (NPs) for the codelivery of bi-adjuvant [Toll-like receptor (TLR) 7/8 agonist R848 and TLR9 agonist CpG] and peptide neoantigens (neoAgs) to draining LNs for efficient Ag presentation in a broad range of APC subsets. These NVs potentiated the immunogenicity of peptide Ags and elicits robust antitumor T cell responses with memory, and remodeled the tumor immune milium with reduced tumor immunosuppression. As a result, NVs significantly enhanced ICB therapeutic efficacy for murine colorectal tumors and orthotopic glioblastoma multiforme (GBM). These results suggest marked potential of bi-adjuvant/neoAg-codelivering NVs for combination cancer immunotherapy.
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Rheumatoid arthritis (RA) is a systemic autoimmune disease with pathogenic inflammation caused partly by excessive cell-free DNA (cfDNA). Specifically, cfDNA is internalized into immune cells, such as macrophages in lymphoid tissues and joints, and activates pattern recognition receptors, including cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), resulting in overly strong proinflammation. Here, nanomedicine-in-hydrogel (NiH) is reported that co-delivers cGAS inhibitor RU.521 (RU) and cfDNA-scavenging cationic nanoparticles (cNPs) to draining lymph nodes (LNs) for systemic immunosuppression in RA therapy. Upon subcutaneous injection, NiH prolongs LN retention of RU and cNPs, which pharmacologically inhibit cGAS and scavenged cfDNA, respectively, to inhibit proinflammation. NiH elicits systemic immunosuppression, repolarizes macrophages, increases fractions of immunosuppressive cells, and decreases fractions of CD4+ T cells and T helper 17 cells. Such skewed immune milieu allows NiH to significantly inhibit RA progression in collagen-induced arthritis mice. These studies underscore the great potential of NiH for RA immunotherapy.
Assuntos
Artrite Reumatoide , Ácidos Nucleicos Livres , Camundongos , Animais , Nanomedicina , Hidrogéis , Artrite Reumatoide/terapia , Terapia de Imunossupressão , Nucleotidiltransferases , Imunoterapia , Linfonodos , DNARESUMO
Current cancer immunotherapy [e.g., immune checkpoint blockade (ICB)] only benefits small subsets of patients, largely due to immunosuppressive tumor microenvironment (TME). In situ tumor vaccination can reduce TME immunosuppression and thereby improve cancer immunotherapy. Here, we present single-dose injectable (nanovaccines + ICBs)-in-hydrogel (NvIH) for robust immunotherapy of large tumors with abscopal effect. NvIH is thermo-responsive hydrogel co-encapsulated with ICB antibodies and novel polymeric nanoparticles loaded with three immunostimulatory agonists for Toll-like receptors 7/8/9 (TLR7/8/9) and stimulator of interferon genes (STING). Upon in situ tumor vaccination, NvIH undergoes rapid sol-to-gel transformation, prolongs tumor retention, sustains the release of immunotherapeutics, and reduces acute systemic inflammation. In multiple poorly immunogenic tumor models, single-dose NvIH reduces multitier TME immunosuppression, elicits potent TME and systemic innate and adaptive antitumor immunity with memory, and regresses both local (vaccinated) and distant large tumors with abscopal effect, including distant orthotopic glioblastoma. Overall, NvIH holds great potential for tumor immunotherapy.
Assuntos
Hidrogéis , Neoplasias , Humanos , Linhagem Celular Tumoral , Imunoterapia , Terapia de Imunossupressão , Neoplasias/terapia , Imunidade Adaptativa , Microambiente TumoralRESUMO
Current cancer immunotherapy (e.g., immune checkpoint blockade (ICB)) has only benefited a small subset of patients. Cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) activation holds the potential to improve cancer immunotherapy by eliciting type-I interferon (IFN-I) responses in cancer cells and myeloid cells. Yet, current approaches to this end, mostly by targeting STING, have marginal clinical therapeutic efficacy. Here, we report a cGAS-specific agonistic oligonucleotide, Svg3, as a novel approach to cGAS-STING activation for versatile cancer immunotherapy. Featured with a hairpin structure with consecutive guanosines flanking the stem, Svg3 binds to cGAS and enhances cGAS-Svg3 phase separation to form liquid-like droplets. This results in cGAS activation by Svg3 for robust and dose-dependent IFN-I responses, which outperforms several state-of-the-art STING agonists in murine and human immune cells, and human tumor tissues. Nanocarriers efficiently delivers Svg3 to tissues, cells, and cytosol where cGAS is located. Svg3 reduces tumor immunosuppression and potentiates ICB therapeutic efficacy of multiple syngeneic tumors, in wildtype but neither cGas-/- nor goldenticket Sting-/- mice. Further, as an immunostimulant adjuvant, Svg3 enhances the immunogenicity of peptide antigens to elicit potent T cell responses for robust ICB combination immunotherapy of tumors. Overall, cGAS-agonistic Svg3 is promising for versatile cancer combination immunotherapy.
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Glioblastoma multiforme (GBM) is the most common and lethal type of adult brain cancer. Current GBM standard of care, including radiotherapy, often ends up with cancer recurrence, resulting in limited long-term survival benefits for GBM patients. Immunotherapy, such as immune checkpoint blockade (ICB), has thus far shown limited clinical benefit for GBM patients. Therapeutic vaccines hold great potential to elicit anti-cancer adaptive immunity, which can be synergistically combined with ICB and radiotherapy. Peptide vaccines are attractive for their ease of manufacturing and stability, but their therapeutic efficacy has been limited due to poor vaccine co-delivery and the limited ability of monovalent antigen vaccines to prevent tumor immune evasion. To address these challenges, here, we report GBM radioimmunotherapy that combines radiotherapy, ICB, and multivalent lymph-node-targeting adjuvant/antigen-codelivering albumin-binding vaccines (AAco-AlbiVax). Specifically, to codeliver peptide neoantigens and adjuvant CpG to lymph nodes (LNs), we developed AAco-AlbiVax based on a Y-shaped DNA scaffold that was site-specifically conjugated with CpG, peptide neoantigens, and albumin-binding maleimide-modified Evans blue derivative (MEB). As a result, these vaccines elicited antitumor immunity including neoantigen-specific CD8+ T cell responses in mice. In orthotopic GBM mice, the combination of AAco-AlbiVax, ICB, and fractionated radiation enhanced GBM therapeutic efficacy. However, radioimmunotherapy only trended more efficacious over radiotherapy alone. Taken together, these studies underscore the great potential of radioimmunotherapy for GBM, and future optimization of treatment dosing and scheduling would improve the therapeutic efficacy.
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Glioblastoma , Vacinas , Animais , Camundongos , Glioblastoma/radioterapia , Radioimunoterapia , Recidiva Local de Neoplasia , Adjuvantes Imunológicos , Adjuvantes Farmacêuticos , Albuminas , LinfonodosRESUMO
Immune checkpoint blockade (ICB) has significantly advanced cancer immunotherapy, yet its patient response rates are generally low. Vaccines, including immunostimulant-adjuvanted peptide antigens, can improve ICB. The emerging neoantigens generated by cancer somatic mutations elicit cancer-specific immunity for personalized immunotherapy; the novel cyclic dinucleotide (CDN) adjuvants activate stimulator of interferon genes (STING) for antitumor type I interferon (IFN-I) responses. However, CDN/neoantigen vaccine development has been limited by the poor antigen/adjuvant codelivery. Here, pH-responsive CDN/neoantigen codelivering nanovaccines (NVs) for ICB combination tumor immunotherapy are reported. pH-responsive polymers are synthesized to be self-assembled into multivesicular nanoparticles (NPs) at physiological pH and disassembled at acidic conditions. NPs with high CDN/antigen coloading are selected as NVs for CDN/antigen codelivery to antigen presenting cells (APCs) in immunomodulatory lymph nodes (LNs). In the acidic endosome of APCs, pH-responsive NVs facilitate the vaccine release and escape into cytosol, where CDNs activate STING for IFN-I responses and antigens are presented by major histocompatibility complex (MHC) for T-cell priming. In mice, NVs elicit potent antigen-specific CD8+ T-cell responses with immune memory, and reduce multifaceted tumor immunosuppression. In syngeneic murine tumors, NVs show robust ICB combination therapeutic efficacy. Overall, these CDN/neoantigen-codelivering NVs hold the potential for ICB combination tumor immunotherapy.
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Nanopartículas , Neoplasias , Vacinas , Adjuvantes Imunológicos , Animais , Imunoterapia , Camundongos , Neoplasias/terapia , PolímerosRESUMO
Abnormalities of α-synuclein (α-syn) and NMDA receptors (NMDARs) are implicated in the pathogenesis of Parkinson's disease. However, how these proteins interact with each other has not been elucidated. Here, the effect of α-syn on NMDARs was investigated by examining the alterations of surface NMDAR NR1 subunits in MES23.5 dopaminergic cells transfected with the human α-syn gene as well as in cells treated with extracellularly added human α-syn. As demonstrated previously that α-syn can enter cells in a non-endocytic manner without being degraded by the cellular proteolytic systems, the extracellularly added α-syn entered the cytoplasm of MES23.5 cells in a concentration-dependent manner. Both the α-syn-transfected cells and α-syn-treated cells exhibited increased intracellular α-syn levels and reduced surface NR1 without altering the total NR1. The α-syn-induced surface NR1 reduction was accompanied by suppression of NMDA-elicited intracellular Ca(2+) elevation and reductions of NMDA-induced caspase 3 activation and cell death, which was abolished by hypotonic shock and K(+) depletion, a procedure that blocks clathrin-mediated endocytosis, and by suppression of RAB5B expression with anti-RAB5B oligonucleotides. The data obtained provide evidence for the first time that α-syn may promote clathrin-mediated NMDAR endocytosis.
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Clatrina/metabolismo , Dopamina/metabolismo , Endocitose/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas de Filamentos Intermediários/metabolismo , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Transformada , Clatrina/genética , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Imunoadsorção Enzimática/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/farmacologia , Potássio/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genética , Sais de Tetrazólio , Fatores de Tempo , Transfecção/métodos , Regulação para Cima/efeitos dos fármacos , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Although abnormal aggregation of α-synuclein (α-syn) is involved in several neurodegenerative diseases, its biological functions remain poorly understood, which limits our understanding of its pathogenic mechanisms. α-Syn exhibits MAP-like activity and promotes the assembly of microtubules. Since microtubules play a pivotal role in proliferative cell division, it is possible that α-syn affects cell proliferation by facilitating microtubule assembly. The role of α-syn in promoting cell proliferation was reported previously in PC12 dopaminergic cells overexpressing α-syn. Here, we extended this study aiming at finding the association between the cell proliferation effect of α-syn and its microtubule assembly activity, and identifying the potential active domain for the effect of α-syn on cell proliferation. By exploiting the property that the 11-mer repeats of synuclein molecules are able to mediate a rapid intracellular translocation of these proteins across the plasma membrane without being degraded by the cellular proteolytic system, we added recombinant full-length α-syn (wild type and A53T and A30P mutants) and ß-syn to the culture medium of MES23.5 dopaminergic cells, and observed their intracellular translocation, subcellular distribution and effects on cell proliferation. We found that all the synuclein molecules could enter the cells where they were localized in both the cytoplasm and nucleus. However, only the wild-type α-syn, which had been shown to have microtubule assembly activity, was able to promote proliferation of the MES23.5 cells. The A53T and A30P mutant α-syn as well as ß-syn, which had been proved not to possess microtubule assembly activity, did not exhibit any effect on cell proliferation. Since the α-syn activity in microtubule assembly was shown to be related to its specific functional domain, we then generated different functional fragments (N-terminal aa1-65, NAC aa61-95 and C-terminal aa96-140) and tested their activities in cell proliferation. We showed that all the α-syn fragments could enter the cells, but with different subcellular localizations. The N-terminal and NAC fragments were localized in the cytoplasm and the C-terminal fragment mainly in the nucleus. In accordance with the activity for the C-terminal part of α-syn in microtubule assembly, only the NAC and C-terminal fragments exhibited the activity in cell proliferation. The N-terminal fragment without microtubule assembly activity did not promote cell proliferation. The above results suggest that the α-syn function in promoting cell proliferation is associated with its microtubule assembly activity with the functional domain localized in its C-terminal part.
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Proliferação de Células , Neurônios Dopaminérgicos/fisiologia , alfa-Sinucleína/química , alfa-Sinucleína/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Microtúbulos/química , Microtúbulos/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
Mitochondrial drug delivery has attracted increasing attention in various mitochondrial dysfunction-associated disorders such as cancer owing to the important role of energy production. Herein, we report a lysosomal pH-activated mitochondrial-targeting polymer nanoparticle to overcome drug resistance by a synergy between mitochondrial delivery of doxorubicin (DOX, an anticancer drug) and erlotinib-mediated inhibition of drug efflux. The obtained nanoparticles, DE-NPs could maintain negative charge and have long blood circulation while undergoing charge reversal at lysosomal pH after internalization by cancer cells. Thereafter, the acidity-activated polycationic and hydrophobic polypeptide domains boost lysosomal escape and mitochondrial-targeting drug delivery, leading to mitochondrial dysfunction, ATP suppression, and cell apoptosis. Moreover, the suppressed ATP supply and erlotinib enabled dual inhibition of drug efflux by DOX-resistant MCF-7/ADR cells, leading to significantly augmented intracellular DOX accumulation and a synergistic anticancer effect with a 17-fold decrease of IC50 relative to DOX. In vivo antitumor study demonstrates that DE-NPs efficiently suppressed the tumor burden in MCF-7/ADR tumor-bearing mice and led to negligible toxicity. This work establishes that a combination of mitochondrial drug delivery and drug efflux inhibition could be a promising strategy for combating multidrug resistance.
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Polymer microspheres are attracting wide attention in localized cancer therapy owing to the excellent biocompatibility and drug loading capacity, controllable biodegradation speeds, and minimized systemic toxicity. Herein, we presented poly(ester-thioether) microspheres, porous and nonporous, as drug depots for localized therapy of non-small cell lung cancer (NSCLC). Specifically, erlotinib and α-tocopheryl succinate (α-TOS), which are respectively an epidermal growth factor receptor (EGFR) inhibitor and mitochondria destabilizer, were efficiently loaded into porous and nonporous poly(ester-thioether) microspheres for the treatment of EGFR-overexpressing NSCLC (A549 cells). The poly(ester-thioether) microspheres significantly improved the bioavailability of both erlotinib and α-TOS in comparison to the free drug combination, realizing synergistic inhibition of A549 cells both in vitro and in vivo. The porous microspheres displayed faster degradation and drug release than the nonporous counterpart, thereby showing better anticancer efficacy. Overall, our study reported a new anticancer strategy of erlotinib and α-TOS combination for therapy of NSCLC, and established that poly(ester-thioether) microspheres could be a robust and biodegradable reservoir for drug delivery and localized cancer therapy.
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Cloridrato de Erlotinib/química , Microesferas , Polímeros/química , Inibidores de Proteínas Quinases/química , alfa-Tocoferol/química , Células A549 , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Portadores de Fármacos/química , Quimioterapia Combinada , Cloridrato de Erlotinib/metabolismo , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Porosidade , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , alfa-Tocoferol/farmacologia , alfa-Tocoferol/uso terapêuticoRESUMO
Challenges associated with low-drug-loading capacity, lack of active targeting of tumor cells and unspecific drug release of nanocarriers synchronously plague the success of cancer therapy. Herein, we constructed active-targeting, redox-activated polymeric micelles (HPGssML) self-assembled aptamer-decorated, amphiphilic biodegradable poly (benzyl malolactonate-co-ε-caprolactone) copolymer with disulfide linkage and π-conjugated moieties. HPGssML with a homogenous spherical shape and nanosized diameter (â¼150 nm) formed a low critical micellar concentration (10-3 mg/mL), suggesting good stability of polymeric micelles. The anticancer drug, doxorubicin (DOX), can be efficiently loaded into the core of micelles with high-drug-loading content via strong π-π interaction, which was verified by a decrease in fluorescence intensity and redshift in UV adsorption of DOX in micelles. The redox sensitivity of polymeric micelles was confirmed by size change and in vitro drug release in a reducing environment. Confocal microscopy and flow cytometry assay demonstrated that conjugating aptamers could enhance specific uptake of HPGssML by cancer cells. An in vitro cytotoxicity study showed that the half-maximal inhibitory concentration (IC50) of DOX-loaded HPGssML was two times lower than that of the control group, demonstrating improved antitumor efficacy. Therefore, the multifunctional biodegradable polymeric micelles can be exploited as a desirable drug carrier for effective cancer treatment.
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Porous polymer microspheres (PPMs) have been widely applied in various biomedical fields. Herein, the self-assisted preparation of poly(ester-thioether)-based porous microspheres and hierarchical microcages, whose pore sizes can be controlled by varying the polymer structures, is reported. Poly(ester-thioether)s with alkyl side chains (carbon atom numbers were 2, 4, and 8) can generate hollow porous microspheres; the longer alkyl chain length, the larger pore size of microspheres. The allyl-modified poly(ester-thioether) (PHBDT-g-C3 ) can form highly open, hierarchically interconnected microcages. A formation mechanism of these PPMs is proposed; the hydrophobic side chains-mediated stabilization of oil droplets dictate the droplet aggregation and following solvent evaporation, which is the key to the formation of PPMs. The hierarchically interconnected microcages of PHBDT-g-C3 are due to the partially crosslinking of polymers. Pore sizes of PPMs can be further tuned by a simple mixing strategy of poly(ester-thioether)s with different pore-forming abilities. The potential application of these PPMs as H2 O2 -responsive vehicles for delivery of hydrophobic (Nile Red) and hydrophilic (doxorubicin hydrochloride) cargos is also investigated. The microspheres with larger pore sizes show faster in vitro drug release. The poly(ester-thioether)-based polymer microspheres can open a new avenue for the design of PPMs and provide a H2 O2 -responsive drug delivery platform.
Assuntos
Doxorrubicina , Microesferas , Oxazinas , Poliésteres , Preparações de Ação Retardada/síntese química , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Oxazinas/química , Oxazinas/farmacocinética , Oxazinas/farmacologia , Poliésteres/síntese química , Poliésteres/química , Poliésteres/farmacocinética , Poliésteres/farmacologia , PorosidadeRESUMO
Biodegradable and stimuli-responsive polymers have been widely explored due to their great potential in various biomedical applications. Here, biodegradable and oxidation-responsive poly(ester-thioether)s with different backbones were prepared by polymerization of dithiols and diacrylates. Two isomeric dithiol monomers, 2,3-dimercaptobutane (DMB) and 1,4-butanedithiol (BDT), were employed to synthesize poly(ester-thioether)s in the presence of 1,6-hexanediol diacrylate (HDA). The polymerization and oxidation kinetics of poly(ester-thioether)s were found to be controllable by tuning the polymer backbones, which were prepared using different monomers. The polymerization kinetics demonstrated that BDT showed a faster polymerization rate than DMB due to less steric hindrance. Poly(ester-thioether)s PHBD and PHDM, which were prepared from BDT and DMB with HDA, respectively, showed the fastest and slowest oxidation-responsiveness both in THF solution and in the form of polymer films. Finally, the potential application of poly(ester-thioether)s as drug vehicles for anticancer therapy was confirmed by using doxorubicin (DOX) as a model drug. The DOX-loaded micelle DOX/mPEG-PHBD showed much faster H2O2-responsive drug release and better anticancer efficacy in both MCF-7 and 4T1 cells due to the higher sensitivity of PHBD to H2O2.
Assuntos
Antineoplásicos/química , Poliésteres/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Humanos , Peróxido de Hidrogênio/química , Cinética , Micelas , Oxirredução , Poliésteres/farmacologia , PolimerizaçãoRESUMO
Conventional photodynamic therapy used for cancer treatment is incapable of achieving satisfactory therapeutic effects because of a low encapsulation efficiency and uncontrolled photosensitizer leakage. Herein, we introduce a novel nanoparticle system with reduction-induced decomposition and photosensitizer self-aggregation capability to upregulate reactive oxygen species (ROS) level under UV irradiation in cancer cells, leading to cancer cells apoptosis. The nanoparticles could be self-assembled with low critical micellar concentration using amphiphilic polymers composed of polyethylene glycol segment and a bis(pyrene) molecule with a disulfide bond as a reduction linker. The 1HNMR and transmission electron microscopy results showed that the responsive disulfide bonds in the nanoparticles were specifically cleaved in the reductive environment, which resulted in in situ self-aggregation of the pyrene residues, which was confirmed by the results of UV and fluorescence spectra. Furthermore, confocal laser microscopy and flow cytometry demonstrated that the ROS level was upregulated in cancer cells exposed to nanoparticles under UV irradiation compared to insensitive group, causing cells apoptosis. Therefore, this strategy opens a new method for cancer therapy.