PROJECT CODE: PN-III-P2-2.1-PED-2019-2638, contract number 272PED ⁄ 2020
Biocatalytic synthesis of new polyesteramides as nanocarriers for bioactive compounds
Procedeu biocatalitic pentru sinteza de noi poliesteramide ca nanosuporturi pentru compuși bioactivi
PROJECT COORDINATOR: Politehnica University of Timişoara
PROJECT DIRECTOR: Prof. Dr. Eng. Francisc Peter

BUDGET: 600.000 RON (Public budget)
START TIME: 03/08/2020
END TIME: 31/07/2022
Industrial biotechnology using isolated enzymes has emerged as a viable alternative for the synthesis of high added-value products, including polymeric materials. Amino acids could represent important bio-based raw-materials for the synthesis of new polymers due to their universal availability, low price and biodegradability.
The aim of this project is to develop the experimental demonstration model for an efficient and biobased route for biocatalytic synthesis of new polyesteramides, starting from amino acids and ɛ-caprolactone, or hydroxy acids and ɛ-caprolactam, using green solvents. The enzyme stabilization will be performed by substrate-directed immobilization methods, including covalent binding onto magnetic particles and magnetic sol-gel entrapment. The selectivity of different lipases, proteases and esterases will be evaluated in terms of catalytic efficiency, to increase the productivity of the process. Several amino acids and hydroxy acids will be tested, and the optimal reactions conditions will be determined by experimental design. The structural analysis and assessment of the physico-chemical properties of the reaction products will be accomplished by advanced analytical techniques. The synthesized oligoesters will be used as starting materials for novel nanoparticles and bioactive compounds carriers.

Project leader: Prof. Dr. Eng. Francisc Peter
Ș.l. dr. ing. Anamaria Todea
Ș.l. dr. ing. Cristina Paul
Ș.l. dr. ing. Iulia Maria Păușescu
Ș.l. dr. ing. Valentin Badea
Dr. Ing. Emese Biro
Postdoctoral researchers:
Asist. Dr. chim. Diana Maria Aparaschivei
PhD Students:
Drd. Ing. Ioana Cristina Benea
Drd. Ing. Ionut-Mihai Tănase

OBJECTIVE: The main objective of the project is to develop a demonstration model of a new biocatalytic approach to synthesize polyesteramides based on renewable sources and suitable as nanosized carriers for bioactive compounds.

Stage 1 (2020, 5 months)- Enzymatic synthesis of novel oligoesteramides/ polyesteramides of epsilon-caprolactone and epsilon-caprolactam

Stage 2 (2021, 12 months)- Optimization of polyesteramide synthesis using an immobilized enzyme, reuse of the biocatalyst and product characterization

Stage 3 (2022, 7 months)- Study of biodegradation and use of polyesteramide-based nanoparticles.

Stage 1
Experimental protocol of enzymatic synthesis;
Methodology for identifying the components of the polymerization product by MALDI-TOF MS and NMR;
Immobilization protocol for each of the type of studied enzymes;
Quantities of the order of grams of the immobilized enzyme with the highest activity;
A scientific paper sent for publication
Stage 1 report.
Stage 2
Experimental protocol for the synthesis of at least three types of polyesteramides;
Laboratory methodology for the characterization of polyesteramides;
Targeted immobilization protocol and characterization of a selected enzyme;
Quantities of the order of grams of the immobilized enzyme with the highest activity;
Validated experimental design protocol;
Quantities of the order of grams of the oligoester with the optimal composition;
Experimental protocol for obtaining nanoparticles by different techniques;
A paper submitted for publication in a journal;
Abstracts for two presentations at scientific conferences in the field.
Stage 2 report.
Stage 3
Experimental protocol for biodegradation evaluation;
Gram quantities of the nanoparticles obtained;
Experimental methodology for nanoparticle characterization;
Laboratory methodology for the kinetic study of the controlled release of a selected bioactive compound;
A paper sent for publication in the journal
Stage 3 report
Final report.


Abstract Stage 1 (2020)

The main scientific objectives of this stage were (i) preliminary study of the enzymatic synthesis of oligo/ polyesteramides and oligo/ polyesters; (ii) characterization of the obtained products; (iii) stabilization study of enzymes with possible uses as biocatalysts in the synthesis of polyesters and polyesteramides. For the synthesis of polyesteramides and polyesters, three reaction systems with one or two monomers were investigated, using as biocatalysts native and immobilized lipases from various microbial sources, along with an esterase and a protease. The monomers used were ɛ-caprolactam and ɛ-caprolactone, in combination with hydroxy acids and amino acids, respectively. The effect of several reaction parameters was studied: molar ratio of monomers, reaction medium, temperature, etc. The reaction products were characterized by MALDI-TOF MS spectrometry, FT-IR spectroscopy, and thermal analysis (TG and DSC). Oligo-esteramides and oligoesters of ɛ-caprolactone with L-arginine and malic acid were obtained for the first time during this project. The immobilization of Candida antarctica B lipase has been directed towards the sol-gel technique, which offers great operational flexibility and high retention of enzymatic activity but requires fine-tuning of the properties of the biocatalyst depending on the reaction system. The sol-gel immobilization methodology was developed for both binary and ternary systems of precursor silanes, to improve the availability of the biocatalyst in the synthesis of high molecular weight products. Based on these results, two manuscripts were prepared for submission in Web of Science - ranked scientific journals. Overall, the objectives of this stage have been accomplished.


Abstract Stage 2 (2021)

In the second stage, the studies initiated in Stage 1 (2020) were continued regarding (i) synthesis of oligoesteramides and oligoesters by an enzymatic route; (ii) characterization of the obtained products; (iii) the study of the stabilization of selected enzymes by immobilization using the sol-gel technique with or without combination with adsorption. The activities initiated in this stage were (iv) the optimization of the synthesis of polyesteramides using an experimental factorial program; (v) studies of the biodegradability of polyesteramides; (vi) production of polyesteramide nanoparticles. For the synthesis of polyesteramides and polyesters, five reaction systems with two monomers were investigated, using immobilized lipases from various microbial sources as biocatalysts. The effect of several reaction parameters was studied, including molar ratio of the monomers, reaction medium, and temperature. The reaction products were characterized by MALDI-TOF MS spectrometry, FT-IR spectroscopy, TG and DSC thermal analysis, demonstrating the formation of oligomers and their thermal stability. All these products are innovative, as they were not previously reported in the scientific literature. The immobilization study of Candida antarctica B lipase was continued by entrapment in sol-gel matrices using a precursor that also allows covalent binding. The sol-gel immobilization methodology has been developed for binary precursor systems, as well as for the combined immobilization method with adsorption on magnetic particles. The activity of the obtained biocatalysts was evaluated comparatively for the synthesis of a model ester, obtaining promising results for the forthcoming utilization of  these hybrid magnetic biocatalysts for the synthesis of oligomers. The characterization of the selected immobilized lipases was achieved by studying the temperature stability and operational stability in repeated uses, proving excellent properties. The optimization of polyesteramide synthesis was carried out considering three essential parameters for the reaction engineering: temperature, molar ratio of the monomers, and enzyme/ monomer ratio. Biodegradation studies were performed with native microbial lipases, but also in a natural aquatic environment, for 2 types of polyesteramides. After 20 days, a mass loss of approximately 40% was observed for the linear polyesteramides. The production of nanoparticles by the emulsification method, based on enzymatically synthesized polyesteramides, was also investigated. Particles with an average size of 375 nm and a polydispersity index of 0.36 were obtained, these results being promising in the perspective of developing efficient encapsulation variants of biologically active compounds, which will be achieved in Stage 3. The dissemination of the results includes 3 journal papers (two in a journal ranked Q2 at the date of publication, Web of Science) and 7 conference presentations (one of them won the best poster award at the 11th IFIB Conference in Trento, Italy). Consequently, all objectives of this stage have been accomplished.


  1. D. Dreavă, I.C. Benea, I. Bitcan, A. Todea, E. Sisu, M. Puiu, F. Peter , Biocatalytic approach for novel functional oligoesters of ε-caprolactone and malic acid, Processes 2021, 9(2), 232. I.F. 2.847 (2020).
  2. A. Todea, D.M. Dreavă, I.C. Benea, I. Bîtcan, F. Peter, C.G. Boeriu, Achievements and trends in biocatalytic synthesis of specialty polymers from biomass-derived monomers using lipases, Processes 2021, 9 (4), 646. I.F. 2.847 (2020).
  3. I. Kántor, D. Aparaschivei, A. Todea, F. Peter, Z. May, E. Biró, T. Feczkó, Co-entrapment of Sorafenib and Cisplatin in poly[ε-caprolactone-co-(12-hydroxystearate)] copolymer for dual drug delivery application, The 1st International Electronic Conference on Biomedicine, Session Nanomaterials and its Application in Biomedicine, 1–26 June 2021, Proceedings available in


  1. I.C. Benea, D. Aparaschivei, I. Bîtcan, A. Todea, E. Șișu, M. Puiu, F. Peter, Enzymatic synthesis and characterization of biobased copolymers of ε-caprolactone and DL-malic acid, The 6th International Conference on Biocatalysis in Non- Conventional Media, University of Milan, May 7-9, 2021 (online event).
  2. C. Vasilescu, C. Paul, F. Peter, Tailoring the sol-gel entrapment method of enzymes by design of experiments for solvent-free ester synthesis, The 15th International Symposium on Biocatalysis and Biotransformations (Biotrans 2021), University of Graz, July 19-22, 2021 (online event)
  3. I.C. Benea, D.M. Dreavă, A. Todea, L. Nagy, S. Kéki, Francisc Péter, A green alternative for the synthesis of polyesteramides from ε-caprolactam and hydroxy acids, The 11th Edition of the International Forum on Industrial Biotechnology and Bioeconomy (IFIB), Italian Association for the Development in Biotechnology, September 30 - October 1, 2021 Trento, Italy. Best Poster Award
  4. C. Vasilescu, C. Paul, F. Peter, Optimization of the sol-gel entrapment of Candida antarctica lipase B through experimental design methods, EFB Virtual Conference, European Federation of Biotechnology, May 10-14, 2021.
  5. I.C. Benea, D. Dreavă, A. Todea, L. Nagy, S. Kéki, F. Péter, Enzymatic approach for the synthesis of ε-caprolactam and hydroxy acids polyesteramides, The 13th Edition of the Symposium - New Trends And Strategies In The Chemistry Of Advanced Materials With Relevance In Biological Systems, Technique and Environmental Protection, "Coriolan Drăgulescu" Institute of Chemistry, October 7-8, 2021, Timișoara, Romania (online event).



Abstract Stage 3 (2022)

In this stage, several studies initiated in the previous stage were continued regarding (i) the study of the degradation of polyesteramides by enzymatic and microbial means; (ii) obtaining nanoparticles based on enzymatically synthesized polyesteramides; (iii) characterization of the obtained nanoparticles. Other activities were initiated in this stage, focused on (iv) biodegradation of the synthesized polyesteramides; (v) encapsulation and (vi) controlled release of a selected bioactive compound. Biodegradation studies were performed in liquid culture media, both in the presence of a native lipase or a consortium of microorganisms collected from a freshwater river. Considering the polyesteramides based on ε-caprolactam and fatty hydroxy acids (one linear and one branched) selected for the enzymatic degradation studies, better results were obtained for the copolymer containing the linear hydroxy acid inserted into the polycaprolactam chain, which presented a mass loss of over 50% after 60 days of incubation at 37°C in the presence of pancreatic lipase. For the microbial degradation studies, polyesteramides based on ε-caprolactam and short-chain hydroxy acids were selected, as they are soluble in water, a necessary condition to be used in OxiTop systems, equipped with sensors that measure the biochemical oxygen demand (BOD). Promising results were obtained with both polyesteramides, showing high biodegradation degrees after only 21 days of incubation with a consortium of microorganisms taken from a natural environment. Nanoparticles were obtained by the solvent evaporation method, in average sizes between 180-350 nm. In order to obtain particles with controlled size distribution around 200 nm, the effect of several process parameters was studied, such as: polymer concentration, oil/water phase ratio, and emulsifier (polyvinyl alcohol) concentration. Based on the obtained results, four combinations of experimental conditions were selected to obtain nanoparticles able to encapsulate sorafenib, a bioactive compound with cytotoxic activity. The optimal amount of the encapsulated bioactive compound was determined to be 1mg/10 mg polymer. The nanoparticles with encapsulated  bioactive compound were characterized by average size, size distribution, encapsulation efficiency, yield, and zeta potential. The sample that showed the best characteristics was used for in vitro release studies in blood plasma (pH 7.4) and sodium acetate buffer (pH 5.5). The in vitro drug release test ended with promising results, allowing to continue the experiments. The dissemination of the results of this stage included the publication of two journal papers (in top 25% journals, considering the WoS ranking at the time of publication), a submitted manuscript, and participation with 3 oral presentations at important international conferences. Consequently, we appreciate that all objectives of this stage have been accomplished.


  1. I. Kántor, D. Dreavă, A. Todea, F. Péter, Z. May, E. Biró, G. Babos, T. Feczkó, Co-entrapment of Sorafenib and Cisplatin drugs and iRGD tumour homing peptide by poly[ε-caprolactone-co-(12-hydroxystearate)] copolymer, Biomedicines 2022, 10, 43, F.I. 4,757,
  2. I. Păușescu, I. Kántor, G. Babos, Z. May, A. Fodor-Kardos, Z. Miskolczy, L. Biczók, F. Péter, M. Medeleanu, T. Feczkó, Halochromic behavior and anticancer effect of new synthetic anthocyanidins complexed with β-cyclodextrin derivatives, Int. J. Mol. Sci. 2022, 23, 8103, F.I. 6,208,
  3. C. Vasilescu, C. Paul, S. Marc, I. Hulka, F. Péter, Development of a tailored sol-gel immobilized biocatalyst for sustainable synthesis of the food aroma ester n-amyl caproate in continuous solventless system, Foods, 2022. F.I. 5,561, în curs de evaluare.


  1. I.C. Benea, D.M. Dreavă, I. Bîtcan, C. Paul, A. Todea, L. Nagy, S. Kéki, I. Kántor, T. Feczkó, F. Péter, Biocatalytic synthesis of novel polyesteramide nanoparticles derived from ε-caprolactam and hydroxy acids, The 5th Edition of Biotech France 2022 International Conference and Exhibition, 15-17 June 2022, Paris, France (oral presentation).
  2. I.C. Benea, D.M. Dreavă, I. Bitcan, C. Paul, A. Todea, L. Nagy, S. Kéki, F. Péter, A biocatalytic strategy for the synthesis of novel polyesteramides from ε-caprolactam and biobased hydroxy acids, RomCat Conference 2022, The 13th International Symposium of the Romanian Catalysis Society, 22-24 June 2022, Băile Govora, România (oral presentation).
  3. I.C. Benea, A. Todea, D.M. Dreavă, F. Peter, Synthesis of biobased oligomeric esteramides and esters by sustainable green pathways, The 5th International Conference on Green Chemistry and Sustainable Engineering (GreenChem-22), 20-22 July 2022, Rome, Italy, Abstracts E-Book, ISBN 978-84-09-42560,, pp. 67 (oral presentation).


Summary of the project results

The project "Biocatalytic synthesis of new polyesteramides as nanocarriers for bioactive compounds" targeted as main objective the realization of a demonstrative model for the biocatalytic synthesis of new esteramide-type oligomers and polymers, mainly by the reaction between ε-caprolactam and hydroxy acids. Using renewable raw materials and biocatalysts, the current requirements of the circular bioeconomy are met, the synthesis pathways being green, and the products biodegradable, as demonstrated in the project's research. The experimental studies were carried out in the Biocatalysis Laboratory at the Politehnica University of Timişoara.

The enzymes used were microbial lipases, being immobilized by entrapping them inside a sol-gel polysiloxane macromolecular network, with the aim of improving their stability and favoring repeated use, thus reducing process costs. The new polyesteramides, obtained on gram scale, were characterized by MALDI-TOF mass spectrometry, gel permeation chromatography and NMR, having after purification molecular masses about 3000 Da. The possibility of making particles with sizes between 200-250 nm based on these new polymers using the solvent evaporation method was demonstrated, particularly in the case of polyesteramide obtained from ε-caprolactam and 16-hydroxypalmitic acid. These nanoparticles were proved as effective for the encapsulation of selected bioactive compounds, e.g., the drug Sorafenib, used to block tumor cell growth. The possibility of controlled release of this compound was demonstrated in vitro, opening the way for further development of this field.

Experimental setup for the enzymatic synthesis of polyesteramides, allowing temperature and stirring control (a); The product obtained after the removal of the enzyme (b, top) and after purification (b, bottom); Assay of microbial degradation in natural aquatic environment (OxiTop system) (c); Poly(e-caprolactam-co-16-hydroxypalmitate) before (d, left) and after microbial degradation (d, right)

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