Team:SSTi-SZGD/Applied Design

What's the problem & Our vision?

After more than twenty years of rapid development, China's cosmetics market, known as "beauty economy", has become the world's second largest cosmetics market after the United States [1] . According to the "analysis of market size and development trend of China's cosmetics industry in 2017" [2] , CAGR (Annual compound growth rate) of China's cosmetics industry was 9.1% in the past five years, far higher than the global aver age CAGR of 4.1%. It is estimated that the market size of China's cosmetics industry could reach 490.6 billion in 2019. (see Fig.1) From the current situation of the cosmetics industry, China's cosmetics market has a broad prospect.

Fig.1. Analysis chart of development trend of Chinese cosmetic market.

At present, as the living standard improves, demand for anti-aging products is growing, and the use of natural polymer ingredients in cosmetics to promote skin repair and wrinkle removal increases every year. Among the commonly used polymer ingredients, hyaluronic acid (HA), a type of macromolecular mucopolysaccharide, is extensively used in cosmetics due to its excellent permeability, moisturizing, and biocompatibility properties, so as to improve skin humidity and achieve the effects of skin rejuvenation.

The biological activity and water retention effects of hyaluronic acid is directly related to its relative molecular weight (Mr). High and middle molecular weight HA (Mr≥1-2×106 Da) with great viscoelasticity, lubrication, and moisturizing effects are widely used in cosmetics, while low molecular weight HA (Mr≤1×104 Da) has potential anti-tumor effects, and promotes wound healing and immune regulation [3] .

HA has prominent effects on promoting tissue remodeling and reducing scar formation, thereby it is a star material in cosmetic products. Most products on the market containing HA and its derivative sodium hyaluronate are facial masks and cream formulations. Although these products make skin appear softer and smoother, due to the barrier effect of skin epidermis, it is difficult for active ingredients to efficiently penetrate through the stratum corneum and release into the deeper skin for a long-term effect, unless using surgical methods (i.e. injection). Surgical methods, on the other hand, usually result in facial bruising, skin necrosis and intense pain, which could seriously affect facial nerve system and result in rigid facial expressions. Injection may also trigger immune reactions, resulting to temporary swelling, headaches, mild nausea and minor numbness [4] . Therefore, searching for an alternative method for effectively and safely delivering HA into skin dermis has become necessary.

In addition, compare to high molecular weight HA, low molecular weight HA can be more efficiently absorbed across skin epidermis. However, its moisturizing and viscoelastic properties are far inferior to high molecular weight HA [5] .

Thereby, we endeavor to develop a new cosmetic application method that combines the advantages of high and low molecular weight HA, as well as eliminating the tedious and potentially dangerous operation of injection and surgery, to achieve lasting effects of skin rejuvenation and wrinkle removal.

Comparison of products in the market

Based on the understanding of the current cosmetic market, and refer to relevant resources, we came across a new method that has the potential to replace the traditional injection approach -- "microneedle delivery system", which represents a new stage of technology development in cosmetics industry. The main advantage of microneedle system is that it can pierce through the skin in a non-invasive and painless way, and help with the efficient delivery of macromolecular ingredients. It is safer than surgery or injections, while being more effective than ordinary moisturizing cream or masks.

The current available microneedle delivery products are made of silicon wafer, metal materials or natural sugars [6] . Silicon wafer based microneedles, fabricated using MEMS (micro-electromechanical system) microfabrication technology, are sharper than those made of polymer, sugar or metal [7] . It is conducive to penetrate through the skin epidermis and reducing discomfort. However, because silicon absorbs proteins, when use in deeper skin, white blood cells are drawn to the material to induce stress response on the affected area [7] . Microneedles made of silicon wafer (see Fig.2) also have drawbacks such as complex preparation procedures, high processing cost and requirements, and fragile proposition. The nature of the material can lead to easy breakage of the tips under the skin, combining with a low biocompatibility property, tip residues can result to skin infection [8] .

Metal based microneedles (see Fig.2) , generally made of stainless steel, titanium, nickel, etc., are fabricated by laser cutting, laser etching and wet etching [8] . Metal material, usually used in combination with serum, can cause painful sensation when use, and frequent use may cause pore enlargement. Also, because metal-based microneedles are usually re-used, it may lead to the contamination of blood-transferrable diseases, i.e. AIDS, hepatitis and so on [8] . With regards to industrial production, metal based microneedle, with its relatively complex preparation requirement, is not prone to mass production. Sugar based microneedles are usually prepared from maltose, trehalose, sucrose, fructose, etc. These sugar materials require stringent preparation and storage conditions. If not stored properly, they can absorb moisture easily and result to reduced hardness [9]

In recent years, the preparation of microneedles with natural and degradable polymer biomaterials has become a hot topic. (see Fig.2) There have been a variety of raw materials, i.e. polyvinyl alcohol, polylactic acid, chitosan, carboxymethylcellulose, HA, silk protein, etc. being used in microneedle fabrication [9] . Among them, HA, with its excellent moisturizing property, biocompatibility, and relatively simple preparation method, has become a popular microneedle biomaterial.

Fig.2.(A) silicon wafer microneedle, (B) metal microneedle, (C) macromolecule microneedle.

There are existing HA micro-needle eye patch products on the market, for example, Quanis(R) from Japan and Acropass(R) from South Korea. (see Fig.3) The manufacturing process for these products is air blast stretching technology. In this way, microneedles are of uniform shape, but with weak mechanical strength and short in needle size, which hamper the efficient penetration into the skin. In addition, these products are used in combination with the water retention serum to assist the better absorption of the serum. In this regard, microneedle is only used for its auxiliary function, which greatly undermines its value and potential.

Figure.3. Various types of microneedles in current market

How to change the situation?

We would like to formulate a new type of HA based microneedle with both the penetration function and moisturizing ability, that is, these microneedles are made of HA material with sufficient strength to pierce through skin epidermis, then release HA molecules to exert the water retention effect.

HA is one of the most functional macromolecules in nature. As an important part of the natural extracellular matrix, HA plays an important role in various biological processes. However, because of its easy degradation and poor mechanical properties, natural HA is not suitable for microneedle preparation. Compared with natural HA, chemical cross-linking of HA can efficiently enhance its stability, mechanical strength, in-situ swelling ability of the microneedle within skin, and decrease its biodegradability. Also, as cross-linking procedures result to a hydrogel like material with stable 3D structures (see Fig.4) , it is better suitable for crafting microneedles.

Fig.4. Hyaluronic acid cross-linking

At present, the mainstream chemical cross-linking agents for HA are diethylsulfone (DVS) and 1, 4-butanediol diglycidyl ether (BDDE). DVS, although widely used in R&D, has high cytotoxicity. Not only this toxic substance is likely to be accumulated in the body after implantation, but also it may affect normal tissue growth due to calcification of the implant. In comparison, BDDE is considered to be the mostly commonly used cross-linking agent in preparation of cosmetic HA fillers. BDDE is biodegradable, much less toxic and more reactive than DVS, thereby safer for biomedical applications.

How to achieve it?

  1. We engineered Bacillus subtilis bacteria to produce HA (HMW and LMW separately) by transgenic methods. Once the secreted raw HA are separated and purified by ethanol precipitation, HA molecules are freeze-dried to form powder as a starter material for the following cross-linking procedures.
  2. We cross-linked LMW HA with less toxic and better reactive crosslinking agent, BDDE, to obtain a hydrogel form of HA. This hydrogel containing HA nanoparticles possesses stable structure, improved mechanical strength and would be less sensitive to enzymatic degradation.
  3. Cross-linked HA and HMW HA molecules are blended in certain ratio to obtain a mixture, which is then poured into the a specially designed mould for microneedle fabrication. Our mould for microneedle fabrication is a 3D printed photosensitive resin, which is a high-strength and high-temperature proof material with features of energy saving, low pollution, fast curing speed and high production efficiency.

Production formulation

HA crosslinking process

Cross-linked HA (cHA) reagent solution was prepared by mixing 200μl of 1, 4-butanediol diglycidyl ether (BDDE) into 9.8ml of 0.25M NaOH (pH13). Approximately 1.0g of HA powder was added to the cHA reagent solution and mixed thoroughly at 40 ℃ for 2h. The prepared hydrogel was ground, squeezed out and screened with 170 mesh sieve to get smaller particles.

Preparation of HA microneedle

HA molecules and crosslinked HA hydrogel are mixed and dissolved in ultra-pure water in different proportions, and the viscous polymer solution is mixed uniformly with magnetic agitator at room temperature. Apply the viscous solution to the mold under vacuum pressure and use the blade to peel off the remaining solution. After curing at room temperature overnight, the transparent microneedle patch is successfully prepared. (see Fig.5)

Fig.5. Flow chart of preparation of microneedle

Characterization of microneedle

In order to make HA more effectively absorbed by the skin, we crafted a mould with cone shaped needles. This particular shape is believed to provide the needles with sufficient mechanical strength and stability, which enables HA entering into the deeper skin efficiently. (see HA-MN results) Given sufficient reaction time, eventually the entire patch would be absorbed completely by the skin with no waste produced. In case of shorter reaction time, the remaining patch can be disposed as ordinary kitchen garbage to undergo natural degradation.

In the future, we plan to craft the HA microneedle patches into several shapes to accommodate different needs.

Shape Purpose
Triangle Used for frown lines, crow's-feet
Crescent Used for nasolabial folds or surrounding eye area
Rectangle Used for forehead wrinkles

Product Design Considerations

We have three aspects to consider in the product design process: product safety, product material and product effectiveness.

Firstly, product safety aspect is related to the source of HA. Most of HA material and its derivatives used in the market are of Streptococcus origin . Streptococcus strain is a conditional pathogenic bacterium that poses potential human and environmental risks. Because of it endogenous endotoxin, its by-products, i.e. HA, are likely to carry the toxin. In the current project, we use food-grade B.subtilis to produce HA. This bacterium strain is well studied with genome fully sequenced, has no endotoxin, and requires low nutrition to reduce production cost. Therefore the raw material for our product is of less biosafety concern.

Secondly, product material refers to the cross-linking process of HA. Our HA microneedle using cross-linked low molecular weight (~17 kDa) HA to obtain a hydrogel. Because the high molecular weight (~1500 kDa) HA has longer polymer chain structure, which when cross-linked, results to more coiled structure formation and greater self-association. This somewhat “packed” structure is unstable and unable to form a homogeneous hydrogel. In contrast, low molecular weight HA, because of its shorter polymer chain structure, has a greater chance of forming inter-molecular bonds, so that hydrogel with stable 3-D structure is more likely to be formed[10]. (see Fig.6)

Fig.6. The cross-linking state of hyaluronic acid

Thirdly, the product effectiveness is decided by microneedle fabrication technology. In order for the microneedle to enter the skin epidermis, needle shape are needed to be effectively produced to assist HA entering the skin. Currently we used resin material to craft the mould, although needles can reasonable mechanical strength, the film is difficult to demould due to impermeability. After consulting the industry experts and relevant resources, we found that a material called polydimethylsiloxane (PDMS), which is a polymer organosilicon compound, may facilitate a better demoulding process [11] . Therefore, we plan to try the PDMS mould in the next round of product fabrication testing.

Evaluation of Impact

Areas for Improvement
Environmental Impact
Biosafety Impact

Microneedle technology provides a way to deliver active ingredients into deeper skin in a non-invasive and painless way. Currently, there are some challenges in the design and preparation of macromolecular microneedles. Because of the elastic characteristics of the skin, the latter will first deform and "surround" the microneedle when the microneedle pierces the skin, so the force applied to the microneedle may be greater than the force required to pierce the microneedle itself. In addition to skin characteristics, the geometrical shape, length, density and sharpness of microneedles influence the puncture performance of microneedles. And how to ensure the safety of the use of microneedles and how to improve the drug load and the efficiency of drug delivery.

Because HA is a natural polymer material that is highly biocompatible, its products can be degraded by a variety of microorganisms in the soil or humid environment after being abandoned. Therefore, it is an environmental friendly material, and its degradation products can form a benign cycle of nature after being returned to the nature, which is conducive to environmental protection. HA macromolecules are prone to degradation. Studies have shown that degradation is mainly caused by hydrolysis and reactive oxygen species on hydroxyl. Ultraviolet, ultrasonic, Co60 ray and some metal ions can degrade HA [12] .

In our final product, there is no bacteria at all. Although we use B.subtilis to manufacture HA, B.subtilis itself is a food-grade safe strain and HA is expressed extracellularly. After expression, we carry out the steps such as separation and purification of HA through ethanol precipitation to remove the impurities, including the remaining bacterial cells, from the cultural media, which is then heat inactivated (or autoclaved) to ensure the full elimination of live bacteria (refer to our Integrated HP ). Before releasing to the market, the final product, HA microneedle, will also undergo a series of safety testing processes by cosmetic authorities. These tests include microbial contamination testing, new material testing, chemical and physical testing, etc. to ensure that our product is safe enough to be used by customers. Therefore, GMO application in our project is for manufacturing purpose only, it is very unlikely that the final product would incur any GMO concerns.

Social Value

In general, beauty needs are relatively concentrated, and for the middle-age population, effective beauty products can be used to delay aging. It may give people younger looking appearance therefor give them more confidence and security. On the other hand, for younger generation, a more vibrate skin condition may help improving their physical and mental well-being, in turn may also help with their career development thus social status. Overall speaking, cosmetics and skin care products may have a positive influence on social and economic development.

Reference

[1] Yansi Li. Development status and strategy analysis of Chinese cosmetics industry[J].Modern Economic information,2015(04):394-396.

[2] The next explosion in the beauty and cosmetics industry [DB/OL]. http://biz.jrj.com.cn/2018/08/28174825014773.shtml.

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

[4] What are the risks of wrinkle removal by injection? [DB/OL]. https://jingyan.baidu.com/article/0964eca2d5f7f58284f53676.html.

[5] Jing Xue, Shanyue Huang. Study on the function and application of different relative molecular weight hyaluronic acid[J]. Chinese Journal of Dialysis and Artificia,2010,04:22-25.

[6] Kim Y C, Park J H,Prausnitz M R. Microneedles for drug and vaccine delivery[J].Pharmaceutics,2015,7(3):90-105.

[7] Koen van der Maaden, Wim Jiskoot. Microneedle technologies for (trans)dermal drug and vaccine delivery[J]. Journal of Controlled Release,2012,161(02):645-655.

[8] Dongyin Shi. Innovative development of medical microneedle[R]. Beijing: Beijing Yanjing electronics co. LTD, 2016:1-3.

[9] Leiqi Wang. Design, developing and microstructure control of polymer microneedle[D].Beijing: Beijing University of Chemical Technology,2017:1-6.

[10] Jie Zhang, Senfeng Ma. Matrix Materials and Their Composites for Dissolvable Microneedle Construction: a Review[J]. Material review,2017,31(19):129-134.

[11] Qilei Wang, Dandan Zhu, Yang Chen. A fabrication method of microneedle molds with controlled microstructures[J]. Materials science,2016:135-142.

[12] Yun He. Degradation of hyaluronic acid[D]. Nanjing, Nanjing university of science and technology, 2010.1-8.

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