Preparation of Low-Molecular-Weight and Oligomeric Hyaluronic Acid
1. Low-Molecular-Weight and Oligomeric Hyaluronic Acid
Hyaluronic acid is a natural linear polysaccharide occurring in the human body. Its molecular weight varies from several thousand to several million Daltons. High-molecular-weight hyaluronic acid (HMW-HA) has excellent moisture-retention and lubrication properties; thus, it can be widely used in cosmetics and medicine. However, recent research has revealed that low-molecular-weight hyaluronic acid (LMW-HA) and oligomeric hyaluronic acid (Oligo-HA), degradation products of HA, possess distinct biological activities compared with HMW-HA and thereby substantially expand the application range of HA.
1.1 Biological Activities of Low-Molecular-Weight and Oligomeric Hyaluronic Acid
The biological effects of LMW-HA and Oligo-HA depend on their molecular weight.
Fig 1. Comparison of Hyaluronic Acids with Different Molecular Weights
- Angiogenesis and Wound Healing: High-Molecular-Weight Hyaluronic Acid (HMW-HA) inhibits blood vessel formation. In contrast, Low-Molecular-Weight Hyaluronic Acid (LMW-HA), especially oligomeric fragments, promotes the proliferation and migration of endothelial cells. Therefore, LMW-HA accelerates angiogenesis. This process supports wound healing and tissue repair.
- Immune Regulation: HMW-HA has anti-inflammatory and immunosuppressive properties. However, LMW-HA acts as an agonist of Toll-like receptors, which activate dendritic cells and macrophages. This activation stimulates the release of pro-inflammatory cytokines and triggers immune responses. Because of this, LMW-HA is a promising candidate for antitumor immunity and vaccine adjuvants.
- Antioxidant Stress: Oligo-HA can scavenge free radicals to protect cells from oxidative damage. This property is generating growing interest in anti-aging and neuroprotection research.
- Promotion of Cell Proliferation and Migration: LMW-HA penetrates the skin barrier more effectively than HMW-HA. It encourages the proliferation of keratinocytes and fibroblasts, which helps in skin repair and regeneration.
1.2 Preparation for Low-Molecular-Weight and Oligomeric Hyaluronic Acid
Preparation of LMW-HA and Oligo-HA mainly relies on scission of intra-glycosidic bonds in the hyaluronic acid backbone. The three major approaches include physical, chemical, and enzymatic degradation. And each possesses different advantages and disadvantages regarding the mechanism of degradation, product molecular weight distribution, cost, and ecological impact.
2. Physical Degradation
Physical methods rely on external energy to disrupt chemical bonds in the HA polymer chain.
2.1 Thermal Degradation
This method relies on the principle of high temperature causing random cleavage of HA chains. The technique is quite simple and inexpensive, since no additional reagents are required. However, the process cannot be precisely controlled, and products obtained often have broad distributions of molecular weight. High temperatures may also lead to some changes in structure, affecting product purity and bioactivity.
2.2 Radiation Degradation
Gamma radiation or electron beams are used on HA solutions to create free radicals that cleave the glycosidic bonds. This method enables one to perform both degradation and sterilization in a very effective and concurrent manner. The main drawbacks of this method are high investment costs of equipment, safety risks, and complicated mechanisms of degradation that are very likely to be followed by side reactions, impairing the reproducibility of the structure of the final product.
2.3 Ultrasonic Degradation
Ultrasound causes cavitation in liquids, leading to the formation of microenvironments with very high temperature and pressure. These shear forces can effectively fragment HA chains. This approach is mild, fast, and environmentally friendly. What’s more, adjusting ultrasound power, duration, and solution concentration enables partial control over the molecular weight of the product. Given these advantages, ultrasonic degradation becomes a common choice for lab-scale and small-scale production.
3. Chemical Degradation
Chemical methods introduce reagents that trigger hydrolysis or redox reactions to break HA chains.
3.1 Acid or Alkaline Degradation
Under strong acid or alkaline conditions, such as HCl and NaOH, respectively, hydrolysis of the glycosidic bond proceeds in HA. Acidic hydrolysis primarily cleaves β-1,4 linkages of glucuronic acid, whereas alkaline hydrolysis predominantly breaks β-1,3 bonds of N-acetylglucosamine. Although this route is cheap and fast, it suffers from being harsh, and the process control is difficult, generally resulting in over-degradation products such as monosaccharides. The subsequent neutralization step and desalination further complicate the process and involve the generation of chemical waste.
3.2 Oxidative Degradation
Oxidation agents, including hydrogen peroxide and sodium periodate, can degrade HA effectively. The Vc/H₂O₂ redox system has attracted widespread interest because of its relatively mild and controllable reaction. The hydroxyl radicals produced in the process may attack the glycosidic bonds. Molecular weight could be reasonably well-controlled by adjusting the ratio, concentration, and reaction time of Vc and H₂O₂. However, it is possible that oxidation not only cleaves the glycosidic bonds but may also change the structure of hydroxyl groups on the HA chain, which may affect the chemical structure and bioactivity of the final product.
4. Enzymatic Degradation
Enzymatic degradation employs highly specific hyaluronidases to catalyze HA breakdown, representing the most promising approach for industrial production.
4.1 What are Hyaluronidases
Hyaluronidases are enzymes that specifically hydrolyze β-N-acetylhexosaminidic bonds in HA. Hyaluronidases are mainly classified by their origin. One type comes from microbes like Streptococci. They act as endoenzymes, which means they cut hyaluronic acid chains at random internal positions. As a result, they break down HA very effectively and are often used in industrial production.
Another type is derived from animals. A common example is testicular hyaluronidase. These animal-derived hyaluronidases are also endoenzymes. They are, however, widely used in scientific research and for making medicines.
Enzymatic degradation offers high specificity, requires milder reaction conditions, and produces fewer by-products. What’s more, by controlling the enzyme amount, reaction time, and temperature, products with narrow molecular weight distributions can be obtained.
4.2 Industrial Enzymatic Production for Low-Molecular-Weight and Oligomeric Hyaluronic Acid
Enzymatic degradation is the preferred method for industrial production of LMW-HA and Oligo-HA. A typical process involves several steps.
Fig 2. Enzymatic degradation [1]
- First, the substrate is prepared. High-molecular-weight HA raw material is dissolved in a suitable buffer solution, forming a uniform mixture.
- Next step, the enzymatic reaction takes place. A specific amount of microbial hyaluronidase is added to the HA solution. Stirring the mixture under controlled conditions. The temperature is maintained between 37–50°C, and the pH is kept at an optimal level.
- During the reaction, the process is monitored. Viscosity changes are tracked in real time. Alternatively, molecular weight changes are followed. This is done using a viscometer or high-performance liquid chromatography.
- The reaction is stopped when the target molecular weight is reached. Termination is achieved by raising the temperature. For example, heating above 80°C denatures the enzyme. Alternatively, changing the pH can also stop the reaction.
- Finally, purification and drying are performed. The enzymatic hydrolysate is filtered. It is decolorized using activated carbon. Alcohol precipitation is then carried out. Centrifugation separates the product. The final powder is obtained through spray drying or freeze-drying.
This process can be precisely controlled. Suitable enzyme types are selected, reaction parameters are optimized, and immobilized enzyme technology may be used. Such controls enable reproducible results. Product molecular weight can be regulated from thousands to hundreds of thousands of Daltons to ensure the products meet various application requirements.
5. Conclusion
Low-molecular-weight hyaluronic acid and oligomeric hyaluronic acid have tremendous potential in pharmaceuticals, cosmetics, and functional foods, owing to their unique biological activities. The three major routes presently developed for their production include physical, chemical, and enzymatic degradations. Of these, enzymatic degradation of hyaluronic acid presents the best option for industrial precision production on a large scale because of its high efficiency, high specificity, mild conditions, and excellent controllability.
[1] Enzymatic Production of Low-Molecular-Weight Hyaluronan and Its Oligosaccharides: A Review and Prospects. Bo Pang, Hao Wang, Hao Huang, Lizhi Liao. Journal of Agricultural and Food Chemistry 2022 70 (44), 14129-14139. DOI: 10.1021/acs.jafc.2c05709
