INTRODUCTION:

TXA is a synthetic lysine derivative that was discovered in 1962 by Japanese scientists Shosuke and Okamoto¹. It is mainly employed as a fibrinolytic agent, inhibiting the conversion of plasminogen to plasmin, lowering prostaglandin and fibroblast growth factor synthesis, and as a result, decrease the melanin synthesis.

The effect of TXA in melasma was a serendipitous discovery by Nijo Sadako²in 1979. TXA can be administered orally, topically, intradermally, or by micro-needling³. The current oral dose for melasma is 250 mg twice daily, which is much less than the 3900 mg daily dose used to treat hemophilia, severe menstrual bleeding, or other hemorrhagic diseases⁴. In multiple studies, TXA have been shown to be effective in patients with melasma^5,6,7. TXA has a bioavailability of 30% to 50% when used orally. Hence topical delivery system could be a good alternative. TXA is regarded as a pregnancy category B drug as no mutagenic activity has been detected in in-vitro and in-vivo test systems. As it is only minimally excreted in breast milk, breastfeeding may be continued, if required⁸.TXA is available in the market as a monotherapy or in a combination with other skin whitening agents in conventional dosage forms but it is not available alone in nano dosage form, hence a novel approach was made to incorporate TXA into nano ultra-deformable vesicles such as transethosomes (TEL) to study its stability and effectiveness for melasma.

Formulations available^9,
10

· Tablets: Only TXA-250 mg, 500 mg; Combination of 250 mg TXA with proanthocyanadin.

· Injections: 100mg/mL

· Topical creams: (0.05g/50 ml) in combination with whitening agents like kojic acid, mulberry extract, arbutin, vitamin E, etc.

A chronic, therapeutically challenging, universally relapsing hyperpigmentation disorder causing the appearance of brown or grey patches on the skin, primarily on the face called Melasma¹¹. It involves mainly the cheeks, chin, nose bridge, forehead, and commonly the upper lip³. Although melasma can occur in both sexes, races, and ethnicities, the overwhelming majority of the patients are women and individuals with Fitzpatrick’s skin types three to five which includes darker white skin, light brown, and brown skin, especially those who live in areas of intense UV radiation, including Hispanics, Asians, and African Americans¹². Melasma seems to be less common in men¹³. Affected individuals had emotional and psychological disruption, according to data from numerous quality-of-life studies¹⁴. Patients often feel irritated, humiliated, worried, and depressed about their skin appearance and suffer from significant emotional impact¹⁴. Etiological factors include intense exposure to sunlight, genetic predisposition, and hormonal imbalances¹⁵. Treatments approaches for melasma include UV protecting agents, topical skin lighteners, some newer oral and topical agents, chemical peeling agents, dermabrasion, and laser therapy¹⁵. All of these conventional creams, gels, and tablets have significant drawbacks, including low bioavailability, unpleasant taste, gastrointestinal side effects, and scarring. To overcome the drawbacks of conventional delivery systems, novel ultra-deformable vesicles (UDV) such as TEL have been developed, which have the advantage of being non-toxic, thermodynamically stable, and used as a tool for the delivery of large proteins and peptides¹⁶. TEL are composed of a phospholipid, ethanol, edge activator, and water. The use of ethanol and edge activators makes them extremely flexible, allowing them to easily cross the stratum corneum. This novel dosage form uses less dose of TXA thus has the potential to have less toxicity and can increase the patient compliance due to decreased dosing frequency¹⁶.

The aim and objective of the present study was to enhance the skin permeation of TXA by incorporating it into TEL and to evaluate for its characteristics such as PS, EE, drug-excipient incompatibility studies (IR and DSC analysis), SEM analysis, in-vitro release study, and ex-vivo skin permeation study into the systemic circulation for 24 hours. Optimized TEL batch is then incorporated into a transdermal patch to increase patient compliance due to ease of application.

MATERIALS AND METHODS:

TXA was obtained as a gift sample from Hunan Dongting Pharmaceutical Co. Ltd, China. Phospholipon 90G [Phosphotidylcholine (PC)] was a gift sample from Lipoid Ludwigshafen, Germany. Sodium cholate, Ethanol 99.8%, and Tween 80 were procured from S.D. Fine chemicals, Mumbai. PEG 400 was obtained from BASF, India. HPMC K 15M was obtained from Chemtech speciality Pvt. Ltd. India. Distilled water used throughout the study was procured from J.K. Laboratories, Thane. All other chemicals and solvents used for the study were of analytical grade.

Preparation of TXA loaded TEL:

TXA-loaded TELs were prepared using the cold method¹⁷. This method is easy to scale up and can be used for both thermolabile and thermostable drugs¹⁸. In conical flask A, phospholipon 90G was dissolved in ethanol with constant stirring at 700 rpm on a magnetic stirrer. TXA and Sodium cholate (edge activator) were dissolved in water in conical flask B, both the vessels were maintained at 30℃. Then the aqueous mixture B is added very slowly in ethanolic mixture A in a fine stream with constant stirring at 700 rpm in a closed vessel. It was stirred for additional 5 min. The system was kept at 30℃ throughout the process. Size reduction was done by probe sonication (Oscar Ultrasonics, India) for five min at RT.

Optimization of TXA loaded TEL:

TXA-loaded TEL were optimized by varying the concentrations of Phospholipon 90G and Sodium cholate over a range of concentrations to create 13 experimental batches. For Phospholipon 90G and sodium cholate, the lowest and highest actual value ranges were 400-600 mg and 40-80 mg, respectively. The required goals were to achieve the smallest PS and the highest E.E. The optimized batch was chosen from among the 13 experimental batches for further characterization studies.

Preparation of adhesive layer¹⁹:

The transdermal patch was designed to deliver optimized TXA-loaded TEL and to control TXA release over a prolonged period of time. To begin, 2.5 % PVA (Polyvinyl alcohol) was dissolved in the required amount of distilled water by intermittently heating at 60°C for a few seconds to form a PVA solution as a backing membrane, which was then sonicated to remove air bubbles. The solution was poured into a petri dish and dried in a hot air oven at a temperature not greater than 60°C until it formed a uniform layer.

Preparation of TXA loaded TEL patch:

TEL patches were prepared by solvent evaporation method²⁰. HPMC K-15 M was soaked in a 1:1 hydroalcoholic solution until a smooth gel was formed. PEG, Tween 80, and lyophilized TEL were dissolved in a beaker using ultrasonication. This solution was slowly poured into a HPMC polymer base. The gel was then ultrasonicated for about 15-20 minutes to remove all of the air bubbles. This final solution was poured into a petri dish, covered with aluminum foil, and allowed to dry at room temperature for 24 hours.

Optimization of transdermal patch²⁰:

TXA-TEL loaded transdermal patch was optimized by varying the concentration of permeation enhancer (Tween 80) and plasticizer (propylene glycol) to get an optimized patch. The hit-and-trial method was used for optimization. By varying the concentrations of permeation enhancer (0.1%-0.3%) and plasticizer (1-3 ml), ten formulations were developed. The best one was selected based on the physical appearance of the transdermal patch created by varying concentrations of independent variables.

VESICULAR CHARACTERIZATION:

PS, Polydispersity Index (PDI), and Zeta potential:

The optimized batch was analyzed for PS and PDI by nanoparticle tracking analysis (NTA 3.1) using Nano sight NS500 with automated sample introduction, the computer-controlled motorized stage with CCD camera and red (638nm) laser. It works on the principle of dynamic light scattering through He-Ne laser having scattering angle of 90˚. Samples were diluted 100 times to get good results. A PDI value close to 0.25 indicates that the particle distribution is narrow and homogeneous²⁰.

The electrophoretic light scattering method was used to examine the optimized batch for Zeta potential. A current is applied across a pair of electrodes at either end of a cell containing the particle dispersion to measure the zeta potential. Particles get charged and attracted to the oppositely charged electrode, and their velocities were measured. Stable formulations have zeta potential values that are not very close to 0 mV. For both particle size and zeta potential measurement, approximately 0.2 μL sample was diluted with 2 mL of distilled water and results were obtained in triplicate^{21, 22}.

Microscopic morphology by scanning electron microscopy (SEM)²³:

The SEM was used to determine the shape and morphology of the TEL. The TEL were directly mounted in SEM aluminum stub, using double-sided sticking tape and scanned in a low vacuum chamber with a focused electron beam at 20 kV and magnification of 12, 000X. Secondary electrons, emitted from the samples were detected as the image formed.

Fourier transform infrared spectroscopy (FTIR):

FT-IR analysis was performed using a Perkin Elmer Infrared spectrophotometer to investigate the interaction between the drug and the excipients in an optimized formulation. TXA and individual excipients were analyzed by the potassium bromide (KBr) disk method over the scanning wavelength range of 4000- 400 cm^-1. The spectrum was analyzed using the software Perkin Elmer Spectrum Express.

DSC studies for excipient interaction:

A precisely weighed sample of drug and individual excipients were placed in aluminum pans, which were then closed with an aluminum cap and sealed. Thermograms were obtained by heating the sample at a constant rate of 10°C/minute with a Mettler Toledo DSC 60. For the run, a dry purge of nitrogen gas at a rate of 20ml/min was used. The sample was heated to temperatures ranging from 35°C to 400°C. DSC thermograms revealed the melting point and peak maxima. The thermograms were acquired and analyzed using STAR^e SW 9.00 software.

% Entrapment Efficiency (EE):

The ultracentrifugation method was used to determine the drug EE of the formulations. The vesicles were separated using ultracentrifugation at 15000 rpm for 90 minutes at 4^oC. The supernatant liquid was separated and diluted with methanol, and the drug concentration was measured spectrophotometrically at 567 nm. Results were obtained in triplicate. EE was determined by the following equation¹⁷.

% EE = (Q_T) - (Q_S) × 100

(Q_T)

Where,

Qt is the amount of the drug added; Qs is the amount of drug found in the supernatant

% Loading capacity:

It finds out the ability of lipids to encapsulate drugs in nanoparticles. The supernatant was used to determine free or un-entrapped TXA using a UV Spectrophotometer at 567 nm, and the loading capacity was calculated using the following formula²⁰.

% Loading capacity = (Q_T) - (Q_S) × 100

(Q_P)

Where,

Qt is the amount of the drug added; Qs is the amount of drug found in the supernatant; Q_Pis the total amount of lipid used.

Percentage yield of nanoparticles:

From an economic standpoint, it is a critical parameter. The percent yield was calculated using lyophilized powdered TEL. The powder was precisely weighed and the total amount of TXA and lipid used in the formulation was compared²⁰.

% Yield = Quantity of powdered TEL’s X 100

Quantity of lipid + TXA

CHARACTERIZATION OF TEL PATCH:

Physical appearance:

All transdermal patches were assessed for different parameters like clarity, color, smooth surface, and homogeneity.

Thickness and weight variation:

The thickness of TEL transdermal patches was measured using a vernier Calliper. The thickness of the prepared patch was measured at various locations, and mean values were calculated. To determine the weight variation, three patches from the same batch were weighed using electronic balance and the standard deviation was calculated^{24, 25}.

Folding endurance:

Three stripes of 2 × 2 cm from each individual batch were cut precisely. The folding endurance of optimized patches was measured manually. It determines the capability of the sample to resist folding, a number of times patch was folded from the same place to develop any cracks or to break the patch²⁶.

% Moisture content:

To determine % moisture content, a 2 × 2 cm patch strip was cut and weighed from the TEL patch. The moisture content of the weighed TEL-transdermal patch was determined by placing it in a desiccator containing calcium chloride at room temperature for 24 hours. Then, the films were weighed repeatedly. This process is continued until and unless a constant weight is achieved²⁶. The process was repeated three times from each batch and the average was determined which was represented as ±SD. Then % moisture content was calculated by using the following formula

% moisture content = (Initial weight-Final weight) × 100

Final weight

Drug content²⁰:

The TXA content in the patch was determined by dissolving it in a buffer (pH 5.8). After filtration, it was spectrophotometrically examined at a λmax of 567 nm. TXA contentuniformity was also evaluated by calculating an average and comparing it to the TXA content of individual patches.

In-vitro drug release study^{27, 28}:

In-vitro drug release was studied using Franz Diffusion cell. A cellophane dialysis membrane with a molecular weight cut off of 8000 - 12000 Dalton (Hi media) was hydrated with receptor medium phosphate buffer pH 5.8 overnight before being fastened between the donor and receptor compartments. The soaked membrane was clamped precautiously to the diffusion tube at one end of of 2 cm diameter (22.7 cm²), which served as the donor section. Vesicular formulation of 4 ml of the TEL or 2*2 cm of the transdermal patch was kept in the donor compartment. The receptor compartment was filled with 13 ml of Phosphate buffer pH 5.8 and stirred with a magnetic bead at 200-300 rpm and the temperature of the system was maintained at 37 ±1℃ to mimic the human skin. The effective permeation area of the Franz diffusion cell was 2.60 cm². At predetermined time intervals of 0, 1, 2, 3, 4, 5, 6, 7, and 24 hours, 1 ml of aliquot was withdrawn and immediately replaced with an equal volume of fresh buffer. All samples were analyzed for TXA content by UV spectrophotometer at 567 nm. The results were obtained in triplicates and the graph was plotted on Microsoft Excel between time and % cumulative release.

The release mechanism of the TXA can be explained through various kinetic models that are applied to in-vitro release profile of a drug. In the current research, various release models; KorsmeyerPeppas²⁹, Higuchi³⁰, Hixon Crowell³¹, first-order³², and zero-order³³were applied on the release data of TXA from TEL loaded transdermal Patch.

Ex-vivo skin permeation study:

Porcine abdomen skin was procured from the local slaughterhouse. The hair and subcutaneous fat adhering to the dermal side of the skin were then removed from the upper portion of the skin surface. Because of its lipid content and permeability similarity to human skin, porcine abdomen skin was used as a model membrane for skin permeation study. The sample of was mounted between the donor and receptor compartments of the diffusion cell. The dorsal surface of the skin was placed in contact with the donor chamber which was filled with 4 ml of TEL or 2*2 cm of the transdermal patch was kept in the donor compartment. The receptor compartment was filled with 13 ml of Phosphate buffer pH 5.8 and stirred with a magnetic bead at 200-300 rpm and the temperature of the system was maintained at 37 ±1℃ to mimic the human skin. The Franz diffusion cell had an effective permeation area of 2.60 cm². 1 ml of aliquot was withdrawn at predetermined time intervals of 0, 1, 2, 3, 4, 5, 6, 7, and 24 hrs respectively and was immediately replaced with an equal volume of fresh buffer. All samples were analyzed for TXA content by UV spectrophotometer at 567 nm and the cumulative amount of drug was plotted against a function of time. The steady-state flux was determined as the slope of linear portion of the plot.

STABILITY STUDY:

The optimized patch was subjected to an accelerated stability study at two different temperatures 25± 3℃ and 5 ± 3℃ at 60 % RH. Optimized patches were wrapped in Alu-Alu pouch for stability studies for 6 months as per the ICH guidelines³⁴. After 0, 1, 3, and 6 months of storage, the patch was tested for weight variation, percent moisture content, folding endurance, drug content, and in-vitro drug release study.

RESULTS AND DISCUSSION:

Analytical method development of TXA:

UV-Vis Spectrophotometric analysis:

The λmax of TXA was found to be 567 nm in water and in Phosphate buffer pH 5.8, which was in the agreement with data in the literature. The UV spectrum of TXA was obtained by derivatizing it with Ninhydrin solution following 30 min heating till bluish-purple color developed³⁵. The UV spectrum of TXA at 18 μg / ml in phosphate buffer pH 5.8 is given below in Fig 1.

Fig 1: UV spectrum of TXA

Preparation of standard plot of TXA in Phosphate buffer pH 5.8:

Serial dilution of TXA from 2 μg /ml- 20 μg /ml in phosphate buffer was prepared. The standard plot as illustrated in Fig 2 was obtained by plotting the absorbance Vs concentration. The R² value for the standard plot for phosphate buffer was 0.9989 indicating a good linear relationship. The method was found to be precise for intraday as well as inter-day. Standard calibration curve of TXA is showed in Fig 2.

Fig 2: Standard calibration curve of TXA

Optimization of TXA loaded TEL:

Different concentrations of independent variables i.e. Phospholipon 90G and sodium cholate along with their responses i.e. PS and E.E are presented in Table 1. Batch number 7 was selected based on the goal of the smallest size of nanoparticles and maximum E.E. the formula for optimized batch is showed in Table 2.

Table 1: Result of PS and EE of different batches

Sr. No.	Amt of Phospholipid 90G (mg)	Amt of Sod. Cholate (mg)	Avg Particle size (nm)	Entrapment efficiency(%)
1	400	40	152	83
2	400	50	111	75
3	400	60	63	58
4	500	40	152	83
5	500	50	312	65
6	500	60	78	76
7	600	40	72	94
8	600	50	59	59
9	600	60	574	79
10	450	55	52	69
11	550	45	101	89
12	450	40	210	90
13	550	55	225	85

Table 2: Formula for optimized batch of TEL (Batch no 7)

INGREDIENTS	QUANTITY
Tranexamic acid	250 mg
Phospholipon 90G	600 mg
Sodium cholate	40 mg
Ethanol	30%
Water	Upto 20 ml

Characterization of Txa Loaded Tel:

Particle size (PS) and polydispersity index (PDI):

The average PS was found to be 72 nm as shown in Fig 3. PDI shows the dispersity of the nanoparticles in the solvent system. It was found to be 0.221 indicating good monodisperse system. PDI more than 0.3 consider as a polydisperse system²⁶.

Fig 3: Particle size of optimized batch (Batch no 7)

Zeta Potential Analysis

The zeta potential of the optimized batch shown in Fig 4 was found to be -16.4 mV which was in good agreement in literature due to the net charge of the lipid composition in the formulation. This is possibly due to steric stabilization by lipid. Phospholipon 90G is a zwitterionic compound with an isoelectric point between 6 and 7. Under experimental condition pH 5.8, Phospholipon carried a net negative charge. The edge activators used were anionic in nature. Therefore a net negative charge in all formulation was observed. Also, the negatively charged liposome formulation strongly improved skin permeation of drugs in Transdermal delivery³⁶. The skin also has a slight negative charge. Therefore, the negative zeta potential of the optimized TEL containing TXA might cause little influence in improved drug permeation through porcine skin due to electrostatic repulsion between the same charge of the skin surface and the TEL^{37, 38}.

Fig 4 : Zeta potential of optimized batch (Batch no 7)

Freeze drying of TEL:

The TEL and blank were pre-frozen for 24 hours at -40°C in a deep freezer (Thermo Scientific, India). The pre-frozen samples were then vacuum dried for 24 hours at -80 °C at 1 Pa. (Labocon, UK). The lyophilized samples were collected under anhydrous conditions and stored in a desiccator in a refrigerator. The lyophilized samples were used for SEM studies.

Scanning Electron Microscopy (SEM):

SEM was used to provide information on the morphology and sizes of the particles. It also confirmed the formation of lipid vesicles. Figure 5 depicts a spherical and uniform particle distribution with no aggregates. The size of all particles is similar, indicating that the system is well dispersed. The nanoparticles ranged in size from 0 to 100 nm.

Fig 5: SEM images of optimized batch (Batch no 7)

Fourier transform infrared spectroscopy:

Figure 6 depicts the FTIR spectra of TXA, Phospholipon 90G, Sodium cholate, and TXA-loaded TEL. It was carried out to examine the interaction between the various components of the optimized formulation, as well as the compatibility of TXA with other components in the TEL formulation. TXA characteristic peaks at 2980 cm-1, 1600 cm-1, 2850 cm-1, and 1650 cm-1 were retained in the nanoparticles³⁵, indicating that no chemical interaction occurred between TXA and other TEL components. This was in accordance with the IR data in the literature^{39, 40}.

DSC Study:

The thermal behavior of the final optimised batch of TXA, Sodium cholate, Phospholipon 90G, and TEL was studied using DSC, and the thermographs are shown in Fig 7. TXA exhibits no peaks until 240°C and appears to be stable. Degradation begins at temperatures above 240°C. The thermogram of the optimized formulation showed a broadening curve and a single peak at 146°C, almost overlapping each individual component. This could be due to lipid component melting and interaction with TXA. This indicated that the drug was entrapped in the lipid vesicles and that the formulation was stable¹⁷.

% Entrapment efficiency (E.E), % loading capacity, and % yield:

The average EE of the optimized TXA loaded batch was found to be 94% by the ultracentrifugation method. Also, the drug loading capacity and % yield were found to be 19.2 % and 91.27 % respectively.

Optimization of transdermal patch:

To improve the physical appearance of the TXA-loaded transdermal patch, the concentration of permeation enhancer and plasticizer was varied. Formulation no.6 given in Table 3 was chosen because it exhibited the most appealing physical characteristics, namely transparency, smoothness, flexibility, and adhesiveness. Table 4 gives the optimized formula for transethosomal patch.

Table 3: Optimization of TXA loaded TEL patch

Sr. No	Permeation enhancer (%)	Plasticizer (mL)	Physical appearance
1.	0.1	1	Non- flexible
2.	0.15	2	Flexible and non-adhesive
3.	0.2	1.5	Transparent and too oily
4.	0.3	3	Opaque, non-flexible and too oily
5.	0.2	0.5	Opaque and non-adhesive
6.	0.1	2	Transparent, smooth, flexible and adhesive.
7.	0.25	2.5	Opaque and non-flexible
8.	0.3	2	Non-adhesive
9.	0.15	1	Non-flexible
10.	0.1	3	Oily and opaque

Table 4: Optimized batch formula for transdermal patch (Batch no 6)

Ingredient	Quantity	Role
HPMC-K15 M	3.5%	Polymer
PEG-400	2 ml	Plasticizer
Tween 80	0.1 %	Penetration enhancer
Hydroalcoholic mixture (1:1)	Qs to 20 ml	Solvent system

EVALUATION OF TXA-TEL LOADED TRANSDERMAL PATCHl

Physicochemical characterization:

The visual appearance of the optimized transdermal patch showed good characteristics. It had a clear and uniform appearance that demonstrated homogeneity. It had a smooth surface and was very flexible. The optimized transdermal patch was evaluated for parameters such as thickness, moisture uptake, drug content, weight uniformity, and folding endurance. Based on the reported method, acceptable results were obtained and depicted in Table 5.

Table 5: Characteristic of optimized transdermal patch (batch no 6)

Parameters	Results
Thickness (mm)	0.27±0.3
Moisture uptake (%)	8.67
Uniformity of weight ( mg)	21±3
Folding endurance	200 ± 5.773
Drug content	94.23%

In-vitro drug release study:

The diffusion studies were carried out in non-occlusive conditions at skin pH (P.B. 5.8) to allow the driving force provided by the osmotic gradient. TXA encapsulation in TEL resulted in a significant prolongation in TXA release across the artificial membrane. According to the above results, TEL dispersion and patch released more drug than conventional formulation. High permeation of TEL may be due to a combination of both ethanol and edge activators. The release profile of the patch indicates slow release as compared to TEL dispersion. This is explained by the fact that drug diffusion from the TEL carrier was followed by diffusion from the patch matrix, resulting in sustained release effects. The lower cumulative release of the plain drug solution can be attributed to its lower solubility. To understand the mechanism of drug release from nanoparticles, various models such as KorsmeyerPeppas²⁹, Higuchi³⁰, Hixon Crowell³¹, first-order³², and zero-order³³were applied. The best fit model was found to be the KorsmeyerPeppas model having R²value 0.9756 suggesting the diffusion-controlled release mechanism.

The improved release of TEL formulation in comparison to plain drug solution can be conventionally attributed to drug: lipid ratio¹⁷. The release from the TEL patch, TEL dispersion, Marketed formulation and plain drug was found to be 95.21%, 89.21%, 73.91 %, and 54.23% respectively as shown in Fig 8.

● TXA patch, ●TEL dispersion, ●Marketed formulation, ●Plain drug

Fig 8: In-vitro drug release

● TXA patch, ●TEL dispersion, ●Marketed formulation, ●Plain drug

Fig 9: Ex-vivo drug release

Ex-vivo skin permeation study:

The ex-vivo skin permeation studies provides information about the product behaviour pattern in vivo since they indicates the amount of drug available for absorption.

The plain drug solution had the lowest skin permeability, with a flux of 7.25 g/cm2 /h, whereas the TXA patch, TEL dispersion, and marketed formulation had fluxes of 35.14 g/cm2 /h, 32.85 g/cm2 /h, and 19.54 g/cm2 /h, respectively. The percent cumulative release in 24 hours was found to be 93.97% for TXA patch, 91.11% for TEL dispersion, 81.19 % for Akira marketed cream, and 65.14% for plain drug solution, respectively as shown in Fig 9. Ethanol gives phospholipid vesicles flexibility and fluidizes the SC lipids, allowing the vesicles to squeeze themselves out of the stratum corneum and thus improve drug penetration.

Stability studies:

The drug retention ability of the TEL-transdermal patch was evaluated using a stability study. The results showed in Table 6 indicated that, TXA-transdermal patches were stable at all temperature and humidity levels over a 6-month period, indicating that the transdermal patch was successfully developed.

CONCLUSION:

Transethosomes loaded with tranexamic acid were successfully prepared and loaded into the transdermal patch. Permeation enhancers and edge activators were used to improve transdermal drug delivery. Optimized transethosomes were having excellent characterization results. TXA loaded TEL’s were magnificently loaded into the transdermal patch and a patient-friendly delivery system was developed. The release and permeation studies concluded that the transdermal patch was capable of efficiently and significantly transporting nano-carriers into the systemic circulation, while also demonstrating excellent stability under various stress conditions. This results in the successful development of a potential carrier for tranexamic acid and other similar drugs, owing to their ease of production and scalability.

CONFLICT OF INTEREST:

There is no conflict to declare.

REFERENCES

1. Grimes PE, et al. New oral and topical approaches for the treatment of melasma. International Journal of Women's Dermatology. 2018 Nov 20; 5(1): 30-36. doi:10.1016/j.ijwd.2018.09.004.

2. George A. Tranexamic acid: An emerging depigmenting agent. Pigment Int 2016; 3(2): 66–71.doi: 10.4103/2349-5847.196295.

3. Taraz M, et al. Tranexamic acid in treatment of melasma: A comprehensive review of clinical studies. Dermatol Ther 2017; 30(3): e12465.doi:10.1111/dth.12465

4. Cai J, et al. The many roles of tranexamic acid: An overview of the clinical indications for TXA in medical and surgical patients. European journal of haematology 2020; 104(2): 79-87.doi:10.1111/ejh.13348.

5. Sheu SL. Treatment of melasma using tranexamic acid: what's known and what's next. Cutis 2018; 101(2):E7–E8.

6. Xu Y, et al. Efficacy of functional microarray of microneedles combined with topical tranexamic acid for melasma: A randomized, self-controlled, split-face study. Medicine (Baltimore) 2017; 96(19): e6897.doi:10.1097%2FMD.0000000000006897

7. Del Rosario, et al. Randomized, placebo-controlled, double-blind study of oral tranexamic acid in the treatment of moderate-to-severe melasma. Journal of the American Academy of Dermatology 2018; 78(2): 363-369.doi:10.1016/j.jaad.2017.09.053

8. Kaur A, et al. Tranexamic acid in melasma: a review. Pigment Int 2020; 7(1):12-25.doi: 10.4103/Pigmentinternational.Pigmentinternational_

9. Tan AWM, et al. Oral tranexamic acid lightens refractory melasma. Australas J Dermatol 2017; 58(3):e105-e108.doi:10.1111/ajd.12474.

10. Zhang L, et al. Tranexamic acid for adults with melasma: a systematic review and meta-analysis. BioMed research international 2018; doi:10.1155/2018/1683414.

11. McKesey J, et al. Melasma treatment: an evidence-based review. American journal of clinical dermatology 2020; 21(2):173-225.doi:10.1007/s40257-019-00488-w.

12. Rodrigues M, Pandya A. Melasma: Clinical diagnosis and management options. Australas J Dermatol 2015; 56(3):151–63.doi:10.1111/ajd.12290.

13. Sarkar R., et al. Melasma in men: A review of clinical, etiological, and management issues. The Journal of clinical and aesthetic dermatology 2018 11(2), p.53-59.

14. Ikino JK, et al. Melasma and assessment of the quality of life in Brazilian women. An Bras Dermatol 2015; 90(2):196–200.doi:10.1590/abd1806-4841.20152771

15. Aishwarya K, et al. Current concepts in melasma - A review article. J Skin Sex Transm Dis 2020; 2(1):13-17.doi: 10.25259/JSSTD_34_2019

16. Beedha. S. et al. Formulation and Characterization of Tramadol HCl Transdermal Patch. Asian J. Pharm. Tech. 2018; 8 (1):23-28. doi: 10.5958/2231-5713.2018.00004.1

17. Shaji J, Bajaj R. Formulation Development of 5-Fluorouracil Transethosomes for Skin Cancer Therapy. Ijppr. Human 2017; Vol. 11 (1): 453-464.

18. Borkar S, et al. Tamoxifen Citrate Loaded Solid Lipid Nanoparticles- A Novel Approach In The Treatment of ER+ Breast Cancer. Research J. Pharma. Dosage Forms and Tech. 2009; 1(2): 143-149.

19. Duan XD, et al. Formulation and Development of Dendrimer-Based Transdermal Patches of Meloxicam for the Management of Arthritis, Trop J Pharm Res 2015; 14(4): 583-590.doi: https://doi.org/10.4314/tjpr.v14i4.4.

20. Talib S., et al. Chitosan-chondroitin based artemether loaded nanoparticles for transdermal drug delivery system. Journal of Drug Delivery Science and Technology 2021; 61:102281.doi: 10.1016/j.jddst.2020.102281

21. Attama, A. A., Momoh, M.A., Builders, P.F. Lipid nanoparticulate drug delivery system: a revolution in dosage form design and development. Recent adv. Novel drug carrier Syst. 2012; 5:107-140.

22. Gavini, MV, et al. Formulation and Invitro Evaluation of Nanoparticulate Drug Delivery System Loaded With 5-Fluorouracil. Res. J. Pharm. Dosage Form. and Tech. 2014; 6(4): 243-248.

23. Jessy Shaji, Monika Kumbhar. Linezolid Loaded Biodegradable Polymeric Nanoparticles Formulation and Characterization. Res. J. Pharm. Dosage Form. and Tech. 2018; 10(4): 272-278. doi: 10.5958/0975-4377. 2018.00040.X

24. Panigrahi L., Ghosal S. K. Formulation and evaluation of pseudo latex transdermal drug delivery system of terbutaline sulphate, Indian J Pharm Sci. 2002; 64(1): 79.

25. Patel R, et al. Formulation and Evaluation of Transdermal Patch of Aceclofenac. Research J. Pharma. Dosage Forms and Tech. 2009; 1(2): 108-115.doi: 10.5958/0975-4377.

26. Salve P, et al. Formulation and Evaluation of Solid Lipid Nanoparticle Based Transdermal Drug Delivery System for Alzheimer’s Disease. Res. J. Pharm. Dosage Form. and Tech. 2016; 8(2):73-80. doi: 10.5958/0975-4377.2016.00011.2

27. Tipre DN, Vavia PR. Formulation optimization and stability study of transdermal therapeutic system of nicorandil. Pharm. Dev. Technol. 2002; 7:325–32.doi:10.1081/PDT-120005729.

28. Singh V, et al. Formulation and Evaluation of Transdermal Patches of Nebivolol Hydrochloride. Research J. Pharma. Dosage Forms and Tech. 2012; 4(5): 272-277.

29. Wu IY, et al. Interpreting non-linear drug diffusion data: Utilizing Korsmeyer-Peppas model to study drug release from liposomes. Eur J Pharm Sci. 2019 October 1; 138: 105026.doi: 10.1016/j.ejps.2019.105026.

30. S. B. Gondkar, et al. Formulation Development and Characterization of Etodolac Loaded Transethosomes for Transdermal Delivery. Research J. Pharm. and Tech. 2017; 10(9): 3049-3057. doi: 10.5958/0974-360X.2017.00541.8

31. Rehman Q, et al. Role of Kinetic Models in Drug Stability. In: Akash M.S.H., Rehman K. (eds). Drug Stability and Chemical Kinetics. Springer, Singapore.2020 Nov 02; 155-165.doi:10.1007/978-981-15-6426-0_11.

32. Singhvi G, Singh M. In-vitro drug release characterization models. Int J Pharm Stud Res. 2011; 2:77-84.

33. Rathore S, Pathak B. Formulation and Evaluation of Aceclofenac-loaded Nanoparticles by Solvent Evaporation Method. Res. J. Pharma. Dosage Forms and Tech.2020; 12(4):237-244. doi: 10.5958/0975-4377.2020.00039.7

34. I.H.T. Guideline, Stability testing of new drug substances and products, Q1A (R2) 2003; current step. 4:1-24.

35. Arayne, M. S., et al. Spectrophotometric techniques to determine tranexamic acid: Kinetic studies using ninhydrin and direct measuring using ferric chloride. Journal of Molecular Structure 2008; 891(1-3), 475–480.doi:10.1016/j.molstruc.2008.04.026

36. Sinico, C, et al. Liposomes as carriers for dermal delivery of tretinoin: in vitro evaluation of drug permeation and vesicle–skin interaction. Journal of Controlled Release 2005; 103(1):123–136.doi:10.1016/j.jconrel.2004.11.020.

37. T. Venkateswara Rao, Owku Ravi Kiran. Review: Transdermal patch. Research J. Pharma. Dosage Forms and Tech. 2013; 5(1): 12-16.

38. Shaji, J., and S. Garude. “Transethosomes and ethosomes for enhanced transdermal delivery of ketorolac tromethamine: a comparative assessment”. International Journal of Current Pharmaceutical Research 2014; 6(4):88-93.

39. J. Charles, Pouchert. The Aldrich library of infrared spectra. 2nd ed. ACS publication; 1972.

40. Silverstien R, Webster F, Kiemle D. Spectrometric identification of organic compounds.1^st ed. New York Wiley; 2004.

Received on 11.07.2021 Modified on 22.10.2021

Res. J. Pharma. Dosage Forms and Tech.2022; 14(1):7-16.

DOI: 10.52711/0975-4377.2022.00002

Temperature	Conditions	0 Month	1 Month	3 Month	6 Month
At temp 25±3℃/60%RH	Physical appearance	Clear and no sedimentation	Clear and no sedimentation	Clear and no sedimentation	Clear and no sedimentation
	Weight variation	22 ±5 mg	22 ±3 mg	21 ±3 mg	20 ±1 mg
	Drug content	94.23%	94.12%	93.54%	93.11%
	% moisture content	7.97 ±0.02%	8.25 ±0.04%	8.67 ±0.02%	8.93 ±0.03%
	Folding endurance	215±5	211±3	208±3	201±6
	%Entrapment efficiency	93.97%	93.45%	92.89%	92.15%
At temp 5 ± 3℃/60%RH	Physical appearance	Clear and no sedimentation	Clear and no sedimentation	Clear and no sedimentation	Clear and no sedimentation
	Weight variation	21 ±3 mg	21 ±2 mg	20 ±3 mg	19 ±5 mg
	Drug content	93.48%	93.21%	92.74%	92.16%
	% moisture content	8.23±0.02%	8.51±0.05%	8.61±0.03%	8.75±0.02%
	Folding endurance	217±3	214±5	210±2	203±1
	%Entrapment efficiency	94%	93.10%	92.56%	92%