Thermoresponsive and mucoadhesive nanogels for intramammary administration of antimicrobial compounds to be employed in bovine mastitis treatment

Project Information

THERMUCNA

Grant agreement ID: 896775

DOI

10.3030/896775

Project closed

EC signature date 9 April 2020

Start date 1 March 2021

End date 15 March 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 172 932,48

EU contribution

€ 172 932,48

Coordinated by

UNIVERSIDAD DEL PAIS VASCO/ EUSKAL HERRIKO UNIBERTSITATEA
Spain

Periodic Reporting for period 1 - THERMUCNA (Thermoresponsive and mucoadhesive nanogels for intramammary administration of antimicrobial compounds to be employed in bovine mastitis treatment)

Reporting period: 2021-03-01 to 2023-02-28

Summary of the context and overall objectives of the project

Dairy activity is vital for the European economy, producing almost a quarter of the world's available milk. Bovine mastitis (BM) is a widespread disease that affects dairy cows and causes significant economic losses in the industry worldwide. The current method of controlling BM is through antibiotic therapies. However, these treatments are often ineffective due to acquired drug resistance and the survival strategies of bacteria. In order to combat this issue, new therapeutic approaches are needed. That's why this research project proposes using smart nanogels (NGs) to deliver antimicrobial agents directly into the bovine udder for BM prophylactic treatment. The overall goal of this project is to synthesize a library of NGs that can effectively encapsulate antimicrobial cargos and have tissue-adhesive capabilities to increase their residence time in the bovine udder. NGs are also expected to (i) trigger cargo release upon the infection environment, (ii) ability to promote internalization into host cells to kill intracellular bacteria, and (iii)the ability to avoid bacterial biofilm formation. By conferring at least one of these features, we expect to develop a novel therapy that effectively reduces bacterial resistant development and optimizes antimicrobial use.
During the project, pH-sensitive thermoresponsive NGs were developed. Various monomers and monomer ratios were evaluated to identify the most efficient NGs that respond specifically to an infection environment. Nisin and ceftiofurwere chosen as antimicrobial cargos, which were successfully encapsulated inside the NGs. Also, many efforts were made to set up relevant in vitro models to test the performance of all synthesized formulations. As a general conclusion of this project, smart NGs can be used to specifically release antimicrobials under infection conditions. This approach can reduce the exposure of host animals to antimicrobials and potentially avoid the development of antimicrobial resistance.
It's important to note that this strategy has not been explored yet in bovine intramammary infections, which highlights the novelty of our research findings.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Over the course of 24 months, we conducted the following experimental activities
Synthesis of pH-sensitive thermoresponsive NGs.
NGs were synthesized using N-isopropyl acrylamide (NIPAM) copolymerized with acidic monomers such as acrylic acid (AA), methacrylic acid (MAA), and itaconic acid (IA) NGs. Partially acrylated dendritic polyglycerol (Ac-dPG) was used as a crosslinker. NIPAM-based NGs are thermoresponsive materials since they undergo a volume phase transition when heated above a specific temperature (Cloud point temperature; TCP). Above TCP, NGs shrink, allowing the release of the cargo. Ac-dPG as a crosslinker enhances the NGs stability when the temperature exceeds the TCP.
Incorporating an acidic monomer enables NGs to undergo a volume phase transition triggered by acidic conditions. The synthesis conditions were optimized to obtain NGs with low-polydisperse size distributions. In the case of IA, we found that the different monomer polymerization kinetics (compared to NIPAM and Ac-dPG) produced polydisperse NGs. Other synthetic approaches like semi-batch and core-shell-like NGs synthesis were tested. However, any of them allowed to obtain NGs with acceptable polydispersity index. Following up, we further characterized NIPAM-co-AA and NIPAM-co-MAA NGs by HNMR and FT-IR to confirm the actual composition of each monomer in the NGs. Also, the morphology and mechanical properties were explored by AFM, which revealed that our NGs were highly hydrophilic, with a spherical sponge-like appearance. The thermoresponsive properties were explored by UV-Vis at pH 5 and 7, corresponding to the pH of an infected and healthy udder, respectively. We found an exact percentage of MAA and AA monomers in the feed optimum to obtain thermo-response only in acidic conditions. Small angle X-ray scattering studies have been performed in Alba synchrotron to confirm the structure of the NGs. These results revealed notable structural changes with the temperature in acidic conditions. Then, we used the two specific samples with thermoresponsive at only acidic conditions for further experiments, like in-vitro drug release and antimicrobial activity.
2. In vitro release studies of antimicrobial-loaded pH-sensitive thermoresponsive NGs.
Two antimicrobials were encapsulated inside synthesized NGs:
*Nisin, an FDA-approved antimicrobial peptide currently used for dairy product preservation.
*Ceftiofur, a broad-spectrum cephalosporin commonly used in veterinary.
The Ceftiour encapsulation efficiency was around 70% in both NGs samples, and NGs showed higher Ceftiour release in acidic conditions. Nisin's encapsulation and release studies are still in optimization because it is impossible to follow the peptide concentration by UV-Vis or using a colorimetric test like Bradford. We are optimizing the Nisin quantification by HPLC.
3. Antimicrobial activity of CF-loaded NGs
For antimicrobial testing, Staphylococcus aureus isolates BMs isolates (characterized by VITEK 2) were provided by Dra. Elena Garcia-Fruitós and Dra. Ana Aris from- IRTA - Spain. Also, commensal bacteria were included in this study. The MIC and MBC of the formulations were studied. Also, a methodology was set up to evaluate if the NGs formulation produces the adaptation of bacteria for long-term treatment.
The effect of the empty NGs on the bacteria biofilm formation and pre-formed biofilms was studied. All these data are being processed and are protected until we finish all experimental analysis
4. Ex vivo models to study adhesion to udder tissue
We designed an in vitro model to test the adhesion of the NGs to bovine udder tissue. Bovine udders were obtained from the local slaughterhouse. Slices of fresh tissue were cut using McIlwain Tissue Chopper (1 mm of thickness). The tissue slices were placed onto a glass slide with a 45 grade of inclination and fluorescent-labeled NGs were added dropwise using a syringe pump, and the non-adhered NGs were collected and further quantified. After setting up the model, we have to continue measuring replicates for each sample to report an accurate result for the adhesion of NGs to bovine udder tissue.
To disseminate the results to the scientific community, the researcher attended the 8th International Galenus Workshop, Valencia, Spain. 2022 (Poster presentation). XVII Simposio Latinoamericano de Polímeros. San Sebastián, Spain. 2022 (Flash oral presentation and poster). Also, she published a perspective article as the first author in Nanomedicine (IF= 6.06) and also there are three research articles in preparation. The researcher also disseminated the project results to the general public in three events (DSS_WeekINN 2022. “En guerra con la superbacterias”, Donostia-San Sebastian, Spain. Date: 22/10/2022; 10º Encuentro vidas científicas. 25 de Octubre y 8 de Noviembre 2021. Eureka! Zientzia Museoa. San Sebastián, España;La Noche Europea de los investigadores e investigadoras.24 de septiembre, Bilbao, Spai).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

We have developed pH-sensitive thermoresponsive NGs that can simultaneously respond to two infection stimuli. So far, this approach was not been explored for treat BM. We consider that the probe of concept addresed in this porject is a starting point for a generation of new profilactic therapies for BM management in dairy farms.

Modeof action of smart NGs for antimicrobial delivery