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Tasks - WP1

1. Characterisation and optimization of the properties of the polysaccharide building blocks (chitosan and hyaluronic acid).
   Team: Dr. V. Milkova, Dr. K. Kamburova
2. Development and optimization of the procedure for synthesis of O/W emulsion (microfluidic approach) and their functionalization through drug incorporation.
   Team: Dr. V. Milkova, Dr. K. Kamburova
3. Design of shell-core capsules (layer-by-layer approach) through subsequent electrostatic adsorption of oppositely charged chitosan and hyaluronic acid on produced drug loaded O/W emulsion droplets.
   Team: Dr. V. Milkova, Dr. K. Kamburova
4. Expected results from WP1: Development of stable functional microformulations from chitosan and hyaluronic acid able to co-encapsulate and co-deliver more than one type of payload (aptamer and therapeutic agent) and protect them from chemical or enzymatic degradation at physiological conditions.
5. Current results (first and second quarters): Our studies so far are focused on the characterization and the optimization of the properties of the building blocks (polysaccharides chitosan and hyaluronic acid) in regard to their ability to form stable structures, their cytotoxicity and antiviral activity against coronavirus. A preliminary analysis was performed over the dependency of the properties of polymer coated particles on the physicochemical characteristics of the chitosan (molecular weight and degree of acetylation).
   To this end, a procedure for obtaining of stable complexes curcumin/chitosan was developed. In addition, complexes of the type shell-core are formed by an electrostatic adsorption of hyaluronic acid on the mentioned structures. The conducted electrokinetic studies showed an existence of dependency of the electric properties and the stability of the complexes on the concentration and the molecular weight of the polysaccharides, as well as on the experimental conditions (pH and ionic strength). The shape and the size of the structures are visualized by using transmission electron microscopy (TEM).
   A selection and synthesis of an aptamer, which is suitable for the purpose of the investigation, was performed. Preliminary experiments on the obtaining and the characterization of the chitosan/aptamer complexes are carried out.

Tasks - WP2

1. Characterization of surface properties and internal structure of the produced polysaccharide-based delivery platforms.
   Team: Dr. S. Vladimirova, Dr. K. Kamburova, I. Piroeva
2. Estimation of the encapsulation efficiency (EE%), kinetics of release of selected therapeutics and capsule stability at physiological conditions.
   Team: Dr. V. Milkova, Dr. K. Kamburova
3. Expected results from WP2: Development of fully characterized polysaccharide-based drug delivery platforms for combined aptamer-therapeutic delivery.
4. Current results (first and second quarters): An analysis of the structure and the size of curcumin/chitosan complexes was made by Light Scattering in electric field, Dynamic Light Scattering (DLS) and TEM.

Tasks - WP3

1. Model investigation of the interactions between selected loaded capsules (from WP 1 and WP 2) and spike glycoproteins involved in a lipid bilayer.
   Team: Dr. V. Milkova, Dr. I. Dimitrov, Dr. A. Gyurova
2. Expected results from WP3: To obtain more information about the mechanism of interaction between the proteins and aptamer. To perform model trial of the working hypothesis laid in the concept of the proposal. The results will provide feed-back to the optimization strategies in WP 1 and WP 4.
3. Current results: check for updates

Tasks - WP 4

1. Examination of the activity of the selected substances on diploid cell culture MRC-5 (Medical Research Council cell strain 5), and determination of their cytotoxic concentration 50 (CC50).
   Team: Dr. N. Vilhelmova-Ilieva, V. Rashev
2. Study of the effect of synthetic products and natural extracts on the replication of HCV/229E in MRC-5 cells.
   Team: Dr. N. Vilhelmova-Ilieva, V. Rashev
3. Expected results from WP4: To obtain information on the cytotoxicity of the substances on the basis of which the following studies will be performed in doses that will be non-toxic to MRC-5 cells. The presence of anti-coronavirus activity of the selected therapeutics is evidence that they are specific inhibitors of human coronavirus strain 229E replication and could be used as therapeutics in coronavirus infection.
4. Current results (first and second quarters): Studies were conducted for evaluation of the cytotoxicity of the selected polysaccharides and curcumin/chitosan complexes. In this regard, the conditions for growing cell culture lines MRC-5 and HCT-8, as well as the conditions for the replication of the human coronavirus strain HCoV-229E (in cell culture line MRC-5) and the strain HCoV-OC43 (in cell culture line HCT-8) were optimized. It was found out that the solutions of chitosan prepared with participation of acetic acid possess higher cytotoxicity than the ones with hydrochloric acid. It was also established that the increase of chitosan’s molecular weight causes a raise of its cytotoxicity and of the toxicity of the corresponding complexes.

Tasks - WP 5

1. Determination of the effect of co-encapsulated aptamer on extracellular virions and cell membrane during viral adsorption.
   Team: Dr. N. Vilhelmova-Ilieva, Dr. P. Grozdanov, Dr. J. Abashev, V. Rashev
2. Determination of the combined effect of co-encapsulated aptamer and potential drug intended for the treatment of COVID-19.
   Team: Dr. N. Vilhelmova-Ilieva, Dr. P. Grozdanov, Dr. J. Abashev, V. Rashev
3. Expected results from WP5: The specific structure of the aptamers should interact with the envelope of HCV/229E and cause blockage of the viral particle even before contact with sensitive cells, as well as interacting with the cell membrane to make it impermeable to the virus and the viral invasion to be stopped in early stage.
4. Current results (first and second quarters): A comparative analysis was performed on the antiviral activity of the polysaccharides and the complexes against the replication of the human coronavirus strains HCoV-229E and HCoV-OC43 in a suitable cell culture (MRC-5 or HCT-8). The results show that from all the substances studied, a significant antiviral activity against the two human coronavirus strains (HCoV-229E and HCoV-OC43) is registered only in case of the chitosan with the lowest molecular weight, and the curcumin and the hyaluronic acid possess a weak activity.

Tasks - WP 6

1. Determination of the degree of oxidative stress in diploid cell culture MPC-5 in a state of coronavirus infection.
   Team: Dr. M. Mileva, Dr. A. Georgieva, Dr. E. Tsvetanova, A. Dimitrova
2. Determination of the degree of control of oxidative stress in human coronavirus strain 229E against cells MPC-5 after treatment with the selected loaded capsules.
   Team: Dr. M. Mileva, Dr. A. Georgieva, Dr. E. Tsvetanova, A. Dimitrova
3. Expected results from WP6: Reduction of the level of oxidative stress as a result of application of the aptamer-therapeutic loaded capsules.
4. Current results: check for updates

Tasks - WP 7

1. Team: All partners in the project
2. Expected results from WP7: Dissemination of the innovation results of the project.
3. Current results (first and second quarters): A website of the project was developed presenting the most important information about the progress so far.

Tasks - WP 8

1. Team: Dr. V. Milkova ( IPC-BAS ), Dr. N. Vilhelmova-Ilieva ( IMB-BAS )
2. Expected results from WP8: Administrative and financial aspects of managing of the project.
3. Current results (first and second quarters): A scientific report was prepared for the current progress for the first and the second quarters of the project, formally accepted by Bulgarian National Science Fund. An oral presentation was given at the colloquium “A. Scheludko”, IPC-BAS, which pointed out the purpose, the main directions and the approaches for fulfilling the tasks set in the project, some experimental results obtained during the first quarter of the work program were shown either. A financial report for the first six months of the project implementation was prepared. Regular meetings and discussions (real or virtual) were organized for the members of the project team.