Search

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.

Cancer is the second leading cause of death in the world and is often untreatable. Protein-based therapeutics, such as immunotherapeutics, show promising results in the fight against cancer, resulting in their market share increasing every year. Unfortunately, most protein-based therapeutics suffer from fast degradation in the blood, making effective treatment expensive, causing more off-target effects (due to the high doses necessary), and often require repeated injections to stay within the correct therapeutic range. Encapsulation of these proteins inside nanocarriers is prompted to overcome these problems by enhancing targeted drug delivery and, thus, leading to a less frequent administration and lower required dose. However, most current protein encapsulation methods show very low loading capacities (LC). This leads to even more expensive treatments and might pose a further risk for the patient caused by systemic toxicity against high concentrations of the carrier material. We investigated and optimized protein nanoprecipitation as a method to obtain a high protein LC and encapsulation efficiency (EE) inside poly(lactic-co-glycolic acid; PLGA) nanoparticles via a simple two-step process. In this work, we used model proteins to investigate the influence of various parameters such as precipitation solvent, addition speed, and protein concentration on protein activity. Our work is a critical step towards the high-loading encapsulation of immunotherapeutics.

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.

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 (

High-throughput emulsion electrospinning is a technology that can enable practical nanofiber application for drug delivery. Core-shell structure of the electrospun fibers allows the encapsulation of the active pharmaceutical ingredients (APIs), protects their activity, and controls their release rate. However, electrospinning using high flow rates usually requires high electric fields that may negatively affect the activity of the biomolecules. Moreover charged APIs tend to migrate to the surface of the fibers during the electrospinning process leading to the high burst release. That is disadvantageous when long-term sustained release is needed. We have investigated the influence of the electrospinning parameters such as distances between the electrode and collector and the applied voltages to both, activity of the encapsulated proteins and their burst release. We have also tested the influence of number of the stabilizers, e.g. trehalose, pluronic, polyvinylpyrrolidone, on their ability to preserve the protein activity, and the influence of the different molecular weights of polyvinyl alcohol on the ability to sustain the release. Our results demonstrate the importance of the water phase composition to both activity and release and are critical for further understanding of the processes taking place during the emulsion electrospinning.

Nowadays, an electrospinning process is one of the most promising technique to produce nanofibers. The popularity of this method is still significant, due to its simplicity and relatively low cost. Moreover, many electrospun compounds can be used in numerous applications in different areas, for instance medical and pharmaceutical fields, textile engineering, environmental remediation. However, a design of an efficient electrospinning system remains challenging, due to high number of parameters strongly affecting fibers properties and productivity of their formation. Lately, a great attention is given to development of needleless configurations, characterized by multi-jets system, thanks to their enhanced output. Indeed, a surface of spinneret, where the liquid jet formation is initiated, plays crucial role to improve the process. The electric field profile of the spinning electrode directly influences the size and uniformity of the produced fibers and should be strongly considered in further development. Our study presents a comprehensive theoretical analysis of the most effective needleless electrospinning configurations and compares their electrostatic features. Our extensive analysis was prepared in COMSOL Multiphysics software, known as a great tool to describe in details physical phenomena and it can strongly help to select the proper electrospinning set up, depending on desired results.

Among the most promising technologies for sustained drug delivery systems are core-shell nanofibres prepared by electrospinning. However, the most common method for the production of those, coaxial electrospinning, suffers from extremely low flow rates limiting the practical applications of such fibres. Emulsion electrospinning, on the other hand, enables the use of the high-throughput needle-less electrospinning devices for the production of the core-shell nanofibres with active pharmaceutical ingredients embedded and protected in their core. The development of such drug delivery systems for the applications in bone regeneration is further challenged by the need for the inorganic additives meant to stimulate the regeneration of the bone. The main objective of this work is to develop a high-throughput electrospinning method for the production of hybrid (organic-inorganic) and bioactive scaffolds needed for bone regeneration. We demonstrate the importance of the formulation, e.g., presence of surfactants, on the stability of the emulsion, and thus on electrospinning. Our work is an important step forward towards the high-throughput production of complex multi-material scaffolds for sustained drug delivery.