It is well known that the venom from the Hymenoptera insect class (bees, wasps, ants) are potent neurotoxic due to secreted phospholipase A2 enzyme (PLA2). For instance, bee venom PLA2 enzyme acts synergistically with the polyvalent cations (toxins) in the venom creating an increased haemolytic effect and a quick access of toxins into the blood flow, targeting important organs like the brain, kidney and liver. In spite of recent technological developments, effective and safe therapies are currently not available for treating the victims of mass insect attacks. Yet, thanks to advances in the fields of Nanotechnology, removing of PLA2 enzyme from sting zone can be accomplished by targeted delivery systems called ligand-free nanogels. The proposed concept for preparing ligand-free nanogels is original and uses bifunctional macromonomers, small molecule cross-linkers and PLA2 template molecules to create antibody-like recognition sites for PLA2 subsequent retention. Hence, PLANano holds significant influence upon the scientific community by new concepts and methodologies for free-ligand nanogel targeted delivery systems (short-term impact: 3 ISI papers, minimum 3 communications, 1 national patent claim, web site of project) and by opening new research directions associated with the side-impacts of the research (like new imuno-therapies) as long term impact. Implementation of this project will also bring specific scientific, economic and social benefits at the national level and at the international level. The greatest impact is given by the health value, if taken in consideration the advantages of the innovative ligan-free nanogels over conventional antivenom production. Plus, the existing infrastructure of the Host Institution is appropriate to sustain the proposed tasks for PLANano successful implementation. But most importantly, the young research team composed of young specialist, post-docs and PhD fellows is highly qualified in this direction.

The pharmacological activity of PLA2 is quite unique. PLA2 binds specifically to various phospholipidic substrates. This targeting ability of PLA2 is conferred by the presence of a 'pharmacological site' on its surface that can direct the molecule towards specific phospholipidic locations. As a result, if this "pharmacological site" can be counteracted by complementary cavities created artificially in nanoparticles, with the use of molecular imprinting techniques (MIT), the system can be called a ligand-free targeted delivery system. This type of systems called ligand-free delivery nanogels, which are free of drugs, can retain various compounds using the matrix itself for targeting. Unfortunately, the studies related to targeted delivery systems for PLA2 from Hymenoptera venom were not found.

Molecular imprinting however, has been used for decades to produce various adsorbent materials and nanomaterials and itconsist of polymerization/cross-linking of one or more functional monomers in the presence of particular template molecules. Further template removal leads to molecularly imprinted cavities with complementary shape, size and functionalities towards target molecules (creating artificial acceptors). The fact that the nanogel matrix prepared in draft will present high affinity only for PLA2 makes the construction of the nanogels less complicated, without magnetic, temperature or pH properties as targeting mechanisms. Conclusively, the proposed concept of ligand-free nanogels targeted delivery systems for PLA2s retention represents a completely original solution in the Nanobiomedicine.

The overall aim of the “PLANano” project refers to developing original, cost-effective and biocompatible ligand-free targeted delivery nanogels for retaining venom-originated PLA2, as potential Hymenoptera insect antivenom.

            For reaching the project goal,specific objectives were established, as follows:

O1. Synthesis Trials for establishing proper Experimental Models for the preparation of ligand-free nanogels.  

O2. Optimization of PLA2 retention performance for ligand-free nanogels to establish Functional Models for their production.

O3. Developing and validating the Laboratory technology for ligand-free nanogels production. 

O4. Dissemination of project scientific output using specific and eligible routes, protection of intellectual property rights and risk management

PLANano is a multidisciplinary project proposal with high influence upon the scientific community by new concepts and methodologies for free-ligand nanogel targeted delivery systems (short term impact of dissemination) and by opening new research directions associated with the side-impacts of the research (long term impact). Short-term impact: at least 3 publication in specific ISI journals - 1 publication in a journal having 3 points Impact Factor and other 2 in journals with over 1.0 Impact Factor, 1 national patent claim, and minimum 3 communications at international Symposia or Congresses, plus website of PLANano project. Long-term impact: new research directions in the Nanotechnology/ Biomedicine field and transferable knowledge to lead-users to manufacture the ligand-free nanogels.

Phases 1. Trials for preparing of the ligand-free nanogels.

                  Perioada: 02.05.2018 – 30.12.2018

SUMMARY OF THE STAGE

Hymenoptera insects exhibit good neurotoxic activity and are mainly composed of phospholipase A2 (PLA2). The PLA2 enzyme, extracted from bee venom, acts synergistically with the polyvalent cations (toxins) existing in the venom, resulting in a strong hemolytic effect and in a rapid access of the toxins in the blood stream, with harmful effects on important organs, such as the brain, the kidneys and the liver. The present project is aimed at synthesizing and at characterizing innovative non-ligand nanogel structures, starting from polyethylene glycol diacrylate (PEGDA) macromonomers, which exhibit high biocompatibility, a new nanogel structure for the recognition and retention of PLA2 being developed. Thus, the proposed concept is for the retention of PLA2, through non-ligand nanogels, is a completely original alternative in the nanobiomedicine field. The proposed concept was feasible because it refers to a typical reverse mini-emulsion polymerization approach, in which the hydrophobic / hydrophilic interface will be controlled even by the presence in the reaction system of the enzyme itself. In Stage I / 2018, studies were conducted on the obtaining and on the physic-chemical and morphological characterization of the non-ligand nanogel structures, developed using polyethylene glycol diacrylate (PEGDA) macromonomers, which exhibit high biocompatibility, with the end of getting a new nano-gel structure able to recognize and retent bee venom phospholipase A2 (PLA2). The new non-ligand nanogels were characterized both physically and physicochemically, using appropriate techniques and equipment. Regarding FTIR spectra of non-ligand nanogels, they showed theoccurrence peaks characteristic of the polymer consisting of polyethylene glycol and of acrylic functional groups. Moreover, the TGA / DTG curves confirmed the occurrence of both polyethylene glycol and acrylic functional groups in the final structure of synthesized nanogels. Regarding the size of the particles obtained from synthesis, DLS measurements revealed, not only in the case of non-imprinted nanogels, but, also, for PLA2-imprinted ones, that the values remained within a suitable range for the final application (120-200 nm ± 10). TEM images, recorded for nanogels, which were molecularly imprinted with the enzyme, showed the formation of molecularly imprinted nano-cavities for NH-MIP (Tris) and NH-MIP (H2O) samples. Thus, we can state that the existence of nano-cavitie,s formed on the surface of polymer nanogel, has been physically proven.

The degree of accomplishment of the aimed objectives and results was over 100%. 

Phases 2. Optimization of specificity and cross-reactivity of ligand-free nanogels by a feed-     back   process.

              Perioada: 01.01.2019 – 30.12.2019

PHASE 2 MAIN OBJECTIVES

The main objective of the research study was to optimize the specificity and reactivity of polyethylene glycol ligand-free nanogels with acrylate end groups (PEGDA), through a feed-back process, as follows:

• Elaboration of the experimental model (EM) for obtaining the polyethylene glycol nanogels with acrylate end groups (PEGDA) in order to determine the specificity for the PLA2 molecule;

• Determining the efficiency of nanogels in phospholipid media (in vitro) by measuring the decrease of PLA2 activity, while they are retained;

• Quantitative determination of the selectivity of nanogels in terms of cross-reactivity through bee venom adsorption measurements.

PHASE 2 SPECIFIC OBJECTIVES

Activity 2.1 Elaboration of the experimental model (EM) for obtaining the nanogels based on polyethylene glycol with acrylate end groups (PEGDA) and diamine in order to determine the specificity of the PLA2 molecule.

Activity 2.2 Determining the efficiency of nanogels in phospholipid media (in vitro) by measuring the decrease of PLA2 activity, while they are retained.

Activity 2.3 Quantitative determination of the selectivity of nanogels in terms of cross-reactivity by bee venom adsorption measurements.

PHASE 2 SUMMARY

The experimental model (EM) for obtaining the polyethylene glycol nanogels with acrylate end groups (PEGDA) was developed in order to determine the specificity of the PLA2 molecule. Within this activity, studies have been carried out on the obtaining and physico-chemical and morphological characterization of the structure of ligand free nanogels based on macromonomers of the poly(ethylene glycol)diacrylate (PEGDA), with proved biocompatibility, in order to create new structures of nanogels for recognition and rebinding of phospholipase A2 (PLA2) from bee venom.

The new structures of the obtained ligand free nanogels were characterized both physically and physico-chemically using different techniques and equipment, such as: infrared spectroscopy (FTIR) and by TGA/DTG analysis for chemical composition and possible interactions between compounds, transmission electron microscopy (TEM) for highlighting particle morphology and size and light diffusion technique (DLS) for particle size, nano-associations. Regarding the FTIR, the spectra of the ligand free nanogels presented the characteristic peaks of the polymer formed from polyethylene glycol and acrylic groups. TGA/DTG analyzes also confirmed the presence of both polyethylene glycol and acrylic groups in the final structure of the synthesized nanogels. The presence of PLA2 enzyme in the structure of imprinted nanogels was not detected, being partially removed from the surface of the nanogels during the centrifugation process. Regarding the size of the particle obtained from the synthesis, the DLS measurements revealed that the size of both the non-imprinted and PLA2-imprinted nanogels remained within a suitable range for the final application (100-200 nm). Images obtained by transmission electron microscopy (TEM) showed a homogenous morphology, in the form of spherical and individual nanoparticles, for all the synthesized ligand-free nanogels. Yet, for the imprinted nanogels taken directly from the emulsion, the presence of emulsifiers (in the form of needles) and the presence of the enzyme (in the form of crystals) was observed on their surface. In conclusion, TEM micrographs obtained on structures of molecularly imprinted nanogels with the enzyme indicated the presence of molecularly imprinted nanocavities. Thus, we can say that we have physically demonstrated the existence of nanocavities formed on the surface of polymeric nanogels. As for the efficiency in phospholipidic environments, it was confirmed that the activity of PLA 2 decreases more than two times after contact with the nanogels.

Phases 3. Laboratory technology for the ligand-free nanogels.

                Perioada: 01.01.2020 – 30.04.2020

PHASE 3 MAIN OBJECTIVES

The main objective of the research study was to establish the laboratory technology for obtaining polyethylene glycol ligand-free nanogels with acrylate end groups (PEGDA), in the presence of phospholipase A2 (PLA2) from bee venom used as a template, by inverse mini-emulsion polymerization, as follows:

• Reproducibility of recipes for obtaining ligand-free nanogels following morphological and dimensional parameters as well as the specificity and reproducibility of in vitro measurements.
• Optimization and validation through biocompatibility studies of laboratory technology for obtaining ligand-free nanogels.

PHASE 3 SPECIFIC OBJECTIVES

Activity 3.1 Reproducibility of recipes for obtaining ligand-free nanogels following morphological and dimensional parameters as well as the specificity and reproducibility of in vitro measurements.

Activity 3.2 Optimization and validation through biocompatibility studies of laboratory technology for obtaining ligand-free nanogels.

PHASE 3 SUMMARY

Within the last phase of the project, the ligand-free nanogels based on macromonomers of the poly(ethylene glycol)diacrylate (PEGDA), in the presence or absence of phospholipase A2 (PLA2), were synthesized via inverse mini-emulsion polymerization. The structures of the obtained ligand free nanogels were characterized both physically and chemically using different techniques and equipment, such as: infrared spectroscopy (FTIR) for chemical composition and possible interactions between compounds, transmission electron microscopy (TEM) for highlighting particle morphology and size and light diffusion technique (DLS) for particle size, nano-associations. The obtained results were reproducible and thus the synthesis method was validated. Furthermore, measurements in phospholipid media (in vitro) of ligand-free nanogels proved to be reproducible, the best results being recorded for imprinted nanogels prepared in Tris solution – with a high adsorption capacity of 75.84 mg PLA2 / g nanogel and an imprinting factor of 2.62. Plus, the data were validated by morphological and dimensional parameters. The high cell viability of imprinted nanogels confirmed the biocompatibility of ligand-free nanogels and, implicitly, sufficiently low cytotoxicity for nanobiomedicine applications. In conclusion, the reproducibility of imprinted nanogels properties led to the elaboration of the laboratory technology for obtaining ligand-free nanogels.

PHASE 1

1 article sent for publication - Bogdan Cursaru, Anita-Laura Radu, Andrei Sarbu, Mircea Teodorescu, Ana-Mihaela Florea, Celina-Maria Damian, Teodor Sandu, François-Xavier Perrin, Tanta-Verona Iordache, Anamaria Zaharia; „ Poly(ethylene glycol) Composite Hydrogels with Natural Zeolite as Filler  for Controlled Delivery Applications”, Polymer Journal, under evaluation.

1. “COMPOSITE NANOGELS FOR CONTROLLED DRUG DELIVERY” - ZAHARIA Anamaria, SPATARELU Catalina Paula, RADU Anita-Laura, CURSARU Bogdan, IORDACHE Tanta-Verona, FLOREA Ana-Mihaela, SANDU Teodor, TRICA Bogdan, SARBU Andrei, TEODORESCU Mircea, PERRIN François-Xavier - Simpozion international Prioritatile Chimiei pentru o Dezvoltare Durabila PRIOCHEM - editia XIV , Bucuresti, 10 - 12 Octombrie 2018.

2. Synthesis and characterization of new PEG composite hydrogels for controlled delivery of bioactive substances,  Bogdan Cursaru, Anita-Laura Radu, Anamaria Zaharia, Andrei Sarbu, Mircea Teodorescu,  Celina-Maria Damian, Tanta-Verona Iordache, Ana-Mihaela Florea, Teodor Sandu, Raluca Ianchis, Bogdan Trica, François-Xavier Perrin, Conferinta internationala "Bucharest Polymer Conference 1st Edition– BPC 2018" 6 - 8 June 2018, The Center for Advanced Research on New Materials, Products and Innovative Processes (CAMPUS) of University POLITEHNICA of Bucharest.

3. NEW CANDIDATES FOR CONTROLLED DRUG DELIVERY BASED ON POLYETHYLENEGLYCOL DIACRYLATE AND NANOZEOLITE COMPOSITE NANOGELS, Anamaria Zaharia, Catalina Paula Spatarelu, Anita-Laura Radu, Bogdan Cursaru, Tanta-Verona Iordache, Ana-Mihaela Florea, Bogdan Trica, Andrei Sarbu, Mircea Teodorescu, Vlad Tofan, François-Xavier Perrin; A- XXXV-a Conferin?? Na?ional? de Chimie, perioada 02-05 octombrie 2018, C?ciulata, jude?ul Vâlcea, Romania.

PHASE 2

The primary results and those obtained in stages 1 and 2 of the project were disseminated through 2 papers published in ISI ranked journals and 6 communications presented during international conferences. Moreover, a patent application was also filed regarding the nanogels preparation.

Patent application

1. Zaharia Anamaria; Iordache Tanta- Verona; Sârbu Andrei; Botez R?zvan-Edward; Cojocaru Crina Thea; Radu Anita Laura, Gavril? Ana- Mihaela, Sandu Teodor; “Imitatori de anticorpi din polimeri sintetici imprentati molecular cu fosfolipaza A2 si procedeu de obtinere a acestora”/ Imitators of antibodies from synthetic polymers molecularly imprinted with phospholipase A2 and process for obtaining them”, OSIM A/00513 din data 27.08.2019.

 Articles

1. Bogdan Cursaru, Anita-Laura Radu, Francois.-Xavier Perrin, Andrei Sarbu, Mircea Teodorescu, Ana-Mihaela Gavrila, Celina-Maria Damian, Teodor Sandu, Tanta-Verona Iordache, Anamaria Zaharia, “Poly(ethyleneglycol) Composite Hydrogels with Natural Zeolite as Filler for Controlled Delivery Applications”, Macromolecular Research, DOI 10.1007/s13233-020-8029-9, (IF = 1.758)
2. Anita- Laura Radu, Ana Mihaela Gavrila, Bogdan Cursaru, Catalina Paula Spatarelu, Teodor Sandu, Andrei Sarbu, Mircea Teodorescu, Francois Xavier Perrin, Tanta-Verona Iordache, Anamaria Zaharia; Poly(ethyleneGlycol) Diacrylate-Nanogels Synthesized by Mini-emulsion Polymerization; Materiale Plastice, 56, No. 3, 2019, 514-519. (IF = 1.393)

Conferences

1. “Innovative ligand-free nanogel structures able to recognize and retain phospholipase A2 from bee venom”; Anamaria Zaharia, R?zvan Edward Botez, Crina Thea Cojocaru, Tanta-Verona Iordache, Andrei Sârbu, Celina Maria Damian, Ana-Mihaela Gavril?, Anita-Laura Radu, Teodor Sandu, Mircea Teodorescu; World Congress on Functional Materials and Nanotechnology, May 13-14, 2019,Valencia, Spain
2. “A new generation of ligand-free nanogels capable of recognizing and retaining phospholipase A2 from bee venom” C.-T. Cojocaru, A. Zaharia, R. E. Botez,T.-V. Iordache, A. Sarbu, C. M. Damian, A.-L. Radu, A.-M. Gavrila, T. Sandu, M. Teodorescu. Graduate Student Symposium on Molecular Imprinting Berlin, Gemania, 28 august -30 august 2019
3. “New achievments in hybrid hydrogels obtaining” Anamaria Zaharia, Andrei Sârbu, Anita Laura Radu, Teodor Sandu, Tanta Verona Iordache, Mircea Teodorescu, Francois- Xavier Perrin, Ana Mihaela Gavril?, Steluta Apostol, R?zvan Edward Botez, Global Chemical Engineering and Chemistry Expo 25-27.03.2019, Valencia, Spain
4. “Nanohydrogels for the Controlled Release of Drugs and a Process for their Preparation”; Anamaria Zaharia, Anita- Laura Radu, Andrei Sârbu, Mircea Teodorescu, Bogdan Cursaru, C?t?lina-Paula Sp?t?relu, Tanta-Verona Iordache, Teodor Sandu, Ana- Mihaela Florea, Patent application: A/00620/07.09.2016, Clasa: M; Cel de-al 45-lea Salon International de Inventii de la Geneva-ELVETIA, 29 martie - 2 aprilie 2017- premiat cu medalie de Argint.
5. “Nanogels composites for controlled-drug delivery” R?zvan Edward Botez, Anamaria Zaharia, Anita-Laura Radu, Crina Thea Cojocaru, Tanta-Verona Iordache, C?t?lin Zaharia, Ionut Cristian Radu, Ana-Mihaela Gavril?, Teodor Sandu,Monica Duldner, Andrei Sârbu. 9th International Conference of the Chemical Societies of the South-East European Countries, 5mai -11 mai 2019
6. “Advances in hydrogelsand micro/nanohydrogels and their applications“, Cojocaru Crina-Thea, Botez Razvan Edward, Zaharia Anamaria, Radu Anita-Laura, Sarbu Andrei, Iordache Tanta-Verona, Gavrila Ana-Mihaela, Sandu Teodor, Teodorescu Mircea. Next – Chem Tehnologii Inovatoare Trans-Sectoriale, 7 mai 2019.

PHASE 3 

Dissemination and communication of the results

The results obtained in PLANANO project were disseminated through 1 paper published in ISI ranked journal; also, a laboratory technology for obtaining polyethylene glycol ligand-free nanogels with acrylate end groups (PEGDA), in the presence of phospholipase A2 (PLA2), by inverse mini-emulsion polymerization, was established.

Article

1. Catalina Paula Spatarelu, Anita-Laura (Radu) Chiriac, Bogdan Cursaru, Tanta-Verona Iordache, Ana-Mihaela Gavrila, Crina-Thea Cojocaru, Razvan-Edward Botez, Bogdan Trica, Andrei Sarbu, Mircea Teodorescu, Vlad Tofan, Francois-Xavier Perrin and Anamaria Zaharia*, Composite Nanogels Based on Zeolite-Poly(ethylene glycol) Diacrylate for Controlled Drug Delivery, Nanomaterials 2020, 10, 195-215; doi:10.3390/nano10020195 (IF = 4.034)