Nanoparticle-encapsulated baicalein markedly modulates pro-inflammatory response in gingival epithelial cells

Xuan Li a, Wei Luo a, Tsz Wing Ng b, Ping Chung Leung c, Chengfei Zhang a, Ken Cham-Fai Leung *b and Lijian Jin *a
aFaculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China. E-mail:; Fax: +852 2858-7874
bDepartment of Chemistry and Partner State Key Laboratory of Environmental and Biological Analysis, The Hong Kong Baptist University, Hong Kong SAR, China. E-mail:; Fax: +852 3411-7348
cInstitute of Chinese Medicine and Partner State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong SAR, China

Received 10th April 2017 , Accepted 4th June 2017

First published on 26th June 2017

Severe gum disease (periodontitis), which is one of the major global oral diseases, results from microbe-host dysbiosis and dysregulated immuno-inflammatory responses. It seriously affects oral health and general wellbeing with significant socio-economic implications. It has been well documented that natural flavonoids such as baicalin (BA) and baicalein (BE) possess potent anti-inflammatory effects. However, their intrinsic poor solubility and low bioavailability severely limit their biomedical applications. In the present study, BA and BE were encapsulated in our synthesized and amine-modified mesoporous silica nanoparticles (MSNs) (Nano-BA and Nano-BE, respectively), and their loading efficiencies and releasing profiles were investigated. Their cytotoxicity was examined on primary human gingival epithelial cells (hGECs), and the cellular uptake of Nano-BA or Nano-BE was visualized via a transmission electron microscope. Their anti-inflammatory effects were evaluated in IL-1β-treated hGECs using the cytokine array and enzyme-linked immunosorbent assay. The present study shows that the amine-modified MSNs could encapsulate BA and BE, and nano-encapsulation greatly enhances the drug delivery rate and prolongs the release of BA and BE up to 216 h. Moreover, both Nano-BA and Nano-BE could be internalized by hGECs and retained intracellularly in nanoparticle-free media for at least 24 h. Note that Nano-BE pre-treatment effectively down-regulates the IL-1β-induced expression of IL-6 and IL-8 in hGECs. In conclusion, nanoparticle-encapsulated BE exhibits notable anti-inflammatory effects through effective release and cellular internalization approaches. This study may facilitate the development of novel drug delivery systems for improving oral care.

1. Introduction

Severe gum disease (periodontitis) is one of the major global diseases and currently remains the leading cause of multiple tooth loss and edentulism in adults worldwide, which has significant socio-economic impacts.1,2 It not only significantly affects oral health and function, but also is closely linked to systemic diseases and disorders such as cardiovascular disease and diabetes.3 Periodontitis results from the plaque biofilm-induced dysregulated immuno-inflammatory response. Porphyrornonas gingivalis is currently recognized as the keystone periodontal pathogen, which drives host-microbe symbiosis to dysbiosis and thereby mediates destructive inflammation at the subgingival niches including gingival epithelia.4 The treatment strategy for tackling this serious oral disease currently focuses on both effective control of pathogenic plaque biofilms and the resultant dysregulated immuno-inflammatory response.5

It has been well documented that a traditional Chinese herb called Scutellaria baicalensis exhibits remarkable anti-inflammatory, antioxidant and anticancer effects, and it has therefore been extensively addressed in biomedical research and application.6–12 The flavonoids extracted from the radix of S. baicalensis, such as baicalin (BA) and baicalein (BE), demonstrate potent anti-inflammatory effect both in vitro and in vivo studies.13–20 However, their poor solubility, low bioavailability and short half-life severely restrict their biomedical applications.21,22

The increasing application of nanotechnology in the biomedical arena has shed new light on refining the administrative approach to effective use of BA and BE for health care. Several solid lipid nanoparticles have been developed to encapsulate the flavonoids of S. baicalensis in order to improve their delivery rate and bioavailability.23–25 Likewise, BE has been loaded into polymer or lipid nanoparticles to enhance its stability and efficiency.26,27 Currently, mesoporous silica nanoparticles (MSNs) are emerging from various nanomaterials and broadly applied in biomedical research and care as promising drug vehicles due to their stable physicochemical property, high biocompatibility and low cytotoxicity.28–30 They are considered to be preferred drug carriers owning to their sponge-like structure, large and easily modifiable surface characteristics and flexible modification for multi-functional purposes.31–33 Hence, herein, BA and BE are encapsulated in our synthesized and surface-modified MSNs, and then their loading and releasing profiles as well as cellular uptake and internalization are investigated. The present study shows the first evidence that nano-encapsulated BE exhibits a potent anti-inflammatory effect in primary human gingival epithelial cells (Scheme 1).

image file: c7nr02546g-s1.tif
Scheme 1 Schematic of the preparation of nano-encapsulated BA and BE using amine-modified mesoporous silica nanoparticles (MSNs) (A) and assessment of their effects on the IL-1β-induced inflammatory response in human gingival epithelial cells (hGECs) (B).

2. Materials and methods

2.1 Chemicals and agents

Cetyltrimethylammonium bromide (CTAB), 3-aminopropyltriethoxy silane (APTES), and BA (95%) were obtained from Sigma-Aldrich (St Louis, MO, USA), and tetraethyl orthosilicate (TEOS) was purchased from Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA). BE (99%) was obtained from Winherb Medical Technology (Shanghai, China). Aqueous ammonia and hydrochloric acid (37%) were purchased from VWR (Radnor, PA, USA) and RCI Labscan (Bangkok, Thailand), respectively. Penicillin streptomycin was obtained from Life Technologies (Thermo Fisher Scientific, Waltham, USA). Acridine orange (AO) and propidium iodide (PI) were procured from Solarbio (Beijing, China).

2.2 Fabrication and surface-modification of MSNs

MSNs (MCM-41) were synthesized as described in our recent study.34 Briefly, CTAB (0.36 g) was dissolved in distilled water (180 mL) at 30 °C, and aqueous ammonia (5 mL) as a catalyst was added dropwise to the solution. The silica source was prepared by suspending TEOS (1.56 mL) in ethanol (10 mL), and then it was added to the CTAB solution at a concentration of 1 mL min−1 with vigorous stirring for 5 h at room temperature. After overnight aging, the MSNs were collected by centrifugation at 6000 rpm for 10 min (Centrifuge 5804, Eppendorf, Hamburg, Germany). After removal of the surfactant with acidic ethanol, the MSNs were oven-dried for assessment of their morphology using an LEO1530 field emission scanning microscope (FE-SEM; Zeiss, Oberkochen, Germany) and Tecnai G2 20 S-TWIN transmission electron microscope (TEM; FEI, Hillsboro, USA). Afterwards, 0.5 g of MSNs was refluxed with 1.25 mL of APTES in ethanol (100 mL) overnight. The amine-modified MSNs (APTS-NPs) were harvested by centrifugation and subsequently oven-dried at 90 °C for further use. Their size (MSNs) and zeta potential (MSNs and APTS-NPs) were analyzed on a Nanotrac Wave (Microtrac, San Diego, CA, USA).

2.3 Loading and releasing efficacies of BA and BE

Briefly, 0.12 g of BA was dissolved in 80 mL of hot ethanol to obtain a clear solution with a light yellow color. Then, 0.2 g of APTS-NPs was dispersed in the solution which was left overnight with magnetic stirring. The nano-encapsulated BA (Nano-BA) was collected by centrifugation at 6000 rpm for 10 min and washed with ethanol twice. Referring to BE, its ethanol solution was freshly prepared by dissolving 0.05 g of BE in 40 mL of ethanol. 0.2 g of APTS-NPs was then dispersed in the BE solution following the procedures for BA loading. The final product was washed with ethanol twice and denoted as Nano-BE. The oven-dried Nano-BA and Nano-BE were assessed for their loading efficiencies on a TGA-6 thermogravimetric analyzer (TGA; PerkinElmer, Waltham, USA). With respect to their releasing profiles, Nano-BA (10 mg) and Nano-BE (10 mg) were each dispersed in 5 mL of distilled water and then shaken at 37 °C. At each time point, the supernatant was collected by centrifugation at 4000 rpm min−1 for 5 min and the Nano-BA or Nano-BE was re-suspended in 5 mL of fresh distilled water. In order to determine the concentrations of BA and BE, the absorbance of their supernatants was measured at 278 and 274 nm on a Cary UV-100 UV/visible absorption spectrometer (Agilent, Santa Clara, USA), respectively.

2.4 Cell cultures

Primary human gingival epithelial cells (hGECs) and their culture media (CnT-prime) were obtained from CELLnTEC (Stauffacherstrasse, Switzerland). The hGECs were cultured in CnT-PR media with 100 units per mL of penicillin and 100 mg per mL of streptomycin at 37 °C with 5% CO2 in a humidified incubator.

2.5 Cytotoxicity assays

The hGECs at passage 4 were seeded in a 96-well plate (2 × 104 cells per well) and 8-well chamber slides (NuncTM Lab-TekTM II Chamber SlideTM System, Thermo Scientific, Waltham, MA, USA) (4 × 104 cells per well) and further incubated for 2 days until reaching 80% confluence. Then, the cells were treated with various concentrations of APTS-NPs (12.5, 25, 50, 100 and 200 μg mL−1), Nano-BA (12.5, 25, 50, 100 and 200 μg mL−1), BA (3.6, 7.1, 14.2, 28.4 and 56.8 μg mL−1), Nano-BE (12.5, 25, 50, 100 and 200 μg mL−1) and BE (1.2, 2.4, 4.7, 9.4 and 18.8 μg mL−1) for 24 h (200 μL per well in a 96-well plate and 400 μL per well in 8-well chamber slides). Cellular viability was assessed using the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich, St Louis, USA) and the permeability of the cell membrane was evaluated using the Pierce Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (Thermo Fisher Scientific, Waltham, USA). Briefly, after 24 h incubation, 10× Lysis Buffer was added to the cells in the maximum LDH activity control groups and incubated for 45 min. 50 μL of the media in all the groups was then transferred into a new plate and incubated with 50 μL of reaction mixture for 30 min. After incubation, another 50 μL of stop solution was added in each well and the absorbance of the media was analyzed at 490 nm and 680 nm using a SpectraMax M2 Microplate Reader (Molecular Devices, California, USA). For the CCK-8 assay, the media containing nanoparticles or free drugs were replaced by fresh media containing CCK-8 (100 μL of media and 10 μL of CCK-8 reagent per well) and incubated with the cells for 2 h. The absorbance of the media was read at 450 nm using the same micro-plate reader. Three independent experiments were undertaken in triplicate. For the cells in the 8-well chamber slides, a live/dead assay was conducted using AO/PI staining. Briefly, AO and PI were dissolved in ethanol to prepare stock solutions with concentrations of 3 and 5 mg mL−1, respectively. 1 μL of each stock solution was added to 1 mL of phosphate buffered saline (PBS) to make a dye mixture, which was subsequently diluted ten times using cell culture medium to prepare the working solution. Prior to staining, the cell media containing nanoparticles or free drugs were discarded, and the cells were gently washed with Hanks’ Balanced Salt Solution (HBSS) three times. Subsequently, 300 μL of working solution was added to each chamber and incubated with the cells for 30 min. A Nikon Eclipse Ti–S microscope with 495 and 515 nm filters (Nikon, Tokyo, Japan) was used to observe the fluorescence of the stained cells.

2.6 Transmission electron microscopy

The hGECs were seeded in 60 mm TC-Treated Culture dishes (Corning, New York, USA) at a density of 5 × 105 cells per dish and incubated for 2 days until reaching 80% confluence. 5 mL of nanoparticle dispersion (APTS-NPs, Nano-BA or Nano-BE at 50 μg mL−1) or free drug solution (BA at 14.2 μg mL−1 or BE at 4.7 μg mL−1) was applied to the hGECs, respectively. Each treatment was repeated in two dishes. After 24 h pre-treatment of the nanoparticles or free drugs, the media were discarded and the cells were washed with HBSS twice. The cells in one dish were fixed in Karnovsky fixative for 1 h at room temperature, and the remaining cells were incubated in fresh medium containing IL-1β (1 ng mL−1) for another 24 h. The cells cultured in the blank medium for 48 h were considered as the control group. After incubation, all the cells were washed again and fixed in Karnovsky fixative as described previously. Afterwards, the fixed cells were scraped, centrifuged and washed with 0.1 M cacodylate buffer, and re-suspended with 1% osmium tetroxide in cacodylate buffer for post-fixation. The cells were subsequently embedded in agar, followed by dehydration in ethanol and infiltration with epoxy resin/propylene oxide (1[thin space (1/6-em)]:[thin space (1/6-em)]1) mixture. After polymerization at 60 °C overnight, the well-embedded samples were sectioned at 100 nm using Leica Ultracut UCT ultramicrotome (Leica, Wetzlar, Germany) and then observed under a Philips CM100 transmission electron microscope (Philips, Amsterdam, Netherlands).

2.7 Cytokine assays

The hGECs were seeded in a 24-well plate at a density of 4 × 105 cells per well and incubated for 2 days until reaching 80% confluence. The cells were then pre-treated with Nano-BA (100 and 200 μg mL−1), Nano-BE (25 and 50 μg mL−1), APTS-NPs (25, 50, 100 and 200 μg mL−1), BA (28.4 and 56.8 μg mL−1) and BE (2.4 and 4.7 μg mL−1). After 24 h, the media containing nanoparticles or free drugs were collected, and HBSS was applied to gently wash the cells twice. Then, the hGECs were sequentially cultured in the media with IL-1β (1 ng mL−1) for another 24 h. All the treatments were performed on three different occasions in triplicate. The cells cultured in blank media for 24 h with subsequent stimulation of IL-1β for 24 h were denoted as the controls. Those pre-treated with Nano-BA (200 μg mL−1) or Nano-BE (50 μg mL−1) followed by IL-1β stimulation were subjected to the cytokine array (Human XL Cytokine Array Kit, R&D Systems, Minneapolis, USA). Some of the supernatant from the three biological repeats was collected and pooled for the assay according to the array procedures. The data were analyzed using HLImage++ (Western Vision Software, US). Moreover, the levels of IL-6 and IL-8 in the supernatants from all groups were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Minneapolis, USA).

2.8 Statistical analysis

The significance of difference between the treated and control groups was determined by one-way analysis of variance with multi-comparisons by Tukey's test using GraphPad Prism 6.

3. Results and discussion

3.1 Characterization of the nanoparticles

The synthesized MSNs with a spherical shape presented regular and ordered pore structures in the TEM images, and exhibit relatively uniform sizes under SEM (Fig. 1A–E). In our recent study we measured the surface area and pore size of the synthesized MSNs (860.52 m2 g−1 and 2.86 nm), which could provide enough vacancies for encapsulating small drug molecules.34 The average diameter of the MSNs was 367 ± 94 nm and their zeta potential was −7.5 mV in water (Fig. 1F).
image file: c7nr02546g-f1.tif
Fig. 1 TEM images of the synthesized MSNs with a spherical shape (A and B) and ordered tunable pores (C and D) and their SEM (E) image. Their size distribution was analyzed by dynamic light scattering (DLS) (F).

After surface modification, the functional groups on the MSNs changed from hydroxyl to amine groups, which switched from a negative to a positive zeta potential (+7.16 mV in water). BA as a natural flavonoid compound with the carboxyl group could be attracted and then self-assembled with the amine centers of the modified MSNs via electrostatic interactions and hydrogen bonds.35,36 Its aglycone (i.e. baicalein) could also form hydrogen bonds with the amine nitrogen atom (Fig. 2A). These molecular interactions provide bases for drug loading and releasing from the amine-modified nanoparticles. After the loading, both Nano-BA and Nano-BE exhibited increased yellow color intensities with reference to the APTS-NPs, which indicates the successful nano-encapsulation of BA and BE (Fig. 2B). As shown in Fig. 2C, the weight loss of APTS-NPs was higher than that of MSNs (18.6% vs. 11.6%) between 100 °C and 700 °C, which is mainly due to the decomposition of alkanes and amine groups induced by surface-modification. The weight loss of Nano-BA increased to 47.0%, whereas the weight loss of pure BA was relatively stable above 560 °C as a result of complete decomposition. Thus, the loading efficiency of Nano-BA was 28.4% (loading efficiency = Weight loss%Nano-BA − Weight loss%APTS-NPs). Meanwhile, the weight loss of Nano-BE was 28.0% between 100 °C and 700 °C, which was eventually used to calculate the BE loading efficiency (9.4%), as described above.

image file: c7nr02546g-f2.tif
Fig. 2 Molecular structures of baicalin (BA) and its aglycone (baicalein, BE) (A). They could be encapsulated in the amine-modified MSNs (APTS-NPs), and display different color intensities (B). Thermal gravimetric analyses were carried out to evaluate the weight loss (%) of MSNs, APTS-NPs, Nano-BA, BA, Nano-BE and BE between 100 and 750 °C (C), whereas the cumulative release profiles of Nano-BA and Nano-BE were examined via UV/visible absorption spectroscopy up to 216 h (D).

Regarding the releasing mode, an initial burst of Nano-BA was observed at the first 48 h, and the nano-encapsulation prolonged the BA releasing efficiently up to 216 h (9 d) with a cumulatively released percentage of 89.8%, whereas, BE released from Nano-BE placidly and its cumulatively released percentage reached 41.7% at 216 h (Fig. 2D). The different releasing patterns observed might be relevant to the drug solubility. It is worthy to note that BA has very low water solubility (0.052 mg mL−1), and BE is almost insoluble in water.37 Their poor solubility severely limits the bioavailability of BA and BE both in in vitro and in vivo conditions. The notable effect of nano-encapsulation may shed new light on tackling these problems. BA was encapsulated into mesoporous amine-modified SBA-15 with a higher loading efficiency and releasing rate than the bare SBA-15, which shows that the amine modification could increase the delivery rate of BA from the mesoporous nanoparticles.38 The present study shows that the amine-functionalized MSNs (MCM-41) could facilitate drug delivery over 200 h. Similarly, nano-encapsulation also overcame the solubility problem of BE and greatly enhanced its delivery rate in water (Fig. 2D).

3.2 Cell viability

All the synthesized nanoparticles were evaluated for their cytotoxicity using the LDH assay, CCK-8 and AO/PE staining, and the cell status was observed by optical microscopy to verify the results of the cytotoxicity assay. As shown in Fig. 3A–C, APTS-NPs, BA and Nano-BA in a range of concentrations did not cause any cytotoxicity on the hGECs, and there was no significant increase in LDH level and decrease in cell viability in the treated groups. The bright-field cell images further support the observation of non-cytotoxic effects of APTS-NPs, BA and Nano-BA. Our previous study shows that bare MSNs at 200 μg mL−1 could significantly reduce the viability of hGECs,39 whereas APTS-NPs at the same concentration did not affect the cell viability (Fig. 3A-b). These findings reveal that amine surface modification with particle size enlargement and shift in the functional groups is basically cell-friendly. It has been reported that the amine-modification could increase the extent of cellular association by altering the surface charge of silica nanoparticles and subsequently reduce their cytotoxicity.40
image file: c7nr02546g-f3.tif
Fig. 3 The Viability of hGECs was assessed using lactate dehydrogenase (LDH) release (a), CCK-8 assay (b) and bright-field cell images (c). The cells were cultured with APTS-NPs (12.5–200 μg mL−1) (A), BA (3.6–56.8 μg mL−1) (B), Nano-BA (12.5–200 μg mL−1) (C), BE (1.2–8.8 μg mL−1) (D) and Nano-BE (12.5–200 μg mL−1) (E) for 24 h. Each assay was performed on three different occasions in triplicate, and the data are presented as mean ± SD. The asterisk indicates the significant differences between the treatment and control groups (p < 0.05). The scale bars in the bright-field cell images are 100 μm.

Interestingly, in the present study, BE at the concentrations of 9.4 and 18.8 μg mL−1 resulted in a dramatic decline in the production of the water-soluble orange colored formazan dye in the cells, which reflects an approximately 50% reduction in cell viability measured by the CCK-8 assay (Fig. 3D-b). Whereas, LDH release did not increase synchronously, which implies that there was no obvious change in cellular permeability (Fig. 3D-a). Moreover, there was no significant difference in the number of cells and their status and morphology between the BE-treated and control groups (Fig. 3D-c). Similar to BE, Nano-BE inhibited the formation of orange formazan within the cells in a concentration-dependent manner with a significant decrease in cell viability (Fig. 3E-b) but relatively stable LDH release (Fig. 3E-a). The bright-field images of the cells indicate that there was no remarkable change in cell numbers in Nano-BE-treated groups. Nonetheless, abundant cellular vacuoles were observed in the Nano-BE-treated groups (200 μg mL−1) and less in the groups treated with a low concentration of 100 μg mL−1. Therefore, Nano-BE with an even lower concentration (e.g. ≤50 μg mL−1) was deemed to be appropriate for the subsequent biological tests.

Apart from the assessment outcomes presented in Fig. 3, a fluorescence-based live-dead assay was conducted in all the testing groups. No significant difference was found between the control and test groups (Fig. S1). Regarding the reduction of cell viability analyzed by the CCK-8 assay in the BE- and Nano-BE-treated groups, it may not fully reflect the cytotoxicity because the other tests (e.g. LDH release, cell images and live/dead staining assay) did not show consistent changes in cell viability. Moreover, the WST-8 based assay (CCK-8) is an indirect approach to evaluate cell viability by analyzing the cellular activity of dehydrogenases and electron transfers.41 Hence, any factors that could affect the dehydrogenases and electron transport may interfere with the results of the CCK-8 assay. There are few studies on the potential variables accounting for the interference with the CCK-8 assay. We could speculate the possible reasons from the documented interference effects on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, since MTT and WST-8 have similar chemical structures and working mechanisms. It has been shown that a group of compounds with reductive potentials such as certain plant extracts and antioxidants could interfere the MTT assay. Additionally, the glycosides of flavones are less active than the aglyca, and consequently could induce a weak interference on the MTT assay.42 Moreover, a recent study has confirmed that graphene could function as an electron transfer agent and thereby disturb its cytotoxicity evaluation by the MTT and CCK-8 assays.43 Taken together, it could be speculated that the reductive structure of BE may to some extent affect the electron transfer to CCK-8, and its glycoside (BA) has relatively lower antioxidant activity with less interference with the CCK-8 assay. For BE, the notable interference with CCK-8 was only observed at the high concentrations of 9.4 and 18.8 μg mL−1, whereas all the Nano-BE treated groups displayed the interferential phenomenon. These accumulated findings suggest that Nano-BE presents favourable solubility and delivery rate as well as insignificant effects on cell viability.

3.3 Cellular uptake of Nano-BA and Nano-BE

Over the years, the cellular uptake and excretion of nanomaterials have been widely reported in different human cell lines. Generally, nanoparticles could enter and leave cells via endocytosis and exocytosis during incubation.44,45 The shapes, sizes and surface functional groups of these particles may affect their interactions with cells.46–48 It has been shown that the accumulation of nano-drug vehicles within cells could enhance therapeutic efficiency.49 Our recent study shows that hGECs could internalize the fluorescent-labelled MSNs and excrete them gradually in the nanoparticle-free medium.39 In the present study, the endocytosis of Nano-BA and Nano-BE by hGECs was confirmed via TEM (Fig. 4). After co-culture for 24 h, both Nano-BA and Nano-BE were presented as dark dots which could be internalized by the cells and trapped in the endosomes (Fig. 4A-i and ii and C-i and ii). Notably, none of the nanoparticles was found within the cell nuclei. After further incubation in nanoparticle-free media for 24 h, a certain amount of Nano-BA and Nano-BE remained in the cells (Fig. 4B-i and ii and D-i and ii). It is apparent that some Nano-BA or Nano-BE were capable of escaping the endosomes and appeared in the cytoplasm (Fig. 4B-ii and D-ii), and a similar observation was recorded for the cells treated with APTS-NPs (Fig. S2A and B). The endosomal escape could be due to the amine modification of these nanoparticles with a cationic ammonium surface. It has been proposed that such observation could be due to the proton sponge effect, fusion and photochemical disruption of the endosomal membrane.50 Therefore, Nano-BA, Nano-BE and APTS-NPs with a cationic surface have a buffering capacity and induce an osmotic pressure and thereby cause endosomal rupture, and eventually escape from the endosomes.51 Since cellular endocytosis and exocytosis usually occur simultaneously and dynamically, these activities may depend on the concentrations of nanoparticles both inside and outside the cells.52 Moreover, the cells pre-treated with BA and BE did not exhibit any abnormality with reference to the controls (Fig. S2C–F).
image file: c7nr02546g-f4.tif
Fig. 4 TEM images of hGECs incubated with Nano-BA (50 μg mL−1) and Nano-BE (50 μg mL−1) for 24 h (A and C, respectively) and then cultured in nanoparticle-free media containing IL-1β (1 ng mL−1) for another 24 h (B and D, respectively). The cells cultured in blank medium for 24 h followed by 24 h incubation in nanoparticle-free media containing IL-1β were used as controls (E). Afterwards, a large amount of Nano-BA and Nano-BE (black dots pointed by red arrows) appeared in the cells (A and B). Following another 24 h incubation in nanoparticle-free media containing IL-1β, a small amount of Nano-BA and Nano-BE exists (black dots pointed by red arrows) within the cells.

3.4 Nano-BA and Nano-BE modulated the expression of cytokines in hGECs

Cytokines critically contribute to cellular signaling and communication, thus the cytokine array with 105 targets was utilized to assess the anti-inflammatory effects of Nano-BA and Nano-BE in IL-1β-stimulated hGECs (Table S1). It is noteworthy that seven cytokines were remarkably down-regulated by both Nano-BA and Nano-BE, which were highlighted on the array membranes (Fig. 5A). These cytokines are to different extents involved in immuno-inflammatory responses. Among them, ENA-78, MCP-1, IL-8, MIP-3α and IP-10 function as chemokines and contribute to inflammation or tissue injury,53–57 whereas G-CSF and GM-CSF could stimulate the differentiation and proliferation of immune cells from hematopoietic stem cells.58 GROα acts as a chemokine with potent angiogenic activities and high expression in melanoma cells.59 The Nano-BA pre-treatment exhibited a stronger inhibitory effect on this chemokine than Nano-BE; however, more studies should be performed to clarify this point. Unlike Nano-BE, pre-treatment with Nano-BA was unable to suppress the IL-1β-stimulated expression of IL-6, IL-8 and G-CSF (Fig. 5B). Therefore, these two pro-inflammatory cytokines (IL-6 and IL-8) were further analyzed by ELISA kits.
image file: c7nr02546g-f5.tif
Fig. 5 Relative Expression of 105 cytokines in the supernatants of cells cultured for 24 h before (control) and after IL-1β treatment (control/IL-1β), and those pre-treated with Nano-BA or Nano-BE and then stimulated by IL-1β (Nano-BA/IL-1β and Nano-BE/IL-1β). Ten inflammatory cytokines with notable intensity change are highlighted in the array membrane (A). Their mean pixel densities were calculated using HLImage++, and the values were normalized to the reference spots. The fold changes of these cytokines in the control/IL-1β, Nano-BA/IL-1β and Nano-BE/IL-1β groups to the controls are plotted in a histogram (B).

As shown in Fig. 6A and B, the APTS-NPs at 100 and 200 μg mL−1, free BA and Nano-BA did not significantly induce an inflammatory response in hGECs, and it was also verified that the pre-treatment with Nano-BA did not significantly affect the IL-1β-induced expression of IL-6 and IL-8. On the other hand, APTS-NPs at 25 and 50 μg mL−1, free BE and Nano-BE did not stimulate the expression of IL-6 and IL-8 (Fig. 7A). However, Nano-BE exhibited potent inhibitory effects on the IL-1β-induced expression of IL-6 and IL-8 (Fig. 7B). Surprisingly, the expression of IL-6 was markedly down-regulated (around 90%) by 50 μg mL−1 of Nano-BE (p < 0.05), and IL-8 expression was dramatically suppressed by an even lower concentration (25 μg mL−1, p < 0.01) (Fig. 7B). In addition, APTS-NPs alone (25 and 50 μg mL−1) could to some extent inhibit IL-8 expression (p < 0.05) (Fig. 7), and free BE at 4.7 μg mL−1 enhanced the expression of IL-6 (p < 0.05). The original datasets of the IL-6 and IL-8 levels are presented in Fig. S3 and S4.

image file: c7nr02546g-f6.tif
Fig. 6 The fold changes of IL-6 (A) and IL-8 (B) in the supernatants from the cells with pre-treatments of APTS-NPs (100 and 200 μg mL−1), BA (28.4 and 56.8 μg mL−1) and Nano-BA (100 and 200 μg mL−1), and subsequent IL-1β stimulation (1 ng mL−1), with reference to the controls with (+) or without (−) such stimulation. The histograms represent the results of three independent experiments in triplicate. The data are presented as mean ± SD.

image file: c7nr02546g-f7.tif
Fig. 7 The fold changes of IL-6 (A) and IL-8 (B) in the supernatants from the cells with pre-treatments of APTS-NPs (25 and 50 μg mL−1), BE (2.4 and 4.7 μg mL−1) and Nano-BE (25 and 50 μg mL−1), and subsequent IL-1β stimulation (1 ng mL−1), with reference to the controls with (+) or without (−) such stimulation. The histograms represent three independent experiments in triplicate. Data are presented as mean ± SD. The asterisk and pound sign indicate the significant differences with a p-value less than 0.05 or 0.01 between the control and treatment groups, respectively.

BA and BE are well recognized as a pair of the most active components of S. baicalensis, and both have been extensively studied in various biomedical areas. Their anti-inflammatory capacity has attracted great attention for promising clinical application via novel delivery approaches. Over the years, a number of in vitro and animal studies have been carried out to elucidate their effects and working mechanisms in various inflammation models.60–66 Both BA and BE enable the alleviation of lipopolysaccharides (LPS)-induced inflammation through the NF-κB pathway.12,67 In the present study, hGECs were not highly responsive to Escherichia coli and P. gingivalis LPSs according to our preliminary experiments. Therefore, a pro-inflammatory cytokine (IL-1β) was used to treat hGECs. A previous study on the human retinal epithelial cell line utilized a similar approach to evaluate the anti-inflammatory effects of BA, BE and wogonin.68 Similarly, BE efficiently suppresses the expression of IL-6 and IL-8 mRNAs and proteins. It should be noted that special agents or solvents such as Na2CO3 and dimethyl sulfoxide have been used to prepare well-dissolved BA or BE stock solutions on account of their poor solubility.68–72 Thus, the utilization of nanoparticles could appropriately address this critical issue and greatly enhance the feasibility and efficiency of drug delivery.73–75 Our present study has indeed shown the great advantage of MSNs-encapsulation of BE for the development of potential immuno-inflammatory modulators through a refined delivery system.

4. Conclusions

In the present study, both BA and BE were encapsulated by amine-modified MSNs, and their nano-encapsulated forms can facilitate drug delivery through a sustained release mode. Nanoparticle-encapsulated BA and BE could be internalized and retained in hGECs for at least 24 h. Notably, Nano-BE effectively alleviates the IL-1β-induced expression of pro-inflammatory cytokines. This pioneering study may contribute to the development of nano-based novel drug delivery systems for improving oral healthcare.


This work was supported by the General Research Fund (GRF) from the Hong Kong Research Grants Council (HKU767512 and 17155216 to L. J. Jin and HKBU201213 to K. C.-F. Leung), and the Modern Dental Laboratory/HKU Endowment Fund to L. J. Jin. We thank Ms. Xinmiao Lan from The University of Hong Kong as well as Dr Elaine Wat and Prof. Clara Bik San Lau from The Chinese University of Hong Kong for their technical support.

Notes and references

  1. L. J. Jin, I. B. Lamster, J. S. Greenspan, N. B. Pitts, C. Scully and S. Warnakulasuriya, Oral Dis., 2016, 22, 609–619 CrossRef CAS PubMed.
  2. B. L. Pihlstrom, B. S. Michalowicz and N. W. Johnson, Lancet, 2005, 366, 1809–1820 CrossRef.
  3. M. Tonetti and K. S. Kornman, J. Clin. Periodontol., 2013, 40, S1–S214 CrossRef PubMed.
  4. G. Hajishengallis, Nat. Rev. Immunol., 2015, 15, 30–44 CrossRef CAS PubMed.
  5. P. M. Bartold and T. E. Van Dyke, Periodontol. 2000, 2013, 62, 203–217 CrossRef PubMed.
  6. Z. Gao, K. Huang and H. Xu, Pharmacol. Res., 2001, 43, 173–178 CrossRef CAS PubMed.
  7. Z. Gao, K. Huang, X. Yang and H. Xu, Biochim. Biophys. Acta, Gen. Subj., 1999, 1472, 643–650 CrossRef CAS.
  8. M. Himeji, T. Ohtsuki, H. Fukazawa, M. Tanaka, S. Yazaki, S. Ui, K. Nishio, H. Yamamoto, K. Tasaka and A. Mimura, Cancer Lett., 2007, 245, 269–274 CrossRef CAS PubMed.
  9. W. H. Huang, A. R. Lee and C. H. Yang, Biosci., Biotechnol., Biochem., 2006, 70, 2371–2380 CrossRef CAS PubMed.
  10. T. Kumagai, C. I. Müller, J. C. Desmond, Y. Imai, D. Heber and H. P. Koeffler, Leuk. Res., 2007, 31, 523–530 CrossRef CAS PubMed.
  11. D. Y. Zhang, J. Wu, F. Ye, L. Xue, S. Jiang, J. Yi, W. Zhang, H. Wei, M. Sung and W. Wang, Cancer Res., 2003, 63, 4037–4043 CAS.
  12. G. W. Fan, Y. Zhang, X. Jiang, Y. Zhu, B. Wang, L. Su, W. Cao, H. Zhang and X. Gao, Inflammation, 2013, 36, 1584–1591 CrossRef CAS PubMed.
  13. C. P. Chang, W. T. Huang, B. C. Cheng, C. C. Hsu and M. T. Lin, Neuropharmacology, 2007, 52, 1024–1033 CrossRef CAS PubMed.
  14. S. F. Chen, C. W. Hsu, W. H. Huang and J. Y. Wang, Br. J. Pharmacol., 2008, 155, 1279–1296 CrossRef CAS PubMed.
  15. C. J. Hsieh, K. Hall, T. Ha, C. Li, G. Krishnaswamy and D. S. Chi, Clin. Mol. Allergy, 2007, 5, 5 CrossRef PubMed.
  16. H. A. Lim, E. K. Lee, J. M. Kim, M. H. Park, D. H. Kim, Y. J. Choi, Y. M. Ha, J.-H. Yoon, J. S. Choi and B. P. Yu, Biogerontology, 2012, 13, 133–145 CrossRef CAS PubMed.
  17. L. Zeng, J. Dong, W. Yu, J. Huang, B. Liu and X. Feng, Pulm. Pharmacol. Ther., 2010, 23, 411–419 CrossRef CAS PubMed.
  18. Y. C. Shen, W. F. Chiou, Y. C. Chou and C. F. Chen, Eur. J. Pharmacol., 2003, 465, 171–181 CrossRef CAS PubMed.
  19. L. Yang, H. Sun, L. Wu, X. Guo, H. Dou, M. O. M. Tso, L. Zhao and S. Li, Invest. Ophthalmol. Visual Sci., 2009, 50, 2319–2327 Search PubMed.
  20. J. Zhu, J. Wang, Y. Sheng, Y. Zou, L. Bo, F. Wang, J. Lou, X. Fan, R. Bao and Y. Wu, PLoS One, 2012, 7, e35523 CAS.
  21. W. Wang, M. Xi, X. Duan, Y. Wang and F. Kong, Int. J. Nanomed., 2015, 10, 3737–3750 CAS.
  22. J. Xing, X. Chen and D. Zhong, Life Sci., 2005, 78, 140–146 CrossRef CAS PubMed.
  23. J. Hao, F. Wang, X. Wang, D. Zhang, Y. Bi, Y. Gao, X. Zhao and Q. Zhang, Eur. J. Pharm. Sci., 2012, 47, 497–505 CrossRef CAS PubMed.
  24. Z. Liu, X. Zhang, H. Wu, J. Li, L. Shu, R. Liu, L. Li and N. Li, Drug Dev. Ind. Pharm., 2011, 37, 475–481 CrossRef CAS PubMed.
  25. Z. Liu, H. Zhao, L. Shu, Y. Zhang, C. Okeke, L. Zhang, J. Li and N. Li, Drug Dev. Ind. Pharm., 2015, 41, 353–361 CrossRef CAS PubMed.
  26. V. N. Babu and S. Kannan, Int. J. Biol. Macromol., 2012, 51, 1103–1108 CrossRef PubMed.
  27. M. J. Tsai, P. C. Wu, Y. B. Huang, J. S. Chang, C. L. Lin, Y. H. Tsai and J. Y. Fang, Int. J. Pharm., 2012, 423, 461–470 CrossRef CAS PubMed.
  28. I. I. Slowing, B. G. Trewyn, S. Giri and V. S. Y. Lin, Adv. Funct. Mater., 2007, 17, 1225–1236 CrossRef CAS.
  29. I. I. Slowing, J. L. Vivero-Escoto, C. W. Wu and V. S. Y. Lin, Adv. Drug Delivery Rev., 2008, 60, 1278–1288 CrossRef CAS PubMed.
  30. F. Tang, L. Li and D. Chen, Adv. Mater., 2012, 24, 1504–1534 CrossRef CAS PubMed.
  31. Q. L. Li, Y. Sun, Y. L. Sun, J. Wen, Y. Zhou, Q. M. Bing, L. D. Isaacs, Y. Jin, H. Gao and Y. W. Yang, Chem. Mater., 2014, 26, 6418–6431 CrossRef CAS PubMed.
  32. N. Song and Y. W. Yang, Chem. Soc. Rev., 2015, 44, 3474–3504 RSC.
  33. Y. W. Yang, MedChemComm, 2011, 2, 1033–1049 RSC.
  34. X. Li, C. H. Wong, T. W. Ng, C. F. Zhang, K. C. F. Leung and L. J. Jin, Int. J. Nanomed., 2016, 11, 2471–2480 CrossRef CAS PubMed.
  35. M. Manzano, V. Aina, C. Arean, F. Balas, V. Cauda, M. Colilla, M. Delgado and M. Vallet-Regi, Chem. Eng. J., 2008, 137, 30–37 CrossRef CAS.
  36. S. Budi Hartono, S. Qiao, K. Jack, B. P. Ladewig, Z. Hao and G. Lu, Langmuir, 2009, 25, 6413–6424 CrossRef PubMed.
  37. H. Chen, Y. Gao, J. Wu, Y. Chen, B. Chen, J. Hu and J. Zhou, Cancer Lett., 2014, 354, 5–11 CrossRef CAS PubMed.
  38. H. Wang, X. Gao, Y. Wang, J. Wang, X. Niu and X. Deng, Ceram. Int., 2012, 38, 6931–6935 CrossRef CAS.
  39. X. Li, K. Y. Pang, T. W. Ng, P. C. Leung, C. F. Zhang, K. C. F. Leung and L. J. Jin, Nanomaterials, 2016, 6, 192 CrossRef PubMed.
  40. T. Yu, A. Malugin and H. Ghandehari, ACS Nano, 2011, 5, 5717–5728 CrossRef CAS PubMed.
  41. M. Ishiyama, Y. Miyazono, K. Sasamoto, Y. Ohkura and K. Ueno, Talanta, 1997, 44, 1299–1305 CrossRef CAS PubMed.
  42. R. Bruggisser, K. von Daeniken, G. Jundt, W. Schaffner and H. Tullberg-Reinert, Planta Med., 2002, 68, 445–448 CrossRef CAS PubMed.
  43. G. Jiao, X. He, X. Li, J. Qiu, H. Xu, N. Zhang and S. Liu, RSC Adv., 2015, 5, 53240–53244 RSC.
  44. X. Huang, X. Teng, D. Chen, F. Tang and J. He, Biomaterials, 2010, 31, 438–448 CrossRef CAS PubMed.
  45. I. I. Slowing, J. L. Vivero-Escoto, Y. Zhao, K. Kandel, C. Peeraphatdit, B. G. Trewyn and V. S. Y. Lin, Small, 2011, 7, 1526–1532 CrossRef CAS PubMed.
  46. Arnida, A. Malugin and H. Ghandehari, J. Appl. Toxicol., 2010, 30, 212–217 CAS.
  47. S. Jambhrunkar, Z. Qu, A. Popat, J. Yang, O. Noonan, L. Acauan, Y. Ahmad Nor, C. Yu and S. Karmakar, Mol. Pharm., 2014, 11, 3642–3655 CrossRef CAS PubMed.
  48. F. Lu, S. H. Wu, Y. Hung and C. Y. Mou, Small, 2009, 5, 1408–1413 CrossRef CAS PubMed.
  49. Z. Xu, L. Chen, W. Gu, Y. Gao, L. Lin, Z. Zhang, Y. Xi and Y. Li, Biomaterials, 2009, 30, 226–232 CrossRef CAS PubMed.
  50. A. K. Varkouhi, M. Scholte, G. Storm and H. J. Haisma, J. Controlled Release, 2011, 151, 220–228 CrossRef CAS PubMed.
  51. C. Lin and J. F. Engbersen, J. Controlled Release, 2008, 132, 267–272 CrossRef CAS PubMed.
  52. Z. Q. Chu, Y. J. Huang, Q. Tao and Q. Li, Nanoscale, 2011, 3, 3291–3299 RSC.
  53. S. L. Deshmane, S. Kremlev, S. Amini and B. E. Sawaya, J. Interferon Cytokine Res., 2009, 29, 313–326 CrossRef CAS PubMed.
  54. R. E. Gerszten, E. A. Garcia-Zepeda, Y. C. Lim, M. Yoshida, H. A. Ding, M. A. Gimbrone, A. D. Luster, F. W. Luscinskas and A. Rosenzweig, Nature, 1999, 398, 718–723 CrossRef CAS PubMed.
  55. A. Sauty, M. Dziejman, R. A. Taha, A. S. Iarossi, K. Neote, E. A. Garcia-Zepeda, Q. Hamid and A. D. Luster, J. Immunol., 1999, 162, 3549–3558 CAS.
  56. M. Schmuth, S. Neyer, C. Rainer, A. Grassegger, P. Fritsch, N. Romani and C. Heufler, Exp. Dermatol., 2002, 11, 135–142 CrossRef CAS PubMed.
  57. A. Walz, P. Schmutz, C. Mueller and S. Schnyder-Candrian, J. Leukocyte Biol., 1997, 62, 604–611 CAS.
  58. Y. Zhan, G. J. Lieschke, D. Grail, A. R. Dunn and C. Cheers, Blood, 1998, 91, 863–869 CAS.
  59. P. Dhawan and A. Richmond, J. Leukocyte Biol., 2002, 72, 9–18 CAS.
  60. Y. C. Chen, S. C. Shen, L. G. Chen, T. J. F. Lee and L. L. Yang, Biochem. Pharmacol., 2001, 61, 1417–1427 CrossRef CAS PubMed.
  61. T. Hong, G. B. Jin, S. Cho and J. C. Cyong, Planta Med., 2002, 68, 268–271 CrossRef CAS PubMed.
  62. W. Lee, S. K. Ku and J. S. Bae, Inflammation, 2015, 38, 110–125 CrossRef CAS PubMed.
  63. B. Q. Li, T. Fu, W. H. Gong, N. Dunlop, H. F. Kung, Y. Yan, J. Kang and J. M. Wang, Immunopharmacology, 2000, 49, 295–306 CrossRef CAS PubMed.
  64. C. C. Lin and D. E. Shieh, Am. J. Chin. Med., 1996, 24, 31–36 CrossRef CAS PubMed.
  65. Y. C. Shen, W. F. Chiou, Y. C. Chou and C. F. Chen, Eur. J. Pharmacol., 2003, 465, 171–181 CrossRef CAS PubMed.
  66. K. J. Woo, J. H. Lim, S. I. Suh, Y. K. Kwon, S. W. Shin, S. C. Kim, Y. H. Choi, J. W. Park and T. K. Kwon, Immunobiology, 2006, 211, 359–368 CrossRef CAS PubMed.
  67. S. J. Dong, Y. Q. Zhong, W. T. Lu, G. H. Li, H. I. Jiang and B. Mao, Inflammation, 2015, 38, 1493–1501 CrossRef CAS PubMed.
  68. N. Nakamura, S. Hayasaka, X.-Y. Zhang, Y. Nagaki, M. Matsumoto, Y. Hayasaka and K. Terasawa, Exp. Eye Res., 2003, 77, 195–202 CrossRef CAS PubMed.
  69. C. C. Lai, P. H. Huang, A. H. Yang, S. C. Chiang, C. Y. Tang, K. W. Tseng and C. H. Huang, Am. J. Chin. Med., 2016, 44, 531–550 CrossRef CAS PubMed.
  70. X. Ma, W. Yan, Z. Dai, X. Gao, Y. Ma, Q. Xu, J. Jiang and S. Zhang, Drug Des., Dev. Ther., 2016, 10, 1419 CrossRef PubMed.
  71. A. Palko-Labuz, K. Sroda-Pomianek, A. Uryga, E. Kostrzewa-Suslow and K. Michalak, Biomed. Pharmacother., 2017, 88, 232–241 CrossRef CAS PubMed.
  72. L. L. Yang, N. Xiao, J. Liu, K. Liu, B. Liu, P. Li and L. W. Qi, Biochim. Biophys. Acta, Mol. Basis Dis., 2017, 1863, 598–606 CrossRef CAS PubMed.
  73. S. Guo, Y. Wang, L. Miao, Z. Xu, C. M. Lin, Y. Zhang and L. Huang, ACS Nano, 2013, 7, 9896–9904 CrossRef CAS PubMed.
  74. J. Kipp, Int. J. Pharm., 2004, 284, 109–122 CrossRef CAS PubMed.
  75. R. Singh and J. W. Lillard, Exp. Mol. Pathol., 2009, 86, 215–223 CrossRef CAS PubMed.


Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nr02546g

This journal is © The Royal Society of Chemistry 2017