Synthesis and Functionalization of β-Glucan Particles for the Effective

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Synthesis and Functionalization of β‑Glucan Particles for the Effective Delivery of Doxorubicin Molecules Kyungwoo Lee,†,‡ Yejin Kwon,† Jangsun Hwang,† Yonghyun Choi,† Kyobum Kim,§ Hyung-Jun Koo,∥ Youngmin Seo,‡ Hojeong Jeon,‡ and Jonghoon Choi*,† †

School of Integrative Engineering, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea Center for Biomaterials, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea § Division of Bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea ∥ Department of Chemical and Biomolecular Engineering, Seoul National University of Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 01811, Republic of Korea ACS Omega 2019.4:668-674. Downloaded from pubs.acs.org by 91.200.82.193 on 01/10/19. For personal use only.

‡

ABSTRACT: β-Glucan, a polysaccharide biopolymer, is one of the constituents of cell walls of microorganisms, basidiomycetes, and plants. It has pathogen-associated molecular patterns, recognizing specific immune cell receptors and can induce innate immunity and control acquired immunity. β-Glucan has properties of biocompatibility, biodegradability, high stability, and low toxicity; therefore, it can be used as a drug-delivery system and therapeutic target. Taking into account the advantages of β-glucan, we designed porous, hollow β-glucan microparticles (YGlu) with doxorubicin (DOX), an anticancer drug. We confirmed the successful loading of the drugs in YGlu by chitosan and alginate electrostatic attraction (Mat) through scanning electron microscopy, UV−vis spectroscopy, and Fourier transform infrared spectrometry. We also examined the cytotoxicity and efficiency of YGlu/Mat/DOX in MDA-MB-231 cells, HUVEC cells, and human peripheral blood mononuclear cells (PBMCs). In addition, we investigated the effects of YGlu on the secretion of cytokines in PBMCs. These results suggest that the YGlu/Mat/DOX may be utilized as a promising platform for drug delivery.

1. INTRODUCTION The development of platforms using biomaterials for drug delivery or detection of target proteins is being increasingly studied.1 The optimal biomaterial platform should be capable of delivering drugs, proteins, or DNA into cells stably. It should also show low cytotoxicity, moderate size to provide support, reactivity with the host, good biocompatibility, and in vivo degradability. Recently, β-glucan has been reported as a material to satisfy these requirements.2−5 β-Glucan is a polysaccharide with a basic skeleton of β(1,3)-glycosidic bonds, extracted from bacteria, mushrooms, yeast, or grains. It is also a food additive acknowledged by the U.S. Food and Drug Administration as being safe at the Generally Recognized as Safe level. Importantly, β-glucan can be recognized as an antigen by macrophages via pathogenassociated molecular patterns,6 has the ability to activate peripheral immune cells, and has immunity-enhancing properties.7 Therefore, β-glucan has been well studied.8 Current methods for treating cancer include surgery, radiation therapy, and treatment through administration of anticancer drugs. However, most of them show very strong cytotoxicity, and both cancer cells and normal cells are affected. Also, various side effects are observed in the course of administration of anticancer drugs.9 Therefore, in this study, as a method to reduce the side effects of anticancer drugs, β© 2019 American Chemical Society

glucan particles were prepared and loaded with antitumor drugs.10 These β-glucan particles were expected to assist in drug delivery, help raise self-immunity, and activate latent immune cells that develop under a persistent cancer condition. At the same time, we tried to address the above-mentioned cancer treatment problems by utilizing the synergistic effect of immune cell activity and chemotherapy by combining drug effects and β-glucan immune assistance. β-Glucan microparticles (YGlu) of size 2−4 μm derived from the cell wall of Baker’s yeast have been effectively used for the delivery of small molecules, siRNA/DNA, proteins, and nanoparticles.11,12 Baker’s yeast used in the study was treated by acid−base extraction. The final product provides a hollow central cavity suitable for encapsulation and transport of pharmaceutical ingredients, with a porous cell wall of β-glucan as the main constituent.13 However, in utilizing such a porous particle as a drug-delivery carrier, there is a possibility that the loaded drug easily leaks out as a result of external environmental conditions. To overcome this disadvantage, it is possible to stabilize the drug by coating the surface of βglucan microparticles by forming a matrix (Mat) through the Received: October 8, 2018 Accepted: December 27, 2018 Published: January 9, 2019 668

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Figure 1. Schematic illustration of preparing doxorubicin (DOX)-loaded hollow, porous β-glucan microparticles (YGlu/Mat/DOX). The matrix combining chitosan and alginate is used to protect pores and prevent leaking of loaded DOX molecules.

Figure 2. Characterization of YGlu and YGlu/Mat using field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM). (A, B) SEM images of bare YGlu. (C) TEM image of YGlu. (D, E) SEM images of YGlu/Mat. (F) TEM image of YGlu/Mat.

500−600 nm were present on the surface (Figure 2A,B). When observed through transmission electron microscopy (TEM) imaging, it could be seen that there were several holes of 500− 600 nm in one particle, rather than one hole, and a hollow structure (Figure 2C). The biocompatible polymers, cationic chitosan, and anionic alginate, used to enhance the delayed release, sustained release, and loading efficiency of the drug, can form a Mat through their electrostatic attraction. The matrix formed can be used as a coating material on the YGlu surface. The YGlu/Mat particle size of about 3−5 μm was confirmed by SEM and TEM imaging (Figure 2D−F). Figure 3 shows that when DOX was encapsulated in YGlu/ Mat, a peak indicating the amide bond of DOX at 1730.09 cm−1, which was not present in neat YGlu/Mat, was found in YGlu/Mat/DOX, confirming its successful loading.18 2.2. Loading and Encapsulation Efficiency of the YGlu/Mat/DOX Complex Particles. To confirm the efficiency of the use of the matrix with chitosan and alginate in loading the DOX into YGlu, we examined the supernatant after centrifugation, as described above. There was a difference in the absorbance of YGlu/DOX of 77.4% and YGlu/Mat/ DOX of 47.6% compared to the Free DOX of the supernatant (Figure 4A,B). Since the remaining DOX in the supernatant is the residual amount of the drug that has not been loaded into the carrier, using the matrix of chitosan and alginate was 30%

electrostatic attraction of cationic chitosan and anionic alginate, which are natural polymers.14 The advantages of these coating materials are excellent biocompatibility, biodegradability, antibacterial action, and cell adhesion. In particular, the sustained and delayed release of the internal drug can be controlled (Figure 1). In this study, the model drug was an antitumor drug, doxorubicin (DOX);15 β-glucan microparticles containing DOX and coated by chitosan and alginate were prepared and their drug-release behavior and cytotoxicity were evaluated. The immune response of β-glucan microparticles was measured using human peripheral blood mononuclear cells (PBMCs). It was confirmed that the β-glucan microparticles can be used as an anticancer drug-delivery system and can be used synergistically to promote immunity enhancement effects.

2. RESULTS AND DISCUSSION 2.1. Characterization of YGlu/Mat/DOX. The hollow, porous β-glucan microparticles (YGlu) derived from the yeast, which were formed through acid and base extraction methods, showed the three-dimensional structure of the yeast cells with the intracellular components removed, and were called “ghost” cells. This reconstructed YGlu is a microparticle with a diameter of 2−4 μm. Field emission scanning electron microscopy (FE-SEM) showed that large holes of about 669

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Figure 3. Fourier transform infrared (FTIR) spectra of DOX, YGlu/ Mat, and DOX-loaded YGlu/Mat/DOX.

Figure 5. In vitro DOX release profiles from the samples of YGlu with or without Mat. The released amounts of Dox at three pH conditions (at 5.2, 6.8, and 7.4) were obtained at 37 °C for different time points (up to 48 h).

more efficient for drug loading. After loading 5 mg of the same amount of YGlu/Mat particles with different concentrations of DOX, absorbance values were measured at 481 nm, the absorption wavelength of DOX. When the dose was quantified using a standard calibration curve for DOX, the loading efficiency (LE) was 1.05% at 1 mg/mL concentration of DOX, and the entrapment efficiency (EE) was 52.4% (Figure 4C,D). 2.3. In Vitro Release Behavior of Loaded DOX. To confirm whether the YGlu/Mat particles prepared in this study have proper drug-release characteristics for chemotherapy, DOX was loaded in YGlu and YGlu/Mat particles, and the drug-release behavior of each carrier was evaluated (Figure 5).

It has been reported that the immunological activity of βglucan starts from endocytosis and binding to receptors of immune cells such as macrophages.16,17 To simulate macrophage lysosomes, drug-release experiments were performed at three different pH conditions (i.e., at 5.2, 6.8, and 7.4). Under all pH conditions, initial bursts of DOX release were observed for the first 6 h with or without matrix. The cause of this initial release may be that the drug is loosely adsorbed on the surface of YGlu or YGlu/Mat particles or weakly trapped in the particles. The release behavior after the initial discharge

Figure 4. (A) UV−vis spectra of the supernatant of free DOX solutions YGlu/DOX and YGlu/Mat/DOX. (B) Color of the supernatant after harvesting YGlu/DOX and YGlu/Mat/DOX. Percentages refer to the relative absorbance of the supernatant at 481 nm. Color loss indicates that the supernatant has a lower amount of DOX, and the DOX loading in particles is higher. (C) Standard curve of DOX (250, 125, 62.5, 31.25, and 15.625 μg/mL in deionized water). (D) EE and LE of YGlu/Mat/DOX with different DOX concentrations. 670

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Figure 6. (A) Cytotoxicity of YGlu and YGlu/Mat in HUVECs. Dimethyl sulfoxide (DMSO) (50%) was used as positive control. (B) Cytotoxicity of YGlu/Mat in PBMCs. Both results indicate no significant cytotoxicity of YGlu/Mat.

Figure 7. Antitumor effect of YGlu/Mat/DOX. (A) Cytotoxic effect of DOX on MDA-MB-231 cells at 24 and 48 h at different concentrations of DOX. Cell viability tested with different free DOX concentrations at 24 and 48 h. The IC50 of free DOX was 0.496 μg/mL. (B) Cytotoxic effects of YGlu/Mat, free DOX, and YGlu/Mat/DOX on MDA-MB-231 cells at 24 and 48 h. At the same IC50 concentration of DOX, there was growth inhibition of MDA-MB-231 cells.

Figure 8. Measurement of (A) TNF-α and (B) IL-2 secretion from PBMCs exposed to different concentrations of YGlu or positive control with phytohormoneglutinin (PHA) stimulation (*P < 0.05, **P < 0.01, ns means no significance, n = 3).

2.4. Cytotoxicity Evaluation of YGlu and YGlu/Mat. The biocompatibilities of YGlu and YGlu/Mat particles were evaluated (Figure 6). First, HUVECs, human endothelial cells, were treated with various concentrations of YGlu and YGlu/ Mat particles and cultured for 24 h. The survival rate of the cells was maintained up to a concentration of 1 mg/mL. We also evaluated the cytotoxicity of YGlu/Mat with PBMCs, human serum immune cells, and found no toxicity at the highest concentration of 50 μg/mL. Therefore, the YGlu/Mat particles in this study have excellent biocompatibility.18 2.5. Antitumor Activity of YGlu/Mat/DOX. The CCK-8 assay was performed to evaluate the antitumor activity of

showed that most of the DOX was released within 24 h for the matrix-free YGlu particles, while the low release rate for the YGlu/Mat particles continued for 48 h (Figure 5). In addition, at lower pH, faster release was observed. The solubility of chitosan is increased by the low stability of alginate at low pH, and it induces rapid release with increasing matrix porosity. However, at higher pH, the solubility of chitosan decreases and the stability of alginate increases. It is also possible to consider the properties of the drug: DOX has a stronger pH dependence due to its decreased solubility at basic pH values and low-pH media can promote drug release. 671

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patient’s immune system as well as drug delivery. Therefore, it is expected that a drug can be selectively loaded in accordance with a patient’s disease with lower drug concentration, and there will be a lot of applications in the future.

YGlu/Mat/Dox on MDA-MB-231 cells. Cytotoxicity was assessed for different concentrations of DOX (Figure 7A). It was confirmed that the concentration of DOX causing 50% cell death at 24 h was 0.496 μg/mL, and this value was selected as the IC50 value. Considering that the LE of YGlu/Mat/DOX is about 1%, the YGlu/Mat/DOX concentration at 49.6 μg/mL is the IC50 concentration value for MDA-MB-231. The YGlu/ Mat/DOX showed a difference in anticancer activity against the same concentration of DOX for the same IC50. The inhibition rates of cancer cell growth of DOX were 19.18 and 59.12% for YGlu/Mat/DOX and 51.5 and 89% for Free DOX, respectively, for 24 and 48 h (Figure 7B). Hence, the matrix of YGlu/Mat/DOX delays and sustains the release of encapsulated DOX. 2.6. Cytokines (IL-2 and Tumor Necrosis Factor-α (TNF-α)) Released from PBMCs. For 24 h, as the matrix of YGlu/Mat formed by chitosan and alginate is biodegraded, gradually particles are exposed on the surface of the YGlu. The exposed surface of the β-glucan microparticles induces activation of immune cells by becoming a target of macrophages. The immunological activity of β-glucan microparticles was evaluated using PBMCs.19 In the case of TNF-α, 3980 pg of TNF-α expression was induced in YGlu at a concentration of 500 μg/mL compared to the control without any treatment. On the other hand, in the case of IL-2, a YGlu concentration of 500 μg/mL showed only a low significant result. The reason for this could be that the effect of β-glucan for immunological activity is considered to be somewhat weak in PBMCs because the population of macrophages in PBMCs is not high (Figure 8). In this study, the incubation time for monocyte PMBCs was short to maintain their maximum survival rate in ex vivo condition, so it is expected that the differentiation as macrophage was very minimal. Therefore, the current study conditions that induce the expression of IL-2 relative to TNF-α are not sufficient in monocytes because they are not completely differentiated in the experimental setups.

4. EXPERIMENTAL SECTION 4.1. Materials. Baker’s yeast and dialysis tube (MW cutoff, 1.4 kDa) were purchased from Fleischmann (Guelph, Canada) and Spectrum Labs (Seoul, Korea), respectively. Doxorubicin (DOX; Sigma-Aldrich, PHR1789), chitosan (Sigma-Aldrich, 448869), sodium alginate (Sigma-Aldrich, 9005-38-3), and triphosphate (Sigma-Aldrich, 7758-29-4) were purchased from Sigma-Aldrich (St. Louis, MO). The enzyme-linked immunosorbent (immunoadsorbent) assay (ELISA) kit was purchased from BioLegend (San Diego, CA) for the detection of TNF-α and IL-2, and all other chemicals were purchased from SigmaAldrich (St. Louis, MO) unless otherwise indicated. 4.2. Preparation of β-Glucan Particles (YGlu). In general, the cell walls in yeast account for 20−30% of the dry cell mass. The main constituents of these cell walls are mannoprotein, β-glucan (85−90%), trace chitin (1−3%), and other fatty acids (2−5%).20 The hollow, porous β-glucan microparticles (YGlu) used in this study were prepared by following the modified method described previously.13 YGlu was subjected to alkaline treatment by homogenizing Saccharomyces cerevisiae yeast (10 g of Baker’s yeast) for 2 h at 90 °C under 100 mL of 1 M NaOH using a homogenizer. After the reaction, the supernatant was removed by centrifugation at 2000g for 10 min, washed twice with deionized water, and then treated with HCl, pH 4−5 at 55 °C. The supernatant was removed by centrifugation at 2000g for 10 min as before, and the insoluble material was washed once with 40 mL of deionized water, four times with isopropanol, and twice with acetone to remove the insoluble material except the β-glucan and the cytoplasmic content of the yeast.21 Thereafter, the final product obtained by freeze drying was 8.12 g of hollow white β-glucan powder. 4.3. Preparation of YGlu/Mat/DOX. The encapsulation of DOX using chitosan and alginate on the dried YGlu was performed following the method described previously.22 After 5 mg of dried YGlu was dissolved in 200 μL of deionized water, 100 μL of DOX solution (1 mg/mL in deionized water) was added and reacted at room temperature for 10 min. After that, 100 μL of chitosan solution (0.4% w/v, pH 5.0 in 0.1 M acetic acid) was added and allowed to react at room temperature for 2 h so that DOX and chitosan were sufficiently mixed in YGlu. After the addition of 1.0 mL of TPP/alginate solution (1.0 mg/mL TPP, 0.4 mg/mL sodium alginate), the resulting particles were dispersed by ultrasonication and kept in an orbital shaker for 1 h. The resulting particles were centrifuged at 5000g for 5 min to remove DOX and impurities remaining in the supernatant and washed three times with deionized water and 70% ethanol. The final product was refrigerated at 4 °C after lyophilization. 4.4. Characterization of YGlu/Mat/DOX. 4.4.1. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). For surface characterization of the dried YGlu and YGlu/Mat, 1 mg of the corresponding sample was dissolved in 1 mL of deionized water. The prepared sample solution was dropped enough to cover the surface of the silicon wafer substrate and then spin-coated at 1500 rpm for 30 s. Silicon wafers coated with a sample were vacuum-coated with platinum for 3 s using an ion coater and then subjected to field

3. CONCLUSIONS The aim of this study was to reduce the side effects caused by indiscriminate aggression of cancer drugs in the conventional chemotherapy for cancer patients. Drug delivery through βglucan particles has been carried out to restore the immune system in the human body, which is neutralized by cancer cells, and to exert a synergistic effect with only a small dose of DOX, thereby improving the efficiency of tumor treatment. Through DOX release profiling, it was found that the coated matrix formed by chitosan and alginate enabled delayed and sustained release by controlling the release rate of the drug from the YGlu. Matrix and porous β-glucan showed almost no cytotoxicity, and YGlu/Mat has excellent biocompatibility. The antitumor ability against breast cancer cells due to delayed and sustained release of DOX from YGlu/Mat was also confirmed. This study confirmed the feasibility of using βglucan microparticles derived from yeast as a drug-delivery system in vitro. It is necessary to reexamine the immune activation ability, which is one of the main objectives in future work, to culture immune cells and cancer cells together, and to examine the cytotoxic effect by the enhanced immunological activity by YGlu/Mat/DOX. The sustained release of DOX would collaborate with exposed YGlu surface that is antigenic to immune cells around the tumor, synergistically activating immunological responses. Reported β-glucan drug delivery systems has excellent biocompatibility and may activate the 672

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release behavior was confirmed by measuring the fluorescence intensity of doxorubicin (emission and excited wavelengths of 590 and 480 nm, respectively).25 4.6. In Vitro Cell Experiments. 4.6.1. Cytotoxicity Assays. To evaluate the cytotoxicity of YGlu and YGlu/Mat microparticles, HUVEC and PBMCs were used. HEK293T cells were cultured in EGM-2 containing 5% fetal bovine serum (FBS) and 1% penicillin and streptomycin in a 5% CO2 incubator at 37 °C. PBMCs were cultured under the same conditions with RPMI 1640 medium containing 10% FBS and 1% penicillin and streptomycin. The cells were seeded at a density of 5 × 103 cells/well in a 96-well plate and cultured at 37 °C with 5% CO2 for 1 day. The final concentrations of YGlu and YGlu/Mat as drug carriers were 1, 0.5, 0.25, and 0.125 mg/mL for HUVECs and 50, 25, 5, 2.5, and 0.5 μg/mL for PBMCs. The cells were cultured in a 5% CO2 incubator at 37 °C, and a CCK-8 assay was performed to measure cytotoxicity after 24 h. 4.6.2. Antitumor Activity of YGlu/Mat/DOX. To evaluate the anticancer effect of DOX released from YGlu/Mat/DOX, the MDA-MB-231 cell line, a breast cancer cell line, was assessed by the CCK-8 assay. The cells were seeded at a density of 5 × 103 cells/well in a 96-well plate at 37 °C in Dulbecco’s modified Eagle’s medium containing 10% FBS and 1% penicillin and streptomycin. YGlu/Mat, YGlu/Mat/DOX, and free DOX samples were added at the target concentrations, and cytotoxicity was measured at 24 and 48 h in a 5% CO2 incubator at 37 °C. 4.6.3. Assay of IL-2 and TNF-α for Immunological Response. PBMCs were cultured for 16 h and then seeded at a concentration of 2 × 105 cells/well in a 24-well plate. PHA (5 μg/mL) and YGlu microparticles as PBMC stimulants were added at final concentrations of 500, 250, and 125 μg/mL, and then the cells were treated for 24 h at 37 °C in 5% CO2. The secretion of cytokines (IL-2, TNF-α) in PBMC cultures was measured using an ELISA kit (BioLegend, Inc.). As a control, 5 μg of phytohemagglutinin (PHA), a PBMC stimulant, was added.26

emission scanning electron microscopy (FE-SEM) (Carl Zeiss) analysis. 4.4.2. Fourier Transform Infrared (FTIR) Analysis. YGlu, matrix-coated YGlu, and YGlu/Mat/DOX samples were analyzed via Fourier transform infrared spectrometry (FTIR) (Jasco) to analyze the surface chemistry of each sample. 4.5. Evaluation of YGlu/Mat/DOX. 4.5.1. Determination of DOX Loading and Encapsulation Efficiency. For quantitative analysis of DOX, 1 mL of DOX solution was diluted to concentrations of 250, 125, 62.5, 31.25, and 15.62 μg/mL and used as a standard material for UV/vis absorbance analysis. Absorbance was measured at 481 nm to obtain a standard calibration curve with a correlation coefficient (R2) value of 0.9958. To confirm the effect of the matrix of chitosan and alginate on the loading efficiency of the drug, 100 μL of DOX (1 mg/ mL in deionized water) was added to the same 5 mg of YGlu and YGlu/Mat and reacted under the same conditions. In addition, DOX alone (free DOX) was reacted under the same conditions without carriers (YGlu or YGlu/Mat). After that, three kinds of DOX solutions (YGlu + DOX, YGlu/Mat + DOX, and free DOX) were centrifuged at 5000g for 5 min to remove the DOX remaining in the solution. A volume of 1 mL of supernatant was removed, and the absorbance values of YGlu and YGlu/Mat versus the absorbance values of free DOX at 481 nm and the 400−600 nm absorption band were compared using the following formula23 (eq 1) free DOX supernatant ratio (%) unloaded DOX absorbance = × 100% free DOX absorbance

(1)

The drug loading efficiency (LE) and entrapment efficiency (EE) of YGlu/Mat were also measured by absorbance analysis.24 The amount of free DOX in the supernatant was calculated using a standard calibration curve for the previously obtained DOX standard by using 1 mL of the previously removed supernatant. The mass of the drug loaded in YGlu/ Mat was calculated by subtracting this value from the amount of 100 μg of DOX initially loaded. LE and EE were obtained using the following formula (eqs 2 and 3)

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*E-mail: [email protected]. Tel: +82-2-820-5258.

drug loading efficiency, LE (%) the amount of drung loaded into YGlu/Mat = × 100% the amount of initial drug

ORCID

Kyobum Kim: 0000-0003-3678-2078 Hyung-Jun Koo: 0000-0001-6804-741X Jonghoon Choi: 0000-0003-3554-7033

(2)

Notes

drug entrapment efficiency, EE (%) the amount of drug loaded into YGlu/Mat = the amount of loaded materials into YGlu/Mat × 100%

AUTHOR INFORMATION

Corresponding Author

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Nano-Material Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. 2017M3A7B8061942).

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4.5.2. In Vitro Release Tests of YGlu/Mat/DOX. To compare the release behavior of DOX from YGlu/DOX and YGlu/Mat/DOX, 1 mg of the lyophilized sample was dissolved in phosphate-buffered saline (pH values 7.4, 6.8 and 5.2), added to 0.02% v/v Tween 80, and incubated at 100 rpm (37.5 ± 5 °C). The release behavior was evaluated at predetermined time intervals. A sample dispersion containing DOX was placed in a dialysis bag. This was transferred to 20 mL of the same buffer (in the phosphate-buffered saline, pH at 5.2, 6.8, or 7.4), the drug release over time was recorded, and the

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