on the excess of thiol groups. Biological safety of the polymers and 6-mercaptonicotinic acid was assessed and no harmful effects were found. Further supporting the earlier results, a prolonged release of ofloxacin was observed.
Mucoadhesive properties are largely defined by the secondary interactions between the mucin proteins and the mucoadhesive polymers. A part of the thesis work was devoted to gain a deeper understanding of mucoadhesive interactions and to assess the potential mucoadhesive properties of cationic polyaspartamide derivatives. Three cationic polyaspartamides with primary, secondary and tertiary amine groups were synthesized and an array of physicochemical methods (turbidimetric titration, dynamic light scattering, zeta potential measurement) were used to probe the interactions with porcine gastric mucin. It was found that the addition of cationic polyaspartamides cause the aggregation of mucin particles similar to the well-known mucoadhesive chitosan indicating a strong interaction. Further addition of the polymers caused disaggregation of mucin particles. It was proven that electrostatic interactions are dominant in the aggregation-disaggregation mechanism, and the strongest interaction was found in the case of the polyaspartamide with primary amines. These findings might help the design of strongly mucoadhesive, cationic and thiolated PASP derivatives in the continuation of the work.
The reverse direction of sol-gel transition, the stimuli-responsive degradation of the hydrogels also holds significant possibilities in the field of smart drug delivery and regenerative medicine. The presence of certain enzymes is a particularly promising stimulus due to their substrate specificity. A synthesis method was developed to prepare a trypsin-degradable poly(aspartic acid) hydrogel. The gel is chemically cross-linked by a tetrapeptide sequence containing a trypsin-specific cleavage site. The hydrogel degrades in the presence of trypsin and neither the polymer nor the cross-linker is cytotoxic according to the cellular assays performed. The encapsulated macromolecular drug is released from the gel in a sustained manner in the presence of trypsin, while the gel restrained the release of the drug in the absence of the enzyme.
The most important conclusions of this thesis can be briefly summarized in the following points (the corresponding papers are indicated for each thesis point):
1. I developed a new method to synthesize thiolated poly(aspartic acid) to avoid premature disulfide formation during synthesis. The method allows synthesizing polymer derivatives with a controlled and remarkably higher free thiol content compared to that of reported in the literature. The repeating units of the polymer were identified, and I confirmed that the oxidation of the thiol groups during synthesis was avoided. The sol-gel transition of the aqueous solutions of the polymers is triggered by chemical oxidation. I demonstrated that the gelation time of the gels shortens and their stiffness increases with increasing thiol content of the polymers. I proved that the thiolation increases the work and force of adhesion of the polymers measured on porcine conjunctive tissue owing to
the formation of a cohesive gel matrix. [1]
2. I synthesized a poly(aspartic acid) derivative that is modified with thiol and β-cyclodextrin moieties at the same time. I showed that the polymer is able to form an inclusion complex with lipophilic drugs such as prednisolone and the stability constant of the complex formation of the polymer do not differ significantly from that of the β-cyclodextrin small molecule. The β-β-cyclodextrin moieties did not hinder the sol-gel transition of the polymer. [2]
3. I developed a synthesis method to prepare S-protected thiolated poly(aspartic acid) derivatives by the conjugation of free thiol groups of the polymer with 6-mercaptonicotinic acid. I proved that the polymer is able to form covalent bonds with mucin protein by thiol-disulfide exchange reaction. I showed that the aqueous solution of S-protected poly(aspartic acid) yields cohesive, chemically cross-linked hydrogels upon the addition of the aqueous solution of thiolated poly(aspartic acid) with no additional reagents. I found that the hydrogels that contain no residual thiol groups are stable and reach their equilibrium swelling degree at physiological pH, while the presence of free thiols causes the dissolution of the hydrogels as a result of a series of thiol-disulfide exchange reactions [3]. I proved that ofloxacin as a water-soluble drug can be encapsulated into the poly(aspartic acid) hydrogel cross-linked with disulfides, and it is released with a sustained kinetics, which can be characterized with the Korsmeyer-Peppas model. The parameters of the model were found to be independent of the amount of chemical net-points [1,3].
4. I found that mucin particles aggregate in aqueous dispersion upon the addition of cationic polyaspartamides due to a bridging effect. Adding the cationic polyaspartamides in mass excess causes the disaggregation of the particles due to the cessation of polymer bridges between mucin particles and the complete coverage of the particles by polymer chains. I showed that the phenomenon is primarily caused by the electrostatic interaction between the positively charged side groups of polyaspartamides and mucin particles bearing a net negative charge. The strongest interaction was found in the case of the polyaspartamide with primary amine pendant groups. [4]
5. I developed the synthesis of a poly(aspartic acid) hydrogel chemically cross-linked with a tetrapeptide. The hydrogel consists exclusively of amino acid residues. The cross-linker peptide contains a trypsin-specific cleavage site causing the degradation (gel-sol transition) of the hydrogel in the presence of trypsin resulting in the release of the macromolecular drug encapsulated in the hydrogel. By choosing the appropriate cross-linker peptide sequence, the enzyme-specific degradation of the hydrogel can be achieved. [5]
List of publications
Publications used for the preparation of the Thesis
(IF: impact factor, IC: independent citation)
1. B.Á. Szilágyi, B. Gyarmati, G. Horvát, Á. Laki, M. Budai-Szűcs, E. Csányi, G.
Sandri, M.C. Bonferoni, A. Szilágyi, The effect of thiol content on the gelation and mucoadhesion of thiolated poly(aspartic acid), Polym. Int. 66 (2017) 1538–
1545. (IF 2017: 2,352, IC: 8)
2. M. Budai-Szűcs, E. L. Kiss, B.Á. Szilágyi, A. Szilágyi, B. Gyarmati, S. Berkó, A.
Kovács, G. Horvát, Z. Aigner, J. Soós, E. Csányi, Mucoadhesive cyclodextrin-modified thiolated poly(aspartic acid) as a potential ophthalmic drug delivery system, Polymers. 10 (2018). (IF 2018: 3,771, IC: 6)
3.
a) B. Á. Szilágyi, B. Gyarmati, E. L. Kiss, M. Budai-Szűcs, E. Csányi, A. Szilágyi, Thiolated and S-protected thiolated poly(aspartic acid) derivatives for in situ gelling drug delivery, 4th International Conference on Bio-Based Polymers and Composites, Balatonfüred, Hungary 2018, poster presentation
b) B.Á. Szilágyi, B. Gyarmati, E. L. Kiss, M. Budai-Szűcs, E. Csányi, A. Szilágyi In situ gelation of thiolated poly(aspartic acid) derivatives through oxidant-free disulfide formation for ophthalmic delivery submitted
4. B.Á. Szilágyi, A. Mammadova, B. Gyarmati, A. Szilágyi, Mucoadhesive interactions between synthetic polyaspartamides and porcine gastric mucin on the colloid size scale, Colloids Surf., B. 194 (2020) 111219. (IF 2019: 4,389, IC: 0) 5. B.Á. Szilágyi, Á. Némethy, A. Magyar, I. Szabó, S. Bősze, B. Gyarmati, A.
Szilágyi, Amino acid based polymer hydrogel with enzymatically degradable cross-links, React. Funct. Polym. 133 (2018) 21–28. (IF 2018: 3,074, IC:5)
Other publications
6. E. Krisch, B. Gyarmati, D. Barczikai, V. Lapeyre, B.Á. Szilágyi, V. Ravaine, A.
Szilágyi, Poly(aspartic acid) hydrogels showing reversible volume change upon redox stimulus, Eur. Polym. J. 105 (2018) 459–468. (IF 2018: 3,621 IC:5) 7. B. Gyarmati, B.Á. Szilágyi, A. Szilágyi, Reversible interactions in self-healing
and shape memory hydrogels, Eur. Polym. J. (2017) 642-669. (IF 2017:3,741 IC:35)
8. M. Budai-Szűcs, G. Horvát, B. Gyarmati, B.Á. Szilágyi, A. Szilágyi, S. Berkó, R.
Ambrus, P. Szabó-Révész, G. Sandri, M.C. Bonferoni, C. Caramella, E. Csányi, The effect of the antioxidant on the properties of thiolated poly(aspartic acid) polymers in aqueous ocular formulations, Eur. J. Pharm. Biopharm. 113 (2017)
178-187 (IF 2016:4,159 IC:3)
9. M. Budai-Szűcs, G. Horvát, B. Gyarmati, B.Á. Szilágyi, A. Szilágyi, T. Csihi, S.
Berkó, P. Szabó-Révész, M. Mori, G. Sandri, M.C. Bonferoni, C. Caramella, E.
Csányi, In vitro testing of thiolated poly(aspartic acid) from ophthalmic formulation aspects, Drug Dev. Ind. Pharm. 42 (2015) 1–6. (IF 2016:2,295 IC:3) 10. M. Budai-Szűcs, G. Horvát, B.Á. Szilágyi, B. Gyarmati, A. Szilágyi, S. Berkó, P.
Szabó-Révész, G. Sandri, M.C. Bonferoni, C. Caramella, J. Soós, A. Facskó, E.
Csányi, Cationic thiolated poly(aspartamide) polymer as a potential excipient for artificial tear formulations, J. Ophthalmol. (2016) Article ID 2647264.
(IF 2016:1,712 IC:3)
11. G. Horvát, B. Gyarmati, S. Berkó, P. Szabó-Révész, B.Á. Szilágyi, A. Szilágyi, J.
Soós, G. Sandri, M.C. Bonferoni, S. Rossi, F. Ferrari, C. Caramella, E. Csányi, M.
Budai-Szűcs, Thiolated poly(aspartic acid) as potential in situ gelling, ocular mucoadhesive drug delivery system, Eur. J. Pharm. Sci. 67 (2015) 1-11.
(IF 2015:3,773 IC:42)
12. G. Horvát, M. Budai-Szűcs, S. Berkó, P. Szabó-Révész, B. Gyarmati, B.Á.
Szilágyi, A. Szilágyi, E. Csányi, Application of in situ gelling mucoadhesive thiolated poly(aspartic acid) in ophthalmic drug delivery Acta Pharm. Hung. 85 (2014) 115-121 (IF - IC:-)
Conference presentations
Oral presentations as presenting author
1. B. Á. Szilágyi, B. Gyarmati, G. Horvát, M. Budai-Szűcs, E. Csányi, A. Szilágyi, Effect of thiol content on mucoadhesive character of thiolated poly(aspartic acid)s, 3rd International Conference on Bio-Based Polymers and Composites, Szeged, Hungary, 2016
2. B. Á. Szilágyi, Enzimatikusan lebomló poliaszparaginsav hidrogélek szintézise és vizsgálata, XXXVI. Kémiai Előadói Napok, Szeged, Hungary, 2013
3. B. Á. Szilágyi, Á. Némethy. A. Magyar, A. Szilágyi, Enzimatikus úton lebomló poliaminosav gélek szintézise és vizsgálata / Synthesis and study of enzymatically degradable polyaminoacid gels, 17th International Conference on Chemistry:
XVII. Nemzetközi Vegyészkonferencia. Cluj, Romania, 2011 Additional oral presentations
4. E. L. Kiss, M. Budai-Szűcs, B. Á. Szilágyi, A. Szilágyi, B. Gyarmati, E. Csányi, Cyclodextrin-modified thiolated poly(aspartic acid) as a potential mucoadhesive ophthalmic drug delivery, 4th International Conference on Bio-Based Polymers and Composites, Balatonfüred, Hungary, 2018
5. Budai-Szűcs, G. Horvát, T. Csihi, Sz. Berkó, B. Gyarmati, B. Á. Szilágyi, A.
Szilágyi, G. Sandri, M. C. Bonferoni, C. Caramella, E. Csányi, Development of
thiolated poly(aspartic acid) from ophthalmic formulation aspects, 3rd International Conference on Bio-Based Polymers and Composites, Szeged, Hungary, 2016
6. B. Gyarmati, B. Á. Szilágyi, G. Horvát, M. Budai-Szűcs, E. Csányi, A. Szilágyi, In situ gelling poly(aspartic acid)s for pharmaceutical applications, 16.
Österreichische Chemietage, Innsbruck, Austria, 2015
7. B. Gyarmati, B. Á. Szilágyi, A. Szilágyi, Poly(aspartic acid) hydrogels with reversible response to redox stimulus, 2nd International Conference on Bio-based Polymers and Composites, Visegrád, Hungary, 2014
8. M. Budai-Szűcs, B. Gyarmati, G. Horvát, Sz. Berkó, P. Szabó-Révész, B. Á.
Szilágyi, G. Sandri, M. C. Bonferoni, C. Caramella, A. Szilágyi, E. Csányi, In situ gelling mucoadhesive drug delivery system for ophthalmic use, 2nd International Conference on Bio-based Polymers and Composites, Visegrád, Hungary, 2014 9. B. Á. Szilágyi, Á. Némethy, B. Gyarmati, I. Szabó, Sz. Bősze, A. Magyar, A.
Szilágyi, Enzymatically degradable poly(amino acid) gels, 15. Österreichische Chemietage, Graz, Austria, 2013
Poster presentations
10. B. Á. Szilágyi, B. Gyarmati, G. Horvát, M. Budai-Szűcs, E. Csányi, A. Szilágyi, Highly thiolated and S-protected thiolated poly(aspartic acid) derivatives as in situ gelling ophthalmic drug delivery system, 30th Conference of the European Colloid and Interface Society, Rome, Italy, 2016
11. B. Á. Szilágyi, B. Gyarmati, G. Horvát, M. Budai-Szűcs, E. Csányi, A. Szilágyi, Thiolated poly(aspartic acid): an in situ gelling mucoadhesive polymer, 16.
Österreichische Chemietage, Innsbruck, Austria, 2015
12. K. Solti, B. Á. Szilágyi, B. Gyarmati, A. Szilágyi, Solvent-induced shape memory of poly(N-isopropylacrylamide)-Laponit® nanocomposite gels, 2nd International Conference on Bio-based Polymers and Composites, Visegrád, Hungary, 2014 13. B. Á. Szilágyi, Á. Némethy, A. Magyar, A. Szilágyi, Synthesis and study of
enzyme-responsive amino acid-based hydrogels, International Symposium on Advanced Macromolecular Systems Across the Length Scales - AMSALS 2012, Siófok, Hungary, 2012
Acknowledgements
I would like to thank my supervisor, András Szilágyi for supporting my studies and research. I am grateful to Benjámin Gyarmati for his valuable advice.
I would like to thank all colleagues and students at Soft Matters Group especially Enikő Molnárné Krisch, Csaba Németh, and Katalin Solti for their friendship, help and the fun and inspiring working environment they created.
I am grateful to the colleagues at the Department of Physical Chemistry and Materials Science. I owe my thanks to Béla Pukánszky for his advice during my work and Dávid Kun for his help with statistical analysis
I very much appreciate the work of all my co-authors especially Mária Budai-Szűcs, Gabriella Horvát, Eszter L. Kiss and Erzsébet Csányi at the Institute of Pharmaceutical Technology and Regulatory Affairs, University of Szeged; Anna Magyar, Szilvia Bősze, and Ildikó Szabó at MTA-ELTE Research Group of Peptide Chemistry.
I appreciate the help of Dániel Szekrényes, Szilárd Pothorszky, and András Deák at the Institute of Technical Physics and Materials Science, Hungarian Academy of Sciences with the DLS measurements.
I apologize to my family and friends for all the time I have not spent with them because of writing this thesis. I can only hope that it was not in vain. I cannot be thankful enough that they – especially my wife and my mother – supported me and endured my never-ending whining in the last not-so-few years.