RESUMO
Nisin ZP is an antimicrobial peptide (AMP) produced by the bacterium Lactococcus lactis, and we have previously demonstrated anticancer activity in NSCLC (A549) cells. In this study, we formulated a nisin ZP dry powder (NZSD) using a spray dryer to facilitate inhaled delivery for the treatment of NSCLC. Nisin ZP was spray-dried with mannitol, l-leucine, and trehalose in a ratio of 75:15:10 using Büchi mini spray-dryer B-290 in different drug loadings (10, 20, and 30% w/w). NZSD powder revealed a good powder yield of >55% w/w with ≤3 % w/w moisture content and high nisin ZP drug loading for all the peptide ratios. The NZSD powder particles were irregularly shaped with corrugated morphology. The presence of an endothermic peak in DSC thermograms and attenuated crystalline peaks in PXRD diffractograms confirmed the semi-crystalline powder nature of NZSD. The anticancer activity of nisin ZP was maintained after fabricating it into NZSD powder and showed a similar inhibitory concentration to free nisin ZP. Stability studies indicated that NZSD powders were stable for three months at 4 and 25 â with more than 90% drug content and semi-crystalline nature, as confirmed by DSC and PXRD. Aerosolization studies performed using NGI indicated an aerodynamic diameter (MMAD) within the desired range (1-5 µm) and a high fine particle fraction (FPF > 75%) for all peptide ratios, suggesting powder deposition in the lung's respiratory airways. In conclusion, a dry powder of nisin ZP was formulated using a spray dryer with enhanced storage stability and suitable for inhaled delivery.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Nisina , Humanos , Administração por Inalação , Peptídeos Antimicrobianos , Pós/química , Aerossóis e Gotículas Respiratórios , Pulmão , Tamanho da Partícula , Inaladores de Pó SecoRESUMO
Three prophylactic vaccines are approved to protect against HPV infections. These vaccines are highly immunogenic. The most recent HPV vaccine, Gardasil-9, protects against HPV types associated with ~90% of cervical cancer (worldwide). Thus, ~10% of HPV-associated cancers are not protected by Gardasil-9. Although this is not a large percentage overall, the HPV types associated with 10% of cervical cancer not protected by the current vaccine are significantly important, especially in HIV/AIDS patients who are infected with multiple HPV types. To broaden the spectrum of protection against HPV infections, we developed mixed MS2-L2 VLPs (MS2-31L2/16L2 VLPs and MS2-consL2 (69-86) VLPs) in a previous study. Immunization with the VLPs neutralized/protected mice against infection with eleven high-risk HPV types associated with ~95% of cervical cancer and against one low-risk HPV type associated with ~36% of genital warts & up to 32% of recurrent respiratory papillomatosis. Here, we report that the mixed MS2-L2 VLPs can protect mice from three additional HPV types: HPV51, which is associated with ~0.8% of cervical cancer; HPV6, which is associated with up to 60% of genital warts; HPV5, which is associated with skin cancers in patients with epidermodysplasia verruciformis (EV). Overall, mixed MS2-L2 VLPs can protect against twelve HPV types associated with ~95.8% of cervical cancers and against two HPV types associated with ~90% of genital warts and >90% recurrent respiratory papillomatosis. Additionally, the VLPs protect against one of two HPV types associated with ~90% of HPV-associated skin cancers in patients with EV. More importantly, we observed that mixed MS2-L2 VLPs elicit protective antibodies that last over 9 months. Furthermore, a spray-freeze-dried formulation of the VLPs is stable, immunogenic, and protective at room temperature and 37 °C.
Assuntos
Anticorpos Antivirais/sangue , Bacteriófagos/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Condiloma Acuminado/prevenção & controle , Proteção Cruzada/imunologia , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Papillomaviridae/classificação , Papillomaviridae/patogenicidade , Vacinas contra Papillomavirus/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/administração & dosagemRESUMO
Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug-target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.
Assuntos
Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Pirazóis/farmacologia , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Meia-Vida , Humanos , Camundongos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Loss of vaccine potency due to extreme temperature exposure during storage and transport remains a significant obstacle to the success of many vaccines, including the Bacille Calmette-Guérin (BCG) vaccine, the only vaccine available against Mycobacterium tuberculosis. BCG is a live, attenuated vaccine requiring refrigerated storage for viability. In this study, we formulated a temperature-stable BCG dry powder using the spray drying technique. METHODS: We employed a factorial design to optimize our formulation of stabilizing excipients that included L-leucine, bovine serum albumin, polyvinylpyrrolidone, mannitol, and trehalose. Powders were characterized for their particle size, yield, water retention and uptake, glass transition temperature, and aerosol performance. Three optimal powder carrier mixtures were selected from the factorial design for BCG incorporation based on their stability-promoting and powder flow characteristics. Vaccine powders were also assessed for BCG viability and in vivo immunogenicity after long-term storage. RESULTS: Live BCG was successfully spray-dried using the optimized carriers. Dry powder BCG showed no loss in viability (25°C, up to 60% relative humidity; RH) and ~2-log loss in viability (40°C, 75% RH) after one year of storage. The aerodynamic size of the powders was in the respirable range. Further, when healthy mice were immunized intradermally with reconstituted BCG powders (storage for 2 years), the vaccine retained its immunogenicity. CONCLUSION: We developed a spray-dried BCG vaccine that was viable and antigenic after long-term storage. To our knowledge, this is a first study to show room temperature stability of live BCG vaccine without any loss in viability for 12 months.
Assuntos
Vacina BCG/química , Vacina BCG/farmacologia , Composição de Medicamentos/métodos , Excipientes/química , Pós/química , Aerossóis/química , Animais , Linhagem Celular , Sobrevivência Celular , Dessecação/métodos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Feminino , Humanos , Leucina/química , Manitol/química , Camundongos Endogâmicos C57BL , Mycobacterium bovis/citologia , Povidona/química , Soroalbumina Bovina/química , Temperatura , Distribuição Tecidual , Trealose/químicaRESUMO
Ambient particulate matter (PM) is associated with increased mortality and morbidity, an effect influenced by the metal components of the PM. We characterized five sediment samples obtained near a tungsten-molybdenum ore-processing complex in Zakamensk, Russia for elemental composition and PM toxicity with regard to pulmonary, vascular, and neurological outcomes. Elemental and trace metals analysis of complete sediment and PM10 (the respirable fraction, < 10 µm mass mean aerodynamic diameter) were performed using inductively coupled plasma optical emission spectrometry (ICP-OES) and mass spectrometry (ICP-MS). Sediment samples and PM10 consisted largely of silicon and iron and silicon and sodium, respectively. Trace metals including manganese and uranium in the complete sediment, as well as copper and lead in the PM10 were observed. Notably, metal concentrations were approximately 10 × higher in the PM10 than in the sediment. Exposure to 100 µg of PM10 via oropharyngeal aspiration in C56BL/6 mice resulted in pulmonary inflammation across all groups. In addition, mice exposed to three of the five PM10 samples exhibited impaired endothelial-dependent relaxation, and correlative analysis revealed associations between pulmonary inflammation and levels of lead and cadmium. A tendency for elevated cortical ccl2 and Tnf-α mRNA expression was induced by all samples and significant upregulation was noted following exposure to PM10 samples Z3 and Z4, respectively. Cortical Nqo1 mRNA levels were significantly upregulated in mice exposed to PM10 Z2. In conclusion, pulmonary exposure to PM samples from the Zakamensk region sediments induced varied pulmonary and systemic effects that may be influenced by elemental PM composition. Further investigation is needed to pinpoint putative drivers of neurological outcomes.
Assuntos
Poluentes Atmosféricos/toxicidade , Aorta Torácica/efeitos dos fármacos , Cádmio/toxicidade , Córtex Cerebral/efeitos dos fármacos , Poeira , Chumbo/toxicidade , Mineração , Material Particulado/toxicidade , Pneumonia/induzido quimicamente , Animais , Aorta Torácica/fisiopatologia , Córtex Cerebral/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Exposição por Inalação , Masculino , Camundongos Endogâmicos C57BL , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Pneumonia/genética , Pneumonia/metabolismo , Medição de Risco , Sibéria , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
High-risk human papillomavirus (HPV) types are responsible for nearly all cases of cervical cancers. Cervarix® and Gardasil® 9 are the current prophylactic vaccines available that protect against the majority of HPVs associated with cancer. Although these vaccines are highly effective, HPV vaccine implementation has been slow, particularly in low-and-middle income countries. Major barriers to the widespread availability of the HPV vaccines is its cost and the requirement for continuous refrigeration (2-8°C). Here, we used spray drying along with stabilizing excipients to formulate a thermostable Gardasil® 9 vaccine. We evaluated the immunogenicity and protective efficacy of the vaccine in mice immediately after spray drying and following storage for three months at 4°C, 25°C, and 40°C. The immunogenicity studies were performed using Gardasil® 9 as a whole antigen, and not individual HPV types, for ELISA. At the dose tested, the spray dried vaccine conferred protection against HPV following storage at temperatures up to 40°C. In addition to the spray-dried vaccine, our studies revealed that the Gardasil® 9 vaccine, as currently marketed, may be stored and transported at elevated temperatures for up to 3 months without losing efficacy, especially against HPV16. This study is critical, as a thermostable vaccine will decrease vaccine cost associated with cold-chain maintenance and could increase vaccine access and coverage, especially in remote regions of the world.
Assuntos
Anticorpos Antivirais/sangue , Vacinas contra Papillomavirus/química , Vacinas contra Papillomavirus/imunologia , Temperatura , Animais , Química Farmacêutica , Feminino , Higroscópicos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Pós , Refrigeração , VacinaçãoRESUMO
Human papillomaviruses (HPVs) are the most common sexually transmitted infections. HPVs are transmitted through anogenital sex or oral sex. Anogenital transmission/infection is associated with anogenital cancers and genital warts while oral transmission/infection is associated with head and neck cancers (HNCs) including recurrent respiratory papillomatosis. Current HPV vaccines protect against HPV types associated with â¼90% of cervical cancers and are expected to protect against a percentage of HNCs. However, only a few studies have assessed the efficacy of current vaccines against oral HPV infections. We had previously developed a mixed MS2-L2 candidate HPV vaccine based on bacteriophage MS2 virus-like particles (VLPs). The mixed MS2-L2 VLPs consisted of a mixture of two MS2-L2 VLPs displaying: i) a concatemer of L2 peptide (epitope 20-31) from HPV31 & L2 peptide (epitope 17-31) from HPV16 and ii) a consensus L2 peptide representing epitope 69-86. The mixed MS2-L2 VLPs neutralized/protected mice against six HPV types associated with â¼87% of cervical cancer. Here, we show that the mixed MS2-L2 VLPs can protect mice against additional HPV types; at the genital region, the VLPs protect against HPV53, 56, 11 and at the oral region, the VLPs protect against HPV16, 35, 39, 52, and 58. Thus, mixed MS2-L2 VLPs protect against eleven oncogenic HPV types associated with â¼95% of cervical cancer. The VLPs also have the potential to protect, orally, against the same oncogenic HPVs, associated with â¼99% of HNCs, including HPV11, which is associated with up to 32% of recurrent respiratory papillomatosis. Moreover, mixed MS2-L2 VLPs are thermostable at room temperature for up to 60 days after spray-freeze drying and they are protective against oral HPV infection.
Assuntos
Proteção Cruzada , Papillomaviridae/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Proteção Cruzada/imunologia , Epitopos/imunologia , Feminino , Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/prevenção & controle , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Imunização/métodos , Levivirus/imunologia , Camundongos , Testes de Neutralização , Proteínas Oncogênicas Virais/imunologia , Vacinas contra Papillomavirus/imunologia , Infecções Respiratórias/prevenção & controle , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia , Vacinação/métodosRESUMO
Exposure to windblown particulate matter (PM) arising from legacy uranium (U) mine sites in the Navajo Nation may pose a human health hazard due to their potentially high metal content, including U and vanadium (V). To assess the toxic impact of PM derived from Claim 28 (a priority U mine) compared with background PM, and consider the putative role of metal species U and V. Two representative sediment samples from Navajo Nation sites (Background PM and Claim 28 PM) were obtained, characterized in terms of chemistry and morphology, and fractioned to the respirable (≤ 10 µm) fraction. Mice were dosed with either PM sample, uranyl acetate, or vanadyl sulfate via aspiration (100 µg), with assessments of pulmonary and vascular toxicity 24 h later. Particulate matter samples were also examined for in vitro effects on cytotoxicity, oxidative stress, phagocytosis, and inflammasome induction. Claim 28 PM10 was highly enriched with U and V and exhibited a unique nanoparticle ultrastructure compared with background PM10. Claim 28 PM10 exhibited enhanced pulmonary and vascular toxicity relative to background PM10. Both U and V exhibited complementary pulmonary inflammatory potential, with U driving a classical inflammatory cytokine profile (elevated interleukin [IL]-1ß, tumor necrosis factor-α, and keratinocyte chemoattractant/human growth-regulated oncogene) while V preferentially induced a different cytokine pattern (elevated IL-5, IL-6, and IL-10). Claim 28 PM10 was more potent than background PM10 in terms of in vitro cytotoxicity, impairment of phagocytosis, and oxidative stress responses. Resuspended PM10 derived from U mine waste exhibit greater cardiopulmonary toxicity than background dusts. Rigorous exposure assessment is needed to gauge the regional health risks imparted by these unremediated sites.
Assuntos
Coração/efeitos dos fármacos , Exposição por Inalação/efeitos adversos , Pulmão/efeitos dos fármacos , Nanopartículas/toxicidade , Material Particulado/toxicidade , Urânio/toxicidade , Compostos de Vanádio/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/análise , Sedimentos Geológicos/química , Humanos , Pulmão/imunologia , Masculino , Camundongos Endogâmicos C57BL , Mineração , Nanopartículas/análise , Estresse Oxidativo/efeitos dos fármacos , Tamanho da Partícula , Material Particulado/análise , Células THP-1 , Urânio/análise , Compostos de Vanádio/análise , Vasodilatação/efeitos dos fármacosRESUMO
This brief communication evaluates the cytotoxicity and targeting capability of a dry powder chemotherapeutic. Nano-in-microparticles (NIMs) are a dry powder drug delivery vehicle containing superparamagnetic iron oxide nanoparticles (SPIONs) and either doxorubicin (w/w solids) or fluorescent nanospheres (w/v during formulation; as a drug surrogate) in a lactose matrix. In vitro cytotoxicity was evaluated in A549 adenocarcinoma cells using MTS and LDH assays to assess viability and toxicity after 48 h of NIMs exposure. In vivo magnetic-field-dependent targeting of inhaled NIMs was evaluated in a healthy mouse model. Mice were endotracheally administered fluorescently labeled NIMs either as a dry powder or a liquid aerosol in the presence of an external magnet placed over the left lung. Quantification of fluorescence and iron showed a significant increase in both fluorescence intensity and iron content to the left magnetized lung. In comparison, we observed decreased targeting of fluorescent nanospheres to the left lung from an aerosolized liquid suspension, due to the dissociation of SPIONs and nanoparticles during pulmonary administration. We conclude that dry powder NIMs maintain the therapeutic cytotoxicity of doxorubicin and can be better targeted to specific regions of the lung in the presence of a magnetic field, compared to a liquid suspension.
Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Compostos Férricos/química , Nanopartículas de Magnetita/química , Células A549 , Aerossóis , Animais , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Campos Magnéticos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Nanosferas/química , Pós , Traqueia/efeitos dos fármacos , Traqueia/metabolismoRESUMO
HPV infections are associated with human cancers. Although three prophylactic vaccines have been approved to protect against HPV infections, the vaccines require cold-chain storage and may not be suitable for third world countries with less developed refrigeration facilities. We previously developed a bacteriophage L2 virus-like particle (VLP)-based candidate vaccine, which elicited broadly protective antibodies against diverse HPV types. Spray-drying of MS2-16L2 VLPs into a dry powder enhanced the stability of these VLPs. Building on these studies, we assessed the long-term stability and immunogenicity of the spray-dried VLPs. Mice immunized with a single dose of spray-dried MS2-16L2 VLPs, which had been stored for 14 months at room temperature (RT), were partially protected from challenge with a high dose of HPV16, one year after immunization. However, immunization with two doses of MS2-16L2 VLPs stored at RT for 34 months elicited high titer anti-HPV antibodies. More importantly, this group of mice showed significant protection from HPV16, 4 months after immunization. These results suggest that spray-dried MS2-16L2 VLPs retain their effectiveness after long-term storage at RT, and may be suitable in third world countries with less developed refrigeration facilities.
RESUMO
Lung cancer has the highest mortality rate of any tissue-specific cancer in both men and women. Research continues to investigate novel drugs and therapies to mitigate poor treatment efficacy, but the lack of a good descriptive lung cancer animal model for preclinical drug evaluation remains an obstacle. Here we describe the development of an orthotopic lung cancer animal model which utilizes the human sodium iodide symporter gene (hNIS; SLC5A5) as an imaging reporter gene for the purpose of non-invasive, longitudinal tumor quantification. hNIS is a glycoprotein that naturally transports iodide (I-) into thyroid cells and has the ability to symport the radiotracer 99mTc-pertechnetate (99mTcO4-). A549 lung adenocarcinoma cells were genetically modified with plasmid or lentiviral vectors to express hNIS. Modified cells were implanted into athymic nude mice to develop two tumor models: a subcutaneous and an orthotopic xenograft tumor model. Tumor progression was longitudinally imaged using SPECT/CT and quantified by SPECT voxel analysis. hNIS expression in lung tumors was analyzed by quantitative real-time PCR. Additionally, hematoxylin and eosin staining and visual inspection of pulmonary tumors was performed. We observed that lentiviral transduction provided enhanced and stable hNIS expression in A549 cells. Furthermore, 99mTcO4- uptake and accumulation was observed within lung tumors allowing for imaging and quantification of tumor mass at two-time points. This study illustrates the development of an orthotopic lung cancer model that can be longitudinally imaged throughout the experimental timeline thus avoiding inter-animal variability and leading to a reduction in total animal numbers. Furthermore, our orthotopic lung cancer animal model is clinically relevant and the genetic modification of cells for SPECT/CT imaging can be translated to other tissue-specific tumor animal models.
Assuntos
Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico , Simportadores/genética , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Tomografia Computadorizada por Raios X/métodos , Células A549 , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Iodetos/metabolismo , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Pertecnetato Tc 99m de Sódio/metabolismo , Simportadores/metabolismo , Transplante Heterólogo , Carga Tumoral/genéticaRESUMO
Existing vaccines against human papillomavirus (HPV) require continuous cold-chain storage. Previously, we developed a bacteriophage virus-like particle (VLP)-based vaccine for HPV infection, which elicits broadly neutralizing antibodies against diverse HPV types. Here, we formulated these VLPs into a thermostable dry powder using a multicomponent excipient system and by optimizing the spray-drying parameters using a half-factorial design approach. Dry-powder VLPs were stable after spray drying and after long-term storage at elevated temperatures. Immunization of mice with a single dose of reconstituted dry-powder VLPs that were stored at 37 °C for more than a year elicited high anti-L2 IgG antibody titers. Spray-dried thermostable, broadly protective L2 bacteriophage VLPs vaccine could be accessible to remote regions of the world (where â¼84% of cervical cancer patients reside) by eliminating the cold-chain requirement during transportation and storage.
Assuntos
Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/química , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Química Farmacêutica/métodos , Humanos , Imunização/métodos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Pós/administração & dosagem , Pós/química , Temperatura , Vacinação/métodosRESUMO
Three-dimensional encapsulation of cells within nanostructured silica gels or matrices enables applications as diverse as biosensors, microbial fuel cells, artificial organs, and vaccines; it also allows the study of individual cell behaviors. Recent progress has improved the performance and flexibility of cellular encapsulation, yet there remains a need for robust scalable processes. Here, we report a spray-drying process enabling the large-scale production of functional nano-biocomposites (NBCs) containing living cells within ordered 3D lipid-silica nanostructures. The spray-drying process is demonstrated to work with multiple cell types and results in dry powders exhibiting a unique combination of properties including highly ordered 3D nanostructure, extended lipid fluidity, tunable macromorphologies and aerodynamic diameters, and unexpectedly high physical strength. Nanoindentation of the encasing nanostructure revealed a Young's modulus and hardness of 13 and 1.4 GPa, respectively. We hypothesized this high strength would prevent cell growth and force bacteria into viable but not culturable (VBNC) states. In concordance with the VBNC state, cellular ATP levels remained elevated even over eight months. However, their ability to undergo resuscitation and enter growth phase greatly decreased with time in the VBNC state. A quantitative method of determining resuscitation frequencies was developed and showed that, after 36 weeks in a NBC-induced VBNC, less than 1 in 10,000 cells underwent resuscitation. The NBC platform production of large quantities of VBNC cells is of interest for research in bacterial persistence and screening of drugs targeting such cells. NBCs may also enable long-term preservation of living cells for applications in cell-based sensing and the packaging and delivery of live-cell vaccines.
Assuntos
Dessecação/métodos , Escherichia coli/fisiologia , Nanoestruturas/química , Preservação Biológica/métodos , Trifosfato de Adenosina/metabolismo , Cápsulas Bacterianas/fisiologia , Módulo de Elasticidade , Escherichia coli/metabolismo , Lipídeos/química , Pós/química , Dióxido de Silício/químicaRESUMO
An ideal prophylactic human papillomavirus (HPV) vaccine would provide broadly protective and long-lasting immune responses against all high-risk HPV types, would be effective after a single dose, and would be formulated in such a manner to allow for long-term storage without the necessity for refrigeration. We have developed candidate HPV vaccines consisting of bacteriophage virus-like particles (VLPs) that display a broadly neutralizing epitope derived from the HPV16 minor capsid protein, L2. Immunization with 16L2 VLPs elicited high titer and broadly cross-reactive and cross-neutralizing antibodies against diverse HPV types. In this study we introduce two refinements for our candidate vaccines, with an eye towards enhancing efficacy and clinical applicability in the developing world. First, we assessed the role of antigen dose and boosting on immunogenicity. Mice immunized with 16L2-MS2 VLPs at doses ranging from 2 to 25 µg with or without alum were highly immunogenic at all doses; alum appeared to have an adjuvant effect at the lowest dose. Although boosting enhanced antibody titers, even a single immunization could elicit strong and long-lasting antibody responses. We also developed a method to enhance vaccine stability. Using a spray dry apparatus and a combination of sugars & an amino acid as protein stabilizers, we generated dry powder vaccine formulations of our L2 VLPs. Spray drying of our L2 VLPs did not affect the integrity or immunogenicity of VLPs upon reconstitution. Spray dried VLPs were stable at room temperature and at 37 °C for over one month and the VLPs were highly immunogenic. Taken together, these enhancements are designed to facilitate implementation of a next-generation VLP-based HPV vaccine which addresses U.S. and global disparities in vaccine affordability and access in rural/remote populations.
Assuntos
Proteínas do Capsídeo/imunologia , Proteínas Oncogênicas Virais/imunologia , Vacinas contra Papillomavirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Técnicas de Visualização da Superfície Celular , Química Farmacêutica , Reações Cruzadas , Estabilidade de Medicamentos , Feminino , Levivirus/genética , Camundongos Endogâmicos BALB C , Proteínas Oncogênicas Virais/genética , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Temperatura , Vacinação/métodos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
We propose the use of novel inhalable nano-in-microparticles (NIMs) for site-specific pulmonary drug delivery. Conventional lung cancer therapy has failed to achieve therapeutic drug concentrations at tumor sites without causing adverse effects in healthy tissue. To increase targeted drug delivery near lung tumors, we have prepared and characterized a magnetically responsive dry powder vehicle containing doxorubicin. A suspension of lactose, doxorubicin and Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) were spray dried. NIMs were characterized for their size and morphological properties by various techniques: dynamic light scattering (DLS) and laser diffraction (LS) to determine hydrodynamic size of the SPIONs and the NIMs, respectively; next generation cascade impactor (NGI) to determine the aerodynamic diameter and fine particle fraction (FPF); scanning (SEM) and transmission (TEM) electron microscopy to analyze particle surface morphology; electron dispersive X-ray spectroscopy (EDS) to determine iron loading in NIMs; inductively coupled plasma atomic emission spectroscopy (ICP-AES) and superconducting quantum interference device (SQUID) to determine Fe3O4 content in the microparticles; and high performance liquid chromatography (HPLC) to determine doxorubicin loading in the vehicle. NIMs deposition and retention near a magnetic field was performed using a proof-of-concept cylindrical tube to mimic the conducting airway deposition. The hydrodynamic size and zeta potential of SPIONs were 56 nm and -49 mV, respectively. The hydrodynamic and aerodynamic NIM diameters were 1.6 µm and 3.27±1.69 µm, respectively. SEM micrographs reveal spherical particles with rough surface morphology. TEM and focused ion beam-SEM micrographs corroborate the porous nature of NIMs, and surface localization of SPIONs. An in vitro tracheal mimic study demonstrates more than twice the spatial deposition and retention of NIMs, compared to a liquid suspension, in regions under the influence of a strong magnetic gradient. We report the novel formulation of an inhaled and magnetically responsive NIM drug delivery vehicle. This vehicle is capable of being loaded with one or more chemotherapeutic agents, with future translational ability to be targeted to lung tumors using an external magnetic field.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Compostos Férricos/química , Magnetismo , Nanopartículas/química , Administração por Inalação , Pulmão/metabolismo , Nanopartículas/ultraestrutura , Tamanho da Partícula , Traqueia/metabolismoRESUMO
Inhalable microparticles containing a large payload of isoniazid (INH) and rifabutin (RFB) in equal proportions show extremely high efficacy against experimental animal tuberculosis (TB). It was investigated whether inhaled microparticles affect the cytokine environment in the lung lumen, and cytokine secretion by airway and lung macrophages recovered from mice infected with Mycobacterium tuberculosis (Mtb). We attempted to determine whether the cytokine environment of the mouse lung receives significant contribution by lung macrophages, and whether these macrophages maintain a profile of cytokine secretion that is consistent with the cytokine environment of the lung. Groups of mice were infected intravenously with Mtb H37Ra and treated with (a) 5 mg/Kg each of INH and RFB administered by oral gavage; or, (b) 2.5 mg/Kg of the same and an additional â¼2.5 mg/Kg in the form of inhaled microparticles; or, (c) â¼2.5 mg/Kg by inhalation alone. Bronchioalveolar lavage (BAL) was carried out and recovered macrophages cultured. BAL Fluid and culture supernatants were assayed for tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin (IL)-12 and IL-10 by ELISA and amounts compared with both infected and uninfected, untreated controls. Inhaled microparticles enhanced secretion of TNF-α and supported IFN-γ secretion despite upregulated IL-10. Oral chemotherapy with the same drugs enhanced IL-12 and downregulated TNF-α. Differences in cytokine profiles suggest distinct effects of drug delivery modalities on innate immune strategies mobilized during host response. These differences might account, in part, for the extraordinary efficacy of the microparticles.
Assuntos
Citocinas/efeitos dos fármacos , Isoniazida/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Rifabutina/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Administração por Inalação , Animais , Líquido da Lavagem Broncoalveolar , Ensaio de Imunoadsorção Enzimática , Interferon gama/efeitos dos fármacos , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Macrófagos Alveolares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/patologia , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
This study aims to evaluate immunization with polymeric microparticles containing recombinant antigen 85B (rAg85B) delivered directly to the lungs to protect against tuberculosis. rAg85B was expressed in Escherichia coli and encapsulated in poly(lactic-co-glycolic acid) microparticles (P-rAg85B). These were delivered as dry powders to the lungs of guinea pigs in single or multiple doses of homologous and heterologous antigens. Bacille Calmette-Guérin (BCG) delivered subcutaneously was employed as the positive control and as part of immunization strategies. Immunized animals were challenged with a low-dose aerosol of Mycobacterium tuberculosis (MTB) H37Rv to assess the extent of protection measured as reduction in bacterial burden (CFU) in the lungs and spleens of guinea pigs. Histopathological examination and morphometric analysis of these tissues were also performed. The heterologous strategy of BCG prime-P-rAg85B aerosol boosts appeared to enhance protection from bacterial infection, as indicated by a reduction in CFU in both the lungs and spleens compared with untreated controls. Although the CFU data were not statistically different from the BCG and BCG-BCG groups, the histopathological and morphometric analyses indicated the positive effect of BCG-P-rAg85B in terms of differences in area of tissue affected and number and size of granulomas observed in tissues. P-rAg85B microparticles appeared to be effective in boosting a primary BCG immunization against MTB infection, as indicated by histopathology and morphometric analysis. These encouraging observations are relevant to boosting adults previously immunized with BCG or exposed to MTB, commonly the case in the developing world, and should be followed by further assessment of an appropriate immunization protocol for maximum protection.
Assuntos
Antígenos de Bactérias/administração & dosagem , Ácido Láctico/administração & dosagem , Pulmão , Microesferas , Mycobacterium tuberculosis/imunologia , Ácido Poliglicólico/administração & dosagem , Tuberculose/imunologia , Aerossóis , Animais , Ensaio de Imunoadsorção Enzimática , Cobaias , Hipersensibilidade Tardia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas Recombinantes/administração & dosagemRESUMO
Activation of human macrophages infected with Mycobacterium tuberculosis was investigated following exposure to microparticles (MP) possessing high anti-tubercular efficacy in mice. A small set of innate responses (cytokine profiles, NO production, Annexin-V staining and caspase-8, caspase-9 and caspase-3 activities) of differentiated THP-1 cells or human monocyte-derived macrophages infected 1:10 in vitro were compared. Cytokines of THP-1 macrophages were comparable in trends, but not in magnitude, with five human genotypes studied. MP reversed suppression of tumor necrosis factor induced by infection, and transiently upregulated gamma-interferon. Drug-free MP surprisingly induced gamma-interferon, but not tumor necrosis factor. Primary cells responded to MP, regardless of drug content, by upregulation of NO; but THP-1 cells did not respond to drug-free MP. About 19% of infected cells exposed to MP underwent apoptosis compared to approximately 11% cells treated otherwise. Cell death induced by drug-free MP was caspase independent. Intracellular bacterial survival varied between individuals. Untreated infection resulted in survival of 900+/-141 cfu; exposure to soluble drugs, drug-containing and blank microparticles respectively, reduced CFU counts to <10, <10 and 102+/-139. These observations indicate that despite variations in magnitude between cells from different sources, innate responses conducive to killing intracellular bacteria were evoked by inhalable MP.
Assuntos
Micropartículas Derivadas de Células/imunologia , Citocinas/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/microbiologia , Mycobacterium tuberculosis , Animais , Anexina A5/metabolismo , Apoptose , Caspases/metabolismo , Linhagem Celular , Citocinas/imunologia , Interferon gama/metabolismo , Camundongos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Macrophage responses to infection with Mycobacterium tuberculosis (MTB) and treatment with soluble isoniazid (INH) plus rifabutin (RFB) versus microparticles containing equivalent amounts of drugs were compared. It was investigated whether macrophages driven to alternative activation upon infection with MTB could be rescued to display the classical activation phenotype. It was established that microparticles sustain high levels of drugs in cytosol of macrophages for longer period as compared to soluble drugs. Microparticles co-localized with intracellular bacteria, and induced a variety of innate bactericidal responses, including induction of free radicals, alteration of mitochondrial membrane potential and apoptosis. The data strongly suggest that additional benefit may be derived from the nature of the drug delivery system, which fulfils Koch's dictum 'stimulate the phagocyte' for curing tuberculosis.
Assuntos
Antituberculosos , Isoniazida , Macrófagos/efeitos dos fármacos , Fagócitos/efeitos dos fármacos , Rifabutina , Administração por Inalação , Antituberculosos/administração & dosagem , Antituberculosos/farmacologia , Fragmentação do DNA , Humanos , Isoniazida/administração & dosagem , Isoniazida/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Mycobacterium tuberculosis/imunologia , Tamanho da Partícula , Fagócitos/metabolismo , Rifabutina/administração & dosagem , Rifabutina/farmacologia , Tuberculose/imunologiaRESUMO
Tuberculosis (TB) infects one third of the world's population, and new infections occur at a rate of 1/s. Better vaccines are needed than the live mycobacterium Bacille Calmette-Guérin (BCG). Alveolar macrophages (AMPhis) play a central role in pulmonary manifestations of TB. Targeting immunomodulators to AMPhis, the first line of defense against Mycobacterium tuberculosis (Mtb), may initiate a potent cell-mediated immune response. Muramyl dipeptide (MDP) and trehalose dibehenate (TDB) have elicited strong immune response when delivered to the lungs as aerosols. AMPhis show toxicity in response to some immunomodulators. The objective of this work was to screen the immunomodulators MDP and/or TDB encapsulated in microparticles (MPs) and to evaluate certain indicators of toxicity in human AMPhi-like cells. Poly(lactide-co-glycolide) (PLGA) MPs containing MDP and/or TDB were prepared by spray-drying. The morphology, particle size distribution, and immunomodulator encapsulation efficiency of MPs were examined. THP-1 cells were exposed to these MPs for 24 h and characteristics of cell morphology, tumor necrosis factor-alpha (TNF-alpha) release, lactate dehydrogenase (LDH), N-acetyl-beta-D: -glucosaminidase (NAG) and alkaline phosphatase (ALP) activity in AMPhi culture supernatants were measured. MTT assay was used to assess the viability of cells. Spray-drying produced low-density MPs having volume median diameters between 4 and 6 microm as measured by laser diffraction and projected area diameter between 3 and 5 microm calculated by microscopy. More TNF-alpha was produced by THP-1 cells exposed to MPs composed of PLGA-MDP or PLGA alone than PLGA-TDB. LDH release following exposure to MPs of PLGA-MDP and PLGA alone was greater than controls. NAG release was higher following exposure to MPs of PLGA alone or PLGA-MDP 0.1% than PLGA-TDB (0.1% and 1.0%). Cells remained viable after exposure to MPs as per MTT assay. PLGA-MDP MPs demonstrated statistically elevated indicators of biochemical responses in cell culture compared to PLGA-TDB MPs, but the extent of their potential to elicit adverse effects in vivo must be studied independently.