Fabrication and optimization of itraconazole-loaded zein-based nanoparticles in coated capsules as a promising colon-targeting approach pursuing opportunistic fungal infections

Application of central composite face-centered design for the optimization of ITZ-ZNP formulation

Based on the unique ability of zein to self-assemble into nanoparticles that are highly stable in gastric and intestinal fluids while start to lose their integrity upon reaching the colon due to colonic microbiota, thus zein was used as colon targeting microbially triggered polymer [17, 45]. The selection of the most suitable ratios of zein: drug and aqueous:organic phases that were used during formulation is critical as they affect NPs size, stability, drug entrapment efficiency, along with its release from the formulated ZNPs. Thus, to achieve the optimized formulation, with accepted lowest PS and PDI values together with high ZP and EE%, central composite face-centered design (CCFD) was used. The independent variables constituted three ratios of zein:drug (A: 1:1, 5.5:1, 10:1) and three ratios of aqueous:organic media (B: 1:1, 5.5:1, 10:1), and the results of the observed dependent variables (responses) were PS (Y1, nm), PDI (Y2), ZP (Y3, mV), and EE (Y4, %) are illustrated in Tables 1 and 2.

Statistical evaluation of ITZ-loaded ZNPsParticle size (PS), polydispersity index (PDI), and zeta potential (ZP)

The particle size of the formulated ITZ-ZNPs ranged between 169.8 ± 6.03 and 1537 ± 6.17 nm, as shown in Table 2. The 2FI model was suggested with R2 coefficient of 0.9274. The difference between predicted R2 (0.7944) and adjusted R2 (0.9032) was reasonable, indicating the ability of the adopted model to navigate the design space [35, 46, 47]. The generated polynomial equation computes the significance of the independent factors on PS:

$$\mathrm= 332.51 + 243.18\mathrm- 251.33\mathrm- 341.15\mathrm$$

(2)

ANOVA statistical analyses revealed that both variables; zein:drug ratio (A) and aqueous:organic ratio (B) as well as the interaction between them have significant effects on ZNPs PS with P-values of 0.0001, 0.0002, and 0.0002, respectively. Figure 1a illustrates that zein: drug ratio (A) has significant positive effect as increasing zein concentration resulted in correlated increase in the particle size. Bisharat et al. proved that high zein content might enhance clustering of zein nanoparticles and formation of larger size aggregates [48]. Also, Nunes et al. had similar finding during the formulation of resveratrol-zein nanoparticles [49]. This can be explained by high zein concentration enhancing hydrophobic intermolecular interactions that hampers zein diffusion from ethanol (solvent) into water (antisolvent); thus, when ethanol evaporates, these massive intermolecular interactions led to formation of large aggregates instead of small ZNPs [50].

Fig. 1figure 1

3-D Response surface plots showing the effects of the continuous independent variables; A: zein:drug ratio and B: aqueous:organic ratio on, PS (a), PDI (b), ZP (c), and EE% (d)

On the other hand, aqueous:organic phases ratio (B) proved significant negative effect on PS as lowering aqueous phase ratio enhances formation of NPs of large size NP. In accordance with li et al. explanation, at low aqueous ratio, precipitation takes place through a nucleation-growth mechanism. In this case, precipitation is an irreversible phenomenon in which an extensive rapid expulsion of water molecules occurs resulting in formation of large size NP. However, high aqueous ratio enhances coacervation of the particles through a nucleation-aggregation mechanism, whereas zein particles grow gradually as ethanol evaporates. Such gradual growth boosts the formation of NP with relatively smaller PS [51]. Thus, increasing aqueous phase ratio had a profound influence in formulating smaller ZNPs.

In addition, the interaction between A and B showed a significant negative effect on PS, where high zein concentration along with high aqueous ratio resulted in small size of ZNP. During self-assembly of ZNP, the presence of huge number of zein molecules in highly hydrophilic aqueous medium resulted in enhancement of hydrophobic interactions between zein molecules that assembled into small spherical NP due to hydrophilicity of the medium [52].

Polydispersity index (PDI) is a parameter that demonstrates either uniformity or diversity of NP size distribution. It was ascertained by international standards organizations (ISOs) that PDI values between 0.1 and 0.5 implies monodispersed systems, while PDI values more than  0.7 implies existence of large aggregates that led to polydispersity of the system [53, 54]. Herein, PDI values of the prepared ITZ-ZNPs ranged from 0.224 ± 0.008 to 0.887 ± 0.017 (Table 2). Statistical analysis of the results suggested 2FI model as the best-fitting model for PDI (Y2) with model R2 coefficient of 0.8345. The generated polynomial equation was:

$$\mathrm= 0.4 + 0.2\mathrm- 0.1\mathrm- 0.1\mathrm$$

(3)

ANOVA statistical analyses presented by the 3-D graphical surface plot (Fig. 1b) proved that incorporation of different ratios of zein: ITZ (A) had significantly positive impact on the PDI (P-value = 0.0012). As previously mentioned, there are hydrophobic intermolecular interactions occur between zein molecules, thus increasing zein concentration led to formation of larger zein aggregates that affect dispersity of the system, and consequently, ZNPs of high PDI were obtained.

On the other hand, both aqueous:organic ratio (B) and the interaction between A and B showed significant negative impact on the PDI (P-value of B = 0.0029, P-value of AB = 0.0242). Similar results by Gajera et al. demonstrated that low aqueous:organic ratio enhanced the agglomeration of particles during nanoparticles formulation leading to increased particle size dispersity throughout the preparation [55]. As well, Danaei et al. found that decreasing aqueous ratio along with increasing zein ratio resulted in formulating NP with large agglomerates and high PDI values [56].

Zeta potential (ZP) is an essential criterion when evaluating the physical stability of nanoparticles; it is important to quantify NP surface charges and reveal the physical stability of pharmaceutical nano-systems. As, high ZP values (about ± 30 mV or higher) indicate the presence of enormous charges on the NPs surface that generate electric repulsion force, thus prevent aggregation of NP and increase system stability; however, values around ± 20 mV are still considered as deflocculated stable systems [57,58,59,60]. Herein, zeta potential study of the ITZ-ZNPs showed positive values ranged from 27 ± 1.72 to 46.5 ± 0.57 mV, as illustrated in Table 2 indicating that the prepared ITZ-ZNPs are of very good stability as all the prepared formulations were positively charged with around 85% of them had ZP > 35 mV.

The 2FI model was the best-fitting model for ZP (Y3) with R2 of 0.7174. The difference between the predicted R2 (0.537) and the adjusted R2 (0.6232) was reasonable, as it is less than 0.2 and implies good ability of model to predict accurate responses values with the ZP polynomial equation:

$$\mathrm= 39.1 - 2.2\mathrm- 5.3\mathrm- 3.5\mathrm$$

(4)

The polynomial equation together with ANOVA statistical analyses presented by the 3-D surface plot (Fig. 1c) clarified that the aqueous:organic media ratio (B) showed a significant negative effect on the ZP with a P-values equals to 0.0033 while zein:drug ratio (A) and interaction between the two independent factors (AB) has no significant effect on the ZP values as its P-value was 0.1359 and 0.0654, respectively.

The significant effect of (B) could be explained based on the fact that zein protein acquires positive charges at low pH media (pH 2) [61, 62]. So that increasing the ratio of the aqueous medium to the acidic organic phase (HCl-ethanolic solution) results in diluting the acidity and increasing the media pH causing a corresponding reduction in ZNP-positive charges. While at low aqueous to acidic organic phase ratio, the nanoparticles dispersion pH is more acidic leading to increased positive charges on the ZNPs and accordingly higher ZP.

Entrapment efficiency (EE%)

The efficiency of zein protein to entrap ITZ and self-assemble to form ITZ-ZNPs is displayed in Table 2. ITZ entrapment in ZNPs ranged between 31.35 ± 3.25 and 81.54 ± 4.15% w/w. The response surface quadratic model was used to best fit EE% (Y4) with regression coefficient R2 of 0.9773. The predicted R2 was 0.8678 and the adjusted R2 was 0.961, implying good model prediction for the response. The EE% polynomial equation was:

$$\mathrm= 77.6 + 4.3\mathrm+ 0.5\mathrm- 19.7\mathrm- 11.7\mathrm2 - 10.2\mathrm2$$

(5)

The above equation together with ANOVA statistical analyses revealed that zein:drug ratio (A) had significant positive effect on the EE% with a P-value = 0.01 while aqueous:organic ratio (B) has insignificant effect (P-value = 0.702). From Eq. (5) and Fig. 1d, it is obvious that increasing zein concentration led to significant increase in the EE% which might be due to the increased hydrophobic interactions between ITZ and zein upon increasing zein concentration, leading to higher percentage of ITZ encapsulated effectively inside ZNPs [63]. Similar findings were observed by Cai et al. in formulating pectin-zein nanoparticles for encapsulating the hydrophobic curcumin, where increasing zein content enhanced the interaction between the hydrophobic drug molecule and zein protein resulting in more drug molecules to be encapsulated into the hydrophobic core of ZNPs [62].

Statistical optimization and physicochemical characterization of the optimized formulation

Stat-Ease Design-Expert® software (Version 10.0.0, Stat-Ease Inc., Minneapolis, USA) was used for optimization of formulation parameters and election of the optimized ITZ-ZNP formulation based on the predetermined criteria (minimum PS and PDI, maximum ZP, and EE%). Following optimization, the optimum formulation with the highest desirability value was selected. As presented in Table 3, formulation with 5.5:1 (zein:drug ratio) and 9.5:1 (aqueous:organic ratio) was considered the optimum formulation with 0.755 desirability as it had the most acceptable combination of responses (expected PS, PDI, ZP, and EE were found to be 196.19 nm, 0.309, 34.47 mV, and 70.22%, respectively).

Table 3 ANOVA results of the responses including predicted and adjusted R2, composition of the optimized formulation and its desirability, expected values, observed values, and residual errors of the responses

Following formulation of the optimized formulation, its observed response values were PS of 208 ± 4.29 nm, PDI of 0.35 ± 0.04, ZP of 35.7 ± 1.65 mV, and EE of 66.7817 ± 3.89%. A comparison between the predicted and observed response values was performed and the residual for each response was calculated to enhance validity of the performed statistical optimizations. The residual errors were found to be 11.81 nm, 0.041, 1.23 mV, and 3.44% for PS, PDI, ZP, and EE, respectively. Such small residual error values indicate that the observed results were very close to the predicted ones, thus ensuring good model fitting and efficient optimization [64, 65].

Differential scanning calorimetry (DSC)

DSC studies were carried out to show the physical state of ITZ-ZNP constituents and their thermotropic properties, besides, to ensure their degree of purity and check crystallinity [66]. To understand the crystalline nature of ITZ before and after formulation, DSC study for ITZ, zein, their physical mixture, and ITZ-ZNP-optimized formulation was performed, and their thermograms were illustrated in Fig. 2. ITZ thermogram (Fig. 2a) showed a single sharp endothermic peak at 167 °C, such a sharp peak corresponding to its melting that indicates the typical crystalline nature of pure ITZ. Similar endothermic peak for ITZ was observed with Alves-Silva et al. who observed that ITZ melting point represented by sharp endothermic peak at 167.9 °C due to ITZ crystallinity [67].

Fig. 2figure 2

DSC thermograms of pure ITZ (a), zein (b), ITZ-zein physical mixture (c), and ITZ-ZNP-optimized formula (d). The curves have been displaced vertically for better visualization

Figure 2b showed the DSC thermogram of zein showing broad endothermic peak at around 80 °C might be due to zein protein degradation from heating during the study. Wang et al. had similar observations as zein powder sample showed broad endothermic peak ranging around 82.09 °C indicating protein degradation without phase transition, as well, such broad peak, means a longer melting process, suggesting a non-crystalline state [68].

In ITZ-zein physical mixture DSC thermogram (Fig. 2c), zein broad endothermic peak was clearly prominent while ITZ peak was present with decreased intensity such decrease in the intensity of the characteristic endothermic peak may be due to the dilution effect due to relatively high zein:drug ratio in the optimized formulation (5.5:1 zein:drug ratio).

On the other hand, Fig. 2d showed complete disappearance of ITZ peak in the optimized formulation, proving ITZ transformation from crystalline to amorphous form which was then molecularly dispersed throughout the ZNP matrix [69, 70].

X-ray diffractometry (XRD)

XRD analysis was performed in order to examine the inner nanocrystalline structure of ITZ during nanoparticle formulation. Potential changes of ITZ crystalline structure may occur according to its chemical nature and physical hardness inside ZNPs [71]. X-ray diffraction is a well-established tool to study crystal lattice arrangements and it yields particularly useful information on the degree of sample crystallinity that might affect various characteristics such as solubility. The X-ray diffractograms of ITZ, zein, physical mixture of them, and optimized ITZ-ZNPs are presented in Fig. 3. ITZ diffractogram (Fig. 3a) showed several sharp high intensity peaks at 8.75, 10.75, 14.51, 17.54, 20.38, 23.51, 25.42, and 25.11 (2θ) that indicates the presence of well-crystallized pure ITZ of sharp defined diffraction peaks [72]. Figure 3b showed two broad scattering bands, instead of sharp peaks, due to α-helices backbone of the amorphous zein [73]. The physical mixture diffractogram (Fig. 3c) maintained the diffraction patterns of both ITZ and zein, but it was observed that ITZ characteristic diffraction peaks were of lower intensities probably due to the dilution effect (zein:ITZ ratio in the physical mixture was the same as that of the optimized formulation (5.5:1)) [27]. The permanence of these characteristic peaks confirmed ITZ crystalline state and excluded the existence of possible drug–zein interaction in the physical mixture.

Fig. 3figure 3

XRD diffractograms of pure ITZ (a), zein (b), ITZ-zein physical mixture (c), and ITZ-ZNP optimized formulation (d). The curves have been displaced vertically for better visualization

On the other hand, the X-ray diffractogram of the optimized formulation (Fig. 3d) showed disappearance of ITZ diffraction peaks. Typically, such diffusive pattern is commonly observed following the conversion of the crystalline ITZ into its amorphous states and its dispersion within zein matrix in amorphous form.

The XRD studies confirmed the DSC results and assured the complete loss of ITZ crystallinity and suggested its inclusion into the ZNP matrix.

Fourier transform infrared spectroscopy (FT-IR)

FT-IR spectroscopy was employed to elucidate the main structural properties of pure ITZ and zein and then detect any possible chemical interaction by pointing the characteristic modifications of their functional group occurred due to chemical interactions between them upon nanoparticle formulation [74, 75]. FT-IR spectra of ITZ, zein, ITZ-zein physical mixture, and ITZ-ZNP-optimized formulation are displayed in Fig. 4. Figure 4a demonstrates ITZ characteristic peaks at 3000–3100 cm−1 (aromatic C-H), 1701 cm−1 (carbonyl group), 1550 cm−1 (C = N), 1450 cm−1 (C = C), and 794 cm−1 (C–Cl) [76]. While Fig. 4b shows that zein characteristic peaks are 3305.99 cm−1, 2931.8 cm−1 (-COOH), 1651 cm−1 (C = O stretching), 1539 cm−1 (N–H bending), and 1242 cm−1 (C-N stretching) [77,78,79].

Fig. 4figure 4

FT-IR of pure ITZ (a), zein (b), ITZ-zein physical mixture (c), and ITZ-ZNP optimized formula (d)

It was observed that the spectra of both physical mixture (9.5:1 zein:drug ratio; same ratio as the optimized formulation) (Fig. 4c) and ITZ-ZNP-optimized formulation (Fig. 4d) showed very close IR behaviors to that of zein spectra (Fig. 4b) due to dilution effect on ITZ due to very high zein:drug ratio (9.5:1) in both samples, consequently, ITZ peaks are less observed in both physical mixture and the optimized formulation.

Furthermore, Fig. 4c and d show the characteristic ITZ peaks at 1701 cm−1 (carbonyl group) and 1550 cm−1 (C = N) disappeared, suggesting coupling of NH of zein with the carbonyl group of itraconazole via hydrogen bond [80, 81]. However, this coupling does not affect the drug antifungal activity as triazole moiety of itraconazole is the functional group responsible for its antifungal activity [82]. To further prove that this interaction did not affect antifungal activity of ITZ, a specific antifungal activity study was performed.

In vitro release of ITZ from the optimized ITZ-ZNP formulation

In order to evaluate ITZ release from the formulated ZNPs in colon, in vitro release study of the optimized formulation, in comparison to that of pure ITZ suspension, was undergone in phosphate buffer (pH = 7.4) containing 2% SLS at 37 ± 0.5 °C and their release profiles are elucidated in Fig. 5. It was found that within the first 1 h, ITZ release was 15.8 ± 7.5% and 15.3 ± 3.9% from drug suspension and optimized formulation, respectively. These results attributed to initial burst release of adsorbed ITZ on the surface of the optimized ITZ-ZNPs [83]. After 5 h, 50.127 ± 5.6% ITZ was released from drug suspension while only 31.4 ± 9.8% ITZ was released from optimized formulation indicating the ability of ZNPs to initially protect the encapsulated ITZ; however, with time, ZNPs lose its integrity due to formation of aqueous channels leading to swelling of ZNPs facilitating gradual drug release through such pores [84, 85]. After 24 h, 94.5 ± 3.7% ITZ was released from optimized ITZ-ZNP formulation while only 78.7 ± 3.7% ITZ was released from drug suspension. Thus, in vitro release results ensured suitable ITZ release upon reaching the colon. Karthikeyan et al. had similar findings during their study of encapsulating a hydrophobic drug (aceclofenac) in zein microspheres where a gradual drug release occurred due to the presence of hydrophobic interactions between the hydrophobic drug aceclofenac and zein protein [86]. Also, zein hydrophobic matrix prevents its erosion but swell in a hydrophilic like pattern that allow gradual drug release [18].

Fig. 5figure 5

release profile of the optimized ITZ-ZNP formulation compared to pure ITZ suspension in phosphate buffer (pH = 7.4) at 37 ± 0.5 °C. Each point represents mean ± SD (n = 3)

Morphological evaluation using transmission electron microscopy (TEM)

The morphology of the optimized ITZ-ZNP formulation and ITZ-free ZNPs were observed via TEM and presented in Fig. 6a and b, respectively. The photomicrographs showed that ITZ-ZNPs and ITZ-free ZNPs all have solid dense structures, with homogenous spherical shape, smooth surfaces, and uniform sizes. Moreover, the core–shell structure of ITZ-loaded ZNPs was elucidated in Fig. 6a, indicating the presence of itraconazole in the inner hydrophobic core of zein nanoparticle [87].

Fig. 6figure 6

Transmission electron micrographs of a ITZ-ZNPs with magnification power of 50,000 × . b ITZ-free ZNPs with a magnification power of 80,000 × . c Size of ITZ-ZNPs with magnification power 80,000 × , core (red arrows) shell (black arrows)

It was interesting to notice that PS range measured by TEM was slightly lower than that measured by zeta sizer, as shown in Fig. 6c. This might be due to hydration of the sample before zeta sizer measurement. Upon hydration of the sample prior to zeta sizer measurements, aqueous channels in the hydrated ZNPs were formed by time, resulting in swelling of ZNPs and increasing its PS during measurement. While for TEM, dried ITZ-ZNPs were observed without any hydration; thus, no swelling occurs to the ZNPs [84, 85].

Tolerance studies of the selected optimized formulation Ex vivo histopathological examination

Microscopic histological examination of rabbit colon mucosa after treatment with either ITZ-ZNP optimal formulation, saline (negative control), or DMSO (positive control) was performed and presented in Fig. 7 to ensure the tolerance and safety of the formulated optimized ITZ-ZNPs and determine any possible destructive or toxic effects of various ingredients used in their formulation on the rabbit colon tissues.

Fig. 7figure 7

Photomicrograph of rabbit colon mucosa after treatment with optimized ITZ-ZNP formulation (a), saline (negative control) (b), and DMSO (positive control) (c)

Microscopic examination of rabbit colon revealed that application of ITZ-ZNPs as well as saline (negative control group), presented in Fig. 7a and b, respectively, showed normal histological structure of the colon wall, normal mucosa with intact goblet cells. Normally, colon wall mucosa is differentiated into lamina muscularis mucosa, glands, and connective tissues. In depth, this structure is constituted of tunica mucosa that was formed of simple columnar epithelium and lamina propria that contained glands with basally situated nuclei and numerous goblet cells [88]. Such structure was totally maintained following administration of either saline as negative control or ITZ-ZNPs indicating that the optimized ITZ-ZNP formulation has harmless innocuous effect on the colon tissue and thus confirmed the safety of their administration.

On the contrary, the DMSO (positive control group, Fig. 7c) exhibited marked histopathological deterioration of the normal histological structure of the colon wall. Necrosis of the colon mucosa was detected in numerous sections associated with inflammatory cells infiltration with accumulation of eosinophilic tissue debris. In conclusion, the optimal ITZ-ZNP formulation was not accompanied by any marked inflammation or necrosis emphasizing its tolerance and safety on colon cells.

Cell viability and cytotoxicity

SRB assay was preformed to assess biosafety and cytotoxicity of pure ITZ, optimized ITZ-ZNP formulation, and drug-free ZNPs, at different concentrations, against HT-29 colon cancer cells. Figure 8 demonstrates HT-29 colon cancer cell viability after treatment with either ITZ, optimized ITZ-ZNP formulation, and ITZ-free ZNPs for 72 h. The results proved that both ITZ-ZNPs and ITZ-free ZNPs had no cytotoxic effect on HT-29 colon cancer cells at any concentration time point indicating safety of zein as a matrix for encapsulating ITZ. Similar results were proved by Nunes et al., where zein had a low cytotoxic profile, making it a safe matrix for drug delivery systems [49]. However, pure ITZ processed a concentration-dependent cytotoxicity on HT-29 colon cancer cells at 72 h post-treatment. This may be due to ability of ITZ to suppress tumor growth and inhibit cell proliferation by inducing Hedgehog signaling pathway that mediates autophagy cell death of colon cancer cells [38].

Fig. 8figure 8

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