Encapsulation of Acetyl Ginsenoside Rb1 within Monodisperse Poly(dl

Article pubs.acs.org/IECR

Encapsulation of Acetyl Ginsenoside Rb1 within Monodisperse Poly(DL-lactide-co-glycolide) Microspheres Using a Microfluidic Device Raziye Samimi,†,∥ Mehrnaz Salarian,‡,∥ William Z. Xu,† Edmund M. K. Lui,§,∥ and Paul A. Charpentier*,†,∥ †

Department of Chemical and Biochemical Engineering, ‡Biomedical Engineering Graduate Program, §Department of Physiology and Pharmacology, and ∥The Ontario Ginseng Innovation & Research Consortium, University of Western Ontario, London, Ontario, Canada, N6A 5B9 S Supporting Information *

ABSTRACT: 6″-O-Acetylginsenoside Rb1 (ac-Rb1) isolated from North American ginseng was encapsulated within biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres. Both a conventional double emulsion and a microfluidic technique were examined for microsphere formation. The ac-Rb1 encapsulation within PLGA microspheres was characterized by various physicochemical techniques including FTIR, DSC, and XRD. The experimental data showed that the PLGA microspheres produced from the microfluidic technique were uniform with tunable mean diameters from 7 to 59 μm and standard deviations less than 10%. The size was independent of the capillary number and could be tuned by altering the continuous and disperse phase flow rate ratios. Release profiles from the uniform microspheres were investigated, showing controlled release of ac-Rb1 which followed a Fickian diffusion while retaining potency toward immunosuppressive activity using macrophages in vitro. This study shows that ac-Rb1 encapsulated PLGA microspheres obtained using the microfluidic approach are promising for the development of next-generation biomedical agents.

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INTRODUCTION Of the well-known traditional Chinese medicines with biological activity, ginsenosides, which are the main active components of the plant ginseng, have been widely used as a medicinal herbal plant extract.1 Ginsenosides have been shown to have various beneficial health effects including antiinflammatory, antistress, and anticancer properties.2−4 For instance, ginsenosides Rb1 and Rg1 have been shown as efficient neuroprotective agents for spinal cord neurons, while ginsenoside Re did not exhibit such activity.5 We recently showed that ac-Rb1 ginsenoside, which was isolated and characterized from North American ginseng (Scheme 1), has potent immunosuppressive effects.6 This investigation was based on previous work on the extraction and isolation of ginsenosides in supercritical fluids.7,8 It has also been reported that transformed ginsenosides and ginsenoside metabolites have shown more potency toward cytotoxic activity than neutral ginsenosides.9,10 However, the oral bioavailability of ginsenosides from the gastrointestinal (GI) tract is known to be low, i.e., 5% or less. This is due to biotransformation of ginsenosides in the human GI tract by enzymes and intestinal bacteria to other ginsenoside-like compounds such as compound K, which is also biologically active.11 Due to the cytotoxicity and poor bioavailability of ginsenosides, there is a growing need for more advanced delivery vehicles for ginseng extracts. Over the past several years, significant progress has been directed toward developing novel delivery systems for bioactive compounds from plant extracts and natural health products including nutraceuticals, probiotics, and traditional Chinese medicines.12 Currently, traditional approaches using tablets, capsules, teas, creams, oils, and liquids are used for the mixtures of generally low concentrations of biologically active com© 2014 American Chemical Society

Scheme 1. Chemical Structure of 6″-O-Acetylginsenoside Rb1 (ac-Rb1)6

pounds. Although some initial work has started investigating various formulations for bioactive and plant extracts such as polymeric nanoparticles, nanocapsules, and liposomes,13−15 very little has been done on investigating ginsenoside Received: Revised: Accepted: Published: 11333

March 15, 2014 June 5, 2014 June 17, 2014 June 17, 2014 dx.doi.org/10.1021/ie501118u | Ind. Eng. Chem. Res. 2014, 53, 11333−11344

Industrial & Engineering Chemistry Research

Article

Griess reagent were purchased from Sigma-Aldrich (USA). Cell culture medium and reagents were purchased from Gibco laboratories (USA). Preparation of ac-Rb1-Loaded PLGA Microspheres. A microfluidic chip with channel dimensions of 100 μm in height and 200 μm in width was fabricated by soft lithography techniques28 in collaboration with Dr. Jun Yang, Mechanical and Material Engineering Department, Western University, Canada. The microfluidic chips were molded by a mixture of PDMS elastomer base/curing agent in 10:1 ratio from SU-8 mold and bonded to a flat PDMS substrate under plasma treatment for 35 s in a PX-250 plasma chamber (March Instruments, Concord, MA, USA) and then immediately placed in contact to bond the surfaces irreversibly. The devices were then bonded using a homemade UV-ozone generator. A 10 mg sample of ac-Rb1 was dissolved in 5 mL of solution (methanol:deionized water (2:3 v/v)) and 100 mg of PLGA was dissolved in 10 mL of DCM. The ac-Rb1 solution was emulsified in PLGA organic solution (oil phase) using a magnetic stirring bar at a speed of 1200 rpm to form the disperse phase. The microchannels were first filled with a continuous phase of 2% PVA aqueous solution before introduction of the organic (disperse) phase.24,26 Digitally controlled syringe pumps (KD scientific pump, Inc., Legato 2200, 1100) delivered liquid phase into the microfluidic device at constant flow rates using Teflon tubing (1.6 mm o.d., 0.6 mm i.d.). The flow rate of the continuous phase was in the range between 0.05 and 0.2 mL/min while that of the disperse phase was kept at a constant value of 0.01 mL/min as summarized in Table 1. At the end of the device outlet, the

controlled release. Microspheres are of significant current interest in several areas of drug delivery including inhalation, nasal, oral, transdermal, and implantation, in which both dosing and control release are important.16−20 Of the microspheres that may be suitable for ginsenosides, PLGA-based microspheres are of particular interest due to their long clinical experience, U.S. Food and Drug Administration (FDA) compliance, high biocompatibility, appropriate degradation characteristics, and possibility for sustained drug delivery. PLGA is one of the most well-known biodegradable polymers and delivery carriers for drugs, proteins, peptides, and steroids due to its ability to encapsulate a variety of materials,21,22 although it has not been examined with ginsenosides, which have unique structures as shown in Scheme 1, with both sugar and steroid moieties. To prepare microspheres, various techniques are possible such as emulsion solvent evaporation/extraction methods, phase separation, spray drying, and supercritical fluids.14,23 Conventional microencapsulation methods generate microspheres with broad size distributions which influences the kinetics of encapsulant release and limits their clinical application.24 Recently, microfluidic devices have attracted attention due to their easy adjustability to create microspheres with controlled morphologies.25 Microfluidic devices utilize low-priced and convenient systems for the control of fluid flow.26 Monodisperse microspheres made from biodegradable polymers have been generated by microfluidic devices widely used in drug delivery systems,24 which may enhance drug release kinetics, reproducibility, and bioavailability. Several polymers such as poly(lactic acid) (PLA) and PLGA, chitosan, poly(N-isopropylacrylamide) (pNIPAAM), hydrogelator, silk protein, pectin, hydrazide and aldehyde-functionalized carbohydrates, dextranhydroxyethyl methacrylate (dex-HEMA), and silica materials have been used for microsphere fabrication via the microfluidic approach.27 However, there is no study available regarding making monodisperse microspheres containing bioactive plant extract with a microfluidic device. In this study, we investigate both a microfluidic technique and a conventional double emulsion technique for ac-Rb1loaded PLGA microspheres. Their encapsulation, release, mechanism of release, and bioactivity are measured.

Table 1. Formulation Parameters and Characteristics of acRb1-Loaded PLGA Microspheres with Microfluidic (MF) and Conventional Double Emulsion (DE) Methods disperse phase flow (mL/min)

continuous phase flow (mL/min)

av size (μm)

SD (μm)

EE (%)

0.05 0.08 0.1 0.15 0.2

n.a.a 59 57 40 7

n.a.a 6 5 9 2

n.a.a 96.7 96.5 93.3 75.2

formuln

0.01 0.01 0.01 0.01 0.01 first emulsion stirrer speed (rpm)

second emulsion stirrer speed (rpm)

av size (μm)

SD (μm)

EE (%)

DE-6 DE-7 DE-8

800 1000 1200

400 400 400

48 41 39

19 24 15

77.5 78.5 78.8

formuln MF-1 MF-2 MF-3 MF-4 MF-5

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EXPERIMENTAL SECTION Materials. 6″-O-Acetylginsenoside Rb1 (ac-Rb1) was isolated and fractionated from North American ginseng root as previously described.6 Poly(DL-lactide-co-glycolide) (PLGA) (75:25, MW 66−107 kDa), poly(vinyl alcohol) (PVA) (87− 89% hydrolyzed, MW 13−23 kDa), 0.1 mol/L phosphate buffered solution (PBS; pH 7.4), dichloromethane (DCM) of HPLC grade (>99.9%), methanol, ethanol, acetic acid, and ammonium hydroxide were purchased from Sigma-Aldrich, Canada, and used as received. Poly(dimethylsiloxane) (PDMS; Sylgard 184) was obtained from Dow Corning (Midland, MI, USA) and used for preparation of the microfluidic microchip. Purified water was produced from a Milli-Q water purification system (18.2 MΩ·cm resistivity, Barnstead EasyPureII, Thermo Scientific, USA). RAW 264.7 (ATCC TIB 67) murine macrophage cell lines were provided by Dr. Jeff Dixon (Department of Physiology and Pharmacology, Western University, Canada). BD OptEIA ELISA kits tumor necrosis factor-α (TNF-α) were provided by BD Biosciences (Bedford, MA, USA). LPS (lipopolysaccharides) from Escherichia coli (0111:B4) of purity >99%, dexamethasone of purity >98%, and

a

Under this experimental condition, no microsphere was formed.

Teflon tubing was submerged into a 10 mL beaker containing 1% PVA aqueous solution at 40 °C to collect the droplets. Next, the solvent was removed from the droplets by evaporation. The formed microspheres were then centrifuged at 4500 rpm for 5 min followed by washing with deionized water three times to remove excess PVA and freeze-drying, and then they were stored at −20 °C. As a comparison to the microfluidic technique, the widely applied water-in-oil-in-water (w/o/w) double emulsion method29 was utilized for the formation of ac-Rb1-loaded PLGA microspheres. Briefly, 10 mg of ac-Rb1 was dissolved in 5 mL of solution (methanol:deionized water (2:3 v/v)) and 100 mg of PLGA was dissolved in 10 mL of DCM. The ac-Rb1 solution was emulsified in the PLGA solution using a magnetic stirring 11334

dx.doi.org/10.1021/ie501118u | Ind. Eng. Chem. Res. 2014, 53, 11333−11344


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Figure 1. PLGA/ac-Rb1 microspheres scanning electron microscopy results from (a, b) double emulsion method and (c, d) microfluidic method where the disperse phase flow included ac-Rb1 solution emulsified in PLGA organic solvent solution, and continuous phase flow contained 2% PVA aqueous solution. The continuous phase flow rate was 0.1 mL/min, while the disperse phase flow rate was 0.01 mL/min.

bar at three different speeds as summarized in Table 1. The entire mixture (w/o) was added to a 2% PVA aqueous solution and emulsified again to generate the double emulsion (w/o/w), which was subsequently poured into 40 mL of deionized water and stirred at 400 rpm for 3 h. After removal of DCM by evaporation, the formed microspheres were centrifuged at 4500 rpm for 5 min, followed by washing with deionized water three times to remove PVA and freeze-drying to remove water. The ac-Rb1-loaded PLGA microspheres were stored at −20 °C. Determination of ac-Rb1 Content in PLGA Microspheres. The ac-Rb1 concentration encapsulated inside the PLGA microspheres was measured using a UV spectrophotometer (Shimadzu UV−vis 3600) in triplicate at 203 nm. First, a standard calibration curve of ac-Rb1 was established using the UV spectrophotometer by measuring the absorbance as a function of concentration. To measure the ac-Rb1 content after PLGA encapsulation, 5 mg of ac-Rb1-loaded microspheres was dissolved in 1 mL of DCM, which was subsequently evaporated with the residue and then dissolved in 1 mL of methanol. The ac-Rb1 content was analyzed by the UV spectrometer based on the calibration curve established from ac-Rb1 standard solution mixtures. The ac-Rb1 encapsulation efficiency (EE%) was calculated via the following equation: EE% =

drug loading (DL%) ·100 theoretical loading (TL%)

theoretical loading (TL%) wt ac‐Rb1 added to system = ·100 wt ac‐Rb1 + wt PLGA

(3)

ac-Rb1 Delivery Carrier Characterization. The surface morphology of the PLGA microsphere systems was measured using a scanning electron microscope (SEM; Hitachi S-4500). Samples were sputtered with gold coating, and placed on a copper stub. Melting points were measured by a differential scanning calorimeter (DSC) Q200 (TA Instrument, New Castle, DE, USA). The scanning rate was 10 °C/min, with a scanning temperature range of 25−250 °C. X-ray diffraction (XRD) data were obtained for ac-Rb1, placebo microspheres, and ac-Rb1-loaded microspheres to evaluate the crystallinity after encapsulation (Inel CPS powder diffractometer). Scanning was done up to a 2Θ angle of 43° using a powder diffractometer instrument which was equipped with a Cu Xray radiation tube, an Inel XRG3000 generator, and an Inel CPS 120 detector. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) (Nicolet 6700, Thermo Scientific) was used to investigate the encapsulation of ac-Rb1 within PLGA polymer microspheres in the range 500− 4000 cm−1. Kinetic Release Studies of ac-Rb1. For the release studies, 5 mg of ac-Rb1-loaded PLGA microspheres was suspended in 10 mL of PBS (pH 7.4) at 37 °C with shaking at 50−100 rpm. At predetermined time intervals, the solution was centrifuged at 2000 rpm for 5 min, with 2 mL of supernatant then collected and replaced with 2 mL of fresh PBS. The ac-Rb1 content in the supernatant was determined at a wavelength of 203 nm using a UV spectrophotometer (Shimadzu UV−vis 3600). All experiments for each time interval were repeated in triplicate. The cumulative release of ac-Rb1 profiles were acquired with the volume loss correction.

(1)

where drug loading (DL%) wt ac‐Rb1 in PLGA measured by UV spectrom = ·100 wt PLGA microspheres (2) 11335



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Figure 2. ImageJ number of size distributions for ac-Rb1-loaded PLGA microspheres: (a) DE-6, (b) DE-7, (c) DE-8, (d) MF-2, (e) MF-3, (f) MF-4, and (g) MF-5.

Bioactivity of Released ac-Rb1. The mouse macrophage cell line RAW 264.7 was cultured in Dulbeccos modified Eagle’s medium supplemented with 10% FBS, 25 mM HEPES, 2 mM glutamine, 100 IU penicillin/mL, and 100 μg/mL streptomycin. The pH of the medium was around 7.4. The cells were kept at 37 °C in a humidified incubator with 5% CO2, and seeded in 96-well tissue culture plates at a density of 1.5 × 105 cells per well. Our previous study showed that ac-Rb1 has potency toward immunosuppressive activity on murine macrophages.6 In this study, the inhibitory effect of released ac-Rb1 from the

microspheres in vitro was evaluated. To examine the inhibitory effect of released ac-Rb1, 5−150 μg/mL ac-Rb1 and released acRb1 were added to the macrophages 2 h prior to the addition of LPS. In this study, dexamethasone (DEX) was used as a positive control for the suppression of LPS-induced stimulation of macrophages. Therefore, cultured macrophages were incubated with DEX (5 μM) for 2 h prior to the addition of LPS. The 24 h cytokine production induced by LPS was determined by measuring NO and TNF-α levels in the culture medium. TNF-α concentrations in supernatants from cultured 11336



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formed. The relationship between the average microsphere size and the continuous phase flow rate is given in Figure 3. As the

cells were analyzed with ELISA. Samples were assessed with mouse cytokine-specific BD OptEIA ELISA kits (BD Biosciences, USA) according to the manufacturer’s protocol. Sample nitrite concentrations in culture medium were determined by means of the Griess reagent (0.5% sulfanilic acid, 0.002% N-1-naphtyl-ethylenediamine dihydrochloride, 14% glacial acetic acid). Culture supernatant of each sample and the Griess reagent (50 μL each) were added to a 96-well plate. The measurements of absorbance were conducted at a wavelength of 550 nm using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific, Finland) with SkanIt software (version 2.4.2, Thermo Fisher Scientific, Finland). Nitrite concentrations were calculated from a sodium nitrite standard calibration curve. Statistics. All experiments were performed at least three times with statistical analysis accomplished using GraphPad prism 4.0a Software (GraphPad Software Inc., USA). Data were reported based on mean ± standard deviation (SD) of three sets of experiments. Data sets with multiple comparisons were evaluated by one-way analysis of variance (ANOVA) with Tukey’s and Dunnett’s posthoc test. p < 0.05 was considered to be statistically significant. Model Fitting of in Vitro Release of ac-Rb1. In order to investigate the kinetics of ac-Rb1 release from the PLGA microspheres, four commonly used empirical models were used to fit the in vitro release data, i.e., the Hixson−Crowell, Korsemeyer−Peppas, Weibull, and first-order models.12,30−32 The curve fitting toolbox of Matlab R2010a software was used to determine both the regression coefficients, r2, and the best fit model parameters to understand the release mechanism for the studied systems.

Figure 3. Effect of continuous phase flow rate on the microsphere size at disperse phase flow rate of 0.01 mL/min.

flow rate of the continuous phase increased from 0.08 to 0.20 mL/min, while that of the disperse phase was kept at 0.01 mL/ min, the average size of the formed microspheres decreased from 59 to 7 μm. However, by lowering the flow rate of the continuous phase to 0.05 mL/min, no microspheres were formed. Our experimental results show that the microsphere size obtained with the microfluidic method are tunable by changing the flow rate, although only a narrow experimental window enabled microsphere formation. In order to provide a better understanding of these results, the Reynolds number (Re) and the capillary number (Ca) need to be considered. Flow in microfluidic systems usually has a low value of Reynolds number which indicates that the flow is laminar. The Reynolds number is defined as Re = ρud/μ, where ρ is the density of the continuous phase, u is the velocity of the continuous phase, d is the characteristic length scale (e.g., the hydraulic diameter of the channel cross section), and μ is the viscosity of the continuous phase. The dimensionless capillary number has a significant role in determining the droplet breakoff. The capillary number is defined as Ca = μu/γ, where μ is the viscosity of the continuous phase, u is the velocity of the continuous phase, and γ is the interfacial tension between the oil and water phases. In the microfluidic system, the formation of microspheres is governed by the competition between the viscous forces and the interfacial forces.34 At low values of Ca (typically less than 0.01), interfacial forces dominate over viscous forces. As a result, the viscous forces on the interface of the emerging droplet are not adequate to distort it significantly. Consequently, the droplet blocks almost the entire cross section of the main channel. If the flow rate of the disperse phase is higher than the flow rate of the continuous phase, the droplet is elongated in the main channel and the developing neck creates a thin film. Ultimately, breakup occurs after the detachment of the dispersed phase from the junction due to the pressure drop across the droplet. However, when the disperse phase flow rate is lower than the continuous phase flow rate, the droplet does not have time to elongate and breakup happens immediately after the droplet blocks the entire cross section of the main channel. In this study, we chose the disperse flow rate lower than the continuous phase flow rate so that the detachment occurs at the junction which is believed to be a more stable process for droplet formation.34 Table 2 shows the values for Ca and Re numbers used in this work which were calculated based on the utilized microfluidic device geometry and the continuous phase properties. The low values of

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RESULTS AND DISCUSSION Characterization of Microspheres. The experimental parameters and characteristics of the examined formulations of microspheres are listed in Table 1. Figure 1 shows the size and morphology of typical samples produced from the double emulsion method and the microfluidic method by SEM. The samples from the double emulsion method have a spherical morphology with a broad size distribution of 10−70 μm. The size distribution of the microspheres was analyzed by using ImageJ software and are displayed in Figure 2a−c, revealing an average size of 41−48 μm with a standard deviation of 15−24 μm for microspheres obtained from the double emulsion method. The size of the microspheres decreased with increasing stirring speed, agreeing well with the results reported in the literature.33 As can be seen in Figure 1c,d, the samples from the microfluidic method have a similar spherical morphology to the microspheres prepared using the double emulsion method; however, the microfluidic approach gave much more uniform microspheres (50−65 μm) compared to the double emulsion method (10−70 μm). Also, Figure 2d−g shows the size distributions of the microspheres which were obtained using ImageJ software, showing a uniform distribution with standard deviations between 2 and 9 μm. In the microfluidic approach, the flow rate ratio of the continuous phase to the disperse phase had a significant effect on the formation of microspheres. It should be pointed out that increasing the disperse phase flow rate from 0.001 to 0.01 mL/ min led to blocking of the inlet microchannel, preventing the formation of microspheres. Also, when the disperse phase flow rate was higher than 0.01 mL/min and higher than the continuous phase flow rate, microsphere particles were not 11337



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of the dispersed phase can prevent drug loss into the continuous phase and increase the encapsulation efficiency.38 The double emulsion technique produced both smaller and larger sizes as shown in their size distribution plots. According to Fu et al.39 smaller microspheres have higher encapsulation efficiency as more time is required to make the smaller emulsion droplets than the larger ones in the emulsion process; therefore, our active pharmaceutical ingredient, ac-Rb1, had more time to diffuse to the aqueous phase before polymer precipitation/sphere formation. The reason the encapsulation efficiency of microspheres from the microfluidic method increases with size is that the utilized low ratio of disperse phase flow rate to continuous phase flow rate may result in fast solidification of microspheres and subsequently high encapsulation efficiency.38 The major advantages of microspheres obtained from the microfluidic method is the existence of a high surface-to-volume ratio and rapid mixing within the droplet at low Reynolds numbers,35 both of which would help increase the encapsulation efficiency as experimentally observed. These results (Table 1 and Figure 2) reveal that, compared to the double emulsion method, more uniform microspheres with higher encapsulation efficiency were obtained using the microfluidic method. These results are in agreement with those reported in the literature where the poly(L-lactic acid) polymeric microspheres obtained from microfluidics were significantly better in terms of encapsulation efficiency than the microspheres fabricated by other methods such as the conventional double emulsion method, spray drying, and supercritical antisolvent methods.24 Encapsulation efficiency is an important factor in drug delivery, especially for valuable and expensive bioactive compounds. Formulation MF-3 generated the most uniform microspheres with the narrowest size distribution (Figure 2e) with an average size of 57 μm and EE% of 96.5, which we examine further below for drug delivery. To confirm the presence of ac-Rb1 in the PLGA microspheres, the FTIR spectra of ac-Rb1, pure PLGA, and ac-Rb1loaded microspheres are compared in Figure 6. In the FTIR spectrum of ac-Rb1 (Figure 6a), the peaks at 1726 and 1021 cm−1 are attributed to the CO and C−O stretching vibrations of an acetic ester group, respectively. Also, the peaks at 3304 and 1643 cm−1 are assigned to the OH of the sugar moieties in ac-Rb1.6 In the FTIR spectrum of pure PLGA (Figure 6b), the peak at 1740 cm−1 is assigned to the absorbance of CO in PLGA.40−42 The spectrum of ac-Rb1loaded PLGA microspheres (Figure 6c) showed all the peaks appearing in the spectrum of pure PLGA and additional hydroxyl peaks from ac-Rb1 at 3303 and 1643 cm−1, indicating successful encapsulation of ac-Rb1 in the PLGA matrix. It should be noted that, in the encapsulation process of ac-Rb1 in PLGA microsphere, the products were freeze-dried at the end to eliminate any residual water which may have similar peaks in the region of 3304 and 1643 cm−1.

Table 2. Dimensionless Parameters for Microfluidic Experiments Qd (mL/min)

Qc (mL/min)

0.01 0.01 0.01 0.01 0.01

0.05 0.08 0.1 0.15 0.2

Ca 1.7 2.7 3.4 5.1 6.8

× × × × ×

10−3 10−3 10−3 10−3 10−3

Re 6.18 9.89 12.37 18.55 24.74

Reynolds number confirm that the flow is laminar. The results show that Ca is lower than 0.01, which according to the literature35 implies that the size of microspheres is independent of Ca and is mainly determined by the ratios of the disperse and continuous phase flow rates. There are several numerical methods using computational fluid dynamics (CFD) that have been developed to model microsphere formation and the mechanism of droplet formation.35−37 Accurate simulation however remains challenging as it deals with microsphere formation of a disperse phase within a continuous phase which is an unsteady process of moving fluid−fluid interfaces and is therefore a complex flow problem.34 Figure 4 represents our CFD results showing the above explained phenomena in this studied condition when the disperse flow rate is lower than the continuous phase flow rate. For this case, the droplet does not have time to elongate and therefore breakup happens immediately after the droplet blocks the entire cross section of the main channel as shown in Figure 4. However, future investigation is needed to simulate the process and mechanism of microsphere formation, which is a complex phenomenon. Encapsulation of ac-Rb1 within PLGA Microspheres. In order to measure the ac-Rb1 concentration encapsulated within the PLGA microspheres, UV spectrometry was utilized to determine the encapsulation efficiency and subsequent drug release. A standard calibration curve of ac-Rb1 in the range 100−300 μg/mL was plotted by measuring the absorbance as a function of concentration. The ac-Rb1 has a maximum absorbance at 203 nm (Supporting Information). Figure 5 shows the encapsulation efficiency calculated for microspheres obtained from both the double emulsion and microfluidic methods. According to eq 1 and Figure 5, ac-Rb1 encapsulation efficiencies of 96.7, 96.5, 93.3, and 75.2% were obtained using four formulations from microfluidics of MF-2, MF-3, MF-4, and MF-5, respectively. For the microfluidic method, the ac-Rb1 encapsulation efficiency increased with increasing average size of the microspheres. However, microspheres obtained from the conventional double emulsion method gave a lower encapsulation efficiency of 77.5−78.8% with the narrowest size distribution obtained using DE-8 at 10−70 μm. Briefly, it is believed that the mechanism of polymer precipitation and microsphere formation are different for ac-Rb1 encapsulation for the microfluidic and double emulsion methods. The surface

Figure 4. Schematic of T-junction microchannel, where the disperse phase flow included ac-Rb1 solution emulsified in PLGA organic solvent solution (red) and continuous phase flow contained 2% PVA aqueous solution (blue). The continuous phase flow rate was 0.1 mL/min, while the disperse phase flow rate was 0.01 mL/min. The droplet does not have time to elongate and breakup happens immediately after the droplet blocks the entire cross section of the main channel. 11338



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Figure 5. Encapsulation efficiency of the microspheres obtained from (a) double emulsion method and (b) microfluidic method.

Figure 6. FTIR results for (a) ac-Rb1, (b) pure PLGA, and (c) ac-Rb1 encapsulated in PLGA microsphere obtained from microfluidic method.

Figure 7. (A) DSC thermograms of (a) ac-Rb1, (b) ac-Rb1-loaded PLGA microspheres obtained from microfluidic method, and (c) placebo PLGA microspheres obtained from microfluidic method. (B) XRD patterns of (a) ac-Rb1-loaded PLGA microspheres obtained from microfluidic method, (b) placebo PLGA microspheres obtained from microfluidic method, and (c) ac-Rb1.

In order to investigate the physical state of the encapsulated ac-Rb1 in the microspheres, DSC and XRD were employed.43 DSC thermograms of ac-Rb1, ac-Rb1-loaded PLGA micropheres, and placebo PLGA microspheres are shown in Figure

7A. The ac-Rb1 shows a sharp endothermic peak (Figure 7A-a) at 54 °C attributed to the melting point of ac-Rb1. A small peak at 99 °C is due to the loss of moisture. In Figure 7A-c, the placebo microspheres show two endothermic peaks at 51 and 11339



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100 °C which are due to the PLGA glass transition temperature (Tg)44,45 and the loss of moisture, respectively. In Figure 7A-b, the ac-Rb1-loaded microspheres demonstrate all the endothermic peaks available in the thermograms of placebo microspheres; however, there is no peak corresponding to ac-Rb1, which is consistent with molecular-level dispersion of ac-Rb1 in the polymer microspheres. The amorphous state of ac-Rb1 in the microspheres was also confirmed by XRD (Figure 7B), as the crystalline peak at 2θ = 4°, which shows the semicrystalline nature of ac-Rb1, disappeared after being encapsulated within the microspheres. In Vitro ac-Rb1 Release and Release Kinetics. Release kinetics of ac-Rb 1 from both the monodisperse and polydisperse microspheres are compared in Figure 8. Each

point in Figure 8 represents the average of three experimental data points. The ac-Rb1 release profile shows faster release kinetics in the first 100 h followed by a slow steady release from 100 to 400 h. The observed burst effect may be due to ac-Rb1 molecules that are at or near the PLGA microsphere surface. Our FTIR results showed that ac-Rb1 was observable, which would indicate some surface enrichment, although the XRD indicated molecular integration; thus either complete micromixing or some surface dispersion cannot be ruled out. As can be seen in Figure 8B, the initial burst release of ac-Rb1 from the monodisperse microspheres in the first 100 h is considerably smaller than that from the polydisperse microspheres.26 From Figure 8A, the total cumulative ac-Rb1 release is 46% from the monodisperse microspheres and 81% from the polydisperse microspheres. The release profile of ac-Rb 1 from the monodisperse microspheres prepared by the microfluidic method is slower than that from the polydisperse samples. Although the large burst effect can release the therapeutic agent ac-Rb1 relatively quickly, it might also damage tissues around the treatment site.29 Therefore, the smaller burst effect may be advantageous. It is believed that any difference in surface morphology could be due to the drug distribution within the microspheres.26 For a better understanding of the difference between the kinetics of these two microspheres, both samples were treated for 1 h in a 1 M aqueous solution of HCl which led to drug release, providing us with additional information about the surface distribution of the drug. ac-Rb1 is soluble in acidic aqueous solution. The results are shown in Figure 9. In order to better understand the difference in kinetics between microspheres obtained from the microfluidic method and those obtained from the double emulsion method, the morphologies of microspheres have been examined.26 The high number of small microspheres obtained from using the double emulsion method is likely a significant factor to both the initial burst release and faster overall release kinetics. Any significant change

Figure 8. (A) Release profiles of ac-Rb1 from (○) PLGA microspheres produced by double emulsion method (DE-8) and (●) PLGA microspheres produced by microfluidic method (MF-3). (B) Enlargement of release profile to magnify the burst effect. Releasing medium was PBS (pH 7.4) at 37 °C with shaking at 50−100 rpm.

Figure 9. Surface morphology of microspheres before (a,c) and after (b,d) treatment with 1 M HCl for 1 h. (a, b) Microspheres produced using microfluidic method and (c, d) microspheres obtained from double emulsion method. 11340



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Figure 10. Inhibitory effect of ac-Rb1 released from microspheres obtained via (first row) microfluidic method and (second row) double emulsion method. Inhibitory effect of ac-Rb1 on LPS-stimulated 24 h macrophage production of (a, c) NO and (b, d) TNF-α. Murine macrophages (RAW 264.7 cells) were pretreated with ac-Rb1 ginsenoside and dexamethasone (DEX) for 2 h after which LPS (1 μg/mL) was added, and the culture supernatants were analyzed for NO and TNF-α by Griess reaction assay and ELISA, respectively. Dexamethasone (DEX) at 5 μM was used as a positive control. Three independent experiments were performed and the data were shown as mean ± SD. Data sets were evaluated by ANOVA. (#) Values p < 0.05 significantly different from LPS-induced group.

Table 3. Release Model Parameters of Different Carriers for ac-Rb1 at pH 7.4a model poly-MS

mono-MS

a

r2

equation

first order

⎛ Mt ⎞ ln⎜1 − ⎟ = − 0.0064t M∞ ⎠ ⎝

Hixon−Crowell

SSE

−0.454

6.447

1/3 ⎛ Mt ⎞ ⎜1 − ⎟ = 0.778 − 0.0007t M∞ ⎠ ⎝

0.642

0.091

Korsmeyer−Peppas

⎛M ⎞ ln⎜ t ⎟ = − 1.392 + 0.226 ln t ⎝ M∞ ⎠

0.898

0.323

Weibull

⎡ ⎤ 1 ⎢ ⎥ ln ln⎢ = − 1.178 + 0.3114 ln t Mt ⎥ 1 − ⎣ M∞ ⎦

0.963

0.211

first order

⎛ Mt ⎞ ln⎜1 − ⎟ = 0.0033t M ⎝ ∞⎠

Hixon−Crowell

1/3 ⎛ Mt ⎞ ⎜1 − ⎟ = 0.936 − 0.0004t M∞ ⎠ ⎝

0.849

0.008

Korsmeyer−Peppas

⎛M ⎞ ln⎜ t ⎟ = − 2.639 + 0.3301 ln t ⎝ M∞ ⎠

0.994

0.0367

Weibull

⎡ ⎤ 1 ⎢ ⎥ ln ln⎢ = − 2.646 + 0.3779 ln t Mt ⎥ 1 − ⎣ M∞ ⎦

0.995

0.0488

−135.5

7.679

poly-MS, polydisperse microsphere; mono-MS, monodisperse microsphere.

could not be seen on the surface of the microspheres obtained from the microfluidic method (Figure 9b). However, several pores formed on the surface of the microspheres obtained using the double emulsion method (Figure 9d). More uniform drug distribution might be obtained in the microspheres produced by the microfluidic technique as the mixing in microfluidics is

homogeneous as mentioned above. These results help reveal that there may be less ac-Rb1 trapped or adsorbed near the surface of the microspheres obtained using the microfluidic method than for those produced using the double emulsion method. The faster drug release rate from microspheres 11341



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Figure 11. Release profiles (left) and fitting regression lines (right) by Weibull kinetic model for (a, b) ac-Rb1 release from PLGA microspheres produced by conventional method and (c, d) ac-Rb1 release from PLGA microspheres produced by microfluidic method.

Analysis of Release Data. To better understand the release mechanism from the microspheres, we fit the ac-Rb1 release curves in Matlab to determine if the release behavior is controlled by Fickian diffusion, erosion, or a combination of both. Four well-known empirical models were examined, i.e. the Hixon−Crowell, Korsmeyer−Peppas, Weibull, and firstorder models.12,30−32 According to the r values shown in Table 3, the Weibull model provides the best fit for the ac-Rb1 release experimental data for all the examined delivery systems. The curves of the best fitting and the linear regression lines by the Weibull model are shown in Figure 11. Almost comparable fitting with the Weibull model was observed in the case of using the Korsmeyer−Peppas model for ac-Rb1 release from mono-MS. A poor fitting was obtained by using the first-order and Hixson−Crowell equations, which showed that they are not proper models for the systems of this study. From Table 3 and Figure 11, the slope of the regression line of the Weibull model is the constant b. According to the literature,48 for values of b lower than 0.75, the release follows Fickian diffusion, whereas for b > 0.75, other mechanisms such as erosion better describe the release. In this study the results indicate that this value for the poly-MS and mono-MS is 0.31 and 0.38, respectively, as can be seen in Table 3. Therefore, the diffusion mechanism controlled the release of ac-Rb1 within the studied time which can be described by both the Weibull and Peppas models24,32 and can be used for long-term drug delivery applications. These results are in accordance with the reported results on the other steroid based drugs such as dexamethasone. In this study, dexamethasone was used as a control for studying the inhibitory effect of ac-Rb1. According to the literature,49,50 the main mechanism of dexamethasone release from polymeric microspheres is believed to be diffusion, which is confirmed by the mathematical models.

produced by the double emulsion method may be due to the larger amount of the drug adsorbed near the surface.26 Bioactivity of Released ac-Rb1. Our results previously showed that ac-Rb1 ginsenoside has potent immunosuppressive effects,6 whereas it was earlier thought that mainly ginseng polysaccharides are active in the modulation of macrophage immune function.46,47 For the current study, the immunosuppressive activity of released ac-Rb1 from microspheres obtained via both the microfluidic method and the double emulsion method was examined by bioassay analysis using macrophages in vitro. The effect of ac-Rb1 treatment on LPS-stimulated NO and TNF-α production in macrophages can be seen in Figure 10. Figure 10 shows that ac-Rb1 released from the PLGA microspheres from both the microfluidic and double emulsion methods significantly inhibits the production of NO and TNFα, in LPS-induced RAW 264.7 macrophages. This inhibition occurs in a dose-dependent manner within the dose range of 5−150 μg/mL released ac-Rb1 extract. The immunosuppressive activity of ac-Rb1 did not change significantly before encapsulation within the carrier and after release from the delivery carrier. Also, the microsphere preparation method does not have a significant effect on the immunosuppressive activity of ac-Rb1. Figure 10 also shows that the influence of ac-Rb1 is inflammatory mediator specific; i.e., the magnitude of inhibition by ac-Rb1 before encapsulation and after release from the delivery carriers was much greater with respect to NO production. The released ac-Rb1 inhibited the LPS-induced NO production more significantly with the lowest levels of NO production around 5 and 7 μM in the presence of 150 μg/mL released ac-Rb1 from PLGA microspheres produced by the microfluidic method and the double emulsion method, respectively. 11342



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(4) Keum, Y. S.; Park, K. K.; Lee, J. M.; Chun, K. S.; Park, J. H.; Lee, S. K.; Kwon, H.; Surh, Y. J. Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng. Cancer Lett. 2000, 150, 41−48. (5) Liao, B.; Newmark, H.; Zhou, R. Neuroprotective effects of ginseng total saponin and ginsenosides Rb1 and Rg1 on spinal cord neurons in vitro. Exp. Neurol. 2002, 173, 224−234. (6) Samimi, R.; Xu, W. Z.; Lui, E. M. K.; Charpentier, P. A. Isolation and Immunosuppressive Effects of 6″-O-Acetylginsenoside Rb1 Extracted from North American Ginseng. Planta Med. 2014, 80, 509−516. (7) Wood, J. A.; Bernards, M. A.; Wan, W.-k.; Charpentier, P. A. Extraction of ginsenosides from North American ginseng using modified supercritical carbon dioxide. J. Supercrit. Fluids 2006, 39, 40−47. (8) Samimi, R.; Xu, W. Z.; Alsharari, Q.; Charpentier, P. A. Supercritical fluid chromatography of North American ginseng extract. J. Supercrit. Fluids 2014, 86, 115−123. (9) Bae, E.-A.; Han, M. J.; Choo, M.-K.; Park, S.-Y.; Kim, D.-H. Metabolism of 20 (S)-and 20 (R)-ginsenoside R g3 by human intestinal bacteria and its relation to in vitro biological activities. Biol. Pharm. Bull. 2002, 25, 58−63. (10) Bae, E.-A.; Han, M. J.; Kim, E.-J.; Kim, D.-H. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch. Pharm. Res. 2004, 27, 61−67. (11) Qi, L.-W.; Wang, C.-Z.; Yuan, C.-S. American ginseng: potential structure−function relationship in cancer chemoprevention. Biochem. Pharmacol. 2010, 80, 947−954. (12) Chen, Z.; Liu, W.; Liu, D.; Xiao, Y.; Chen, H.; Chen, J.; Li, W.; Cai, H.; Cai, B. Development of brucine-loaded microsphere/ thermally responsive hydrogel combination system for intra-articular administration. J. Controlled Release 2012, 162, 628−635. (13) Ajazuddin; Saraf, S. Applications of novel drug delivery system for herbal formulations. Fitoterapia 2010, 81, 680−689. (14) Chiellini, F.; Piras, A. M.; Errico, C.; Chiellini, E. Micro/ nanostructured polymeric systems for biomedical and pharmaceutical applications. Nanomedicine (London, U. K.) 2008, 3, 367−93. (15) Gupta, P.; Vermani, K.; Garg, S. Hydrogels: from controlled release to pH-responsive drug delivery. Drug Discovery Today 2002, 7, 569−579. (16) Mahajan, H. S.; Gundare, S. A. Preparation, characterization and pulmonary pharmacokinetics of xyloglucan microspheres as dry powder inhalation. Carbohydr. Polym. 2014, 102, 529−536. (17) Feng, C.; Song, R.; Sun, G.; Kong, M.; Bao, Z.; Li, Y.; Cheng, X.; Cha, D.; Park, H.; Chen, X. Immobilization of Coacervate Microcapsules in Multilayer Sodium Alginate Beads for Efficient Oral Anticancer Drug Delivery. Biomacromolecules 2014, 15, 985−996. (18) Lin, K.; Zhai, D.; Zhang, N.; Kawazoe, N.; Chen, G.; Chang, J. Fabrication and characterization of bioactive calcium silicate microspheres for drug delivery. Ceram. Int. 2014, 40, 3287−3293. (19) Gabikian, P.; Tyler, B. M.; Zhang, I.; Li, K. W.; Brem, H.; Walter, K. A. Radiosensitization of malignant gliomas following intracranial delivery of paclitaxel biodegradable polymer microspheres: Laboratory investigation. J. Neurosurg. 2014, 120, 1078−1085. (20) Lam, P. L.; Gambari, R. Advanced progress of microencapsulation technologies: In vivo and in vitro models for studying oral and transdermal drug deliveries. J. Controlled Release 2014, 178, 25−45. (21) Liu, J.; Meisner, D.; Kwong, E.; Wu, X. Y.; Johnston, M. R. A novel trans-lymphatic drug delivery system: Implantable gelatin sponge impregnated with PLGA−paclitaxel microspheres. Biomaterials 2007, 28, 3236−3244. (22) Khaled, K. A.; Sarhan, H. A.; Ibrahim, M. A.; Ali, A. H.; Naguib, Y. W. Prednisolone-loaded PLGA microspheres. In vitro characterization and in vivo application in adjuvant-induced arthritis in mice. AAPS PharmSciTech 2010, 11, 859−869.

CONCLUSION ac-Rb1 was encapsulated in PLGA microspheres by using different formulations of the microfluidic method and the conventional double emulsion method, with the first method generating microspheres of more uniform size. The microspheres were characterized by SEM, FTIR, DSC, and XRD to examine the morphology and the physical state of ac-Rb1 in the microspheres. The monodisperse microspheres produced by a microfluidic method (50−65 μm) were found to exhibit a slower release rate and a smaller burst effect than the polydisperse microspheres prepared using a double emulsion method (10−70 μm). Moreover, the released ac-Rb1 from delivery systems showed immunosuppressive effect on LPSinduced macrophages, revealing that the microsphere preparation method does not change the bioactivity of ac-Rb1 in vitro. The in vitro release kinetics data followed a Fickian trend with the best fit observed with the Weibull model. The bioactive plant extract ac-Rb1 encapsulated within PLGA microspheres could provide many potential applications in pharmaceutical industries.

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ASSOCIATED CONTENT

S Supporting Information *

UV−vis absorption spectra and the calibration curve of standard ac-Rb1, and an image of the microfluidic chip. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: (519) 661-3466. Fax: (519) 661-3498. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Tingjie Li and Prof. Jun Yang, Department of Mechanical and Material Engineering, Western University, for assistance with PDMS microfluidic chip preparation. Moreover, they thank Dr. Alireza Mohammadi, Department of Mechanical and Material Engineering, Western University, for assistance with CFD modeling study. Also, they thank Ms. Hua Pei, Department of Physiology and Pharmacology, Western University, for assistance with the reported bioassay work and Dr. Jeff Dixon, Department of Physiology and Pharmacology, Western University, for his donation of RAW 264.7 (ATCC TIB 67) murine macrophage cell lines. Financial funding was provided from OGIRC and the Canadian Foundation for Innovation (CFI).

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REFERENCES

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Encapsulation of Acetyl Ginsenoside Rb1 within Monodisperse Poly(dl

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