Synergistic Action of Electroporation and Controlled Release of Nanoparticle Additives to Promote Pathogen Lysis

NON-TECHNICAL SUMMARY: At present, there are no technologies available for low energy, reliable and rapid destruction of pathogens without the alteration of food freshness and quality. Even though thermal heating or pasteurization is the most common method used for pathogen elimination and food preservation processes, it suffers from high energy cost and low shelf life. Hence, more fundamental studies exploring the mechanism and rate of bacterial cell lysis (disruption of the bacterial cell wall) are needed to develop a cost effective and reliable technology for food safety. This research is aimed at the improvement of food safety and biosecurity through the incorporation of preservative-loaded, biocompatible nanoparticles into liquid and paste foods. The research targets a low temperature, low electric field and synergistic method for pathogen lysis in food products. The outcomes of this 'proof of concept' study will guide the development of a new technology for the elimination of pathogens for improved and contamination-free food and agricultural products.
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APPROACH: The research program utilizes a high precision microfluidic platform which integrates various components such as microscope detection, flow, dilution and applied electric fields. The microfluidic platform will allow measurements of E. coli lysis kinetics, on-line control over lysis conditions and integration fluorescence detection, high electric fields and nanoparticle dilution. From the fundamental point of view, such a platform is ideal for exploring the transport and reaction kinetics parameters due to fast diffusion times and precise flow and electric control. In addition, nanoencapsulation of lysozyme in chitosan-coated calcium-alginate nanoparticles proceeds by the ionic gelation method, intended to preserve enzyme activity. Since alginate and chitosan are edible, biocompatible polymers, the health risk of introducing nanoparticles of such materials into food is minimal. Fluorescence microscopy, UV absorption spectroscopy and nuclear magnetic resonance imaging (NMRI) will be used in combination for detailed characterization of the encapsulation efficiency, controlled release rates, and subsequent enzyme activity. The research program unifies the multiple skill set of two investigators.
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PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: The major outputs of this project are (a) new protocols for synthesis and characterization of chitosan and chitosan-coated alginate gel nanoparticles, (b) new insights into bacteria-nanoparticle clustering interactions, and (c) new technology for rapid bacteria lysis with 20-fold reduction in applied electric field strength. These outcomes were validated using characterization of nanoparticle size and zeta potential distributions by a combination of zetasizing and TEM, and through observation of bacteria-nanoparticle interactions and measurements of the fraction of bacteria lysed via phase contrast and fluorescence microscopy. Additional advances include the microencapsulation of proteins and development of a microfluidic platform for high precision testing. A key product of this research work is fundamental knowledge about nanoparticle-bacteria interactions and the impact of nanoparticles on bacteria lysis in an applied electric field. The results of the investigation can guide the development of a new, low energy technology for the elimination of pathogens in food and agricultural products. During the project period, four graduate students and three undergraduate students in the two investigators' laboratories have been thoroughly trained in the relevant experimental techniques and exposed to the excitement of independent research. We presented the results of this study at two conferences: the American Chemical Society annual fall meeting (August, 2007) and the Biomedical Engineering Society annual fall meeting (October, 2007). Dissemination of results also occurs through the publications listed below and through our laboratory web sites (Brown University and Columbia University). PARTICIPANTS: The advances of this exploratory project were accomplished in the laboratories of the two investigators. <br>(1a) Nina C. Shapley (Columbia University), Principal Investigator, led the research activities involving nanoparticle synthesis, characterization, and microencapsulation, and also coordinated the efforts of the two laboratories. <br>(1b) Anubhav Tripathi (Brown University), Co-Principal Investigator, led the research activities involving observations of bacteria-nanoparticle interactions and bacteria lysis, and development of a microfluidic platform. <br> (2a) Mona Utne Larsen (Columbia University), Graduate Student, performed the majority of the experiments, data analysis, and interpretation of results regarding nanoparticle synthesis, characterization, and microencapsulation. <br> (2b) Chunguang Xi (Columbia University), Graduate Student, assisted with particle imaging efforts. <br> (2c) Stephanie McCalla (Brown University), Graduate Student, assisted with observations of bacteria-nanoparticle interactions. In addition, several students contributed significantly to the project without being supported directly by the USDA award. <br> (3a) Mathew Seward (Brown University), Graduate Student, performed the majority of the experiments, data analysis, and interpretation of results regarding bacteria-nanoparticle interactions and developed the bacteria lysis experiments. (3b) Andrea Jones (Brown University), Undergraduate Student, performed bacteria lysis experiments and data analysis. > (3c) Alexander Luryi, (Brown University), Undergraduate Student, performed bacteria lysis experiments and data analysis. (3d) Pamela Sundelacruz (Columbia University), Undergraduate Student, synthesized and characterized nanoparticles by zetasizing. <br> The project provided professional development opportunities for the graduate and undergraduate student participants. The students learned new experimental techniques and gained experience in working as part of a highly interdisciplinary team. In addition, the students honed their communication skills while preparing multiple publications for journal submission and conference presentations for national meetings. Columbia University and Brown University are the two collaborating institutions. <br>TARGET AUDIENCES: The main target audience for the Changes in Knowledge achieved by the project is research workers in related areas of study. The results obtained here have been communicated by multiple publications submitted to relevant journals and conference presentations at two national meetings. An additional target audience consists of the student participants of the project, where the goal is to broaden the students' experience regarding laboratory, teamwork, and speaking and writing skills.
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IMPACT: 2007/01 TO 2007/12 <br>
The success of the synergistic technology for pathogen lysis depends on several connected effects, including preferential nanoparticle adsorption on the bacterial cell surface and enhanced electric field-membrane effects due to the presence of nanoparticles. The exploratory study investigated each of these challenges in depth. The following Changes in Knowledge are the main Outcomes/Impacts of this exploratory project. <br>1) Rapid aggregation and microbial arrest occur in Escherichia coli PBS suspensions when chitosan nanoparticles with highly positive zeta potential are added. For strongly negative nanoparticles, no clusters form, while aggregates are small and loose at intermediate conditions. This work establishes the dominant role of electrostatic attraction in bacteria-nanoparticle interactions. In addition, larger and denser clusters form as the nanoparticle or bacteria concentration increases. Moreover, the nanoparticles form significantly larger bacterial clusters (by approximately a factor of 8) than an equal mass of molecular chitosan in solution, indicating the high efficiency of nanoscale flocculants. Experimentally, chitosan and chitosan-alginate nanoparticles are synthesized by ionic gelation and the zeta potential and size distribution are characterized. Nanoparticles are mixed with dilute suspensions of BL21(DE3) derivative fluorescent E. coli in PBS solutions. The bacteria-nanoparticle interactions are observed by phase contrast and fluorescence microscopy. Initial protein encapsulation efforts involving nanoparticles also highlight the importance of opposite charge electrostatic effects. The bacteria-nanoparticle aggregation effect unveiled here promises a rapid separation method for pathogen elimination, and for use in sample preparation for pathogen detection. <br>2) When chitosan nanoparticles with highly positive zeta potential are mixed with E. coli prior to bacteria lysis by an applied electric field, there is an order of magnitude increase in the fraction of the bacteria population lysed compared to the same system in the absence of nanoparticles. The presence of the nanoparticles allows the electric field strength to be an order of magnitude lower (i.e. O(100 V/cm) instead of O(2000 V/cm), the high field intensity typically found in the literature) while achieving the same extent of lysis. Experimentally, E. coli cells are mixed with various concentrations of chitosan nanoparticles, treated with a range of electric field strengths, cultured, and then live/dead populations for each case are compared. These observations indicate extremely strong nanoparticle-cell membrane effects in an electric field that enable synergistic lysis at remarkably low applied voltage and hence low energy conditions. 3) Other results include successful synthesis and characterization of chitosan and chitosan-coated calcium-alginate nanoparticles. In addition, a microfluidic platform has been developed for testing. Therefore, the results of the investigation support the development of a new, low energy technology for the elimination of pathogens for improved and contamination-free food and agricultural products.