1. Introduction
Iron (Fe) is a key micronutrient essential for various biochemical functions in the human body. Its deficiency impacts oxygen transportation and enzymatic reactions involved in various metabolic pathways [
1]. Progressive augmentation in iron deficiency would lead to a medical condition called ‘Anemia’ or more specifically ‘Iron Deficiency Anemia (IDA)’ [
2]. Subjects such as pregnant women and children are highly vulnerable groups to IDA. This medical condition is determined based on the percentage of hemoglobin (Hb) present in red blood cells. As per the World Health Organization, adult males are said to be anemic if the Hb value is <13.0 g/dL. Similarly, females and pregnant women are called anemic if the Hb values are <12.0 g/dL and <11.0 g/dL, respectively [
3]. Various strategies exist to counter IDA that include but are not limited to iron supplementation or iron food fortification using various salts of iron [
4]. Clinically, various non-invasive traditional oral dosage forms (tablets, capsules, and liquid preparations) of iron are commonly used as the first line of therapy [
5]. Nevertheless, they are hard to consume for special populations such as pregnant women, pediatric, and geriatric patients with dysphagia, vomiting tendency, bipolar disorder, oral cancer, and Parkinson’s disease [
6,
7,
8,
9]. Moreover, these dosage forms also cause dose-dependent side effects such as gastric upset, diarrhea, nausea, metallic taste [
10], and changes in the gut microbiome [
11,
12,
13]. At the same time, the parenteral administration of iron is associated with various disadvantages. For instance, intramuscular administration leads to local reactions such as pain, skin staining (brown), atrophy, necrosis, and immediate reaction like anaphylactic shock. Similarly, intravenous administration of iron may lead to phlebitis and various systemic reactions such as fever, dizziness, myalgia, arthralgia, and vomiting [
14]. Nonetheless, in very severe IDA (Hb < 8.0 g/dL), parenteral iron therapy is preferred due to its superior rapidity and efficacy [
15].
The above-mentioned limitations in general and the difficulties of the special population, in particular, highlight the need for a novel drug delivery system. Accordingly, the pharmaceutical and clinical research industries have witnessed a transition in oral drug delivery from traditional dosage forms (tablets or injections) to a new non-invasive concept of rapidly dispersing orodispersible films (ODFs) [
16,
17]. ODFs are ultra-thin, portable, and postage-stamp-sized dosage forms that ‘rapidly disperse when administered to the tongue and swallowed naturally along with the saliva to get absorbed into the systemic circulation via the gastrointestinal tract’ [
17]. Most importantly, compared to traditional dosage forms, ODFs do not need water for administration and are easy to administer to patients across all age groups [
18]. Thus, they are especially well-positioned to meet the requirements of the special population (pediatrics geriatrics, and pregnant women) suffering from dysphagia, nausea/vomiting, and other conditions or diseases stated above [
6,
7,
9].
Various film-forming polymers are employed in the fabrication of ODFs, such as maltodextrin [
19], polyvinyl alcohol [
20], hydroxypropyl methylcellulose [
21], and pullulan [
22]. These polymers offer tremendous support in maintaining the film’s mechanical properties, disintegration and dissolution, and good quality with an acceptable mouthfeel. Of particular interest, pullulan is a natural, biocompatible, biodegradable, non-ionic, water-soluble material obtained from the black yeast
Aureobasidium pullulan [
23,
24]. Pullulan has been used in film formulations of various drugs and has been found to be a safe and non-toxic material [
25,
26]. Accordingly, in this study pullulan was employed as a polymer in the formulation of i-ODFs.
Generally, the most commonly used source of iron supplements for therapeutic and prophylactic use is ferrous sulfate, which is freely soluble in water [
27,
28]. However, its use is associated with common side effects such as the darkening of teeth, nausea, abdominal pain, and black/ dark stools [
29,
30,
31,
32]. To overcome these issues, compounds such as ferrous succinate, ferrous fumarate, and ferric saccharate have evolved, which are poorly soluble in water but soluble in dilute hydrochloric acid, have less organoleptic issues compared to ferrous sulfate, and readily enter the common iron pool during digestion [
33]. Nevertheless, these salts are not bioavailable and need effective taste masking, since the acceptability of any ODF depends predominantly on its taste. Hence, the taste of the ODF is a crucial factor to gain end-user acceptance and thereby compliance. Therefore, effective strategies to taste-mask the active substance need to be developed to formulate an ideal ODF [
34].
Accordingly, in light of the above rationale and background of iron and pullulan, the objective of the present study was to develop a palatable, rapidly dispersing, non-irritant, and readily bioavailable pullulan-based iron-loaded ODF (i-ODF) for prophylactic use in subjects across all age groups and especially for people with special needs.
2. Materials and Methods
2.1. Materials
Ferric saccharate was obtained from Biodeal Pharmaceuticals (New Delhi, India). The film-forming polymer, pullulan, was purchased from Kumar Organics (Bangalore, India). Sodium alginate, calcium acetate, and all other pharmaceutically acceptable excipients employed in the formulation of the ODFs were purchased from S.D. Fine Chemicals Ltd. (Mumbai, India). All other chemicals and solvents used were of analytical grade.
2.2. Preparation of Microencapsulated Iron Particles
Table 1 provides details of the composition to fabricate microencapsulated or microparticles (MPs) of iron (Fe). The fabrication steps include dissolving sodium alginate and ferric saccharate in 100 mL of distilled water to obtain a viscous mixture. The mixture obtained was added dropwise under controlled magnetic stirring (at a temperature of 25 °C) to a solution of calcium acetate to obtain MPs of Fe [
35,
36]. The obtained particles were separated by filtration under vacuum and resuspended in distilled water to remove soluble salts, if any. This step was repeated to remove all the soluble salts. Finally, the particles were filtered under a vacuum to obtain Fe MPs. Thereafter, the MPs were dried at room temperature and subjected to further characterization studies or stored in a well-closed container with a small amount of talc till further use.
2.3. Characterization of the Iron Microparticles
2.3.1. Optical Microscopy
The size of the Fe MPs was estimated using an optical microscope (Nikon Eclipse, E800, Tokyo, Japan). A small portion of the MPs (aggregate) along with a drop of water was placed on a slide and covered with a coverslip for microscopic examination using an optical microscope. The size was measured using a calibrated and superimposed scale on each image.
2.3.2. Inductively Coupled Plasma Optical Emission Spectroscopy
The amount of calcium and iron in the Fe MPs was estimated using inductively coupled plasma optical emission spectroscopy (ICP-OES) (iCAP Pro, ICP-OES, Thermo Fisher Scientific, Waltham, MA, USA). A small portion (200 mg) of the MPs of Fe was combined with nitric acid and digested in a microwave oven and subjected to ICP-OES analysis.
2.3.3. Scanning Electron Microscopy (SEM) with X-ray Energy Dispersion (EDS)
A high-resolution SEM (Carl Zeiss, Neon Crossbeam, Berlin, Germany) equipped with a spectrophotometer with an EDS (EDAX company, Unterschleissheim, Germany) was employed for the study. A voltage of 5 to 10 kV was employed as an acceleration voltage to obtain SEM images of the Fe MPs. A semi-quantitative detection was performed to detect the elemental composition of Fe MPs using an acceleration voltage of 10 kV.
2.4. Fabrication of Pullulan-Based ODFs Loaded with Fe MPs
A solvent casting method with minor modifications was employed to fabricate iron ODFs.
Table 2 lists the ingredients and quantities employed in the preparation. First, the film-forming polymer, pullulan, was dissolved in water and left overnight to obtain a clear solution. Similarly, lecithin was dissolved separately in a portion of the solvent. Fe MPs and beta-cyclodextrin were mixed in water under continuous stirring followed by the addition of mannitol and sweetening agents. Other ingredients, namely, calcium carboxymethylcellulose, ascorbic acid, and malic acid, were also added under continuous stirring for 15 min. Other excipients, namely, polyethylene glycol, sorbitol, and flavoring agents were then added under continuous stirring. Thereafter, the obtained solution along with the lecithin solution was added to the solution of pullulan under continuous stirring for about 20 min to obtain a homogenous slurry and subjected to deaeration under vacuum (pressure between 600 to 700 mm of Hg) for 2 to 3 h to remove air bubbles, if any. Finally, the solution was cast as a layer using an automatic layering machine with a predetermined thickness. Thereafter, the film is removed carefully and dried at a temperature of 60 °C followed by cutting and packaging in a tri-laminate aluminum pouch. The packed films are stored in a desiccator to prevent moisture or microbial attack and were used for further characterization.
2.5. Characterization of the ODFs
2.5.1. Physical Examination
The fabricated pullulan-based i-ODFs were physically examined with respect to their appearance (using the naked eye), handling property, and texture.
2.5.2. Weight
The ODF samples were cut into a size of 2 × 2 cm2 and weighed on an electronic balance (Mettler-Toledo, Mumbai, India). The average weight was considered as a mean weight variation.
2.5.3. Thickness
The thickness of the film was measured using a screw gauge having an accuracy of 0.001 mm. Measurements were taken from the center and also from the four corners of the film having the size of 2 × 2 cm2. The thickness measured is reported as mean ± SD.
2.5.4. Drug Content
To ensure the drug payload in the film, drug content analysis was done. A premeasured region of the film was dissolved in phosphate buffer (50 mL) by stirring followed by filtration through a filter paper (0.45 µm) and the amount of drug was determined by high-performance liquid chromatography (HPLC) method. The method employed a mobile phase comprising phosphate buffer (pH 2.5) and methanol in a ratio of 970:30 (flow rate: 1.0 mL/min). The detection wavelength was 210 nm and the run time was 15 min.
2.5.5. Disintegration Time
The disintegration time of the ODFs was measured by a Petri dish method [
37], wherein a film of 2 × 2 cm
2 was placed in a Petri dish having 10 mL of phosphate buffer having pH of 6.8 (simulated salivary fluid;
Table S1). The time taken for the ODF to disintegrate completely was measured using a stopwatch and the same experiment was repeated thrice and the mean value is reported.
2.5.6. Folding Endurance
The fabricated ODF was folded repeatedly at a predetermined spot until it broke. The number of times the film can be folded without breaking is taken as the value of folding endurance. The value was measured in triplicate.
2.5.7. Tensile Strength
To measure tensile strength, the ODF having a size of 2 × 2 cm
2 was clamped at one end and the other end was attached to a hanging pan for loading weights. The total weight required for the breakage of the film was estimated. The experiment was performed in triplicate and the tensile strength is reported as an average.
2.5.8. Surface pH
An ODF sample was moistened with distilled water (0.5 mL) and left for 2 min. The pH of the moistened film was measured using a pH meter (IKON Instruments, Delhi, India). In this method, the surface of the pH meter electrode was touched with the moistened surface of the film to measure the pH. Readings were taken in triplicate for each sample and the average value of the readings is reported.
2.5.9. Karl–Fischer Titration (Water Content)
To determine the water content, Karl–Fisher titration was performed using a DL37 coulometric titrator (Mettler-Toledo, Mumbai, India). One ODF sample (2 × 2 cm2) was added to about 5 mL methanol and the titration was continued until it reaches an electrometric endpoint. The water content determination was performed in triplicate corrected for solvent water content. The moisture content was calculated using the formula:
2.5.10. Morphology by Scanning Electron Microscopy (SEM)
The overall surface morphology of ODFs loaded with Fe MPs was evaluated by scanning electron microscopy (SEM) (Carl Zeiss, Neon Crossbeam, Berlin, Germany). The ODF sample (size: 1 mm2) was positioned on a circular aluminum stub and sputter coated with Au/Pd under argon atmosphere using a vacuum evaporator followed by scanning using SEM.
2.5.11. Fourier Transform Infrared Spectroscopy (FTIR)
FTIR spectra (scanned between 4000 and 400 cm
−1; resolution: 4 cm
−1 for 20 scans) of the film-forming material, pullulan, ferric saccharate, iron-loaded microparticles, and the physical mixture for ODFs consisting of the active substance (iron) was carried out. The spectra were obtained by the traditional potassium bromide disc method followed by FTIR analysis (Shimadzu, model 8400, Tokyo, Japan) [
38].
2.5.12. Microbial Load
The ODF samples were tested for microbial burden (aerobic colony count, molds, yeasts, and coliforms) as per ISO 11737-1 for determining the microbial load (population) in a product.
2.6. Dissolution Studies
An in vitro dissolution study of pullulan-based i-ODFs was performed in triplicate using a USP XXXIV paddle apparatus (type II) with 900 mL of 0.1 N HCl as dissolution media at 50 rpm and 37 ± 0.5 °C. Samples (aliquots of 5 mL) were collected periodically and replenished with equivalent volumes of fresh dissolution medium to maintain sink conditions. The collected samples were filtered through a filter paper (0.45 µm) and the amount of drug released was determined by high-performance liquid chromatography (HPLC) as aforementioned.
2.7. Stability Studies
The optimized ODF formulation was subjected to stability studies at a temperature of 25 ± 2 °C and relative humidity of 60% ± 5% for 90 days. The ODFs were packed in a tri-laminate aluminum pouch and kept in a stability chamber. After the stipulated periods of 30, 60, and 90 days, the i-ODF samples were analyzed for their appearance (physical), thickness, drug content, folding endurance, surface pH, disintegration time (seconds), and dissolution profile.
2.8. In Vivo Biocompatibility Study Using Hamster Cheek Pouch Model (Irritation Study)
The mucosal irritation caused, if any, by i-ODF was assessed using a hamster cheek pouch model. This model was employed to study the safety of i-ODFs in the oral cavity. Hamsters of each sex were housed individually and provided with controlled environmental conditions of 12 h light/dark cycle with free access to both food and water. After acclimatizing for seven days, hamsters weighing between 100 to 150 g were selected for the study. The complete experimental procedures were carried out as per The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines. The entire study was reviewed and approved by the Institutional Animal Ethics Committee (IAEC/JSSCPM/349/2020), Mysuru, Karnataka, India.
Study design: Hamsters were divided into two groups; Group I and Group II. Group I was kept on placebo film while Group II was kept on i-ODFs. Respective films were administered (positioned in the hamster’s cheek pouch) for a time period of about 10 min followed by rinsing with distilled water. Thereafter, the pouch was immediately observed for redness (irritation), if any, and also after 24 h period. The placebo and i-ODFs were administered to hamsters twice a day for a time period of 4 to 5 days.