TiO2 hybrid nanocomposite as a new oral drug-delivery system

Accepted Manuscript Study on montmorillonite/insulin/TiO2 hybrid nanocomposite as a new oral drug-delivery system

Younes Kamari, Payam Ghiaci, Mehran Ghiaci PII: DOI: Reference:

S0928-4931(16)32058-6 doi: 10.1016/j.msec.2017.02.115 MSC 7449

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

5 November 2016 6 December 2016 21 February 2017

Please cite this article as: Younes Kamari, Payam Ghiaci, Mehran Ghiaci , Study on montmorillonite/insulin/TiO2 hybrid nanocomposite as a new oral drug-delivery system. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.02.115

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ACCEPTED MANUSCRIPT Study on montmorillonite/insulin/TiO2 hybrid nanocomposite as a new oral drug-delivery system Younes Kamaria, Payam Ghiacib, Mehran Ghiacia,* a

Department of Chemistry, Isfahan University of Technology, Isfahan,8415683111,Iran

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Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden

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[email protected]

Abstract

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This study was conducted in two main stages. In the first stage, drug-loaded montmorillonite nanocomposites were prepared by intercalation of insulin into the montmorillonite layers in

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acidic deionized (DI) water. In the second stage, to increase the release of insulin from the prepared nanocomposites they were coated with TiO2, an inorganic porous coating, by using

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titanium (IV) butoxide, as precursor. The prepared nanocomposites were characterized by FT-IR, XRD, FE-SEM, BET, DLS and Zeta potential analysis. After investigating the release behaviour

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of the nanocomposites by UV-Vis absorbance technique, the results revealed that incorporation of porous TiO2 coating increased the drug entrapment noticeably, and decreased the amount of

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drug release, so that nanocomposites without and with TiO2 coating released the drug after 60 min and 22 h in pH 7.4, respectively. These results could be used in converting the insulin

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utilization from injection to oral.

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Keywords: Montmorillonite, Insulin, hybrid nanocomposite, Intercalation, TiO2, Oral drug delivery.

ACCEPTED MANUSCRIPT 1. Introduction Diabetes is a high prevalence and one of the most severe and lethal diseases in the world. The international diabetic federation reported that 366 million people were affected by diabetes in 2011 and estimated that by 2030 this number will raise up to 552 million [1]. Insulin (Ins) is commonly used to treat diabetes in order to give patients a better life condition. As a protein drug

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used to treat diabetes, insulin has been conventionally administered via subcutaneous injection

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[2]. This type of administration have many disadvantages such as the inconvenience of multiple injections, occasional hypoglycemia due to insulin overdose, local tissue necrosis, microbial

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contamination, and most importantly, poor patient compliance with injections [3-5]. To resolve these problems, in recent years scientists and researchers have replaced the other methods [3, 6-

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10]. Among the proposed methods, the oral administration is considered the most convenient alternative to deliver insulin [11], but it faces important challenges. The low stability of insulin

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in the gastrointestinal tract and its low permeability across biological membranes in the intestine, are drawbacks to overcome [2]. Therefore, the encapsulation or intercalation of insulin into

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suitable matrices is considered as a good strategy to improve insulin oral bioavailability [12]. The disadvantage of using organic matrices in oral insulin delivery such as polymer-based

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nanocarriers and nanoparticles is degradation of the matrix and then releasing insulin in acidic and enzymatic medium of gastrointestinal tract [13-15]. This problem could be obviated by

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utilizing the inorganic carriers, because these carriers could be more stable in the gastrointestinal tract, compared to organic matrices.

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Layered silicate clays are particularly interesting because of their geometric platelet shapes and natural abundance. Among them, montmorillonite (Mt) is well known due to its large surface

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area and a high cation exchange capacity [16]. Montmorillonite has layered structure with tunable interlayer distance that can be used for intercalation of a variety of drugs [17-19]. Montmorillonite has hydrophilic and hydrophobic regions that could interact with different functional groups of the insulin. Moreover, montmorillonite is low cost, nontoxic and stable in the low pH environment of the stomach, so it could be a good carrier for intercalation of insulin. In our previous works, we introduced a new technique using a simple and cost-effective procedure in drug delivery systems [20, 21]. In the present study, to develop this technique we used montmorillonite as carrier for controlled release of insulin. After preparation of Mt/Ins

ACCEPTED MANUSCRIPT nanocomposites, they were coated by different amounts of titanium dioxide and in this way Mt/Ins/TiO2 hybrid nanocomposites were prepared (Scheme 1). In vitro drug release of nanocomposites were studied by UV–Vis absorbance in pH 7.4 and the results showed that Mt/Ins nanocomposites released the whole insulin after 60 min, but the coated nanocomposites released about 70% after 10 h.

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Scheme 1

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2. Experimental

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2.1. Materials

Insulin (regular, human insulin, 1 Vial of 10 mL, 100 units per mL) was obtained from Exir

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pharmaceutical Co. of Iran. Hydrochloridric acid (HCl, 37%), n-hexane, Na2HPO4, KH2PO4 and H3PO4 were obtained from Merck. Sodium montmorillonite (with cation-exchange capacity of

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92 meq/100 g) and titanium (IV) tetra-n-butoxide (97%) were supplied from Sigma-Aldrich. All of reagents were used without any further purification. Human breast cancer cell line (MCF-7

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cells) and human normal kidney cell line (Hek293T cells) were supplied from pasture

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institute (Tehran, Iran). 2.2. Characterization techniques

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FT-IR spectrophotometer (Jasco 680plus) in the range of 400–4000 cm−1 was used to record the IR spectra. A diffractometer with Cu anode (Philips X’pert, PW3040, Netherland), scanning

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from 2° to 20° at 3°/min, was used for XRD analyses. The content of drug in buffer solution was quantified using UV-Vis absorbance (UV-2100, UNICO Instrument Corp.). The surface

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morphology of samples were observed by FESEM (TESCAN MIRA3) using an acceleration voltage of 15.0 kV. Adsorption-desorption isotherm of the selected nanocomposite was measured by NOVAWin2, version 2.2 (Quantachrome instruments). Surface area and mesoporosity of the nanocomposites were measured by the BET equation and BJH method, respectively [22, 23]. The zeta potential and size distribution of samples were determined by Zeta sizer (Ver 6.00, Malvern Instrument Ltd., UK). 2.3. Preparation of Mt/Ins nanocomposites

ACCEPTED MANUSCRIPT For this purpose, initially 0.1 g montmorillonite was added to 50 mL deionized water and it was stirred for 2 h to be dispersed completely. Then, a calculated amount of insulin (10, 30, 50 wt% relative to the Mt) was added to the prepared suspension at pH 2 under vigorous stirring. Because insulin is difficult to dissolve in water, we decreased the pH of the solution with a diluted acidic solution until it reaches pH = 2. Also, at pH below 5.4, the charge of the hormone is positive and because Mt is a cation exchanger, the pH in which the intercalation

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takes place has a profound effect on the intercalation process, thus all the intercalation was

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performed without changing the pH (∼2). After 3 days the products were filtered and rinsed

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several time with deionized water and dried under vacuum at 25 °C overnight to obtain montmorillonite/insulin (Mt/Ins) nanocomposites. The results of XRD analysis confirmed the

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intercalation of insulin between the montmorillonite layers. The information of prepared nanocomposites is summarized in Table 1.

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Table 1

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2.4. Preparation of Mt/Ins/TiO2 hybrid nanocomposites

In the first step, 0.1 g of the nanocomposite (Mt/Ins wt% = 50) was dispersed in 50 mL of

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dry n-hexane under vigorous stirring at 25 °C. Then given quantity of titanium tetra butoxide (30, 50, 100 wt%) dissolved in 5 mL of n-hexane and added dropwise to the mixture. After 1 h of

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stirring, to hydrolyse the titanium tetra butoxide the required amount of deionized water was added to the suspension. After 24 h, the resulting product was filtered by filter paper and washed

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few time with water and dried at room temperature. The information of synthesized hybrid nanocomposites is summarized in Table 2.

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Table 2

2.5. Drug release studies In order to simulate the biological condition of body, the drug release of nanocomposites was investigated at the physiological temperature of 37 °C and pH 7.4. The content of Ins in buffer solution was quantified using UV-Vis absorbance (UV-2100, UNICO Instrument Corp.) at λmax = 280 nm. For establishing environments similar to the gastric juice, intestine and blood, we used the buffer solutions with pH values of 1.2, 5.3 and 7.4, respectively. These buffer solutions were prepared according to the same procedure in our previous works [20, 21].

ACCEPTED MANUSCRIPT 2.6. Kinetic and mechanism of drug release In this section, the prepared hybrid nanocomposites were studied kinetically using various important mathematical models to investigate the in vitro drug release behavior [24]. The Korsmeyer–Peppas model has been described for different dissolution processes as the

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equation(1):

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(1)

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In this equation, Mt/M∞ is a fraction of drug released at time t, k is the release rate constant and n is the release exponent.

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2.7. Cytotoxicity and cell viability assays

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The in vitro cytotoxicity of montmorillonite/insulin/TiO2 hybrid nanocomposite (B2) was investigated with MMT assay. For this purpose, MCF-7 and Hek293T cells were cultured in complete RPMI-1640 (Gibco, Scotland) medium supplemented with 10% fetal

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bovine serum and penicillin/streptomycin (100 units/mL). 200 μL of cell suspension (at

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concentration of 4×104cells/well) was seeded into a 96-well U-bottom microplate (Nunc, Denmark) and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO 2. Then,

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serial dilutions of B2 (20 μL; final concentration: 0.5, 1, 2 and 4 μM) were added and incubated for another 48 h. In the last step, to evaluate the cell viability, 20 μL of MTT

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solution (0.5% w/v) was added followed by incubation for 4 h. Then, the media was replaced with 200 μL of DMSO and the plate shaken for 20 min at 37 ºC. Absorbance was

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quantified at 540 nm by an ELISA plate reader (Awareness, USA). Each experiment included six wells for each condition and results expressed as mean ± standard deviation.

3. Results and discussion 3.1. FTIR spectroscopy analysis Fig. 1 represents the FTIR spectra of Mt, Ins, Ins/Mt nanocomposites and Mt/Ins/TiO2 hybrid nanocomposites. As can be seen, in the spectrum of Mt, the broad band around 3400 cm-1 is attributed to the –OH stretching vibrations of adsorbed water. Moreover, the peaks at 3620 cm-1

ACCEPTED MANUSCRIPT and 3698 cm-1 are assigned to the –OH stretching vibration in the Al–OH and Si–OH, respectively. The overlaid peak at 1640 cm−1 is assigned to –OH bending vibration of silicate and adsorbed water. The appeared peaks at 1116 and 1035 cm−1 are attributed to Si–O stretching and Si–O–Si bending vibrations relative to the layered silicates, respectively. Also, the Al–Al–OH, and Al–Mg–OH bending vibrations were appeared at 915, 874, and 836 cm−1, respectively [17, 25]. In the spectrum of insulin, the characteristics peaks at 3375 attributed to the stretching

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vibrations of OH and NH bonds. Also, stretching vibrations of aliphatic C−H bonds were

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observed in the region of 2880–3000 cm-1. The absorption peaks at the 1658, 1543 cm-1 are

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ascribed to amide I and II bands in the structure of insulin. The existence of characteristic amide bands of insulin at the ∼1500 cm-1 region in the Mt/Ins nanocomposites (A1-A3) spectra can

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confirm the presence of insulin in the composites [8]. Furthermore, in these spectra there were not prominent shifts of the IR bands of amide I and II to the other frequencies that probably couls

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state that there was no significant denaturation of the insulin after preparation of nanocomposites. The FTIR spectra of Mt/Ins/TiO2 hybrid nanocomposites are shown in Fig. 1b. According to the observed characteristic peaks for TiO2, the broad peak appearing at 450–600

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cm−1 in these spectra can be attributed to the vibration of the Ti−O−Ti bonds [20]. As can be

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seen, by increasing the amount of TiO2 from B1 to B3, the intensity of this broad peak is also

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increasing that is in agreement with the XRD patterns. Fig. 1

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3.2. X-ray diffraction patterns

XRD patterns of Mt, Mt/Ins nanocomposites (A1-A3) and the chosen Mt/Ins/TiO2 hybrid

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nanocomposite (50%, 50%) (B2), i.e., the composite with highest loading and a reasonable release rate, are shown in Fig. 2. In the XRD pattern of Mt, a characteristic diffraction peak at 2θ = 7.4° indicated a basal spacing of 11.8 Å [17, 26]. As a rule, this distance should increase by intercalation of insulin between the layers of Mt that would be appeared in the XRD analysis. According to the XRD patterns of the intercalated nanocomposites (A1 to A3), it can be found that by increasing the wt% of insulin the intensity of characteristic diffraction peak of Mt was decreased gradually and shifted to lower angles that means increasing in interlayer distance of Mt layers (12.3 to 14.5 Å from A1 to A3). Also, it is obvious that a new phase has formed in these patterns at 2θ = 2.3-2.9°. These appeared peaks correspond to interlayer distance of about

ACCEPTED MANUSCRIPT 29.6, 30.4, and 31.6 Å for the A1, A2 and A3, respectively. Since the minimum interlayer distance of Mt for intercalation of insulin is 27.9 Å, one could imagine that the obtained distance in Mt layers would be enough space for intercalation of insulin [8]. XRPD pattern of Mt/Ins/TiO2 hybrid nanocomposite (B2) in comparison with A3 probably shows a little shrinkage in the Mt layers as a result of formation an amorphous TiO2 around the Mt/Ins nanocomposite.

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Fig. 2

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3.3. Zeta potential and DLS measurements

Usually to determine the stability of a colloidal suspension, the zeta potential analysis

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could be used. In this regard, zeta potential of the Mt/Ins/TiO2 hybrid nanocomposite (B2) was measured in deionized water at room temperature. As can be seen in the Fig. 3a, the zeta

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potential of B2 was obtained -54.5 mV with area of 100%. This amount of zeta potential is greater than ±30 mV and it can be concluded that prepared hybrid nanocomposite was stable in water [27]. In the other words, B2 does not tend to coagulate in the water and so it can be utilized

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as a good carrier in drug delivery systems [28, 29]. Moreover, to determine the size

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distribution of Mt/Ins nanocomposite (A3) and Mt/Ins/TiO2 hybrid nanocomposite (B2), we applied the dynamic light scattering (DLS) analysis with experimental conditions similar to

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the zeta potential analysis. As can be seen in the Fig.3b the average particle size of B2

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sample was 46.4 nm and for A3 sample was 32.7 nm (unpublished data). Fig. 3

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3.4. FE-SEM images

Fig. 4 demonstrates the morphology of the uncoated Mt/Ins nanocomposite (A3), and that of the hybrid nanocomposite (B2), i.e., Mt/Ins/TiO2. The images of A3 (Fig. 4a,b) clearly illustrate the sheets of Mt [30]. After coating this nanocomposite with a porous TiO2, the morphology of the sample has changed in a way that one could imagine that the montmorillonite sheets were decorated with TiO2. In other words, the morphology of the B2 sample presents clearly a uniform coating of a mesoporous TiO2 around the surface of Mt/Ins nanocomposite (Fig. 4c,d) [31]. It could be expected that by formation a porous shell of TiO2 around Mt/Ins nanocomposite, the diffusion rate of the insulin through the pores would be reduced.

ACCEPTED MANUSCRIPT Fig. 4 3.5. Nitrogen adsorption–desorption isotherms Figs. 5a and 5b represented the nitrogen adsorption–desorption isotherm and the pore size distribution of B2, respectively. By comparing the obtained isotherm and IUPAC definitions, it can be concluded that the selected hybrid composite, i.e., Mt/Ins/TiO2 (50%, 50%) (B2), depicts

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a type II adsorption–desorption isotherm and has a specific surface area of 146.2 m2/g [32]. Also, this composite exhibits a H3 hysteresis loop which contributed to the materials comprised of

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aggregate of plate-like particles forming slit shape pores [33]. As can be observed in the size distribution diagram of B2 (Fig 5b), it is adapted by a bi-modal distribution with a fraction of

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small pores (centred on 2.53 nm) and a fraction of bigger pores almost between 3-20 nm.

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Therefore, the prepared hybrid composite could be classified as a mesoporous solid [34]. 3.6. Drug release studies

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In this section, the insulin release tests were studied by suspending the prepared nanocomposites in different buffer solutions at 37 °C. The release was monitored with UV-Vis

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spectrophotometry by observing the change in absorbance of the characteristics band of insulin at λmax= 280 nm. All experiments were done in triplicate and the results were averaged. Initially,

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Mt/Ins nanocomposites with 10, 30 and 50 wt% of insulin (A1-A3) were tested for controlled drug-release. Fig. 6a depicts the release profiles of insulin from the Mt/Ins nanocomposites in pH

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7.4 over 60 min. As can be seen, almost all of the insulin released in 60 min from all of the composites. According to the obtained results, clearly one could say that montmorillonite

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alone should not be used a carrier for controlled release of insulin because the obtained release time for it is double greater than the obtained time for zirconium phosphate in the work of Díaz et al [8]. Nevertheless, as we demonstrated in our previous studies the drug release time of composites can be increased by formation of a porous inorganic coating like TiO2 around them. Among the uncoated nanocomposites, the sample containing 50 wt% of Ins (A3) had the most regular release profile with highest correlation coefficient, and was selected as the optimum formulation for coating. Therefore, in the second step the best nanocomposite (A3) was coated by 50 wt% of TiO2 and in this way the Mt/Ins/TiO2 hybrid nanocomposite (50%, 50%) (B2) was prepared, according to the method mentioned in section 2.4. It was expected that the coated

ACCEPTED MANUSCRIPT nanocomposite should release the insulin in a longer period of time, compared to uncoated nanocomposite. The release behavior of new nanocomposite was studied in pH 7.4 and the release profile was plotted in Fig. 6b. Fortunately, the results were as expected and the coated hybrid nanocomposite (B2) released about 65% of insulin over of 10 h (Fig. 6b) and whole of insulin was released after 22 h (data not shown). To interpret this difference in the microscopic scale, one could imagine that TiO2 coating remains strongly on the Mt/Ins nanocomposite

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probably through the formation of covalent bonds between the Mt/Ins matrix with TiO2 (during

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the hydrolysis of titanium tetra butoxide) and also due to strong hydrogen bonding interactions

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between Mt/Ins nanocomposite with OH groups on the surface of TiO2.

In addition, to investigate the effect of amount of TiO2 on the release time, the selected

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nanocomposite (A3) was coated with different amounts of TiO2. Hence, other Mt/Ins/TiO2 hybrid nanocomposites with different amounts of TiO2 were prepared (B1 and B3) (Table 2).

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The drug release profiles of the B1 and B3 were added to the Fig. 6b for better comparison. As is clear, the amount of released insulin for B1 with 30 wt% of TiO2 coating was higher than that for

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the B2 sample (with 50 wt% TiO2) while the performance of B3 (100 wt% TiO2) was about similar to B2. According to the obtained results, the hybrid nanocomposite Mt/Ins/TiO2 (50%,

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50%) (B2) was selected as the optimum composite. In the last step, to study the effect of pH, the release behaviour of B2 was tested in different

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buffers with pH 1.2, 5.3 and 7.4 (Fig. 6c). The obtained results revealed that the release of insulin from B2 is pH sensitive. As can be seen, in pH 1.2 (simulated gastric juice) the release

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rate of insulin was much faster than neutral conditions, but in pH 5.3 release was slightly lower than pH 7.4. This difference can be interpreted by reasons such as; faster exchange of insulin

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with H+ in pH 1.2 (Mt is a cation exchanger in lower pH) and also hydrolysis of TiO 2 in acidic conditions which leading to faster release of insulin from B2. However, as can be seen in the Fig. 6c, release of insulin in the simulated gastrointestinal juices with pHs of 1.2 and 5.3 was continued for 6 h and >10 h, respectively. It can be said, by using this method insulin could be protected from enzymatic digestion, thus the method is recommended for oral insulin application. In this research, we showed that using the hybrid of a carrier and a porous inorganic coating can be applied as a controlled-drug delivery system. This study has advantages compared to the previous works such as better performance of montmorillonite as a carrier for protection of

ACCEPTED MANUSCRIPT insulin compared to zirconium phosphate [8] and also increasing shelf-life of insulin and time of release to 22 h by incorporation of TiO2, compared to the other works [8, 12]. Generally, the satisfactory results of this research and our previous studies could approve that introduced method is applicable for various drugs and carriers in drug-delivery systems. By using the various important mathematical models like zero order, first order, Higuchi and Korsmeyer–Peppas, the in vitro drug release behaviour of B2 were investigated kinetically.

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According to the obtained R2 values of the curves for various mathematical models, it was shown

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that there was a very good fitting between experimental data and the Korsmeyer–Peppas model.

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Fig. 6 3.7. Cell viability assays

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The cytotoxicity of Mt/Ins/TiO2 hybrid nanocomposite (B2) toward MCF-7 and Hek293T cells was explored at different concentrations of B2 after 48 h incubation time

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and obtained data are given in Fig. 7. When MCF-7 cells were treated with B2, they generally showed lower viability compared to Hek293T cells. As can be seen, different

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concentrations of B3 (0.5-4 μM) reduced viability of MCF-7 and Hek293T cells down to 76 and 90%, respectively. As a whole, the Mt/Ins/TiO2 hybrid nanocomposite showed very low

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4. Conclusions

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as a cancerous cell line.

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cytotoxic effect on Hek293T as a normal cell and relatively low cytotoxic effects on MCF-7

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successfully

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nanocomposites

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montmorillonite/insulin/TiO2 with pharmaceutically approved, biocompatible and non-polluting materials and also by using a simple and cost-effective procedure. Intercalation of insulin into Mt and then incorporation of TiO2 as a porous inorganic coating on the composite, could prevent the insulin from digestive degradation, and increased drug passage time through its matrix into intended media. Results of in vitro drug release are very promising and the samples have drug release even after 22 h. In addition, due to increasing the shelf life of hormone, this type of

ACCEPTED MANUSCRIPT encapsulation could alert the insulin administration from injection to oral and presents a painless and more comfortable treatment for diabetics. Acknowledgments Thanks are due to the Research Council of Isfahan University of Technology for supporting

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of this work.

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[23] E.P. Barrett, L.G. Joyner, P.P. Halenda, The determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms, J. Am. Chem. Soc. 73 (1951) 373-380. [24] S. Dash, P.N. Murthy, L. Nath, P. Chowdhury, Kinetic modeling on drug release from controlled drug delivery systems, Acta Pol Pharm 67 (2010) 217-223. [25] J. Zheng, L. Luan, H. Wang, L. Xi, K. Yao, Study on ibuprofen/montmorillonite intercalation composites as drug release system, Appl. Clay Sci. 36 (2007) 297-301. [26] Q. Wu, Z. Li, H. Hong, K. Yin, L. Tie, Adsorption and intercalation of ciprofloxacin on montmorillonite, Appl. Clay Sci. 50 (2010) 204-211. [27] R. Greenwood, Review of the measurement of zeta potentials in concentrated aqueous suspensions using electroacoustics, Adv. Colloid Interface Sci. 106 (2003) 55-81. [28] R. Greenwood, K. Kendall, Selection of suitable dispersants for aqueous suspensions of zirconia and titania powders using acoustophoresis, J. Eur. Ceram. Soc. 19 (1999) 479-488. [29] K. Ofokansi, G. Winter, G. Fricker, C. Coester, Matrix-loaded biodegradable gelatin nanoparticles as new approach to improve drug loading and delivery, Eur. J. Pharm. Biopharm. 76 (2010) 1-9. [30] N. Sarier, E. Onder, S. Ersoy, The modification of Na-montmorillonite by salts of fatty acids: An easy intercalation process, Colloids Surfaces A Physicochem. Eng. Asp. 371 (2010) 40-49. [31] Y. Kamari, M. Ghiaci, Incorporation of TiO2 coating on a palladium heterogeneous nanocatalyst. A new method to improve reusability of a catalyst, Catal. Commun. 84 (2016) 16-20. [32] G. Leofanti, M. Padovan, G. Tozzola, B. Venturelli, Surface area and pore texture of catalysts, Catal. Today 41 (1998) 207-219. [33] M. Kruk, M. Jaroniec, Gas adsorption characterization of ordered organic-inorganic nanocomposite materials, Chem. Mater. 13 (2001) 3169-3183. [34] L. Albarran, T. López, P. Quintana, V. Chagoya, Controlled release of IFC-305 encapsulated in silica nanoparticles for liver cancer synthesized by sol–gel, Colloids Surfaces A Physicochem. Eng. Asp. 384 (2011) 131-136.

ACCEPTED MANUSCRIPT Figures Legend Scheme 1. Overall schematic of Mt/Ins/TiO2 hybrid nanocomposite as a new oral drug delivery system. Fig. 1. FT-IR spectra of (a) Mt, Ins, A1, A2, and A3 nanocomposites; (b) TiO2, B1, B2, and B3

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Fig. 2. XRD patterns of Mt, A1, A2, A3 and B2 nanocomposites.

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nanocomposites.

Fig. 4. FESEM images of A3 (a,b) and B2 (c,d).

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Fig. 3. Zeta potential (a) and size distribution (b) of B2 nanocomposite.

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Fig. 5. Nitrogen adsorption–desorption isotherms (a) and Pore size distributions (b) of

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B2.

Fig. 6. Cumulative Insulin release (%) of A1-A3 at pH 7.4 (a); B1-B3 at pH 7.4 (b) and

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B2 in different pHs (c).

Fig. 7. Cell viability of (a) MCF-7 and Hek293T cells exposed to different concentrations of B2

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after 48 h incubation.

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Scheme 1.

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Fig. 1.

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Fig. 2.

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Fig. 3.

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Fig. 4.

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Fig. 5.

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Fig. 6.

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Fig. 7.

ACCEPTED MANUSCRIPT Table Legend Table 1- Feed composition and drug content of Mt/Ins nanocomposites.

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Table 2- Different amounts of TiO2 coated on Mt/Ins nanocomposites.

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Table 1 Abbreviation

Mt (g)

Ins (g)

Mt/Ins (10%)

A1

0.1

0.01

Mt/Ins (30%)

A2

0.1

0.03

Mt/Ins (50%)

A3

0.1

0.05

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Entry

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Table 2 Entry

Abbreviation

Mt/Ins(g)

TiO2 (g)

B1

0.1

0.03

Mt/Ins/TiO2 (50%, 50%)

B2

0.1

0.05

Mt/Ins/TiO2 (50%, 100%)

B3

0.1

0.1

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Mt/Ins/TiO2 (50%, 30%)

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Graphical abstract

ACCEPTED MANUSCRIPT Research Highlights 

Montmorillonite/Insulin nanocomposites were coated with different amounts of TiO2.



Incorporation of TiO2 enhanced the drug entrapment, and reduced the drug release.

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 This method could be used in altering the insulin utilization from injection to oral.