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Project Description

Our project aims to develop a more effective and efficient way to treat chronic wounds - reduce the time of healing, alleviate pain, and reduce cost. Our designed E.coli is able to produce molecules that can prevent bacterial infection and promote the healing of chronic wounds. We construct the plasmids with the coding sequence of both Yebf - LL37 and SDF1α-ELP proteins. SDF1α-ELP is a fusion protein with SDF1α and ELP (elastin-like peptide), which the introduction of ELPs can enable the fusion protein to automatically form into nanoparticles that increase the stability of the molecule.

Chronic Wounds

Chronic wounds, unlike most wounds, do not heal in an orderly set of stages or in a predictable amount of time. Wounds that do not heal within three months are often considered as chronic wounds[1], which in some cases may never heal or take years to do so. These wounds cause patients severe emotional and physical stress while creating a significant financial burden on patients. In addition, bacterial infection and colonization can damage tissue by causing a greater number of neutrophils entering the wound site[2]. Due to the immense time needed for chronic wounds to heal, antibiotic-resistant bacteria have more time to develop[3]. Infection can not only aggregate chronic wounds but also lead to gangrene, loss of the infected limb, and death of the patient.

Antimicrobial Peptide

Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), are part of the innate immune response found among all forms of life. These peptides are potent, broad spectrum antibiotics. Human antimicrobial peptide LL-37 is the only member of Cathelicidin AMP family found in the human body. LL-37 plays a vital role in decreasing skin infection, due to its ability to kill Gram negative and Gram positive bacteria, enveloped viruses, fungi, and even transformed or cancerous cells. It can also neutralize endotoxin and reduce the damage caused by LPS infection.

SDF1α & ELP

Stromal cell-derived factor1 (SDF1) is a key mediator of the wound healing response. It can recruit endothelial progenitor cells that proliferate and differentiate into mature vascular endothelium, which contributes to the revascularization and re-epithelialization of skin wounds[4].

However, the clinical application of such bioactive peptides is limited because they would be rapidly degraded in the wound environment by proteases. In response to this issue, a solution needs to be found in order to promote the effect of such bioactive peptide.

Elastin-Like-Peptide: Nanoparticles for Wound Healing

Elastin-like peptides (ELPs) are non-immunogenic, non-pyrogenic and biologically compatible derivatives of tropoelastin with pentapeptide repeats of Valine-Proline-Glycine-(Xaa)Glycine, where Xaa can be any natural amino acid except Proline. ELPs can reversibly form into nanoparticles and become insoluble above a transition temperature which may be due to the repeated sequence of the pentapeptide (when the sequence is repeated 50 times, the transition temperature is around 40℃). ELPs can be also be expressed as fusion proteins together with a wide range of bioactive peptides. ELP-based fusion proteins, by forming nanoparticles, have been shown to protect biomolecules from proteolysis and ELPs can act as "drug depots" that supply the biomolecules over an extended period of time[4].

Experiment Result

Experiments conducted in diabetic mice indicated that SDF1-ELP nanoparticles in vivo were significantly superior to that of free SDF1. When applied to full thickness skin wounds in diabetic mice, wounds treated with SDF1-ELP nanoparticles were 95% closed by day 21, and fully closed by day 28, while wounds treated with free SDF1, ELP alone, or vehicle were only 80% closed by day 21 and took 42 days to fully close. In addition, the SDF1-ELP nanoparticles significantly increased the epidermal and dermal layer of the healed wound, as compared to the other groups. These results indicate SDF1-ELP fusion protein nanoparticles are promising agents for the treatment of chronic skin wounds[4].]

Description of Improved parts

We improved the part BBa_K875009, human antimicrobial peptide LL-37, by fusing it with a motor protein yebf (The improved part is BBa_K1892004). Therefore, LL-37 can be carried out through cell membranes by yebf and is much more convenience for use. We have also proved with our experiments that BBa_K1892004 is effective against Gram-positive and Gram-negative bacteria, thus to characterize BBa_K875009 further. We documented our characterization in the Experience Page of BBa_K875009.

Reference

[1]Mustoe T (March 17–18, 2005). "Dermal ulcer healing: Advances in understanding" (PDF).Tissue repair and ulcer/wound healing: molecular mechanisms, therapeutic targets and future directions. Paris, France: EUROCONFERENCES. Archived from the original (PDF) on October 27, 2005.

[2]Snyder, Robert J. (2005). "Treatment of nonhealing ulcers with allografts". Clinics in Dermatology. 23 (4): 388–95.doi:10.1016/j.clindermatol.2004.07.020.PMID 16023934.

[3]Halcon, L; Milkus, K (2004). "Staphylococcus aureus and wounds: A review of tea tree oil as a promising antimicrobial". American Journal of Infection Control. 32 (7): 402–8.doi:10.1016/j.ajic.2003.12.008. PMID 15525915.

[4]YANG Hao*(2012). "Prokaryotic expression and purification of gene multimers from human antimicrobialpeptide LL-37 and its variant based on sequence reformation" 1004-5503（2012）05-570-04

[5]Agnes Yeboah, Rick I. Cohen, ReneaFaulknor, Rene Schloss, Martin L. Yarmush, Francois Berthiaume, The development and characterization of SDF1α-elastin-like-peptide nanoparticles for wound healing, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.04.020

[6]S.A. Eming, T. Krieg, J.M. Davidson, Inflammation in wound repair: molecular and cellular mechanisms, J Invest Dermatol, 127 (2007) 514-525.

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