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).
