Team:SSTi-SZGD/Description

Background

According to the “Analysis of the development scale of China's cosmetics industry and the forecast of the development prospect of online channels” released by the Chinese Industrial Information Online, the overall retail sales statistics showed that the proportion of retail sales of cosmetic products continued to increase in China [1] . On the other hand, wit h the development of the national economy and improvement of living standard, it is likely that the annual spending on cosmetic products continues to incline in the coming future. (see Fig.1)

Fig.1. The proportion of cosmetics consumption continues to increase

In the past decade, demand for skin rejuvenation and anti-aging products is growing, and the use of natural polymer ingredients in cosmetics to help with skin repair and wrinkle removal is increasing every year. Hyaluronic acid (HA) is one of the natural polymer ingredients widely used in cosmetic products. (see Fig.2) HA is a mucopolysaccharide composed of disaccharides unit of N-acetyl glucosamine and glucuronic acid polymerizat ion, has excellent permeability, biocompatibility and water solubility, and is excellent to improve skin humidity and skin rejuvenation.

The effect of HA is associated with its molecular weight; extensive researches have demonstrated that different molecular masses HA are associated with unique and irreplaceable functions. High molecular mass (HMW) HA has important physiological roles in living organisms owing to its associated unique viscoelastic and rheological properties, as well as being extensively used in cosmetics and pharmaceutics; low molecular mass (LMW) HA (include oligosaccharides) has been commonly used in angiogenesis, wound healing, cell differentiation and proliferation, apoptosis, and tumor cell migration [2] . In addition, HA oligosaccharides can be easily absorbed by the body and serve as precursors for the synthesis of HMW HA molecules. Therefore, efficient production of HA oligosaccharides with narrow size distribution has an important implication in cosmetics, medicine, and food industries.

Fig.2. Molecular model of hyaluronic acid

Existing problems

With importance roles in medical and cosmetics applications, commercial production of high yield and cost - effective HA of multiple molecular masses has become a sought-after field for industrial researchers. Currently, besides traditional animal extraction and chemical synthesis approaches, microb ial biosynthesis is a common approach for HA production, given the advances in microbiology, industrial fermentation and genetic engineering in recent years. The majority of microbial biosynthesized HA in the market is through the use of S.zooepidemicus species, which, despite relatively high production rate, is a gram-positive bacterium with high risk of pathogenicity and fewer genetic manipulation techniques available. HA produced in this way has the potential to carry endotoxin. Therefore construction of heterologous HA-producing strains with known genetic background, i.e. B.subtilis or Lactic acid bacteria, provides an attractive alternative.

Microbial biosynthesis of HA is generally HMW HA that requires additional steps to become LMW HA. Currently, LMW HA is mainly produced by degradation of HMW HA via physical and chemical methodsHA [3] . Physical methods using heating, ultrasonic or radiation that induces HA to fracture randomly. Physical degradation is simple and the product is easy to recycle, However, products generated by these methods have a broad range of molecular weights, making it difficult to obtain HA oligosaccharides with specific molecular weights. Also, pitfalls of physical methods include easy discoloration of HA (heating), low efficiency (ultraviolet and ultrasonic), and poor stability of the products. Common chemical methods for HA oligosaccharide synthesis include hydrolysis and oxidative degradation using strong acid or strong alkali. Chemical degradation result in chemical pollution and complex reaction that affect the properties of HA and making the purification process rather difficult, as well as producing a large amount of wastewater. Another method, the enzymatic production of HA oligosaccharides with well-characterized hyaluronidases (HAase), is promising and attractive because of its unique advantages, such as mild operation conditions, high degradation rates and high product uniformity [4] . (see Fig.3)

Fig.3. HAase 3D structure model

Zooming to HA’s application in cosmetics, due to the barrier effect of skin epidermis, it is difficult for active ingredients, especially high molecular weight polymers, to efficiently penetrate the epidermis and release it under the skin unless surgery or injection is used. However, intolerable pain along with series of complications, such as inflammatory, temporary swelling, and facial stiffness, are common problems of surgical or injection methods. Alternative methods for efficient and safer delivering of HA across skin epidermis are required. (see Fig.4)

Fig.4. Cosmetics stay in the epidermis

How to solve problems

This year, our iGEM team proposed to construct recombinant B.subtilis strains that can secrete large amount of HA of both high and low molecular weights. While different molecular mass HAs are already of great commercial values, we also proposed to use the low molecular weight HA in the development of a novel form of cosmetic product - HA microneedles.

HA production

Given the existing problems, in order to ensure that our final product is safe, we select B. subtilis as the chassis, because it is not only a food grade safe bacterium (GRAS) whose genetics are fully decoded, but also has endogenous glycolytic pathway that synthesizes the two precursors ( UDP - GlcNAc and UDP - GlcA) for biosynthesis of HA. Due to the lack of HA synthase gene, wildtype B.subtilis cannot produce HA. Furthermore, a number of native genes (tuaD, gtaB, glmU, glmM, glmS) that regulate the biosynthesis of UDP - GlcNAc and UDP - GlcA are present in the glycolysis pathway of B. subtilis. Production level of HA in recombinant B. subtilis strain maybe elevated by elevating the expression level of these native genes.

In order to hydrolyze HA from high molecular mass to low molecular mass, we selected the Leech HA hydrolysis gene (LHyal), which has high substrate specificity and no trans-glucoside effect. By cloning into the recombinant B.subtilis strain secreting HA, hyaluronidase can be expressed and performs enzymatic hydrolysis on the high molecular weight HA molecules directly in the cell culture environment. By completing the above steps, we can obtain a single B.subtilis cell that produces large quantity of low molecular weight HA.(refer to )

HA microneedles

Through understanding the current cosmetic market and identifying the existing problems, we came across a new method - microneedles, which may replace the traditional injection approach to facilitate the delivery of HA into deeper skin. Microneedles have the advantages of being painless when deliver polymers into the skin, safer than surgery and injections, and more effective than ordinary cream or masks.

Because of the easy degradation of HA and poor mechanical properties, covalent cross-linking is necessary to improve stability of HA. We used BDDE, a cross-linking agent, to covalently cross-link HA to form hydrogel with stable 3D structure. In the same time, in order to facilitate the entering of HA into the skin, skin patch with multiple micro-sized needles are prepared using cross-linked HA and HA molecule mixture. A specially crafted microtemplate is used to solidify HA hydrogel mixture (refer to Applied design ). This proposed HA microneedle patch has the great potential in providing moisturizing and anti-wrinkle effects for the skin.

Project overview

Four key products from the project:

① High-molecular-weight purified HA product (MW>1000000 Da)
② Leech HA hyaluronidase enzyme product (58kDa)
③ Low-molecular-weight purified HA product(MW~10000-100000 Da)
④ HA microneedle patch

Reference

[1] Analysis of the development scale of China's cosmetics industry and the forecast of the development prospect of online channels. [DB/OL].

[2] Yueshan Huang, Jing Xue. Research on the function and application of HA with different molecular weights[J]. Chinese Journal of Dialysis and Artificia | Organs,2011(02):10-13.

[3] Tengfei Li. Basic and experimental research of low molecular weight HA by ultrasonic-chemical method[D]. Chongqing: Chongqing university,2011.1-9.

[4] Jin P, Kang Z, Zhang N, et al. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers [J]. Sci. Rep., 2014, 4: 4471.

Copyright © 2018 SSTi-SZGD All Rights Reserved.
Copyright © 2018 SSTi-SZGD All Rights Reserved.