RESUMO
Retinitis pigmentosa (RP) is a heterogeneous disease and the main cause of vision loss within the group of inherited retinal diseases (IRDs). IRDs are a group of rare disorders caused by mutations in one or more of over 280 genes which ultimately result in blindness. Modifier genes play a key role in modulating disease phenotypes, and mutations in them can affect disease outcomes, rate of progression, and severity. Our previous studies have demonstrated that the nuclear hormone receptor 2 family e, member 3 (Nr2e3) gene reduced disease progression and loss of photoreceptor cell layers in RhoP23H-/- mice. This follow up, pharmacology study evaluates a longitudinal NR2E3 dose response in the clinically relevant heterozygous RhoP23H mouse. Reduced retinal degeneration and improved retinal morphology was observed 6 months following treatment evaluating three different NR2E3 doses. Histological and immunohistochemical analysis revealed regions of photoreceptor rescue in the treated retinas of RhoP23H+/- mice. Functional assessment by electroretinogram (ERG) showed attenuated photoreceptor degeneration with all doses. This study demonstrates the effectiveness of different doses of NR2E3 at reducing retinal degeneration and informs dose selection for clinical trials of RhoP23H-associated RP.
Assuntos
Modelos Animais de Doenças , Receptores Nucleares Órfãos , Degeneração Retiniana , Retinose Pigmentar , Animais , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Camundongos , Receptores Nucleares Órfãos/genética , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/etiologia , Eletrorretinografia , Retina/metabolismo , Retina/patologia , Terapia Genética/métodosRESUMO
MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression in many diseases, although the contribution of miRNAs to the pathophysiology of lung injury remains obscure. We hypothesized that dysregulation of miRNA expression drives the changes in key genes implicated in the development of lung injury. To test our hypothesis, we utilized a model of lung injury induced early after administration of intratracheal bleomycin (0.1 U). Wild-type mice were treated with bleomycin or PBS, and lungs were collected at 4 or 7 days. A profile of lung miRNA was determined by miRNA array and confirmed by quantitative PCR and flow cytometry. Lung miR-26a was significantly decreased 7 days after bleomycin injury, and, on the basis of enrichment of predicted gene targets, it was identified as a putative regulator of cell adhesion, including the gene targets EphA2, KDR, and ROCK1, important in altered barrier function. Lung EphA2 mRNA, and protein increased in the bleomycin-injured lung. We further explored the miR-26a/EphA2 axis in vitro using human lung microvascular endothelial cells (HMVEC-L). Cells were transfected with miR-26a mimic and inhibitor, and expression of gene targets and permeability was measured. miR-26a regulated expression of EphA2 but not KDR or ROCK1. Additionally, miR-26a inhibition increased HMVEC-L permeability, and the disrupted barrier integrity due to miR-26a was blocked by EphA2 knockdown, shown by VE-cadherin staining. Our data suggest that miR-26a is an important epigenetic regulator of EphA2 expression in the pulmonary endothelium. As such, miR-26a may represent a novel therapeutic target in lung injury by mitigating EphA2-mediated changes in permeability.
Assuntos
Endotélio Vascular/patologia , Lesão Pulmonar/patologia , MicroRNAs/genética , Receptor EphA2/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Permeabilidade da Membrana Celular , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Humanos , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/genética , Lesão Pulmonar/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor EphA2/genéticaRESUMO
For vision-threatening retinitis pigmentosa and dry age-related macular degeneration, there are no United States Food and Drug Administration (FDA)-approved treatments. We identified, biosynthesized, purified, and characterized lens epithelium-derived growth factor fragment (LEDGF1-326) as a novel protein therapeutic. LEDGF1-326 was produced at about 20 mg/liter of culture when expressed in the Escherichia coli system, with about 95% purity and aggregate-free homogeneous population with a mean hydrodynamic diameter of 9 ± 1 nm. The free energy of unfolding of LEDGF1-326 was 3.3 ± 0.5 kcal mol(-1), and melting temperature was 44.8 ± 0.2 °C. LEDGF1-326 increased human retinal pigment epithelial cell viability from 48.3 ± 5.6 to 119.3 ± 21.1% in the presence of P23H mutant rhodopsin-mediated aggregation stress. LEDGF1-326 also increased retinal pigment epithelial cell FluoSphere uptake to 140 ± 10%. Eight weeks after single intravitreal injection in Royal College of Surgeons (RCS) rats, LEDGF1-326 increased the b-wave amplitude significantly from 9.4 ± 4.6 to 57.6 ± 8.8 µV for scotopic electroretinogram and from 10.9 ± 5.6 to 45.8 ± 15.2 µV for photopic electroretinogram. LEDGF1-326 significantly increased the retinal outer nuclear layer thickness from 6.34 ± 1.6 to 11.7 ± 0.7 µm. LEDGF1-326 is a potential new therapeutic agent for treating retinal degenerative diseases.
Assuntos
Atrofia Geográfica/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Retinose Pigmentar/tratamento farmacológico , Animais , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Clonagem Molecular , Eletrorretinografia , Atrofia Geográfica/patologia , Humanos , Injeções Intraoculares , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Luz , Peso Molecular , Tamanho da Partícula , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/isolamento & purificação , Fagocitose/efeitos dos fármacos , Estabilidade Proteica , Estrutura Secundária de Proteína , Ratos , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Retinose Pigmentar/patologia , Espalhamento de Radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estresse FisiológicoRESUMO
Retinitis Pigmentosa is a leading cause of severe vision loss. Retinitis Pigmentosa can present with a broad range of phenotypes impacted by disease age of onset, severity, and progression. This variation is influenced both by different gene mutations as well as unique variants within the same gene. Mutations in the nuclear hormone receptor 2 family e, member 3 are associated with several forms of retinal degeneration, including Retinitis Pigmentosa. In our previous studies we demonstrated that subretinal administration of one Nr2e3 dose attenuated retinal degeneration in rd7 mice for at least 3 months. Here we expand the studies to evaluate the efficacy and longitudinal impact of the NR2E3 therapeutic by examining three different doses administered at early or intermediate stages of retinal degeneration in the rd7 mice. Our study revealed retinal morphology was significantly improved 6 months post for all doses in the early-stage treatment groups and for the low and mid doses in the intermediate stage treatment groups. Similarly, photoreceptor function was significantly improved in the early stage for all doses and intermediate stage low and mid dose groups 6 months post treatment. This study demonstrated efficacy in multiple doses of NR2E3 therapy.
Assuntos
Modelos Animais de Doenças , Receptores Nucleares Órfãos , Degeneração Retiniana , Animais , Camundongos , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/tratamento farmacológico , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Retinose Pigmentar/tratamento farmacológico , Retina/patologia , Retina/metabolismo , Retina/efeitos dos fármacosRESUMO
Nanoparticles in porous microparticles (NPinPMP), a novel delivery system for sustained delivery of protein drugs, was developed using supercritical infusion and pressure quench technology, which does not expose proteins to organic solvents or sonication. The delivery system design is based on the ability of supercritical carbon dioxide (SC CO2) to expand poly(lactic-co-glycolic) acid (PLGA) matrix but not polylactic acid (PLA) matrix. The technology was applied to bevacizumab, a protein drug administered once a month intravitreally to treat wet age related macular degeneration. Bevacizumab coated PLA nanoparticles were encapsulated into porosifying PLGA microparticles by exposing the mixture to SC CO2. After SC CO2 exposure, the size of PLGA microparticles increased by 6.9-fold. Confocal and scanning electron microscopy studies demonstrated the expansion and porosification of PLGA microparticles and infusion of PLA nanoparticles inside PLGA microparticles. In vitro release of bevacizumab from NPinPMP was sustained for 4 months. Size exclusion chromatography, fluorescence spectroscopy, circular dichroism spectroscopy, SDS-PAGE, and ELISA studies indicated that the released bevacizumab maintained its monomeric form, conformation, and activity. Further, in vivo delivery of bevacizumab from NPinPMP was evaluated using noninvasive fluorophotometry after intravitreal administration of Alexa Fluor 488 conjugated bevacizumab in either solution or NPinPMP in a rat model. Unlike the vitreal signal from Alexa-bevacizumab solution, which reached baseline at 2 weeks, release of Alexa-bevacizumab from NPinPMP could be detected for 2 months. Thus, NPinPMP is a novel sustained release system for protein drugs to reduce frequency of protein injections in the therapy of back of the eye diseases.
Assuntos
Anticorpos Monoclonais Humanizados/química , Nanopartículas/química , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Dióxido de Carbono/química , Olho/efeitos dos fármacos , Ácido Láctico/química , Tamanho da Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Porosidade , Pressão , Ratos , Solventes/químicaRESUMO
Inducing humoral and cytotoxic mucosal immunity at the sites of pathogen entry has the potential to prevent the infection from getting established. This is different from systemic vaccination, which protects against the development of systemic symptoms. The field of mucosal vaccination has seen fewer technological advances compared to nucleic acid and subunit vaccine advances for injectable vaccine platforms. The advent of the next-generation adenoviral vectors has given a boost to mucosal vaccine research. Basic research into the mechanisms regulating innate and adaptive mucosal immunity and the discovery of effective and safe mucosal vaccine adjuvants will continue to improve mucosal vaccine design. The results from clinical trials of inhaled COVID-19 vaccines demonstrate their ability to induce the proliferation of cytotoxic T cells and the production of secreted IgA and IgG antibodies locally, unlike intramuscular vaccinations. However, these mucosal vaccines induce systemic immune responses at par with systemic vaccinations. This review summarizes the function of the respiratory mucosa-associated lymphoid tissue and the advantages that the adenoviral vectors provide as inhaled vaccine platforms.
RESUMO
Since long before the first approval of gene therapy for retinal disease, ocular gene therapy has captured the hopes of patients, clinicians, and scientists alike. Indeed, the retina provides a unique system for studying and treating ocular diseases, and it holds the distinction as the first tissue targeted by an approved gene therapy for inherited disorders in the United States. There are many methods for addressing genetic diseases in the eyes using a wide range of potential delivery systems and vectors. However, despite the immense progress over the last several decades, both old and new challenges remain, such as the long-term effects of treatments, immunogenicity, targeting, and manufacturing. This review provides a discussion of the history of ocular gene therapy, the various gene therapy approaches, methods to deliver a gene directly to ocular tissues (including both routes of administration and vectors), challenges to ocular gene therapy, the current clinical trial landscape, and future directions of the field.
Assuntos
Degeneração Retiniana , Humanos , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Retina , Terapia GenéticaRESUMO
Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100mM Tris buffer containing 6M n-propanol and 2M urea. Around 4 mg/ml of r-hGH from inclusion bodies were solubilized in 6M n-propanol-based buffer containing 2M urea. Existence of native-like secondary structure of r-hGH in 6M n-propanol solution was confirmed by CD and fluorescence spectra. Solubilized r-hGH was subsequently refolded by pulsatile dilution, purified to homogeneity and found to be functionally active. Tris buffer containing 6M n-propanol and 2M urea also effectively solubilized a number of proteins expressed as inclusion bodies in E. coli. Mild solubilization of inclusion body proteins, chaotropic effect of n-propanol at high concentration and kosmotropic effect at lower concentration helped in improved refolding of the solubilized protein. Around 40% of the r-hGH in the form of inclusion body aggregates was refolded into bioactive form while using n-propanol as solubilization agent. Solubilization with 6M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.
Assuntos
1-Propanol/química , Hormônio do Crescimento Humano/química , Corpos de Inclusão/química , Proteínas Recombinantes/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Escherichia coli/metabolismo , Hormônio do Crescimento Humano/metabolismo , Hormônio do Crescimento Humano/farmacologia , Humanos , Redobramento de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , SolubilidadeRESUMO
The world has responded to the COVID-19 pandemic with unprecedented speed and vigor in the mass vaccination campaigns, targeted to reduce COVID-19 severity and mortality, reduce the pressure on the healthcare system, re-open society, and reduction in disease mortality and morbidity. Here we review the preclinical and clinical development of BBV152, a whole virus inactivated vaccine and an important tool in the fight to control this pandemic. BBV152, formulated with a TLR7/8 agonist adjuvant generates a Th1-biased immune response that induces high neutralization efficacy against different SARS-CoV-2 variants of concern and robust long-term memory B- and T-cell responses. With seroconversion rates as high as 98.3% in vaccinated individuals, BBV152 shows 77.8% and 93.4% protection from symptomatic COVID-19 disease and severe symptomatic COVID-19 disease respectively. Studies in pediatric populations show superior immunogenicity (geometric mean titer ratio of 1.76 compared to an adult) with a seroconversion rate of >95%. The reactogenicity and safety profiles were comparable across all pediatric age groups between 2-18 yrs. as in adults. Like most approved vaccines, the BBV152 booster given 6 months after full vaccination, reverses a waning immunity, restores the neutralization efficacy, and shows synergy in a heterologous prime-boost study with about 3-fold or 300% increase in neutralization titers against multiple SARS-CoV-2 variants of concern. Based on the interim Phase III data, BBV152 received full authorization for adults and emergency use authorization for children from ages 6 to 18 years in India. It is also licensed for emergency use in 14 countries globally. Over 313 million vaccine doses have already been administered in India alone by April 18th, 2022.
Assuntos
COVID-19 , SARS-CoV-2 , Adjuvantes Imunológicos , Adolescente , Adulto , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Criança , Pré-Escolar , Humanos , Pandemias/prevenção & controle , Receptor 7 Toll-Like , Desenvolvimento de Vacinas , Vacinas de Produtos Inativados/efeitos adversosRESUMO
Crystallins, small heat shock chaperone proteins that prevent protein aggregation, are of potential value in treating protein aggregation disorders. However, their therapeutic use is limited by their low potency and poor intracellular delivery. One approach to facilitate the development of crystallins is to improve their activity, stability, and delivery. In this study, zinc addition to αB-crystallin-D3 (αB-D3) formed supramolecular nano- and micro- assemblies, induced dose-dependent changes in structure (beta-sheet to alpha-helix) and increased surface hydrophobicity and chemical stability. Further, crystallin assemblies exhibited a size-dependent chaperone activity, with the nano-assemblies being superior to micro-assemblies and 4.3-fold more effective than the native protein in preventing ß-mercaptoethanol induced aggregation of insulin. Insulin rescued by crystallin assemblies retained the activity as evidenced by glucose uptake in 3T3-L1 cells. The most active nano-assemblies enhanced protein stability, in the presence of urea, by 1.6-fold, whereas intracellular delivery was enhanced by 3.0-fold. The αB-D3 crystallin nano-assemblies exhibit uniquely enhanced stability, activity, and delivery compared to the native protein.
Assuntos
Insulinas , Cadeia B de alfa-Cristalina , Agregados Proteicos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismoRESUMO
Carbohydrate hydrogels are extensively used in pharmaceuticals and engineered biomaterials. Molecular conformations, assembly, and interactions of the carbohydrate strands with stabilizers such as clay minerals in aqueous solution are difficult to quantify in experiments and the hydrogel properties remain largely a result of trial-and-error studies. We analyzed the assembly of gellan gum in aqueous solution and interactions with dispersed clay minerals in all-atomic detail using molecular dynamics simulation, atomic force microscopy (AFM), and comparisons to earlier measurements. Gellan strands associate at low pH values of 2 and gradually disassemble to double strands with weak association of -0.4 kcal per mole carbohydrate ring as the pH values increases to 9. Ionization of the carbonic acid side groups in the backbone extends the chains and accelerates the conformational dynamics via rapidly changing intramolecular ion bridges. Gellan interactions with clay minerals depend on the strength of electric triple layers between clay, cations, and anionic polymer strands, as well as weaker hydrogen bonds along the edges, which are tunable as a function of the clay surface chemistry, local ionic strength, and pH values. Interaction energies range from -4 to +6 kcal per mol carbohydrate ring and were most favorable for electric triple layers with high charge mobility, which can be achieved for intermediate cation exchange capacity of the clay mineral and high pH values to increase ionization of the clay edges and of the polymer. The findings provide understanding and help control the dynamics and stabilization of carbohydrate hydrogels by clay minerals.
RESUMO
Ephrin type-A receptor 2 (EphA2) is a transmembrane receptor which is upregulated in injured lungs, including those treated with bleomycin. YSA peptide (YSAYPDSVPMMS), a mimic of ephrin ligands, binds to EphA2 receptors on cell surface with high affinity. In this study, we assessed the ability of YSA-functionalized and non-functionalized poly (dl-lactide-co-glycolide) (PLGA) nanoparticles to enhance delivery to bleomycin treated cultured vascular endothelial cells and, in a bleomycin induced lung injury mouse model. Nanoparticles were loaded with a lipophilic fluorescent dye. Human umbilical vein endothelial cells (HUVEC) with or without 2-day bleomycin pretreatment (25 µg/ml) and adult mice with or without intratracheal instillation of bleomycin (0.1 U) were dosed with nanoparticles. Mice received nanoparticles via tail vein injection 4 days after bleomycin treatment. Three days after nanoparticle injection, tissues (lung, heart, kidney, spleen, liver, brain, eyes and whole blood) were harvested and quantified for fluorescence using IVIS imaging. Mean particle uptake increased with time and concentration for both types of particles in HUVEC, with the uptake being higher for YSA-functionalized nanoparticles. Bleomycin treatment increased the 3-h uptake of both types of nanoparticles in HUVEC by about two-fold, with the YSA-functionalized nanoparticle uptake being 1.66-fold compared to non-functionalized nanoparticles (p < .05). In mice, bleomycin injury resulted in 2.3- and 4.7-fold increase in the lung levels of non-functionalized and YSA-functionalized nanoparticles (p < .05), respectively, although the differences between the two particle types were not significant. In conclusion, PLGA nanoparticle delivery to cultured vascular endothelial cells and mouse lungs in vivo is higher following bleomycin treatment, with the delivery tending to be higher for YSA functionalized nanoparticles.
Assuntos
Bleomicina/efeitos adversos , Efrina-A2/agonistas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Lesão Pulmonar/tratamento farmacológico , Pulmão , Nanopartículas , Peptídeos , Animais , Bleomicina/farmacologia , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Camundongos , Nanopartículas/química , Nanopartículas/uso terapêutico , Peptídeos/química , Peptídeos/farmacologia , Receptor EphA2RESUMO
A tetrameric protein of therapeutic importance, Escherichia coli L-asparaginase-II was expressed in Escherichia coli as inclusion bodies (IBs). Asparaginase IBs were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps by ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from IBs was around 50%. The melting temperature (Tm) of the purified asparaginase was found to be 64°C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 IU/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the IB aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of IB aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. Our mild solubilization technique retaining native-like structures, improved recovery of asparaginase in bioactive tetrameric form.
RESUMO
Particle size, antigen load and its release characteristic are the three the main attributes of polymer particles based vaccine delivery systems. The present studies focus on the formulation of spray dried polylactide microparticles entrapping pneumococcal surface protein A (PspA). Influence of process variables during polymer particle formation were optimized by using half-factorial design. Feed rate and atomization pressure during spray drying were found to be the most important parameters for achieving uniform size particles. Spray drying of preformed particles from different stages of solvent evaporation method resulted in formation of particle having different porosity and protein release profile. Presence of polyvinyl alcohol in the external aqueous phase not only contributed towards regulating the size of particles but also influenced the burst release of protein from particles. Polymer particles entrapping PspA elicited robust IgG responses both in mice and in rats. Antigen load in microparticles correlated with the antibody titer indicating the maintenance of protein integrity during particle formation using spray drying. Both, process engineering and formulation parameters during spray drying influenced the particles in terms of size, load and antigen release characteristics.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Poliésteres/química , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Química Farmacêutica , Composição de Medicamentos , Feminino , Imunização , Masculino , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Pós , Ratos Wistar , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Reologia , Propriedades de SuperfícieRESUMO
Empty adenovirus serotype 5 (Ad5) capsids devoid of viral genome were developed as a novel delivery system for nanoparticles, proteins, and nucleic acids. Ad5 capsids of 110 nm diameter undergo an increase in particle size to 1637 nm in 1mM acetic acid at pH4.0 and then shrink to 60 nm, following pH reversal to 7.4. These pH shifts induced reversible changes in capsid zeta potential and secondary structure and irreversible changes in tertiary structure of capsid proteins. Using pH shift dependent changes in capsid size and structure, 20 nm fluorescent nanoparticles, FITC-BSA, and Alexa Fluor® 488 conjugated siRNA were encapsulated with high efficiency in Ad5 capsids, as confirmed by electron microscopy and/or flow cytometry. HEK cell uptake with capsid delivery system was 7.8-, 7.4-, and 2.9-fold greater for nanoparticles, FITC-BSA, and Alexa-siRNA, respectively, when compared to plain solutes. Physical mixtures of capsids and fluorescent solutes exhibited less capsid associated fluorescence intensity and cell uptake. Further, unlike physical mixture, pH shift assembled Ad5 capsids protected siRNA from RNase degradation. Ad5 capsids before and after pH shift exhibited endolysosomal escape. Thus, empty Ad5 capsids can encapsulate a variety of solutes based on pH shift assembly, resulting in enhanced cellular delivery.
Assuntos
Adenoviridae/química , Proteínas do Capsídeo/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes/administração & dosagem , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Soroalbumina Bovina/administração & dosagem , Capsídeo/química , Portadores de Fármacos/química , Fluoresceína-5-Isotiocianato/administração & dosagem , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Tamanho da Partícula , Estrutura Secundária de ProteínaRESUMO
The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low ß-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.