Preparation of PHT-loaded chitosan-alginate nanoparticles

In an effort to improve PHT poor aqueous solubility, the hydrophobic drug was incorporated into the NPs after being dissolved in methanol prior to its addition to the ALG solution [39]. After the addition of CS, the two polymers formed discrete NPs which simultaneously entrapped the suspended PHT within their neutralized core. It is believed that, although each individual polymer is a hydrophilic charged polymer, the interaction between oppositely charged polyelectrolytes leads to the formation of particles which consist of a neutralized, relatively compact core, surrounded by a shell of the excess polymer, stabilizing the formed particles by electrostatic repulsion [40, 41]. Therefore, the end product consisted of a colloidal dispersion of CA NPs entrapping PHT. It is worth mentioning that chitosan and alginate polymers were selected in this study because, in addition to their intrinsic wound healing properties, the interaction between their opposite charges formed particles with a neutralized uncharged core which helped increasing the entrapment efficiency of an uncharged hydrophobic drug such as PHT.

Effect of drug loading

Table 2 shows the effect of adding different amounts of PHT on the PS, PDI, and %EE of CAP NPs prepared with ALG:CS weight ratio 1:1, where the weight of the polymer blend equals 22.5 mg. Increasing the PHT amount from 2.25 to 4.5 mg showed no significant increase in both PS and PDI (P > 0.05). On the contrary, the loading of 9 mg (F3) resulted in a significant increase in both PS and PDI (P < 0.001). The incorporation of PHT in the chitosan-alginate nanoparticles may have caused some reorientation of the polymers that form the nanosystem leading to a relative increase in size. This may be attributed to the interaction between PHT and the polymers which occurred at the expense of CS-ALG interactions, thus altering the way the two polymers associate [42]. Increasing the PHT amount to 9 mg might have led to a further alteration in the formation of the nanosystem, leading to a dramatic increase in particle size and the formation of a less compact system. A similar finding was observed with Goycoolea et al. [42] where insulin-loaded chitosan-alginate NPs showed a significantly higher PS than the blank NPs. In addition, Thai et al. [43] also observed a similar increase in losartan-loaded chitosan-alginate nanoparticles’ size when increasing the drug concentration, accompanied by a broader size distribution which might be due to strong drug-drug interaction at high drug concentration that may lead to the formation of drug agglomerates within the nanoparticles, thus causing an increase in the PS and PDI [43].

Concerning %EE, statistical analysis showed that there was no significant difference between formulas F2 (4.5 mg) and F3 (9 mg), while F1 (2.25 mg) showed a significantly lower %EE when compared to F2 and F3. A similar finding was observed by Das et al. [44] who reported that increasing the initial concentration of curcumin resulted in an increase in %EE. A possible explanation of this phenomenon may be as follows: increasing the initial amount of PHT led to an increase in the amount of suspended PHT which would rather be encapsulated in the neutral uncharged environment of the NPs’ core rather than in the aqueous outer environment.

From the previous results, it could be concluded that loading PHT with a drug:polymer weight ratio of 1:5 was the optimum selection for further investigations. This ratio resulted in a high %EE, together with lower particle size and more homogenous particle size distribution than the 1:2.5 ratio.

Effect of alginate to chitosan weight ratio

Different CS concentrations, thus different ALG:CS weight ratios were used in the preparation of PHT-loaded CA NPs. The calculated amount of PHT was dissolved in MeOH and added dropwise to ALG solution, to obtain a drug:polymer ratio of 1:5. Then, a fixed volume of CS (15 mL) with different concentrations (0.05%, 0.075%, 0.1125%) was added dropwise to ALG solution yielding different ALG:CS weight ratios (1:0.67, 1:1, 1:1.5 respectively). As illustrated in Table 2, PS increased significantly when CS concentration was increased from 0.05% (F4) to 0.075% (F2) and 0.1125% (F5) (P < 0.05). On the contrary, there was no significant increase in PS between F2 and F5. The increase in PS at higher CS concentration may be due to the repulsion between the positive charges of the added CS, as previously reported by Mukhopadhyay et al. [45]. The PDI of PHT-loaded CA NPs was not significantly altered by the increase in CS concentration (P > 0.05).

Regarding the drug entrapment, F4 with the lowest CS concentration showed the highest %EE of 90 ± 1.5%, which was significantly higher than the other formulations. Increasing CS concentration led to a decrease in %EE. Similar findings were observed with Motwani et al. [46], where the entrapment of gatifloxacin in chitosan-alginate nanoparticles decreased with increasing polymers concentration. A possible explanation is that at high polymers concentrations, the polymers make the bulk of the nanoparticles’ matrix and less volume is available for drug encapsulation [46].

Table 2 Effect of the variation of the amounts of drug added and ALG: CS weight ratio on particle size (PS), polydispersity index (PDI), zeta potential (ZP), and percent entrapment efficiency (%EE) of the prepared phenytoin-loaded chitosan-alginate nanoparticles (n = 3) In vitro drug release study

The dialysis bag method was used to compare the release behavior of the PHT-loaded NPs to that of pure drug. For the release study, 2 formulas were selected, namely F4 and F5, to evaluate the influence of the presence of either excess chitosan or alginate on the release behavior of PHT from CA NPs. These formulations were compared to PHT suspension and PHT solution (in MeOH:H2O = 60:40).

As shown in Fig. 2, complete release of PHT from solution occurred after 1 h, indicating the good dialysability of the drug.

Fig. 2

In vitro release profile of phenytoin from PHT-loaded chitosan-alginate nanoparticles compared to PHT suspension and PHT solution at 100 rpm and 32 °C in 0.1-M phosphate buffer pH7.4 + 1% SLS for 24 h, n = 3. PHT, phenytoin; CAP − ve, negatively charged chitosan-alginate nanoparticles; CAP + ve, positively charged chitosan-alginate nanoparticles

The release of PHT suspension was relatively slower than the formulations, with 42% released after 24 h. Since PHT is a biopharmaceutical classification system (BCS) class II drug, the slow and incomplete dissolution and release of its crystalline powder can be expected and may be attributed to its hydrophobic nature, poor wettability, and particles agglomeration during the experiment. A negligible dissolution profile of crystalline PHT in phosphate-buffered saline of pH 6.5 containing simulated fasted duodenal solution was also observed with Widanapathirana et al. [47].

Regarding the nanosystems, a burst release was observed after 4 h for both formulas followed by a slow modulated release. A similar result was observed by Motwani et al. [46] during the release of gatifloxacin from chitosan-alginate NPs. This initial rapid release may be due to the rapid hydration of the NPs due to the hydrophilic nature of chitosan and alginate. The initially released PHT is most probably present on the surface or near the surface of the NPs [46, 48].

Both CAP NPs exhibited slightly enhanced extent and rate of release, compared to PHT suspension, with 48% and 61% released after 24 h for CAP + ve and CAP − ve, respectively. The enhanced dissolution rate may be explained by the smaller particle size of nano-sized PHT, thus a higher surface area available for dissolution [49]. When comparing both formulations, it was observed that CAP + ve NPs (F5) showed a relatively slower release than CAP − ve NPs (F4). The cumulative percentage release of PHT from CAP + ve NPs and CAP − ve NPs was 48% and 61% after 24 h. The difference in the release profiles could be attributed to the higher concentration of CS in CAP + ve which may retard the release of PHT from the NPs due to the poor solubility of CS in neutral conditions [50]. Similar results were obtained with Mukhopadhyay et al. [45, 51].

In general, both CAP NPs showed a sustained release pattern, compared to the PHT solution, which would be suitable for topical in vivo applications. In addition, the release of PHT from the CAP NPs indicates that the drug is not highly bound to the system and can be released from the nanoparticles.

Physicochemical characterization Transmission electron microscope

Morphological examination of PHT-loaded negatively charged CAP NPs ( F4, named CAP -ve) and positively charged CAP NPs (F5, named CAP + ve), confirmed the formation of NPs with spherical shape in the nanometric range, as shown in Fig. 3A.

Fig. 3

A Transmission electron microscopy images of phenytoin-loaded chitosan-alginate nanoparticles (a) CAP − ve NPs, negatively charged chitosan-alginate nanoparticles (b) CAP + ve NPs, positively charged chitosan-alginate nanoparticles, stained with uranyl acetate (scale bar = 100 nm); B differential scanning calorimetry thermograms of pure CS, ALG, PHT, and PHT-loaded CA NPs; C Fourier-transform infrared spectra for pure CS, ALG, PHT, their physical mixture, and PHT-loaded CA NPs. CS, chitosan; ALG, alginate; PHT, phenytoin; PHT-loaded CA NPs, phenytoin-loaded chitosan-alginate nanoparticles

Differential scanning calorimetry (DSC)

DSC analysis was carried out to study any changes in the drug crystallinity. DSC thermogram of PHT showed a sharp endothermic peak at 295.6 °C related to its melting point which indicates the crystalline nature of the drug [32]. However, the thermogram of PHT-loaded NPs presented no peak in this region as shown in Fig. 3B. These results suggest that PHT was incorporated in CA NPs and that the crystalline nature of PHT may have been converted to the amorphous form [52, 53].

Fourier-transform infrared spectroscopy

FT-IR spectroscopy was carried out to characterize possible interactions between PHT and the polymers in CA NPs. The IR spectrum of pure PHT (Fig. 3C) showed characteristic absorption bands at 3195 cm−1 (N–H stretching), 1770 cm−1 (C = O stretching), and 744 cm−1 (phenyl ring C-H out of plane vibration). Similar findings were observed by Ali et al. [32]. On the other hand, the spectrum of PHT-loaded CA NPs showed the presence of PHT bands at 1772 and 744 cm−1, which confirms the successful entrapment of PHT in CA NPs. However, the characteristic absorption band of PHT at 3195 cm−1 disappeared. A similar finding was observed with Motawea et al. [53] which may be attributed to the interaction of the drug with the polymers, probably via intermolecular hydrogen bonding, which reflects drug entrapment inside the polymeric matrix.

Wound healing studyEvaluation of ulcer healing

Diabetes induction is confirmed when a sustained hyperglycemic state is reached 48 to 72 h following STZ injection [36]. All rats maintained blood glucose levels > 500 mg/dL during the study. Ulcers were digitally photographed every 3 days until the end of the 14-day study. Figure 4 shows the progression of the healing of the ulcers throughout the experiment. All ulcers exhibited progressive healing; however, healing rates were different. PHT-loaded NPs and PHT suspension showed significantly higher initial healing rates over the first two time intervals (4 and 7 days) compared to the control group (p < 0.05), as shown in Fig. 5. For instance, at day 4, PHT suspension (14.35%) significantly improved the percentage of wound closure compared to the control group (9.24%), followed by the negatively charged NPs (18.25%) and finally the positively charged NPs (22.38%) which showed the highest percentage of wound closure. This observation persisted at day 7, with the positively charged NPs resulting in a significantly higher percentage of wound closure of 56.54% compared to the other groups. It is worth mentioning that PHT-loaded nanoparticles have not been assessed so far for the treatment of diabetic pressure ulcers. However, several clinical studies investigated the use of PHT for the treatment of various wounds and reported faster healing time and more epithelialization in wounds treated with PHT compared to the control group. Among these, reports were Patil et al. [12] who used 100 mg PHT powder mixed with saline for 0–5 cm2 diabetic foot ulcers [12] and Pereira et al. [17] who studied the use of PHT cream 0.5% for excisional wounds on the face and on the back [17], but did not determine the exact daily dose used. Hokkam et al. [14] also reported a shorter healing time of chronic venous ulcers in patients treated with a lotion consisting of 1 g PHT mixed with 25 mL liposomal base, compared to saline. These results are consistent with ours regarding the shorter healing time for PHT-treated groups, compared to control groups. In our study, the daily dose used was 1 mg PHT per ulcer, which had an initial diameter of about 1.2 cm, hence an area of about 1.13 cm2. The positive healing effects obtained with the use of such a low dose suggest the promising effect of using PHT in a nanosystem.

Fig. 4

Stages of ulcers healing with the different treatments for 14 days. PHT, phenytoin; CAP − ve NPs, negatively charged chitosan-alginate nanoparticles; CAP + ve NPs, positively charged chitosan-alginate nanoparticles

Fig.5

Effect of different treatments on percent wound closure of pressure ulcers of rats during 14 days, data are expressed as the mean ± standard deviation (n = 4 rats with a total of 16 wounds per group), PHT, phenytoin; CAP − ve NPs, negatively charged chitosan-alginate nanoparticles; CAP + ve NPs, positively charged chitosan-alginate nanoparticles

Histological analysis

Figure 6A shows the hematoxylin and eosin (H&E)-stained images of the ulcers treated with the different therapies throughout the study.

The results showed that PHT had a great impact on improving the quality of regenerated skin. All PHT-treated groups showed well-structured regenerated epithelium and significantly less inflammation compared to the control untreated group (Fig. 6A (b)). Moreover, the incorporation of PHT in CA NPs resulted in improved skin quality and maturity. For instance, in addition to the re-epithelialization and mild inflammation seen in the PHT suspension group (Fig. 6A (c)), the CAP − ve NP group (Fig. 6A (d)) showed well-organized dermis with marked neovascularization. On the other hand, remodeling of the epidermis and dermis was more mature in the CAP + ve NPs group (Fig. 6A (e)) compared to the other groups. Well-organized dermis structure similar to the normal structure, in addition to the formation of skin appendages, such as hair follicles and sebaceous glands, was also observed in this group. Similar findings were reported by Cardoso et al. [23] who studied the wound healing effect of chitosan hydrogels loaded with either free PHT, PHT in polycaprolactone nanocapsules or PHT nanoemulsion at 0.025% w/v, for the treatment of excisional wounds in rats, over 6 days. They reported that PHT-treated groups showed no necrotic foci, less inflammation, and evident collagen fibers and fibroblast proliferation, compared with the control group and groups treated with unloaded nanocarriers. PHT has been reported to promote wound healing by multiple mechanisms. First, it promotes the maturation of collagen in normal and granulation tissue. In addition, PHT was reported to enhance neovascularization [54]. The above results suggest that our system resulted in favorable wound healing rates and quality of the formed skin while using as low as 1 mg PHT daily for 14 days. This might be attributed to its use as a nano-sized system with a bioactive carrier. It is worth mentioning that PHT-loaded NPs, both CAP − ve and CAP + ve, resulted in less inflamed and more mature skin structure, compared to their corresponding blank CA NPs reported in our previous work [31], suggesting that the incorporation of PHT enhanced the wound healing properties of the chitosan-alginate NPs.

Fig. 6

A Histologic examination using hematoxylin and eosin (H&E) stain of (a) normal skin, (b) control untreated group, (c) treated with PHT suspension, (d) treated with CAP − ve NPs (e) treated with CAP + ve NPs, showing epithelium (white arrow), inflammatory cells (red arrow), edema (yellow arrow), new blood vessels (green arrow), skin appendages (black arrow), HF, hair follicle; SG, sebaceous gland; B histologic examination using Masson’s trichrome stain of (a) normal skin, (b) control untreated group, (c) treated with PHT suspension, (d) treated with CAP − ve NPs, (e) treated with CAP + ve NPs, showing normal random collagen arrangement (red arrow) and fibrosis with parallel condensed collagen bundles (black arrow). PHT, phenytoin; CAP − ve NPs, negatively charged chitosan-alginate nanoparticles; CAP + ve NPs, positively charged chitosan-alginate nanoparticles

Histomorphometric analysis of collagen

During granulation tissue formation in the wounded area, fibroblasts synthesize the new extracellular matrix of which collagen is the main component, thus replacing the provisional matrix consisting initially of fibrin [38]. Figure 6B shows collagen fiber-stained blue with Masson’s trichrome stain after 14 days. Regarding the arrangement of collagen fibers, groups treated with PHT, shown in Fig. 6B (c, d, and e), showed a better organized and distributed collagen fibers compared to the control group shown in Fig. 6B (b).

For each group, four slides were examined and four images per slide were captured to calculate the percentage of blue staining. The percent areas occupied by collagen fibers in the different groups are shown in Fig. 7. Treatment with PHT suspension (50.35 ± 1.34%) caused a significant increase in collagen amount compared to the control untreated group (32.9 ± 1.9%). Incorporation of PHT in CA NPs resulted in a further improvement in the deposition and organization of collagen fibers with % collagen areas of 63.65 ± 1.2% and 57.9 ± 1.55% for CAP + ve and CAP − ve, respectively, which were significantly higher than the other groups. Many previous studies as Habibipour et al. [54] and Cardoso et al. [23] also reported that topical PHT application caused an increase in collagen deposition compared to control groups, possibly by enhancing collagen cross-linking and inhibiting collagenases activity [54].

Fig. 7

Quantification of collagen formation in wound samples of diabetic rats receiving various treatments, on day 14, data are expressed as the mean ± standard deviation (n = 4 rats with a total of 16 wounds per group), PHT, phenytoin; CAP − ve NPs, negatively charged chitosan-alginate nanoparticles; CAP + ve NPs, positively charged chitosan-alginate nanoparticles

When comparing these results with the results of the blank chitosan-alginate nanoparticles in our previous study [31], it is worth mentioning that although PHT-loaded CA NPs did not produce a significant difference in the percentage of wound closure compared to the blank NPs, the addition of PHT significantly enhanced collagen deposition and improved the quality of the newly formed skin compared to blank NPs. Therefore, when evaluating a drug for wound healing, a better judgment of its therapeutic potential would be achieved when healing rates are complemented with histological examination.