Intramuscular delivery is one of the main routes for DNA vaccines administration. However, it requires large amounts of the DNA to be administered and external stimulation to encourage the internalization of the DNA. In this work, we consider alternative routes for less invasive administration and develop drug delivery systems (DDS) for intranasal administration. Chitosan polyplexes using sodium tripolyphosphate (TPP) as a crosslinker were prepared using the ionic gelation method. Our method allowed preparation of nanoparticles with the size below 50 nm, which is at least two times lower than previously reported sizes. Moreover, despite the small sizes, we obtained DNA encapsulation efficiencies of about 70%. Parameters that may affect the encapsulation efficiency were investigated, including different TPP-chitosan ratios and concentrations of DNA. We found that encapsulation efficiency of DNA inside the particles decreases with the increasing TPP-chitosan ratio. Moreover, increasing the DNA concentration leads to a higher encapsulation efficiency. Small (<50 nm) chitosan nanoparticles hold enormous potential as DNA carriers due to their physiological barriers and subsequent internalization.

Conventional bivalent IgG antibodies targeting a subgroup of receptors of the TNF superfamily (TNFSF) including fibroblast growth factor-inducible 14 (anti-Fn14) typically display no or only very limited agonistic activity on their own and can only trigger receptor signaling by crosslinking or when bound to Fcγ receptors (FcγR). Both result in proximity of multiple antibody-bound TNFRSF receptor (TNFR) molecules, which enables engagement of TNFR-associated signaling pathways. Here, we have linked anti-Fn14 antibodies to gold nanoparticles to mimic the “activating” effect of plasma membrane-presented FcγR-anchored anti-Fn14 antibodies. We functionalized gold nanoparticles with poly-ethylene glycol (PEG) linkers and then coupled antibodies to the PEG surface of the nanoparticles. We found that Fn14 binding of the anti-Fn14 antibodies PDL192 and 5B6 is preserved upon attachment to the nanoparticles. More importantly, the gold nanoparticle-presented anti-Fn14 antibody molecules displayed strong agonistic activity. Our results suggest that conjugation of monoclonal anti-TNFR antibodies to gold nanoparticles can be exploited to uncover their latent agonism, e.g., for immunotherapeutic applications.

Immunotherapy is considered as a new pillar of cancer treatment. However, the application of some promising immunotherapeutic antibodies, such as antibodies against certain immune-stimulatory receptors of the TNF receptor superfamily (TNFRs), including CD40, 41BB, CD27 and anti-fibroblast growth factor-inducible 14 (anti-Fn14), are limited due to their low bioactivity. It has been previously shown that the bioactivity of such anti-TNFR antibodies could be improved by crosslinking or attachment to the plasma membrane by interaction with Fcγ receptors (FcγR). Both result in the proximity of multiple antibody-bound TNFR molecules, which allow for the activation of proinflammatory signaling pathways. In this work, we have grafted antibodies on gold nanoparticles to simulate the “activating” effect of FcγR-bound, and thus plasma membrane-presented anti-TNFR antibodies. We have developed and optimized the method for the preparation of gold nanoparticles, their functionalization with poly-ethylene glycol (PEG) linkers, and grafting of antibodies on the surface. We showed here that antibodies, including the anti-Fn14 antibody PDL192, can be successfully attached to nanoparticles without affecting antigen binding. We hypothesize that conjugation of monoclonal anti-TNFR antibodies to the inorganic nanoparticles is a promising technique to boost the efficacy of these immunotherapeutic antibodies.