Development of bioflavonoid containing chemotherapeutic delivery systems for UV-damaged skin and kangri cancer

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, n-butanol, polyethylene glycol, propylene glycol. Surfactant and cosurfactant mixture, also known as Smix^{[26, 27]}. The list of fluorouracil microemulsion ingredients and their respective properties is presented in Table 1.

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: (1) s=(m×l)/t {\rm{s}} = ({\rm{m}} \times {\rm{l}})/{\rm{t}}

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 ex-vivo permeation test was conducted in vertical Franz diffusion cells with a diffusion surface of 0.63 cm² at room temperature. The Franz diffusion cell was applied on a goat skin and on human cadaver skin to test the ex-vivo diffusion of medicament.

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 (n = 9) and managed as follows: group (i) control group (no treatment); group (ii) test formulation (niosomal gel applied); and group (iii) formalin, a standard irritant (0.8% v/v) was applied. The animals were exposed daily with gel or formalin for seven days continuously. The investigation of treated skin consisted of visual observation for erythema and edema. The outcomes were recorded as per modified Draize et al. protocol.

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.

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.

Figure 1

Figure 2

Figure 3

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).

Figure 4

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.

Figure 5

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.

Figure 6

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.

Figure 7

To estimate the potency of the R-5FU microemulsion formulation for permeation on skin, the ex-vivo experiments were performed on both goa skin and human cadaver skin using Franz diffusion cells. In vitro drug release experiments were conducted to measure the release of drug (5-FU) from three RME (RME1, RME2, RME3); all observations showed the same amount of drug. Table 5 shows the efficiency of different R5-FU microemulsions with free 5-FU in goat and human cadaver skin. It was observed that the 5-FU with RME3 formulation for goat skin expressed the highest permeation compared to RME2, RME1, and free 5-FU. The formation microemulsion in free 5-FU is responsible for the enhancement in the permeation; consequently, its surface area along with its bioavailability is also increased. The results obtained for human cadaver skin permeability are of almost the same pattern, indicating that RME3 shows the best skin permeability with negligible side effects for the skin. Generally, when 5-FU was employed alone on the upper layer of the skin, the surface was considerably smaller, and 5-FU could easily penetrate the inner layer of the skin.

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.

Figure 8

Figure 9

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.

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.

Figure 10

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.