Niclosamide encapsulated in mesoporous silica and geopolymer: A potential oral formulation for COVID-19

Abstract

COVID-19 is a rapidly evolving emergency, for which there have been no specific medication found yet. Therefore, it is necessary to find a solution for this ongoing pandemic with the aid of advanced pharmaceutics. What is proposed as a solution is the repurposing of FDA approved drug such as niclosamide (NIC) having multiple pathways to inactivate the SARS-CoV-2, the specific virion that induces COVID-19. However, NIC is hardly soluble in an aqueous solution, thereby poor bioavailability, resulting in low drug efficacy. To overcome such a disadvantage, we propose here an oral formulation based on Tween 60 coated drug delivery system comprised of three different mesoporous silica biomaterials like MCM-41, SBA-15, and geopolymer encapsulated with NIC molecules. According to the release studies under a gastro/intestinal solution, the cumulative NIC release out of NIC-silica nanohybrids was found to be greatly enhanced to ~97% compared to the solubility of intact NIC (~40%) under the same condition. We also confirmed the therapeutically relevant bioavailability for NIC by performing pharmacokinetic (PK) study in rats with NIC-silica oral formulations. In addition, we discussed in detail how the PK parameters could be altered not only by the engineered porous structure and property, but also by interfacial interactions between ion-NIC dipole, NIC–NIC dipoles and/or pore wall-NIC van der Waals in the intra-pores of silica nanoparticles.

Graphical abstract

1. Introduction

World Health Organization (WHO) declared COVID-19 as global pandemic on March 11, 2020, ever since it started spreading, 13 fold higher than just in China's Wuhan, where it was first reported in 2019 [1]. As of July 30, 2021, around 196 M individuals were affected with 4.19 M deaths, showing the necessity to stop the spread as urgent as possible. Lately, there have been many reports and reviews showing the potency of nano-technology [[2], [3], [4], [5]] and re-purposing old drugs [6] for viral diseases such as MERS, and SARS which have been affecting south-east Asian countries in the early 2000s [6].

According to the latest reports, it was suggested that vaccination alone would not be enough to stop the vigorous transmission of COVID-19 [7]. In this context, therefore, nano-aided therapeutic strategies, either alone or in combination with vaccines, to completely eliminate the pandemic are critical for diseases like COVID-19, but no proper medications have yet discovered. On the other hand, re-purposing clinically relevant and FDA approved drug like niclosamide (NIC) seems practically efficient, because it has already been known for its therapeutic benefits on similar but different strains of pathogenesis such as SARS [8], and MERS [9], etc. NIC, an anthelmintic drug, has shown significantly higher potency on many viral diseases such as Zika Virus (ZIKV) [10], Japanese encephalitis virus (JEV) [11], Chikungunya (CHIKV) [12], Human adenovirus (HAdV) [13], Epstein–Barr Virus (EBV) [14], etc. Since the new SARS-CoV-2 virus showed the similar host interaction for its entry, scientists came up with an idea that a nano-engineered NIC could be much beneficial for tackling the ongoing pandemic.

However, the most challenging thing in developing NIC drug is to significantly improve the in-vivo bioavailability, since NIC is hardly soluble in aqueous solution. Though there were some approaches to enhance the bioavailability by polymer encapsulation [15], and chemical modification etc. [16], they turn out to be not satisfactory. More recently, when NIC was formulated as an amorphous solid dispersion by Miguel et al. (2021), an improved pharmacokinetic (PK) parameters could be obtained in rat model, but the mean plasma concentration was far below 100 ng mL⁻¹ to expect the therapeutic effect [17].

It is, therefore, highly required to find a suitable drug carrier that can efficiently load and release therapeutically relevant doses of NIC in a controlled manner. In the case of COVID-19, the virus can interact with the host cells through ACE-2 (angiotensin converting enzyme factor-2) proteins which are mainly expressed in the liver or GIT [18]. NIC is known to exert anti-viral properties by blocking such viral entry through endocytosis and autophagy. In addition, viral replication can be inhibited by NIC, preventing their further maturation in the host cells [19]. However, NIC can undergo faster metabolism in liver as glucuronic acid-NIC metabolite by cytochrome P450 enzymes [20]. Therefore, it is required to have drug delivery carrier to release encapsulated NIC molecules in a sustained manner to delay the metabolism by glucuronidation in liver and/or intestine in a tricky way.

In this context, ordered mesoporous silicas (PSs) such as MCM-41 (Mobil Composition of Matter No. 41) and SBA-15 (Santa Barbara Amorphous-15) have been suggested as drug delivery carriers owing to their meso porosities with ordered hexagonal structure, large surface area and pore volume, allowing efficient drug adsorption [[21], [22], [23], [24], [25], [26], [27]]. However, both MCM-41 and SBA-15 have neutral framework, thereby one can expect only a weak molecular interaction like van der Waals ones with drug molecules. Therefore, we came up with an idea to develop a zeolitic silica with a charged framework, but with the same mesoporous MCM-41 structure.

In this study, an attempt was made to develop mesoporous geopolymer (PG) with the aluminosilicate network with MCM-41 structure. The present PG with a charged framework was accomplished by dissolving clay (kaolinite) as the aluminosilicate source in an aqueous alkali solution [28]. According to our previous studies, various hexagonal type (p6mm) PGs were successfully prepared from metakaolin, a low-cost Si and Al source [29,30]. Basically, the present PG has MCM-41 structure, where the tetrahedrally coordinated Si(IV) atoms are partially substituted by Al(III) ones resulting in negative charge in the pore, where alkali ions like Na⁺ one, depending on the synthetic solution, are stabilized for satisfying the charge neutrality criteria. Since the Na⁺ ions are present in the mesopores of PG with MCM-41 structure, NIC drug molecules can not only be encapsulated in the pore by ion-dipole interaction, but also be released out in a controlled manner upon modifying the pore charge, namely, the cation exchange capacity (CEC). It is, therefore, expected that PG could serve as an efficient therapeutic vehicle with high NIC loading capability and enhancing NIC solubility [[31], [32], [33]]. These are the main reasons why PG was also added as one of the mesoporous silicas (PSs) drug delivery carriers, MCM-41 and SBA-15, for encapsulating NIC, a poorly water-soluble anti-SARS-CoV-2 drug candidate. The inclusion and the release of drug molecules are dependent on surface chemistry within pore (Scheme 1 a). The as made NIC-PSs or PG nanohybrids were further coated with muco-adhesive non-ionic polymer, Tween 60 (Tween 60 coated NIC-PSs or PG nanohybrids), to induce high drug solubility and eventually enhanced bioavailability in-vivo, leading to have improved PK parameters (Scheme 1b). The PK parameter for the Tween 60 coated NIC-PG nanohybrid was compared with those of NIC-MCM-41 and NIC-SBA-15 ones, respectively, exploring the potential benefits as oral NIC drug delivery systems. This study marks as the first in-vivo investigation into the oral delivery performance of NIC-PG nanohybrid as an efficient NIC drug delivery system, which, in fact, is an important step toward understanding the clinical applicability of PG.

Scheme 1.

Schematic diagrams showing the (a) synthesis of NIC-PG nanohybrid and (b) NIC–PG nanohybrid coated with Tween 60 via a simple physical coating method.

Additionally, much emphasis has been given on how the PK parameters of NIC from silica-nanohybrids could be amended by rationally engineering the porous structure, properties and molecular interactions within the intra-pores of the silica nanohybrids in detail.

2. Experimental section

2.1. Materials

Kaolinite (K7375), fumed silica (S5130, 0.007 μm), MCM-41 and SBA-15 were purchased from Sigma-Aldrich (Table S1), South Korea. Cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH), ethyl alcohol (C₂H₅OH) and hydrochloric acid (HCl) were obtained from Daejung Chemicals, South Korea. NIC (C₁₃H₈Cl₂N₂O₄) was purchased from DERIVADOS QUIMICOS, Spain. Tween 60 was purchased from TCI, Japan.

2.2. Preparation of PG

PG was made as per our earlier works with some necessary changes [29,30]. Basically, the initial molar ratio was 0.25 Al: 0.75 Si: 0.50 NaOH: 0.1 CTAB: 100 H₂O to synthesize PG. Firstly, metakaolin (Al₂Si₂O₇) was obtained from dihydroxylation of kaolinite at 750 °C for 10 h. Secondly, metakaolin was dissolved in NaOH solution along with fumed silica as an extra silicon source to induce aluminosilicate fragments, and allowed 24 h stirring at room temperature to get the final viscous suspension. Thirdly, the as made suspension was treated to CTAB solution at 40 °C with 2 h vigorous stirring while keeping the pH ~10.5, thereafter it was treated at 100 °C for 24 h hydrothermal condition. After cooled down to room temperature, the particles were finally washed with water, then dried at room temperature to get CTAB/geopolymer nanohybrids. These were calcined to remove the CTAB in an air flowing environment at 600 °C to finally get the well-formed PG.

2.3. Preparation of NIC- PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids

NIC was loaded into the PG (hereafter referred as NIC-PG) using the solvent evaporation method. Accordingly, NIC was dissolved in EtOH (99.9%) to obtain a concentrated solution (10 mg mL⁻¹). The ratio of NIC/PG in the loading solution was dependent upon the pore volume of PG. The volume occupied by the amorphous NIC in PG was assessed using the following equation (1).

$$V_{PG}\left(c{m}^3{g}^{-1}\right)=W_{NIC}\left(g\right)/M{w}_{NIC}\left(gmo{l}^{-1}\right)\times V_{NIC}\left(c{m}^3\right)\times NA\left(mo{l}^{-1}\right)$$ (1)

Where V_PG is the pore volume of PG (cm³ g⁻¹), W_NIC is the mass of NIC, V_NIC is the minimal molecular dimension of NIC (cm³), NA is the Avogadro's number (6.022 × 10²³ mol⁻¹), and Mw_NIC is the molecular weight (g mol⁻¹) of the respective NIC.

PG was treated with the drug solution under stirring for 12 h at room temperature in order to achieve an adsorption equilibrium. Finally, the solvent was evaporated in vacuum by stirring for 6 h, and then dried at 160 Torr.

The procedure for NIC loading for MCM-41 and SBA-15 nanohybrids were exactly the same as mentioned above. For better understanding, the NIC loaded MCM-41 and SBA-15 nanohybrids will hereafter be referred as NIC-MCM-41 and NIC-SBA-15 nanohybrids, respectively.

2.4. Preparation of Tween 60 coated NIC-PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids

To prepare Tween 60 coated NIC-PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids, the suspension of above mentioned NIC-silica nanohybrids were prepared in EtOH (99.9%), and were physically coated with Tween 60 with the ratio 3:2 w/w (NIC-PG and NIC-PSs nanohybrids : Tween 60). The coated samples were subjected to evaporation using rotary evaporator to get the final powder products.

2.5. Physico-chemical characterization

The powder X-ray diffraction (PXRD) experiments were conducted for various samples using a Bruker D2 Phase diffractometer (Germany) with Cu K_α radiation (λ = 1.5418 Å). The whole analyses were carried with a 30 kV tube voltage and 10 mA current. The FT-IR measurements carried out using a Jasco FT/IR-6100 spectrometer (Japan) using the KBr pellet technique. N₂ adsorption-desorption measurements were conducted on a BELSORP II mini (Japan) machine, at 77 K. The porous parameters were calculated using Brunauer– Emmett–Teller (BET) technique at relative pressure ranges of 0.05–0.25, and the total pore volume was measured at elative pressure of 0.99. The porosity of the material was also determined by the Barrett, Joyner, and Halenda (BJH) and nonlocal density functional theory (NLDFT) technique. The dynamic light scattering (DLS) particle size distribution and zeta potential for PG, MCM-41and SBA-15, and their NIC nanohybrids such as NIC-PG, NIC-MCM-41, and NIC-SBA-15 were evaluated, respectively, by a DLS instrument (ELSZ-2000ZS, Japan) in aqueous solution. The Energy dispersive spectroscopy (EDS) of PG was studied by using a Sigma 300 (Carl Zeiss, Germany) field-emission scanning electron microscope (FE-SEM). Transmission electron microscopy (TEM) images were obtained using a JEOL JEM-2100F at an accelerating voltage of 200 kV.

2.6. NIC content analysis

To evaluate the NIC content in the NIC-PG and NIC-PSs nanohybrids and their Tween 60 coated ones, each sample was dispersed in 99.9% EtOH. The coated samples were further probe-sonicated for 0.5 h, in order to fully take the NIC molecules out of NIC-PG, NIC-PSs nanohybrids and their Tween 60 coated ones. Finally, the samples were filtered with 450 nm sized PVDF filter and the absorbance were checked at 333 nm by UV spectrophotometer (V-630, Japan).

The NIC content in NIC-PG, NIC-MCM-41 and NIC-SBA-15 were determined to be 49.3 ± 2.1%, 48.2 ± 3.0% and 48.3 ± 1.0%, respectively. While in Tween 60 coated NIC-PG, NIC-MCM-41 and NIC-SBA-15, the NIC content was, however, reduced down to 28.0 ± 0.6%, 28.9 ± 2.2% and 29.0 ± 0.3%, respectively, and this could be due to the proper coating of Tween 60 on the samples under study.

2.7. In-vitro drug release study

The NIC release was determined as per the previous reports using a DST-810 dissolution apparatus (Labfine, Korea) [34,35], where the temperature was fixed at 37 °C with a stirring frequency of 50 rpm. The drug release was conducted using solutions with two pH conditions of 1.2 and 6.8, both of them having 2% Tween 60 in it, simulating human's gastric and intestinal fluids (USP, 2002a, pp. 2011–2012). Samples with an identical NIC quantity of 0.015 g was suspended in the dissolution chamber. Thereafter, the aliquot was taken at pre-determined time schedules. For the drug release, all the triplicated samples were evaluated by measuring the absorbance at 333 nm by UV spectrophotometer.

2.8. In-vivo pharmacokinetic study

300g body weighed male Sprague-Dawley rats were used for the in-vivo PK analyses as per our previous protocol [36]. Institutional Animal Care and Use Committee (IACUC No. 20-KE-608) at the KNOTUS Co., Ltd. (Incheon, Korea) sanctioned all the study protocols related to animal experiments. Prior to the in-vivo, the rats underwent fasting for overnight with a free access to water, thereafter 50 mg kg^-1 NIC of Tween 60 coated NIC-PG and NIC-PSs nanohybrids were administered orally. Accordingly, Tween 60 coated NIC-PG; NIC-MCM-41; and NIC-SBA-15 samples were given orally with n = 5. After oral administration, roughly 0.25 mL of blood sample was withdrawn from each of the rats with a pre-determined time schedules (0, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h). Then the plasma was quickly separated by centrifugation at 13,000 rpm for 0.25 h at 4 °C and kept at −70 °C before analysis. Thereafter, the blood samples were analyzed by the HPLC- MS/MS.

3. Results and discussion

3.1. Structure analysis with PXRD and TEM

Fig. 1a–b shows PXRD patterns of unloaded and NIC loaded nanohybrids. According to the small-angle PXRD patterns shown in Fig. 1a, the whole samples showed well-ordered PXRD peaks, such as (100), (110), and (200) diffraction planes assigned to a hexagonal p6mm structure [29,30]. The d spacing value of the (100) peak for PG, MCM-41 and SBA-15 were 3.84, 3.84 and 8.34 nm, respectively, as determined from Bragg's law [equation (2); λ = the wavelength of Cu Ka X-ray]. The lattice parameter ‘a’ was found to be 4.43 nm for PG and MCM-41, and 9.62 nm for SBA-15 [a hexagonal-type lattice equation (3)] (Fig. S1), in agreement with literature data [37,38]. According to the TEM images (Fig. 1c), the present carriers such as PG, MCM-41, and SBA-15 displayed parallel channels with highly ordered honeycomb like 2D hexagonal array of mesopores, those which are in excellent agreement with the PXRD data.

$$n\lambda =2d\sin \theta$$ (2)

$$\frac{1}{d_{hkl}^2}=\left[\frac{4}{3}\times \left(h^2+hk+k^2\right)+l^2\times {\left(\frac{a}{c}\right)}^2\right]\times {\left(\frac{1}{a}\right)}^2$$ (3)

Fig. 1.

(a) Small-angle and (b) wide-angle PXRD patterns of intact NIC, PG, NIC-PG, MCM-41, NIC-MCM-41, SBA-15 and NIC-SBA-15 nanohybrids. (c) TEM images of the pristine silicas PG, MCM-41 and SBA-15 along the pore axis (top) and normal to the pore axis (bottom).

The broad diffraction peaks corresponding to PG, MCM-41 and SBA-15 were almost the same after the NIC loading, demonstrating their structural stability. In addition, wide-angle PXRD patterns (Fig. 1b) showed a broad peak at 2θ = 10°–40°, indicating an amorphous framework of SiO₂ [37,38], whereas, NIC retained its characteristic peaks even after the hybridization, confirming the possibility of its proper distribution predominantly at the intra-pores while partially on the surface.

3.2. FT-IR analysis

Fig. 2 shows the FT-IR spectra of intact NIC, PG, MCM-41, SBA-15, NIC-PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids respectively. The intact NIC had its innate characteristic peaks at 3577 cm⁻¹(-OH), 3490 cm⁻¹ (-NH), 1650 cm⁻¹ (-C=O), 1517 cm⁻¹(-NO₂), and 570 cm⁻¹ (C–Cl) [36]. Whereas, the specific bands at 810 cm⁻¹ and 1090 cm⁻¹were corresponding to PG. Both of these bands were characteristic ones for Si–O–Si/Al symmetric and asymmetric stretching vibrations in the PG [39]. Additionally, the broad band at 3400 cm⁻¹ indicating the –OH stretching vibration, while those at 1640 cm⁻¹ corresponding to the adsorbed water. The Si–O–Si and O–Si–O bands were observed at 479 cm⁻¹, which was seen in all the samples. On the other hand, both MCM-41 and SBA-15 showed their characteristic Si–O bands at ~810 cm⁻¹, and 1090 cm⁻¹ respectively. After the hybridization, all characteristics bands for NIC and porous carriers were overlapped within the same range of wavenumbers for NIC-PG and NIC-PSs nanohybrids.

Fig. 2.

FT-IR spectra of intact NIC, PG, NIC-PG, MCM-41, NIC-MCM-41, SBA-15 and NIC-SBA-15 nanohybrids.

3.3. Porosity analysis

The mesoporous properties for PG, MCM-41, SBA-15 and their NIC-PG and NIC-PSs nanohybrids were characterized with nitrogen adsorption-desorption isotherm as shown in Fig. 3 . The respective surface property parameters such as surface areas (S_BET), total pore volumes (V_p) and average pore diameters (D_p) were evaluated by the BET, BJH and NLDFT methods (Table 1 ). The BET isotherm of PG was a type IV with a specific surface area of 468.9 m² g ⁻¹ and a total pore volume of 0.74 cm³ g⁻¹, and a mesopore size of 3.10 nm (Fig. 3a). Whereas, after NIC loading, the NIC-PG nanohybrid showed a reduced specific surface area of 103.2 m² g⁻¹ and pore volume of 0.29 cm³ g⁻¹, retaining hysteresis loop unchanged, indicating that the pore shape was the same even after drug encapsulation. A similar pattern was observed for MCM-41 and SBA-15 after NIC drug loading (Fig. 3b–c). After drug loading, the pore volume and surface area were decreased not only due to the incorporated NIC molecules in the mesopores but also due to the partly adsorbed NIC ones on the external surface of the nanohybrids. It was found that approximately 30% of pore volume for each NIC-PG, NIC-MCM-41 and NIC-SBA-15 was not filled with NIC molecules, but still remained empty probably due to the condensation of polar drug molecules in the pores.

Fig. 3.

BET nitrogen adsorption-desorption isotherms (top) and BJH pore size distribution (bottom) of (a) PG and NIC-PG nanohybrid, (b) MCM-41 and NIC-MCM-41 nanohybrid, (c) SBA-15 and NIC-SBA-15 nanohybrid, respectively.

Table 1.

Porosity analysis for PG, NIC-PG nanohybrid, MCM-41, NIC-MCM-41 nanohybrid, SBA-15 and NIC-SBA-15 nanohybrid.

	SBET (m2 g−1)a	Dp (nm)b	Vp (cm3 g−1)c	Loading capacity (%)
PG	468.9	3.1	0.74
NIC-PG	103.2	3.1	0.29	49.3 ± 2.1
MCM-41	917.8	3.0	0.74
NIC-MCM-41	241.9	3.0	0.22	48.2 ± 3.0
SBA-15	702.1	5.8	0.86
NIC-SBA-15	228.1	5.8	0.30	48.3 ± 1.0

^aFrom the specific surface area evaluated by fitting with the BET equation.

^bMesopore diameter from NLDFT analysis.

^cTotal pore volume evaluated from the amount of adsorption at P/P₀ = 0.99.

3.4. Surface charge and Al content

Fig. 4 shows the zeta potential values for PG, MCM-41, SBA-15 and their NIC loaded NIC-PG, NIC-MCM-41, and NIC-SBA-15 nanohybrids measured in aqueous solution. It has been already known that the alteration in the pH values can influence the electrical conductivity on the surface of PG, MCM-41 and SBA-15 [[40], [41], [42]]. In an aqueous solution, the surface charge of PG was determined to be −44.36 ± 1.74 mV, which is higher than that of MCM-41(-39.33 ± 2.20 mV) and SBA-15(-39.26 ± 2.18 mV) respectively. This pattern recommended that the Si to Al ratio determines acidic functionality based on their presence at the interfacial site. In short, the bridging (Si–OH–Al) hydroxide groups presented on the outer surface of particles can easily form Si–O by de-protonation, thereby making the surface charge negative [43]. However, the surface charge of NIC loaded NIC-PG, NIC-MCM-41, and NIC-SBA-15 were determined to be −45.67 ± 0.80, −47.74 ± 2.79, and 47.36 ± 1.86 mV, respectively, which were similar or slightly larger than the unloaded porous carriers.

Fig. 4.

Zeta potential of PG, MCM-41, SBA-15, NIC-PG, NIC-MCM-41, and NIC-SBA-15 nanohybrids (n = 3).

It was noted that even after NIC loading, NIC-PG still retained their surfae charge almost same as before, indicating that NIC molecules were preferentially loaded inside the pore due to the ion-dipole interaction between intrapore Na ion and NIC molecules in the pore. On the other hand, the significantly increased negative surface charge, especially in the NIC-MCM-41 and NIC-SBA-15 nanohybrids could be attributed to the adsorbed NIC molecules on internal and external surfaces of those porous materials.

In addition, Al content was evaluated using EDS during FE-SEM study. As shown in Fig. S2, the Si/Al ratio was determined to be 3.03, which was in good agreement with the nominal value (Na_0.25Si_0.75Al_0.25O₂; Si/Al = 3.00). In order to confirm the acidic properties based on the Si/Al ratio, PG was characterized through ammonia-temperature programmed desorption (NH₃-TPD) (Fig. S3). Generally, the low temperature desorption peak represents a weak acid sites, while those at high temperature indicate a strong acid sites. Compared to the MCM-41 and SBA-15 (only one weak broad peak at 100–150 °C) [[44], [45], [46]], PG exhibited four desorption peaks: the first one 163 °C (weak acid sites), the second and third peaks at 321 and 378 °C (intermediate acidic sites) and the last peak at 604 °C (strong acid sites), indicating that the acidic property and the number of acid sites are increased with extra Al content in the PG framework. Similar phenomenon could be observed in the case of zeolite [47]. Therefore, this extra Al content in the PG makes them very special as a charged porous material, which can be further utilized for loading both charged and neutral drug molecules for various biomedical purposes.

3.5. Particle size distribution

The particle size distribution analysis for various samples such as PG, MCM-41, SBA-15 and NIC loaded NIC-silica nanohybrids in aqueous solution were determined by DLS instrument. As shown in Fig. 5 , the approximate particle size of PG, MCM-14, and SBA-15 were determined to be 526 ± 166, 572 ± 86, and 607 ± 74 nm, respectively. Whereas, the particle size distributions of NIC loaded NIC-PG, NIC-MCM-41, and NIC-SBA-15 nanohybrids were determined to be 616 ± 94, 719 ± 32, and 745 ± 74 nm, respectively, which were larger than those of porous carriers. All the results including zeta potential values indicated that NIC molecules were encapsulated mostly inside the pore for NIC-PG nanohybrid, but partially adsorbed on the external surface for cases of NIC-MCM-41 and NIC-SBA-15 nanohybrids.

Fig. 5.

The particle size distribution of (a) PG, (b) MCM-41, and (c) SBA-15 before NIC loading (black line) and after NIC loading (red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.6. In-vitro release study

The in-vitro NIC release properties of NIC-PG and NIC-PSs nanohybrids were investigated under the simulated gastric fluid (pH 1.2) and intestinal fluids (pH 6.8), both containing 2% Tween 60 as shown in Fig. 6 . We found that even after 2h, a very low ~10% NIC was dissolved from intact NIC under the gastric condition, due to its low aqueous solubility (Fig. 6a). Compared to the control NIC, the hybrids such as NIC-PG, NIC-MCM-41 and NIC-SBA-15 significantly enhanced the NIC release from it. Conversely, 15.0% intact NIC was dissolved at the intestinal pH 6.8 (Fig. 6b) and this could be associated with their better dissolvability at higher pH [48]. Meanwhile, all the NIC-PG and NIC-PSs nanohybrids were able to drastically enhanced NIC drug release compared to the intact NIC; furthermore, a dual phase release kinetics was found, wherein, an early and quick NIC release was followed by sustained drug release. NIC-PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids displayed an initial release of 25%, 28% and 30%, respectively, within the first 10 min. This initial release could be attributed to weakly bound drug molecules on the surface of NIC-PG and NIC-PSs nanohybrids. Following this release, more than 97% cumulative release in NIC-PG and NIC-PSs nanohybrids were observed in 24 h, suggesting that, compared to the free drug, NIC-PG and NIC-PSs nanohybrids have sustained-release properties, owing to the large surface area and pore size facilitating the diffusion of drug. Interestingly, the release rate of NIC-PG nanohybrid was slightly slower compared to that of NIC-MCM-41 and NIC-SBA-15 nanohybrids, both in pH 1.2 and 6.8 media. This effect could be understood since there is a strong ion-dipole interaction between Na⁺ and NIC molecules for NIC-PG nanohybrid, which might alter the release kinetics of NIC from the silica nanohybrids.

Fig. 6.

In-vitro NIC release profiles from intact NIC, NIC-PG, NIC-MCM-41, NIC-SBA-15 nanohybrids in (a) pH 1.2 medium and (b) pH 6.8 containing 2% Tween 60.

To get more insight of the NIC release kinetic mechanism, the release profiles of NIC-PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids were mathematically modelled using various kinetic fitting models as shown in Fig. S4. Considering the r² value, the simulation equations and correlation coefficients were calculated and compared in Table S2. The best fitting model among all the nanohybrids was the Freundlich model, indicating that the release of the incorporated NIC from NIC-PG and NIC-PSs nanohybrids were a kind of diffusion-controlled processes [36,49].

On the other hand, we also investigated the effect of surfactant in the release medium and its effect on the NIC release. As presented in Fig. S5, a very slow drug dissolution was found under the pH conditions of 1.2 and pH 6.8 having 0.5% Tween 60, indicating that the Tween 60 content in drug formulation could greatly influence the solubility of NIC and hence the release profile.

3.7. In-vivo pharmacokinetics of NIC

As mentioned in previous sections, Tween 60 plays vital role in enhancing the NIC release. Therefore, to improve the solubility of the sparingly dispersing NIC, nanohybrids such as NIC-PG and NIC-PSs coated homogeneously with Tween 60, to enhance the bioavailability of NIC. In addition, Tween 60 can stabilize the NIC-nanohybrids along with an improved drug release kinetics. As described above, Tween 60 coating was only via a physical mixing, and therefore no chemical modification was expected even after coating with NIC-nanohybrids. The UV analysis (Fig. S6) clearly showed that the NIC peak was not changed even after Tween 60 coating, assuring that it was just a kind of physical adsorption [50].

Single time oral administration of Tween 60 coated NIC-PG, NIC-MCM-41and NIC-SBA-15 nanohybrids resulted in to a PK profile as shown in Fig. 7 , and the main PK parameters were summarized in Table 2 . The AUC_(last) values of NIC-PG, NIC-MCM-41 and NIC-SBA-15 were quite similar, achieving an increased bioavailability compared to the reported values for intact NIC in the literature [48,51,52]. This suggests that the synergistic effect of mesoporous carriers and surfactants contained in NIC-PG, NIC-MCM-41 and NIC-SBA-15 could improve the oral absorption of NIC in the gastro-intestinal tract. However, there was around 1.8 times higher C_max for the NIC-MCM-41 (294.75 ± 46.38 ng mL⁻¹) compared to NIC-PG (159.38 ± 47.07 ng mL⁻¹) and NIC-SBA-15 (166.99 ± 37.65 ng mL⁻¹) nanohybrids. Additionally, t_1/2 value of NIC-SBA-15 (7.56 ± 3.29 h) was approximately 1.8 times higher than NIC-PG (4.10 ± 0.50 h) and NIC-MCM-41 (4.54 ± 0.67 h) nanohybrids. In the case of T_max, there was around 10.7 times higher T_max for the NIC-PG (2.67 ± 1.15 h) compared to NIC-MCM-41 (0.25 ± 0.0 h) and NIC-SBA-15 (0.25 ± 0.0 h) nanohybrids. These difference in PK parameters between the samples could be associated with their different pore characteristics. For example, PG has charged porous structure, which can load NIC drug molecules based on a strong ion-dipole interaction. On the other hand, both MCM-41 and SBA-15 were consisted of neutral porous structures but with different pore volume as described before. These specific characteristic properties could, therefore, alter the overall PK parameters as shown in Table 2.

Fig. 7.

Mean plasma concentration of NIC vs time after oral administration of Tween 60 coated NIC-PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids.

Table 2.

PK parameters of NIC from orally administered Tween 60 coated NIC-PG, NIC-MCM-41 and NIC-SBA-15 nanohybrids at a dose of 50 mg kg^-1 in Sprague-Dawley rats (n = 5).

PK Parameters	NIC-PG	NIC-MCM-41	NIC-SBA-15
AUC (last) (ng·h mL−1)a	873.91 ± 88.82	953.68 ± 82.13	857.20 ± 92.74
Cmax (ng mL−1)b	159.38 ± 47.07	294.75 ± 46.38	166.99 ± 37.65
Tmax (h)c	2.67 ± 1.15	0.25 ± 0.00	0.25 ± 0.00
t1/2 (h)d	4.10 ± 0.50	4.54 ± 0.67	7.56 ± 3.29

^aAUC = area under the plasma concentration–time curve.

^bC _max = maximum plasma concentration.

^cT_max = time required to reach C_max.

^dt_1/2 = elimination half-life.

To further understand the improved T_max associated with the NIC-PG than the other samples, we made a molecular structure model for NIC and its possible interactions with the porous materials such as PG, MCM-41 and SBA-15, as shown in Scheme 2 . It should be noted that, though the pore size for PG was as similar as that of MCM-41 (2.7 nm), the SBA-15 exhibited bigger pore window of 6.3 nm. In addition, the charged pores in the PG could hold NIC molecules majorly by three mechanisms such as 1) Na + -NIC (ion-dipole) interaction; 2) NIC-PG (dipole-pore wall) van der Waals (vW) interaction and 3) NIC–NIC (dipole-dipole) molecular interaction within the pore. Obviously, in the PG pores, the ion-dipole interaction might be dominant, thereby NIC could be released in a sustained manner. This was surely the reason why both t_1/2 and T_max were improved in the NIC-PG nanohybrid. On the other hand, two dominant intra-pore interactions could be expected in the NIC-MCM-41 nanohybrid; 1) vW interaction between dipolar NIC and pore wall, and 2) molecular interactions between NIC and NIC. Since they are fairly weaker than ion-dipole interaction, the sustained in-vivo release could not be expected. Because of this weak NIC–NIC molecular interaction, it was not that surprising why the NIC-MCM-41 nanohybrid showed such a high C_max and a low T_max compared to the NIC-PG one. In the NIC-SBA-15, more NIC molecules could be accommodated in the meso pore of SBA-15 than MCM-41via collective interaction among NIC molecules, since SBA-15 showed bigger pore volume than MCM-41. In addition, vW interaction between NIC dipole and pore wall could also be expected. Because of this large pore volume with collective dipole-dipole interaction energy gain among NIC–NIC, the NIC-SBA-15 nanohybrid showed very high t_1/2 compared to the other two samples, however, the T_max was turned out to be very low.

Scheme 2.

(a) Molecular structure model of NIC considering the van der Waals radii of constituting atoms. The approximate dimensions are obtained by modeling a NIC (C₁₃H₈Cl₂N₂O₄) molecule in a rectangular prism. (b) The types of intra-pore interaction models depending on structural properties of NIC-PG, NIC-MCM-41, and NIC-SBA-15 nanohybrids.

The major difference in PK parameters, in particular T_max and t_1/2, among nanohybrid samples could be interpreted very well on the basis of the NIC interaction chemistry within mesopores as shown in Scheme 2. It is worthy to mention that by controlling the pore size and charge density of internal pores of silica nanohybrids, one could achieve desirable drug release profile. Especially, the pores in PG can be altered by changing the Al content, making them more charged, in such a way that the NIC dosage or loading content can be controlled as required, and thereby, the overall release kinetics can be tuned to achieve maximum outcome towards SARS-CoV-2. In this way we could specifically deliver therapeutic dosages for combating contagious diseases like COVID-19.

Additionally, a recent study postulating the importance of using clay-based nanomaterials for selective targeting to COVID-19 is of great relevance. To exemplify one such study, the montmorillonite clay nanomaterial could interact with the spike proteins of SARS-CoV-2 virus as pseudo antibody, in such a way the protein conformation could be altered, thereby hindering their adhesion to the human ACE2 receptors [53]. Since the present anti-viral nanohybrids are of clay origin, with a NIC loaded in it, it could be acted through synergistic mechanisms via targeting + therapy. Therefore, our future studies will be focused on understanding such fundamentals to bring the NIC silica nanohybrids from bench to market.

4. Conclusions

In conclusion, for the first time, we report successful encapsulation of the poorly soluble drug NIC in mesoporous silicas such as MCM-41, SBA-15 and PG, which were further coated with Tween 60 for oral administration towards COVID-19. The in-vitro NIC release was found to be enhanced under gastro/intestinal solution compared to the intact NIC. Further the in-vivo oral administration of the present NIC-PG and NIC-PSs hybrids showed improved PK parameters, which were dependent on the pore structure/property relation along with their interfacial and molecular interactions within the intra-pores. In a sustained bioavailability aspect, we suggest that NIC-PG might be good formulation, since it improved both T_max and t_1/2 whereas the NIC-MCM-41 and NIC-SBA-15 nanohybrids showed only better t_1/2 with a low T_max. On the other hand, NIC-MCM-41 showed maximum AUC and C_max, along with higher t_1/2 which could also be beneficial for achieving a therapeutic outcome. Conversely, the NIC-SBA-15 had significantly higher t_1/2, showing that the elimination half-life, t_1/2, of NIC can be sustained quite longer than the other two. In summary, we recommend that tuning the Si/Al ratio would allow one to control the charged pores in the silica nanohybrids, thereby regulating the release profile in a sustained fashion to improve the overall therapeutic outcome. In fact, depending on one's own requirements, it is possible to achieve efficient NIC release and PK parameters from our rationally designed silica nanohybrids. The present study therefore, clearly demonstrated that silica based nanohybrids could be an effective mesoporous therapeutic agents to improve the efficacy from orally administered, poorly-soluble drugs such as NIC.

CRediT authorship contribution statement

Huiyan Piao: Methodology, Investigation, Formal analysis, Writing – original draft. N. Sanoj Rejinold: Formal analysis, Writing – review & editing. Goeun Choi: Investigation, Formal analysis, Writing – review & editing, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: