Improving The Anti‑ageing Activity Of Coenzyme Q10 Through Protransfersome‑loaded Emulgel

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Premature skin aging occurs because the skin, as the outermost organ, is always directly exposed to oxidants in the environment and is frequently a determining factor in social life. In addition, with increasing age, the activity of mitochondria in the body as a producer of energy in regenerating cells and tissues decreases', Both these internal and external factors cause impaired tissue function and structural changes culminating in skin aging characterized by thinning of the epidermis and skin dermis and, ultimately, resulting in wrinkles, fine facial lines, and loss of elasticity4. Skin elasticity is largely dependent upon young collagen fibers and fibroblasts, collagen-producing cells in the dermis layer, whose numbers decrease during the aging process5.

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Anti-aging cosmetics have been widely used to promote skin regeneration, especially in the upper skin layers which protect the skin against dehydration, and penetration by various microorganisms, allergens, and irritants, 1Master Program of Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Nanizar Zaman Joenoes Building, Campus C Mulyorejo, Surabaya 60115, Indonesia.'Faculty of Pharmacy, Hospital Administration, Public Health, and Radiology, Study Program of Pharmacy, Institut Ilmu Kesehatan STRADA, JI. Manila 37,Kediri 64133, Indonesia. cistanche cholesterol 3Department of Pharmaceutical Sciences, Faculty of Pharmacy, Universitas Airlangga, Nanizar Zaman Joenoes Building, Campus C Mulyorejo, Surabaya 60115, Indonesia. 'Department of Anatomy and Histology, Faculty of Medicine, Universitas Airlangga, J. Mayjen. Prof.Dr. Moestopo No.47, Campus A Mulyorejo, Surabaya 60132, Indonesia.5Department of Veterinary Pathology, Faculty of Veterinary Medicine, Universitas Airlangga, JI.Mayjen.Prof.Dr. Moestopo No.47, CampusC Mulyorejo, Surabaya 60115, Indonesia.-email: andang-m@ff.unair.ac.id

reactive oxygen species (ROS) and radiation, thereby maintaining healthy skin*. Coenzyme Q10(CoO10) is one of the natural compounds often employed as an antioxidant, which plays a key role in stabilizing plasma and other intracellular membranes that protect against membrane phospholipid peroxidation. CoO10 acts by maintaining skin quality against free radicals" which have been known to activate the mitogen-activated protein kinase(MAPK)pathway that produces matrix metalloproteinases(MMPs) such as collagenase, thus damaging collagen fibers-10. During aging, the levels of CoQ10 in organs, including the skin, also decrease with the result that it is necessary to supply CoQ10 to achieve normal levels of between 0.50 and 1.65 ug/mL within the body. Topical administration of CoQ10 has been shown to be effective in reducing wrinkles in the skin that has been exposed to UV rays3.

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CoQ10 demonstrates low solubility in water (0.193 ug/mL) with a large molecular weight of 863.36 g/mol and high lipophilicity with a log P value of 21. This limits its penetration of the skin and explains its tendency to be deposited in the stratum corneum,12. Moreover, CoQ10 decomposes when exposed to light. Loading CoQ10 into protransfersome, a vesicular carrier would probably constitute an effective strategy to enhance its biological activity within the skin in addition to increasing its stability. Protransfersome, one of the proven secular nanocarriers that provide superior skin penetration and high stability, is widely used in transdermal delivery4. It possesses a flattened liquid crystal structure which is converted into an ultra-flexible vesicle known as transfer some through the absorption of water from the skin during in situ hydration5-17. Transfer some is known to be an ultra deformable vesicle that is highly flexible and deformable, rendering it capable of passing through three skin penetration pathways'8. Transfer some can rapidly penetrate the stratum corneum and enter the deeper skin layers via the intercellular lipid of the stratum corneum. It can fuse with the cell membrane, enabling it to enter the transcellular pathway, and is able to penetrate intact through the hair follicle pathway to penetrate the deeper layers of the skin19-21. Protransfersome is composed of amphipathic lipid components such as phosphatidylcholine which, significantly, form double-layer membrane vesicles， and surfactant as an edge activator that increases the vesicle flexibility or detormability²， In general，protransfersome contains a larger number of phospholipids than that present in transfersomes. During the manufacturing process, the protransfersome does not undergo an extrusion process to produce unilamellar vesicles as observed in the transverse, This is because the protranstersome is a proven secular carrier system which will be converted into transfer some after it comes into contact with water in situ. cistanche deserticola side effects Therefore, under a light microscope, the protransfersome can be seen to possess a palisade crystalline liquid form, whereas transfersomes are vesicular when in liquid media 24

The use of ultra deformable vesicles has successfully improved the skin penetration of drugs and the efficacy of anti-aging properties of certain antioxidant molecules such as tocopherol which, when prepared in transfersome, possess good characteristics with a particle size<100 nm and entrapment efficiency of up to 90%. Moreover, it is well distributed within the skin layer and in vitro tests have proved it biocompatible with keratinocytes and fibroblasts, indicating its protective effect against oxidative damage and the potential for wound healing5. Previous reports have evaluated the use of nanocarriers for CoO10 delivery such as a self-emulsifying drug delivery system (SEDDS)26, ethosomes27, transethosomes28, and microemulsion2. The use of transethosomes successfully encapsulated CoQ10 up to 97% in vesicles and produced>95% drug deposition in different skin layers resulting in high efficacy for androgenic alopecia28. The low water solubility of CoQ10 frequently limits drug encapsulation efficiency in nanocarriers, thus the use of large amounts of lipid phase or ethanol may improve its loading. In this study, a protransfersome containing CoQ10 will be prepared for anti-aging emulgel. The high level of phospholipids contained in protransfersome is intended to improve drug loading. The use of protransfersome in the anti-aging activity and irritation level of Protransf-CoQ10 emulgel was evaluated in vivo using UV-induced aged mice models. This study could represent an attempt to improve CoQ10 anti-aging activity with the result that is effective, safe, and non-irritating.

Results

This study aims to evaluate the potential use of protransfersome for topical delivery of CoQ10 as an anti-aging agent, This study provides a scientific approach to successfully delivering low water solubility and poor permeable lipophilic substances and nanovesicular carriers specifically designed for anti-aging cosmetics. The CoQ10 was loaded into protransfersomal emulgel composed of oleic acid-containing soluble CoQ10, phospholipids as bilayer-forming lipids, and Tween 80 which acts as the edge activator of the bilayer membrane after the protransfersome has been hydrated with skin water in situ, before being loaded into an emulgel base. there were improvements in stability and potential efficacy to inhibit premature aging of the skin in UV-radiation skin aged-induced mice models as demonstrated in this study. Physical characteristics and stability of protransfersome-loaded CoQ10 emulgel. After dissolving the CoQ10 in oleic acid and encapsulating it into protransfersomes composed of phospholipids and Tween 80, the protransfersome-loaded CoQ10 (Protransf-CoQ10)forms a bright orange, viscous, oily liquid, with a distinctive phospholipid smell, and viscous consistency. After hydration with saline, lamellar vesicular structures rapidly formed and were ultimately transformed into transfer some vesicles, as shown in Fig.1.

The dispersion of Protransf-CoQ10 into the emulgel base(Fig. 2A, B) at a weight ratio of 2:1 produced Protransf-CoQ10 Emulgel whose color changes to brownish orange with a reduction in its pungent smell as shown in Fig. 2C.CoQ10 dissolved in oleic acid (CoQ10-Ole) was in the form of a bright orange odorless emulgel (Fig.2D)whose character is identical to that of CoO10 Emulgel except that it is more transparent due to no oleic acid being present in the formula(Fig.2E). cistanche dosage reddit The darkening color of Protransf-CoO10 emulgel is probably due to the large amount of L-a-Phosphatidylcholine content which is dark yellow in color30and easily oxidized when it is exposed to air for lengthy periods31,32.

The particle size and polydispersity index value were further evaluated since they determine the ability of the vesicles to penetrate the deeper layers of the skin. The smaller the particle size of the vesicles, the easier the vesicles are to penetrate. In addition, the smaller the polydispersity index value, the more homogeneous the particle size of the vesicles, thus ensuring that a larger number of vesicles penetrate the skin. From the results, it is evident that the entrapment efficiency value of the CoQ10 in Protransf-CoQ10 is comparatively high at 45 64+752% with a particle size of 201.5+6.] nm (by manual shaking method), polydispersity index value of 0.229±0.047, and (-the potential of -11.26±5.14 mV, as presented in Table 1. The manual shaking method of 5 min duration was reflective of the real situation in which protransfersomes change into transfersomes. The Protransf-CoQ10 Emulgel had the smallest particle size compared to both CoQ10-Ole Emulgel and CoQ10 Emulgel, which were 134.3±4.8 nm<146.9±1.6 nm<238.8±3.1 nm, respectively, with the intensity distribution

of particle presented in Fig. 2F-H. The polydispersity index values for Protransf-CoQ10 Emulgel, CoQ10-Ole Emulgel, and CoQ10 Emulgel were 0.291±0.020<0.298±0.019<0.384±0.010.

In order to evaluate any interaction between CoQ10 and protransfersomal matrix, a Fourier Transform Infra-Red (FTIR) analysis was further observed. As presented in Fig. 3, there were no new absorption bands of functional groups or peak shifts observed for Protransf-CoQ10, which shows similar infrared spectroscopical profiles to Protransfersome blank, while no specific peaks of CoQ10 appear. cistanche extract benefits This result indicates that CoQ10 successfully encapsulated protransfersome and no chemical interaction between the mixtures occurred33-35, Moreover, according to the result of differential thermal analysis, the CoQ10 encapsulation into pro-trans-fearsome produced changes in the structure of crystallinity. CoQ10 and L-a-Phosphatidylcholine showed sharp endothermic peaks at 53.3 and 112.3 °C, respectively; however,protransfersomal CoQ10 showed a weak endothermic peak at 143.9℃C indicating that less ordered crystalline structures were observed as presented in Fig.4.

A physical stability test was subsequently carried out to determine the physical resistance of the system when stored at different temperatures, namely; room temperature and a lower temperature for 28 days. During the study, the parameters of particle size, polydispersity index, and pH were observed. As seen in Fig. 5.the results showed that after a 28-day storage period, there were no significant differences in particle size or polydispersity index(P<0.05). On the other hand, a significant difference was observed in the pH during the same period. although the pH value remained within the pH range of the skin. No significant difference existed in the particle size or particle size distribution of the preparation after 28 days of storage. In vivo anti-aging activity of pro-transfer some-loaded CoQ10 emulgel. cistanche genghis khan To evaluate the ability of pro-transfer comes to topically deliver CoQ10 and produce an effective anti-aging activity, the Protransf-CoQ10 Emulgel was topically applied for 14 days to the back skin of UV-rays-induced subjects who were subsequently observed for skin histopathology. The control group subjects who received UV rays had the lowest collagen density of 52.30±7.87%, indicating that UV rays damage the collagen in the skin dermis. The administration of both Protransf-CoQ10 Emulgel and CoQ10-Ole Emulgel significantly improved the collagen density of UV-ray radiated subjects' skin as indicated in Fig.6. However, there was no significant difference between these groups. The use of protransfersomes successfully delivered CoQ10 providing protection against skin damage and repairing that resulting from exposure to UV rays.

The anti-aging activity test result was further analyzed by observing the number of fibroblast cells capable of producing collagen. Therefore, the higher the number of fibroblasts, the more collagen was formed. In this study, the assessed fibroblasts were young and light purple in appearance. The results showed that the CoQ10 Emulgel had a significantly different number of fibroblasts compared to the control group, with pro-CoQ10

Emulgel produced the highest number of fibroblasts, which was 31.50±9.48 cells per field view, as indicated in Fig.7.This shows that transfer somes delivering CoQ10 successfully increase the number of fibroblasts. In vivo skin irritation test. The safe use of Protransfersome-loaded emulates in this study was also evaluated by conducting an in vivo irritation test. Epidermis liquefaction, subepidermal edema, collagen fiber swell-

ing, inflammatory cells infiltration, and dan appendages degeneration were observed for determining irritation in models' skin. As presented in Fig.8, there are differences in skin histopathology between normal and UV-induced skin. For further evaluation of the severity level of skin irritation, scoring was then determined for each group.

The results of the histopathological scoring of the model's back skin after 24 h of application showed that CoQ10 Emulgel had an irritation score of 0.52, while CoQ10-Ole Emulgel had one of 1.36, and Protransf-CoQ10 Emulgel one of 0.92 as presented in Fig.9. This result shows that the Protransf-CoQ10 Emulgel does not irritate the skin, while the CoQ10-Ole Emulgel induced mild irritation due to the nature of oleic acid. According to the Kruskal Wallis statistical test results, there was no significant difference between these emulgel preparations.

Discussion

In this study, the Protransfersomes and Protransfersomal emulgel preparations for CoQ10 delivery as the active cosmetic ingredient have the potential to inhibit premature aging of the skin. The main purpose of the transfer-some formulation is to significantly encapsulate CoO10 in order to modify the physicochemical characteristics of CoQ10, rendering it more water dispersible and able to penetrate the skin since high lipophilic CoQ10 demonstrates low water solubility and poor skin penetration. However, the high content of oleic acid, which accounted for approximately 37% of the final weight of protransfersomal emulgel, would render it unacceptable for daily use as a skin cosmetic. Therefore, it was added to emulate to increase its appropriateness for use. As far as the functional aspects of vesicles are concerned, the formation of transfer some due to hydration of protransfersome

by water content in the emulgel base produces ultra-deformable vesicles which allow them to easily penetrate the skin. In addition, previous reports showed that the presence of a gelling agent would act as a steric hindrance which would be adsorbed onto the vesicle surface preventing fusion or aggregation, thus increasing physical stability during storage3637. The addition of lipid vesicles to gel is beneficial for increasing vesicle stability, prolonging drug release, improving dermal permeability, and enhancing drug deposition in the skin38.

Protransfersomes have been developed as the nanometer-sized carrier form of transfer some provesicles and have a higher phospholipid content compared to transfersomes. This enables the protransfersome system to demonstrate greater entrapment efficiency due to a higher number of vesicles formed that are subsequently available for encapsulating drugs, thus providing high stability when compared to the transfer some system". Protransfersomes are able to carry active ingredients through the skin pores into the deeper layer. The programs-fearsome system analyzed in this study has positive characteristics including nanometer size, and thick consistency resulting from its large phospholipid content. When the transfer is observed using a light microscope, a palisade lamellar structure appears in the form of liquid crystals. This is due to differences in the degree of hydration of surfactants and phospholipid molecules triggered by solvent limitations. The transfer of some forms is a mixture of flat liquid crystals resembling palisade and vesicular lamellae linked together. The percentage of entrapment efficiency(EE%)of the protransfersome system is a parameter used to predict the stability of the dispersion40 describing the amount of drug present in the vesicle4l.In this study, the EE% value was comparatively large because it corresponded to the phospholipid content in the formula and the tendency of CoO10 to be retained in the phospholipid membrane due to its lipophilic properties.

To improve acceptability, the pro transfer some was formulated as an emulgel preparation incorporating the use of an emulated gel base. In this study, three types of emulates were developed and evaluated for their anti-aging and irritability activity, namely; Protransf-CoQ10 emulgel, emulgel loaded CoQ10 which was previously dissolved in oleic acid(CoQ10-Ole emulate) and CoQ10 dispersed in an emulgelbase (CoO10 emulgel).

During the homogenization method for preparing necessary samples the particle size test involves manual shaking which is considered to closely replicate real-life conditions. The particle size of the emulgel-loaded Co-Q10 remained in the nanometer range, indicating that adding emulgel base to the particle size of Protransf-CoO10 had no effect. The particle size of Protransf-CoO10 Emulgel is smaller than that of Protransf-CoO10 itself. This indicates that the particles have turned into transfer vesicles because they have been partially hydrated by the presence of water in the emulgel base. The decreased vesicle size of protransfersomal CoQ10 after dispersion into the emulgel base is probably due to the shearing stress that occurs during the incorporation of Protransf-CoQ10 into hydrated Carbopol-based emulgel. This causes the small vesicles formed and the emulgel matrix to be adsorbed onto the vesicle surface, preventing vesicle fusion or aggregation36,7, while spontaneous hydration of transfer some produces larger vesicles than those resulting from dispersion into emulgel, When compared to the particle sizes of CoQ10-Ole Emulgel and Co-Q10 Emulgel, those of all three emulgels-loaded CoO10s can be measured in nanometers. The order of particle size from the smallest to the largest is Protransf-CoO10 Emulgel<CoO10-Ole Emulgel<Co-O10 Emulgel. Co-O10 Emulgel is the largest in size because CoO10 is only dispersed in the emulgel base, while Protransf-CoQ10 Emulgel and CoQ10-Ole Emulgel had similar particle size and PDI probably due to CoO10 solubility in Oleic Acid for both formulas, From the results of the polydispersity index, it is evident that all particles have a uniform size distribution. This indicates that the preparation will be stable during storage because it reduces the tendency for particle aggregation which causes the system to become unstable.

A test was carried out to determine the physical stability of Protransf-CoQ10 emulgel when stored at different temperatures, namely; room temperature and colder temperatures for 28 days, and whether differences in particle size, polydispersity index, and pH existed. There was no significant difference in particle size, polydispersity index, and pH of Protransf-CoQ10 emulgel during the study period.

The results of the anti-aging activity of CoQ10 loaded in emulgel and evaluated for skin collagen density confirmed CoQ10-Ole Emulgel as having the highest percentage of collagen density, followed by Protransf-CoQ10 Emulgel. However, no significant difference existed between these groups(P>0.05). These two groups demonstrated significant improvement in collagen density compared with the control group whose subjects had been exposed to UV and who recorded the lowest density value. This is probably due to soluble CoO10 in Oleic Acid loaded into emulgel had been easily released from emulgel than that of Protransf-CoQ10 Emulgel, which the formation of vesicle during hydration results in semipermeable bilayer membrane as water diffusion-limiting barriers for CoQ10 release. The low collagen density has been known caused by an imbalance between collagen synthesis by fibroblasts and collagen degradation of UV irradiation, while collagen synthesis is proportionally related to fibroblasts' residence, Moreover, collagen synthesis by fibroblasts will actively occur on the 4th day of 21 days45. The faster CoQ10 release from CoQ10 Ole Emulgel will stimulate fibroblast proliferation which increase the expression of the collagen matrix, while the late CoQ10 release from Protransf-CoQ10 Emulgel will result in delayed effects on fibroblast-stimulated collagen synthesis.

On the other hand, the Co-Q10 Emulgel-treated group had a similar collagen density to that of normal mice, indicating that UV light damages the collagen in the skin dermis. It has been known that UV-irradiation damages dermal collagen and elastin fibers47, while CoQ10 increases the collagen content through a decrease in MMP-1 protein level in mice exposed to UV-B8. CoQ10 also promotes fibroblast proliferation49, However, it seems that the fibroblast stimulation process to produce collagen matrix between normal and CoQ10-treated groups is different. This situation differed from that of the group treated with CoQ10 in the emulates. From these results, it can be concluded that CoQ10 provides protection against the aging effects of UV rays.

The anti-aging activity test was further evaluated for the number of fibroblasts in the skin tissues. Fibroblasts are cells capable of producing collagen. In this case, the assessed fibroblasts were young and light purple in color. The higher the number of fibroblasts, the more collagen was formed. The results showed that the CoQ10emulgels had a significantly different number of fibroblasts compared to the control group, with the Protransf-CoQ10 Emulgel having the highest number, which was 31.50±9.48% per field view. This indicates that CoO10 is able to increase the number of fibroblasts.

The safety of these anti-aging emulates was further evaluated by an irritancy test. The results indicated that the Protransf-CoQ10 Emulgel produced no signs of irritation in the skin tissues observed, while the CoQ10-Ole Emulgel induced mild skin irritation due to the nature of oleic acid. Protransf-CoQ10 Emulgel has potential as an anti-aging product. However, information is lacking about both the drug release profile and its dermal penetrability which supports the theory that protransfersome and its incorporation into emulgel could prove a useful model for developing skin anti-aging cosmetics. Moreover, both the ability of protransfersome and protransfersomal emulgel to maintain drug stability and the physicochemical properties of the forms of skin dosage need to be evaluated for drug levels during study periods in line with ICH guidelines. Therefore, the product development involved could be comprehensively analyzed.

Conclusions

The results of this study indicate that emulgel-loaded protransfersomes, are employed as delivery carriers of CoO10. possess positive physical properties, thereby increasing anti-aging activity with a low skin irritancy score. Pro-posing the incorporation of protransfersomal emulgel into cosmetics requires further studies, especially on the acceptability test in humans and stability tests for longer storage times. From the results of this study, although the primary nature of CoQ10 severely limits its skin delivery, protransfersome provides potential benefits when used as a delivery system for active cosmetic ingredients within skin aging therapy.

This article is extracted from Scientifc Reports | (2022) 12:906 | https://doi.org/10.1038/s41598-021-04708-4