RESUMO
Herein, a method for synthesizing and utilizing DNA dendrons to deliver biomolecules to living cells is reported. Inspired by high-density nucleic acid nanostructures, such as spherical nucleic acids, we hypothesized that small clusters of nucleic acids, in the form of DNA dendrons, could be conjugated to biomolecules and facilitate their cellular uptake. We show that DNA dendrons are internalized by 90% of dendritic cells after just 1 h of treatment, with a >20-fold increase in DNA delivery per cell compared with their linear counterparts. This effect is due to the interaction of the DNA dendrons with scavenger receptor-A on cell surfaces, which results in their rapid endocytosis. Moreover, when conjugated to peptides at a single attachment site, dendrons enhance the cellular delivery and activity of both the model ovalbumin 1 peptide and the therapeutically relevant thymosin alpha 1 peptide. These findings show that high-density, multivalent DNA ligands play a significant role in dictating cellular uptake of biomolecules and consequently will expand the scope of deliverable biomolecules to cells. Indeed, DNA dendrons are poised to become agents for the cellular delivery of many molecular and nanoscale materials.
Assuntos
DNA/química , Dendrímeros/química , Animais , Linhagem Celular , Dendrímeros/metabolismo , Endocitose , Camundongos , Ovalbumina/química , Peptídeos/química , Timalfasina/químicaRESUMO
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding for the ion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Long considered a lung disease for the devastating impact on the respiratory function, the recent diagnostic and therapeutic advances have shed the light on the extra-pulmonary manifestations of CF, including gastrointestinal, hepatobiliary and pancreatic symptoms. We have previously demonstrated that thymosin alpha1 (Tα1), a naturally occurring immunomodulatory peptide, displays multi-sided beneficial effects in CF that concur in ameliorating the lung inflammatory pathology. In the present study, by resorting to murine models of gut inflammation with clinical relevance for CF patients, we demonstrate that Tα1 can also have beneficial effects in extrapulmonary pathology. Specifically, Tα1 restored barrier integrity and immune homeostasis in the inflamed gut of CF mice as well as in mice with the metabolic syndrome, a disorder that may arise in CF patients with high caloric intake despite pancreatic sufficiency. The protective effects of Tα1 also extended to pancreas and liver, further emphasizing the beneficial effects of Tα1 in extra-pulmonary complications of CF. By performing wide-ranging multi-organ anti-inflammatory effects, Tα1 could potentially integrate current therapeutic approaches to tackle the complex symptomatology of CF disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Fatores Imunológicos/química , Timalfasina/química , Animais , Candida albicans/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Feminino , Homeostase/efeitos dos fármacos , Humanos , Fatores Imunológicos/farmacologia , Cinurenina/metabolismo , Fígado , Pulmão , Camundongos , Mutação , Obesidade/tratamento farmacológico , Pâncreas , Transdução de Sinais , Timalfasina/farmacologiaRESUMO
Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.
Assuntos
Adjuvantes Imunológicos/farmacocinética , Timalfasina/farmacocinética , Acetilação , Acetiltransferases/metabolismo , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/farmacologia , Animais , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Feminino , Meia-Vida , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Neoplasias/tratamento farmacológico , Peptídeos/química , Proteólise , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/ultraestrutura , Proteínas Ribossômicas/metabolismo , Timalfasina/sangue , Timalfasina/química , Timalfasina/genética , Viroses/tratamento farmacológico , Tratamento Farmacológico da COVID-19RESUMO
The use of interferon α-2 in combination with thymosin α-1 shows higher anti-cancer effect in comparison when both are used individually because of their synergistic effects. In this study we produced an important human interferon α-2-thymosin α-1 (IFNα2-Tα1) fusion protein with probable pharmaceutical properties coupled to its high-level expression, characterization, and study of its biological activity. The IFNα2-Tα1 fusion gene was constructed by over-lap extension PCR and expressed in Escherichia coli expression system. The expression of IFNα2-Tα1 fusion protein was optimized to higher level and its maximum expression was obtained in modified terrific broth medium when lactose was used as inducer. The fusion protein was refolded into its native biologically active form with maximum yield of 83.14% followed by purification with â¼98% purity and 69% final yield. A band of purified IFNα2-Tα1 fusion protein equal to â¼23 kDa was observed on 12 % SDS-PAGE gel. The integrity of IFNα2-Tα1 fusion protein was confirmed by western blot analysis and secondary structure was assessed by CD spectroscopy. When IFNα2-Tα1 fusion protein was subjected to its biological activity analysis it was observed that it exhibits both IFNα2 & Tα1 activities as well as significantly higher anticancer activity as compared to IFNα-2 alone.