Immunoengineering with Ginseng Polysaccharide Nanobiomaterials

Article Cite This: ACS Biomater. Sci. Eng. 2019, 5, 2916−2925

pubs.acs.org/journal/abseba

Immunoengineering with Ginseng Polysaccharide Nanobiomaterials through Oral Administration in Mice Kazi Farida Akhter,†,‡ Md Abdul Mumin,† Edmund M. K. Lui,‡ and Paul A. Charpentier*,†,§ †

Chemical and Biochemical Engineering, ‡Physiology and Pharmacology, §Biomedical Engineering, University of Western Ontario, London, Ontario N6A 5B9, Canada

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S Supporting Information *

ABSTRACT: Plant polysaccharides (PS) such as American ginseng polysaccharide (GPS) have drawn immense interest in the field of immunoengineering, as they offer a way to actively control immune cell behavior and stimulation. These pharmacological activities have been limited by PS’s inherent physicochemical properties including large molecular size, heterogeneity, and poor solubility. In this work, we hypothesized that by nanosizing and encapsulating GPSs, we could enhance their immunomodulation by increased penetration and absorption through the GI tract. Herein, GPS nanoparticles (NPs) of average size 20 nm (± 4 nm) were prepared using a microfluidic approach, then encapsulated within porous nanospheres (diameter 180 ± 10 nm) of biodegradable gelatin to enhance their oral delivery. To locate the GPS NPs inside the gelatin, we encapsulated fluorescent-labeled GPS in gelatin and analyzed using confocal microscopy. An in vitro investigation on tumor induced macrophage cell lines showed a concentration dependent enhanced immunostimulation with the encapsulated GPS NPs. The immunomodulation was then studied for different formulations of GPS through oral gavage in Swiss albino mice. The results showed that the production of proinflammatory mediators in blood samples was significantly increased for the encapsulated GPS in a dose- and time-dependent manner compared to other GPS treatments. This study shows that GPS and potentially other PS systems’ immunomodulation properties can be significantly enhanced for use in simple oral drug delivery. KEYWORDS: polysaccharides, nanoparticles, encapsulation, oral delivery, immunomodulation, controlled delivery

1. INTRODUCTION Immunoengineering is an emerging field that links biology, pharmaceutics, and biomedicine with engineering to develop new strategies for enhancing disease control ranging from cancer to infectious diseases to tissue regeneration. The immune system plays a critical role in defending the body against infectious organisms and invaders. It protects the body from the common cold to deadly diseases, from common influenza to HIV and cancer.1 The complexity of the immune system has given rise to an emerging field, immunoengineering, which works at the interface of materials engineering and immunology to develop tunable and modular materials that regulate the cells/organs of the immune system.1,2 The recent state-of-the-art concepts are nanosizing, encapsulation, and bioconjugation of macromolecules or biomaterials to modulate the immune function.2−4 Plant polysaccharides (PS) are an important class of biomaterials of immense interest possessing a wide range of biological and immunological activity. In the last few decades, numerous PS have been isolated from plants showing diverse therapeutic properties including immunomodulatory, antitumoral, hypoglycemic, wound healing, antiaging, and antioxidant effects.5,6 Of the various plants, ginseng PS (GPS) is one © 2019 American Chemical Society

of the most bioactive PS types reported for having diverse biological activities including immunomodulatory, anticancer, antidiabetic, antiviral, antibacterial, cytoprotective, radioprotective, antidepressant, antifatigue, and antioxidant effects.7,8 These bioactive PS have been shown to upregulate the production of cytokines and antibodies, promote the activity of antioxidant enzymes, and scavenge free radicals. They also hinder tumor cell proliferation, viruses entering and replicating in cells, and foster tumor cell apoptosis. All of these biological activities significantly influence the immune response.6 Despite having such significant pharmacological activities, PS and GPS have limited bioavailability because of their physicochemical properties including large molecular size, short biological halflives, and poor water solubility. Oral delivery is one of the most popular and facile methods for drug or bioactive ingredient delivery. Oral administration and pharmacological effects of ginseng extracts have been studied by several research groups.9,10 Moreover, degradation of bioactive compounds by the gastrointestinal (GI) tract Received: March 12, 2019 Accepted: May 7, 2019 Published: May 7, 2019 2916

DOI: 10.1021/acsbiomaterials.9b00348 ACS Biomater. Sci. Eng. 2019, 5, 2916−2925

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ACS Biomaterials Science & Engineering

Scheme 1. Schematic Illustration: (Step 1) Extraction, Nanosizing, Fluorescent Labeling, and Encapsulation of Ginseng PS (GPS); (Step 2) Nanoencapsulation of GPS in Gelatin by Two-Step Desolvation Processa; (step 3): oral administration of GPS on Swiss albino male mice and intracardiac blood collection. Four different treatment groups were used (a) native GPS, (b) GPS NPs (c) labeled GPS NPs and (d) encapsulated GPS NPs

a

GTA, glutaraldehyde; LMW, low molecular weight, HMW-high molecular weight).

enzymes and poor permeability through the intestinal mucosa have been shown to decrease their bioavailability.11 Ginseng extracts have shown high biliary excretion, elimination in the liver, decomposition in the stomach, and metabolism in the large intestine.12 The bioavailability of PS extracted from Radix ophiopogonis (Mw = 4.8 kDa) (a medicinal plant) was studied and it was found that only 1.7% was available in rats after oral administration.13 This low bioavailability was attributed to the large molecular size and hydrophilic characteristics of the extracted PS. By engineering the morphology of GPS and the delivery system, we hypothesize that we can overcome these limitations. Previously, we reported the microfluidic synthesis of GPS nanoparticles (NPs) which showed an enhanced immunostimulating activity of macrophage cell lines compared to native GPS.14 Compared with the larger sized micrometer particles, the NPs were found to more effectively penetrate the submucosal layers, resulting in an enhancement of drug delivery efficiency.15 Herein, for the first time, a facile in situ approach is developed to encapsulate the GPS NPs within a biocompatible nanocarrier of gelatin using a water-soluble cross-linking agent. The morphology, particle size, swelling property, PS release, cross-linking degree, and pH sensitivity of the encapsulated products were characterized using advanced analytical tools and techniques. Furthermore, in vitro biological studies including cytotoxicity and immunostimulating activity of the gelatin encapsulated GPS NPs are investigated using

murine macrophage cell lines. Finally, oral administration of GPS NPs and encapsulated GPS NPs was investigated using an animal model (Swiss albino male mice), and the immunostimulating activity was studied by quantifying the production of reactive oxygen species (NO) and organic cytokine tumor necrosis factor (TNF-α).

2. EXPERIMENTAL SECTION 2.1. Materials, Reagents, and Animals. American ginseng (Panax quinquefolius) roots were supplied by the Canadian Centre for Agri-food Research in Health and Medicine (CCARM). Gelatin (Type A), fluorescein-5-thiosemicarbazide (FTSC), glacial acetic acid, glutaraldehyde (25% aqueous solution), sodium cyanoborohydride, phosphate buffered solution (PBS, pH 7.4), dimethyl sulfoxide (DMSO), Griess reagent, and lipopolysaccharides (Escherichia coli 0111: B4) were purchased from Sigma-Aldrich, Canada. Milli-Q water purification system (Barnstead EasyPureII, Thermo Scientific, USA) was used to produce purified water. All the in vitro studies were carried out using RAW 264.7 (ATCC TIB 67) murine macrophage cell lines in Professor Ed Lui’s Lab (Physiology and Pharmacology, Western University, Canada). ELISA kits (interleukin-1β, tumor necrosis factor-α, Interleukin-6) were purchased from BD Biosciences (Bedford, MA, USA). DMEM (Dulbecco’s modified Eagle’s medium), FBS (fetal bovine serum), penicillin, glutamine, streptomycin were purchased from Gibco Laboratories (USA). Swiss albino male mice (average weight 21g) were purchased from Charles River, St. Constant, QC, Canada and housed in a temperature−humidity controlled facility operating at a 12 h light and dark cycle. Animals were provided food and water ad libitum. Mice were used in 2917


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ACS Biomaterials Science & Engineering accordance with the guidelines of the Canadian Council on Animal Care. The study protocol (protocol no. 2009−070) was approved by the Animal Care Committee of Western University, Canada. 2.2. Nanosizing and Fluorescent Labeling of Ginseng Polysaccharides. Ginseng polysaccharides used in this study was extracted from Panax quinquefolius root as described in our previous work.14,16 A microfluidic device with a T-junction (based on precipitation mechanism) was used to synthesize GPS NPs as previously reported.14 Aqueous GPS and acetone (as an antisolvent) were pumped through two separate infusion syringe pumps. The flow rate of acetone and GPS solution was maintained at 0.2 mL/min and 0.01 mL/min, respectively.14 Labeling of GPS NPs with fluorescence dye (FTSC) in cyanoborohydride solution was carried out following our previously developed method.17 2.3. Gelatin Encapsulation of Ginseng Polysaccharide NPs. Gelatin NPs were fabricated using a modified two-step desolvation method.18,19 At first, gelatin (type A) was dissolved in water (5 wt %/v) at 45 °C. Then, 75 mL of acetone was added to the solution to precipitate high-molecular-weight (HMW) gelatin. This precipitate was separated and used in the second desolvation step, in which 20 mL of acidic solution (pH 2.5) was used to redissolve the HMW gelatin portion at 45 °C under continuous stirring. Thereafter, GPS NPs (0.125g) were added to the gelatin solution. Next, acetone (75 mL) was added dropwise to the gelatin solution. The cross-linker, glutaraldehyde (four different weight ratios of 0.01, 0.02, 0.035, and 0.05% were used) was then added to the reaction mixture to form the stable gelatin NPs. The synthesized NPs were collected via centrifugation at 6000 rpm and 4 °C. The formed gelatin NPs were purified by washing with water and acetone solution (7:3) for five times and finally lyophilized using a freeze-dryer. The process is shown in Scheme 1, step 2. 2.4. Material Characterization. The surface morphology and size of the prepared GPS NPs (encapsulated and nonencapsulated) were characterized using transmission electron microscopy (TEM) (Philips CM10) and Scanning electron microscopy (SEM) (LEO 1530). SEM samples were prepared by depositing the dry powder sample on a carbon adhesive tape mounted on a sample holder. Then, these samples were coated with a thin layer of Osmium in a vacuum chamber. TEM samples were made by applying the suspension of sample on copper grids coated with carbon film. Dynamic light scattering (DLS) (Nano S model ZEN 1600, Malvern, Orsay, France) was used to evaluate the size distribution of the gelatin and GPS NPs. Zeta potential of NPs was investigated using Malvern zeta-sizer 3000HSA. The presence of functional groups in gelatin, GPS NPs and GPS NPs loaded gelatin were examined using Fourier-transform infrared spectroscopy (FTIR) (Nicolet 6700) with wavenumber range (500−4000 cm−1). The Brunauer−Emmett−Teller (BET) method (Micromeritics TriStar II 3020) was used to determine the surface area, pore size and pore volume of different types of gelatin NPs. Before running BET experiments, the tested materials were degassed overnight at 150 °C. To investigate the crystallinity of gelatin NPs, an X-ray powder diffractometer (Bruker D2 phaser) was used. The samples were analyzed using Cu Kα radiation (λ for Kα = 1.54059 Å) at 30 kV and 10 mA, with a scan range of 2θ = 10−80°. The inverted confocal laser scanning microscope (CLSM) (Zeiss LSM 510 Duo) was used to characterize the fluorescent labeled GPS loaded gelatin NPs to confirm the presence of GPS within the carrier. Confocal analysis was carried out using an argon laser at 488 nm. 2.5. Releasing and Swelling Study. The release rate of GPS NPs from gelatin was measured using a UV−vis spectrophotometer (Shimadzu UV-3600). A calibration curve was prepared to represent the correlation of GPS concentration with respective absorbance intensity by UV−vis spectroscopy. The releasing kinetics were studied for different ratios of glutaraldehyde to gelatin, and changing pH (1, 4.5, and 7.4). Gelatin is known to be a “pH-sensitive hydrogel”, so they swell/contract depending on the change of solution pH. Therefore, the swelling study was carried out with different pH conditions and with different cross-linker ratios (during synthesis) using the following equation.

swelling ratio =

Ws − Wd 100% Wd

(1)

Where Wd and Ws represent the weight of dried and swollen gelatin NPs, respectively. The experiments were performed in triplicate. 2.6. Encapsulation Efficiency. The amount of GPS within gelatin NPs was measured using UV−vis spectroscopy. Five mg samples were added to 10 mL PBS solution (pH 7.4) and stirred continuously. After 24 h, the supernatant was collected from the gelatin in PBS dispersion via centrifugation and measured GPS content using UV−vis spectroscopy. The following equations were used to calculate the percentage of GPS loading and their encapsulation efficiency. The experiments were performed in triplicate.

drug‐loading efficiency =

amount of drug in NPs 100% amount of drug‐loaded NPs (2)

=

drug 100% drug + NPs

(3)

encapsulation efficiency amount of encapsulated drug = 100% amount of drug used for NP preparation

(4)

2.7. In Vitro Study. RAW 264.7 macrophages were seeded in a 96-well plate at a density of 1.5 × 105 cells per well and cultured in media at 37 °C and 5% CO2. Macrophages were treated with different concentrations (0, 25, 50, and 200 μg/mL) of encapsulated and nonencapsulated GPS NPs for 24 h to estimate the dose-related immunostimulation response of the cell lines. LPS was used as a positive control during the experiments. Then, macrophage response to encapsulated or nonencapsulated NPs were determined by quantification of nitric oxide (NO), interleukin-1β (IL-1 β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) in the cultured medium. The quantification of NO production in culture medium or blood serum was determined using a Griess assay. Fifty microliters of cell culture supernatant or blood serum was added to the 50 μL of Griess reagent in a 96-well plate. The absorbance of the colored complex was measured at 550 nm using a microplate reader (Thermo Fisher Scientific, Finland) with SkanIt software (version 2.4.2, Thermo Fisher Scientific, Finland). Sample NO concentrations were calculated from the sodium nitrite standard curve. IL-1β, IL-6, and TNF-α production were quantified in cultured supernatants and blood serum with respective ELISA kits based on the “multiple antibody sandwich principle.”20 First, a 96 well-plate was precoated with polyclonal antibody and then cultured supernatants or blood serum was added to the plate. After 2 h incubation, the culture was washed to remove unbound substances. Next, polyclonal antimurine antibody conjugated to horseradish was added to the plate and incubated at room temperature. After 2 h, the culture wash washed again to ensure no unbound antibody-enzyme reagent remained. Finally, peroxidase substrate (tetramethyl benzidine) was added to the plate and the reaction was stopped after 30 min. The absorbance of the colored complex was measured at 450 nm using a microplate reader. Cell viability study was carried out using the 3-(4,5-dimethythiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay, to investigate in vitro toxicity of the gelatin particle. RAW 264.7 macrophages were cultured in a 96-well plate at a density of 1.0 × 104 cells per wall for 24 h at 37 °C and 5% CO2. Next, 100 μL of gelatin NPs were added to the well plate and incubated for another 24, 48, and 72 h. The cultured medium was then transferred to another 96-well plate and 150 μL of MTT solution (0.5 mg/mL) was added to the plate. After 4 h, media was removed from the well and 100 μL of DMSO was added in each well. Finally, the absorbance of the colored complex was measured at 540 nm using the microplate reader. Cellular uptake study was further carried out with the FTSC-labeled GPS-loaded gelatin NPs using flow cytometry for quantitative analysis. Here, the macrophage cell lines 2918


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Figure 1. (a) TEM image of GPS NPs, (b) SEM image of GPS-loaded gelatin nanospheres, (c) TEM image of GPS-loaded gelatin nanospheres, (d) size distribution of GPS NPs using ImageJ, (e) size distribution of gelatin nanospheres using DLS, (f) size distribution of gelatin nanospheres using ImageJ, and (g) a model of the developed drug delivery system. were seeded at a density 5 × 105 cells/ml in a 12 well dish. The NPs were dispersed in DMSO and prepared at concentrations of 10, 20, and 50 μg/mL. Here, cell culture media was used as a control. The cells were stained with a surface marker CD11c. After 24 h, the treated cells were monitored by flow cytometry using a BD BioSciences FACSCalibur and imaged using a Leica DMIRE2 fluorescence microscope. 2.8. In Vivo Study. Adult Swiss albino male mice were divided into six groups containing five mice each; (i) native GPS, (ii) GPS NPs, (iii) labeled GPS NPs, (iv) encapsulated GPS NPs, (v) sham control (vehicle). The treatment group received 50 mg/kg of GPS materials (dissolved in water) once daily for 3 or 6 consecutive days by oral gavage. The sham control group received water, once daily by oral gavage. After the treatment, mice were anesthetized with an intraperitoneal injection of (6 mg/kg) xylazine and (100 mg/kg) ketamine solution. After being anaesthetized, intra cardiac blood samples were collected. 2.9. Statistical Analysis. Data are presented as mean ± standard deviation (SD). The two-way analysis of variance (ANOVA) followed by Bonferroni’s posthoc test was used for comparison of the means with the untreated control group. The one-way analysis of variance (ANOVA) followed by Dunnett’s posthoc test was used for comparison of the means with the untreated control. Statistical analysis was performed using GraphPad Prism 8 software (San Diego, CA). Values P < 0.05 compared between treatments groups were considered statistically significant.

desolvation method for the sustained release of GPS. Nowadays, nanocarriers have shown unique physical and biological properties that can be applied to enhance the efficacy of the drug delivery system. The size and shape of the prepared gelatin NPs were observed using SEM and TEM (Figure 1 b,c). From these micrographs, it is clearly seen that the gelatin NPs are smooth and spherical. There is no significant heterogeneity appearing on the NPs surface. Moreover, there are no differences in size and morphology between the GPS loaded gelatin NPs and bare gelatin NPs. The average size was estimated as 180 nm (±10 nm) in diameter using DLS analysis (Figure 1e) and ImageJ software (Figure 1f). The stability of gelatin NPs under physiological environment was investigated by analyzing the changes in zeta potential and particle size over a period of 7 days. In both cases, the GPS-loaded gelatin NPs were dispersed in the standard cell culture media (RPMI 10% Bovine fetal serum). The zeta potential of the gelatin NPs sample was obtained with an average value of +33.9 ± 2 mV. This higher value of zeta potential confers a higher stability of gelatin NPs by electrostatic repulsion. There was not a significant zeta potential change observed over the investigated time period (Figure S1). The surface potential of the gelatin before and after loading of GPS NPs was observed as 33.7 and 33.9 mV respectively. Therefore, most GPSs were encapsulated inside the gelatin matrices rather than adsorbed onto the surface. According to the DLS analysis, there was not a significant variation observed in the particle hydrodynamic size within 7 days. Chemical functionalities and crystal structures of GPS NPs, gelatin NPs and encapsulated GPS NPs were examined using FTIR and XRD. The spectra and explanation are given in Figure S2. 3.2. Surface Properties and Encapsulation Performance of Nanocarrier. To optimize the cross-linker concentration, we added 0.01, 0.02, 0.035, and 0.05% of glutaraldehyde as a cross-linking agent during the synthesis of gelatin NPs. The morphology of synthesized gelatin NPs was

3. RESULTS AND DISCUSSIONS 3.1. Morphology and Structural Characteristics of Gelatin-Encapsulated Ginseng Polysaccharide NPs. Following our previously reported methodology for the extraction of GPS from American ginseng root and nanosizing using a microfluidic device,14 TEM was employed to examine the size and shape of the resulting GPS NPs. Figure 1a shows the uniformly dispersed NPs formation of GPS in a TEM micrograph. The particle size of the GPS NPs ranged from 15 to 30 nm with an average of 20 nm in diameter (Figure 1d). The size distribution of NPs was estimated from the TEM images using ImageJ software. The GPS NPs were then encapsulated within gelatin nanocarriers using a two-step 2919


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Table 1. Ginseng Polysaccharide NP Loading, Gelatin Encapsulation Efficiency, and Surface Properties of GPS NP-Loaded Gelatin Nanospheres with Changing Cross-Linker Concentration cross-linker (%)

loading efficiency (%)

encapsulation efficiency (%)

maximum PS release (%)

surface area(m2/g)

pore size (nm)

0.01 0.02 0.035 0.05

51.3 ± 6.5 45.7 ± 6.2 42.1 ± 5.5

73.9 ± 2.5 66.1 ± 2.8 60.6 ± 2.5

91.6 81.6 75.2

213 ± 12 184 ± 15 161 ± 14

3.84 ± 0.4 2.72 ± 0.5 1.98 ± 0.5

Figure 2. Confocal laser scanning microscopy (CLSM) images of dye-labeled GPS loaded gelatin NPs: (a) bright-field image, (b) dark-field image, and (c) merge image. CLSM images of macrophage cell lines internalization of dye-labeled GPS NPs loaded after 24 h: (d) bright-field image, (e) dark-field image, and (f) merge image.

maximum swelling and GPS release were found at a crosslinker concentration of 0.02%. 3.3. Ginseng Polysaccharide NP Dispersion in Gelatin and Cellular Uptake. To identify the encapsulation of GPS NPs in gelatin nanocarriers, we encapsulated dye (FTSC)labeled GPS NPs within the gelatin following the same synthesis procedure described earlier (section 2.2 and 2.3). A laser scanning confocal microscope (LSCM) with an argon laser (excitation wavelength 488 nm) and objective 20× magnification was used for the fluorescence imaging of gelatin encapsulated labeled GPS NPs. Figure 2a−c represents the confocal images under different contrast modes; :bright field, dark field, and the merging of bright and dark field. In the case of bright-field imaging (Figure 2a), the small spots represent the gelatin nanocarriers. From this image, it is not possible to locate the exact position of GPS NPs within the gelatin matrix. Figure 2b shows the dark field image, in which the green spots are observed in the same position of gelatin (Figure 2a). These green emissions are due to emission from the dyes under laser excitation. The merged image (Figure 2c) reveals that most of the dye-labeled GPS are loaded within the gelatin nanospheres. In addition, the dye-labeled gelatin NPs were suspended in the liquid well, there is no aggregation detected from the Figure 2b, c.

examined using SEM (images shown in Figure S3). The most isolated and homogeneously sized gelatin NPs (diameter 180 ± 10 nm) were formed at a cross-linker ratio of 0.02%. The loading and encapsulation efficiency of GPS NPs were measured for different cross-linker concentrations (Table 1). For the very low cross-linker concentration (0.01%), because of formation of no stable NPs, the loading and encapsulation efficiencies, and surface properties were not determined. However, these were significantly prominent after a small increase of cross-linker concentration to 0.02%. Further increasing the cross-linker concentration from 0.02 to 0.05% led to the loading percentage decreasing, which in turn reduced the encapsulation efficiency of gelatin from 74 to 60.6%. The surface properties of gelatin NPs including surface area and pore size (prepared with different amounts of crosslinker) were measured by BET and summarized in Table 1. The results from Table 1 reveal that NPs prepared with 0.02% glutaraldehyde showed the best surface properties and porosity. The surface area and porosity of the gelatin nanostructure decreased with increasing concentration of glutaraldehyde, attributed to the increased molecular entanglement inside the gelatin matrix.19 The swelling and GPS release behavior of gelatin nanocarrier with variation of cross-linker and pH (1.0, 4.5 and 7.4) were also studied with the results and related discussion provided in the Figure S4. The 2920


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Figure 3. (a) Flow cytometry analysis of FTSC-GPS-loaded gelatin NP uptake by macrophage cell lines at a concentration of 10 μg/mL. The orange box outlines the cells population that is FTSC positive and the number in the bottom right corner is the percentage of the total cell population that is FTSC positive (cells have taken up nanoparticles) and (b) percentage of cells that uptake media (as control), FTSC only and FTSC-GPS -loaded gelatin NPs at different concentrations (10, 20, and 50 μg/mL). Three independent experiments were performed, and the data are shown as mean ± SD, n = 3. Data sets were evaluated by one-way ANOVA followed by Dunnett’s post hoc test. * Values P < 0.05 compared to the control were statistically significant.

Figure 4. Immunostimulatory effect of encapsulated GPS NPs within gelatin nanospheres, nonencapsulated GPS NPs, and released GPS from gelatin matrix on macrophage cell lines. (a) NO production, (b) TNF-α production, (c) IL-1β production, and (d) IL-6 production. Murine macrophages were treated with four different concentrations of the treatment (25, 50, 100, 200 μg/mL), LPS (1 μg/mL) for 24 h and the culture supernatants were analyzed for production of inflammatory mediators. Three independent experiments were performed, and the data were shown as mean ± SD, n = 3. Data sets were statistically analyzed by two-way ANOVA followed by Bonferroni’s post hoc test. * Values P < 0.05 and ** values P < 0.001 compared to the control were statistically significant.

after 24 h.17 Figures 2d−f shows the cellular uptake and distribution of the encapsulated GPS NPs within the cell lines. Bright-field images represent the cell lines (Figure 2d). The dark field image (Figure 2e) shows the fluorescent signal, indicating that the labeled GPS loaded gelatin NPs was uptaken by the cell lines. The overlay image (merge of bright and dark field) illustrates that all the cell lines uptake the encapsulated-labeled GPS. Before conducting the confocal analysis, the treated cell lines were washed with PBS to remove any gelatin NPs not uptaken by the investigated cell lines.

Cellular uptake was further carried out using the FTSC labeled GPS loaded gelatin NPs. Here, 200 μg/mL of the labeled GPS loaded gelatin NPs was treated with macrophage cell lines (RAW264.7) for an incubation period of 24 h. The incubation time was chosen based on our previously reported study.17 In that work, macrophage cell lines were treated with 200 μg/mL of nonencapsulated GPS NPs (FTSC labeled) and then examined at 0, 2, 4, 6, 24, and 48 h using an inverted confocal microscope. The results reveal that cellular uptake of GPS NPs began at 4 h of incubation and reached a maximum 2921


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Figure 5. Effect of GPS treatment on the production of NO and TNF-α within blood sample of Swiss albino male mice (n = 5 mice per group). Mice were given GPS treatments or control (vehicle) for 3 consecutive days through oral gavage and sacrificed. Different GPS materials used: (i) GPS1- native GPS, (ii) GPS2- GPS NPs, (iii) GPS3- labeled GPS NPs, and (iv) GPS4- encapsulated GPS NPs. Blood serum was used for NO and cytokines analysis. (a) NO production, (b) TNF-α production. Three independent experiments were performed, and the data were shown as mean ± SD, n = 3. Data sets were evaluated by one-way ANOVA followed by Dunnett’s post hoc test. * Values P < 0.05 compared to the control are statistically significant.

incubation, the production of proinflammatory mediators was measured from the culture supernatants. The cell lines without treatment and LPS (lipo-polysaccharides) were considered as a negative and positive control respectively. As seen in Figure 4, all proinflammatory mediator production was significantly increased for both the encapsulated and nonencapsulated GPS NPs. These mediators upregulation followed a dose-dependent manner. In the case of encapsulated GPS NPs, NO and cytokines production was lower compared to the nonencapsulated GPS NPs. From the releasing study, we observed that there was around 50% GPS release from the gelatin nanocarrier in the first 24 h (Figure S4). These 50% released GPSs contribute to the production of cytokines. This steady increase in cytokine production is also ideal for the controlled drug/nutraceutical delivery. To examine the efficacy of nonencapsulated GPS NPs and the released GPS from gelatin nanoparticulate system, we also carried out an in vitro study for the released GPS NPs. Herein, GPS-loaded gelatin NPs were suspended in PBS solution (pH 7.4) and incubated at 37 °C in a water bath for 120 h (maximum release of GPS obtained, Figure S4). An aliquot of the eluted GPS was collected after centrifugation and lyophilized. All investigated mediator production including NO, TNF-α, IL-1β, using released GPS were almost the same as produced from the nonencapsulated GPS NPs. Therefore, the encapsulation procedure prolongs the mediator production without diminishing the “stimulatory” property of GPS NPs. 3.5. In Vivo Study: Immunostimulation through Oral Administration in Swiss Albino Male Mice. Swiss albino mice were treated with 50 mg/kg of native GPS, GPS NPs, labeled GPS NPs, and encapsulated GPS NPs for 3 days through oral gavage. The blood serum was collected, analyzed, and the production of the reactive oxygen species NO and organic cytokine TNF-α were measured. The results revealed that the production of both NO and TNF-α were significantly higher than the sham control (mice without any treatment) as shown in Figure 5a, b. All types of GPS treatments significantly enhanced the production of NO and TNF-α. In agreement with the in vitro results of section 3.4 and previous reports,14 here we also observed that GPS NPs produced an increased amount of NO and TNF-α compared to the native GPS. On the other hand, labeled and nonlabeled GPS NPs produced an almost similar magnitude of NO and TNF-α production. The

Quantitative analysis of the cellular uptake of the FTSClabeled GPS-loaded gelatin NPs was further studied using flow cytometry. The results reveal that the macrophage cell lines are labeled in a dose dependent manner (Figure 3). The higher the concentration of the treatments (10, 20, and 50 μg/mL of FTSC-GPS-loaded gelatin NPs), the more macrophage cells labeling (30.7, 43.9, and 68.4%, respectively) were observed. The cells treated with labeled GPS loaded gelatin NPs showed a significant increase in fluorescence intensity compared to the control group (media only). Moreover, the cells treated with only FTSC dye showed a similar response (6.1%) to the control group (5.2%). These data indicate that the free FTSC dye was not able to be transported into the living cells compared to the cell internalization of gelatin NPs. Therefore, the formed FTSC-GPS-loaded gelatin NPs exhibit potential for the quantitative determination of target saccharide metabolized or transported into the other living cells. The cytotoxicity of the gelatin NPs were examined using the MTT assay. Five different concentrations (50, 200, 500, 1000, 2000 μg/mL) of nonencapsulated GPS NPs and gelatin encapsulated GPS NPs were investigated for 24, 48, and 72 h of incubation period. The results show that for the nonencapsulated GPS NPs over the examined concentration range, no significant difference in cell viability was observed compared to the control (Figure S5). The encapsulated GPS NPs also showed no substantial cytotoxicity to macrophage cells up to a concentration of 500 μg/mL with an incubation time 24−72 h. However, compared to the control group there was only a minor (less than 12%) cell death observed for high concentrations (more than 1000 μg/mL) of gelatin encapsulated GPS NPs after 72 h of treatment. 3.4. In Vitro Study: Effect of Nanosizing and Encapsulation on Immunostimulation. According to previous reports, GPS shows an immune-stimulatory activity on macrophage cell lines.14,16,21 In this work, our focus was toward examining the changes of the immune-stimulatory activity after gelatin encapsulation of GPS NPs and a better understanding of the underlying absorption mechanism. Here, we investigated and compared the production of proinflammatory mediators (e.g., NO, TNF-α, IL-1β, IL-6) on RAW264.7 macrophage cell lines treated with both encapsulated and nonencapsulated GPS NPs (Figure 4). The treatment loading was varied as 25, 50, 100, and 200 μg/mL. After 24 h 2922


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Figure 6. Effect of GPS treatment on the production of NO and cytokine within blood sample of Swiss albino mice (n = 5 mice per group). Mice were given GPS treatments or control (vehicle) for 3 and 6 consecutive days through oral gavage and sacrificed. Two different GPS materials used: (i) GPS2- GPS NPs and (ii) GPS4- encapsulated GPS NPs. Blood serum was used for NO and cytokine analysis. (a) NO production, (b) TNF-α production. Three independent experiments were performed, and the data were shown as mean ± SD, n = 3. Data sets were evaluated by one-way ANOVA followed by Dunnett’s post hoc test. * Values P < 0.05 compared to the control were statistically significant.

Figure 7. Effect of different dosages of GPS NPs treatment on the production of NO and cytokine within blood sample of Swiss albino male mice (n = 5 mice per group). Mice were given GPS treatments or control (vehicle) for 3 days through oral gavage and sacrificed. Different GPS materials used: (i) GPS2_L- GPS NPs with 50 mg/kg of treatment, (ii) GPS2_H- GPS NPs with 75 mg/kg of treatment and (iii) GPS4_L- encapsulated GPS NPs with 50 mg/kg of treatment and (iv) GPS4_H- encapsulated GPS NPs with 75 mg/kg of treatment. Blood serum was used for NO and cytokines analysis. (a) NO production, (b) TNF-α production. Three independent experiments were performed and the data were shown as mean ± SD, n = 3. Data sets were evaluated by one-way ANOVA followed by Dunnett’s post hoc test. * Values P < 0.05 compared to the control were statistically significant.

15 and 25% higher than the 3 day treatment. These results indicate that the immunostimulatory response of both GPS NPs and encapsulated GPS NPs are time-dependent. To determine whether the treatment dosage has any impact on NO and cytokine production, two different dosages of GPS NPs and encapsulated GPS NPs were used for oral gavage. As shown in Figure 7, 50 mg/kg GPS NP-treated mice produced 22 μg/mL NO and 47 pg/mL TNF-α, whereas 75 mg/kg GPS NP-treated mice produced 32 μg/mL and 58 pg/mL, respectively. Similar observations were noticed for the encapsulated GPS NPs. Hence, 75 mg/kg treated mice blood serum produced higher NO and TNF-α levels compared to 50 mg/kg treated mice blood serum. Therefore, the immunostimulatory effects of GPS NPs and encapsulated GPS NPs showed a dose−response relationship, exhibited by a marked increase in production of NO and TNF-α in effect at the high dosage treatment. The oral route is the most popular and facile way to administer drugs and bioactive compounds (e.g., GPS). To obtain enhanced pharmacological effects, the bioactive materials need to be absorbed across the intestinal barrier

maximum NO and TNF-α production obtained using the encapsulated GPS NPs (Figure 5a, b), where encapsulation enhances cytokines production. From these observations we can conclude that compared to the native GPS, GPS NPs are more effective as an immunostimulating agent and the encapsulated GPS NPs are even better compared to the nonencapsulated ones. Another experiment was further performed to investigate the effect of NO and TNF-α production with time. To examine this, Swiss albino male mice were given GPS NPs and encapsulated GPS NPs for 3 and 6 consecutive days. Oral treatment of Swiss albino mice with GPS NPs and encapsulated GPS NPs for 3 and 6 day treatment showed increased production of NO and TNF-α which are higher than the sham control (Figure 6a-b). Compared to the 3 day treatment, 6 day treatment of GPS NPs produced 2× higher NO levels. A similar response was also observed with the encapsulated GPS treatment, 6 day treatment of mice showed increased NO production compared to 3 day treatment. In addition, both the GPS NPs and encapsulated GPS NPs for 6 day treatment showed increased TNF-α production, which is 2923


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Scheme 2. (a) Gastrointestinal Absorption of Oral Delivered Bioactive Compounds into Systemic Circulation; (b) Schematic Illustration of the Intestinal Epithelium and the Pathways Available for Bioactive Compound Absorption: (i) Paracellular Pathway (in between adjacent cells), (ii) Transcellular Pathway (through the epithelial cells), (iii) Absorption by M-Cells of Peyer’s Patches into the Lymphatic Circulation; (c) Schematic Illustration of Swelling of Hydrogel Nanocarrier, Biodegradtion, and Releasing of Drug; (d) Cellular Internalization

obtained and then encapsulated to obtain nanosphere carriers of 180 ± 10 nm diameter. These nanovesicles were examined for GPS loading, encapsulation efficiency, surface properties, swelling and GPS release behavior by varying the cross-linker concentration and pH. Both in vitro (macrophage cell lines) and in vivo (Swiss albino mice) investigation helped confirm that the GPS NPs produced more immunostimulating mediators compared to the native GPS. The orally administered GPS NPs and encapsulated GPS NPs significantly increased the production of NO and TNF-α in a dosedependent and time-dependent manner compared to the Sham control. This study shows that orally administered GPS NPs can be considered as a therapeutic agent for immunostimulation with gelatin as a potential carrier for the prolonged delivery for enhanced GI absorption for bioactive polysaccharides.

into systematic circulation as shown in Scheme 2a. The intestinal absorption of bioactive compounds is limited because of their inherent physicochemical properties including large molecular size, short biological half-lives and poor solubility. Moreover, poor permeability through the intestine mucosa and degradation by gastrointestinal tract enzymes causes poor bioavailability of bioactive compounds.22−24 Our results show that nanosized GPS are more effective in bypassing the GI barrier compared to the native GPS. The possible mechanism of GPS NPs passing through the GI tract include: (1) paracellular passage: when particles are extremely small (

Immunoengineering with Ginseng Polysaccharide Nanobiomaterials

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