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

Inflammatory bowel diseases (IBD) patients receiving immunosuppressive drugs usually suffer from opportunistic infections in various areas of the gut, including colonic fungal infections. Such colonic infections usually result in clinical manifestations such as diarrhea, fever, rectal abscess, abdominal pain, and colon ulcers. Accordingly, IBD treatments might frequently incorporate antifungal drugs like itraconazole for patients suffering from opportunistic colonic fungal infections [1,2,3]. Interestingly, colon cancer patients receiving anticancer drugs (such as quercetin, methotrexate, doxorubicin, and paclitaxel) might also suffer from opportunistic fungal infections due to the immunosuppressive effect of such therapy; therefore, they are at high risk of colon fungal infections. Such fungal infections promote the progress and deterioration of colon cancer; consequently, antifungal treatment might also indirectly aid in amending colon cancer [4, 5].

Poor targeting capabilities of antifungal drugs usually lead to increasing the need for high doses to exert their antifungal activity in the colon; however, this might possibly result in complications and toxicities such as gastrointestinal, kidney, and liver disorders [6, 7]. Consequently, colon targeting and localization of antifungal drugs can significantly reduce the drugs’ doses and so diminish the expected accompanying complications and side effects.

Itraconazole (ITZ), a triazole broad-spectrum antifungal, is used orally for treatment of colonic fungal infections in high doses (200–400 mg/day), which might lead to potential complications such as pseudo-hyperaldosteronism, gastrointestinal, and liver disorders along with induction of congestive heart failure [6,7,8]. Therefore, reducing ITZ doses achieved by localizing it via colon-targeting therapeutic approach will surely result in reduced complication.

Colon targeting can be classified according to the system unit into single and multiple-unit delivery systems. Various multiple-unit systems, such as liposomes, microspheres, and nanoparticles, have been exploited for colon targeting due to their superior performance over single unit dose systems [9]. Liposomes are multi-particulate lipid bilayer vesicles, used successfully for colon targeting and encapsulating either hydrophilic or lipophilic drugs. For example, curcumin-loaded colon-targeting liposomes successfully delivered the encapsulated drug via selective targeting to the colorectal cancer [10]. Also, microspheres showed effective colon-targeting capabilities due to their short gastric transition time, fast drug release, especially at the targeting site, resulting in enhanced oral bioavailability of colon-targeted lipophilic drugs [11].

For IBD therapy, it is suggested that nanoparticulate colon-targeting systems are ideal due to their ability to be accumulated exclusively in the inflamed colon tissues [12]. According to their surface modification, they can be classified into pH-dependent, microfold cell-targeted, reactive oxygen species (ROS)-responsive, mucus-permeative, and active targeting-based nano-delivered systems. Such systems are capable distinctively to keep their encapsulated drug from the gastric and intestinal pH and enzymes and releasing it exclusively in the colon [10].

Moreover, colon-targeting delivery systems can be classified according to the targeting technique and the used colon-targeting polymers into time-dependent polymers, microbially triggered polymers, as well as using pH-dependent polymers (such as Eudragit) or a combination of them [13]. Eudragit^® (poly-methacrylate) has different grades that include ionic polymers whose solubility is affected by medium pH such as Eudragit® S100, an anionic polymer, that dissolve only at pH > 7 [14]. Unfortunately, although being simple, pH-dependent colon-targeting systems are doubtful due to the highly variable range of physiological and pathological gastrointestinal tract pH. Also, time-dependent colon-targeting systems sometimes show poor colon-targeting capabilities due to the high variability of the transition time throughout the gastrointestinal tract that might lead to imprecise time estimation of the drug release. Recently, a combination of both mechanisms was suggested to ensure an ideal colon-targeting system. For example, indomethacin pellets presented promising colon-targeting system as they targeted indomethacin and sustain its release inside the colon through using Eudragit^® FS30D, as a pH-dependent polymer, and Eudragit^® RS100, as a time-dependent controlled release polymer [12].

Additionally, the presence of an enormous number of microbiomes and enzymes in the colon promotes using microbially triggered colon-targeting prodrugs. Azo polymer- based hydrogels are microbially triggered prodrugs that succeeded in delivering curcumin effectively to the colonic cancer cells and therefore provided promising colon- targeted cancer treatment [15]. Also, pH–enzyme-sensitive microparticles were fabricated as a specific delivery system for the treatment of ulcerative colitis, based on mesalamine-loaded chitosan microparticles coated with methacrylic acid copolymers. By coating mesalamine-loaded chitosan microparticles with methacrylic acid copolymer, they were able to target mesalamine to the colon efficaciously and promote remedy of ulcerative colitis through using a combination of pH-dependent and microbially triggered colon-targeting polymers [16].

Zein, a natural biodegradable and biocompatible corn protein, is recently used as a colon-targeting polymer for its distinctive ability in protecting drugs during its passage through the gastrointestinal (GI) tract and targeting drug release in the colon [17]. Zein is an amphiphilic protein that contains about 75% hydrophobic amino acids; such hydrophobic regions control the drug release, and additionally, the presence of hydrophilic regions cause its swelling in aqueous media without being eroded making it a suitable excipient for controlled drug delivery systems [18]. All types of zein are insoluble in water but soluble in 60–95% aqueous ethanol solutions. Therefore, it is suggested that zein’s poor water solubility (at pH > 11), in addition to its digestion by enzymes of intestinal fluids, aids in encapsulating lipophilic drugs such as itraconazole, targeting them into the colon, and control their release there [17, 19, 20].

It was expected that using a combination of pH-dependent polymer (Eudragit^® S100) in combination with microbially-triggered polymer (zein) can be promising and highly effective for achieving colon-targeting drug delivery [17].

Based on the aforementioned, this study aimed to alleviate colon fungal infections orally through fabrication, characterization, and optimization of colon-targeting coated capsules containing ITZ-loaded zein nanoparticles (ZNPs). Herein, antisolvent precipitation technique was used for preparation of ITZ-loaded ZNPs. Central composite face-centered design (CCFD) was utilized to determine the significant effects of different ratios of zein: drug and aqueous:organic solvents on particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (EE %), and to elicit the optimized formulation. Afterward, the optimized formulation was in vitro and ex vivo evaluated to ensure its efficacy and safety. Then, lyophilization was done, and the lyophilized formulation was loaded into capsules that were coated with a pH-dependent polymer (Eudragit^® S100) to enhance colon targeting. Finally, X-ray examination of the optimized formulation was performed on human volunteers to ensure the ability of the prepared coated capsules to target ITZ to the colon.

Materials

Pure itraconazole (ITZ) powder was kindly granted from DBK Pharma Co. (Cairo, Egypt). Zein (protein from corn, pharmaceutical-grade F4400C, approximate molecular weight: 35 KDa) was a kind gift from Flo Chemical Corporation (Qiagen, Germany). Eudragit^® S100 was a kind gift from Evonik Industries AG. (Darmstadt, Germany). Sodium lauryl sulfate (SLS) was obtained from oxford lab fine chem (Maharashtra, India). Sodium dihydrogen phosphate, disodium hydrogen phosphate, and absolute ethanol were supplied by El-Nasr Pharmaceutical Chemicals Company (Cairo, Egypt). All other chemicals and solvents were of analytical grade and were used without further purification.