Drugs based on oligoribonucleotides from yeast RNA have a reasonably low solubility in the range of 0.5-3 mg/ml. Our previous studies have shown that ORN has anti-inflammatory properties, and ORN with D-mannitol has anti-inflammatory and antiviral properties. The mechanism of this phenomenon is unclear. D-mannitol itself does not have these activities, but increases the solubility by 1.5-2 times and stabilises the structure of the ORN, which probably leads to new biological effects. In the course of our research, we found that D-mannitol affects the binding of ORN to interferon and the thermodynamic and conformational parameters of this interaction .

We studied the ability of yeast RNA (ORN), yeast RNANa salt (ORN Na), and yeast ORN: D-mannitol complex (ORN: D-M) and AMP to effect on fluorescence quenching and conformational changes of Interferon α2b. To determine the energy parameters of protein-ligand interactions, we use isothermal titration nanocalorimetry Nano ITC.

It is showing that when using ORN and ORN: D-M quenching of the fluorescent INF were 25% and 28%, AMP and AMP: DM - 15 and 21%. Quenching INF fluorescence in the titration of ORNsNa and ORNsNa: DM was 16% and 17%, AMPNa and AMPNa: DM - 8% and 10%. INF has a life-time of 2.95 ns. When interacting with ORN and ORN: D-M INF has fluorescence time of 2.37 and 2.32 ns, respectively, AMP and AMP: D-M 2.01 and 1.92 ns. When interacting with ORNsNa and ORNsNa: D-M INF has a fluorescence time of 2.73 and 2.49 ns, respectively, AMPNa and AMPNa: D-M 2.31 and 2.43 ns. Thus, ORN, and especially ORN: D-M and AMP: D-M leads to a change in the conformational mobility of interferon α-2b by increasing the content of disordered regions. At the same time, salt analogues increase the number of structured secondary elements, such as α-helices, β turns and β antiparallel sheets and probably increase the conformational stiffness of interferon α-2b. The results of the study of enthalpy changes in the titration of interferon α-2b acid form of ORN and ORN: D-M was -63.28 kJ/mol and -96.61 kJ/mol, respectively, and for the ORNNa and ORNNa: D-M respectively 4,516 and 5,139 kJ/mol. The change in entropy when adding the ORN to interferon α-2b was -38.72 and in the case of the ORN: D-M -63.53 kJ/mol*K, respectively. The difference in entropy when adding the ORNNa to interferon α-2b was 17.05 kJ/mol*K, and the ORNNa: D-M, respectively, 17.58 kJ/mol*K. A similar pattern demonstrated when studying the change in Gibbs energy during titration of interferon α-2b with ORNORN and ORN: D-M, and it was -24.56 and -33.07 kJ/mol, respectively. And when titrated with ORNNa and its ORNNa: D-M, respectively -12.9 and -12.43 kJ/mol.

These results of studying the effects of thermodynamics of different forms of RNA and their complexes with D-mannitol in the titration of interferon α-2b may indicate different sites of binding of different forms of ORN to protein, as well as other modes of binding and various types of conformational changes in the protein.

Immunotherapy is considered to be one of the most promising cancer treatments. However, applications of some promising immunotherapeutic, such as anti-fibroblast growth factor-inducible 14 (anti-Fn14), are limited due to their low bioactivity. Anti-Fn14 antibodies mimic TWEAK, a member of the tumor necrosis factor superfamily TNFSF ligands, which stimulate Fn14 (TNFRSF) which results in the induction of necrotic and apoptotic cell death.

It has previously shown that the bioactivity of such antibodies, could be improved by their crosslinking, that is immobilization of multiple antibodies in close proximity. In this work, we have developed a method to increase the activity of the anti-Fn14 antibodies by physical cross-linking on gold nanoparticles. We have developed and optimized the method for the preparation of gold nanoparticles, their functionalization with poly-ethylene glycol (PEG) linkers, and grafting of the antibodies on the surface.

We showed that antibodies can be successfully attached to nanoparticles without affecting their activity. Most importantly, the bioactivity measured of the grafted antibodies was increased in comparison to non-grafted antibodies as verified by the triggering HT-1080 cell line to produce the inflammatory cytokine IL-8 which was characterized by ELISA assay.

Our results suggest that conjugation of monoclonal IgG1 antibody on the inorganic nanoparticles is a very promising technique to boost the efficacy of the immunotherapeutic.

FUNDING:

This project has received funding from the European Union´s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 813871.

REFERENCES:

1. Medler, J. et al. (2019) ‘TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity’, Cell Death and Disease. Springer US, 10(3). doi: 10.1038/s41419-019-1456-x.
2. Kelley, S. K. et al. (2001) Preclinical Studies to Predict the Disposition of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Humans: Characterization of in Vivo Efficacy, Pharmacokinetics, and Safety. Available at: http://jpet.aspetjournals.org (Accessed: 19 September 2019).
3. Alerts, E. (2019) ‘Cachectin / tumor necrosis factor : production , distribution , and metabolic fate in vivo . Information about subscribing to The Journal of Immunology is online at : METABOLIC FATE IN VIVO ’’.
4. Winkles JA. The TWEAK-Fn14 cytokine-receptor axis: Discovery, biology and therapeutic targeting. Nat Rev Drug Discov. 2008;7(5):411–25.

Electrospinning is a powerful and cost-effective method that applies a strong electric field and polymer solution to fabricate versatile nanofiber using a wide array of raw materials for different biological applications. Electrospun nanofibers have developed as potential materials for the manufacturing of nanocarriers for therapeutic platforms due to controllable surface decoration, large surface areas, complex pore structure, good surface functionalization with high biocompatibility. The prominence of this technique is the possibility of merging low soluble drugs loaded in the nanofibers to enhance drug release and bioavailability. Incorporation of metal nanomaterials with electrocatalytic and/or plasmonic features into electrospun polymer nanofiber enhances thermodynamic and mechanical properties to obtain synergetic function and properties. This review discussed an overview on the theory of electrospinning, achievements and concerns, and challenges currently face in fabricating an efficient drug delivery system. Additionally, the drug-loaded into electrospun nanocarriers for drug delivery applications and opportunities, and insights into future prospects were explained.

Having started my career in pharmaceutical industry, my research focuses on the control and use of solid-state forms of API. The solid state of API has an important impact on properties of solid drug forms such as tablets, influencing hygroscopicity, biodisponibility. My research focuses on engineering the solid-state of API adding multiple components to the same solid phase. We showed how multi-component crystallization (co-crystallization) can be used to control the solid-state properties of drug compounds (hygroscopicity, melting point stabilization, …)¹ but can be used as well as to expand the patent life-time of a given drug.² In parallel, we also specialize in developing multi-component crystallization processes, upscaling it to the kg scale.³ Doing so, requires a careful control of underlying thermodynamics and kinetics. In a final part of my research, we go one step further, using the specific properties of multi-component systems to develop novel crystallization-based applications. In particular my group has developed a novel type of resolution based on enantiospecific co-crystallization from solution.⁴ We recently expanded this to resolve mandelic acid using preferential co-crystallization.⁵ In our latest ground-breaking work, we used the underlying thermodynamics of these systems, to pull them towards a transformation of the racemate into an enantiopure drug in a so-called deracemization process.⁶

Rosuvastatin calcium (Rsv) is one of the most recent and effective statins, with a potent antihyperlipidemic effect. However; it suffers poor bioavailability owing to its poor solubility. Thus; encapsulating Rsv into a nanovesicular structure could overcome this problem. The aim of this work is to investigate the potential of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in enhancing the solubility of Rsv, using Quality by Design (QbD) concept to develop a better quality product. A complete risk assessment study has been conducted, where the critical process parameters (CPP), material attributes (MA), and critical quality attributes have been identified using the ishikawa diagrams. Selected CPP/MA were screened and further upgraded to a 2⁴ full factorial design to develop the design space with the optimized formula. The screened CPP/MA were tested on each of the particle size, polydispersity index (PDI), zeta potential and the entrapment efficiency (EE%). A comprehensive approach for Rsv nanovesicular carriers has been conducted, where the NLC showed better results than the SLN. The optimized formula was prepared with 3 % total lipid content, 0.154% surfactant, and 9.4 mg drug. The optimized formula had a particle size 310.5 nm, with 0.243 PDI, a zeta potential of -24.7 mV and EE% of 93.87%, and showed a sustained release of the drug up to 72 hours. It successfully lowered each of the total cholesterol, low density lipoprotein, and triglycerides and elevated the level of the high density lipoprotein of high fat fructose diet-induced hyperlipidemia/hypercholesterolemia rats, with better results as compared to the standard drug. Thus, a complete QbD study was conducted to explore experimental regions for many successful nanovesicular carriers for the enhancement of the solubility of poorly soluble drugs.