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.

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.

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.

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