Kangri cancer, a type of skin cancer, occurred only in part of the Kashmir Valley of India. It is caused by the application of a kangri, a ceramic vessel covered with wicker-work, utilized as a source of heat during the winter season. It is kept closely near the abdomen to warm the body in cold weather. As a result of their continuous exposure to kangri, the abdomen and inner thighs are most likely to become affected by kangri cancer. Currently, the cases of kangri cancer have declined, but it has not been eradicated completely. The main reasons for this are the limited resources and irregular power supply as poor residents are forced to use only the low-cost kangri in order to get relief from extreme cold. Strong social rituals are another reason for the frequent use of kangri in Kashmir, as it is a custom to give kangri as a gift during festivals and weddings. Thus, kangri cancer is caused as a consequence of multiple factors—regional, socio-economic, and environmental. To eradicate such cancers, an innovative drug delivery system is required that is enables drugs to penetrate more deeply into the layers of skin. Microemulsion technology is expected to be the most promising technology because of its small droplet size, fluid character, significant connection with the skin cells, effective permeation capability, and ability to deliver even dermally irritating, high-molecular-weight, and volatile molecules[1]. The fluid nature and emulsifier/surfactant interface provide smooth and rapid interaction of microemulsions with skin cells. Microemulsions are stable, transparent dispersions of oil and water lowered by an interfacial film composed of surfactant and co-surfactant molecules. The oil phase plays a dynamic role in microemulsion formulation, as it solubilizes lipophilic drugs destined to be employed to cure a variety of diseases. Rutin 5-fluorouracil (R-5FU), with antiproliferative properties, has been widely employed as topical chemotherapy for skin diseases, such as cutaneous premalignant lesions, malignant lesions, and skin cancer[2]. Hence, a transformed 5FU microemulsion formulation with the required level of permeation might be beneficial in the cure of various skin diseases. As such, the drug delivery approach should be designed to confine a high concentration of drug inside the epidermis and dermis layers for the most effective treatment[3]. Recently, the use of topical vehicle systems having permeation enhancers has attracted considerable attention[4], but the employment of these chemical enhancers may be unsafe, especially as long-term applications. Therefore, the investigation of harmless topical vehicle systems without chemical enhancers is the need of the day for the administration of drug permeation through the skin[5]. Although skin cells have a well-organized antioxidant system, significant or long-term subjection to UV radiation may enhance their antioxidant properties, resulting in oxidative disturbances[6]. Therefore, for the protection of skin from the detrimental impacts of UV exposure, discovering a photoprotective compound of natural origin is desirable. Although several vitamins, such as A, C, E, and β-carotene, are an important constituent of skincare products, the antioxidant and photoprotective properties of polyphenolic compounds have attracted much attention in the expectation that they could be used as nutrients or for relevant application. Flavonoids with antioxidant properties contain a large group of polyphenols; among them, rutin (a quercetin glycoside) is one of the important flavonoids extracted mainly from the citrus plant[7].
Rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) is a flavonoid that is also present in apple, green tea, buckwheat, and Betula leave[8]. Literature reports have been proven clearly that rutin exhibits antioxidant as well as anti-inflammatory activities[9]. Extensive research has been done to elucidate its oral and topical route administration. Its oral administration has demonstrated some adverse effects, therefore an alternative route such as transdermal administration of rutin would be advantageous, and the systemic bioavailability of rutin is also enhanced after transdermal administration[10,11]. Oil-based microcarriers have not been well explored for transdermal/topical delivery of hydrophilic drugs[12,13,14]. Many attempts, namely, microemulsions, transferosomes, ethosomes, niosomes, liposomes, microparticles, phonophoresis, iontophoresis, electroporation, and use of chemical enhancers, have been screened for promotion of transdermal/topical delivery of 5-FU[15,16,17,18,19]. Oil-based microcarriers like microemulsions demonstrated notable superiorities over other unstable dispersion like suspensions and emulsions, in terms of enhanced permeation, solubilization, and bioavailability as well as thermodynamic stability for transdermal/topical delivery of several lipophilic drugs both
In the present study, several formulations of 5FU with rutin, made with oil, surfactant, and cosurfactant, are well characterised using various parameters to closely analyse the effect of microemulsion formulations. Therefore, the objective of this report is to develop and examine microemulsion formulations of hydrophilic R-5FU for topical prevention of kangri cancer using oil, surfactant, and cosurfactants.
5-Fluorouracil (gift samples from Neon laboratories Mumbai, India) rutin, tween 80 (Molychem, Mumbai, INDIA), isopropyl alcohol (Molychem, Mumbai), oleic acid (Central Drug House Pvt. Ltd., New Delhi, India), and dimethyl sulphoxide (DMSO) (Sisco research Laboratory, Mumbai, India), ethanol,
The solubility of R-5FU in various oils was investigated by the addition of a high amount of medicament in 2 mL of oils separated in 5-mL vials and mixing them with the use of a shaker. The sample was put on hold for 72 hours to attain equilibrium. The materials were separated and centrifuged for approximately 15 min at 3,000 rpm. Finally, the supernatant was separated using a membrane filter, and the percentage of medicament was determined by the help of UV spectroscopy. Surfactants were separately dissolved in water: 2.5 mL of 15% surfactant solution was prepared, and 4 μL of oil was added with uninterrupted stirring to obtain a cloudy solution[25]. Cosurfactant acted as a solubilizing agent during the generation of the microemulsion. Cosurfactant was attached with surfactants that assisted their solubility, and a clear, transparent solution was obtained. A number of cosurfactants were screened: ethanol, isopropyl alcohol,
Ingredients list for preparation of FU microemulsion.
5-FU | Drug |
Tween 20 | Surfactant |
Isopropyl alcohol | Cosurfactant |
Isopropyl myristate | Oil |
DMSO | Penetration enhancer |
Rutin | Antioxidant |
Distilled water was added with gentle stirring to a beaker containing all other reactants with gentle stirring. 5-FU was added, and all ingredients were homogenized at a constant temperature to form the microemulsion. To get a clear, transparent microemulsion formulation, the mixture was sonicated for 25 min.
The concentrations of surfactant, cosurfactant, oil and water were determined for the preparation of the microemulsion with the help of a pseudo-ternary phase diagram. The surfactant and cosurfactant were mixed at three ratios—1:1, 2:1, and 3:1. Each phase diagram had a specific weight ratio for Smix and oil mixtures, and they were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. Phase diagrams were constructed for each ratio of mixture of surfactant-cosurfactant (Smix) and oils. The combinations of Smix and oil at the above weight ratios were mixed dropwise, with uninterrupted stirring. The amount of water needed to create the microemulsions was monitored visually. The phase diagram was optimized with the help of triangular graph constructor software, and the microemulsion was prepared on the basis of the phase diagram. An assessment was estimated to calculate the percentage of water, oil, and Smix present at the point of turbidity. After calculating individual percentages, a pseudo-ternary phase diagram was designed with the marked clear-solution region, and the best emulsification region was defined.
After the formulation of R-5FU microemulsions in different ratios based on 5FU, rutin, tween 80, oleic acid, isopropyl alcohol, and dimethyl sulphoxide, further characterization was performed, in other words, visual inspection and thermodynamic stability including stress test as well as centrifugation. In the visual inspection, the colour, homogeneity, and odour of the formulation of FU microemulsion was assessed in each of the different ratios[28–29].
Thermodynamic stability reveals the phase separation, cracking, and coalescence with the help of the stress test and centrifugation[30]. The stress test was performed at 5°C and 46°C with frequent intervals of 8 hrs for a duration of 48 hours to maintain an effective microemulsion system under suitable conditions. The final formulation was evaluated for phase separation, cracking, and coalescence[31]. The prepared microemulsion batches were centrifuged at 1,000 rpm for 1 hour and at 3,000 rpm for 1 hour, and afterwards the phase separation was evaluated[32]. A dynamic light-scattering technique was used for the determination of particle size using the instrument Zetasizer (Malvern Zetasizer ver. 7.03; serial number: mal1023461).
Transmission electron microscopy (TEM) studies were performed in which the microemulsion sample was kept in a 300-mesh, carbon-coated copper grid and 2% phosphotungstic acid was used as a negative stain. Finally, the samples were examined using an FEI Tecnai Spirit Biotwin G2 microscope. The microscope was procured from AIIMS (All India Institute of Medical Sciences, New Delhi) and operated at 80 kV.
The pH of the microemulsion sample was measured with a pH meter: 1 g of microemulsion was dissolved in 100 mL of water for 2 hours; the electrodes were completely immersed into the microemulsion solutions, and the pH determined. The pH determination was performed three times for each sample, and average values were calculated[33].
The viscosity of the microemulsion formulations was measured with a Brook Field Viscometer Model (wdv-8). An appropriate amount of each microemulsion formulation (each formulation by surfactant-cosurfactant ratio) was decanted into a wide-mouthed jarand the spindle of the viscometer was immersed completely into the liquid.
The apparatus used for the spreadability study consisted of a marked wooden block and two glass slides with a pan mounted on a pulley. To obtain a uniform thickness of the formulation, the desired amount of microemulsion was kept between two glass slides and a 100-g weight was put on the upper glass slide for 2 min. A 100-g weight was also added to the pan. The spreadability measurement was defined as the time (s) required to separate the two glass slides.
The following equation was used for the determination of spreadability:
Where s denotes spreadability; m is weight placed on the upper slides; l is the length of the glass slide and; t is the time to separate the slides in seconds.
The
The cell contains two chambers, donor and receptor. The donor chamber was open at upper portion. The receptor chamber was encased in a water jacket to control the temperature, and it was also used as a sample port. The diffusion solution was phosphate buffer (pH 6.4), which was stirred with magnetic beads operated by a magnetic stirrer. Either human cadaver or goat skin was used as a membrane between the compartments. The diffusion sample was continuously stirred so that the sample of highly medicated formulation did not come in direct contact with membrane. The formulation sample loaded into the receptor chamber was observed frequently over a period of 3 hours, and UV spectrophotometry was utilized to determine the concentration of the formulation sample using a standard curve. Different quantities of the samples were distributed at different time intervals, and further concentration calculations were done. A graph of these results was plotted against time.
A skin irritation test was performed at Shri Rawatpura Sarkar Institute of Pharmacy (Kumhari, Chhattisgarh, India) using the method developed by Draize et al. over 75 years ago. Wistar albino rats were acclimated to laboratory conditions until one week before the experiment (34). The room temperature was kept constant at 25°C, and its humidity was monitored to maintain up to 40–45% relative humidity (RH). A 5-cm patch of the dorsal part of the rat was marked for the experiment and hair was removed by trimming. Animals were categorized into three groups (
The solubility of 5FU, various oils, co-surfactants, and surfactants was assessed for the preparation of microemulsions, and the results are shown in Table 2. The solubility of isopropyl myristate, tween 20, and isopropyl alcohol are 165.28, 93.12, and 107.28 mg/mL, respectively; therefore, they were selected for the preparation of microemulsion formulations.
Solubility of R-5FU in different oils, surfactants and co-surfactants.
Isopropyl myristate | 165.28 |
Oleic acid | 183.12 |
Castor oil | 44.26 |
Olive oil | 32.61 |
Tween 80 | 105.28 |
Tween 20 | 93.12 |
Span 80 | 58.27 |
Span 20 | 31.62 |
Isopropyl alcohol | 107.28 |
Ethanol | 43.12 |
N-butanol | 58.27 |
Polyethylene glycol | 69.62 |
Propylene glycol | 79.62 |
A phase diagram was drafted on the basis of parameters such as solubility of drug, oil, surfactants, co-surfactants, and aqueous phase. The percentageS of surfactant, cosurfactant, water, and oil at The 1:1, 2:1, and 3:1 weight ratio of Tween 20 : isopropyl alcohol, respectively, are shown in supporting Tables 1, 2, and 3. The aqueoustitration was conducted with the addition of water (5% increment of up to 95%). The area of the microemulsion region in the phase diagram decreased minimally when the Smix ratio increased, as shown in Figures 1, 2, and 3. The Smix 1:1 (Figure 1) indicates a larger microemulsion region than that of 3:1 (Figure 3). Notably, during aqueous titration, the Smix 1:1 ratio took up a greater quantity of water and still remained a clear mixture.
Viscosity of prepared microemulsion batches.
RME1 | 14.68 |
RME2 | 16.52 |
RME3 | 17.25 |
Phase diagram of 1:1 weight ratio of microemulsion.
Phase diagram of 2:1 weight ratio of microemulsion.
Phase diagram of 3:1 weight ratio of microemulsion.
The thermodynamic stability of a prepared drug's microemulsion discloses its stability in terms of various temperatures, energy, or the equilibrium state of the prepared drug's microemulsions with the environment. We observed the thermodynamic stability of the R-5FU microemulsions at different temperatures (i.e., 2–4°C, 20–25°C, 35–50°C) for a duration of 3 months. No phase separation or color change was seen during this time period. The sample formulations were then subjected to a stress test at 4°C and 45°C for 48 hours each for a period of six cycles, after which centrifugations were performed at 1,000 rpm and 3,000 rpm for 1 hour. The stress tests were done to optimize the microemulsion formulation under extreme conditions. Centrifugation was performed to assess phase separation, coalescence, or cracking, and no changes in microemulsion after centrifugation was seen. These results indicate that no phase separation, flocculation, or precipitation was observed in the microemulsion preparations.
The shape and morphology of the prepared R-5FU microemulsion under study was assessed by transmission electron microscopy (TEM). The surface morphology was determined to be an oval shape. The solubility of microemulsions increases, as the size decreases. TEM images show the micro size and uniform distribution of the prepared R-5FU (Figure 4).
Transmission electron microscopic surface area of prepared microemulsion batches.
Viscosity is the friction between the intermolecular layers in microemulsions; it also indicates the thickness of the prepared sample. For the study of viscosity of prepared microemulsions, the Brookfield viscometer was used. The viscosity of RME3 was found to be 17.25 ± 0.22 Pa·s as mentioned in Table 3. The highest viscosity was that of RME3, as is clearly shown on the graph. (Figure 5). Because of its high viscosity, RME3 was chosen for topical application; and hence it was considered to be the best microemulsion formulation for the undergoing studies.
Viscosity of microemulsion batches.
The pH of the prepared 5-FU was measured with a pH meter (Table 4). Figure 6 indicates the pH values of microemulsion samples. Nearly all batches of the microemulsions demonstrate a pH of 5.24–5.93, which is close to neutral pH and favorable to use on skin for medical purposes.
pH of prepared microemulsion batches.
RME1 | 5.24 |
RME2 | 5.93 |
RME3 | 5.67 |
Graph of pH values of microemulsion batches.
The globule size of the prepared microemulsion was assessed by using Malvern Zetasizer ver. 7.03 (serial number: mal1023461). The average globule size was 100–300 nm for all three RME. Figure 7 indicates three peaks with the smallest size found at 105.29 nm for RME3. Thus, observation shows the homogeneous distribution of microemulsion globules, and it is within the required range. Consequently, a translucent microemulsion can be formulated efficaciously.
Globules size range of prepared microemulsion batches.
To estimate the potency of the R-5FU microemulsion formulation for permeation on skin, the
Values of drug permeation of prepared microemulsion batches.
0.128 | 0.128 | 0.135 | 0.138 | |
0.130 | 0.145 | 0.152 | 0.156 | |
0.161 | 0.165 | 0.174 | 0.178 | |
0.182 | 0.193 | 0.203 | 0.208 | |
0.217 | 0.232 | 0.244 | 0.251 | |
0.275 | 0.291 | 0.305 | 0.312 | |
0.358 | 0.386 | 0.406 | 0.416 | |
0.510 | 0.581 | 0.61 | 0.625 | |
1.012 | 1.161 | 1.22 | 1.25 | |
1.012 | 1.161 | 1.22 | 1.25 |
Figure 8 depicts the comparative skin retention profiles for all formulations of R-5FU microemulsion with free 5-FU. The skin retention value for free 5-FU was found to be approximately 4.86 mg/cm2. However, the microemulsion of 5-FU demonstrated delightful results, that is, RME1, RME2, and RME3 are 5.45 mg/cm2, 6.89 mg/cm2, and 8.12 mg/cm2, respectively. The release of 5-FU from RME1, RME2, and RME3 formulations after 30 min was highly important when compared to free 5-FU (Figure 9). The percentage of 5-FU that was released from RME1, RME2, and RME3 was found to be 99.4%, 99.6%, and 99.8%, respectively. The smallest droplet size, lowest pH, lowest viscosity, and eventually higher surface area for RME3 are the factors responsible for the highest drug delivery. on the bases of the experimental observations above, we came to the conclusion that microemulsion-based drug expresses better drug deposition on skin compared to free 5-FU.
Comparative skin retention profile for the all the formulation 5-FU
Percentage (%) mean cumulative 5-FU microemulsions penetration in skin.
The HET-CAM assay estimates effective skin irritation from chemicals as observed through investigating a chemical's capacity to initiate irritation and toxicity in the chorioallantoic membrane of chicken. The formulations RME1, RME2, and RME3 were with the HET-CAM assay, and the irritation score was determined. Notably, upon application of the RME3 and RME1 formulations, no changes were noticed in the chorioallantoic membrane. All three formulations indicated no irritation score (IS=0), as no lysis, hemorrhage, or coagulation were observed after their application. In contrast, a very high irritation score (IS = 23) was observed for irritant (Table 6). The scoring scheme for the irritation test using the HET-CAM assay is explained in supporting Table 4. The degradation of the membrane initiated in just 0.5 min. Descriptions of irritating effects of the test formulations with respect to a specific time limit are well shown in Table 7.
Scoring for irritation testing (HET-CAM assay).
Lysis | -- | -- | -- | 0 | Non-irritant | |
Hemorrhage | -- | -- | -- | |||
Coagulation | -- | -- | -- | |||
Lysis | - | -- | -- | 0 | Non-irritant | |
Hemorrhage | -- | -- | -- | |||
Coagulation | -- | -- | -- | |||
Lysis | -- | -- | -- | 0 | Non-irritant | |
Hemorrhage | -- | -- | -- | |||
Coagulation | -- | -- | -- | |||
Lysis | -- | -- | -- | 0 | non-irritant | |
Hemorrhage | -- | -- | -- | |||
Coagulation | -- | -- | -- | |||
Lysis | -- | 7 | -- | 23 | Severe-irritant | |
Hemorrhage | 7 | -- | -- | |||
Coagulation | 9 | -- | -- |
Effects of RME1, RME2, RME3, control and irritant on chorioallantoic membrane with different time interval.
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RME1, RME2, and RME3 formulated materials demonstrated zero erythema and edema index scores for negative and test-controlled values. The microscopic observations of the controlled mouse skin sections (without treatment) and the sections of skin treated with all formulations are depicted in Figure 10. Mean erythemal score was evaluated for different formulations mentioned in Table 8 on the basis of Table 5 (supporting). Anatomical and pathological transformations estimated in this study did not offer the safety of tested formulations on mouse skin. A steady observation of skin tumor treated with test formulations of 5-FU microemulsions was performed, and it was observed that the skin recovered its usual physiologic condition. Skin sections of mice exhibited normal physiology, thus verifying the efficacy of prepared microemulsions as compared to that of controls used in the experiment.
Acute skin irritation test of prepared microemulsion batches (a) RME 1, (b) RME 2, (c) RME 3 and control (d).
Mean erythemal score evaluated for different formulation.
2 | 5 | 7 | 10 | 14 | 18 | 21 | |
0 | 0 | 0 | 0 | 0 | 0 | 0 | |
0 | 0 | 0 | 0 | 0 | 0 | 1 | |
0 | 0 | 0 | 0 | 0 | 1 | 1 | |
0 | 0 | 0 | 0 | 0 | 1 | 1 | |
0 | 0 | 1 | 1 | 2 | 3 | 3 |
The MTT assay, a high-throughput cell-oriented assay, was employed to investigate the cytotoxic effects of various cell lines to a number of oncology products. MTT assay is a test performed in lab and is a normal colorimetric assay (an assay that estimates color changes) for assessing growth in a cell. In our study, the MTT assay was adjusted to act as a chemosensitivity test, and its efficiency was evaluated. This technique also has many advantages regarding speed, managment of various samples, quantitation, and cell number. The exploration of this assay for chemosensitivity testing seems to be significant and beneficial. MTT evaluates cell respiration: the degree of formazan made is proportionate to the quantity of living cells existing in the culture. A reduction or escalation in cell number leads to concurrent change in the quantity of formazan made, showing the extent of cytotoxicity caused by the drug. An IC50 (concentration) of the tested drug is capable of causing the death of 50% of the cells and can estimate the extent of its cytotoxic response. As the value decreases, the cytotoxicity of the substance increases. Figure 11 depicts a graph of the IC50 of selected chemotherapeutic drugs against human cancer cell lines.
The present study focused on the preparation of a topical microemulsion of R-5FU using the phase titration method. Smix ratios were developed using different component ratios for the microemulsions. The medication was dissolved in oil, and then the mixture was added in Smix followed by uninterrupted stirring until the generation of a clear formulation. Water was added dropwise until a clear solution appeared. The formulations of the FU microemulsion were characterized by physicochemical investigations. The result reveals globule sizes within 100 nm – 300 nm, pH values ibetween 5.64 and 5.97, viscosity iwithin 13.52 Pa·s and 18.23 Pa·s, which was a significant result for the formation of the preparation. The quantity of 5-FU released from all formulations after 3 hourrs ranges from 95.57% to 83.67%. HET-CAM assay studies showed no irritation score (IS = 0) in RME1, RME2, and RME3 formulations, demonstrating no hemorrhage, lysis, or coagulation appeared after application. For irritant studies, in contrast, very high score (IS = 23) was obtained. The physicochemical characteristics of all other formulations were also shown to be equally good. R-5FU microemulsion demonstrates an efficient drug delivery system with good stability and release profile. Rutin has increased its permeability, which would be very beneficial for the treatment of kangri cancer.