20.109(F12): WF Blue pre-proposal

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==Introduction==
==Introduction==
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A biofilm, often formed in living systems, is an aggregate of microorganisms embedded in a matrix of extracellular polymeric substances (EPS)[3]. Medically, biofilms account for over 80 percent of microbial infections in the body such as infections in the middle ear, urinary tract, and lethal infections such as cystic fibrosis. Bacteria within biofilms exhibit increased antibiotic resistance (up to 1000-fold) which presents a problem in conventional antibiotic treatments[4]. As an alternative method to control biofilm-associated infections, bacteriophage have been successfully engineered to produce EPS degrading enzymes in vitro[2]. Despite this promising evidence, the use of engineered bacteriophage therapy is in the preliminary stages and has yet to be established for clinical usage. In order to develop a therapeutic treatment against biofilms in the body, it is crucial to test these bacteriophages under physiological conditions. The use of the engineered bacteriophages in vivo would benefit from a carrier to deliver the phage to targeted biofilms. Graphene oxide (GO) functionalized with water-soluble and biocompatible polymers has been studied as a tool for drug delivery and observed to enhance GO biocompatibility, suggesting its potential as a carrier for delivering and releasing phages[5,6]. Therefore, the use of GO in biofilm degradation could be a novel component to phage therapy and progressing the field towards clinical applications.
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A biofilm, often formed in living systems, is an aggregate of microorganisms embedded in a matrix of extracellular polymeric substances (EPS)[3]. Medically, biofilms account for over 80 A biofilm, often formed in living systems, is an aggregate of microorganisms embedded in a matrix of extracellular polymeric substances (EPS)[3]. Medically, biofilms account for over 80 percent of microbial infections in the body such as infections in the middle ear, urinary tract, and lethal infections such as cystic fibrosis. Bacteria within biofilms exhibit increased antibiotic resistance (up to 1000-fold) which presents a problem in conventional antibiotic treatments[4]. As an alternative method to control biofilm-associated infections, bacteriophage have been successfully engineered to produce EPS degrading enzymes in vitro[2]. Despite this promising evidence, the use of engineered bacteriophage therapy is in the preliminary stages and has yet to be established for clinical usage. In order to develop a therapeutic treatment against biofilms in the body, it is crucial to test these bacteriophages under physiological conditions. The use of the engineered enzymatic bacteriophages in vivo would benefit from a carrier to deliver the phage to targeted biofilms. Graphene oxide (GO) functionalized with water-soluble and biocompatible polymers has been studied as a tool for drug delivery and observed to enhance GO biocompatibility, suggesting its potential as a carrier for delivering and releasing phages[5,6]. Its antibacterial properties and minimal cytotoxic effects on mammalian cells have also been established [6].Therefore, we believe the use of GO in biofilm degradation could be a novel component to phage therapy and progressing the field towards clinical applications.
==Idea==
==Idea==

Current revision

20.109(F12): Laboratory Fundamentals of Biological Engineering

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Contents

Investigators

20.109(F12) Pre-Proposal: Degrading biofilms by engineered enzymatic bacteriophage via functionalized graphene oxide

Project Summary

Bacterial biofilms, composed of microorganisms embedded in a matrix of extracellular polymeric substances (EPS), exhibit antibiotic resistance, presenting a problem in controlling biofilm-associated clinical infections[1]. Previous research on biofilm control has shown phage therapy to be effective in disrupting the biofilm[2]. We propose to use modified graphene oxide (GO) as a carrier to facilitate the delivery of engineered bacteriophages in order to improve the degradation of biofilms.

Introduction

A biofilm, often formed in living systems, is an aggregate of microorganisms embedded in a matrix of extracellular polymeric substances (EPS)[3]. Medically, biofilms account for over 80 A biofilm, often formed in living systems, is an aggregate of microorganisms embedded in a matrix of extracellular polymeric substances (EPS)[3]. Medically, biofilms account for over 80 percent of microbial infections in the body such as infections in the middle ear, urinary tract, and lethal infections such as cystic fibrosis. Bacteria within biofilms exhibit increased antibiotic resistance (up to 1000-fold) which presents a problem in conventional antibiotic treatments[4]. As an alternative method to control biofilm-associated infections, bacteriophage have been successfully engineered to produce EPS degrading enzymes in vitro[2]. Despite this promising evidence, the use of engineered bacteriophage therapy is in the preliminary stages and has yet to be established for clinical usage. In order to develop a therapeutic treatment against biofilms in the body, it is crucial to test these bacteriophages under physiological conditions. The use of the engineered enzymatic bacteriophages in vivo would benefit from a carrier to deliver the phage to targeted biofilms. Graphene oxide (GO) functionalized with water-soluble and biocompatible polymers has been studied as a tool for drug delivery and observed to enhance GO biocompatibility, suggesting its potential as a carrier for delivering and releasing phages[5,6]. Its antibacterial properties and minimal cytotoxic effects on mammalian cells have also been established [6].Therefore, we believe the use of GO in biofilm degradation could be a novel component to phage therapy and progressing the field towards clinical applications.

Idea

Phage therapy is a promising method to target harmful bacteria in biofilms given increased bacterial resistance to antibiotics. However, the use of phage therapy in clinical settings is limited by several factors, including the current lack of knowledge on phage toxicity on mammalian cells. To eradicate biofilms without harming surrounding cells, we will use the previously engineered T7 bacteriophage introduced by Lu and Collins that produce enzymes to degrade the adhesin protein of the EPS [2]. In order to progress towards in vivo application of this engineered T7 for eliminating biofilms, we hope to achieve three goals in this project.

First, we want to determine the cytotoxicity of engineered T7 bacteriophage on human endothelial cells as an initial measure of bacteriophage compatibility with physiological non-bacterial cells. Through examination of cell morphology and viability, we hope to observe minimal cytotoxic effects of the engineered bacteriophages on human cells. Secondly, we will attempt to deliver the phage to biofilms by using graphene oxide as a carrier. Carbon nanomaterials including graphene have been explored as potential drug delivery carriers. It has been shown that graphene oxide can be functionalized with polyethylene glycol (PEG) and was used to facilitate targeted drug delivery to cancer cells[5]. We hope to assemble this modified graphene oxide with the engineered bacteriophages in order to allow targeted delivery of the phage to biofilms. Lastly, we will test the efficacy and toxicity of the GO-Phage assembly on a biofilm layer in vitro. Accomplishments of these steps would allow us to make progress towards the ultimate, long-term goal of using the GO - phage in clinical applications in the human body.

Figure

References

1. N. Hoiby, T. Bjarnsholt, M. Givskov, S. Molin, O. Ciofu. Antibiotic resistance of bacterial biofilms. International Journal of Antimicrobial Agents Vol 35, 322-332

2. T. K. Lu and J.J. Collins. Dispersing biofilms with engineered enzymatic bacteriophage. Proceedings of the National Academy of Sciences Vol 104. No 27.

3. M.E. Cortes, J.C. Bonilla, R.D. Sinisterra. Biofilm formation, control and novel strategies for eradication. Science against microbial pathogens: communicating current research and technological advances.

4. National Institute of Health. Research on Microbial Biofilms. 2002, 12.20.

5. Z. Liu, J.T. Robinson, S.M. Tabakman, K. Yang, H. Dai. Carbon materials for drug delivery& cancer therapy. Materialstoday Vol 14, 316-323

6. Y. Pan, N.G. Sahoo, L. Li. The application of graphene oxide in drug delivery. Expert Opin. Drug Delivery (2012) 9(11):1365-1376

7. J. Azeredo and I.W. Sutherland. The Use of Phages for the Removal of Infectious Biofilms. Current Pharmaceutical Biotechnology, 2008, 9, 261-266.

8. A. Gajewicz, B. Rasulev, T.C. Dinadayalane, P. Urbaszek, T. Puzyn, D. Leszczynska, J. Leszczynski. Advancing risk assessment of engineered nanomaterials: Application of computational approaches. Advanced Drug Delivery Reviews Vol 64, 1663-1693 L. Gravitz. Turning a new phage. Nature Medicine 18, 1318-1320.

9. E.M. Ryan, M.Y. Alkawareek, R.F. Donnelly, B.F. Gilmore. Synergistic phage-antibiotic combination for the control of Escherichia coli biofilms in vitro. FEMS Immunology & Medical Microbiology Vol 65 Issue 2.

10. T. Kaur, N. Nafissi, O. Wasfi, K. Sheldon, S. Wettig, R. Slavcev. Immunocompatibility of Bacteriophages as Nanomedicines. Journal of Nanotechnology Vol 2012, Articile ID 247427
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