RESUMO
Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2'-3' cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.
Assuntos
Administração Intranasal , Nanopartículas , SARS-CoV-2 , Animais , Nanopartículas/química , Nanopartículas/administração & dosagem , Camundongos , Cricetinae , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Modelos Animais de Doenças , Humanos , Proteínas de Membrana/agonistas , Proteínas de Membrana/metabolismo , Feminino , Nucleotídeos Cíclicos/farmacologia , Ratos , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Masculino , Antivirais/farmacologia , Antivirais/administração & dosagem , Camundongos Endogâmicos C57BLRESUMO
Immunization programs against SARS-CoV-2 with commercial intramuscular vaccines prevent disease but are less efficient in preventing infections. Mucosal vaccines can provide improved protection against transmission, ideally for different variants of concern (VOCs) and related sarbecoviruses. Here, we report a multi-antigen, intranasal vaccine, NanoSTING-SN (NanoSTING-Spike-Nucleocapsid), eliminates virus replication in both the lungs and the nostrils upon challenge with the pathogenic SARS-CoV-2 Delta VOC. We further demonstrate that NanoSTING-SN prevents transmission of the SARS-CoV-2 Omicron VOC (BA.5) to vaccine-naïve hamsters. To evaluate protection against other sarbecoviruses, we immunized mice with NanoSTING-SN. We showed that immunization affords protection against SARS-CoV, leading to protection from weight loss and 100% survival in mice. In non-human primates, animals immunized with NanoSTING-SN show durable serum IgG responses (6 months) and nasal wash IgA responses cross-reactive to SARS-CoV-2 (XBB1.5), SARS-CoV and MERS-CoV antigens. These observations have two implications: (1) mucosal multi-antigen vaccines present a pathway to reducing transmission of respiratory viruses, and (2) eliciting immunity against multiple antigens can be advantageous in engineering pan-sarbecovirus vaccines.
Assuntos
Administração Intranasal , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Animais , SARS-CoV-2/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/transmissão , COVID-19/virologia , Camundongos , Cricetinae , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Feminino , Camundongos Endogâmicos BALB C , Humanos , Mesocricetus , Antígenos Virais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangueRESUMO
Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3+ KLRG1- lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4+ T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4+ T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille-Calmette-Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis.
RESUMO
Although glutamine addiction in cancer cells is extensively reported, there is controversy on the impact of glutamine metabolism on the immune cells within the tumor microenvironment (TME). To address the role of extracellular glutamine, we enzymatically depleted circulating glutamine using PEGylated Helicobacter pylori gamma-glutamyl transferase (PEG-GGT) in syngeneic mouse models of breast and colon cancers. PEG-GGT treatment inhibits growth of cancer cells in vitro, but in vivo it increases myeloid-derived suppressor cells (MDSCs) and has no significant impact on tumor growth. By deriving a glutamine depletion signature, we analyze diverse human cancers within the TCGA and illustrate that glutamine depletion is not associated with favorable clinical outcomes and correlates with accumulation of MDSC. Broadly, our results help clarify the integrated impact of glutamine depletion within the TME and advance PEG-GGT as an enzymatic tool for the systemic and selective depletion (no asparaginase activity) of circulating glutamine in live animals.
RESUMO
Activated macrophages are the primary targets in rheumatoid arthritis (RA) management. So, we report efficacious, dual-functional Methotrexate (MTX) loaded folate-conjugated pH-responsive glycol-chitosan nanoparticles (MFGCN) prepared by nano-precipitation and zero-order cross-linking reaction for targeting inflamed arthritic tissue. Physical characterization by DLS, SEM and TEM indicated a spherical, smooth morphology with a diameter ~ 300 nm. 1H NMR and FTIR indicated folic acid conjugation to GC by zero-order cross-linkers. In vitro release kinetics in PBS showed pH-responsive and sustained release behaviour of MFGCN. Enhanced cellular uptake and cytotoxicity of MFGCN in LPS(+)RAW and activated peritoneal macrophages (MÏ) were observed when compared to LPS(-)RAW cells. MFGCN-induced mitochondrial membrane potential (MMP) perturbations indicated apoptosis. Oxidative stress was evident by significant increase in ROS and RNS, 4 h post incubation with MFGCN. Negligible hemolysis by FGCN and MFGCN on rat RBC's indicated biocompatibility. In vivo biodistribution of MFGCN in adjuvant-induced arthritis (AIA) rats indicated RA targetability. Prolonged blood circulation coupled with higher concentrations of 99mTc-MFGCN at the arthritic site was observed post 24 h of injection. The gamma scintigraphic image confirmed accumulation of radiolabelled MFGCN in arthritic paw when compared to the non-inflamed paw, confirming the selective uptake of 99mTc-MFGCN by folate-overexpressing macrophages in the arthritic synovium thereby proving its targeted efficacy and theranostic potential. In AIA rats, MFGCN lowers arthritic signs, improves antioxidant response and decreases pro-inflammatory cytokines, suggesting its potential in targeting activated macrophages of synovium. Graphical abstract.
Assuntos
Antirreumáticos/administração & dosagem , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Quitosana/administração & dosagem , Ácido Fólico/administração & dosagem , Metotrexato/administração & dosagem , Nanopartículas/administração & dosagem , Animais , Sobrevivência Celular/efeitos dos fármacos , Quitosana/química , Liberação Controlada de Fármacos , Feminino , Ácido Fólico/química , Células HEK293 , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Metotrexato/química , Camundongos , Nanopartículas/química , Células RAW 264.7 , Ratos WistarRESUMO
Lignin nanoparticles synthesis is among recent developments in lignin valorization especially for biomedical applications. In this study, a new technique where complete self-assembling of lignin was ensured by simultaneous solvent displacement and flash pH change was used to optimize particle size of blank lignin nanoparticles (BLNPs) for suitability in cell uptake along with maximized yield. To establish BLNPs as drug carrier, safety studies including hemocompatibility, cytotoxicity and elaborate genotoxicity studies on Drosophila melanogaster as a model organism were done. Finally, irinotecan loaded lignin nanoparticles (DLNPs) were synthesized to establish their drug carrying potential and thorough in vitro characterization was performed. BLNPs with controllable size (â152 nm), low polydispersity (<0.2), maximized yield (>65%), negative surface charge (-22 to -23 mV), spherical shape and smooth surface were obtained with acceptable %hemolysis (<2%). In vitro cytotoxicity studies revealed that BLNPs were significantly toxic (74.38 ± 4.74%) in human breast adenocarcinoma (MCF-7), slightly toxic (38.8 ± 4.70%) in human alveolar epithelial adenocarcinoma (A-549) and insignificantly toxic (15.89 ± 2.84%) to human embryonic kidney (HEK-293) cells. BLNPs showed concentration dependent early neuronal defects in Drosophila, but nuclei fragmentation and gut cell damage were absent. Sustained release DLNPs with high drug loading reduced the IC50 value of irinotecan by almost 3 folds.
Assuntos
Portadores de Fármacos/efeitos adversos , Portadores de Fármacos/química , Lignina/efeitos adversos , Lignina/química , Nanopartículas/efeitos adversos , Nanopartículas/química , Células A549 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Drosophila melanogaster/efeitos dos fármacos , Células HEK293 , Humanos , Células MCF-7 , Tamanho da Partícula , Ratos , Ratos WistarRESUMO
PURPOSE: The present investigation was aimed at developing Teriflunomide (TEF) and Methotrexate (MTX) loaded hydroxyapatite nanoparticles and increasing tolerability towards combination therapy against rheumatoid arthritis by reducing hepatotoxicity. METHODS: Drug-loaded HAp-NPs were synthesized by wet-chemical precipitation method and optimized by Box-Behnken experimental design. The developed NPs were subjected to in vitro and in vivo characterization. In-vivo pharmacodynamics and biochemical studies were performed on adjuvant- induced arthritis model treated with different formulations; MTX-TEF-SOL, TEF-HAp-NP, MTX-HAp-NP, TEF-MTX-HAp-NP, FOLITRAX-10 and AUBAGIO. RESULTS: The size of the optimized formulations, TEF-HAp-NP and MTX-HAp-NP, was found to be 224.3 ± 83.80 nm and 268.3 ± 73.86 nm with drug loading 53.11 ± 0.84% and 67.04 ± 1.12% respectively. In vitro release of TEF from TEF-HAp-NP (70.41 ± 1.22%) and MTX from MTX-HAp-NP (82.43 ± 1.31%) up to 24 h revealed sustained release pattern. Results of the arthritic assessment study showed a significant (P < 0.05) reduction in ankle diameter (61.30 ± 7.42) and arthritis score (2.35 ± 0.24) with a marked restoration of ankle joint micro-architecture in TEF-MTX-HAp-NP treated group. During Hepatotoxicity studies, liver histopathology revealed that the formulation MTX-TEF-HAp-NP was least hepatotoxic with less hepatocyte swelling and fibrous connective tissue proliferation while Folitrax-10 was found to be most hepatotoxic. Biochemical studies revealed that Folitrax-10 significantly (P < 0.05) increased the GOT (313.64 ± 16) and GPT level (334.46 ± 13) while insignificant (P > 0.05) change in GOT (263.68 ± 17) and GPT (229.38 ± 10) level was recorded with TEF-MTX-HAp-NP. CONCLUSIONS: We report that the subcutaneous delivery of TEF-MTX-HAp-NP was most effective as it successfully reduced the dosage by half for maximizing therapeutic efficacy and minimizing side effects. Graphical Abstract á .
Assuntos
Antirreumáticos/administração & dosagem , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Crotonatos/administração & dosagem , Durapatita/química , Metotrexato/administração & dosagem , Nanopartículas/química , Toluidinas/administração & dosagem , Animais , Antirreumáticos/farmacocinética , Antirreumáticos/farmacologia , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Crotonatos/farmacocinética , Crotonatos/farmacologia , Portadores de Fármacos , Combinação de Medicamentos , Composição de Medicamentos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Feminino , Concentração de Íons de Hidrogênio , Hidroxibutiratos , Cinética , Metotrexato/farmacocinética , Metotrexato/farmacologia , Nitrilas , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Ratos , Ratos Wistar , Distribuição Tecidual , Toluidinas/farmacocinética , Toluidinas/farmacologiaRESUMO
Raloxifene, an anti-osteoporotic drug, is recently approved for prevention of breast cancer in postmenopausal women and thus the drug may be employed to combat the bony adverse effects of letrozole, another anticancer drug. However, the cytotoxic effect of their combination on human breast cancer (MCF-7) and human embryonic kidney (HEK) cell lines is not known. MCF-7 and HEK cell lines were treated with different graded doses of letrozole, raloxifene and their combination, then incubated for 24-48 h. MTT assay was performed to check the cytotoxicity of the drugs. The study indicates that the combination of letrozole and raloxifene possess additive effect in terms of cytotoxicity of cancer cell lines (MCF-7) and negligible effects in normal cell lines (HEK). Our study indicates that the addition of raloxifene doesn't interfere with anticancer efficacy of letrozole rather the combination acted additively for the treatment of breast cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Letrozol/farmacologia , Cloridrato de Raloxifeno/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Feminino , Humanos , Letrozol/uso terapêutico , Nitrilas/farmacologia , Cloridrato de Raloxifeno/uso terapêuticoRESUMO
PURPOSE: The study purposes to evaluate nanocrystalline biopolymeric nanoparticles encapsulating methotrexate and dexamethasone with high biocompatibility, enhanced therapeutic efficacy and reduced toxicity. METHODS: Chitosan nanoparticles were prepared by ionic gelation, and Methotrexate (MTX) and Dexamethasone (DEX) were loaded during the preparation and screened for their in vitro efficacy in HEK and RAW264.7 cells, ex vivo and in vivo efficacy. RESULTS: FTIR confirmed the involvement of phosphoric group of sTPP with amine groups of chitosan and also role of hydrogen bonding involved in the preparation of MTXCHNP and DEXCHNP. Controlled release patterns coupled with diffusion of drug were observed in two different buffers (PBS) at pH 7.4 and pH 5.8. The IC50 for MTXCHNP for HEK was 26.1 µg/ml and 7.7 µg/ml for RAW 264.7 cells. In DEXCHNP, the IC50 was 20.12 µg/ml for HEK and 7.37 µg/ml for RAW264.7 cells. Enhanced uptake of FITC-CHNP by RAW cells indicated internalization of nanoparticles by phagocytosis. The enhanced release of drug at lower pH justified increased cytotoxicity. Negligible ex-vivo hemolysis indicated the higher biocompatibility of the nanoparticles. 99mTc-CHNP exhibited maximum absorption in blood circulation in 3 h, followed by hepatic metabolism and renal clearance. Higher in-vivo anti-arthritic activity and antioxidant activity was observed post-intraperitoneal (i.p.) injections by both MTXCHNP and DEXCHNP when compared to MTX (0.75 mg/Kg by i.p. route) and DEX (0.2 mg/Kg/i.p./daily) per se. CONCLUSION: The nanocrystalline biopolymeric nanoparticles were stable, biocompatible and have potential to be administered through i.p. route with minimal toxicity and high efficacy.
Assuntos
Antirreumáticos/administração & dosagem , Artrite Reumatoide/tratamento farmacológico , Quitosana/química , Dexametasona/administração & dosagem , Metotrexato/administração & dosagem , Nanopartículas/química , Animais , Antioxidantes/administração & dosagem , Antioxidantes/química , Antioxidantes/farmacologia , Antirreumáticos/química , Antirreumáticos/farmacologia , Materiais Biocompatíveis , Técnicas de Cultura de Células , Linhagem Celular , Dexametasona/química , Dexametasona/farmacologia , Sistemas de Liberação de Medicamentos , Estabilidade de Medicamentos , Excipientes/química , Humanos , Concentração de Íons de Hidrogênio , Metotrexato/química , Metotrexato/farmacologia , Tamanho da Partícula , Ratos Wistar , Propriedades de Superfície , Distribuição TecidualRESUMO
PURPOSE: Cervical cancer is the second most prevalent cancer in women worldwide. Survival of patients has been improved by cisplatin-based chemotherapy, but its effectiveness is limited due to its adverse effects on many tissues, especially nephrotoxicity. To optimize the efficacy of CDDP, we propose a combination therapy using natural products with minimal side effects. Vitamin C being a natural antioxidant is capable of selectively targeting cancer cells at pharmacological concentrations. Vitamin C synergistically enhances the activity of chemotherapeutic agents without increasing toxicity to normal cells. Therefore, we exploited co-therapy with cisplatin and vitamin C to kill cervical cancer cells. METHODS: We elucidated the role of CDDP and VC on cervical cancer cell line (SiHa) by using cell growth assays, DNA fragmentation analysis, comet assay, in vitro morphological assessment of apoptosis (AO/EB and DAPI staining), ROS analysis by DCFDA, flow cytometry, biochemical assays (GST, GSH, NO, catalase, TPA) and Western blotting. RESULTS: Our results clearly demonstrated that CDDP and VC treatment exhibited ameliorative effect on induction of cell death by p53 overexpression and generation of hydrogen peroxide in SiHa cells, thereby reducing the dosage of CDDP required to induce cell death in cancer cells. CONCLUSIONS: These studies provide novel approaches to combat cisplatin resistance in cervical cancer.
Assuntos
Ácido Ascórbico/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/patologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Ácido Ascórbico/administração & dosagem , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Cisplatino/administração & dosagem , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Oxirredução , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismoRESUMO
The present work evaluates the synergistic anticancer efficacy of bioactive Hydroxyapatite (HA) nanoparticles (HA NPs) loaded with Bendamustine HCl. Hydroxyapatite is a material with an excellent biological compatibility, a well-known fact which was also supported by the results of the Hemolytic studies and a high IC50 value observed in the MTT assay. HA NPs were prepared by the chemical precipitation method and loaded with the drug via physical adsorption. In-vitro release study was performed, which confirmed the sustained release of the drug from the drug loaded HA NPs. MTT assay, Cell Uptake and FACS studies on JURKAT E6.1 cell line and in-vivo pharmacokinetic studies in Wistar rats revealed that the drug loaded HA NPs could be easily internalized by the cells and release drug in a sustained manner. The drug loaded HA NPs showed cytotoxicity similar to the drug solution at 1/10th of the drug content, which indicates a possible synergism between the activity of the anticancer drug and calcium ions derived from the carrier. An increase in intracellular Ca(2+) ions is reported to induce apoptosis in cells. Tumor regression study in Balb/c mice Ehrlich's ascites model presented a similar synergistic efficacy. The drug solution was able to decrease the tumor volume by half, while the drug loaded HA NPs reduced the tumor size by 6 times.