Taste masking of a drug by pH-responsive coordination polymer-coated mesoporous silica nanoparticles

Abstract

We developed a simple and efficient method for fabricating a taste-masked oral drug delivery system (DDS) that regulates the release of unpleasant drug taste via the change in pH value in the physiological environment of the alimentary canal. In this drug delivery system, a pH-sensitive metal–organic coordination polymer (CP), Fe-4,4′-bipyridine (Fe-bipy) complex, works as a taste-masker. The pH-sensitive Fe-bipy was grafted onto the mesoporous silica nanoparticles (MSNs) containing the model bitter drug mequindox (MEQ) in its mesopores through metal–organic coordination cross-linking, resulting in the CP-coated nanodrug MSN-NH₂-MEQ@Fe-bipy. In artificial saliva (pH 6.6), Fe-4,4′-bipyridine CPs effectively prevent the leaking of the loaded guest molecule MEQ. On the other hand, in artificial gastric fluid (pH 1.0), the coordination bonds of the Fe-4,4′-bipyridine complex were broken, leading to the release of MEQ molecules from MSN-NH₂-MEQ@Fe-bipy. The results indicate that the simple and efficient preparation method for taste-masked MSN-NH₂-MEQ@Fe-bipy could provide new insights into taste-masking technology by using pH-responsive smart nanomaterials.

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Introduction

The oral administration of drugs is the most popular route for systemic effects due to its safety, convenience, and high patient compliance.¹ Particularly, antimicrobial and antiparasitic drugs are usually administered orally via water or feed for ease of delivery on a population basis and to reduce the stress of animal handling in the veterinary care of both farm and companion animals.² However, many drugs have unpleasant qualities such as bitterness, saltiness, sourness, metallic or spicy taste, or causing oral numbness,³ so that the unpleasant taste of drugs is a serious problem affecting the adherence of a patient to the medication.⁴ Although traditional tablets and capsules might mask the taste of drugs, they are difficult for geriatric and pediatric patients and infected animals to swallow.^5–7 Therefore, taste-masking technologies are required for patient-oriented multiparticulate dosage forms (powders, granules, or suspensions) that are available to be orally administered via water or feed. These technologies could improve not only the compliance of sick animals and the convenience of administering medication to groups of animals, but also the stability of drugs.

Nanomaterials, which provide new multiparticulate formulation options for both dispersed liquid droplet dosage forms and dry powder formulations, have many advantages over traditional dosage forms, such as enhanced dissolution properties and the potential for intracellular drug delivery.⁸ Among the many nanomaterials, highly ordered mesoporous silica nanoparticles (MSNs) have attracted active research interest as excellent drug-delivery vehicles.^9–20 This is partly driven by their fascinating properties such as remarkable biocompatibility,^21–26 low toxicity, available degradation pathways in the biological milieu,^27–32 biochemical and physicochemical stability,³³ and high guest molecular loading capability.^20,34–37 Their advantages, such as high surface area and uniform mesoporous structure^38,39 and facility of surface-functionalization,^40–42 which allow the loading of drugs and fine control of release kinetics,^24,43,44 have provided a powerful impetus and guarantee for developing taste-masking nanodrugs.

Acidity in animal bodies is different between various interstitial fluids; for example, gastric fluid is more acidic (pH 0.9–1.5) compared with saliva (pH 6.6–7.1) and with blood and normal tissue (pH 7.4). Thus, the pH-targeting approach is regarded as a more feasible strategy than many other targeting approaches.⁴⁵ Many studies on pH-responsive MSN drug delivery systems (DDS) for injectable antineoplastic agents have been reported; however, studies on pH-responsive MSN DDS for taste-masked oral drugs are few.^46–48 Moreover, the most reported stimuli-responsive MSN drug delivery systems (DDS) involve fussy multistep chemical synthesis. Therefore there is great demand for the development of a simple method for preparing pH-sensitive MSN, especially for the purpose of taste masking.

As is well known, coordination polymers (CPs) are acid labile. Recently, CP-coated MSN for drug release systems based on the pH-responsive behaviour of metal–organic coordination bonds have been reported.^49–52 4,4′-Bipyridine-metal CPs were extensively investigated as appealing biomedicine materials⁵³ due to the biocompatibility of 4,4′-bipyridine.⁵⁴ Moreover, Long's group has reported that the coordination ability and coordination mode of 4,4′-bipy ligand to the metal ion are affected by pH value.⁵⁵ Therefore, we reasoned that the incorporation of 4,4′-bipy-metal coordination polymers as a pH-responsive shell onto the surface of MSN can create taste-masking nanovehicle systems (Scheme 1).

Scheme 1 Schematic illustration of the working protocol for taste-masking drug based on physiological pH-responsive release of unpleasant-tasting drug from mesoporous silica nanoparticles capped with metal–organic coordination shell.

In this respect, we herein report a simple and effective strategy for the construction of CP-coated MSN with the properties of pH-controlled drug release property (Scheme 2), in which MCM-41-type MSN is enwrapped with Fe-4,4′-bipy coordination polymer. First, MCM-41-type MSN material containing the template n-cetyltrimethylammonium bromide (CTAB) in its nanopores was synthesized according to a previously reported method.⁵⁶ Then, the outer surfaces of the as-made CTAB@MSN were selectively functionalized with amino groups by grafting 3-aminopropyltriethoxysilane (APTES) to yield as-synthesized CTAB@MSN-NH₂ because the templates fill the nanopores. After removal of the templates by reflux in a methanolic solution of HCl, the amino-group surface-functionalized MSN with empty nanopores are obtained (denoted MSN-NH₂), which can be harnessed as microstorage rooms to encapsulate the unpleasant-tasting drug. The amino groups are designed to catch the metal ions and further CP growth on the MSN surface after drug loading. Mequindox (MEQ), a popular veterinary drug to improve growth and ameliorate bacterial enteritis in enteron,⁵⁷ was chosen as a typical unpleasant-tasting drug model for this DDS. The MEQ-loaded MSN-NH₂ was then capped by the CPs of the iron-4,4′-bipyridine complex grown on the outer surfaces like a snowball due to the amino groups and the surface free energy ensuring close fusion and preference for epitaxial growth of the CP layer via coordination chemistry, giving rise to MSN-NH₂-MEQ@Fe-bipy architecture. For a detailed description, see Scheme 2 and experimental section. The MSN-NH₂-MEQ@Fe-bipy is stable in neutral saliva and can release MEQ molecules in the digestive system because of the acidic stomach condition. The working principle of this system is schematically described in Scheme 1. Furthermore, this system consists of biocompatible components such as silica particles, FeCl₃·6H₂O, and 4,4′-bipyridine, allowing the basis for pharmacological applications.

Scheme 2 Schematic illustration of the preparation process for MSNs-NH₂-MEQ@Fe-bipy and triggered drug release in gastric juice.

Experimental

Materials

Mequindox (MEQ) was used as purchased from China Animal Husbandry Industry Co Ltd. Biochemical regents, including phosphate-buffered saline (PBS), fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM), and methyl tetrazolium (MTT) were provided by Sigma-Aldrich (St. Louis, MO). Both NIH/3T3 cells and L-929 cells were provided by Stem Cell Bank, Chinese Academy of Sciences. 96-well polystyrene plates were provided by Chinese Sangon Biotech. All other chemicals were purchased from Sinopharm and used as received.

Characterization

Absorption spectra were recorded on a UV-2880 UV-vis spectrometer (Shanghai Sunny Hengping Scientific Instrument Co., Ltd). The N₂ adsorption and desorption isotherms, surface area (SA), and median pore diameter (MPD) were measured using a TriStar II 3020 sorption analyzer. Surface areas and pore size distributions were calculated using the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) method, respectively. The total pore volumes were calculated from the nitrogen adsorption/desorption data. The morphology of mesoporous silica nanoparticles were observed with SEM (Hitachi S4800). TEM images were taken with a FEI Tecnai G20 microscope operating at 200 kV.

Synthesis of mesoporous silica nanoparticle-containing template (CTAB@MSN)

n-Cetyltrimethylammonium bromide (CTAB, 2.00 g, 5.48 × 10⁻³ mol) was first dissolved in 960 mL of double-distilled water. Sodium hydroxide aqueous solution (2.00 M, 7.00 mL) was added to CTAB solution, and the solution temperature was adjusted to 80 °C. Tetraethoxysilane (TEOS, 10.00 mL, 44.8 mmol) was added dropwise to the surfactant solution under vigorous stirring. The mixture was stirred for 2 h to give a white precipitate.⁵⁶ The white solid crude product was filtered with a sintered G4 filter funnel (pore size 3–4 μm), excessively rinsed with double-distilled water and methanol, then methanol, respectively. The sample was dried in vacuum at room temperature overnight to yield 3.7 g of the as-synthesized CTAB@MSN.

Surface amino-functionalization of mesoporous silica nanoparticle-containing template (CTAB@MSNs-NH₂)

20 g as-made CTAB@MSN was firstly suspended in n-hexane (40 mL), then 20 mL 3-aminopropyltriethoxysilane (APTES) was added to the mixture. The solution was stirred at room temperature for 48 h to produce crude CTAB@MSN-NH₂ as a white precipitate. The product was centrifuged and extensively rinsed with methanol to remove the excess APTES. The white product was dried in vacuum at room temperature overnight to yield the as-synthesized CTAB@MSN-NH₂.⁵⁸

Preparation of aminated mesoporous silica nanoparticles (MSN-NH₂)

To remove the surfactant template (CTAB) from the nanopores of CTAB@MSN-NH₂, the as-made CTAB@MSN-NH₂ was refluxed in a mixture of MeOH (500 mL) and concentrated HCl (10 mL) for 12 h. After centrifugation, the solid crude product was again refluxed in another mixture of MeOH (500 mL) and concentrated HCl (10 mL) for 12 h, then centrifuged. The resulting powder was extensively washed with triethylamine–methanol (1 : 1 by volume) and MeOH. The white product was placed in vacuum to remove the remaining solvent in its mesopores. The 13.45 g sample was denoted as amino-functionalized MSN (MSN-NH₂).⁵⁹

Drug loading and coating with coordination polymer (MSN-NH₂-MEQ@Fe-bipy)

Mequindox (MEQ), a popular veterinary drug to improve growth and ameliorate bacterial enteritis, was employed as the unpleasant-tasting model drug in this study. A mixture of MSN-NH₂ (100 mg) and mequindox (100 mg) in EtOH (10 mL) was stirred for 24 h at room temperature, followed by the alternate addition of an ethanolic solution (0.1 mL × 10) of FeCl₃·6H₂O (1.62 mg, 10 μmol) and an ethanolic solution (0.1 mL × 10) of 4,4′-bipyridine (1.56 mg, 10 μmol) every 5 min.⁵¹ After being stirred for another 30 min, the reacting mixture was centrifuged and extensively rinsed with ethanol. The resulting product was placed in vacuum to remove the remaining solvent. 307 mg brown powder was collected as the coordination polymer-coated, MSN-containing mequindox (MSN-NH₂-MEQ@Fe-bipy). The drug-loading content (DLC) was evaluated using a UV/vis spectrophotometer based on the equilibrium absorbance of the MSN-NH₂-MEQ@Fe-bipy particles in 1 M hydrochloric acid solution at 374.5 nm.

Preparation of artificial saliva and simulated gastric juice

Modified Fusayama artificial saliva was used as the immersion buccal test media, which consisted of NaCl (400 mg L⁻¹), KCl (400 mg L⁻¹), CaCl₂·2H₂O (795 mg L⁻¹), NaH₂PO₄·H₂O (690 mg L⁻¹), KSCN (300 mg L⁻¹), Na₂S·9H₂O (5 mg L⁻¹), and urea (1000 mg L⁻¹). The artificial saliva was adjusted to pH 6.6 with sodium hydroxide and maintained at 37 °C.⁶⁰

To prepare simulated gastric juice, pepsin was suspended in sterile saline (0.85% NaCl, w/v) to a concentration of 10 g L⁻¹. The pH was adjusted to 1.0 with sterile 1 M HCl using a pH meter (State Pharmacopoeia Committee of the People's Republic of China, 2015).

In vitro drug release

The release study was performed with cellulose dialysis tubes with MWCO of 8000–14 000. MSN-NH₂-MEQ@Fe-bipy (6.3 mg) particles were suspended in 1 mL artificial saliva (pH 6.6). The dialysis tubes containing MSN-NH₂-MEQ@Fe-bipy particles were immersed into 19 mL artificial saliva (pH 6.6) with stirring at 37 °C. Similarly, MSN-NH₂-MEQ@Fe-bipy (5.8 mg) particles were suspended in 1 mL artificial gastric fluid (pH 1.0). Dialysis tubes containing MSN-NH₂-MEQ@Fe-bipy particles were immersed into 19 mL artificial gastric fluid (pH 1.0) with stirring at 37 °C. Incubation media samples were taken out for analysis at given time intervals and then returned to the original release media. The extracted incubation media were analysed by UV-2880 UV-vis spectrometer at the wavelength of 374.5 nm.

Cell cultures

NIH/3T3 normal cells (a cell line of mouse embryonic fibroblast cells) and L-929 normal cells (a cell line of mouse embryonic fibroblast cells) were cultured in DMEM in a humidified atmosphere containing 5% CO₂ at 37 °C. DMEM was supplied with 10% FBS, penicillin (50 units per mL), and streptomycin (50 units per mL).

Cell viability in vitro evaluated by MTT assay

The vitality of NIH/3T3 normal cells and L-929 normal cells were estimated by the 3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) reduction assay, respectively. In the MTT assay, both fibroblasts were seeded into 96-well plates at 8000 cells per well in 200 μL of culture medium and incubated for 12 h. Then, the fibroblasts were cultured with various concentrations of free MEQ, FeCl₃·6H₂O, 4,4′-bipyridine, Fe-bipy coordination polymer, MSN-NH₂ and MSN-NH₂-MEQ@Fe-bipy nanoparticles for another 72 h. For each concentration, six parallel measurements were carried out at the same time. 20 μL of 5 mg mL⁻¹ MTT solution in PBS was added into each well and incubated for 4 h. After aspirating the solution, 150 μL of DMSO was added into the wells, and the 96-well plates were shaken for 10 min to dissolve purple formazan crystals at the bottom. The absorbance at 490 nm wavelength was recorded on a BioTek Synergy H4 hybrid reader to calculate the fibroblast viability. The vitality of NIH/3T3 normal cells and L-929 normal cells was calculated as a percentage of viable cells after treatment with free MEQ, carrier materials, or MEQ-loaded nanoparticles compared with the untreated cells, which were taken as a control (100% viability).

Results and discussion

The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of MSN-NH₂ and MSN-NH₂-MEQ@Fe-bipy are shown in Fig. 1. As is well known, a clear and readable SEM image shows that the sample surface is electrically conductive and grounded to prevent the accumulation of an electrostatic charge. The higher-resolution SEM image of MSN-NH₂-MEQ@Fe-bipy compared with MSN-NH₂ suggested that the highly conductive coordination polymer Fe-4,4′-bipy was coated on poorly conductive MSN-NH₂ nanoparticles containing the drug. TEM images (Fig. 1c) show that the average diameter of MSN-NH₂ particles is around 100 nm. The highly ordered lattice array over the surface of MSN-NH₂ indicated a uniform, well-defined mesoporous structure. The blurred feature with lattice array of MSN-NH₂-MEQ@Fe-bipy compared with MSN-NH₂ revealed that a CP coating covered the MSN surface (Fig. 1c and d). However, there are no clear differences in shape and average diameter between the MSN-NH₂-MEQ@Fe-bipy and the MSN-NH₂, which indicates that the Fe-bipy CPs deposit an ultrathin pH-sensitive shell coating on the drug-loaded MSN-NH₂ to allow quick drug release from the MSN-NH₂ nanovehicle in artificial gastric fluid.

Fig. 1 (a) SEM and (c) TEM micrographs of MSN-NH₂. (b) SEM and (d) HRTEM micrographs of MSN-NH₂-MEQ@Fe-bipy.

The MSN-NH₂ and MSN-NH₂-MEQ@Fe-bipy were also characterized by Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) analyses, respectively (Fig. 2). N₂ adsorption–desorption isotherms also provide valid evidence of drug loading of the carrier system. Sample MSN-NH₂ exhibits a typical IV isotherm, giving a large pore volume (0.80 cm³ g⁻¹), surface area (871.29 m² g⁻¹), and narrow pore size distribution (2.49 nm). After the drug loading and end-capping process, the mesoporous volume (0.12 cm³ g⁻¹), surface area (149.49 m² g⁻¹), and mesoporous size distribution (2.21 nm) of the product MSN-NH₂-MEQ@Fe-bipy are sharply decreased compared with those (0.80 cm³ g⁻¹, 871.29 m² g⁻¹, 2.49 nm) of MSN-NH₂ nanoparticles, which should be essentially attributed to storage of mequindox in the mesopores of the sample MSN-NH₂-MEQ@Fe-bipy.

Fig. 2 BET nitrogen adsorption/desorption isotherms (a) and BJH pore size distribution (b) of MSN-NH₂ and MSNs-NH₂-MEQ@Fe-bipy.

Furthermore, MSN-NH₂ and MSN-NH₂-MEQ@Fe-bipy were characterized by FTIR spectroscopy. Fig. 3 shows the FTIR spectra of MSN-NH₂ before and after modification in each grafting step. The carbonyl stretching vibration at 1699 cm⁻¹ in MEQ was overlaid by the broad band of water stretching vibration in the MSNs, while the weak signal at 1384 cm⁻¹, which may be assigned to the stretching of N–O, indicates that the MEQ molecules were encapsulated into the pores of the MSNs. The strong signal at 1075 cm⁻¹ and the weak signal at 1535 cm⁻¹ were attributed to the asymmetric stretching of Si–O–Si and bending stretching of NH₂, respectively, confirming the successful functionalization of MSNs with amino groups. The signals at 813, 790, and 636 cm⁻¹, which are related to the characteristic peaks of 4,4′-bipyridine, confirm that the Fe-bipy polymeric complex is coated onto MSN-NH₂-MEQ. These results suggest that MSNs-NH₂-MEQ@Fe-bipy was successfully synthesized.

Fig. 3 FTIR spectra of MSNs-NH₂ (a), MEQ (b), Fe-bipy (c), MSN-NH₂-MEQ@Fe-bipy (d).

The drug loading content (DLC) was estimated to be 0.76 mmol g⁻¹ (165 mg g⁻¹) according to the equilibrium status absorbance of MEQ in 1 M hydrochloric acid solution.

To verify the taste-masking effect of MSN-NH₂-MEQ@Fe-bipy, MEQ release from MSN-NH₂-MEQ@Fe-bipy under artificial saliva environment (pH 6.6) was studied. As shown in Fig. 4, 14.4% of MEQ was released from MSN-NH₂-MEQ within 5 min. On the other hand, only 0.43% of MEQ was released from MSN-NH₂-MEQ@Fe-bipy system within 5 min, and just 9.22% of MEQ seeped out even within 8 h, which indicates the outstanding storage and sealing efficiency of CPs on MSN (pH 6.6), preventing the MEQ drug from contacting with taste buds in the oral cavity. Thus, the bitter taste of MEQ could be hidden. The pH-responsive release behaviour of MSN-NH₂-MEQ@Fe-bipy was then studied in artificial gastric fluid (pH 1.0) at 37 °C. Notably, 31.94% of MEQ was favourably released when the MSN-NH₂-MEQ@Fe-bipy was exposed to artificial gastric fluid (pH 1.0) within 5 min, and almost all the MEQ molecules were released within 8 h, due to the triggering the cleavage of CPs on MSN; thus, the effect of the MEQ drug could be provided in stomach. These results suggest that the MSN-NH₂-MEQ@Fe-bipy is a good taste-masking feature in the oral drug delivery system.

Fig. 4 Time-dependent cumulative release profiles of MEQ from MSN-NH₂-MEQ and MSN-NH₂-MEQ@Fe-bipy in artificial saliva (pH 6.6) and artificial gastric fluid (pH 1.0).

To test for feasibility in biomedical applications, the in vitro cytotoxicity of the free MEQ, carrier materials, and MSNs-NH₂-MEQ@Fe-bipy nanoparticles were synthetically evaluated under identical conditions via routine MTT assay. For this test, both NIH/3T3 normal cells and L-929 normal cells were treated with free MEQ, FeCl₃·6H₂O, 4,4′-bipyridine, Fe-bipy coordination polymer, and MSN-NH₂ and MSN-NH₂-MEQ@Fe-bipy nanoparticles at different concentrations from 0.53125 to 100 μM. The fibroblasts without any treatment were used as the control. As shown in Fig. 5, the viabilities of L-929 normal cells incubated with carrier materials, including FeCl₃·6H₂O, 4,4′-bipyridine, Fe-bipy coordination polymer, were very high even in high concentrations of materials and were in the acceptable range when incubated with low concentrations of the materials including MEQ, MSN-NH₂, and MSN-NH₂-MEQ@Fe-bipy. On the other hand, the viabilities of NIH/3T3 normal cells incubated with carrier materials, including MSN-NH₂, were about 100% even in high concentrations, and those incubated in low concentration of other materials were in the acceptable range. These data indicate that all the carrier materials were of acceptable toxicity to NIH/3T3 normal cells and L-929 normal cells, especially in low concentration.

Fig. 5 Cell viability of L-929 normal cells (A) and NIH/3T3 normal cells (B) cultured with free MEQ, FeCl₃·6H₂O, 4,4′-bipyridine, Fe-bipy coordination polymer and MSN-NH₂ and MSN-NH₂-MEQ@Fe-bipy nanoparticles determined by MTT assay after 72 h, respectively. Results are all presented as mean value (n = 6).

Conclusion

In summary, we have successfully designed and synthesized CP-capped mesoporous silica nanoparticles via a simple preparation method. The nanoparticles serve as pH-sensitive nanoreservoirs for taste-masking in an oral drug delivery system. In this system, MSN serves as drug carrier while in the mouth, and the drug is released in the stomach because of the cleavage of the coordination bond of the CP nanolayer by H⁺ in the more acidic condition. The efficient, smart stomachic drug release system based on pH-responsive nanocarriers provide new insights into taste-masking technology for oral administration of drugs via water or feed, improving the compliance of sick animals and the convenience of administering medication to group animals.

Acknowledgements

We gratefully acknowledge the National Natural Science Foundation of China (No. 31560712, and 21461011), the Natural Science Foundation of Jiangxi Province (20151BAB204014, and 20144BDH80006) for support of this work.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra19789b

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