Progress 05/01/18 to 03/23/23
Outputs
Target Audience:Nanotechnology is currently being pursued as a novel means to enhance the efficacy of diverse chemicals of value to the agriculture industry, such as veterinary drugs, nutrients or vitamins for livestock. This project focus on the application of nanomaterials to deliver antibiotics for livestock. The extensive and widespread use of conventional antibiotics for livestock in prevention and treatment of disease has led to significant environmental and food safety concerns, including the proliferation of antibiotic-resistant microorganisms. Nanomaterials known to provide slow release of drugs and targeted delivery could enhance the effectiveness of antibiotics and reduce the amount of antibiotics used for treatment. The use of these nanoparticles is expected to change the fate and toxicity of antibiotics, as well as the risk for development of antibiotic resistance. However, current limitations in knowledge and understanding of the environmental, health, and safety consequences, either positive or negative, of nano-delivered antibiotics hinders their application in livestock. This research on the synthesis, characterization, fate, and biological interactions of nanoparticles in the gut environment is expected to be broadly applicable to design safe and effective nanomaterials across all sectors of agriculture, from drug and nutrient delivery for livestock to pesticide and fertilizer delivery for crops. Changes/Problems:Justification for replacing zein nanoparticles (ZNPs) with lignin-based polymeric nanoparticles (LNPs) Initially, two types of biodegradable particles, poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) and ZNPs, were proposed in the project. While PLGA NPs with entrapped enrofloxacin were successfully synthesized and tested without any problems, zein polymeric nanoparticles were synthesized both empty and with entrapped enrofloxacin in the reporting period. However, the zein polymeric nanoparticles exhibited an increase in size up to 400 nm and became very polydisperse (PDI >0.5) after freeze-drying. Although attempts were made to address this issue by adding trehalose (cryoprotectant) or excess surfactants (polyvinyl alcohol), the resulting zein polymeric nanoparticles were still not monodisperse (PDI<0.2) after resuspension in buffer solutions. These aggregation-related problems are likely to have significant implications for the stability, drug release, and degradation profiles of nanoparticles, which were crucial for future studies of this project. As such, an alternative to ZNPs was sought, which would still be plant-based. LNPs, which were newly developed with support from NSF EPSCoR, were identified as a promising alternative. These biodegradable and renewable particles were appealing because they were small in size, ranging from 75 to 125 nm depending on the amount of lignin used. More importantly, these new nanoparticles were very monodisperse (PDI<0.15) even after freeze-drying when resuspended in water or buffers. Therefore, LNPs were considered to be a superior alternative to ZNPs for stability and release studies of importance to this project. What opportunities for training and professional development has the project provided?This project has facilitated the education and graduation of two Ph.D. students, Sachin Paudel and Shaida Shakiba, who completed their studies in 2022. Furthermore, this project partially funded the education of two additional Ph.D. students, Haoran Wu, and Genesis Herrera. Additionally, an undergraduate student participated in this project and received training. The project also involved the development of supplementary teaching materials for the courses taught by the principal investigator (PI) to enhance the training and professional growth of graduate and undergraduate students at UH. How have the results been disseminated to communities of interest?As part of the project, there were two outreach events in Spring 2021 and Fall 2021, where high schoolstudents were provided with lab tours and the graduate students presented the project. In addition, Alaia Homawoo, a UH undergraduate PURS student, was also involved in the project. The project's outcomes were also disseminated in the environmental microbiology class for engineers and a graduate-level course taught by the PI. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: We synthesized two nanodrug delivery systems, poly(lactic-co-glycolic) acid (PLGA) and lignin-graft-PLGA, to entrap enrofloxacin. The PLGA nanoparticles were synthesized using a single emulsion evaporation technique, resulting in monodisperse, spherical nanoparticles with a mean size of 102±2.6 nm, and a high repulsive force that enhanced the stability of the nano-delivery system. The lignin-graft-PLGA biopolymer was synthesized using alkaline lignin and confirmed by H-NMR and FTIR. The resulting nanoparticles were small in size (92±2 nm) with a compact spherical structure, negatively charged (-65±3 mV), and suitable for entrapment and delivery of enrofloxacin (ENRO). We assessed the toxicity of both nanoparticles with porcine epithelial cells using the MTT assay, and the results showed concentration-dependent cytotoxicity. PLGA(ENRO) nanoparticles were slightly less toxic than Lignin(ENRO). To determine the minimum bacterial inhibitory concentration (MIC) for pathogenic E. coli O157:H7, we investigated nanocarriers based on the highest concentration that would not induce cytotoxicity in IPEC-J2 cells and would result in more than 50% inactivation of the bacteria. A concentration of 0.14 mg/L of PLGA(ENRO) nanoparticles was selected for the infection experiments, while a concentration of 0.8 mg/L of Lignin(ENRO) was observed to inactivate E. coli O157:H7. In vitro infection experiments were conducted using the method of multiplicity of infection (MOI) with ratios of 1:10 and 1:100. The optimization of the experiment was initially done with PLGA(ENRO), which showed great infection curtailment activity in both MOI compared to free enrofloxacin drugs. The MOI for Lignin(ENRO) demonstrated that the infection efficacy was higher for PLGA(ENRO) nanoparticles at longer incubation times (24 h) compared to shorter incubation times (4 h). Fluorescently labeled PLGA(ENRO) and Lignin(ENRO) nanoparticles were incubated with IPEC-J2 cells and observed in confocal laser microscopy (CLSM), confirming that both nanoparticles were able to enter the cells. Objective 2: We developed an advanced multi-detector AF4 method to monitor enrofloxacin release from nanocarriers. A method validation study was published demonstrating advantages over conventional release assays. A mechanistic study was then published that applies the AF4 method to distinguish the release profile of entrapped enrofloxacin from a surface-adsorbed drug surrogate, coumarin 6. The nanocarriers with entrapped enrofloxacin showed better robustness to hold the drug in 20% methanol, suggesting the drug will not prematurely release before reaching a drug delivery target, even in media with mildly hydrophobic or amphiphilic solubilizing agents (e.g., proteins or biosurfactants). Density field theory modeling provided supporting evidence for the differing polymer-drug interactions, depending on drug hydrophobicity. Enrofloxacin release in biological media was directly evaluated in simulated saliva (pH 7) with or without amylase, as well as simulated gastric fluid (SGF, pH 2.4) with or without pepsin. A novel coupling of the AF4 method with total organic carbon (TOC) and fluorescence detection was developed for complex matrix analysis, where AF4 separates the nanocarriers from high molecular weight proteins, while the detectors probe the drug-protein-nanocarrier interactions. As hypothesized, the entrapped enrofloxacin release rate was not affected by the proteins. However, pH had a significant effect, with faster release in SGF attributed to protonation of the enrofloxacin. A Matlab modeling code was developed to process the multi-dimensional release data from the AF4 method, supporting the mechanism of accelerated diffusive release in SGF, as opposed to degradation of the nanocarrier. In summary, this objective produced a new analytical tool capable of identifying mechanisms for nanocarrier release in biological media. Objective 3: In this study, bioreactors were set up with pig slurry and treated with PLGA and Lignin loaded with Enrofloxacin. RNA extractions were performed on control and treatment samples at 24-, 48-, and 72-hours post-treatment and converted to cDNA to create a DNA library with 16S rRNA 24 PCR Barcoding kit. The DNA library was sequenced in triplicate using the ONT Mk-1C minion device for 6 hours. The focus was on the core microbial community of the pig gut microbiome, and metatranscriptomics analysis was conducted on the RNA to investigate the evolution of antibiotic-resistant genes (ARGs). The functional core microbial composition of the pig gut microbiome was composed of several genera, including Fusobacterium, Ruminococcus, Peptostreptococcus, Clostridium, and Escherichia. Furthermore, we aimed to determine whether the combination of Enrofloxacin with lignin nanoparticles could lead to dysbiosis by targeting important microbial functions compared to PLGA (Enro). We found an overall decrease in alpha diversity of various metabolic functions at 24 hours with free antibiotic treatment, but the slow drug release property of nanoparticles showed a similar alpha diversity index as the control at 24 hours. At 72 hours, all treatments with Enrofloxacin showed a decrease in alpha diversity index. The estimated metabolic functions were associated with lipids and glycan biosynthesis, which have been linked to short chain fatty acids (SCFAs) production. The study found that the diversity of the microbial community is influenced by the microbial community's capacity to consume byproducts or degrade biopolymers. Lignin nanoparticles showed comparable activity in functions associated with SCFAs to the control. The study also found that the microbial community could maintain their biochemical processes at the cellular and molecular levels. The inferred functions from the 16S rRNA microbial community showed that NPs could mitigate free antibiotic-induced changes through the core microbes conserved functions. The Procrustes statistical analysis showed that most metabolic functions for empty and loaded NPs conditions were comparable to the control, despite the distance between the estimated microbiological functions and relative abundance of 16S rRNA genes. Objective 4: This study aimed to investigate the emergence of antibiotic resistance genes (ARGs) using qPCR. However, the targeted ARGs were not detected in the samples converted to cDNA. Hence, instead we used EPI2ME with the Fastq Antimicrobial Resistance workflow to analyze the detected DNA with the CARD and NCBI database. The study expected to detect common fluoroquinolone ARGs in treatments containing Enrofloxacin, but none were found in any experimental condition from 24-72 hours. One possible explanation for this is that the concentration of Enrofloxacin used in the study was lower than commonly used to detect fluoroquinolone resistance. However, the study found an increase in aminoglycoside and tetracyclines ARGs at 24 hours but decreased at 72 hours. The study detected acrF as the only gene that conferred resistance to fluoroquinolones in the control sample at 72 hours. Furthermore, we observed that Proteobacteria, which commonly host fluoroquinolone resistant genes, showed the highest abundance in the control condition, and was significantly reduced in conditions with Enrofloxacin. The study recommends careful selection of bulk lignin materials for drug delivery in human or other mammalian therapeutic purposes. No significant difference was observed between the two biodegradable polymers in ARGs detection between 24-72hrs post treatment.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2023
Citation:
Trif, E., C. Cerbu, C. E. Astete, S. Libi, E. Pall, S. Tripon, D. Olah, A. Valentin Pot�rniche, L. Witkowski, G. Florinel Brudasc?, M. Sp�nu, C. M. Sabliov. Delivery of florfenicol in veterinary medicine through a PLGA-based nanodelivery system: improving its performance and overcoming some of its limitations. Veterinary Research Communications.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Haoran Wu, Alaia Homawoo, Elham H. Fini, Carlos E. Astete, Debora F. Rodrigues, Cristina M. Sabliov, and Stacey M. Louie. Distinction of enrofloxacin release mechanisms in oral and gastric fluids by asymmetric flow field flow fractionation. In preparation for Analytica Chimica Acta.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Sheyda Shakiba, Saba Shariati, Haoran Wu, Carlos E. Astete, Rafael Cueto, Elham H. Fini, Debora F. Rodrigues, Cristina M. Sabliov, and Stacey M. Louie. Distinguishing nanoparticle drug release mechanisms by asymmetric flow fieldflow fractionation. Journal of Controlled Release, 2022, 352, 485-496.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Karin Mattsson, Victor H. da Silva, Amrika Deonarine, Stacey M. Louie, and Andreas Gondikas. Monitoring anthropogenic particles in the environment: Recent developments and remaining challenges at the forefront of analytical methods. Current Opinion in Colloid and Interface Science. 2021, 56, 101513.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Effects of loaded biodegradable nanoparticles with antibiotics on the microbial community from the pig gut microbiome, Genesis Herrera, Dr. Sachin Paudel, Dr. Debora Rodrigues, Dr. Carlos Astete, Dr. Cristina Sabliov, Dr. Stacey Louie, 2022 AEESP Conference, June 28 30, 2022, St.Louis, Missouri.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Wu, H., & Louie, S. M. Drug release from polymeric nanoparticles in complex media analyzed by asymmetric flow field-flow fractionation (AF4). 11th Sustainable Nanotechnology Organization (SNO) Conference, Nov. 2022, Austin, TX.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Louie, S. M.. Separation and multi-detector characterization of polymeric nanoparticles. 11th Sustainable Nanotechnology Organization (SNO) Conference, Nov. 2022, Austin, TX.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Louie, S. M. Multi-detector asymmetric flow field flow fractionation (AF4) for size-resolved release assays on polymeric nanoparticles. 22nd International FFF Symposium, Sep. 2022, Riverside, CA.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Marfua Mowla, Sheyda Shakiba, and Stacey M. Louie. Selective quantification of nanoplastics in environmental matrices by asymmetric flow field-flow fractionation with total organic carbon detection. Chemical Communications, 2021, 57, 12940-12943.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Louie, S. M., Mowla, M., Wu, H., & Shakiba, S. Characterization of polymeric nanoparticles by asymmetric flow field flow fractionation (AF4). 96th ACS Colloid & Surface Science Symposium, Jul. 2022, Golden, CO.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Louie, S. M. Sustainable Agrochemicals: Developing Tools to Predict and Evaluate Environmental Fate. FMC Corporation Stine Research Center, Invited Seminar, Newark, DE, May 2022.
|
Progress 05/01/21 to 04/30/22
Outputs
Target Audience:In this project, four Ph.D. students were recruited to work on the project, of which one was afemale engineering student and the other a biology student. The PI has continued to teach in her graduate class CIVE 6391 - Environmental Engineering Microbiology a module in bioreactors to teach the students how bioreactors can be related to the real gut environment. In this class they learn how to set up a bioreactor and how to perform the proper calculations for a bioreactor. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has allowed the training and graduation of two Ph.D. students (Sachin Paudel and Shaida Shakiba) that have graduated last year. This project is also partially funding the training of two additional Ph.D. students (since the project and funding is ending next year, they will not be able to finish their thesis under this project): Haoran Wu and Genesis Herrera. An undergraduate student has also participated in this project and received training. Additional teaching materials have been developed for the classes taught by the PI to provide additional training and professional development of graduate and undergraduate students at UH. How have the results been disseminated to communities of interest?In this project there were two outreach events (lab tours) for undergraduate engineering students in Spring 2021 and Fall 2021, where the grad students presented on the project. In addition, there is one UH undergraduate PURS student, Alaia Homawoo, who is currently helping on the project. The results were also disseminated in the environmental microbiology class for engineers, which is a graduate level class taught by the PI. What do you plan to do during the next reporting period to accomplish the goals?To complete Objective 2, we will conclude the release experiments on enrofloxacin-loaded PLGA NPs in simulated intestinal fluid (SIF) with bile salts and proteins. We will also conduct comparative studies on enrofloxacin release from the lignin NPs in the various media. To complete Objective 4, we will be using diverse statistical analysis to understand the role of nanoparticles and antibiotics in the changes of the microbial communities and the appearance of ARG genes in the community as well as the evolution of antibiotic resistant genes when exposed to the nanoparticles with ENRO.
Impacts
What was accomplished under these goals?
Objective 1: This objective was completed in the previous reporting period. Objective 2: We have completed the proof of principle of the development of a novel multi-detector AF4 method to monitor enrofloxacin release from the PLGA nanoparticles and the application of this method to determine release rates in PBS. Specifically, the AF4 method was more robust and selective than conventional dialysis methods to distinguish drug entrapment and release from the NPs without interference from background (dissolved) drugs. Our study identified a predominant influence of the glass transition temperature on the release rate. In addition, coupling AF4 size separation with the drug release analysis (denoted as "size-resolved" release analysis) provided strong supporting evidence that the mechanism of release was drug diffusion through the PLGA matrix. The AF4 method was also evaluated in simulated gastric fluid (SGF: 4 mM HCl, pH 2.4) with or without pepsin (3.2 g/L) as the digestive enzyme. Immediate release occurs at 37 °C, so the release in all conditions was tested at 30 °C (near the glass transition temperature of the PLGA NPs), where slow release is achieved. In the simulated saliva (with or without amylase), the release rate was similar to that in PBS (pH 7.4) indicating no significant influence of the type or concentration of salts and no significant adsorptive interactions of amylase with the NPs or the enrofloxacin. In addition, no significant influence of the pepsin was observed when comparing SGF with or without pepsin. The lack of influence of the proteins is consistent with the slow timescale for polymer degradation (typically on the order of days to weeks) compared to the more rapid timescale of the drug release (hours to days). In contrast, an accelerated release was observed in the SGF when compared to the neutral pH media (simulated saliva or PBS), indicating a significant influence of pH on the drug release. To further investigate the mechanism for the accelerated release in the acidic SGF, several additional analyses were conducted. First, the NP size was evaluated and found to shrink immediately in the SGF. However, evaluation of the size-resolved release data using the AF4 method showed that the release was much faster than could be explained by the change in size. The release in SGF was also compared at room temperature (below the glass transition temperature) and 30 °C (near the glass transition temperature). No acceleration of drug release was observed at room temperature, indicating that diffusion of the acidic media into the NP matrix is required to accelerate the release of the enrofloxacin. Considering the chemical structure and pKa of enrofloxacin, the drug is expected to be zwitterionic (with a net neutral charge) at pH 7 to cationic at pH 2.4. Taking this information altogether, the mechanism for accelerated release was identified to be enhanced solubilization of enrofloxacin, upon diffusion of the acidic media into the PLGA NP matrix (which occurs more rapidly at the glass transition temperature). These results highlight the combined importance of the media, polymer, and drug properties on the overall drug release behavior. The data collection for this study is being concluded and data analysis is in progress for manuscript preparation. Objective 3: We set up the bioreactors with fresh pig slurry and we extracted the RNA of control (No nanoparticle or antibiotic), loaded PLGA and Lignin with Enrofloxacin, and emptied PLGA and Lignin in the bioreactors. The RNA extractions were done for 24-, 48-, and 72-hour post-treatment and were converted to cDNA and then created the DNA library with 16S rRNA 24 PCR Barcoding kit from Oxford Nanopore Technologies (ONT). The DNA library was sequenced in the ONT Mk-1C minion device for 6 hours. The sample collection and sequencing were conducted in triplicates. Our focus was on the core microbial community, the collection of microbes consistently present in control and treatment samples, changes occurring in active microorganisms; therefore, metatranscriptomics analysis was conducted on the RNA converted to cDNA of the 16S rRNA region. The functional core microbial composition of the pig gut microbiome was composed of Fusobacterium, Ruminococcus, Peptostreptococcus, Clostridium, Cloacibacillus, Roseburia, Escherichia, Blautia, Alistipes, and Desulfovibrio, which are most abundant and accounted for more than 95% pig microbial community in this study. These common genera have been reported as part of the gut microbiome of pigs in other studies and present in the gut microbiome of humans. We also investigated the evolution of antibiotic resistant genes (ARG) in the reactors exposed to the nanoparticles and antibiotics. We attempted to determine the antibiotic resistance emergence via qPCR. Still, we could not detect with RNA extractions converted to cDNA the targeted ARGs (qnrA, gyrA, or parC) in our samples, but the DNA was detected. Since our focus was on the RNA (gene expressed), we chose to analyze the ARGs of the microbial community by ONT sequence-based-metagenomics rapid-barcoding kit on each condition for 24- and 72-hours post-treatment. The ARGs were analyzed with EPI2ME from the ONT desktop agent with their Fastq Antimicrobial Resistance workflow that blasts the reads with the CARD and NCBI database. Objective 4: Analysis of alpha and beta diversity of the microbial community was done. Results showed that Fusobacterium, a commensal bacterium in the human gut, and other animals, which have been typically associated with increased inflammation and the development of colorectal cancers in high abundance, increased over 150% in the case of Lignin (Enro) treatment compared to the control. Furthermore, the population of essential bacteria changed significantly depending on the treatment. The loss of crucial bacteria such as Clostridium, Desulfovibrio, and Ruminococcus by the effect of Enro drug was deemed more prominent with Lignin (Enro) than PLGA(Enro). This suggests that after a combinatorial treatment with enrofloxacin drug and lignin nanoparticles, the microbial community does not resist the effects compared to PLGA(ENRO) and suggests potential gut dysbiosis after the use of drug Lignin(Enro) for bacterial infection treatment. The analysis of antibiotic resistant genes (ARG) is ongoing, to determine the relationship between the ARGs expressed in each condition. For all time periods, we are using the NMDS plot and PERMANOVA test.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2021
Citation:
Paudel, S. and Rodrigues, D.F. (2021) Chapter 10: Biosynthesis of Metallic Nanoparticles by Extremophiles and Their Applications In S. K. Khare, R. Sinha & M. Gupta (Eds.), Interfaces Between Nanomaterials and Microbes: Taylor & Francis Group.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Shakiba, S.; Astete, C. E.; Cueto, R.; Rodrigues, D. F.; Sabliov, C. M.; Louie, S. M. (2021). Asymmetric flow field-flow fractionation (AF4) with fluorescence and multi-detector analysis for direct, real-time, size-resolved measurements of drug release from polymeric nanoparticles. Journal of Controlled Release, 338, 410-421.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Mowla, M.; Shakiba, S.; Louie, S. M. (2021). Selective quantification of nanoplastics in environmental matrices by asymmetric flow field-flow fractionation with total organic carbon detection. Chemical Communications, 57, (96), 12940-12943.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Mattsson, K.; da Silva, V. H.; Deonarine, A.; Louie, S. M.; Gondikas, A. (2021). Monitoring anthropogenic particles in the environment: Recent developments and remaining challenges at the forefront of analytical methods. Current Opinion in Colloid and Interface Science, 56, 101513.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
G Herrera, J Pe�a-Bahamonde, S Paudel, DF Rodrigues. 2021. The role of nanomaterials and antibiotics in microbial resistance and environmental impact: an overview. Current Opinion in Chemical Engineering 33, 100707
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Paudel, S., Pe�a-Bahamonde, J., Shakiba, S., Astete, C. E., Louie, S. M., Sabliov, C. M., & Rodrigues, D. F. (2021). Prevention of infection caused by enteropathogenic E. coli O157:H7 in intestinal cells using enrofloxacin entrapped in polymer based nanocarriers. Journal of Hazardous Material, Journal of Hazardous Materials 414, 125454.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Paudel, S. 2021. Polymer-Based Nanocarriers to Treat Intestinal Infection and Reduce Impact on Gut Microbiome. Ph.D. dissertation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Herrera, Genesis, Paudel, S., S., Astete, C. E., Louie, S. M., Sabliov, C. M., & Rodrigues, D. F., Protective effects of loaded biodegradable nanoparticles with antibiotics on microbial community from the pigs gut microbiome. 1st Annual TMC Microbe Symposium Feb 10th, 2022, Cyberspace-Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Herrera, Genesis, Paudel, S., S., Astete, C. E., Louie, S. M., Sabliov, C. M., & Rodrigues, D. F., Protective effects of loaded biodegradable nanoparticles with antibiotics on microbial community from the pigs gut microbiome. 10th SNO conference Nov3-Nov5, 2021, Cyberspace-Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Louie, S. M.; Shakiba, S. Achieving a Complete Picture of the Fate of Polymeric Nanoformulations through Complementary Particle Fractionation and Mass Spectrometry Analysis. ACS Spring 2021 National Meeting, Apr. 2021, Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Shakiba, S.; Louie, S. M. Investigation of the effect of material and matrix properties on active ingredient release from polymeric nanoparticles by asymmetric flow field-flow fractionation (AF4). ACS Spring 2021 National Meeting, Apr. 2021, Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Louie, S. M.; Shakiba, S. A New Analytical Framework to Elucidate Mechanisms of Nanocarrier Release. Sustainable Nanotechnology Organization (SNO) 10th Annual Conference, Nov. 2021, Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Mowla, M.; Louie, S. M. Asymmetric flow field flow fractionation (AF4) with total organic carbon (TOC) detection for selective quantification of nanoplastics in environmental matrices. Sustainable Nanotechnology Organization (SNO) 10th Annual Conference, Nov. 2021, Virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Rodrigues, D.F. 2021.Enrofloxacin entrapped in polymer based nanocarriers for gut infection prevention. European Advanced Materials Congress 2021. 23-25 August 2021. Virtual
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Rodrigues, D.F. 2021. Enrofloxacin entrapped in polymer based nanocarriers for gut infection prevention. Sustainable Nanotechnology Organization (SNO) 10th Annual Conference, Nov. 2021, Virtual.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2021
Citation:
Shakiba, Sheyda. Surface and Molecular Level Characterization of Nanomaterials for Water Treatment and Drug Delivery, PhD Dissertation, May 2021.
|
Progress 05/01/20 to 04/30/21
Outputs
Target Audience:In this project, two Ph.D. students continued to participate in the project, of which one is a female engineering student. Additional outreach was not possible this last reporting period because of the COVID pandemic. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph.D. students were hired to work on the project. The first Ph.D. is mainly working in objective 2 and assisting in objective 4, where the technique A4F-MALS-FLD-UV-RI is being developed to analyze the behavior of both nanoparticles in complex environmental conditions, such as the simulated gut reactor of pig. The second Ph.D. student is mainly working on objective 3 and assistingwith objective 4. Objective 1 is being accomplished by the LSU team. How have the results been disseminated to communities of interest?The results of this work have been presented at national and international conferences as well as invited talks, such as: Pannano 2020 in Brazil and the SNO conference 2020. The results have also been disseminated in the graduate classroom at UH, by the PD through the development of a teaching module in bioreactors, as presented in the last progress report. Five manuscripts were published, and two others are currently being prepared for publication from the results obtained in this reporting period. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan on working on the objectives 2, 3 and 4 of this project. In objective 3, we will run the optimized reactors with or without the loaded PLGA and lignin nanoparticles. We will also perform qPCR and sequencing of the 16S rRNA and the antibiotic resistant genes. We also plan to optimize the measurements of drug delivery in the complex gut reactor with theA4F-MALS-FLD-UV-RI method (Objective 2). The data collected from the previous objectives, as well as, objective 3 will be used to do the Objective 4 of this project.
Impacts
What was accomplished under these goals?
After the completion of the synthesis of the biocompatible nanoparticles (objective 1), progress was made towards objectives 2,3 and 4 in this reporting period. We operated a batch anaerobic reactor to study antibiotic-loaded nanoparticles' effects on the microbial diversity and development of antibiotic resistance in vitro. Objective 2: Develop advanced analytical methods to characterize transformations and biological interactions of antibiotic loaded nanoparticles in complex media. We have made significant progress toward completing Objective 2. The novel A4F-MALS-FLD-UV-RI method was fully developed and validated in this project periodto characterize release from the enrofloxacin-loaded PLGA NPs in PBS media at three different temperatures (below, near, and above the glass transition temperature). This method is facile and rapid, requiring no sample pre-purification. We also identified that the new AF4 generates much more accurate and reliable release profiles than conventional release assays (dialysis) because it selectively monitors the drug within the NPs. Other assays available only measure the dissolved drug or the total drug, and therefore are susceptible to interferences of the "burst" release. The AF4 method is more capable to track the actual loading of the NP when they are recovered from the gut reactors. A manuscript on this method development has been submitted to the Journal of Controlled Release. We have also made further progress in identifying the NPs and their transformations in complex media. First, we have supplemented the AF4 method with another online detector, total organic carbon (TOC), that is capable to quantify all the components (biomolecules, dissolved polymers, and polymer NPs) being separated by the AF4. With this detector, we are able to observe the presence of excess polymer surfactant in the unpurified NPs and removal of this surfactant upon purification of the NPs, while simultaneously monitoring the drug loading and release. We have further complemented this analysis with liquid chromatography - quadrupole time-of-flight mass spectrometry (LC-QTOF MS) to validate the drug release measurements and to identify the macromolecular species present in the NP extracts. A manuscript on this study is in preparation for submission to Chemical Communications (invited publication). Finally, we have validated the capability of the AF4 to separate proteins from the NPs. All of these methods will now be combined to monitor NP transformations (protein binding, NP degradation) and enrofloxacin release during incubation with the simulated gut media. Objective 3: Evaluate antibiotic-loaded nanoparticles' effects on the microbial diversity and development of antibiotic resistance in vitro gut reactors. We started the batch bioreactor setup to understand the effects of Enrofloxacin loaded PLGA nanoparticles and lignin nanoparticles on the microbial diversity. The fecal slurry from the pig's gastrointestinal (GI) tract was collected from JJ Packing, Brookshire, TX, and was carefully transported to UH under refrigeration. Upon arrival to the laboratory, the slurry sample was incubated to enrich the microbial community in anaerobic conditions to acclimate the slurry microbial community before the experiments can start. In this period, we standardized theDNA and RNA extraction protocols for further downstream application. We also standardized the PCR for our extracts.The methodology to conduct polymerase chain reaction (PCR) for our sample was standardized to amplify various genes such as the 16S rRNA gene and plasmid-mediated antibiotic-resistant (PMQR) genes. The 16S rRNA gene amplification for our control was successfully achieved at an annealing temperature of 55°C. The 16S rRNA gene amplification will be used for genomic sequencing using Oxford nanopore MinIon sequencing instrument to study microbial communities' change before introducing free enrofloxacin drug and enrofloxacin loaded nanoparticles. To standardize the PCR amplification for the gyrA , parC,qepA, and qnrA genes, gradient PCR amplifications were conducted between the temperature range of 47.6 to 51.6°C. Theoptimal amplification of the gyrA and parC genes occurred at 48.6°C and 50°C, respectively. For the qepA, and qnrA genes athe bestannealing temperatures were at57 and 51°C, respectively. In addition to the standardization of DNA/RNA extraction andPCR methodologies for the sludge, a methodology for genomic sequencing was standardized. Sequencing was performed in a control sample where no drug or nanoparticles were introduced. The top five genera with the most relative abundance in the sequenced control sample were Fusobacterium (28%), Shigella (5%), Enterococcus (4.5%), Escherichia (1%), Faecalicatena(1%), and others (60%). We expected to have a higher abundance of anaerobic microorganisms, and the results confirm enrichment of the anaerobic community. The top five most abundant species were Fusobacterium perfoetens, Fusobacterium varium, Enterococcus faecium, Shigella sonnei, andFusobacterium mortiferum. Additional analysis and interpretation of the results are needed to furtherunderstand their role in the intestinal flora. In the next reporting period, we will have the results of the microbial community changes with and without exposure to Enrofloxacin and Enrofloxacin in loaded PLGA and lignin nanoparticles. We will also study the development of PMQR and quinolone resistance genes. Objective 4:Model the effects of the NPs and antibiotic on cytotoxicity and on changes of the gut microbiome diversity and antibiotic resistance. For this objective, we have made progress collecting data on the pig cell toxicity and optimized the gut microbiome reactor to run the experiments. We also have optimized the DNA/RNA extraction in the slurry as well as the qPCR reactions and sequencing reactions. Once we get the data from Objective 3, we will be able to perform the modeling of the effects of the NPs. More progress on this objective is expected in the next reporting period.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2021
Citation:
Paudel, S. and Rodrigues, D.F. (2021) Chapter 10: Biosynthesis of Metallic Nanoparticles by Extremophiles and Their Applications In S. K. Khare, R. Sinha & M. Gupta (Eds.), Interfaces Between Nanomaterials and Microbes: Taylor & Francis Group.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Paudel, S., Pe�a-Bahamonde, J., Shakiba, S., Astete, C. E., Louie, S. M., Sabliov, C. M., & Rodrigues, D. F. (2021). Prevention of infection caused by enteropathogenic E. coli O157:H7 in intestinal cells using Enrofloxacin entrapped in polymer based nanocarriers. Journal of Hazardous Material.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Paudel, S., Pe�a-Bahamonde, J., Shakiba, S., Astete, C. E., Louie, S. M., Sabliov, C. M., & Rodrigues, D. F., Augmented In Vitro Bacterial Infection Curtailment in Porcine Epithelial Cell using Enrofloxacin loaded Poly(lactic-co-glycolic) acid (PLGA) Nanocarriers. 9th SNO conference Nov12-Nov13, 2020, Cyberspace-Virtual.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Astete, C. E., J. U. De Mel, S. Gupta, Y. Noh, M. Bleuel, G. J. Schneider, C. M. Sabliov. 2020. Lignin graft poly(lactic-co-glycolic) acid biopolymers for polymeric nanoparticle synthesis. ACS Omega. 5 (17): 98929902. https://doi.org/10.1021/acsomega.0c00168.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Byrne, C. E., C. E. Astete, M. Vaithiyanathan, A. T. Melvin, M. Moradipour, S. E. Rankin, B. L. Knutson, E. C. Martin, C. M. Sabliov. 2020. Novel Lignin-graft-PLGA Drug Delivery System Improves Efficacy of MEK1/2 Inhibitor in Triple Negative Breast Cancer. Nanomedicine. 15(10): 9811000. doi: 10.2217/nnm-2020-0010.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Shakiba, S.; Astete, C.E.; Cueto, R.; Rodrigues, D.F.; Sabliov, C. M.; Louie, S. M. Asymmetric flow field-flow fractionation (AF4) with fluorescence and multi-detector analysis for direct, real-time, size-resolved measurements of drug release form polymeric nanoparticles. Submitted to Journal of Controlled Release.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Shakiba, S.; Astete, C. E.; Paudel, S.; Sabliov, C. M.; Rodrigues, D. F.; Louie, S. M., Emerging investigator series: polymeric nanocarriers for agricultural applications: synthesis, characterization, and environmental and biological interactions. Environmental Science: Nano 2020, 7, (1), 37-67.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Louie, S.M.; Shakiba, S.; Astete, C. E.; Sabliov, C. M.; Rodrigues, D. F. Sensitive and Accurate Real-Time Determination of Drug Release Rates from Polymer Nanoparticles by Asymmetric Flow Field - Flow Fractionation. 2nd Pan-American Nano Conference, Mar 2020, Aguas de Lindoia, SP, Brazil.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Shakiba, S.; Astete, C. E.; Sabliov, C. M.; Rodrigues, D. F.; Louie, S.M. Capturing Release Rates from Polymeric Nanoparticles by Asymmetric Flow Field-Flow Fractionation (AF4) with Fluorescence Detection. Sustainable Nanotechnology Organization (SNO) Conference, Nov 2020, Virtual.
|
Progress 05/01/19 to 04/30/20
Outputs
Target Audience:In this project, two Ph.D. students continued to participate in the project, of which one is a female engineering student. During the summer, the PD was involved in the UH summer camp for STEM high school students. In this summer camp, the PD prepared hands on activities about nanomaterials and the experiments related to the project to generate excitement and interest to the STEM students. In total, there were 44 high school students participating in the UH summer camp and in the outreach activities developped by the PD. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two Ph.D. students were hired to work on the project. The first Ph.D. is mainly working in objective 2 and assisting in objective 4, where the technique A4F-MALS-FLD-UV-RI is being developed to analyze the behavior of both nanoparticles in complex environmental conditions, such as the simulated gut reactor of pig. The second Ph.D. student is mainly working on objective 1 and 3 and assist with objective 4. Objective 1 is being accomplished by the LSU team. In addition to the training and professional development of the Ph.D. students in this reporting period, 44 high school students benefited of the findings in this project at the UH summer program. How have the results been disseminated to communities of interest?The results of this work have been presented at national and international conferences as well as invited talks, such as: Pannano 2020 in Brazil, USDA Grantees Meeting, and atIowa State University. The results have also been disseminated in the graduate classroom at UH, by the PD through the development of a teaching module in bioreactors, as presented in the last progress report. Two manuscripts were published, and two others are currently being prepared for publication from the results obtained in this reporting period. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, the focus will be mainly on performing the MOI in the PLGA and Lignin nanoparticles and finalize the MIC for the lignin NPs with E. coli and Salmonella. We will also perform the analysis of reactive oxygen species for the lignin nanoparticles that is still missing to understand their toxic properties. These final experiments will allow us to finalize the Objective 1 and have enough data for a publication in a scientific journal. In addition to work on Objective 1, we expect to also conclude the experiments of Objective 2 by finalizing the development of the method under simple (DI water) conditions and start experiments with Pig slurry to determine the suitability of the method to quantify antibiotic release from the nanoparticles for complex systems. In the next report period, we expect to obtain preliminary results for Objective 3 by investigating the effects of the antibiotic-loaded NPs on the gut microbiome and proliferation of antibiotic resistance genes in the gut bioreactor. We have setup already the reactor and optimized some of the procedures to be done in this Objective, we expect to be able to obtain preliminary data for this Objective for the next reporting period.
Impacts
What was accomplished under these goals?
In this reporting period, further progress were made to complete objectives 1 and 2. In the last progress report, we were able to successfully synthesize PLGA (ENRo) NPs designed for prolonged delivery of enrofloxacin by a single emulsion evaporation technique and preliminary toxicity experiments were performed. The new findings of this reporting period are described below: Objective 1:Synthesize, characterize, and evaluate the cytotoxicity of naked and antibiotic-loaded nanoparticles (NPs) In this task, instead of zein, lignin nanoparticles were produced (as explained in the last progress report, we were unsuccessful in encapsulating the antibiotic in the zein nanoparticles). A lignin-graft-PLGA biopolymer was synthesized with alkaline lignin (ALGN) at 1:2 w/w ALGN: PLGA ratios. H-NMR and FTIR confirmed the conjugation of PLGA to LGN. The ALGN-PLGA biopolymer was used to form nano delivery systems suitable for entrapment and delivery of enrofloxacin. The ALGN-PLGA NPs with entrapped enrofloxacin were small in size (92±2 nm, PDI of 0.254±0.012), and negatively charged (-65±3 mV). Small-Angle Scattering (SAS) data showed that particles encompass a relatively smooth surface and a compact spherical structure with distinct core and shell. The particles were freeze-dried and shipped to the University of Houston for further testing. In this reporting period, in addition to the synthesis of PLGA and Lignin nanoparticles produced by LSU and shipped to UH, two activities of drug delivery were investigated, which involved the investigation of their antibacterial activity and cytotoxicity. The initial assessment of toxicity was done for both nanoparticles with porcine epithelial cells (IPEC-J2). The results showed concentration-dependent cytotoxicity using the MTT assay. The PLGA(ENRO) NPs of enrofloxacin equivalent concentration beyond 0.16 mg/L were observed to be toxic.While for enrofloxacin loaded lignin nanoparticles imposed cytotoxicity at minimum concentration of 0.12 mg/L, which demonstrated that PLGA(ENRO) nanoparticles wereless toxic than Lignin( ENRO). In the case of the antimicrobial investigation, the minimum bacterial inhibitory concentration (MIC) was investigated with the nanocarriers based on the highest concentration that would not induce cytotoxicity to IPEC-J2 cells and would ensure more than 50% of pathogenic E. coli O157: H7 inactivation. The concentration of 0.14 mg/L of PLGA(ENRO) NPs was selected as the desired concentration for infection curtailment experiments. The concentration of 0.8 mg/L (enrofloxacin equivalent) of Lignin (ENRO) was observed to have an inhibitory concentration for E. coli O157: H7 inactivation. In vitro infection experiments were conducted incubating IPEC-J2 cell lines and pathogenic E.coli using the methodmultiplicityof infection (MOI) with a ratio of 1:10 and 1:100. The optimization of this experiment was done initially with PLGA(ENRO). In both MOI, PLGA(ENRO) NPs demonstrated great infection curtailment activity compared to free enrofloxacin drugs.The MOI for Lignin(ENRO) is still being analyzed, and therefore, it is not reported here. As a result of the MOI, the infection curtailment efficacy was higher for PLGA(ENRO) NPs at longer incubation time (24 h) compared to shorter incubation times (4 h) where free enrofloxacin was observed to be more effective. The higher infection prevention efficacy of PLGA(ENRO) NPs at longer incubation times was postulated for NPs being able to enter the intestine cells via endocytosis and release the drug slowly to prevent infection. To confirm this hypothesis, the synthesized PLGA(ENRO) NPs and Lignin (ENRO) were modified by fluorescently labeling with tetramethylrhodamine (TRITC). These labeled nanoparticles were incubated with IPEC-J2 cells and observed in confocal laser microscopy (CLSM). The results clearly showed that both nanoparticles enter the cells. So far, we have used E. coli O157: H7 as a model pathogen for curtailment of infection, further work will focus on curtailment of infection involving Salmonella typhimurium as well for both nanoparticles. Objective 2:Develop advanced analytical methods to characterize transformations and biological interactions of antibiotic-loaded nanoparticles in complex media The antibiotic release profile from the polymeric nanoparticles (NPs) is a key factor in assessing their effectiveness. We developed an advanced method to obtain the release profile of enrofloxacin from ENRO-PLGA NPs by the use of asymmetric field flow fractionation (A4F) coupled with various detectors (UV, fluorescence (FLD), multi-angle light scattering (MALS), and dynamic light scattering (DLS)). Several fundamental advantages of the A4F method over traditional methods of characterization have been verified. First, the A4F method rapidly purifies the NPsin situof any released or dissolved compounds, and this enables more selective and reliable analysis of only the entrapped drug compared to traditional dialysis methods, where the lag time for the separation of the free and entrapped drug can lead to misleading interpretations of results. Hence, A4F is highly advantageous to characterize the release of entrapped drug when the background concentration of the free drug is high and when the release rate is rapid relative to the dialysis rate. Secondly, the NP can be measured in aqueous media matching the release media to preserve the size and structure of the NP, which is measured by the MALS and DLS detectors simultaneously to the release profile. An effect of temperature could also be easily identified through the A4F measurements, where a more rapid release was observed as the temperature approached and surpassed the glass transition temperature of the ENRO-PLGA NPs. A manuscript is currently in preparation for the method development. Future work will then apply the method to evaluate the effects of interactions in complex media, e.g. adsorption of biomolecules, on the release rate. Objective 3:Evaluate the effects of the antibiotic-loaded nanoparticles on the microbial diversity and development of antibiotic resistance in vitro gut reactors To understand the effects of enrofloxacin loaded PLGA nanoparticles and lignin nanoparticles on the microbial diversity we started the batch bioreactor setup. The fecal slurry from pig's gastrointestinal (GI) tract was collected from JJ Packing, Brookshire, TX and was carefully transported to UH under refrigeration. Upon arrival to the laboratory, the slurry sample was incubated for enrichment of the microbial community in anaerobic conditions to acclimate the slurry microbial community before the experiments can start. We are now in the process of standardizing a protocol to extract the DNA and RNA from the microbial community to improve the DNA and RNA extracted yields for further downstream analysis of sequencing and reverse transcriptase Real Time PCR. After standardization of the extraction protocol, we will further perform experiments to identify the changes in microbial abundance with and without presence of drug and drug loaded nanoparticles in the system using 16 S rRNA sequencing. We will also use real-time PCR (RT-PCR) to determine whether there is an increase in presence of drug resistant genes in the reactors containing the free drugs and the encapsulated drug.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Paudel, S., C. Cerbu, C. E. Astete, S. M. Louie, C. Sabliov, D. F. Rodrigues. 2019. Enrofloxacin-Impregnated PLGA Nanocarriers for Efficient Therapeutics and Diminished Generation of Reactive Oxygen Species. ACS Appl. Nano Mater. 2 (8): 5035-5043.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Shakiba, S., C. E. Astete, S. Paudel, C. M. Sabliov, D. F. Rodrigues, and S. M. Louie. 2019. Polymeric Nanocarriers: Synthesis, Characterization, and Environmental and Biological Interactions. Environmental Science: Nano. 1. DOI: 10.1039/C9EN01127G. Environ. Sci.: Nano, 2020, Advance Article. DOI:
10.1039/C9EN01127G
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2020
Citation:
Paudel, S. and Rodrigues, D.F. (2020) Biosynthesis of Metallic Nanoparticles by Extremophiles and Their Applications; in: Interfaces between Microbes and Nanomaterials , CRC Press/Taylor & Francis Group. (submitted)
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Astete, C. E., J. U. De Mel, F. S. Gonzales, S. Gupta, Y. Noh, M. Bleuel, G. J. Schneider, C. M. Sabliov. Lignin graft poly(lactic-co-glycolic) acid biopolymers for polymeric nanoparticle synthesis. ACS Omega. To be submitted.
- Type:
Journal Articles
Status:
Other
Year Published:
2020
Citation:
Byrne, C. E., C. E. Astete, M. Vaithiyanathan, A. T. Melvin, M. Moradipour*, S. E. Rankin, B. L. Knutson, E. C. Martin, C. M. Sabliov. Novel Lignin-graft-PLGA Drug Delivery System Improves Efficacy of MEK1/2 Inhibitor in Triple Negative Breast Cancer. Nanomedicine. To be submitted.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Sabliov, C. M. September 2019. Polymeric nanoparticles- a versatile delivery system in medicine and agriculture. Mechanical Engineering Seminar. Iowa State University. Aimes, IA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Louie, S. M., Shakiba, S., Astete, C. E., Sabliov, S., and Rodrigues, D. F. Asymmetric Flow Field-Flow Fractionation (AF4) Methods for Rapid Characterization of Polymeric Nanocarriers. USDA Grantees Meeting, Vanderbilt University, Nashville, TN, May 2019.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Paudel, S. and Rodrigues, D.F. Poly(lactic-co-glycolic) Acid (PLGA) Nanoparticle for controlled drug delivery: Synthesis, Characterization and Toxicity. USDA Grantees Meeting, Vanderbilt University, Nashville, TN, May 2019
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2020
Citation:
Paudel, S., C. Cerbu, C. E. Astete, S. M. Louie, C. Sabliov, D. F. Rodrigues. 2020. Enrofloxacin transporting PLGA nanocarrier for reduced ROS generation and efficient therapeutics. PANNANO 2020, Sao Paulo, Brazil.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Sofia K. Fanourakis, S. K.; Pe�a-Bahamonde, J.; Bandara, P.C.; Rodrigues, D.F. 2020. Nano-based adsorbent and photocatalyst use for pharmaceutical contaminant removal during indirect potable water reuse. npj Clean Water (2020) 3:1 ; https://doi.org/10.1038/s41545-019-0048-8
|
Progress 05/01/18 to 04/30/19
Outputs
Target Audience:In this project, two Ph.D. students were recruited to work on the project, of which one is a female engineering student. In addition to the Ph.D. students, a female undergraduate student (Odalys Najera) is being mentored. She is the recipient of the UH Provost's undergraduate research scholarship (PURS). She is investiating the impact of nanoparticles on the photo-degradation of antibiotics. The PI has also incorporated last semester in her graduate class CIVE 6391 - Environmental Engineering Microbiology a module in bioreactors to teach the students how bioreactors can be related to the real gut environment. They learned how to set up a bioreactor and how to perform the proper calculations for a bioreactor. Changes/Problems:Justification for replacing ZNPs with LNPs Two types of particles were initially proposed in the study, 1. poly(lactic-co-glycolic) acid nanoparticles, and 2. zein nanoparticles, both biodegradable. PLGA NPs with entrapped enrofloxacin were synthesized and tested without any encountered issues. Zein polymeric nanoparticles were synthesized both empty and with entrapped enrofloxacin in this reporting period. The PLGA NP mean diameter was in the 150-165 nm (by dynamic light scattering) before freeze drying. Unfortunately, the zein polymeric nanoparticles increased in size after freeze drying up to 400 nm and became very polydisperse (PDI >0.5). Attempts were made to overcome the size increase by adding trehalose (cryoprotectant) or excess surfactants (polyvinyl alcohol), but we were not able to obtain a monodisperse zein polymeric nanoparticles (PDI<0.2) after resuspension in buffer solutions. The aggregation during freeze-drying is expected to impact nanoparticles properties (stability, drug release, degradation profiles) of importance in future studies of this project, hence we looked for an alternative that would still be plant-based like the zein nanoparticles. We propose to replace zein nanoparticles with lignin-based polymeric nanoparticles, newly developed over the past year with support from NSF EPSCoR. These biodegradable and renewable particles are appealing because they are small in size 75 to 125 nm, depending of the amount of lignin used. More importantly, these new nanoparticles are very mono dispersed PDI<0.15 even after freeze-drying when resuspended in water or buffers. The lignin based polymeric nanoparticles offer a better alternative to the protein-based zein nanoparticles for stability and release studies of importance to this project. What opportunities for training and professional development has the project provided?Two Ph.D. students were hired to work on the project. The first Ph.D. will mainly work in objective 2 and assist in objective 4, where the techniqueA4F-MALS-FLD-UV-RI is being developped to analyze the behavior of both nanoparticles in complex environmental conditions, such as the simulated gut reactor of pig. The second Ph.D. student will mainly work on objective 3 and assist with objective 1 and 4. Objective 1 is being accomplished by LSU. Most of the work done in this reporting period was done with the PLGA nanoparticles. In addition to the training and professional development of the Ph.D. students in this reporting period, 11 graduate students also benefitted from a new teaching module in the graduate class developped by the project PD at UH. In addition to the graduate students receiving training, one undergraduate student was also engaged in this project to do research. How have the results been disseminated to communities of interest?The results of this work have been presented at national conferences, such as: the Sustainable Nanotechnology Conference, the Gordon Conference on Nanoscale Science and Engineering for Agriculture and Food Systems, as well as the Sloan Foundation Workshop on Nanochemistry. The results have also been disseminated in the graduate classroom at UH, by the PD through the development of a new teaching module in bioreactors. Two manuscripts are currently being prepared for publication from the results obtained in this reporting period. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, the focus will be mainly on the synthesis and characterization of the novel lignin nanoparticles. We will initially investigate the minimum inhibitory concentration of these nanoparticles toward E. coli O157 and Salmonella typhimurium to understand the toxic effects (if any) of these new nanoparticles. We will also determine the cytotoxicity of the NPs to gastrointestinal (GI) epithelial cells from swine. We have already obtained the epithelial cells from swine and we also have created the frozen stock of cells. Using the MIC, we will also determine the mutation rates and frequencies caused by the pure antibiotic and by the antibiotic encapsulated with PLGA and Lignin nanoparticles. In addition to these investigations, we will also continue to develop advanced analytical methods to characterize transformations and biological interactions of antibiotic-loaded nanoparticles in complex media. The asymmetric flow field - flow fractionation with online multi-angle light scattering, fluorescence, UV-vis absorbance, and refractive index detection (A4F-MALS-FLD-UV-RI), will continue to be established for the purpose of monitoring NP fate (e.g., degradation and antibiotic release) and the biological interactions of the antibiotic-loaded NPs in complex GI samples. For the extension and outreach component, LSU will share the findings of this project via the Co-PI Sabliov and Astete, with the LSU Agricultural Center extension agents. The county agents will be provided with materials they will further share with the farmers to educate them on advantages, risks, and challenges of nano-enabled antibiotic delivery systems for prevention and treatment of livestock.
Impacts
What was accomplished under these goals?
In this reporting period, most of the work was done with the poly(lactic-co-glycolic) acid (PLGA) nanoparticles. PLGA (ENRo) NPs designed for the prolonged delivery of enrofloxacin were made by a single emulsion evaporation technique. The findings of this reporting period are described below: Characterization of the PLGA NPs The PLGA(ENRO) Nps showed a spherical shape with narrow size distribution and mean size of 102±2.6 nm. The polydispersity index (PDI) and zeta potential were observed to be 0.095±0.021 and -32±3 mV at pH 7.3. With PDI of 0.095, the particle is considered nearly monodispersed. Absence of aggregates indicated a uniform local distribution of the particles synthesized. Zeta potential of highly negative values signifies high repulsive force, which prevents the agglomeration and enhances the stability of the nano-delivery system. With the zeta potential of -32.3 mV, long-term stability of nanoparticle is expected. The drug entrapment efficiency (EE) and loading capacity of synthesized PLGA(ENRO) NPs was 43.8±8.3% and 14.1±2.7 µg/mg of Nps. Development of A4F method for advanced nanoparticle characterization The release profile of the ingredients from polymeric nanoparticles (NPs) is one of the determining factors in assessing their effectiveness. We developed an advanced method to obtain the release profile of enrofloxacin from ENRO-PLGA NPs by the use of asymmetric field flow fractionation (A4F) coupled with various detectors (UV, fluorescence (FLD), multi-angle light scattering (MALS), and dynamic light scattering (DLS)). One advantage of this method, in comparison to traditional methods (e.g., degrading the polymeric matrix and quantification of ingredients inside), is preserving the NPs. Therefore, the size distribution or degradation of the NPs can be determined simultaneously with the antibiotic release profile. After an initial burst release, the ENRO-PLGA NPs show a slow release profile at room temperature, but the size of the NPs does not change significantly during 6 hrs. These observations, as well as constant UV area during the run, suggest that diffusion is the release mechanism for these particles (not degradation). Another important result obtained by the A4F method is that temperature significantly affects the release profile of enrofloxacin, while other factors like stock concentration, dialysis, and pH have minimal effects. We also demonstrated that the A4F method is capable of separating the ENRO-PLGA NPs in samples containing protein (BSA), enabling simultaneous evaluation of interactions of biomolecules with the NPs or released antibiotic. In future work, separation of NPs in more complex media, as well as their interactions with different macromolecules, will be investigated. Determination of Reactive Oxygen Species One of the major parameters to be studied in the development of nanoparticle is its ability to generate reactive oxygen species (ROS). We studied the generation of major ROS such as H2O2, OH•, and 1O2 in the newly synthesized PLGA nanoparticles. We observed significant generation of H2O2 by native enrofloxacin. However, loading the enrofloxacin drug onto PLGA nanoparticles reduced the generation of H2O2. The insignificant amount of OH•, and 1O2 was detected for both native enrofloxacin and enrofloxacin loaded nanoparticles. These results showed that PLGA has negligible ROS production and can even reduce the ROS production of certain antibiotics, such as the enrofloxacin. This reduction on ROS has implications in the toxicity of the nanoparticles, which could lead to a much safer nanoparticle for applications in drug delivery. Antimicrobial Activity Enrofloxacin has been reported to act against a wide range of bacteria and is concentration dependent, its clinical efficacy is determined by dose and type of bacteria. We exposed native enrofloxacin and PLGA(Enro) to E.coli and S. aureus, a Gram-negative and Gram-positive bacteria. Native enrofloxacin and PLGA(Enro)'s antibacterial activity was compared. For the Gram-negative bacteria, E.coli, minimum inhibitory concentration (MIC) value was 23% smaller for PLGA(Enro) than enrofloxacin alone. However, higher dosing was required to observe similar MIC value for S. aureus. The antibiotic activity was still very effective, even after encapsulation of the antibiotic. Another observed result from this study was that PLGA(ENRO) NPs had the capability to inhibit visible growth of E.coli for five days. This result with the NP gives multiple advantages over the classical parenteral administration of antibiotics, as the treatment procedure becomes easier, with just one administration every 5 days. Moreover, the increased bioavailability of the Enro nanoparticles could eventually minimize enrofloxacin excretion, thus contributing to minimizing the overall antibiotic resistance phenomenon. Cell Viability Assay Cytotoxicity is considered an important aspect of any nanoparticle study to determine the applicability of nanomaterial as a carrier for antibiotic delivery. For a cargo delivery, using nanocarrier to be considered safe, it is essential to ensure that nanoparticle does not harm mammalian cellular structure and can receive a status of generally regarded as safe (GRAS). In vitro cell viability investigation demonstrated approximately 10% loss in cell viability by PLGA(ENRO) and ENRO. At the highest concentration tested, 100 µg/ml for PLGA(ENRO) and ENRO, loss in cell viability was observed to be 10% and 12% respectively. Whereas, loss in cell viability by PLGA(ENRO) and ENRO for concentration less than 100 µg/ml were insignificant. The small loss in cell viability can be accredited to the ability of ENRO to produce ROS causing oxidative stress in cells. PLGA NPs are inert and are not toxic to the cells. Conclusions Encapsulation of enrofloxacin in PLGA nanoparticles by single emulsion evaporation technique was successfully done. The release of enrofloxacin from PLGA NPs was biphasic, i.e. there was a initial burst release, followed by a uniform release later. Encapsulation of ENRO into PLGA NPs was able to preserve the therapeutic efficacy. Enrofloxacin by itself generated a significant amount of ROS (H2O2), however, the encapsulation of the drug into PLGA NPs reduced the ROS generation from the antibiotic. Thus, encapsulation of enrofloxacin in PLGA nanoparticles provided a viable option for safer administration of the antibiotic to mammalian cells.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Shakiba, S., Astete, C.E., Sabliov, C., and Louie, S.M. Characterizing the fate of drug-loaded nanoparticles in complex biological media. Sustainable Nanotechnology Organization (SNO) conference, Washington, DC, November 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Louie, S.M. Nanoparticle interactions with macromolecules and small organic molecules. Oral presentation at an invited workshop. Sloan Foundation Workshop on NanoChemistry of Indoor Environments, New York City, NY, July 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Astete, C.E., Cerbu, C., Sondh, S.K., Shakiba, S., Paudel, S., Louie, S.M., Rodrigues, D.F., and Sabliov, C.M. Fate of antibiotic loaded nanoparticles in digestive systems and effects on livestock gastrointestinal microbiome. Gordon Research Conference (GRC) on Nanoscale Science and Engineering for Agriculture and Food Systems, Mount Holyoke, MA, June 2018.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Kacso, T., I. O. Neaga, A. Erincz, C. E. Astete, C. M. Sabliov, R. Oprean, E. Bodoki. November 2018. Zein-based polymeric delivery systems with programmed wear down for sustainable agricultural applications. SNO Annual Conference. Washington DC, USA.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Pe�a-Bahamonde, J.; Nguyen, H.N.; Fanourakis, S.; Rodrigues, D.F. 2018. Recent advances in graphene-based biosensor technology with applications in life sciences. Journal of Nanobiotechnology 16 (1), 75.
|
|