Hyaluronic Acid Intensified: Cross-linking Improves Stability, Functionality

Hyaluronic acid (HA) is well-known for the benefits it provides in topical formulations, including moisturization, delivery of water and actives to the skin, film formation and antioxidant effects.^1-4 HA also is critical to the functional well-being of normal physiological processes of the skin;^5-7 notably protection via said antioxidant effects, hydration, stabilization of the tissue matrix structure and cellular repair.^8-12 Its linear structure consists of repeating dimers of N-acetyl glucosamine and Na-D-glucuronate, which are linked together to form a long, unbranched chain having a high molecular weight (HMW) of 2-4 ×10⁶ Da.¹³ HMW HA chains form hydrated random coils, which interact to produce highly viscoleastic solutions.¹⁴

To enhance the beneficial properties of topical HMW HA and increase its functionality, stability and use, derivatives were developed using divinyl sulfone as a cross-linker to react with HA’s primary hydroxyl groups and covalently bond the chains together via sulfonyl-bis-ethyl cross-links.¹⁵ Using this approach, the backbone of the HA chain remains unchanged, which allows the formation of soft, viscoelastic hydrated gels that maintain the biocompatibility of the HA molecule (see Figure 1). Such cross-linked HA gels^a were developed first for use in medical devices as soft tissue augmentation agents, to correct soft tissue deformities, e.g., facial wrinkles, via intradermal injection;¹⁶ they therefore have been established as safe and biocompatible.¹⁷

A unique feature of these cross-linked HA gel matrices is the ability to form “nonequilibrium” or non-fully swollen gels,¹⁸ with a range of concentrations and rheological parameters that are effective delivery vehicles for different actives.^{19, 18} This property is a direct result of their structure and water-binding capacity. Non-equilibrium gels are typically better able to incorporate and entrap actives, as they may be further swollen in the presence of a solution of the active. The actives therefore become part of the hydrated molecular cage of the cross-linked HA gel.

As is described here, the cross-linked HA gels were assessed for potential personal care applications by testing their water-binding capacity, delivery of actives, and stability against heat, free radicals, other chemicals and pH changes, as compared with non-cross-linked native HA.

Water Binding

The water-binding properties of a polymer greatly influence its structure, stability and macroscopic properties. In formulations, water-binding acts as a reservoir for water that, upon application, enables the formation of a protective and hydrating film. A high water-binding capacity also facilitates and enhances the loading of actives. The water-binding capacity and characteristics of cross-linked HA gel, HA and other natural polymers were thus measured, using differential scanning calorimetry (DSC) thermograms (transition temperature heating curves).²⁰

DSC can distinguish between different types of bound water, including freezable, non-freezable and free water in a polymer.²⁰ In the case of HA, the primary goal was to measure the total bound water, i.e., nonfreezing bound water, plus freezing bound water since this has a critical influence on the structure of the HA and HA derivatives and the formation of protective and lasting hydrating films. Free water is influenced by the amount of true “bound” water and will have a reduced rate of diffusion that is relevant to the delivery of water and other actives (prolonged).

Non-freezable bound water: Non-freezable bound water is highly structured, and tightly associated with and attached to the hydroxyl groups and hydrophilic portions of the HA and HA gel structures through hydrogen bonding. Non-freezable bound water in hydrophilic polymers does not crystallize to form hexagonal ice due to steric constraints—even when cooled to -100°C.²¹

Freezable bound water: Also referred to as semi-bound water, freezable bound water is structured and characterized as having a phase transition temperature lower than bulk water due to weaker interactions with the polymer. It contributes significantly to the physical properties of the polymer such as viscoelasticity, hydration capabilities and loading capacity of actives (including water).

DSC revealed five times more freezable water was present in the cross-linked HA gel than the HMW HA, and three to 200 times more water than the other natural polymers (see Table 1).²⁰ The cross-linked structure enabled the formation of clusters or “traps” of freezable bound water, which behaves as water confined within pores in the reticulated polymer network. 

Free water: Free water does not form hydrogen bonds with polymers. It can be distinguished from freezable bound water by the transition temperature measured in DSC heating curves, since its freezing temperature is the same as bulk water. However, free water may be compartmentalized or held within the polymer matrix, and it will exhibit restricted diffusion, which is influenced by the amount and type of bound water. This, in turn, contributes to the hydration properties and delivery capabilities of the polymer.

Ex vivo Delivery to the Stratum Corneum

As noted, the capacity to form stable clusters or traps of freezable bound water enhances the ability of cross-linked HA gels to deliver water and other actives to stratum corneum (SC). The gels form a permeable hydrated microlayer—in essence, a water-filled polymer barrier—that provides hydration and moisturization.

This enhanced hydrating activity of cross-linked HA gel (non-equilibrium form) was demonstrated in an independent study by Rutgers,²² in which scientists measured the water content of human SC after treatment with cross-linked HA gel (NEF) or HMW HA, each having identical polymer content. After 24 hr, the skin sample treated with the cross-linked gel contained six times more moisture in the total sample, and five times more moisture in the SC than the HA-treated skin. Deuterated water (D₂O) was used to quantify water content via confocal Raman spectroscopy (see Figure 2).

Free Radical-Scavenging

A natural scavenger of free radicals, HA protects against both cell damage and damage to healing tissues while also inhibiting lipid peroxidation.^23-25 These effects are molecular weight-dependent; i.e., HMW HA provides greater protection against free radical-induced damage than lower MW HA. This is most likely due to the ability of the larger molecule to absorb, neutralize and consume more free radicals as a result of the 3D matrix structure, greater space occupation, surface area, etc. It also exhibits greater stability due to covalent cross-links and higher water-binding capacity; hence, it has a greater capacity for free radical consumption.

In relation, cross-linked HA gel and HMW HA were evaluated in vitro for antioxidant activity using xanthine oxidase/hypoxanthine as a source of free radicals (superoxide, O₂-). The amount of free radicals consumed was determined by monitoring the increase in absorbance, at 550 nm, caused by the reduction of cytochrome c. In the presence of a free radical scavenger, absorbance is decreased due to the removal (scavenging) of free radicals by a particular agent.^{26, 27}

In the present studies, the cross-linked HA gel matrix was ~ 4 × more effective in scavenging free radicals in vitro than the same concentration of HMW HA (> 1 × 10⁶) for the same 30 min duration (see Figure 3). Additionally, studies were conducted using polymorphonuclear leukocytes (PMN), i.e., white blood cells associated with inflammation, that were stimulated with phorbal myristate acetate and compared with superoxide dismutase as the positive control (100% inhibition). The cross-linked HA gel inhibited the generation of free radicals (O₂-) by PMN (data not shown) in a dose-dependent manner.²⁴

Delivery of Actives

The delivery of a particular substance is influenced by its chemical nature and mechanisms including prolonged release, absorption and surface contact. A good example of this latter case is sunscreen, whose retention on skin’s surface is desired to reduce both penetration and loss of efficacy. Studies suggest the cross-linked HA gel matrix may impart these effects by either functioning as a reservoir, slowly releasing active over time; or complexing with the active, to maintain/retain it on the skin surface.^{2, 18, 19}

Glycerol deposition: Cross-linked HA gel matrices were assessed for their capability to deliver incorporated/entrapped substances to the surface of skin.^{18, 19} In a study of 12 human subjects over the course of eight days, the deposition of glycerol on skin from a shower gel containing cross-linked HA gel was measured.²⁹ Subjects’ forearms were preconditioned for one week without use of lotions, creams or oils. Before the application of test samples, subjects were acclimated for 20 min in a controlled environment (40 + 5% RH; 68 + 2°C). Then, 0.1 mL of test article was applied to the test site. The sample was lathered for 15 sec, left on the arm for 30 sec, and rinsed for 2 min. The arm was dried and subjects were re-acclimated for 20 min before glycerol content was evaluated.

Ethanol extractions of the skin sites were taken, the extracts were dried, and the glycerol concentrations were analyzed by gas chromatography/mass spectrometry (GC/MS). In the presence of the cross-linked HA gel, a statistically significant increase in glycerol deposition was observed (p = 0.01) on the skin after rinse-off (see Figure 4).²⁰

Release kinetics: In addition to deposition, the release kinetics of lactic acid, an alpha hydroxyl acid, from non-equilibrium cross-linked HA gel were evaluated and compared with those from a control solution. Alpha hydroxyl acids such as lactic acid are popular in anti-aging products due to their effects of increasing epidermal firmness and thickness, and improving skin smoothness. To measure the release rate, a radio-labeled form of lactic acid was used; experiments were carried out in dialysis chambers.

The cross-linked HA gel slowly released the lactic acid active from the matrix over a 24-hr period (see Figure 5). This slow release rate of lactic acid, a surface-modifying agent, in the presence of a highly hydrated environment on the skin surface may serve to minimize potential irritation and/or inflammation from such actives.

Sunscreen retention: Previous studies¹⁸ also were conducted on a UV filterb combined with cross-linked HA gel in order to form a sunscreen product with increased surface retention and reduced absorption into the skin. Test samples, i.e., 4% sunscreen with cross-linked HA gel, and control samples, 4% sunscreen solution only, were applied to skin sites at a rate of 10 µL per site, and allowed to dry for 2 hr. The penetration of the sunscreen was measured by tape-stripping each skin site of application.

This was followed by isopropanol elution of each tape, and measurements of absorption of the eluted solution at 311 nm; i.e., the maximum absorbance wavelength of the test sunscreen^b. The data showed higher levels of sunscreen present in the uppermost layers of skin in the samples incorporating the cross-linked HA gel, compared with the control; approximately 30% more sunscreen was retained in the “surface” layers of skin, with a trend toward lower levels of sunscreen in deeper layers (see Figure 6).¹⁸

Stability and Resistance to Degradation

Basic studies also were carried out to determine the effects of specific conditions, i.e., chemical interactions, heat and free-radical exposure, on HA vs. the cross-linked HA gel. Exposure of HA to free radicals (XO/HX, 1U/mL) caused notable degradation within 5 min, with maximum degradation after 60-90 min.²⁸ The viscosity of the free radical-treated HA decreased to approximately that of the buffer (water) alone; i.e., 1 cps. In contrast, exposing the cross-linked HA gel to the same concentration of free radicals for 1 hr had a minimally degradative effect; a 2.3% increase in the formation of degradation products was observed, as shown by soluble, 0.2 µm filterable polysaccharide.

Intact cross-linked HA gel does not pass through a 0.2 µm filter, thus the presence of filterable material following exposure to agents such as free radicals is indicative of, and a measure of, degradation. However, at higher free radical concentrations and prolonged exposure periods (48 hr), significant degradation was observed, with a notable reduction in viscosity (visual) and increase in the amount of filterable degradation product (data not shown).

Conclusion

Cross-linked HA gels provide a practical means to enhance the benefits associated with using HA in topical skin care formulations. The major properties shown here include: the ability to load the gels with active ingredients including water; the high water-binding capacity; and the capability of forming a hydrated, oxygenated film on skin to protect, conform and moisturize the surface of the SC.

References

1. T Pavivic, et al, Efficacy of cream-based novel formulation of hyaluronic acid of different molecular weights in anti-wrinkle treatment, J Drugs Dermatol 10(9) 990-1000 (2011)
2. MB Brown and SA Jones, Hyaluronic acid: A unique topical vehicle for the localized delivery of drugs to the skin, J Eur Acad Dermat and Venerol 19 308-318 (2005)
3. US Patent 4303676, Hyaluronate based compositions and cosmetic formulations containing same, EA Balazs (Dec 1, 1981)
4. R Albertini, A Passi, PM Abuja and G DeLuca, The effect of glygosaminoglycans on lipid peroxidation, Int J Mol Med (6)126-136 (2000)
5. JP Bentley, in Repair and Regeneration, JE Dunphy and W Van Winkle, Jr, eds, McGraw-Hill, NY 151-160 (1968)
6. EA Balazs et al, Matrix engineering, Blood Coag Fibrinol 2 173-178 (1991)
7. EA Balazs and DA Gibbs, The rheological properties and biological function of hyaluronic acid, in Chemistry and Molecular Biology of the Intercellular Matrix, New York Academy 1241-1253 (1970)
8. N Rydell, Decreased granulation tissue reaction after installment of hyaluronic acid, Acta Orthop Scand 41 307-311 (1970)
9. G Abatangelo, M Martelli and P Vecchia, Healing of hyaluronic acid-enriched wounds: Histological observations, J Surg Res 35
10. SR King, WL Hickerson, KG Proctor and AM Newsome, Beneficial actions of exogenous hyaluronic acid on wound healing, Surgery 109 76-84 (1991)
11. BA Mast, LC Flood and JH Haynes, Hyaluronic acid is a major component of the matrix of the fetal rabbit skin and wounds: implications for healing by regeneration, Matrix 11 63-68 (1991)
12. MT Longaker, DJ Whitby and RW Jennings, Adult skin in the fetal environment heals with scar formation, Surg Forum 41 639-641 (1990)
13. K Meyer, Chemical structure of hyaluronic acid, Fed Proc 17 1075-1077 (1958)
14. DA Gibbs, EW Merrill and KA Smith, Rheology of hyaluronic acid, Biopolymers 6 777-791 (1968)
15. EA Balazs and E Leshchiner, Hyaluronan, its cross-linked derivative hylan and their medical applications, in Cellulosics Utilization: Research and Rewards in Cellulosics, H Inagaki and GO Phillips, eds, Elsevier Applied Science, NY 233-241 (1989)
16. DL Piacquadio, NE Larsen, JL Denlinger and EA Balazs, Hylan B gel (Hylaform) as a soft tissue augmentation material, in Tissue Augmentation in Clinical Practice, AW Klein, ed, Marcel Dekker, NY 269-291 (1998)
17. USFDA, www.fda.gov, Hylaform; and hylan B and hylasome HA gels are chemically identical polymers, Ref: PMA P030032 (2004)
18. US Pat 8,679,470, Compositions using cross-linked hyaluronic acid for topical cosmetic and therapeutic applications, AK Leshchiner, NE Larsen and EG Parent (Mar 25, 2014)
19. NE Larsen, EA Leshchiner, EG Parent and EA Balazs, Hylan and hylan derivatives in drug delivery, in Cosmetic and Pharmaceutical Applications of Polymers, CG Gebelein, F Cheng and V Yang, eds, Plenum Press, NY 147-157 (1991)
20. S Takigami, M Takigami and GO Phillips, Effect of preparation method on the hydration characteristics of hylan and comparison with another highly cross-linked polysaccharide, gum arabic, Carbohydrate Polymers 26 11-18 (1995)
21. ZH Ping, QT Nguyen, SM Chen, JQ Zhou and YD Ding, Studies of water in different hydrophilic polymers–DSC and FTIR studies, Polymer 42 8461-8467 (2001)
22. C Flach, LipoChemicals Internal Report, study conducted by Rutgers University (2008)
23. D Presti and JE Scott. Hyaluronan-mediated protective effect against cell damage caused by enzymatically produced hydroxyl (OH-) radicals is dependent on hyaluronan molecular mass, Cell Biochem Funct 12(4) 281-288 (1994)
24. NE Larsen, KM Lombard, EG Parent and EA Balazs, Effect of hylan on cartilage and chondrocyte cultures, J Orthop Res 10 23-32 (1992)
25. D Foschi, L Casatoldi, E Radaelle, P Abelli, G Calderini, A Rastrella, C Mariscotti, M Marazzi, E Trabucchi. Hyaluronic acid prevents oxygen free-radical damage to granulation tissue: A study in rats, Int J Tiss Reac XII(6) 333-339 (1990)
26. McNeil et al, Depolymerization products of hyaluronic acid after exposure to oxygen-derived free radicals, Annal Rheum Dis 44 780-789 (1985)
27. WH Betts and LG Cleland. Effect of metal chelators and anti-inflammatory drugs on the degradation of hyaluronic acid, Arthritis Rheum 25 1469-76 (1982)
28. RA Greenwald and SA Moak, Degradation of hyaluronic acid by polymorphonuclear leukocytes, Inflammation 10 15-30 (1986)
29. Colgate Palmolive Company, internal report (2005)