Novel Transethosomal Gel Loaded With 5 Fluorouracil: A Promising Approach for Enhanced Skin Cancer Treatment

Abstract

Introduction:-5-Fluorouracil (5-FU) is an antimetabolite chemotherapeutic agent used primarily to treat various cancers, such as actinic keratosis, breast and skin cancer. It has a biological half-life of 10 – 20 mins. The conventional cream/Ointment dosage may have present challenges such as poor skin penetration, necessitating frequent application for effective treatment. Additionally, they can cause significant skin irritation and discomfort, with some risk of systemic absorption leading to unwanted side effects. To address these issues, a transethosomes gel formulation of 5-FU was developed. This gel enhances skin penetration, provides sustained drug release, improves bioavailability etc, making it a more effective and patient-friendly alternative to conventional creams. Therefore, transethosomes containing 5 - Fluorouracil embedded in appropriate gel delivery systems can effectively deliver this drug for a long time ( slow and sustain drug release) in the treatment of actinic keratosis. Methodology:- 5 - Fluorouracil loaded Transethosomes were formulated by using Cold Method. Total 12 formulations were prepared using Soya phosphotidylcholine, ethanol and with varying concentration of Surfactants/Edge activators such as Span 60, Tween 80, Sodium deoxycholate and Kolliphor RH 40. The formulations were further characterized for various parameters such as, particles size, polydispersity index, zeta potential, entrapment efficiency and in-vitro drug release. The promising transethosomal formulation (TET3) was incorporated into a suitable gel base and evaluated for physical appearance, pH, rheological study, spreadability test, content uniformity, in- vitro drug release and kinetic studies. Results and Discussions:- Among all formulations, the formulation (TET3) prepared with Tween 80 showed Smaller vesicular diameter (167.9 nm) with Excellent PDI ( 0.110 ) and good zeta potential (-21.6 mV), good drug content (98.12 %), better entrapment efficiency (65.15%), optimum drug release for 8 hrs (62.53%).So, Formulation TET3 Was Considered As A Promising Formulation. The Results Revealed That 5 - Fluorouracil Transethosomal Gel Was Successfully Developed As A Transdermal Drug Delivery System. The Gel Formulation TET3 – 1% Showed Optimum Drug Release That Is 58.78% For 8 Hrs. Conclusion:- In Conclusion, The Development Of 5- Fluorouracil - Loaded Transethosomes Using The Cold Method Proved Effective In Enhancing Transdermal Delivery For The Treatment Of Skin Cancer. The Optimized Formulation Exhibited Desirable Characteristics, Superior Skin Permeation And Improved Prolonged Efficacy Compared To The Commercially Formulation, Showcasing Its Potential As A Promising Approach In Treatment.

Keywords

Fluorouracil, Transethosomes, Cold method, Edge activator, Soya phosphotidylcholine, Increased permeability.

Introduction

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Table No 1: Composition of 5 - Fluorouracil Transethosomes prepared by Cold method

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TPGS – D-alpha-Tocopheryl Polyethlene Glycol Succinate.

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Evaluation of 5 - Fluorouracil-loaded transethosomes: -

1. Particle size, polydispersity index & zeta potential: The Malvern zeta sizer (Malvern Instruments, Worcestershire, UK) was utilized to ascertain the particle size, polydispersity index and zeta potential of the transethosomes. The measurements were conducted at a constant angle of 90° and a temperature of 25 ± 2°C. To facilitate the analysis, the samples were diluted in millipore water and the measurements were carried out in triplicate. These parameters provide information regarding the size, homogeneity and stability of the vesicles.¹³

2. Entrapment efficiency: The Ultra-centrifugation technique was used for estimating the entrapment efficiency of transethosomal formulations. 2ml of each formulations was transferred into a microcentrifuge tube and was subjected to ultracentrifugation at 1,00,000 at 4°C by an Ultracentrifuge (Kubota, Japan) for 1 hour. Following that, the supernatant was extracted with a micropipette and diluted with methanol to rupture the vesicles. A UV spectrophotometer (Shimadzu -1900, Japan) was used to measure the quantity of drug in the supernatant at 295.0 nm.¹⁴

The following formula was used to compute the ?.

?=Total drug content-Unentrapped drugTotal drug content×100

 

 

Evaluations of transethosomal gel

1. Determination of pH: In order to ascertain the pH of multiple gels, a digital pH meter was employed. Firstly, 500 mg of pre-prepared transethosomal gels were dissolved in 20 millilitres of distilled water. The resulting mixture was stirred for a duration of 15 minutes at room temperature using a magnetic stirrer. Subsequently, the pH sensor probe electrode was immersed in the dissolved gel and the pH value of the formulation was recorded from the digital screen.¹⁵

2.Determination of viscosity:The viscosity of the optimized Transethosomal gel formulation was estimated using a Brookfield viscometer. After applying the Transethosomal gel formulation, it was allowed to settle for 5 minutes. After that, spindle number one was revolved at 50 revolutions per minute at a temperature of 25±2°C.¹⁶

3. Determination of spreadability: The spreadability of the Transethosomal gel was assessed using the glass slide technique. A precisely measured 2 grams of gel was positioned at the center of a glass slide measuring 10x5 cm. Another slide of the same dimensions was placed on top of it. To ensure uniform compression and maintain a consistent thickness, a weight of 100 grams was applied to the upper slide for a duration of 5 minutes. The time required for the glass slide to move 6 cm and separate from the lower glass slide was recorded.^17,18 The spreadability of the gel was then calculated using the following formula: S=M.L/T

Where, ‘'S' represents Spreadability, 'M' represents the weight applied to the upper slide, 'L' represents the distance travelled by the slide (6cm) and 'T' represents the time taken in seconds.¹⁹

4. Determination of drug Content: In a 50 ml volumetric flask, 1 gm of gel was dissolved with 50 ml water. The solution was vortexed until a transparent solution was achieved. Subsequently, the solution was filtered through a 0.45µm filter and appropriately diluted using water. The drug concentration was determined by measuring absorbance with a UV spectrophotometer, using water as the reference solution.²⁰

Drug Content=Actual drug content in vesiclesTheoretical drug content in vesicles×100

 

5. In vitro drug release study: The Franz diffusion cell was utilized to determine the in vitro diffusion of drugs from different transethosomal gel formulations. To activate the dialysis membrane, it was previously soaked in pH 7.4 buffer. The receptor chamber was then filled with 12 ml of phosphate buffer at pH 7.4, which served as the medium for diffusion in the receptor compartment. The gel formulation, equivalent to 2mg of the drug, was accurately weighed and thronged in the preactivated dialysis membrane. Throughout the experiment, the receptor medium was maintained at a temperature of 37 ±2 °C and stirring was consistently performed at 100 rpm. At specific time intervals of 0.5, 1, 2, 4 and 8 hours, 0.5 ml samples were withdrawn from the cell and replaced with fresh medium. The quantification of drug release was carried out using a UV spectrophotometer at a wavelength of 266nm.²¹

RESULTS

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Table No 3: Standard Calibration Curve of 5- Fluorouracil in Distilled Water (?max=262.70 nm)

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Graphs: In-Vitro Release Plot, First Order Plot, Higuchi Diffusion Plot & Peppas Log-Log Plot of Transethosomes of TES3, TET3, TEC3 and TEK3

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Table No 5: pH, Viscosity and Spreadability, drug content of Transethosomal Gel

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DISCUSSIONS

Characterization of Transethosomal Formulations:

1.Vesicles of 5 – Fluorouracil, PDI & Zeta Potential.

The vesicle size of all 12 formulation were below 500d.nm. The results were within the limits .  The PDI of 12 formulations was below 0.5. The result were within the limit. The Zeta Potential of 12 formulations were from ranged -40.7mV to -21.6mV within the official limit. The zeta potential is the critical characteristic that influences vesicular features such as the interaction of vesicles with the skin and the stability of transethosomes. According to the findings presented in Table 9, it has been observed that all the formulated transethosomal preparations exhibited a zeta potential characterized by a negative charge. Negatively charged vesicles exhibit superior skin permeability compared to positively charged vesicles. The negative zeta potential observed in transethosomes primarily arises from the presence of ethanol within these nanocarriers. Ethanol induces negative charges on the polar head groups of phospholipids, leading to electrostatic repulsion. This would limit vesicle agglomeration and hence increase the stability of these transethosomal nanocarriers. The size of transethosomes, formulated by using Tween 80 were smaller compared with the Transethosomes formulated by using other surfactants/permeation enhancers such as Span 60, Sodium Deoxycholate & Kolliphor RH 40. Effect of surfactants on the size of transethosomes were studied using a different concentration of surfactant. The results were showed that transethosomes size decreased respectively with increased Surfactants concentration and this was due to decrease in surface energy with increasing hydrophobicity leading to smaller vesicles and favorable interactions (hydrogen bonding or van der waal forces) between transethosomes matrix and drugs. It was observed that the relative amount of surfactant was found to play an important role in the determination of vesicle size. The results were revealed that the sizes of vesicle are dependent on type and concentration of surfactants used.

2. Drug Content and Entrapment Efficiency: The content of all the transethosomes were determined at 262.80 nm against blank by using the Shimadzu UV/visible spectrophotometer. The drug content were found in the range of 95.23 to 98.47 % (Table15). The results were within the official limits. Among all, formulation TET3 (98.12 %) showed maximum drug content. The percent entrapment efficiency of all the formulations were found in the range of 53.06 to 68.22 %. Among all, formulation TET1 (68.22 %) showed maximum entrapment efficiency.The Entrapment Efficiency % were more for Tween 80 compared to all other surfactants. The Entrapment Efficiency of formulations containing Tween 80 decreases with Increase in concentration of Tween 80. This was because of Lower concentration of Tween 80 favour higher entrapment efficiency due to better vesicle stabilization and Higher concentration lead to destabilization of vesicles and a drop in the entrapment efficiency, as the non-ionic surfactant disrupts the lipid bilayer structure.

3. In Vitro Drug Release Studies: The in-vitro release profiles of 5 - Fluorouracil formulations were examined in PBS (pH 7.4) for 8 h. The % drug release after 8 h from all formations were found in the range of 42.91 to 65.76 % (Table 11 to 14 ). The order of % drug release in the 8 hour in Sodium Deoxycholate > Tween 80 > Kolliphor RH 40 > Span 60. The impact of different surfactants on the release profile was observed. Span 60 Transethosome formulation provided slowest release due to rigidity of the vesciles, Tween 80 transethosome formulation exhibited significant faster drug release and release rate is optimal for moderate and controlled drug release, Sodium Deoxycholate transethosome formulation showed fastest drug release due to high permeability and lastly Kolliphor RH 40 offered a release profile same as tween 80, though slightly slower.

4. Kinetics Studies: The release profile of all the formulations were subjected to various kinetic equations like first-order plots, Higuchi-diffusion plots and Peppas log-log plots (Table 14 ). The regression coefficient values of these kinetic equations are very nearer to 1, suggesting that plots are fairly linear. So, it confirms that the drug release mechanism was diffusion mechanism. • Slope values of Peppas log-log plots were in the range of 0.1224 to 0.1529, indicates that the drug release by a Fickian release mechanism i.e., the drug was released by diffusion controlled alone.

DISCUSSIONS OF TRANSETHOSOMAL GEL

Characterization of Transethosomal Gel Formulations:

1. Physical Appearance: The Gel formulation was first examined physically for appearance, homogeneity and texture. The prepared formulations was found to be Transparent clear gel, smooth and homogeneous. There was no sign of grittiness.

2. pH measurement, Rheological study, Spreadability test: pH of the gel formulations was measured by digital pH meter and found to be 6.64 in the physiological skin surface pH range. The spreadability of the formulation was recorded. The results was 26.05g/cm/sec, the gel has good spreadability with little force applied. These assured that the formulation could maintain a good wet contact time when applied at the target site. The viscosity of the gel formulations was 11480cps.

3. Content Uniformity: The drug content was determined at 262.70 nm against blank by using the Shimadzu UV/visible spectrophotometer. The drug content is 97.41 %. The results were within the official limits.

4. In-Vitro Drug Release Profile: The in-vitro drug release data showed that 15.23 % of drug released from marketed cream ( Flonida Cream) after 1 hr and percent drug release was significantly increased from 15.23% to 84.67 % up to 8 hrs, whereas in case of optimized transethosomal gel (TET3 Gel) only 10.529% of drug release was observed after 1 hr and continuous and appreciable increase in cumulative drug releases was reported up to 08 hrs. At the end of 08 hrs, 58.788 % of drug release was recorded. These results indicated that the Controlled and sustained drug release of drug through the membrane for a prolonged period was obtained in case of TET3 Gel (Transethosomal gel) formulation as compared to marketed cream.

CONCLUSION

In the present study, it was concluded that the transethosomal formulation of 5 - Fluorouracil could be successfully synthesized by Cold Method using different surfactants such as Span 60, Tween 80, Sodium Deoxycholate and Kolliphor RH 40. The study also showed that all prepared formulations showed a Controlled and sustained drug release rate for 8 hrs. The Cold Method is a promising technique for the preparation of 5 - Fluorouracil transethosomes. Among all formulations, the formulation (TET3) prepared with Tween 80 showed Smaller vesicular diameter (167.9 nm) with Excellent PDI ( 0.110 ) and good zeta potential (-21.6 mV), good drug content (98.12 %), better entrapment efficiency (65.15%), optimum drug release for 8 hrs (62.53%).From the FTIR studies, it was concluded that the drug structure is undisturbed in the transethosomal formulation, thus revealing no interaction between the selected drug and excipients used.The results showed that 5 - Fluorouracil Transethosomal gel was successfully developed as a transdermal drug delivery system with 1?rbopol 980.The prepared gels were found to be Transparent and clear gel which had a cool and smooth feeling on application. From the results, it is evident that the gel formulations showed good homogenesity, viscosity and spreadability. There was no sign of grittiness. The drug content and pH were found to be within the acceptable limit. In vitro diffusion studies revealed that the transethosomal gel containing 1?rbopol 980 exhibited Controlled and sustained drug release of drug through the dialysis membrane for a prolonged period. The findings of the study indicate that the transethosomal gel of 5 - Fluorouracil is better in terms of pH, Viscosity, Spreadability, In vitro drug release compared to marketed formulation ( Flonida Cream ). These findings suggest the potential of 5 Fluorouracil -loaded transethosomes as a promising approach for the treatment of actinic keratosis.

REFERENCES

1. Skin cancer – symptoms and causes by mayo clinic. https://www.mayoclinic.org/diseases-conditions/skin-cancer/symptoms causes/syc-20377605.
2. Galal H. Elgemeie, Reham A. Mohamed-Ezzat, Chapter 4-Pyrimidine-based anticancer drugs, New Strategies Targeting Cancer Metabolism, Elsevier,2022:107-142.
3. Prince GT, Cameron MC, Fathi R, Alkousakis T. Intralesional and Laser-Assisted 5-Fluorouracil in Dermatologic Disease: A Systematic Review. J Drugs Dermatol. 2018; 17(3):274-80.
4. Entezar-Almahdi E, Mohammadi-Samani S, Tayebi L, Farjadian F. Recent Advances in Designing 5-Fluorouracil Delivery Systems: A Stepping Stone in the Safe Treatment of Colorectal Cancer. Int J Nanomedicine. 2020 ;30(15):5445-58.
5. Abraham W, Downing DT. Deuterium NMR investigation of polymorphism in stratum corneum lipids. Biochim Biophys Acta. 1991; 1068(2):189-94.
6. Aggarwal N, Goindi S. Preparation and evaluation of antifungal efficacy of griseofulvin loaded deformable membrane vesicles in optimized guinea pig model of microsporumcanis dermatophytosis. Int J of pharm. 2012; 437(1):277-87.
7. Garg U, Jain K. Dermal and Transdermal Drug Delivery through Vesicles and Particles: Preparation and Applications. Adv Pharm Bull. 2022; 12(1):45-57.
8. Ravindran Vinod K, Suneel Kumar M, Anbazhagan S, Sandhya S, Saikumar P, Tera Rohit R, et al. Critical Issues Related to Transfersomes-Novel Vesicular System [Internet]. Acta Sci. Pol., Technol. Aliment. 2012;11(1): 67-82.
9. Kesharwani R, Patel DK, Sachan A, Kumar V, Mazumdar B, Assam D. Ethosomes: a Novel Approch for Transdermal and. World J Pharm Pharm Sci. 2015;4(6): 348–59.
10. Bajaj KJ, Parab BS, Shidhaye SS. Nano-transethosomes: A novel tool for drug delivery through skin. Indian Journal of Pharmaceutical Education and Research. 2021;55(1): 1–10.
11. Prow TW, Grice JE, Lin LL, Faye R, Butler M, Becker W, Wurm EM, Yoong C, Robertson TA, Soyer HP, Roberts MS. Nanoparticles and microparticles for skin drug delivery. Advanced drug delivery reviews. 2011; 63(6):470-91.
12. Srinivas S, Kumar YA, Hemanth A, Anitha M. Preparation and evaluation of niosomes containing aceclofenac. Dig J Nanomater Biostructures. 2011;5(1):249-54.
13. Aggarwal Ajay, Saroha Kamal, Nanda Sanju. Formulation, evaluation and comparison of ketorolac tromethamine transdermal gel containing natural and synthetic permeation enhancers. Pharm Sin. 2014;5(3):41-5.
14. Zaki RM, Seshadri VD, Mutayran AS, Elsawaf LA, Hamad AM, Almurshedi AS, et al. Wound Healing Efficacy of Rosuvastatin Transethosomal Gel, I Optimal Optimization, Histological and In Vivo Evaluation. Pharmaceutics. 2022;14(11): 2521-32.
15. Abdulbaqi IM, Darwis Y, Assi RA, Khan NAK. Transethosomal gels as carriers for the transdermal delivery of colchicine: Statistical optimization, characterization, and ex vivo evaluation. Drug Des Devel Ther. 2018;12: 795–813.
16. Abdulbaqi IM, Darwis Y, Assi RA, Khan NAK. Transethosomal gels as carriers for the transdermal delivery of colchicine: Statistical optimization, characterization, and ex vivo evaluation. Drug Des Devel Ther. 2018;12: 795–813.
17. Naga Sravan Kumar Varma V, Maheshwari P V., Navya M, Reddy SC, Shivakumar HG, Gowda D V. Calcipotriol delivery into the skin as emulgel for effective permeation. Saudi Pharmaceutical Journal. 2014;22(6): 591–9.
18. Shivhare UD, Jain KB, Mathur VB, Bhusari KP, Roy AA. Formulation development and evaluation of diclofenac sodium gel using water soluble polyacrylamide polymer. Dig J Nanomater Biostructures. 2009;4(2):285-90.
19. Sabale V. Kunjwani1 H, Sabale P. Formulation and in vitro evaluation of the topical antiageing preparation of the fruit of Benincasahispida. J Ayurveda Integr Med. 2011;2(3):1-5.
20. Rahangdale M, Pandey P. Development and characterization of apremilast transethosomal gel for transdermal delivery. International Journal of Pharmaceutical Sciences and Nanotechnology. 2021;14(3): 5508–18.
21. Adin SN, Gupta I, Rashid MA, Alhamhoom Y, Aqil M, Mujeeb M. Nanotransethosomes for enhanced transdermal delivery of mangiferin against rheumatoid arthritis: formulation, characterization, invivo pharmacokinetic and pharmacodynamic evaluation. Drug Deliv. 2023;30(1): 1-15.