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The Alg-5FU nanoparticles were prepared using a spray drying technique;5FU can be encapsulated in the matrix of the polysaccharides via bonds with functional groups in the alginate chain [31]. Particle size and zeta potential are the principal criteria for evaluating effective anticancer drug delivery targeting tumor tissue [32]. Studies have shown that nanoparticles in the size range of 20–200 nm have the maximum retention in the tumor cell [33].The particle size of the prepared nanoparticles was determined in an aqueous solution. It was found that the particle size of nanoparticles is in the range 202–254 nm. Nanoparticles 50–500 nm in size are acceptable for transdermal drug delivery [34]. The formulated nanoparticles were of sizes within acceptable range, and thus suitable for skin delivery. The particle size of nanoparticles slightly increases with the addition of 5FU (t-test; p < 0.05), confirming the encapsulation of 5FU into the matrix of crosslinks formed by alginate chains in nanoparticles. Table 1 showsthe zeta potential results of the prepared nanoparticles. The prepared nanoparticles had negative zeta potential, because of the negative zeta potential of the alginate component [32]. The blank nanoparticle had a high surface charge of −43.67 mV while the presence of 5FU slightly increased the surface charge (−38.72 mV). The highly negative zeta potential induced repulsion between nanoparticles, greatly reducing the propensity to aggregate and making the nanoparticles highly stable in an aqueous solution [35]. The zeta potential values greater than +30 or less than −30 mV indicate the stability of nanoparticles, and helps to prevent nanoparticles aggregation [36]. Thus, the alginate-based nanoparticles protect the drug, and is an ideal carrier for a drug delivery system. The SEM images (Figure 1) show that the nanoparticles possess a spherical shape with a smooth surface.
eparation and Characterization of Hydrogels Table 1. Size, zeta potential, and %DC of nanoparticles. F. Code Size (nm) Zeta Potential The 5FU solution and 5FU-Alg-Np-loaded chitosan-gelatin-based hydrogels were prepared. Chitosan and gelatin solutions when mixed together, electrostatic interaction occurs, which results in interpenetrating self-assemble soft aqueous pores as a result of PDI %DC Alg-Np 5FU-Alg-Np 202.35 ± 3.57 254.71 ± 3.98 * −43.67 ± 2.58 −38.72 ± 2.91 0.33 ± 0.27 0.38 ± 0.24 - amide bonds formation [37]. These soft pores were filled with 5FU solution and/or 5FU78.23 ± 3.26 Note: * p < 0.05. Alg-Np to form drug-loaded hydrogels. 2.2.1. ATR-FTIR Analysis 2.2. Preparation and Characterization of Hydrogels The ATR-FTIR spectra of drug 5FU, alginate, chitosan, nanoparticles, and hydrogels are shown in Figure 2. We found that 5FU has the characteristic peaks at 2992 (N–H stretchThe 5FU solution and 5FU-Alg-Np-loaded chitosan-gelatin-based hydrogels were prepared. Chitosan and gelatin solutions when mixed together, electrostatic interaction occurs, which results in interpenetrating self-assemble soft aqueous pores as a result of amide bonds formation [37]. These soft pores were filled with 5FU solution and/or 5FUAlg-Np to form drug-loaded hydrogels. 2.2.1. ATR-FTIR Analysis ing vibrations), 2883 (C=H stretching vibrations), 1723 (C=O group of ketone), 1505 and 1253 cm−1 (stretching vibration of C=N and C=F, respectively) [38]. The characteristic FTIR peaks of chitosan were at 3390, 2977, 1665, and 1401 cm−1, and for alginate were at 3121, 2928, 1404, and 1050 cm−1. The characteristics peaks of gelatin were observed at 3292 cm−1 (N–H stretching), 2947 cm−1 (C–H stretching), and 1637 cm−1 (C=O stretching). The FTIR spectrum of nanoparticles have the characteristic absorption peaks at 3339, 2924, 1450, and 1060 cm−1, showing the absorption bands shift to new positions as compared 5FU. This indicates the successful encapsulation of 5FU into alginate nanoparticles. The absorption peaks of nanoparticles and 5FU were weaken in FTIR spectrum of hydrogels (Figure 2). This confirms the incorporation of nanoparticles into the chitosan-based hydrogels. 2.2.2. pH and Visual Transparency of Hydrogels The ATR-FTIR spectra of drug 5FU, alginate, chitosan, nanoparticles, and hydrogels are shown in Figure 2. We found that 5FU has the characteristic peaks at 2992 (N–H stretching vibrations), 2883 (C=H stretching vibrations), 1723 (C=O group of ketone), 1505 and 1253 cm−1 (stretching vibration of C=N and C=F, respectively) [38]. The characteristic FTIR peaks of chitosan were at 3390, 2977, 1665, and 1401 cm−1, and for alginate were at 3121, 2928, 1404, and 1050 cm−1. The characteristics peaks of gelatin were observed at 3292 cm−1(N–H stretching), 2947 cm−1(C–H stretching), and 1637 cm−1(C=O stretching). The FTIR spectrum of nanoparticles have the characteristic absorption peaks at 3339, 2924, 1450, and 1060 cm−1, showing the absorption bands shift to new positions as compared 5FU. This indicates the successful encapsulation of 5FU into alginate nanoparticles. The absorption peaks of nanoparticles and 5FU were weaken in FTIR spectrum of hydrogels (Figure 2). This confirms the incorporation of nanoparticles into the chitosan-based hydrogels. The pHisanimportant parameter for topical formulations. The pH of all the prepared hydrogels was checked using digital pH meter (Table 2). The pH of the hydrogel (6.83) was slightly higher than its sol form (6.27). The results show that there is no drastic change in pHvalue after conversion of sol to gel, confirming that there is no undesired chemical reaction in the system. This pH range is also in correspondence to biological skin pH, hence no irritation on skin surface will be caused. Skin pH ranges from 4 to 6, and the topical formulations should possess pH in this range [39]. Visual evaluation of the prepared hydrogel was performed at different temperatures (i.e., 25−37 ◦C) for the assessment of transparency. The chitosan-gelatin hydrogel was transparent at 25 ◦C, which proves that all the added ingredients are homogeneously mixed. It was observed that the hydrogel became turbid at body temperature in comparison to the initial sol
Rheological analysis can provide information regarding the ability of gel formulations to spread on the skin. The rheological properties of the gel can also affect the drug release kinetics and therefore, the study of these properties is essential. The viscosity of the prepared hydrogel remained higher between 15–36 ◦C, and then started to decline until 50 ◦C, showing the viscoelastic behavior of the hydrogels. Viscosity of nanoparticles incorporated hydrogels was higher than the simple hydrogels. This was due to the presence of polymer alginate in 5FU-Alg-Np-HG. The hydrogel becomes more viscous at body temperature as compared to the room temperature. Similar results were also previously reported by [40]. Chitosan a positive charge polymer interacts electrostatically with gelatin and form hydrogels. Gelatin temperature of hydrogels is determined by several factors including GP content, chitosan concentration, polymers molecular weight and its degree of deacetylation [41]. The elastic modulus (G) of the prepared hydrogel increase gradually after 30 ◦C, which confirms the thermosensitive behavior of the prepared hydrogels. On the other hand, the viscous modulus (G) remained constant [42]. Higher values of G indicates mechanical reinforcement of the gel under body temperature (37 ◦C) conditions, showing a stronger hydrogel network [42].
The surface morphology of the prepared hydrogel (5FU-Alb-Np-HG) was investigated using scanning electron microscopy. It was observed that the prepared hydrogels exhibited a denseandporousnetworkstructure asshownbyredarrowsinSEMimage(Figure4). The pores in the hydrogel are due to the existence of nanoparticles in the fabric. The presence of pores helps in the release of drug/nanoparticles from the polymer network. The dense structure shows the formation of more stable hydrogels. 2.2.5. Swelling Studies Swelling is described as a phenomenon in which water and/or biological fluids are retained in a polymer network. Swelling ability of the prepared hydrogels was evaluated by monitoring the mass changes during incubation in the dissolution medium. The swelling studies of chitosan-gelatin-based hydrogels containing 5FU-Alg nanoparticles at temperatures 32 and 37 ◦C and pH at 5.5 and 7.4 were performed. The capability of a hydrogel to swell depends on its chemical structure and the characteristics of its medium. It was found that the swelling of hydrogel increases with increase in temperature from 32 to 37 ◦C (Figure 5). This might be due to the hydrogen bond of the amine group in chitosan may dissociate with the water molecules inside the hydrogel network. As a result, the polymer chains in the hydrogel relax, which leads to increased swelling [43]. Swelling of hydrogels helps in control/sustain the release of 5FU from the hydrogel matrix. Higher swelling at 37 ◦C andpH7.4indicates more control over the release of 5FU in the deeper layers of skin.
3. In Vitro Release Studies The release of drugs from polymeric materials can be controlled by a number of physical processes, such as hydration, swelling, gel formation, drug diffusion from the matrix, and the erosion of the matrix [44]. The release profile of 5FU from the nanoparticles and hydrogel was investigated in phosphate buffer pH 5.5 (in-accordance with skin pH) and phosphate buffer pH 7.4 (in-accordance with blood pH) at a temperature 32 ◦C (inaccordance with skin temperature) and 37 ◦C (in-accordance with blood temperature), The release of drugs from polymeric materials can be controlled by a number of physical processes
respectively(Figure6a,b).Thesimple5FUsolution(inphosphatebufferpH7.4)withouta carrierwasalsoexploredasareference.Theresultsshowthatdrugsolutionreleases5FU upto97%in6h.Alginatenanoparticlessignificantlysloweddown/controlthereleaseof 5FUascomparedtosolution(ANOVA;p