Team:Tufts/app scenarios

Phage Encapsulation in Silk Bandages

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Abstract
Background and Significance
Silk in Therapeutics
Silk and Bacteriophage
Approach
Notebook
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Abstract

Recent developments in the field of biomaterials have demonstrated the efficacy of silk in stabilizing temperature-sensitive compounds like antibiotics, enzymes, and vaccines. Moreover, silk has shown potential in wound healing applications due to its compatibility with human tissues and its ability to dissolve in a directed fashion. These unique characteristics make silk a promising platform for the deployment of bacteriophages to treat chronic wounds and combat the growing threat of antibiotic-resistant bacteria. Tufts Synthetic Biology envisions a biomedical product consisting of a silk film embedded with a lyophilized cocktail of bacteriophage-targeting pathogens responsible for chronic wound infection. This system could be utilized most readily as a bandage, with applications ranging from long-term burn management in the hospital setting to rapid treatment of penetrating wounds on the battlefield. The encapsulation of phage is also a viable antiseptic option for the silk scaffolds used in tissue regeneration. While this system has extensive possibilities for external and internal treatment options, this proposal specifically targets methicillin-resistant Staphylococcus aereus (MRSA) skin infections.

Background and Significance

Bacteriophage Therapy

“The ability of phage to replicate exponentially and kill pathogenic strains of bacteria indicates that they should play a vital role in our armamentarium for the treatment of infectious diseases.”

Perhaps the most critical problem in modern medicine is the increased development of pathogenic bacteria resistant to most, if not all, available antimicrobial agents . The increasing occurrence of such antibiotic resistant strains has bred significant concern that a return to the “pre-antibiotics” era is a very real possibility. As such, expansion of alternative anti-infection therapy options has developed into one of the highest research priorities in biotechnology and modern medicine. When first-line and second-line antibiotic options become limited by resistance, healthcare providers are must resort to antibiotics that may be more toxic, are frequently more expensive, and often less effective . Research has shown that patients with resistant infections are often far more likely to die and those that do survive face significantly longer hospital stays, delayed recuperation, and long-term disability.

A treatment option considered prior to the discovery and spread of antibiotics, bacteriophage therapy has an extensive and controversial history but has recently returned as a research subject of intensive speculation. Bacteriophage therapy faces difficulties of regulation, bacterial resistance to phages, limited host range, manufacturing, delivery, and the side effects of bacterial lysis . Recent advances in macromolecule delivery, biotechnology, synthetic biology, and bacterial diagnostics provide the potential to overcome past obstacles. Thus far, utilization of bacteriophage for the prevention of Listeria monocytogenes growth on meat products has been approved in Europe and the United States and various academic and commercial sectors are currently attempting to successfully bring bacteriophage therapy through the clinical stage. Such efforts must be joined by approaches both rigorous and varied to successfully bring bacteriophage therapy from “the bench to the bedside.”

Methicillin-resistant Staphyloccocus aereus (MRSA)

Each year, in the United States alone, a conservatively estimated 2 million individuals are infected by antibiotic-resistant bacteria. At least 23,000 people die as a direct result of these infections, many others succumb to associated complications. Beyond the human cost, antibiotic-resistant infections add considerable and avoidable costs that the U.S. estimates to be as high as $20 billion in excess healthcare costs and additional $35 billion due to lost productivity each year (in 2008 dollars).

One of the most prevalent of these antibiotic-resistant infections is methicillin-resistant Staphylococcus aureus – more commonly known by the acronym MRSA. Infecting over 75,000 individuals each year, MRSA is one of ten antibiotic-resistant bacteria that the Centers for Disease Control (CDC) has classified as a “Serious Threat” in their 2013 report. Until recently, MRSA was almost exclusively contracted in medical facilities but evolving strains have emerged in the community capable of causing severe infections in otherwise healthy people . These new strains of MRSA infections are classified as community-acquired (CA-MRSA) as opposed to their hospital acquired counterpart (HA-MRSA) . The majority of community-acquired infections are skin infections while HA-MRSA often begin as skin infections but develop into life-threatening bloodstream infections, pneumonia, and surgical site infections.
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Silk in Therapeutics

Silk proteins belong to a class of unique, high molecular weight, block copolymer-like proteins that have found widespread use in biomaterials and regenerative medicine. Silk boasts an impressive array of properties, including: self-assembly, robust mechanical properties, biocompatibility, and biodegradability, each of which may be enhanced through a variety of chemical modifications. Here, we focus specifically on the silk fibroin produced by the Bombyx mori silkworm, one of the most prominent silk proteins utilized for therapeutic and biomedical applications.

Silk fibroin, one of the strongest natural fibers, is formed with glycine-alanine-glycine-alanine-glycine-serine (GAGAGS) repeats which self-assemble into anti-parallel β–sheet structures. These β–sheets are highly crystalline and extensively cross-link the protein, giving the silk its robust mechanical properties. This structure can be further modified with specialized processes in order to manipulate mechanical strength, degradation, and aqueous processibility.

Silk fibroin in both the raw and regenerate forms has been extensively utilized in biomedical applications such as sutures, coatings for cell culture, drug delivery matrices, 3D scaffolds for ligament, bone, cartilage, fat, and vasculature engineering, and, most recently, to model brain function in vitro . Silk has been approved by the Food and Drug Administration (FDA) for various medical applications and recent research has successfully demonstrated their efficacy for orthopedic purposes. These biomaterials are particularly advantageous for orthopedic purposes as they are fully biodegradable, avoid temperature sensitivity, evoke limited inflammatory response, provide a better mechanical match than metallic alternatives, and can be functionalized with antibiotics or other therapeutics to enhance repairs or reduce infections at the surgical site . Silk fibroin has also been developed for skin grafts and shown to accelerate wound healing, improve adhesion and spreading of normal human keratinocytes and fibroblasts, and upgrade the growth and development of skin tissue.

Perhaps the most exciting property of silk fibroin is that the self-assembly process of the proteins ensures the material can be functionalized for controlled release and drug delivery, a quality incredibly beneficial for in vivo structures. These interactive biomaterials not only offer a controlled and predictable therapeutic release, but have been proven to thermostabilize enzymes, antibiotics, and vaccines across a wide range of temperatures at and above 25oC for several months without or with minimal loss of efficacy.
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Silk and Bacteriophage

Silk and bacteriophage are both the focus of extensive research and, while neither have discovered the limits of their application, they have yet to be concurrently examined or utilized. While bacteriophage therapy could be developed and employed in a manner similar to that of antibiotics and other therapies, encapsulation within silk fibroin has the potential advantage of significantly extending shelf-life at or above room temperature while maintaining efficacy. This has not only economic benefits – drug loss results in the loss of billions each year to specialized transport and storage – but also increases the potential for global impact by enabling distribution to developing nations with limited means and infrastructure.

Benefits

- Easy storage; silk stabilizes biologically active substances at higher temperature
- Effective against antibiotic resistant bacteria
- Compatible with human tissue; will dissolve predictably
- Engineered phage can avoid bacterial lysis to prevent endotoxin release
- Specificity means beneficial microflora remain unharmed

Challenges
- Safety; some phages can mutate to attack off-target bacterial strains
- Public perception of bacteriophage; comparisons to human viruses
- FDA regulations
- Generation of a sufficiently diverse phage cocktail
- Bacterial resistance to phage; CRISPR-based immunity
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Approach

This project aims to create a silk bandage functionalized with bacteriophage for methicillin-resistant Staphylococcus aereus skin infections that are thermostable at or greater than 25oC for several months.

Bacteriophage Selection

Many naturally occurring bacteriophages can be quite effective against infectious bacteria and would be worthwhile inclusions as part of a phage cocktail. These can include:
- Sb1 staph-phage (MRSA)
- PAK-P1 (Pseudomonas aeruginosa)
- C1 (group C Streptococci)
- B30 (Streptococcus agalactiae)

The strains above are well-characterized and would likely be effective in clinical applications, but this is nevertheless a partial list. More bacteria should be targeted, and with a much wider range of phage. Phage diversity can be achieved with conventional methods for screening of phage from natural locations such as sewage. Although these isolated phages can be utilized without genetic modification, endotoxin release by lytic phages may pose a problem. The removal of lysis genes and addition of exogenous genes to confer lethality would prove most effective.

Engineering Phage

Engineering a more potent bacteriophage involves two main components.

1. Selecting a bacteriophage platform into which exogenous genes can be inserted
Non-lytic phage, although they do not kill bacteria as aggressively or propagate after infection, would be preferable since endotoxin release would be minimalized and they would not mutate and replicate uncontrollably. However, lytic phage can be adapted to function like a non-replicating strain if their holin and/or endolysin genes are knocked out. Various non-lytic phages which can be readily modified to include exogenous genes are:

- Pf3 (P. aeruginosa)
- M13 (E. coli)
- P954 (S. aureus, created by removing endolysin gene)

2. Identifying exogenous genes which confer lethality or increased antibiotic sensitivity to bacteria

- Endonucleases that cleave bacterial chromosomal DNA (BglII in PA01)
- Expressing lethal genes gef and chpBK via M13 phage (E. coli)
- Expression of lexA3 gene to prevent the repair of DNA damaged induced by antibiotics
- Introducing a CRISPR system to cleave/degrade specific regions of the bacterial genome
- Using small regulatory RNAs to silence antibiotic-resistance genes or genes necessary for cell survival

Specific bacterial strains and their complementary phage may require significant modifications, but the basic workflow can be accomplished with standard microbiology techniques. Although working with an established phage platform can be easier, greater phage diversity will be generated by isolating bacteriophage from the environment. This is typically done by centrifuging sewage samples and then growing the supernatant in LB broth containing the bacteria strain of interest. After a sufficient incubation time, the cells are centrifuged and the supernatant is filtered. This filtrate is then grown on an agar plate with the same bacteria of interest to observe plaque formation. From areas of plaque clearing or preventative bacterial growth, the desired phage are isolated. The endolysin and holin genes of the phage can be identified via sequencing. The preferred method of knocking out one or both of these genes is homologous recombination. Any standard plasmid (with the appropriate origin of replication) can be used as a vector. The 5’ and 3’ ends of the endolysin gene, interrupted by a reporter gene, will be inserted into this vector. This vector is transformed into infected bacteria. Those which are unable to form plaques underwent homologous recombination to remove the endolysin gene.

More extensive genetic modifications complicate the process, especially if the genes introduced to the phage are lethal to the bacteria which are intended to produce the phage particles. This can be avoided by changing codon assignments in the host bacterial strain such that CRISPR-Cas9 or RNA silencing approaches which are lethal to a wild-type bacterium but have no effect on the genetically modified host. For phages which are modified to synthesize lethal proteins, inserting an RNA silencing system into the host will ensure that the phage can replicate within the cell without killing it. Inserting these lethal genes can be done through homologous recombination the same way that endolysin genes are knocked out. Conversely, for phages which have circular genomes, it may be possible to insert a gene via conventional DNA assembly and electroporate it into a host. Additionally, yeast can be a suitable organism for cloning during the construction of phage genomes since they will not express phage genes would might otherwise harm bacterial hosts.

Recovering the lysis-deficient phage can be accomplished by lysing bacteria with small glass beads, pelleting the debris, and filtering the supernatant to obtain purified phage. Similarly, the host bacteria can be transformed with a plasmid harboring an endolysin gene under an inducible promoter to achieve the same effect.
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Capturing Phage in Silk Film

Once the phage particles have been recovered from the host, a solution of them can be mixed with a silk fibroin aqueous solution. This mixture is then freeze-dried. This lyophilization process results in a thin silk film which stably incorporates phage and will prevent their degradation even at high temperatures. A sufficiently large film of this nature can be utilized as a bandage. To achieve better delivery of these phage particles, the incorporation of phage into a silk hydrogel could also be attempted using a very similar procedure.