ABSTRACT
Viral nucleic acids are recognized by specific pattern-recognition receptors of the Toll-like and RIG-I-like receptor families. Synthetic DNA and RNA oligonucleotides can activate the immune system through these receptors and potentiate Ab and CD8 cytotoxic responses to Ags. Systemic application of immunostimulatory oligonucleotides however also results in a generalized, non-Ag-specific stimulation of the immune system. In this study, we have dissociated the induction of an Ag-specific response from the systemic immune activation generally associated with immunostimulatory oligonucleotides. Delivery of CpG oligodeoxynucleotides that bind TLR9 by cationized gelatin-based nanoparticles potentiates the in vivo generation of an Ag-specific cytotoxic T cell and Ab response. Furthermore, immunization with CpG-loaded nanoparticles induces a protective antitumoral response in a murine model of melanoma. The systemic release of proinflammatory cytokines and widespread immunostimulation associated with free CpG is however completely abolished. In addition, we show that gelatin nanoparticle formulation prevents the destruction of lymphoid follicles mediated by CpG. Nanoparticle-delivered CpG, in contrast to free CpG, are selectively targeted to APCs in the lymph nodes where they mediate local immune stimulation. We describe a novel strategy to target immunostimulatory oligonucleotides to the initiation site of the immune response while at the same time protecting from an indiscriminate and generalized activation of the immune system.
Subject(s)
Immunity/drug effects , Lymph Nodes/metabolism , Nanoparticles/administration & dosage , Neoplasms/therapy , Oligodeoxyribonucleotides/administration & dosage , Animals , Drug Delivery Systems/methods , Female , Gelatin , Immunotherapy/methods , Lymph Nodes/immunology , Mice , Neoplasms/immunology , Oligodeoxyribonucleotides/pharmacokinetics , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/immunology , Toll-Like Receptor 9/metabolismABSTRACT
The freeze-drying properties of gelatin nanoparticles were investigated with the goal of providing practicable nanoparticle formulations for in vitro applications or clinical studies. Various excipients and rehydration protocols were assessed, and gelatin nanoparticles loaded with oligonucleotides were successfully freeze-dried and rehydrated. An NF-kappaB decoy oligonucleotide-loaded gelatin nanoparticle formulation was developed and applied in a drug targeting approach in an animal model. The high concentrations of nanoparticles achieved after rehydration with reduced volumes proved to be critical for the in vivo effect. Finally, short term storage stability under accelerated conditions was assessed for dried gelatin nanoparticles formulated in sucrose, trehalose, mannitol, or a mannitol/sucrose mixture. Size, size distribution, and residual moisture content were investigated. Sucrose- and trehalose-containing formulations exhibited the greatest stability, but mannitol-containing formulations also showed notable stabilization despite their crystalline nature.
Subject(s)
Drug Delivery Systems , Gelatin/chemistry , Nanoparticles/chemistry , Oligonucleotides/chemistry , Animals , Chemistry, Pharmaceutical , Freeze Drying , Lipopolysaccharides/pharmacology , Liver/metabolism , Male , NF-kappa B/metabolism , Oligonucleotides/administration & dosage , Rats , Rats, Sprague-Dawley , SuspensionsABSTRACT
PURPOSE: Cationized gelatin nanoparticles (GNPs) were used as carrier to improve delivery of immunostimulatory CpG oligonucleotides (CpG ODN) both in vitro and in vivo. METHODS: Uptake of CpG ODN-loaded cationized gelatin nanoparticles (CpG-GNPs) into murine myeloid dendritic cells (DCs) and their respective immunostimulatory activity was monitored. In vivo, induction of cytokine secretion by CpG-GNPs was measured. For experiments on primary human cells, prototypes of the three CpG ODN classes were adsorbed onto GNPs. Uptake and induction of proinflammatory cytokines were assessed in human plasmacytoid DCs and B cells, the only existing human target cells for CpG ODN. RESULTS: In the murine system, gelatin nanoparticle formulations enhanced the uptake and immunostimulatory activity of CpG ODN both in vitro and in vivo. Furthermore, delivery by cationized gelatin nanoparticles of CpG ODN of the classes B and C to primary human plasmacytoid DCs increased production of IFN-alpha, a key cytokine in the driving of both the innate and adaptive immune responses. CONCLUSION: GNPs can be used as a biodegradable and well tolerated carrier to deliver CpG ODN to their target cells and strongly increase activation of the immune system. This concept may be applied as novel adjuvant for antiviral and antitumoral vaccines.
Subject(s)
Adjuvants, Immunologic/pharmacology , B-Lymphocytes/drug effects , Dendritic Cells/drug effects , Drug Carriers , Gelatin/chemistry , Nanoparticles , Oligodeoxyribonucleotides/pharmacology , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/metabolism , Adult , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cations , Cells, Cultured , Chemistry, Pharmaceutical , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Drug Compounding , Female , Humans , Immunity, Innate/drug effects , Interferon-alpha/metabolism , Male , Mice , Mice, Inbred C57BL , Middle Aged , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Toll-Like Receptor 9/drug effectsABSTRACT
The PEGylation of colloidal drug carrier systems protects them from a rapid clearance from the blood stream and therefore prolongs their plasma half-lives. This fundamental concept is nowadays widely applied whereas the analytical description, i.e., the quantification of the PEGylation process, is still challenging due to the poor spectrophotometrical properties of PEG. The aim of this work is to quantify the PEGylation process of gelatin nanoparticles by utilizing the combination of asymmetrical flow field-flow fractionation (AF4) and refractive index (RI) detection and to demonstrate the potential of AF4 in the work with colloidal drug carrier systems. An AF4 separation mechanism of gelatin nanoparticles and PEG was developed without further sample preparation. After separation, the PEGylation could be directly quantified from the respective RI data and a threshold of a maximum amount of PEG that can be bound onto the surface of the nanoparticles could be determined. The PEGylation could be further visualized by atomic force microscopy (AFM). In sum, the presented results show the successful application of AF4 in the field of colloidal drug carrier systems, and in combination with AFM, both techniques can be stated as promising tools for the future analysis of colloidal drug carrier systems.
Subject(s)
Chemistry, Pharmaceutical , Drug Carriers/chemistry , Fractionation, Field Flow/methods , Gelatin/chemistry , Polyethylene Glycols/analysis , Refractometry/methods , Microscopy, Atomic Force/methods , Nanoparticles , Polyethylene Glycols/chemistryABSTRACT
PURPOSE: The aim of this study was to evaluate cationized gelatin nanoparticles as biodegradable and low cell toxic alternative carrier to existing DNA delivery systems. METHODS: Native gelatin nanoparticles were produced using a two step desolvation method. In order to bind DNA by electrostatic interactions onto the surface of the particles, the quaternary amine cholamine was covalently coupled to the particles. The modified nanoparticles were loaded with different amounts of plasmid in varying buffers and compared to polyethyleneimine-DNA complexes (PEI polyplexes) as gold standard. Transfection ability of the loaded nanoparticles was tested on B16 F10 cells. Additionally, the cell toxicity of the formulations was monitored. RESULTS: Different setups resulted in efficient gene delivery displayed by exponential increase of gene expression. The gene expression itself occurred with a certain delay after transfection. In contrast to PEI polyplexes, cationized gelatin nanoparticles almost did not show any significant cytotoxic effects. CONCLUSIONS: Cationized gelatin nanoparticles have shown the potential of being a new effective carrier for nonviral gene delivery. The major benefit of gelatin nanoparticles is not only the very low cell toxicity, but also their simple production combined with low costs and multiple modification opportunities offered by the matrix molecule.
