Transdermal and moisturizing effects of novel supramolecular hya
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Annals of Experimental Biology

Research Article - Annals of Experimental Biology ( 2023) Volume 11, Issue 3

Transdermal and moisturizing effects of novel supramolecular hyaluronic acid

Haimiao Wang1, Zhongxia Jin2, Xia Lu3, Lei Wang4, Xiyin Sheng2, Shaocheng Wang2, Tongxun Xu2, Peng He2* and Chen Li1
1Department of Plastic and Reconstructive Surgery, First Medical Center, PLA General Hospital, Beijing, China
2Xiangpeng (Beijing) Biotechnology Co. Ltd., Beijing, China
3Department of Nuclear Medicine, Northern Jiangsu People’s Hospital, Yangzhou, China
4Department of Urology, Peking University Shougang Hospital, Beijing, China
*Corresponding Author:
Peng He, Xiangpeng (Beijing) Biotechnology Co. Ltd., Beijing, China, Email:

Received: 21-Apr-2023, Manuscript No. aeb-23-96740; Editor assigned: 22-Apr-2023, Pre QC No. aeb-23-96740 (PQ); Reviewed: 25-Apr-2023, QC No. aeb-23-96740 (Q); Revised: 01-May-2023, Manuscript No. aeb-23-96740 (R); Published: 12-May-2023


Hyaluronic Acid (HA) is an important macromolecular polymer widely existing in the skin, which can lock water molecules in the skin. Due to the barrier effect of skin stratum corneum, high molecular weight HA cannot enter the deep skin, while low molecular weight HA has the ability limited to maintain water. Nowadays, HA injection is widely used as a supplement method, which brings many disadvantages due to its invasiveness. Therefore, in this study, the ionic liquid induction technology was used to transform the macromolecular HA into the supermolecular state. Through in vitro transdermal experiment and human skin moisturizing experiment, it was proved that the super molecular state HA has better transdermal and skin moisturizing ability, which provide a new potential method for non-invasive exogenous supplementation of HA.


Hyaluronic acid, Supramolecular technique, transdermal absorption, moisturizing effect


Skin is the largest organ of the human body, and its basic structure can be divided into the epidermis, dermis, and subcutaneous tissue and epidermis is the outermost layer of skin. According to the differentiation and characteristics of keratinocytes, the epidermis is divided into 5 layers from outside to inside, namely stratum corneum, stratum lucidum (only exists in palm and sole), stratum granulosum, stratum spinosum, stratum basale, the basal layer is connected with dermis using basement membrane.

The skin provides a physical and physiological barrier to the human body, this barrier function mainly depends on the stratum corneum. The keratinocytes that make up the stratum corneum, which are filled with bundles of densely aggregated keratin, play an important role in the barrier [1]. It is because of this barrier effect, isolated a certain size of the material is into the human body through the skin. Studies have shown that only substances with a Molecular Weight (MW) of less than 500 Da, as well as lipophilic compounds, are generally able to penetrate the skin barrier [2].

The chemical name of hyaluronic acid is (1,4)-O-β D-glucuronic acid-(1,3)-2-acetamido-2-deoxy-β-D-glucose.It is a highmolecular linear glycan, a polymer formed by alternating N-Acetylglucosamine (GlcNAc) and Glucuronic Acid (GlcA) disaccharide units repeatedly (Figure 1), with the molecular formula (C14H20NO11Na)n , and the MW of disaccharide units is 401.3 [3]. The MW of HA molecules with different lengths of polymeric linear chains varies widely, ranging from 600 Da to 1,000 kDa [4].


Figure 1. Structural formula of hyaluronic acid

HA is widely involved in various physiological activities of the body, such as tissue homeostasis, cell proliferation, cell migration, cell differentiation, angiogenesis, tumor activity, and anti-apoptosis. Since Meyer and Palmer initially isolated HA from bovine vitreous in 1934, HA has been widely used in medical cosmetology, biomaterials, drug delivery, and prevention of adhesion after abdominal surgery [5-11].

Although HA is widely distributed in the human body, the most concentrated organ of HA in our body is still the skin, which is the endogenous polysaccharide with the highest concentration in skin and connective tissue and an important component of dermal Extracellular Matrix (ECM) [12]. Studies have shown that the content of HA in the skin accounts for more than 50% of the total HA in the body.HA has strong hydrophilicity, and HA in an aqueous solution can combine water molecules with more than 1000 times its mass; In addition, HA aqueous solutions are non-Newtonian fluids with good viscoelasticity and strain ability [13]. Therefore, HA contributes a lot to the morphological support of the skin, moisturizing, and 111 maintaining extensibility and elasticity [14].

With the age increasing and ultraviolet radiating, the HA content in the skin will continue to decrease, and then there will be relaxation, wrinkles, and other outcomes, resulting in a decline in skin barrier function [15,16]. Therefore, exogenous HA supplementation is of great significance in skin moisturizing and wrinkle removal, and facial anti-aging in medical cosmetology.

Due to the skin barrier effect, the High Molecular Weight (HMW) HA can only stay on the skin surface and cannot penetrate deep into the skin. Therefore, HA micro-needle injecting has become one of the main approaches to exogenous HA supplementation nowadays. However, many shortcomings attribute to its invasive characteristics. How to supply HA into the deep skin non-invasively has become a very meaningful research direction.

The creation of supramolecular chemistry is a breakthrough in the field of biology and chemistry in the last century, providing many valuable methods and tools for materials chemistry, physiology, medicine, and so on. At least six scientists have won Nobel Prizes for their work in supramolecular chemistry. Supramolecular chemistry is based on the existence of molecular assemblies and intermolecular forces, and different types of molecules can interact and self-assemble according to their different strengths, orientations, and dependence on distance and angle [17]. According to the principle of hyper molecular self-assembly, the interaction force between molecules can be used as a tool to assemble components or modules with specific structures and functions into new supramolecular compounds in a certain way [18]. These new compounds not only exhibit unique properties that individual molecules do not possess but also greatly increase the variety and number of compounds [19].

To make HA break through the limitation of the skin barrier, this study will use supramolecular technology to prepare HA molecules with different MWs into the supramolecular state in a specific ionic environment, and compare their transdermal effects respectively with non-supramolecular HA molecules, and evaluate their respective skin moisturizing effects on human skin (Figure 2).


Figure 2.Schematic diagram of the supramolecular hyaluronic acid transdermal principle

Materials and Methods

Experimental materials

HA powders of different MW(MW 10 kDa -100 kDa, 100 kDa -1,800 kDa, >1,800 kDa, 95.7%) were purchased from Huaxi Biotechnology Co., Ltd. (Beijing, China); Citric acid, potassium hydroxide, glycerol, panthenol, 1,3-propanediol, phenoxyethanol, 1,2-hexanediol, ascorbyl glucoside, p-hydroxy acetophenone, lipoic acid, carnosine, arginine, triethanolamine, DMSO, fluorescein amine were purchased from Sigma (St. Louis, MO, USA).

HA molecular fluorescence labeling experiment

Weigh a certain amount of HA powder with different MW and dissolve in ultrapure water respectively, adjust pH to about 4.5 with concentrated hydrochloric acid, weigh a certain amount of EDC and NHS, dissolve in DMSO, add them into HA solution after full dissolution, activate for 1h at room temperature, adjust pH to 7-8 with NaOH solution; After a certain amount of fluorescein amine was dissolved in DMSO solution, it was added into the activated HA solution sample to make the ratio of-COOH to-NH2 in the solution be 1:1, and the reaction was carried out for 24 hours at room temperature in the dark; Add 4 times volume of absolute ethanol to the labeled HA solution, react for 5 minutes, centrifuge at 3500 grams for 10 minutes, remove the supernatant, wash the precipitate twice with 3 ml absolute ethanol, centrifuge at 3500 grams for 3 minutes each time, dissolve the precipitate with ultrapure water, and store at room temperature in the dark.

Preparation of supramolecular hyaluronic acid

Adding a certain amount of citric acid into a potassium hydroxide solution to form a citric acid buffer solution system, adding a certain amount of glycerol, panthenol, 1,3-propanediol, phenoxyethanol, 1,2-hexanediol, ascorbic acid glucoside, p-hydroxy acetophenone, lipoic acid, carnosine, arginine and triethanolamine into the citric acid buffer solution, shaking and dissolving to obtain a supramolecular ion induction system; The fluorescence-dyed HA solution and that supramolecular ion induce system are mixed in equal volume and store at room temperature in a dark place.

Laboratory animals

The abdominal skin of the Bama miniature pig was cut immediately after being killed by anesthesia, the subcutaneous fat and connective tissue were carefully peeled off, washed with Normal Saline (NS) and placed in NS, and stored in a low-temperature refrigerator for later use. Thaw naturally before the test, soak in NS for 30 minutes, and blot dry with filter paper for later use. The procedures related to the laboratory animals involved in this study complied with the relevant provisions in the Guidelines for Ethical Review of Laboratory Animal Welfare issued by the General Administration of Pattern Supervision, Inspection, and Quarantine of China and the Standardization Administration of China.

Transdermal penetration of supramolecular HA in vitro

Firstly, the skin (pig skin) was fixed between the supply chamber and the receiving chamber of Franz diffusion cell, the stratum corneum of the skin faced the supply chamber, and the dermis side faced the receiving chamber;adding a receiving liquid into the receiving chamber, tightening the piglet skin and fixing the piglet skin with the receiving pool, adding a certain volume of receiving liquid (PBS) into the receiving chamber, exhausting air, and making the skin dermis layer closely contact with the receiving liquid;supermolecular HA sample and non-supermolecular HA samples with different MW are respectively added to that skin surface in the supply chamber:adding a certain amount of HA sample to be tested to the skin surface of an independent donor, and uniformly spreading the sample from the central part of the skin to the edge in a radial manner by using a disposable gun head;Start the electromagnetic stirrer to stir at the speed of 300 rpm, keep the water bath at constant temperature of (32±1)°C, and ensure that there is no bubble in the interlayer of the water bath, and make transdermal reaction for 2 hours. After the tissues were washed with water, they were fixed with formalin solution overnight, and then dehydrated in different concentrations of ethanol, transparent, waxed, embedded, and sectioned. The fluorescence intensity was observed under a fluorescence microscope.

Volunteers recruitment of human skin moisturizing experiment

The enrollment conditions of the recruited volunteers are by the number of volunteers and the condition standard for “Determination of moisture content of stratum corneum by capacitance method” in the Guidelines for Evaluation of Moisturizing Efficacy of Cosmetics (QB/T 4256-2011) issued by the Ministry of Industry and Information Technology of China, specifically, the number of volunteers is 24-30, and the enrollment age is between 18 years and 65 years old. The basic value of the capacitance skin moisture tester in the forearm test area is between 15 and 45; No use of antihistamines in the past week or immunosuppressants in the past month; Patients who have not applied any anti-inflammatory drugs to the test site within the past two months; Non-insulin-dependent diabetes mellitus; Non-lactating or pregnant women; those who have not participated in other clinical trials at the test site at present or in the past three months; There is no scar, nevus or other factors that may affect the determination of the test results on the skin of the forearm to be tested; Voluntary participation in this trial and ability to complete the specified contents as required by the trial.

Moisturizing experiment of supramolecular HA on human skin

The test is conducted according to the test environment conditions, instruments, and test procedures for “Determination of moisture content in stratum corneum by capacitance method” in the Technical Specification for Safety of Cosmetics (2015 Edition) issued by the former China Food and Drug Administration and the Guidelines for Evaluation of Moisturizing Efficacy of Cosmetics (QB/T 4256-2011) issued by the Ministry of Industry and Information Technology of particular to that method for carrying out real-time dynamic monitoring under the test environment temperature of 20 DEG C to 22 DEG C and the humidity of 40% to 60%. Volunteers should not use any cosmetics or topical drugs 2 - 3 days before the test, and should not touch water 1 hour -3 hours before the test. Before the test, volunteers wiped and cleaned the inner forearms of both hands with dry tissue; selecting three test areas with an area of 3cm * 3cm from the inner side of the forearms of both hands of the volunteer, wherein the test areas have the same area and the interval between each area is at least 1cm, and the test areas are divided into a blank control group, a supramolecular HA group, and a non-supramolecular HA group and marked; After marking, the volunteers sat quietly in the room for 30 minutes, during which time they could not drink water or beverages, and kept their forearms exposed, placed in the test state and kept relaxed.

Before the sample application, the skin capacitance value of the volunteers in the test area was measured by Corneometer® CM 825 skin moisture meter (Courage & Khazaka, Germany), and then the supramolecular HA solution, non-supramolecular HA solution, and ultrapure water were evenly applied to the test area on the inner forearm respectively. The skin capacitance value of the test area was measured 4 hours and 8 hours after the sample application. Each test area is measured in parallel 3 times each time, and the measurement probe is cleaned before each measurement.

Data Statistics

Use EXCEL software t o m ake d escriptive s tatistics f or e ach measured v alue, i ncluding quantity, m ean v alue, m inimum value, maximum value, etc. SPSS analysis software was used to test the significance of the difference in normal distribution by the ShapiroWilk Test. Sig. (Two-sided)> 0.05, the normal distribution is presented, the paired t-test is performed, and the significant difference level α is 0.05.If Sig. If (two-sided)<0.05, it is non-normal distribution, Wilcoxon signed rank test is performed, and the level of significant difference α is 0.05.


Supermolecular HA transdermal experiment

The in vitro transdermal results of fluorescent-labeled supramolecular HA solution and non-supramolecular HA solution are shown in Figure 3. The results of fluorescence staining showed that the transdermal effect of HA decreased with the increase of its MW. At the same MW, the transdermal effect of supramolecular HA was much better than that of non-supramolecular HA, and it could penetrate deep skin. In the moderately polymerized state with an MW of 100 kDa-1,800 kDa and the highly polymerized state with a MW greater than 1,800 kDa, supramolecular HA can still penetrate deep into the skin, while non-supramolecular HA can hardly penetrate deep into the skin.


Figure 3.In vitro transdermal experiment of supramolecular HA and non-supramolecular HA with different MW. After 2 hours of transdermal reaction, the fluorescent-labeled HA (A\B: low molecular weight HA of 10 kDa -100 kDa, C\D: middle molecular weight HA of 100 kDa -1,800 kDa, E\F: high molecular weight HA of more than 1,800 kDa) were cut into paraffin sections and observed under confocal laser scanning microscope. A, C, and E are the transdermal results of supramolecular HA, and B, D, and F are the transdermal results of non-supramolecular HA.

Human skin moisturizing experiment volunteer’s recruitment

A total of 24 valid subjects meeting the volunteer enrollment criteria were finally recruited, including 4 males and 20 females, aged from 20 years to 63 years, with an average age of 50.50 years ± 2.66 years. Information on enrolled volunteers is provided in Table 1.

Table 1.Information of effective volunteers enrolled in human skin moisturizing experiment

 Number Subject Name Gender Age
1 LZR Female 60
2 ZWY Female 59
3 CY Female 44
4 SCX Female 60
5 LG Male 60
6 CZL Female 41
7 HML Female 62
8 HZY Female 61
9 WHW Female 57
10 ZFJ Female 58
11 SZQ Female 24
12 ZYP Male 42
13 ZJ Male 62
14 GJR Female 54
15 CSH Female 58
16 GJG Female 58
17 ZDJ Male 20
18 BCL Female 63
19 DJH Female 52
20 ZHY Female 53
21 HRR Female 53
22 ZLJ Female 49
23 ZCQ Female 41
24 WYJ0 Female 21

Moisturizing experiment of supramolecular HA on human skin

By measuring the skin capacitance value in the test area of volunteers, it was found that the water content of stratum corneum was significantly increased at 4 hours and 8 hours after the application of supramolecular HA and non-supramolecular HA compared with the blank group (Figure 4). The water content of stratum corneum increased by 25.83% (P<0.001) and 17.21% (P<0.001) respectively at 4h and 8 h after the administration of supramolecular HA. The water content of the stratum corneum increased by 12.66% (P<0.001) and 7.83% (P <0.001) at 4 hours and 8 hours after the application of non-supramolecular HA, and the increased the water content of the stratum corneum of supramolecular HA was higher than that of non-supramolecular HA (P<0.001).


Figure 4. Change the trend of moisture content in the stratum corneum of volunteers ‘skin in the test area. The blue histogram represents the area coated with ultrapure water, the orange histogram represents the area coated with supramolecular HA, and the green histogram represents the area coated with non-supramolecular HA.

To compare the moisturizing effect of supramolecular HA and non-supramolecular HA, we calculated the increase in water content of the stratum corneum before the application of HA and 4 and 8 hours after application. The results showed that the increase of skin stratum corneum water content of supramolecular HA was significantly higher than that of non-supramolecular HA at 4 hours and 8 hours after application (Figure 5), and the increase of skin water content of supramolecular HA at 8 hours after application was higher than that of non-supramolecular HA at 4 hours.


Figure 5. The increased moisture content of stratum corneum. The blue histogram represents the area coated with ultrapure water, the orange histogram represents the area coated with supramolecular HA, and the green histogram represents the area coated with non-supramolecular HA.


HA is abundant in the dermis of the skin in humans, providing a spatial framework for the distribution of collagen fibers and elastin, forming together a skin scaffold to maintain skin tissue stability and skin elasticity. If one of the three is missing, it can accelerate the f skin aging and the formation of wrinkles. But studies have shown that human skin epidermal thickness shrinks by an average of 6.4% every 10 years. In the process of skin aging, the production of Extracellular Matrix (ECM) such as HA, collagen, and elastin decreases, while the expression of Matrix Metalloproteinases (MMPs) increases, which increases the degradation of the extracellular matrix and further accelerates the collapse of skin structure [14]. Therefore, exogenous supplementation HA is an important approach to restoring aging skin structure and maintaining skin function.

As one of the components of human connective tissue and synovial fluid, HA is one of the world’s most widely used skin fillers because of its high biocompatibility. Commonly used for periocular wrinkles like crows-feet and glabellum wrinkles, and nasolabium wrinkles [20,21].

However, the 500 Dalton rule for the skin penetration of chemical compounds and drugs, on the one hand, protects the body, but on the other hand, it also sets up obstacles for functional compounds such as macromolecular drugs and cosmetics to enter the skin.

As a polysaccharide, the MW of HA is positively correlated with the number of water bound by HA and negatively correlated with the depth of penetration into the skin. Essendoubi et al. 2016 showed that hyaluronic acid of different MWs can be absorbed by the skin, with HMW (1,000 kDa-1,400 kDa) HA mainly penetrating the stratum corneum of the skin, while LMW (20 kDa-300 kDa) HA penetrates deeper [22].

Thus, microneedle injection has become the most widespread means of dermal exogenous HA supplementation. However, considering the invasiveness of injection, this method has a series of disadvantages such as infection risk, long recovery period, specific operators and locations, and high cost. Therefore, it is of great significance to find a non-invasive method that can make HMW HA penetrate deep into the skin.

Brown et al. (1999) demonstrated that HA is not only absorbed by the skin in a passive diffusion manner but also has an active transport absorption capacity [23]. This suggests that HMW HA has the potential to penetrate deep into the skin, but its physicochemical properties limit this possibility.

Previous studies have also used liposomes to encapsulate HMW HA molecules and deliver them deep into the skin by modifying their lipophilicity [24]. However, this method has a series of disadvantages such as a complicated preparation process, high cost, storage limitation, etc., and cannot be widely applied on a large scale.

In this study, the common HA molecule was transformed into a supramolecular state by using ionic liquid induction, and then the skin penetration mode of the HA molecule, especially the HMW HA molecule, was changed, so that it could penetrate the stratum corneum and entered deeper skin non-invasively. It can be seen from the experimental results that the transdermal ability of HA with HMW is obviously improved after being activated by ionic liquid to present a supramolecular state, and its ability to maintain water molecules and replenish water to the skin is also greatly improved.

The preparation process of supramolecular HA in this study is relatively simple, low cost has simple storage conditions and is convenient for subsequent industrial large-scale production.

As for the comparison of the transdermal ability of supramolecular HA with different MW, it still accords with the rule that the lower the molecular weight, the better the transdermal effect. However, the HMW HA that remains outside the stratum corneum of the skin forms a protective barrier on the skin surface, preventing water loss from the skin. We speculate that if supramolecular HA with different MW is made into a certain proportion of mixed solution, using the strategy, which is MMW and LMW HA transdermal hydration and repair skin tissue, HMW HA to create a protective barrier its moisturizing effect will be better than using a single certain MW supramolecular one. Therefore, the following experiments can compare the hydration effect of mixed supramolecular HA with that of pure HMW, MMW, and LMW supramolecular HA to verify the effectiveness of the above strategy. At the same time, the optimal mixing ratio of supramolecular HA with different MW in the mixed supramolecular HA solution can be explored.

In addition, the results of this study confirmed that HMW HA can penetrate deep skin, suggesting that supramolecular HA has unique physical and chemical characteristics. This also provides ideas and references for the development and improvement of other transdermal functional compounds in a supramolecular state.


In this study, we prepared HA with different MW by ion-induced method and confirmed that HA with different MW can penetrate deep skin. Compared with ordinary HA, supramolecular HA can increase the skin moisture content by more than 2 times and can help the skin lock moisture for a longer time and delay the skin water loss process.