**1. Introduction**

Diabetic mellitus (DM) is a metabolic disorder categorized by hyperglycemia due to inopportune insulin secretion. DM is differentiated as type I (insulin-reliant) and type II

**Citation:** Jermy, B.R.; Salahuddin, M.; Tanimu, G.; Dafalla, H.; Almofty, S.; Ravinayagam, V. Design and Evaluation of Pegylated Large 3D Pore Ferrisilicate as a Potential Insulin Protein Therapy to Treat Diabetic Mellitus. *Pharmaceutics* **2023**, *15*, 593. https://doi.org/10.3390/ pharmaceutics15020593

Academic Editors: Koyo Nishida and Tomáš Etrych

Received: 22 October 2022 Revised: 27 January 2023 Accepted: 1 February 2023 Published: 9 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

diabetes (insulin resistance) [1,2]. Diabetic mellitus, a disease termed as a life style disease, is quickly turning into a global epidemic. The prime reason for this is attributed due to change in life style, unhealthy diets and lack of awareness. In 2019, diabetic severity resulted in 1.5 million mortalities, and notably, 48% of these deaths occurred before 70 years of age. Among the two types of diabetes, type II diabetes is dominant, accounting for 95% (WHO). Recent data show that worldwide, about 537 million people have diabetes, and this number is expected to reach 783 million by 2045 [3]. Such a high percentage and increasing rate of DM is primarily attributed to obesity and changes in life style. The healthcare expenditure spent on diabetic treatment was estimated to be USD 966 billion in 2021.

Insulin was discovered by Frederick Banting in 1921, while Charles Best developed the clinical use of insulin in 1922 [4]. Insulin is administered to control the blood glucose level. Insulin helps to uptake glucose by binding with the insulin receptor and intitiating several protein activations cascades (e.g., Glut-4 transporter to plasma member, influx of glucose, synthesis of glycogen, glycolysis and triglyceride production). Type 1 DM (due to defective pancreatic β cells) depends on the lifelong supply of insulin. In the case of type 2 DM patients, the peripheral cells resist the administration of insulin, while some patients at the lateral life stage also require insulin. In order to treat type 1 DM, a common mode of insulin administration is through the subcutaneous route with about four injections per day. The treatment affects patient compliance and induces several side effects (lipoatrophy). Still, the subcutaneous administration route is preferred due to insulin's low bioavailability and challenges in developing an effective drug delivery system due to low membrane permeability and molecular size constraints.

In order to limit the number of injection cycles, a controlled insulin release strategy was followed using zinc and protamine. The used formulation showed poor reproducible kinetic parameters and the effect between meal periods was minimal and led to hypoglycemic events [5]. Accordingly, similar to insulin, insulin detemir and insulin glargine were shown to subvert the hyproglycemic action, but the required dose level was almost double compared to the normal dose of insulin for one day [6]. It has been reported that diabetic mediators similar to insulin could also alter the mitogenic pathway and can be a potential carcinogen in the long run [7]. The construction of a stimuli-responsive smart drug release system is the most recent attractive research direction involving interdisciplinary research between material science and medical science. An appropriately constructed nanovehicle with controlled insulin delivery using biocompatible nanosilica is proposed to overcome the deficiencies in subcutaneous therapy, improve therapeutic efficiency, enhance the stability of drug release and make ease diet control and exercise regiments. Several glucosesensitive smart drug delivery system based on phenylboronic acid (PBA) and proteins such as concanavalin and glucose oxidase have been reported [8]. However, such a glucosesensitive, sensor-based nanovehicle requires a multi-step synthesis procedure, the use of solvents and an advanced chemical set-up.

Recently, a biocompatible drug delivery system based on structured silica/polymeric nanocomposites are shown to be a promising nanovehicle to carry insulin [9]. A microneedle design based on mesoporous silica capped with zinc oxide in the form of an insulin reservoir has been reported to effectively control insulin delivery for prolonged periods of time [10]. Several studies are ongoing to improve the efficacy of protein delivery using mesoporous silica/chitosan and poly(lactic-co-glycolic) acid nanoformulations and improve their permeability [11]. The isomorphous substitution of biocompatible metals such as Fe, Zn, Ti, etc., into the silica framework is gaining importance in biomedical applications [12,13]. The use of Fe cations (Fe3+ and Fe2+) with particle sizes ranging between 3 and 15 nm is gaining attention in multifunctional therapeutics as contrasting agents for magnetic resonance imaging, in hyperthermia treatments and as drug delivery agents [14]. The presence of iron oxide nanoparticles (FeNPs) favors biocompatibility [15], and as such, they are applied in hyperthermia for their anticancer [16,17] and antibacterial activity [18], as well as in tissue engineering [19]. Previously, we reported a direct hydrothermal synthesis of Iron-incorporated Santa Barbara Amorphous 16 (FeSBA-16) [20]. The presence of large

3D cubic pores of ferrisilicate could be exploited for insulin entrapment/loading capacity and insulin release. The wrapping of the nanocarrier with polyethylene glycol is reported to improve the bioavailability and drug stabilization and facilitate the transport of protein across human gastrointestinal fluid [21]. In this study, we investigated the effect of a pegylated, large 3D porous ferrisilicate/insulin nanoformulation for diabetes management. The textural characteristics are investigated using different physico-chemical characterization techniques. The insulin encapsulation/loading capacity and the pH-based, smart kinetic release behavior in response to stimuli were studied for insulin release. Furthermore, the nanoformulation toxicity in vitro and hypoglycemic effect in vivo were assessed.

### **2. Material and Methods**

The silica source tetraethylorthosilicate (reagent grade, 98%, Sigma Aldrich, Darmstadt, Germany) and non-ionic template Pluronic F127 (BioReagent, suitable for cell culture, BASF, Wyandotte, MI, USA), iron(III) nitrate nonahydrate (≥99.95%, BioReagent, suitable for cell culture, Sigma Aldrich, Saint Louis, MO, USA), n-butanol (≥99%, anhydrous, Sigma Aldrich, Saint Louis, MO, USA), human recombinant insulin (rHu, dry powder, Sigma-Aldrich Chemie Holding GmbH, Taufkirchen, Germany) and polyethylene glycol (BioUltra, MW = 400 Daltons, Sigma-Aldrich Chemie Holding GmbH, Taufkirchen, Germany) were obtained from Sigma Aldrich. All chemicals were used as received without any further purification.

### *2.1. Synthesis*

2.1.1. Synthesis of Ferrisilicate Using Hydrothermal Technique

Fe-SBA-16, termed as ferrisilicate, was prepared using sol–gel technique. The ferrisilicate containing Fe species can be tuned between SiO2/Fe2O3 ratios of 50 and 250. In the present study, the Fe content can reach a SiO2/Fe2O3 ratio of 50. In brief, 5 g of F127 was dissolved in acidic HCl solution (2 M) and stirred for 1 h. Then, 16 g of n-butanol (cosolvent) was added along with 24 g of tetraethylorthosilicate and the iron source (0.186 g of iron nitrate nonahydrate (Si/Fe ratio 250)) and stirred for 24 h. The mixture was stored in a polypropylene bottle (Nalgene, Rochester, NY, USA, Thermo Fisher Scientific, Bohemia, NY, USA) and transferred to an oven to be hydrothermally aged at 100 ◦C for 24 h. The precipitate was filtered, washed several times with excess water and dried at 100 ◦C for 12 h. The as-synthesized sample was finally calcined at 550 ◦C for 6 h.

2.1.2. Synthesis of Iron-Impregnated Structured Silica (10 wt% Fe/KIT-6 and 10 wt% Fe/MSU-F) Using Impregnation Technique

Firstly, 0.7235 g of iron nitrate nonahydrate was dissolved in 80 mL of distilled water. Then, 1.0 g of KIT-6, mesosilicalite, or Mesocellular Silica Foam (MSU-F) was added and stirred for 24 h at room temperature (RT). The solution was dried at 120 ◦C for 3 h and calcined at 500 ◦C for 2 h.

### 2.1.3. Insulin/Ferrisilicate

For insulin loading, 80 mg of insulin was added to 8 mL of 0.01 M HCl solution and stirred for 20 min. Then, 160 mg of ferrisilicate was added and stirred at 300 rpm overnight in an ice-cold environment. After that, the mixture was filtered, washed with 5 mL of distilled water and dried at RT (5 h) and stored at 4 ◦C.

### 2.1.4. Insulin/Ferrisilicate/PEGylation

For PEGylation, 14 μL of PEG (Molecular weight = 400) was added in 3 mL of deionized water, stirred for 20 min under argon atmosphere and then 150 mg of Insulin/Ferrisilicate was added and stirred under an ice-cold environment for 24 h. Then, the mixture was freeze-dried using the lyophilization technique.

### *2.2. Characterization Techniques*

The phase of insulin, insulin/ferrisilicate/PEG, was identified using benchtop XRD (Miniflex 600, Rigaku, Tokyo, Japan). The textural features, including BET surface area, pore size and pore volume, were measured using the nitrogen adsorption technique (ASAP-2020 plus, Micromeritics, Norcross, GA, USA). The ferrite nanoparticles' chemical coordination was analyzed using DRS-UV-visible spectroscopy analysis (V-750, JASCO, Tokyo, Japan). The insulin functional groups of our nanoformulation were determined using FT-IR spectroscopy (L160000A, Perkin Elmer, Waltham, MA, USA). The morphological variations of insulin/ferrisilicate/PEG were investigated using transmission electron microscopy (TEM, JEM2100F, JEOL, Tokyo, Japan).
