RESUMO
The aim of the present study was to improve the immunogenicity of peptide epitope vaccines using novel nanocarriers based on self-assembling materials. Several studies demonstrated that peptide antigens in nanoparticulate form induce stronger immune responses than their soluble forms. However, several issues such as poor loading and risk of inducing T cell anergy due to premature release of antigenic epitopes have challenged the clinical success of such systems. In the present study, we developed two vaccine delivery systems by appending a self-assembling peptide (Ac-AAVVLLLW-COOH) or a thermosensitive polymer poly(N-isopropylacrylamide (pNIPAm) to the N-terminus of different peptide antigens (OVA250-264, HPV-E743-57) to generate self-assembling peptide epitopes (SAPEs). The obtained results showed that the SAPEs were able to form nanostructures with a diameter from 20 to 200 nm. The SAPEs adjuvanted with CpG induced and expanded antigen-specific CD8+ T cells in mice. Furthermore, tumor-bearing mice vaccinated with SAPEs harboring the HPV E743-57 peptide showed a delayed tumor growth and an increased survival compared to sham-treated mice. In conclusion, self-assembling peptide based systems increase the immunogenicity of peptide epitope vaccines and therefore warrants further development toward clinical use.
Assuntos
Epitopos de Linfócito T/metabolismo , Peptídeos/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/metabolismo , Células Cultivadas , Epitopos de Linfócito T/imunologia , Imunoterapia , Camundongos , Nanopartículas/química , Ovalbumina/química , Vacinação/métodosRESUMO
Self-assembling peptides have gained increasing attention as versatile molecules to generate diverse supramolecular structures with tunable functionality. Because of the possibility to integrate a wide range of functional domains into self-assembling peptides including cell attachment sequences, signaling domains, vaccine epitopes, and even therapeutic moieties, complex nanostructures can be obtained with a wide range of applications in the biomedical field. The first part of this Review provides a concise overview of how peptide primary and secondary structure dictate the way such self-assembling peptides organize into higher ordered, supramolecular structures. Next, an overview of the literature will be given on recent studies on peptide self-assembly for application in drug delivery, vaccination, and tissue engineering.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Peptídeos/química , Engenharia Tecidual/métodos , Vacinação/métodos , Animais , Humanos , Nanoestruturas/química , Peptídeos/metabolismo , RegeneraçãoRESUMO
PURPOSE: To investigate the effect of polyethylene glycol (PEG) in nanoparticles based on blends of hydroxylated aliphatic polyester, poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and PEG-PLGHMGA block copolymers on their degradation and release behavior. METHODS: Protein-loaded nanoparticles were prepared with blends of varying ratios of PEG-PLGHMGA (molecular weight of PEG 2,000 and 5,000 Da) and PLGHMGA, by a double emulsion method with or without using poly(vinyl alcohol) (PVA) as surfactant. Bovine serum albumin and lysozyme were used as model proteins. RESULTS: PEGylated particles prepared without PVA had a zeta potential ranging from ~ -3 to ~-35 mV and size ranging from ~200 to ~600 nm that were significantly dependent on the content and type of PEG-block copolymer. The encapsulation efficiency of the two proteins however was very low (<30%) and the particles rapidly released their content in a few days. In contrast, all formulations prepared with PVA showed almost similar particle properties (size: ~250 nm, zeta potential: ~-1 mV), while loading efficiency for both model proteins was rather high (80-90%). Unexpectedly, independent of the type of formulation, the nanoparticles had nearly the same release and degradation characteristics. NMR analysis showed almost a complete removal of PEG in 5 days which explains these marginal differences. CONCLUSIONS: Protein release and particle degradation are not substantially influenced by the content of PEG, likely because of the fast shedding of the PEG blocks. These PEG shedding particles are interesting system for intracellular delivery of drugs.
Assuntos
Portadores de Fármacos/química , Muramidase/administração & dosagem , Nanopartículas/química , Poliésteres/química , Polietilenoglicóis/química , Soroalbumina Bovina/administração & dosagem , Portadores de Fármacos/síntese química , Composição de Medicamentos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Poliésteres/síntese química , Polietilenoglicóis/síntese química , Propriedades de SuperfícieRESUMO
In a broader scenario, the forced degradation studies provided by the ICH guidelines for Q1A, Q1B, and Q2B degradation studies allow to know the CQA of the molecule used as a drug product, to determine the appropriate analytical methods, excipients, and storage conditions ensuring the quality of the drug, its efficacy, and patient safety. In this study, we focused our attention on understanding how oxidative stress is performed by H2 O2 -impacted small synthetic peptides that do not contain residues susceptible to oxidation such as methionine. Among the amino acids susceptible to oxidation, methionine is the most reactive and depending on the structure of the protein where it is exposed, it tends to oxidize by converting into methionine sulfone or methionine sulfoxide by oxidation of its sulfur atom. Scouting experiments obtained by forced oxidative stress conditions are presented on two small synthetic peptides that do not contain any methionine residues spiked with different amounts of H2 O2 , and they are analyzed by LC-MS/MS. Less frequent oxidation products than those commonly observed on proteins/peptides-containing methionine have been characterized on both peptides. The study demonstrated that somatostatin, by means of one residue of tryptophan on the molecule, can generate traces of several oxidized products detected by UPLC-MS. Furthermore, even at a negligible level, oxidation on tyrosine and proline in cetrorelix that does not contain methionine nor tryptophan has been detected by UHPLC-MS/MS. Identification and quantification of oxidized species were achieved by high-resolution MS and MS/MS experiments. Thus, FDSs undoubtedly aid the evaluation of the CQAs as an important component of the characterization package as recommended by HAs and ICH, facilitating the understanding of unforeseen features of the studied molecule used as drugs.
Assuntos
Peróxido de Hidrogênio , Triptofano , Humanos , Cromatografia Líquida , Peróxido de Hidrogênio/química , Triptofano/química , Espectrometria de Massas em Tandem , Proteínas/química , Hormônio Liberador de Gonadotropina/metabolismo , Metionina/química , Somatostatina/metabolismo , Oxirredução , Estresse OxidativoRESUMO
Synthetic biology aims at reprogramming existing, or creating new, biological systems, with the ultimate aim to obtain artificial cells whose functions can be tailored. For the latter, encapsulation of complex biochemical reactions into cell-sized compartments, such as liposomes, is required. Recently, several groups have demonstrated that proteins of interest can be produced de novo within liposomes by entrapping cell-free protein-synthesis systems and DNA templates inside liposomes. Although detectable, intraliposomal protein synthesis was generally poor. Here, we have optimized intraliposomal cell-free protein synthesis by changing several variables, including lipid composition as well as liposome, pyrophosphatase, and T7 RNA polymerase concentration. Further, by using an activity-based assay, we have quantified the amount of full-length protein that was produced from DNA templates inside liposomes before and after optimization of aforementioned variables. Based on the model protein beta-galactosidase, it is demonstrated that liposomal protein synthesis can yield microgram quantities of protein (30-40 microg/mL liposomes).
Assuntos
Lipossomos/metabolismo , Biossíntese de Proteínas , Biologia , Sistema Livre de Células/metabolismo , Células/metabolismo , RNA Polimerases Dirigidas por DNA , Lipossomos/química , Proteínas/metabolismo , Transcrição Gênica , Proteínas Virais , beta-Galactosidase/metabolismoRESUMO
N,N,N-Trimethylated chitosan (TMC) with varying degree of quaternization (DQ) is currently being investigated in mucosal drug, vaccine and in gene delivery. However, besides N-methylation, O-methylation and chain scission occur during the synthesis of this polymer. Since both side reactions may affect the polymer characteristics, there is a need for TMCs without O-methylation and disparities in chain lengths while varying the DQ. In this study, O-methyl free TMC with varying DQs was successfully synthesized by using a two-step method. First, chitosan was quantitatively dimethylated using formic acid and formaldehyde. Then, in the presence of an excess amount of iodomethane, TMC was obtained with different DQs by varying reaction time. TMC obtained by this two-step method showed no detectable O-methylation ((1)H NMR) and a slight increase in molecular weight with increasing DQ (GPC), implying that no chain scission occurred during synthesis. The solubility in aqueous solutions at pH 7 of O-methyl free TMC with DQ<24% was less as compared to O-methylated TMC with the same DQ. On the other hand, O-methyl free TMC with DQ>33% had a good aqueous solubility. On Caco-2 cells, O-methyl free TMCs demonstrated a larger decrease in transepithelial electrical resistance (TEER) than O-methylated TMCs. Also, with increasing DQ, an increase in cytotoxicity (MTT) and membrane permeability (LDH) was observed.
Assuntos
Quitosana/síntese química , Quitosana/farmacologia , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , PolímerosRESUMO
In the search for non-invasive delivery options for the increasing number of therapeutic proteins, pulmonary administration is an attractive route. Supercritical fluid (SCF) drying processes offer the possibility to produce dry protein formulations suitable for inhalation. In this study, insulin-loaded microparticles suitable for pulmonary administration were prepared and characterized. N-Trimethyl chitosan (TMC), a polymeric mucoadhesive absorption enhancer and dextran, a non-permeation enhancer, were used as carriers for insulin. The particles were prepared by spraying an acidic water/DMSO solution of insulin and polymer into supercritical carbon dioxide. The mean size of the particles was 6-10microm (laser diffraction analysis) and their volume median aerodynamic diameter ca. 4microm (time-of-flight analysis). The particles had a water content of ca. 4% (w/w) (Karl-Fischer), and neither collapsed nor aggregated after preparation and storage. In the freshly prepared dried insulin powders, no insulin degradation products were detected by HPLC and GPC. Moreover, the secondary and tertiary structures of insulin as determined by circular dichroism and fluorescence spectroscopy were preserved in all formulations. After one-year storage at 4 degrees C, the particle characteristics were maintained and the insulin structure was largely preserved in the TMC powders. In conclusion, SCF drying is a promising, protein-friendly technique for the preparation of inhalable insulin-loaded particles.
Assuntos
Sistemas de Liberação de Medicamentos , Insulina/administração & dosagem , Pulmão/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Microesferas , Tamanho da Partícula , Espectrometria de FluorescênciaRESUMO
In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of proteins was investigated. TMC nanoparticles were prepared by ionic crosslinking of TMC solution (with or without ovalbumin) with tripolyphosphate, at ambient temperature while stirring. The size, zeta-potential and morphology of the nanoparticles were investigated as a function of the preparation conditions. Protein loading, protein integrity and protein release were studied. The toxicity of the TMC nanoparticles was tested by ciliary beat frequency measurements of chicken embryo trachea and in vitro cytotoxicity assays. The in vivo uptake of FITC-albumin-loaded TMC nanoparticles by nasal epithelia tissue in rats was studied by confocal laser scanning microscopy. The nanoparticles had an average size of about 350 nm and a positive zeta-potential. They showed a loading efficiency up to 95% and a loading capacity up to 50% (w/w). The integrity of the entrapped ovalbumin was preserved. Release studies showed that more than 70% of the protein remained associated with the TMC nanoparticles for at least 3 h on incubation in PBS (pH 7.4) at 37 degrees C. Cytotoxicity tests with Calu-3 cells showed no toxic effects of the nanoparticles, whereas a partially reversible cilio-inhibiting effect on the ciliary beat frequency of chicken trachea was observed. In vivo uptake studies indicated the transport of FITC-albumin-associated TMC nanoparticles across the nasal mucosa. In conclusion, TMC nanoparticles are a potential new delivery system for transport of proteins through the nasal mucosa.
Assuntos
Quitosana/química , Sistemas de Liberação de Medicamentos/métodos , Nanoestruturas/química , Proteínas/administração & dosagem , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Cílios/efeitos dos fármacos , Cílios/fisiologia , Eletroforese em Gel de Poliacrilamida , Géis , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mucosa Nasal/citologia , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologia , Ovalbumina/administração & dosagem , Ovalbumina/química , Ovalbumina/farmacologia , Proteínas/química , Proteínas/farmacologia , Ratos , Ratos Wistar , SolubilidadeRESUMO
The design of particulate vaccine delivery systems, particularly for mucosal surfaces, has been a focus of interest in recent years. In this context, we have previously described the development and the characterization of a new nanosized delivery system, consisting of a model antigen adsorbed to chitosan particles and coated with sodium alginate. In the present work the ovalbumin release profiles from these coated nanoparticles in different pH buffers were investigated and compared to those of the uncoated particles. Cytotoxicity of the polymers and nanoparticles was assessed using the MTT assay. Finally, particle uptake studies in rat Peyer's patches were performed. It was demonstrated that the coating of the nanoparticles with sodium alginate not only avoided a burst release observed with uncoated particles but also increased the stability of the particles at pH 6.8 and 7.4 at 37 degrees C. At neutral pH, the release was lower than 5% after 3.5 h incubation in a low ionic strength buffer. For both, chitosan and alginate polymers, and for the nanoparticles, comparable cell viability data close to 100%, were obtained. Additionally, based on confocal laser scanning microscopy observations, it was shown that alginate coated nanoparticles were able to be taken up by rat Peyer's patches, rendering them suitable carriers for intestinal mucosal vaccination.
Assuntos
Alginatos/administração & dosagem , Quitosana/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanoestruturas , Ovalbumina/administração & dosagem , Nódulos Linfáticos Agregados/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Ácido Glucurônico/administração & dosagem , Ácidos Hexurônicos/administração & dosagem , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/química , Ovalbumina/imunologia , Ratos , Ratos Wistar , Solubilidade , Baço/citologia , Baço/efeitos dos fármacos , VacinaçãoRESUMO
This study investigated the feasibility of the use of polymeric microparticles for sustained and local delivery of immunomodulatory antibodies in immunotherapy of cancer. Local delivery of potent immunomodulatory antibodies avoids unwanted systemic side effects while retaining their anti-tumor effects. Microparticles based on poly(lactic-co-hydroxymethyl-glycolic acid) (pLHMGA) and loaded with two distinct types of immunomodulatory antibodies (CTLA-4 antibody blocking inhibitory receptors on T cells or CD40 agonistic antibody stimulating dendritic cells) were prepared by double emulsion solvent evaporation technique. The obtained particles had a diameter of 12-15 µm to avoid engulfment by phagocytes and were slightly porous as shown by SEM analysis. The loading efficiency of the antibodies in the microparticles was >85%. The in vitro release profile of antiCD40 and antiCTLA-4 from microparticles showed a burst release of about 20% followed by a sustained release of the content up to 80% of the loading in around 30 days. The therapeutic efficacy of the microparticulate formulations was studied in colon carcinoma tumor model (MC-38). Mice bearing subcutaneous MC-38 tumors were treated with the same dose of immunomodulatory antibodies formulated either in incomplete Freund's adjuvant (IFA) or in microparticles. The antibody-loaded microparticles showed comparable therapeutic efficacy to the IFA formulation with no local adverse effects. The biodegradable microparticles were fully resorbed in vivo and no remnants of inflammatory depots as observed with IFA were present in the cured mice. Moreover the microparticles exhibited lower antibody serum levels in comparison with IFA formulations which lowers the probability of systemic adverse effects. In conclusion, pLHMGA microparticles are excellent delivery systems in providing long-lasting and non-toxic antibody therapy for immunotherapy of cancer.
Assuntos
Anticorpos/administração & dosagem , Antígenos CD40/imunologia , Antígeno CTLA-4/imunologia , Preparações de Ação Retardada/síntese química , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Animais , Anticorpos/química , Anticorpos/imunologia , Cápsulas/administração & dosagem , Cápsulas/síntese química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Preparações de Ação Retardada/administração & dosagem , Difusão , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/patologia , Resultado do TratamentoRESUMO
Immunotherapy of cancer is a promising therapeutic approach which aims to eliminate malignancies by inducing or enhancing an immune response against the tumor. Immunotherapy, however, faces several challenges such as local immunosuppression in the tumor area leading to immunological tolerance. To overcome these challenges, particulate formulations such as nano- and microparticles containing immunotherapeutics have been developed to increase therapeutic efficacy and reduce toxicity of immunotherapy. Particulate formulations based on biodegradable aliphatic polyesters such as poly(lactic-co-glycolic acid) (pLGA) have been extensively used with promising results. In this review, we addressed the potential of pLGA-based particulate formulations for immunotherapy of cancer. The discussion was focused on cancer vaccines and delivery of immunomodulatory antibodies. Features and drawbacks of pLGA systems were discussed together with several examples of recently developed therapeutic cancer vaccines and antibody-loaded particulate systems. Various strategies to overcome the drawbacks and optimize the formulations were given. In conclusion, pLGA-based particulate systems are attractive carriers for development of clinically acceptable formulations in immunotherapy of cancer.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Imunoterapia/métodos , Ácido Láctico/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias/terapia , Ácido Poliglicólico/administração & dosagem , Animais , Anticorpos/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Humanos , Neoplasias/imunologia , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
The aim of the current study was to develop a cancer vaccine formulation for treatment of human papillomavirus (HPV)-induced malignancies. Synthetic long peptides (SLPs) derived from HPV16 E6 and E7 oncoproteins have been used for therapeutic vaccination in clinical trials with promising results. In preclinical and clinical studies adjuvants based on mineral oils (such as incomplete Freund's adjuvant (IFA) and Montanide) are used to create a sustained release depot at the injection site. While the depot effect of mineral oils is important for induction of robust immune responses, their administration is accompanied with severe adverse and long lasting side effects. In order to develop an alternative for IFA family of adjuvants, polymeric nanoparticles (NPs) based on hydrophilic polyester (poly(d,l lactic-co-hydroxymethyl glycolic acid) (pLHMGA)) were prepared. These NPs were loaded with a synthetic long peptide (SLP) derived from HPV16 E7 oncoprotein and a toll like receptor 3 (TLR3) ligand (poly IC) by double emulsion solvent evaporation technique. The therapeutic efficacy of the nanoparticulate formulations was compared to that of HPV SLP+poly IC formulated in IFA. Encapsulation of HPV SLP antigen in NPs substantially enhanced the population of HPV-specific CD8+ T cells when combined with poly IC either co-encapsulated with the antigen or in its soluble form. The therapeutic efficacy of NPs containing poly IC in tumor eradication was equivalent to that of the IFA formulation. Importantly, administration of pLHMGA nanoparticles was not associated with adverse effects and therefore these biodegradable nanoparticles are excellent substitutes for IFA in cancer vaccines.
Assuntos
Vacinas Anticâncer/administração & dosagem , Papillomavirus Humano 16/imunologia , Indutores de Interferon/administração & dosagem , Proteínas E7 de Papillomavirus/administração & dosagem , Infecções por Papillomavirus/terapia , Poli I-C/administração & dosagem , Neoplasias do Colo do Útero/terapia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Colo do Útero/virologia , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Adjuvante de Freund/uso terapêutico , Humanos , Indutores de Interferon/imunologia , Indutores de Interferon/uso terapêutico , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Nanopartículas/química , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/imunologia , Proteínas E7 de Papillomavirus/uso terapêutico , Infecções por Papillomavirus/imunologia , Poli I-C/imunologia , Poli I-C/uso terapêutico , Poliésteres/química , Neoplasias do Colo do Útero/imunologia , VacinaçãoRESUMO
Particulate antigen delivery systems aimed at the induction of antigen-specific T cells form a promising approach in immunotherapy to replace pharmacokinetically unfavorable soluble antigen formulations. In this study, we developed a delivery system using the model protein antigen ovalbumin (OVA) encapsulated in nanoparticles based on the hydrophilic polyester poly(lactide-co-hydroxymethylglycolic acid) (pLHMGA). Spherical nanoparticles with size 300-400 nm were prepared and characterized and showed a strong ability to deliver antigen to dendritic cells for cross-presentation to antigen-specific T cells in vitro. Using near-infrared (NIR) fluorescent dyes covalently linked to both the nanoparticle and the encapsulated OVA antigen, we tracked the fate of this formulation in mice. We observed that the antigen and the nanoparticles are efficiently co-transported from the injection site to the draining lymph nodes, in a more gradual and durable manner than soluble OVA protein. OVA-loaded pLHMGA nanoparticles efficiently induced antigen cross-presentation to OVA-specific CD8+ T cells in the lymph nodes, superior to soluble OVA vaccination. Together, these data show the potential of pLHMGA nanoparticles as attractive antigen delivery vehicles.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Raios Infravermelhos , Nanopartículas/química , Ovalbumina/química , Poliésteres/química , Coloração e Rotulagem , Vacinas/imunologia , Animais , Apresentação de Antígeno/imunologia , Morte Celular , Células Dendríticas/metabolismo , Epitopos , Interações Hidrofóbicas e Hidrofílicas , Imunidade , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Poliésteres/síntese químicaRESUMO
A novel, EGFR-targeted nanomedicine has been developed in the current study. Glutaraldehyde crosslinked albumin nanoparticles with a size of approximately 100nm were loaded with the multikinase inhibitor 17864-L(x)-a platinum-bound sunitinib analogue-which couples the drug to methionine residues of albumin and is released in a reductive environment. Albumin nanoparticles were surface-coated with bifunctional polyethylene glycol 3500 (PEG) and a nanobody-the single variable domain of an antibody-(Ega1) against the epidermal growth factor receptor (EGFR). EGa1-PEG functionalized nanoparticles showed a 40-fold higher binding to EGFR-positive 14C squamous head and neck cancer cells in comparison to PEGylated nanoparticles. 17864-L(x) loaded EGa1-PEG nanoparticles were internalized by clathrin-mediated endocytosis and ultimately digested in lysosomes. The intracellular routing of EGa1 targeted nanoparticles leads to a successful release of the kinase inhibitor in the cell and inhibition of proliferation whereas the non-targeted formulations had no antiproliferative effects on 14C cells. The drug loaded targeted nanoparticles were as effective as the free drug in vitro. These results demonstrate that multikinase inhibitor loaded nanoparticles are interesting nanomedicines for the treatment of EGFR-positive cancers.
Assuntos
Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Nanopartículas/química , Inibidores de Proteínas Quinases/administração & dosagem , Albumina Sérica/química , Anticorpos Imobilizados/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/metabolismo , Receptores ErbB/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Nanopartículas/ultraestrutura , Inibidores de Proteínas Quinases/farmacologia , Albumina Sérica/metabolismoRESUMO
As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal/phagosomal CD14 and Toll-like receptors 1 and 2. Humans, however, possess natural antibodies against HSPB5 that block receptor binding. To protect it from these antibodies, we encapsulated HSPB5 in porous PLGA microparticles. We document here size, morphology, protein loading and release characteristics of such microparticles. Apart from effectively protecting HSPB5 from neutralization, PLGA microparticles also strongly promoted macrophage targeting of HSPB via phagocytosis. As a result, HSPB5 in porous PLGA microparticles was more than 100-fold more effective in activating macrophages than free soluble protein. Yet, the immune-regulatory nature of the macrophage response, as documented here by microarray transcript profiling, remained the same. In mice developing cigarette smoke-induced COPD, HSPB5-loaded PLGA microparticles were selectively taken up by alveolar macrophages upon intratracheal administration, and significantly suppressed lung infiltration by lymphocytes and neutrophils. In contrast, 30-fold higher doses of free soluble HSPB5 remained ineffective. Our data indicate that porous HSPB5-PLGA microparticles hold considerable promise as an anti-inflammatory biomaterial for humans.
Assuntos
Anti-Inflamatórios/administração & dosagem , Pulmão/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Pneumonia/complicações , Pneumonia/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/complicações , Cadeia B de alfa-Cristalina/administração & dosagem , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/uso terapêutico , Linhagem Celular , Portadores de Fármacos/química , Proteínas de Choque Térmico Pequenas/administração & dosagem , Proteínas de Choque Térmico Pequenas/imunologia , Proteínas de Choque Térmico Pequenas/uso terapêutico , Humanos , Ácido Láctico/química , Receptores de Lipopolissacarídeos/imunologia , Pulmão/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia/imunologia , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/imunologia , Cadeia B de alfa-Cristalina/imunologia , Cadeia B de alfa-Cristalina/uso terapêuticoRESUMO
Recently we have shown that liposomes can be used as artificial microbes for the production and delivery of DNA-encoded antigens. These so-called antigen-expressing immunostimulatory liposomes (AnExILs) were superior in inducing antigen-specific antibodies compared to conventional liposomal protein or DNA vaccines when tested in mice after i.m. immunization. In this study, we investigated the capacity of AnExILs to induce T-cell responses. By using a plasmid vector encoding a model antigen under control of both the prokaryotic T7 and the eukaryotic CMV promoter we hypothesized that antigen production could lead to CTL activation via two distinct routes: i. production of antigens inside the AnExILs with subsequent cross-presentation after processing by APCs and ii. endogenous production of antigens after AnExIL-mediated transfection of the pDNA. Although we were not able to demonstrate transfection-mediated expression of luc-NP in mice, i.m. injection of AnExILs producing luc-NP resulted in T-cell responses against the encoded NP epitope, as determined by tetramer staining. T-cell responses were comparable to the responses obtained after i.m. injection of naked pDNA. In order to find out whether CTL activation was caused by cross-presentation of the exogenous antigens produced inside AnExILs or by endogenous antigen production from transfection with the same pDNA source a second study was initiated in which the contribution of each of these effects could be separately determined. These results demonstrate that the observed T-cell responses were not exclusively caused by cross-presentation of the AnExIL-produced antigens alone, but were rather a combination of dose-dependent antigen cross-presentation and low levels of endogenous antigen production.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos/genética , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Vacinas de DNA/administração & dosagem , Animais , Antígenos/imunologia , Feminino , Lipossomos , Luciferases/genética , Luciferases/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Linfócitos T/imunologia , Vacinas de DNA/imunologia , beta-Galactosidase/genética , beta-Galactosidase/imunologiaRESUMO
Liposomes are versatile (sub)micron-sized membrane vesicles that can be used for a variety of applications, including drug delivery and in vivo imaging but they also represent excellent models for artificial membranes or cells. Several studies have demonstrated that in vitro transcription and translation can take place inside liposomes to obtain compartmentalized production of functional proteins within the liposomes (Kita et al. in Chembiochem 9(15):2403-2410, 2008; Moritani et al.in FEBS J, 2010; Kuruma et al. in Methods Mol Biol 607:161-171, 2010; Murtas et al. in Biochem Biophys Res Commun 363(1):12-17, 2007; Sunami et al. in Anal Biochem 357(1):128-136, 2006; Ishikawa et al. in FEBS Lett 576(3):387-390, 2004; Oberholzer et al. in Biochem Biophys Res Commun 261(2):238-241, 1999). Such a minimal artificial cell-based model is ideal for synthetic biology based applications. In this study, we propose the use of liposomes as artificial microbes for vaccination. These artificial microbes can be genetically programmed to produce specific antigens at will. To show proof-of-concept for this artificial cell-based platform, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen ß-galactosidase were entrapped inside multilamellar liposomes. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (ß-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal ß-galactosidase or pDNA encoding ß-galactosidase). In conclusion, AnExILs present a new platform for DNA-based vaccines which combines antigen production, adjuvanticity and delivery in one system and which offer several advantages over existing vaccine formulations.
RESUMO
Therapeutic peptides/proteins and protein-based antigens are chemically and structurally labile compounds, which are almost exclusively administered by parenteral injections. Recently, non-invasive mucosal routes have attracted interest for administration of these biotherapeutics. Chitosan-based delivery systems enhance the absorption and/or cellular uptake of peptides/proteins across mucosal sites and have immunoadjuvant properties. Chitosan is a mucoadhesive polysaccharide capable of opening the tight junctions between epithelial cells and it has functional groups for chemical modifications, which has resulted in a large variety of chitosan derivatives with tunable properties for the aimed applications. This review provides an overview of chitosan-based polymers for preparation of both therapeutic peptides/protein and antigen formulations. The physicochemical properties of these carrier systems as well as their applications in protein and antigen delivery through parenteral and mucosal (particularly nasal and pulmonary) administrations are summarized and discussed.
Assuntos
Antígenos/administração & dosagem , Quitosana/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Proteínas/administração & dosagem , Animais , Antígenos/química , Quitosana/química , Humanos , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/imunologia , Proteínas/química , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismoRESUMO
Chitosan derivatives such as N,N,N-trimethylated chitosan (TMC) are currently being investigated for the delivery of drugs, vaccines and genes. However, the influence of the extent of N-acetylation of these polymers on their enzymatic degradability and biological properties is unknown. In this study, TMCs with a degree of acetylation (DA) ranging from 11 to 55% were synthesized by using a three-step method. First, chitosan was partially re-acetylated using acetic anhydride followed by quantitative dimethylation using formaldehyde and sodium borohydrate. Then, in presence of an excess amount of iodomethane, TMC was synthesized. The TMCs obtained by this method showed neither detectable O-methylation nor loss in acetyl groups ((1)H NMR) and a slight increase in molecular weight (GPC) with increasing degree of substitution, implying that no chain scission occurred during synthesis. The extent of lysozyme-catalyzed degradation of TMC, and that of its precursors chitosan and dimethyl chitosan, was highly dependent on the DA and polymers with the highest DA showed the largest decrease in molecular weight. On Caco-2 cells, TMCs with a high DA ( approximately 50%), a DQ of around 44% and with or without O-methylated groups, were not able to open tight junctions in the trans-epithelial electrical resistance (TEER) assay, in contrast with TMCs (both O-methylated and O-methyl free; concentration 2.5mg/ml) with a similar DQ but a lower DA which were able to reduce the TEER with 30 and 70%, respectively. Additionally, TMCs with a high DA ( approximately 50%) demonstrated no cell toxicity (MTT, LDH release) up to a concentration of 10mg/ml.
Assuntos
Quitosana/química , Muramidase/metabolismo , Acetilação , Adenocarcinoma/patologia , Animais , Biopolímeros , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Linhagem Celular Tumoral/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Quitosana/síntese química , Quitosana/farmacologia , Quitosana/toxicidade , Neoplasias do Colo/patologia , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/fisiologia , Humanos , Hidrólise , L-Lactato Desidrogenase/análise , Metilação , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Junções Íntimas/efeitos dos fármacosRESUMO
The potential of N-trimethyl chitosan (TMC) with two degrees of quaternization (DQ), TMC20 (DQ 20%, as a mucoadhesive) and TMC60 (DQ 60%, as a mucoadhesive and a permeation enhancer), and dextran (as a non-mucoadhesive and non-permeation enhancer) microparticles as carriers for pulmonary delivery of insulin was studied in diabetic rats. The impact of the powder formulation on insulin bioavailability and its pharmacological effect was evaluated using a population pharmacokinetic-pharmacodynamic (PKPD) model. Insulin-loaded microparticles were prepared by a supercritical fluid (SCF) drying technique. They had a median volume diameter and median volume aerodynamic diameter of about 6-10 microm and 4 microm, respectively. The PK of insulin in the diabetic rats was analyzed by a one-compartment disposition model and the PD was described by the minimal model of glucose disappearance. The bioavailability of the pulmonarily administered dextran-, TMC20- and TMC60-insulin microparticles relative to subcutaneously (SC) administered insulin, was 0.48, 0.59 and 0.95, respectively. Histological examinations of the rats' lungs did not show any local adverse reactions after single administration of insulin powders. The pharmacodynamic model could describe the insulin-glucose relationship and pharmacodynamic efficiency of insulin formulations, which was about 0.6(*)10(-5) ml/microU, irrespective of the formulations. The current findings suggest that TMC microparticles are a promising vehicle for pulmonary delivery of insulin.