Ethosomes Loaded with Spiranolactone for Acne treatment through Topical Gel Formulation

 

Shalu Verma¹*, Alka Singh², Vikash Jakhmola³

¹Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University,

Dehradun, Uttarakhand - 248007, India.

²School of Pharmaceutical Sciences and Technology, Sardar Bhagwan Singh University,

Dehradun, Uttarakhand - 248161, India.

³Uttaranchal Institute of Pharmaceutical Sciences, Uttarakhand, Dehradun-248007, India.

 

ABSTRACT:

The goal of this work was to create Spiranolactone-loaded ethosomes and use them as a topical acne therapy gel. The size of the vesicle, Spiranolactone filling, and encapsulation function of ethosomes were all created and tested. Improved ethosomes were produced as Carbomer 974 gels and tested for transdermal permeability and porosity, as well as in vitro transplantation, against traditional hydroethanolic gels. With Spiranolactone efficiency of loading and encapsulation of 0.433±0.006 mg/mL and 39.29±0.65 percent, the produced ethosomes had a mean size of vesicle 68.1±1.8 nm. A well-developed ethosomal gel had 2.5 times the transdermal flux and 2.1 times the skin implantation of regular gels. For mild skin irritation, ethosomal gel has a superior anti-acne impact. This research reveals that ethosomal formation is a good way to transfer spiranolactone to the skin, and that spiranolactone ethosomal gels could be used to treat acne in the future.

 

 

 

INTRODUCTION: 

Acne is a complex condition that can affect the upper chest, back, and other parts of the skin. This is the most common skin disorder, affecting 84 percent of children^1,2. Propionibacterium acnes colonisation and excessive androgen production in men have both been associated to the pathophysiology of illness³. Although various compounds are used to treat acne, the majority of them do not all of the underlying reasons are addressed, and adverse effects such as thirst, rashes, and photosensitivity⁴. Spiranolactone, a well-known aldosterone receptor antagonist, has been shown to have therapeutic potential and a variety of therapeutic actions in acne^5,6. Spiranolactone has a limited solubility in water and a total bioavailability in the mouth of only about 2.5 percent in mice, making skin administration a good choice in this combination^7,8.

 

The Corneum Stratum, the outer layer of skin coated in lipid bilayers and containing corneocytes, is a substantial barrier to the transport of medicines to the skin⁹. There are now more topical capsules containing ethosomes available thanks to various methods of getting through the SC barrier¹⁰. The ethosome can move even if the holes are much smaller than their diameter because the ethanol in the ethosome device makes the vesicular membrane flexible.¹¹ Additionally, ethanol works well as a respirator¹². The advantages of ethosomes as a current issue have been supported by numerous studies undertaken over the past ten years¹³. Spiranolactone was combined with ethosomes and used to give acne treatment concepts in the current investigation.^14,15

 

MATERIAL AND METHODS:

Materials:

Spiranolactone was obtained from Aquatic remedies, Pvt. Ltd. (purity 98.2%, India). Soybean phosphatidylcholine (SPC, Lipoid S100) was obtained from Aquatic remedies, Pvt. Ltd (India). Oleic acid and Carbomer 974 was obtained from Aquatic remedies, Pvt. Ltd (India). Polyethylene glycol 400(PEG-400) was obtained from Aquatic remedies, Pvt Ltd. (India). Trypsinase was obtained from Aquatic remedies, Pvt. Ltd (India).

 

Preparation of Spiranolactone ethosomes:

Table 1 shows the composition of the several manufactured ethosomes. A magnetic stimulant mixes an ethanolic solution of soybean phosphatidylcholine and spiranolactone at 700 revolutions per minute. After 15 minutes of mixing, the mixture was submerged in cold bath water for three cycles of 150 seconds each while being powered by a 400Wsonotrode (2s pulses with 3 s interval). To recover the decreased spiranolactone, the mixture was passed through three polycarbonate membrane (220nm) filters. Vesicle dimensions and shape. By using laser scattering, the polydispersity index and vesicle size of the ethosomes were calculated in triplicate.

 

Table 1: Ethosomes preparation

Formulation	F1	F2	F3
Spiranolactone	10mg	10mg	10mg
SPC (soybean phosphatidylcholine)	0.5g	0.5g	0.3g
Ethanol	3ml	2ml	4ml
Water	7ml	8 ml	6ml

 

Efficiency of loading and encapsulation of spironolactone:

Spiranolactone fill up of ethosome suspension was shown by sample purification and HPLC analysis. The effectiveness of encapsulation (EE) was studied by means of ultrafiltration. An Amicon ultra centrifugal filtering unit was hired to isolate ethosomes into free Spiranolactone. Briefly, 500μL ethosome suspension samples were added to the filter unit, centrifuged there at 6000rpm for 15 minutes, and had filters filtered via methanol for the HPLC Spiranolactone measurement method. The ethosomes' EE is determined on the basis of the given formula:

 

EE% = CL – CF ÷ CT × 100.

Where CT was the predicted concentration of Spiranolactone and CL was the ethosome loading with spiranolactone, CF was the Spiranolactone concentration that remained in the filtrate. The outcomes are presented as the averages across three different measurements.

 

Ethosomal gel Preparation:

A 1.0 percent carbomer gel was enhanced by adding 2 mL of 10 percent (w/v) NaOH to a 0.5g carbomer 974 solution in 48mL of water. After the 1.0g of gel had been dissolved in 10mL of filtered water, the pH of the carbomer gel was measured three times with a pH metre. A 1 percent carbomer gel was combined with a 1:1 (v/w) ethosome solution to create the ethosomal gel, which was then homogenised by regeneration. The spiranolactone F1 formulation's equivalent loading of spiranolactone was combined with 3mL of ethanol, 10g of 1percent carbomer gel, and 7mL of water before being swirled until homogenous. Creating a blank ethosomal gel by creating an F1 without spiranolactone and creating empty ethosomes were mixed at a 1:1 (v/w) ratio with 1 percent carbomer gel.

 

In vitro study:

Nine to eleven days after PBRI's Experimental Animal Center in Bhopal, India, gave a pig a deadly dose of sodium barbital, the pig's back and stomach were discovered covered in skin. The skin was cut and trimmed to the same thickness using a drum dermatome (about 1 mm). A firm epidermis and a portion of the dermis made up the restored skin. The skin is kept at or below - 80°C before application.. The PBRI Veterinary Assessment Committee in Bhopal, India, approved all procedures performed on the animals in this study. In vitro percutaneous investigation was conducted using specific distribution cells (distribution area: 2.83 cm2; reception room volume: 6.8 mL).As a reception, a saline solution containing 40% PEG-400 was employed. The skin sample is maintained at 37 °C, covered with the gel, and rotated at 250rpm with the receptor media (1.0 g). The receptor medium (1 ml) was collected, the volume of each sample was measured, and fresh material was added at intervals of up to 24 hours. Each sample's spiranolactone content was calculated using HPLC-UV. The skin was weighed after the test, cleansed with a damp cotton pad, chopped into little pieces, and put in a plastic tube (24 hours). A 1 mL dosage of a 1 percent trypsinase solution was then added, and the tube was kept at 37°C for 4 hours. After adding 4 mL of ethyl acetate to the mixture, it was sealed for 5 minutes before being centrifuged at 3000 rpm for 10 minutes. The ethyl acetate was moved to a fresh tube and allowed to evaporate while being affected by nitrogen gas. With 0.5 mL of methanol, the remaining sample is reconstituted for HPLC analysis. Separation is carried out in a column heater set at 40°C in column C18. Methanol: water (85:15, v: v) was used in the moving phase at a flow rate of 1.0mL/min. The wavelength of the UV detector is 263 nm. With a low quantification rate of 4ng/mL and a low acquisition limit of 1.5ng/mL, the method had a positive line (r2 = 0.9995) at 4 1000ng/mL.

 

Anti-acne effect: The anti-acne efficacy of spiranolactone ethosomal gels was tested using an oleic acne model.The rabbits utilised were male and weighed around 2.5kg. They were supplied to the PBRI Experimental Animal Center in Bhopal, India. Oleic acid (0.2mL) was applied twice day for two weeks to a region outside the ear canal 2 x 2cm below the pinafore. The breed model was created for a total of 12 rabbits, who were divided into three groups. For two weeks, 0.5 g was applied twice a day to the acne region in one-third of the formulation (ethanomal gel loaded with Spiranolactone, a standard gel, and an empty ethosomal gel). A lethal dose of sodium barbital was administered into rabbits. The ear tissues were removed 12 hours after the last treatment and soaked in 4 percent protected formaldehyde before being regularly immersed in paraffin. The 4μm thick segments were cut with a rotary microtome, HM340E, and hematoxylin-eosin contaminated according to the standards. The components were waxed and immersed with a Harris Haematoxylin solution for 10 minutes. The pieces are then rinsed and immersed for 2 minutes in eosin.¹⁶

 

Skin irritation test:

The OECD Guideline 404 "acute cutaneous irritation/ Corrosion"¹⁷ was used to conduct the skin irritation testing. We selected 17-week-old rabbits with robust skin and a healthy weight of 3-3.6 kg. The hair on the back of each rabbit was trimmed without injuring the skin in an area of roughly 10 x 15cm 24 hours before the test. The shaved region is covered with a gauze patch secured in a bandage and spiranolactone ethosomal gels or empty gels are administered. After 24 hours, the patch was removed and the area was cleansed with gauze soaked in warm water. At 1, 24, 48, and 72 hours, the skin reaction was measured according to OECD norms. The primary markers of irritation were discovered using a score of 24, 48, and 72 hours. After the last evaluation, the rabbits were slaughtered, and the treated skin was removed for analytical and histological testing. In each group, three rabbits were subjected to the tests.

 

Statistical analysis:

For studies with three or more repeats, the information was presented as a mean standard deviation. To determine the significance of the mean difference, the Student's t-test was utilised. The limit of statistical significance was established at P<0.05.

 

Fig. 1: Pictures of ethosomes filled with spiranolactone

 

RESULTS:

Evaluation of ethosomes:

Size and shape of vesicles. The ethosomes with spiranolactone were a yellow to semi-transparent scarlet saffron colour (Fig. 1). Table 2 lists the vesicle sizes and polydispersity indexes (PDI) for each shape. The diameter of ethosome vesticle veins ranges from 68.1 to 78.1nm. The overall PDIs were less than 0.3, indicating that the volume of the vesicles was similar. F1 was chosen to fix ethosomal gels because it had the highest drug loading and EE of the three active components.

 

Transdermal In vitro study:

The pH of the empty cabomer gel (n = 3) was 7.19±0.02.The features of ethosomal and conventional genes' transdermal penetration in vitro are examined. Cumulative spiranolactone penetration at each location was structured as a time function to represent transdermal permeation features (Fig. 2). Table 3 shows the total values and stable transdermal variations estimated from the slope of the retrieval operations for the last three points of each curve. Ethosomal gels demonstrate a 2.5-fold increase in volume and transdermal flow above conventional gels, demonstrating that ethosomes were active skin delivery vesicles.

 

Anti-acne effect:

Figures 3 and 4 show the spiranolactone-loaded ethosomal gel's ability to treat acne. Normal rabbit ear skin has a thin, multilayered SC, no bacterial colonisation in the pilosebaceous orifice, and no inflammation in the pilosebaceous aperture because there is no inflammation in the dermis (Figs. 3 and 4A). Significant lymphatic cell infiltration, dermal thickening from bacteria and neutrophil granulocytes, and keratoplasia were all observed in the pilosebaceous orifice, SC, of rabbits treated with ethosomal clear gel and all these changes were visible (Fig. 4B). The deep layer of the skin was still impacted by conventional gel therapy, which decreased lymphatic cell access into the dermis, neutrophil granulocyte and bacterial thickening (Fig. 3B) (Fig. 4C) and the rigidity of the epidermis was adherent but unabated (Fig. 3A). Following treatment with an ethosomal gel containing spiranolactone, the skin's natural structure was restored (Fig. 4D). The pilosebaceous unit was not inflamed, and neither the SC nor the pilosebaceous orifice had keratoplasia.. The deeper layers, or dermis, were not shown to have lymphatic cell infiltration. The size of the epidermis was not significantly different from normal skin (Figure 3A). These results suggest an ethosomal gel containing spiranolactone showed better anti-acne properties.

 

Table 2: Evaluation of ethosomes

Formulation	Vesicle size/ nm	PDI	Spiranolactone loading/ mg/ml	EE%
F1	66±1.94	0.185±0.017	0.445±0.007	40.31±0.67
F2	69±1.94	0.225±0.004	0.251±0.001	23.23±0.09
F3	78±2.2	0.204±0.017	0.181±0.005	9.13±0.50

 

Fig. 2: Ethosomal gel and ordinary gel have different in vitro transdermal profiles and skin depositions, respectively. (n = 6) **, P<0.01.

 

 

There is no inflammation in the dermis (Figs. 3 and 4A). Ethosomal clear gel induced neutrophil granulocyte Significant lymphatic cell infiltration, keratoplasia, bacterial thickening in the pilosebaceous orifice and SC, and thickening of the dermis and epidermis in the acne-causing model rabbits (Fig. 4B). Conventional gel therapy clearly reduced the amount of lymphatic cells that entered the dermis and prevented the thickening of bacteria and neutrophil granulocytes (Fig. 3B), but the deep layer of the skin was still affected (Fig. 4C). The epidermis was adherent but inevitably stiff due to keratoplasia in the pilosebaceous orifice and SC (Fig. 4C) (Fig. 3A). After being treated with an ethosomal gel containing spiranolactone, the skin returned to its normal state (Fig. 4D). Both keratoplasia and inflammation were absent from the pilosebaceous unit, SC, and orifice. There was no evidence of lymphatic cell infiltration into the deeper layers' covering, the dermis. The epidermis' size was comparable to that of healthy skin in terms of size (Figure 3A). These findings demonstrate that spiranolactone-loaded ethosomal gel showed superior anti-acne effects.

 

Table 3: Transdermal characteristics of conventional and ethosomal gels applied to excised pig skin (n = 6)

Formulation	Q24h/ng/cm2	Jss/ng/(h.cm2)
Ethosomal gel	186.6±42.6	18.35±4.18
Conventional gel	74.98±22.16	7.915±1.9.31

 

Skin irritation:

In the spiranolactone there were no obvious cutaneous reactions, such as erythema or edoema, in the groups treated with ethosomal gel or gel. According to OECD norms, the key irritation markers for each group at any given moment are always 0, and spiranolactone ethosomal gel does not cause skin irritation. Empty ethosomal gel groups and spiranolactone ethosomal gel features histological graphs resembled untreated skin (Fig. 5). Only a few lymphatic cells in treated rabbits may be identified (Figures 5B and 5C).

 

Figure 3: Effect of a spiranolactone-filled ethosomal gel on transdermal thickness (A) and lymphatic cell counts in a dermis radon region (10 μm10μ m) (B). *P>0.05, **P0.01. #P>0.05, *P0.05, **P0.01.

 

DISCUSSION:

Our ethosomes are all less than 99 nm long (desk 2), showing that these vesicles are capable of transporting spiranolactone into the epidermis. Vesicles smaller than 395 nm are significant in topical drug delivery mechanisms18. This is because they could carry their payloads deep into the layers of the pores and epidermis. Ethanol was chosen because it formed ethosomal vesicles that were smaller than liposome vesicles and fluidized phospholipid bilayers19.The ethosome length is also affected by phosphatidylcholine concentration, according to our findings. F1 and F2 have a similar recognition on phosphatidylcholine and vesicle size, despite the fact that F3 has substantially less phosphatidylcholine and larger vesicles. The system's transport potential is checked using medication loading and essential EE variables. It was discovered that cutaneous transport was linked to drug exposure in the formula, with only seized capsules showing improved outcomes²⁰. This could be owing to the ethosomal center's heightened awareness of the substance in ethanol gift ²¹. EE is reliant on the focus of ethanol and lipid. Extending EE by increasing ethanol awareness from 30% to 40% and lipid awareness from 2% to 4% was possible due to increased membrane fluidity and membrane fabric. Ethanol concentrations beyond 40% became unprofitable because they may have made the vesicles more leaky^22. Future research will focus on the F1 method, which has a higher spiranolactone and EE burden (table 2). Whether the effects of the formulation are regional or systemic, drugs must pass through the SC barrier.

 

Using specialised distribution cells, we looked at ethosomes' capacity to carry spiranolactone into and out of pig skin. Despite being in the skin, ethosomal gels fully include spiranolactone, which has a significantly higher transdermal flux than regular gels. Spiranolactone deposition in the skin at the conclusion of the test varied significantly depending on whether it was injected into the ethosomal gel or the conventional gel (Table 2). Inflammation, keratoplasia, bacterial colonisation, and epidermal thickness were all found in the histological study of the acne model skin and were all signs of acne in people (Fig. 4B). The spiranolactone-loaded ethosomal gels had a more effective anti-acne effect than ordinary gels, and the skin structure of the treated rabbits was practically normal. The spiranolactone-loaded ethosomal gel may cause some modest skin irritation, but more studies on humans are required to confirm this level of irritation^24,25.

 

Figure 4: Effect of a spiranolactone-loaded ethosomal gel on the histopathology of oleic acid-induced rabbit ear acne.

A: regular rabbit ear, B: an acne model was given a placebo ethosomal gel treatment, C: a spiranolactone gel, and D :a filled with spiranolactone ethosomal gel.

 

Figure 5: Normal rabbit back skin stained with hematoxylin and eosin (A), using empty ethosomal gel to cure rabbit skin (B), and ethosomal gel-treated rabbit skin that contains spiranolactone. (C).

 

CONCLUSION:

Spiranolactone-loaded ethosomes have smaller vesicles, a higher spiranolactone load, and higher Entrappment Efficiency. The penetration of carbomer gel into the skin and the incorporation of ethosomes was higher than that of traditional hydroethanolic gel. In vivo tests revealed that the ethosomal gel containing spiranolactone had a better anti-acne impact than the standard gel, while causing less skin irritation. This study reveals that spiranolactone can be delivered to the skin effectively by ethosomes, and spiranolactone ethosomal gel may one day be used to treat acne.

 

REFRENCES:

1.     Arora MK, Yadav A, Saini V. Role of hormones in acne vulgaris. Clin Biochem. 2011; 44(13): 1035–40. doi: 10.1016/ j.clinbiochem. 2011.06.984

2.     Bloom DF. Is acne really a disease?: a theory of acne as an evolutionarily significant, high-order psychoneuroimmune interaction timed to cortical development with a crucial role in mate choice. Med Hypotheses. 2004; 62(3): 462–469. doi: 10.1016/j.mehy.2003.11.003

3.     Liu PF, Hsieh YD, Lin YC, Two A, Shu CW, Huang CM. Propionibacterium acnes in the pathogenesis and immunotherapy of acne vulgaris. Curr Drug Metab. 2015; 16(4): 245–254. doi: 10.2174/1389200216666150812124801

4.     Valente DS IC. Novel pharmacological approaches for the treatment of acne vulgaris. Expert Opin Investig Drugs. 2014; 23(10): 1389–410. doi: 10.1517/13543784.2014.923401

5.     Cai Y, Zhang W, Chen Z, Shi Z, He C, ChenM. Recent insights into the biological activities and drug delivery systems of tanshinones. Int J Nanomedicine. 2016; 11: 121–30. doi:10.2147/ ijn.s84035

6.     Tang S, Shen XY, Huang HQ, Xu SW, Yu Y, Zhou CH. Cryptotanshinone suppressed inflammatory cytokines secretion in RAW264.7 macrophages through inhibition of the NF-kappaB and MAPK signalling pathways. Inflammation. 2011; 34(2): 111–118. doi: 10.1007/s10753-010-9214-3

7.     Wu CY, Hsieh CY, Huang KE, Chang C, Kang HY. Cryptotanshinone down-regulates androgen receptor signaling by modulating lysine-specific demethylase 1 function. Int J Cancer. 2012; 131: 1423–34

8.     Akhtar N. Vesicles: a recently developed novel carrier for enhanced topical drug delivery. Curr Drug Deliv. 2014; 11(1): 87–97. doi: 10.2174/15672018113106660064

9.      Sneh Priya, Divya Jyothi, Jainey P James, Amala Maxwell. Formulation and Optimization of Ethosomes loaded with Ropinirole Hydrochloride: Application of quality by Design Approach. Research J. Pharm. and Tech, 2020; 13(9): 4339-4345. doi:10.5958/0974-360x.2020.00767.2

10.  Baek JS, Pham CV, Myung CS, Cho CW. Tadalafil-loaded nanostructured lipid carriers using permeation enhancers. Int J Pharm. 2015; 495(2): 701–9. doi: 10.1016/j.ijpharm.2015.09.054

11.  Ashok A. Hajare, Hemalata S. Dol. Screening of effective formulation techniques for Designing and Fabrication of Terbinafine hydrochloride ethosomes. Research J. Pharm. and Tech. 2021; 14(3): 1353-1359. doi:10.5958/0974-360x.2021.00241.9

12.  Monisha C, GNK. Ganesh, Mythili L, Kayalvizhi Radhakrishnan. A Review on Ethosomes for Transdermal Application. Research J. Pharm. and Tech. 2019; 12(7): 3133-3143. doi: 10.5958/0974-360x.2019.00529.8

13.  Moghal. Roohi Shabreen, S. Sangeetha. Ethosomes: A Novel Drug Delivery System And Their Therapeutic Applications -A Review. Research J. Pharm. and Tech, 2020; 13(4): 1970-1978.

14.  OECD. Test No. 404: Acute Dermal Irritation/Corrosion, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris. 2015.

15.  Verma DD, Verma S, Blume G, Fahr A. Particle size of liposomes influences dermal delivery of substances into skin. Int J Pharm., 2003; 258(1-2):141–151. doi: 10.1016/s0378-5173(03)00183-2

16.  Shraddha Pandey, Shashi Kiran Misra, Nisha Sharma. Ethosomes- A Novelize Vesicular Drug Delivery System. Research J. Pharm. and Tech., 2017; 10(9): 3223-3232. doi: 10.5958/0974-360x.2017.00572.8

17.  Amala Maxwell, Sneh Priya. Nanosized Ethosomes – A Promising Vesicular Drug Carrier for Transdermal Drug Delivery. Research J. Pharm. and Tech., 2019; 12(2): 876-880. doi: 10.5958/0974-360x.2019.00150.1

18.  Dubey V, Mishra D, Jain NK. Melatonin loaded ethanolic liposomes: physicochemical characterization and enhanced transdermal delivery. Eur J Pharm Biopharm. 2007; 67(2): 398–405. doi: 10.1016/j.ejpb.2007.03.007

19.  Elsayed MM, Abdallah OY, Naggar VF, Khalafallah NM. Deformable liposomes and ethosomes: mechanism of enhanced skin delivery. Int J Pharm. 2006; 322(1-2): 60–6. doi: 10.1016/j.ijpharm.2006.05.027

20.  Choi EH, Ahn SK, Lee SH. The changes of stratum corneum interstices and calcium distribution of follicular epithelium of experimentally induced comedones (EIC) by oleic acid. Exp Dermatol. 1997; 6(1): 29–35. doi: 10.1111/j.1600-0625.1997.tb00142.x

21.   Yamakoshi T, Makino T, Matsunaga K, Yoshihisa Y, Rehman MU, Seki T. Efficacy of chlorhexidine gluconate ointment (Oronine H((R))) for experimentally-induced comedones. Clin Cosmet Investig Dermatol. 2012; 5: 79–83. doi: 10.2147/ccid.s33361

22.  Kwon HH, Lee JB, Yoon JY, Park SY, Ryu HH, Park BM. The clinical and histological effect of homeuse, combination blue-red LED phototherapy for mild-to-moderate acne vulgaris in Korean patients: a double-blind, randomized controlled trial. Br J Dermatol. 2013; 168: 1088–94.

23.  R.B. Saudagar, S. Samuel. Ethosomes: Novel noninvasive carrier for Transdermal Drug Delivery. Asian J. Pharm. Tech. 2016; 6(2): 135-138. doi: 10.5958/2231-5713.2016.00019.2

24.  Roge Ashish B, Sakhare Ram S, Bakal RL, Channawar MA, Bakde BV, Gawande SR , Chandewar AV. Ethosomes: Novel Approach in Transdermal Drug Delivery System. Research J. Pharma. Dosage Forms and Tech. 2010; 2(1): 23-27.

25.  Anuradha Patel, R.K. Sharma, Mahesh Trivedi, Shivaprakash, Anjana Panicker. Ethosomes: A Novel Tool for Transdermal Drug Delivery. Research J. Pharm. and Tech. 2013; 6(8): 838-841.

 

 

 

Accepted on 02.08.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1219-1224.