Biodegradable Magnesium-Incorporated Poly(l-lactic acid

Jun 7, 2019 - ... of Mg/PLLA microspheres for tailoring drug release in a physiological environment. The animal experiment reveals that Mg particles c...
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Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23546−23557

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Biodegradable Magnesium-Incorporated Poly(L‑lactic acid) Microspheres for Manipulation of Drug Release and Alleviation of Inflammatory Response Fenghe Yang,†,‡ Xufeng Niu,*,†,‡,§ Xuenan Gu,†,‡ Chuanping Xu,†,‡ Wei Wang,∥ and Yubo Fan*,†,‡,⊥

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†

Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China ‡ Beijing Advanced Innovation Centre for Biomedical Engineering, Beihang University, Beijing 100083, China § Research Institute of Beihang University in Shenzhen, Shenzhen 518057, China ∥ Department of Immunology, School of Basic Medical Sciences, NHC Key Laboratory of Medical Immunology, Peking University, Beijing 100191, China ⊥ Beijing Key Laboratory of Rehabilitation Technical Aids for Old-Age Disability, National Research Center for Rehabilitation Technical Aids, Beijing 100176, China S Supporting Information *

ABSTRACT: Poly(L-lactic acid) (PLLA) and magnesium (Mg) are widely concerned biodegradable materials, but during in vivo implantation, the former produces acidic degradation byproducts and can easily induce inflammation in surrounding tissues, whereas the latter is fast corroded and generates alkaline products. The purpose of this study is to develop Mg/PLLA composite microspheres as a novel delivery system, in which Mg particles are used to regulate the drug release profile and suppress PLLA-induced inflammatory response. Morphological observation shows that multiple Mg particles are dispersed both on the surface and in the interior of composite microspheres. In vitro release study indicates that by varying the Mg contents or its particle sizes, the internal connectivity of composite microspheres is changed during hydrolytic degradation, and drug delivery can be facilely manipulated with tunable release patterns. In vivo release study further confirms the feasibility of Mg/ PLLA microspheres for tailoring drug release in a physiological environment. The animal experiment reveals that Mg particles can alleviate macrophage infiltration and inflammatory cytokine expression. These results demonstrate the availability of using biodegradable Mg particles to manipulate drug release as well as alleviate PLLA-induced inflammation. The present Mg/PLLA composite microspheres have potential applications in controlled delivery of various therapeutic agents, especially some growth factors, for bone regeneration. KEYWORDS: controlled release, microspheres, poly(l-lactic acid), magnesium, inflammation

1. INTRODUCTION

them, poly(L-lactic acid) (PLLA) has been widely investigated due to a number of advantageous features. First, PLLA is a biodegradable polymer, which can gradually degrade into water and carbon dioxide in the body.4,5 Second, drug release profiles

As one of the most promising delivery systems of therapeutic agents, polymer microcarriers have gained much attention over the past few decades. Administration of drugs or proteins via such systems is advantageous because the polymer matrix has the potential to protect its contents from degradation, as well as provide the sustained release behavior.1−3 To date, many polymers have been used to fabricate microcarriers, and among © 2019 American Chemical Society

Received: March 1, 2019 Accepted: June 7, 2019 Published: June 7, 2019 23546

DOI: 10.1021/acsami.9b03766 ACS Appl. Mater. Interfaces 2019, 11, 23546−23557

Research Article

ACS Applied Materials & Interfaces Table 1. Mg/PLLA Microsphere Composition and Encapsulation Efficiency of BSAa microsphere type

Mg size (μm)

Mg mass (mg)

PLLA mass (mg)

Mg-free PLLA 1% Mg/PLLA 2.5% Mg/PLLA 5% Mg/PLLA 10% Mg/PLLA 2.5% Mg(L)/PLLA 10% Mg(L)/PLLA

N/A 9.68 ± 4.93 9.68 ± 4.93 9.68 ± 4.93 9.68 ± 4.93 31.02 ± 14.17 31.02 ± 14.17

0 3 7.5 15 30 7.5 30

300 297 292.5 285 270 292.5 270

BSA mass (mg) Mg/(Mg + PLLA) mass ratio (%) 25 25 25 25 25 25 25

0 1 2.5 5 10 2.5 10

encapsulation efficiency (%) 84.2 83.0 88.9 81.2 75.7 86.7 87.1

± ± ± ± ± ± ±

0.6 2.9 4.2 3.0 3.6 1.9 0.8

a The microspheres were formulated such that the whole microsphere and BSA mass were constant among different groups, whereas Mg and PLLA mass were varied. Microsphere type 1% Mg/PLLA means the Mg/(Mg + PLLA) ratio in this type of microspheres was 1%. Microsphere with larger Mg particles (31.02 ± 14.17 μm) inside was represented as Mg(L)/PLLA microsphere.

As a promising biodegradable and biocompatible metallic material, magnesium (Mg) may offer an opportunity to achieve these characteristics.24 In physiological conditions, Mg first transforms into Mg(OH)2. Due to the existence of Cl− in the body, Mg(OH)2 subsequently converts into MgCl2 and produces OH−.25 Hence, the dissolution of Mg can buffer the acid in the surrounding environment and increase the local pH.26 Moreover, Mg is quickly dissolved in body fluids.27 It may offer another choice to control the drug release precisely besides polymer degradation. In addition, many studies have shown that Mg is a bioactive material with good osteogenesis capacity,28−31 which means that it can cowork with some therapeutic agents, such as bone morphogenetic proteins (BMPs),32 to realize better bone regeneration. All of these features imply that Mg is a potential candidate to overcome the issues of PLLA as used in delivery systems. The overall goal of this study is to develop a new kind of Mg/PLLA composite microspheres as a drug delivery system, in which the Mg particle, as a bioactive material, can not only tailor drug release profiles but also regulate PLLA-induced inflammatory response. PLLA with high molecular weight is selected to eliminate the influence of polymer degradation on drug release rates. To investigate the effect of Mg contents and its particle size on drug release behaviors, several types of Mg/ PLLA composite microspheres are synthesized, with bovine serum albumin (BSA) as a model protein drug. Both in vitro and in vivo studies are conducted to monitor the drug release profiles. Cell and animal studies are launched to evaluate the cytotoxicity and inflammatory response of these microspheres, respectively. We hypothesized that the incorporation of a suitable amount of Mg particles into PLLA-based microspheres would tailor drug release kinetics, as well as alleviate the degree of inflammatory reaction.

of PLLA microcarriers can be tailored by numerous ways, including changing the PLLA molecular weight,6 adjusting microcarrier formulation parameters,7 copolymerization or blending with other biodegradable polymers,8,9 fabricating PLLA-based stimuli-responsive carriers,10 etc. Third, the size of PLLA-based carriers can be regulated accurately ranging from micrometers down to only a few nanometers, which can fit different demands for intracellular or extracellular delivery situations.11,12 Furthermore, PLLA microcarriers can be formulated to load a wide range of therapeutic agents, including both hydrophilic and hydrophobic drugs,13,14 showing the great versatility of this material as a delivery system. Despite all of the advantages listed above, some issues still exist for this material as used in delivery systems. First and foremost, the regulation of drug release from a PLLA-based matrix is commonly realized by changing polymer molecular weights or by varying microcarrier formulation parameters. This is a typical “passive control”, mainly based on the hydrolytic degradation of PLLA. To achieve the more advanced “active control”, some stimuli-responsive materials need to be introduced into PLLA.15,16 However, the introduction of stimuli-responsive materials is always accompanied by complex chemical reactions, which might inevitably lead to decreased biocompatibility due to the residue of organic phase in final products. Another common issue is acidic byproducts generated in the process of PLLA hydrolytic degradation, which can decrease local pH and result in acidinduced inflammation in surrounding tissues.17 To solve this problem, some studies attempted to incorporate basic agents like CaCO3 and Mg(OH)2 into the PLLA matrix, and these agents were proved to successfully suppress inflammatory reaction by neutralizing the acidic byproducts.18,19 However, with the presence of such basic agents, the undesirable decreasing mechanical properties of PLLA were also observed because of the low interface strength between the polymer matrix and additives.20 The third issue is the bioinert character of PLLA, making it unable to work synergistically with agents encapsulated inside to achieve the best therapeutic effect. A potential solution was the introduction of some bioactive organic or inorganic materials.21−23 This led to improved therapeutic effect, but many of these components are unable to degrade in the body, which limited their further application in delivery systems. The ideal PLLA-based delivery system should be easily synthesized with the ability of tailoring drug release profiles precisely, buffering acidic byproducts, and synergizing with typical drugs or proteins to enhance their therapeutic effects.

2. MATERIALS AND METHODS 2.1. Materials. PLLA (RESOMER L 207 S, Inherent viscosity: 1.5∼2.0 dL/g) was purchased from Evonik Industries AG (Essen, Germany). Pure Mg particles (>99% purity, with average particle sizes of 9.68 μm and 31.02 μm) were supplied by Tangshan Weihao Magnesium Powder Co., Ltd. (Tangshan, China). Poly(vinyl alcohol) (PVA) hydrolyzed 88% was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). BSA and rhodamine B isothiocyanate labeled BSA (RBITC-BSA) were purchased from Solarbio Co., Ltd. and Biosynthesis Biotechnology Co., Ltd. (Beijing, China), respectively. 2.2. Preparation of Mg/PLLA Microspheres. BSA-containing Mg/PLLA composite microspheres were fabricated using a water-inoil-in-water (w/o/w) double-emulsion solvent extraction/evaporation technique. PLLA and Mg particles were added into dichloromethane and stirred for 1 h to dissolve PLLA thoroughly as well as make Mg 23547

DOI: 10.1021/acsami.9b03766 ACS Appl. Mater. Interfaces 2019, 11, 23546−23557

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ACS Applied Materials & Interfaces particles suspend uniformly. Then, 500 μL of 5% (w/v) BSA solution was mixed with the above solution and emulsified using a probe sonicator at an output power of 100 W for 20 s over an ice bath to form a water-in-oil (w/o) emulsion. This emulsion was gradually added into 250 mL of 1% (w/v) PVA solution under stirring to produce a w/o/w double emulsion. The obtained emulsion was stirred at room temperature for 3 h to evaporate dichloromethane, and the solid microspheres were collected by centrifugation. The resultant microspheres were washed three times with double-distilled water and lyophilized for 2 days. To investigate the effect of Mg content and its particle size on drug release rates, seven types of microspheres were designed using the optimized synthesis parameters as shown in Table 1. 2.3. Characterization of Microspheres. A scanning electron microscope (SEM) (FEI Quanta 250 FEG) was used to observe the surface appearance and internal structure of microspheres. The lyophilized microspheres were mounted onto metal stubs, coated with a layer of platinum, and observed microscopically. For the observation of the internal structure, the microspheres were dispersed in an optimal cutting temperature compound, frozen, and cross-sectioned using a freezing microtome. An energy-dispersive spectrometer (EDS) was used to analyze the chemical composition of microspheres. Besides, the size distribution of microspheres was measured using the image analysis program ImageJ (National Institute of Health, Bethesda). 2.4. BSA Encapsulation Efficiency. The encapsulation efficiency of BSA was tested according to a reported method.33 In brief, 10 mg of microspheres was hydrolyzed in a mixed solvent of NaOH (0.8 mL, 1 M) and phosphate-buffered saline (PBS) (0.2 mL, 0.1 M, pH 7.4) with a vigorous shaking at 37 °C for 12 h. BSA standard solution (0.2 mL) was also mixed with NaOH (0.8 mL, 1 M) and hydrolyzed using the same procedure. Afterward, HCl (1 mL, 0.8 M) was added to neutralize the sample solution. The BSA concentration was examined by using a Micro-BSA protein assay kit. 2.5. In Vitro Degradation Studies. The in vitro degradation of microspheres was investigated by monitoring Mg2+ release, media pH, molecular weight variation of the PLLA matrix, and surface morphology of microspheres during the course of 28 days. Ten milligrams of microspheres were placed in test tubes, immersed in 2.0 mL of PBS (0.1 M, pH 7.4), and kept in a 37 °C incubator shaker. At predetermined time intervals, the samples were centrifuged. Then, 1 mL of the supernatant was collected and replaced with an equal amount of fresh PBS (0.1 M, pH 7.4). Mg2+ concentration in the supernatant was tested by using inductively coupled plasma optical emission spectrometry (Optima 5300DV Spectrometer). Before testing, the collected supernatant was diluted 10× in PBS containing 1% (v/v) HCl to make sure all Mg in the release medium was dissolved. To monitor the pH value during degradation, another 10 mg of microspheres was immersed in 2 mL of PBS (0.1 M, pH 7.4) and kept in a 37 °C incubator shaker. At predetermined time intervals, all of the degradation media were collected and replaced with an equal amount of fresh PBS (0.1 M, pH 7.4). The pH value of degradation media was measured using a pH meter (Leici PHS-3C, China). As for the degradation of PLLA, the microspheres rather than the supernatant were collected by centrifugation at predetermined time intervals, washed with distilled water, and lyophilized for 2 days. Gel permeation chromatography (GPC) (Waters 1515) analysis was performed to measure the molecular weight of PLLA, using a system equipped with a waters 2414 refractive index detector and Agilent PLgel 5 μm MIXED-C columns. Chloroform was used as the mobile phase (flow rate 1 mL/min, column temperature 35 °C). The calibration curve was prepared using monodisperse polystyrene standards. The surface morphology of microspheres during in vitro degradation was also examined on 1, 9, and 28 days using SEM. 2.6. In Vitro Release Studies. Ten milligrams of microspheres were suspended in 2 mL of PBS (0.1 M, pH 7.4) with 0.02% (w/v) NaN3 as the preservative. The suspensions were kept in an incubator shaker maintained at 37 °C. At predetermined time intervals, the samples were centrifuged. Then, 1 mL of the supernatant was

collected and replaced with an equal amount of fresh PBS (0.1 M, pH 7.4). BSA concentration in the supernatant was measured using a Micro-BSA protein assay kit. After reaction following the protocol, the supernatant was placed in a 96-well plate, and the concentration of BSA in all samples was determined at a wavelength of 562 nm using a multimode microplate reader (Varioskan Flash, Thermo Scientific). 2.7. Cell Culture. MC3T3-E1 pro-osteoblasts purchased from Chinese Academy of Medical Sciences & Peking union medical college (Beijing, China) were selected as normal cells for the cytotoxicity assay. The MC3T3-E1 cells were cultured in α-minimum essential medium (α-MEM, Gibco) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2. The medium was changed every 3 days. When cells reached 80% confluence, they were detached with 0.25% trypsin−ethylenediaminetetraacetic acid solution (Solarbio, China), resuspended in α-MEM, and seeded in a 24well plate for further exploration. 2.7.1. Cell Viability. A cell counting kit-8 (CCK-8, Dojindo Laboratory, Japan) was used to assess cell proliferation. Cells were seeded in a 24-well plate at a density of 5 × 103 cells/well. After 24 h culture for cell attachment, three kinds of sterile microspheres (Mgfree PLLA, 2.5% Mg/PLLA, 10% Mg/PLLA) were added in wells (5 mg/well) separately. Wells without microspheres were set as the control group. At predetermined time points (1, 3, and 6 days), the culture medium was removed. Then, 500 μL of fresh α-MEM and 50 μL of CCK-8 solution were added to each well. After 1 h incubation in a 5% CO2 incubator at 37 °C, 100 μL of the supernatant was transferred into a 96-well plate. The absorbance was measured at 450 nm to assess cell proliferation using a multimode microplate reader (Varioskan Flash, Thermo Scientific). 2.7.2. Acridine Orange/Ethidium Bromide (AO/EB) Staining. AO/ EB staining was employed to assess the morphology and density of cells visually. Briefly, cells were washed with PBS (0.1 M, pH 7.4) 2 times. Then, 500 μL of PBS (0.1 M, pH 7.4) containing 1 μg/mL AO (Solarbio, China) and 1 μg/mL EB (Solarbio, China) was added to each well. After 5 min incubation under room temperature, cells were washed with fresh PBS (0.1 M, pH 7.4) again and examined under a fluoresce microscope (IX73, Olympus, Japan). 2.8. In Vivo BSA Release and Degradation Studies. Animals were obtained from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and kept in a specific pathogen-free facility at Peking University Health Science Center (Beijing, China). All of the animal procedures conformed to the Chinese Council on Animal Care Guidelines, and the study was approved by the ethics committee of Peking University Health Science Center with an approval number of LA2018327. 2.8.1. In Vivo RBITC-BSA Release. The microspheres containing RBITC-BSA were implanted into the female BALB/c nude mice (aged 5−7 weeks, 18−21 g). For subcutaneous implantation, each nude mouse was anesthetized by intraperitoneal injection of sodium pentobarbital (0.05 mg/g of body weight). An incision (1 cm) was made on one side of the back to create a subcutaneous pocket. Ten milligrams of RBITC-BSA-loaded microspheres were implanted into the subcutaneous pocket, and the incision was closed with poly(glycolic acid) sutures. On days 0, 7, 14, and 28, the side-view images of nude mice were collected at a wavelength of 620 nm (excitation wavelength 550 nm) using a fluorescence imaging system (Maestro 2 in vivo imaging system). 2.8.2. In Vivo BSA Release from a Cage Implant System. The BSA-containing microspheres were loaded into a cage made of silicone tubing and stainless mesh before surgery. For subcutaneous implantation, each male Sprague-Dawley (SD) rat (age 10 weeks, approximately 300 g) was anesthetized by intraperitoneal injection of sodium pentobarbital (0.05 mg/g of body weight). The dorsal surface of each rat was shaved and sterilized with 75% ethanol and then sterile physiological saline. An incision (2 cm) was made on one side of the back to create a subcutaneous pocket. The cage was implanted into the subcutaneous pocket; then, the incision was closed with sterile wound clips. To eliminate the effect of native protein in vivo, the same amount of BSA-free microspheres was also loaded into the cage and 23548

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Figure 1. SEM photographs of the surface and cross-sectional appearance of some typical microspheres. (A, D, G) Mg-free PLLA microspheres; (B, E, H) 2.5% Mg/PLLA microspheres; (C, F, I) 10% Mg/PLLA microspheres. (J−L) Corresponding mapping EDS analyses of the cross-sectional images. (J) Mg-free PLLA microspheres; (K) 2.5% Mg/PLLA microspheres; (L) 10% Mg/PLLA microspheres. in paraffin and sectioned into a thickness of 5 μm. The sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope (Nikon Eclipse LV100ND, Japan) for tissue inflammatory reaction examination. 2.9.2. Immunohistochemistry. The samples (5 μm) were deparaffinized and stained with antibodies (obtained from Servicebio Co., Ltd., Beijing, China) including anti-F4/80 for monocyte/ macrophage, anti-IL-1β and anti-TNF-α for cytokine expression, anti-Ly6G for neutrophil, anti-CD11b for myeloid cell, and anti-CD3 for T-cell. The stained sections were then observed under a light microscope (Nikon Eclipse LV100ND, Japan). 2.10. Statistical Analysis. Statistical analysis was performed using statistical software SPSS (IBM). All experiments were performed in triplicates, and the results were presented as mean value ± standard deviation of replicate experiments. Statistical significance was assessed using a one-way analysis of variance. Statistical significance was accepted as p < 0.05.

implanted into another side of the back. At predetermined time intervals, the rats were sacrificed by injecting an overdose of sodium pentobarbital (0.5 mg/g of body weight). The cages were removed from the subcutaneous space, and the microspheres inside were collected. The collected microspheres were washed three times with distilled water and lyophilized for 2 days. The remaining BSA in the collected microspheres was calculated by subtracting the protein content in BSA-free microspheres from the protein content in BSAcontaining microspheres. 2.8.3. In Vivo Degradation Studies. In vivo degradation studies were also carried out using the cage implant system. In each group, two rats were set and implanted with cages on both sides of the back. At predetermined time intervals, the microspheres in these cages were collected and used for in vivo degradation studies by GPC (Waters 1515). 2.9. In Vivo Inflammatory Response. Ten milligrams of BSAfree microspheres were directly implanted into the female BALB/c mice (aged 6−8 weeks, 18−22 g) using the same surgery procedures as listed above. Sterile cotton was also implanted into each subcutaneous pocket as the positive control group. Each group had 10 mice totally. On days 10 and 30 postsurgery, 5 mice of each group were sacrificed by injecting an overdose of sodium pentobarbital (0.5 mg/g of body weight). The implants along with their surrounding tissues were retrieved for the following evaluation. 2.9.1. Histological Analysis. The samples were fixed in 4% (w/v) phosphate-buffered paraformaldehyde solution for 2 days and dehydrated in graded series of ethanol. Then, they were embedded

3. RESULTS 3.1. Characterization of Mg/PLLA Microspheres. Various Mg/PLLA composite microspheres were fabricated with changing Mg contents, and the microspheres had protein encapsulation efficiency ranging from 75.7 to 88.9% (Table 1). Figure 1 shows the surface and cross-sectional morphology of the typical microspheres listed in Table 1. It can be seen that all microspheres were spherical in shape and dense in internal 23549

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Figure 2. Effects of Mg contents on in vitro BSA release profiles. (A) BSA release profiles, (B) Mg release profiles, and (C) media pH of the Mgfree PLLA, 1% Mg/PLLA, 2.5% Mg/PLLA, 5% Mg/PLLA, and 10% Mg/PLLA microspheres. Data represent means ± standard deviation of three measurements on one sample. (D−L) SEM images of various microspheres degradation over 1, 9, and 28 days. (D, G, J) Mg-free PLLA microspheres; (E, H, K) 2.5% Mg/PLLA microspheres; and (F, I, L) 10% Mg/PLLA microspheres.

Figure 3. Effects of Mg particle size on in vitro BSA release profiles. (A) BSA release profiles of the 2.5% Mg/PLLA, 2.5% Mg(L)/PLLA, 10% Mg/ PLLA, and 10% Mg(L)/PLLA microspheres. Data represent means ± standard deviation of three measurements on one sample. (B−E) SEM photographs of the cross-sectional view of typical microspheres after 28 days of degradation. (B) 2.5% Mg/PLLA microspheres; (C) 10% Mg/ PLLA microspheres; (D) 2.5% Mg(L)/PLLA microspheres; and (E) 10% Mg(L)/PLLA microspheres.

3.2. In Vitro Release Studies. Both the effects of Mg content and its particle size on BSA release profile were investigated. Figure 2A illustrates BSA release profiles of microspheres with the same Mg particle size but different Mg contents. All microspheres presented sustained BSA release, and the Mg-free group had the lowest release over the 28 days of experiment. With increasing Mg contents, the initial BSA release within 9 days became more and more obvious, which corresponded to the fast dissolution of Mg at this stage (Figure 2B). The dissolution of Mg also increased the pH of degradation media. For high Mg content groups (5 and

structure, with the diameter mainly ranging from 200 to 500 μm (Figure S1, Supporting Information). For the Mg/PLLA microspheres, Mg particles appeared apparently on the surface of microspheres (Figure 1E,F, black arrows). Mapping EDS was employed to further identify Mg distribution within the microspheres. The results revealed that strong and homogeneous Mg signals were detected inside the Mg/PLLA microspheres (Figure 1K,L, red points), indicating the successful encapsulation and wide distribution of Mg particles in the whole microspheres. 23550

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Figure 4. MC3T3-E1 cell proliferation assays. (A) CCK-8 assays of cell proliferation with the different microspheres (* denotes statistical difference with the control group, *p < 0.05). (B−E) AO/EB staining after 6 days of proliferation. (B) Control group; (C) Mg-free PLLA microspheres; (D) 2.5% Mg/PLLA microspheres; and (E) 10% Mg/PLLA microspheres. Scale bar is 300 μm.

Figure 5. Effects of Mg contents on in vivo BSA release profiles. (A−L) In vivo fluorescent images of mouse with subcutaneously implanted microspheres at different time points. (A−D) Mg-free PLLA microsphere-implanted mouse; (E−H) 2.5% Mg/PLLA microsphere-implanted mouse; (I−L) 10% Mg/PLLA microsphere-implanted mouse. (M) In vivo release profiles of BSA from the Mg-free PLLA, 2.5% Mg/PLLA, and 10% Mg/PLLA microspheres. Data represent means ± standard deviation of three measurements on one sample.

PLLA microspheres could maintain the smooth and dense morphology during the whole in vitro degradation period. Figure 3 shows the effects of Mg particle size on BSA release kinetics. As compared with the microspheres with 9.68 μm Mg size particles, the microspheres including the same amount of 31.02 μm Mg size particles exhibited similar but slower BSA release patterns. As a result, at the end of 28 days of the in vitro release study, the cumulative releases of BSA from the 2.5% Mg(L)/PLLA and 10% Mg(L)/PLLA microspheres were 55.5 and 70.8%, respectively, which were approximately 30% lower than those from the corresponding 2.5% Mg/PLLA and 10% Mg/PLLA microspheres. To further clarify how Mg particles affected BSA release profiles, we investigated the internal structure of microspheres. After 28 days of degradation, the Mg-free PLLA microspheres still preserved a dense structure, showing poor internal connectivity (Figure S2, Supporting Information). However, for Mg-containing microspheres, the dissolution of Mg particles resulted in the formation of small cavities in their previous position (Figure 3B−E). With the formation of cavities, the internal structure became unstable, resulting in some cracks emerging in these microspheres. For the 2.5% Mg/PLLA microspheres, only a small number of cavities and cracks was observed, whereas a large number of cavities and cracks was found in the 10% Mg/PLLA

10%), the media pH increased to almost 8.8 on day 9. While for low Mg content groups (1 and 2.5%), the elevation of the pH value was quite milder (Figure 2C). Morphology observation revealed that some micropores formed on the surface of Mg-containing microspheres, and the pore numbers increased with increasing Mg contents (Figure 2H,I). Notably, besides pores, surface cracks were also observed on the surface of 10% Mg/PLLA microspheres on day 9 (Figure 2I and its inset), which was believed to induce the second burst release that appeared in this group at this time. After that, the gradually decreased BSA release rates and media pH were observed in all groups. As a result, most of the BSA was released from the 10% Mg/PLLA group at the end of 28 days of experiment, and the cumulative release amount for the other groups decreased correspondingly with reducing Mg contents in the microspheres. As for the pH level, the 5% Mg/PLLA and 10% Mg/PLLA groups possessed pH values close to 7.4 at the end of this study. The 1% Mg/PLLA and 2.5% Mg/PLLA groups kept a constant pH value of approximately 7.4 since day 9. While for Mg-free PLLA microspheres, the media pH dropped to a value close to 6.8 after 28 days of degradation. Besides, molecular weight analysis indicated that PLLA kept almost the same weight-averaged molecular weight (Mw) (Table S1, Supporting Information), and thus the Mg-free 23551

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Figure 6. Local inflammatory cell infiltration around the cotton, Mg-free PLLA microspheres, 2.5% Mg/PLLA microspheres, and 10% Mg/PLLA microspheres on 10 and 30 days postsurgery. (A) Images of H&E staining showing the inflammatory degree around the implants. (B) Images of F4/80 positive cells staining showing the infiltrating macrophage number. Scale bar is 50 μm.

fluorescence intensity was observed on day 28 (Figure 5E−H). The fastest fluorescence decay was found in the 10% Mg/ PLLA microspheres. Rapid decrease of fluorescence was observed at all time points, resulting in almost negligible fluorescence intensity at the end of the in vivo release study (Figure 5I−L). To quantity BSA release more precisely, we further constructed a cage implant system according to a previous study for in vivo release analysis.34 All three groups exhibited similar BSA release patterns (Figure 5M), and the cumulative BSA release amount increased with improving Mg contents in the microspheres. After experiencing a rapid release period within the first 3 days, slower and near-zero-order release of BSA was observed in the following days. At the end of 28 days of experiment, the cumulative release of BSA for 2.5% Mg/ PLLA and 10% Mg/PLLA microspheres was 84.3 and 92.7%, respectively, whereas that for the Mg-free groups was approximately 60.0%. 3.5. In Vivo Inflammatory Response. To investigate whether the introduction of Mg can inhibit inflammatory response caused by PLLA under the physiological or pathological condition, we conducted a subcutaneous implantation experiment. Cotton was used as the positive control group. The samples of 2.5% Mg/PLLA and 10% Mg/ PLLA microspheres were set as low and high Mg dose groups, respectively. Acute and chronic inflammation were evaluated through H&E staining and immunohistochemistry on 10 and 30 days postsurgery. In H&E staining sections, the degree of local inflammation was assessed semiquantitatively by counting layers of inflammatory cells around the implants (Figure 6A). After 10 days of implantation, the cotton group triggered the most severe inflammatory response and was surrounded by 10−12 layers of cells, and some cells even infiltered into the fibers of cotton. By comparison, the Mg-free PLLA and 10% Mg/PLLA

microspheres. Compared with the 2.5% Mg/PLLA and 10% Mg/PLLA microspheres, the size of cavities was larger in the corresponding 2.5% Mg(L)/PLLA and 10% Mg(L)/PLLA groups, but the internal structure was denser in these two groups with less cavities and cracks observed. 3.3. In Vitro Cell Viability Assays. The cell viability on different microspheres was assessed by the CCK-8 assay and AO/EB staining. Figure 4 shows that cell proliferation viability of the 2.5% Mg/PLLA group was comparable to that of the control group at all time points, indicating the good cytocompatibility of this kind of microspheres. As a comparison, the other two groups showed different degrees of inhibition on cell proliferation. For the 10% Mg/PLLA microspheres, significantly lower cell viability was observed on both day 3 and day 6 as compared with the control group. While for the Mg-free PLLA microspheres, cell viability was only significantly poor on day 3. However, it should be noted that although cell proliferation viability of the Mg-free PLLA and 2.5% Mg/PLLA groups was both comparable to that of the control group on day 6, cell viability of the Mg-free PLLA was still lower than that of the 2.5% Mg/PLLA group. AO/EB staining further revealed that after proliferation for 6 days, cells in all groups maintained good morphology regardless of the proliferation rate. 3.4. In Vivo Release Studies. Fluorescence imaging technique was first employed here to assess the in vivo release profiles of RBITC-BSA-containing microspheres. Overall, microspheres with higher Mg contents had faster RBITCBSA release rates, which were consistent with the results of in vitro release studies. During the whole release period, both the area and intensity of fluorescence decreased slightly in the Mgfree PLLA microspheres, and a high level of fluorescence was still preserved on day 28 (Figure 5A−D). For the 2.5% Mg/ PLLA microspheres, obvious fluorescence signal loss first happened on day 14, and finally less than 50% of the initial 23552

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Figure 7. Local inflammatory cytokine expression around the cotton, Mg-free PLLA microspheres, 2.5% Mg/PLLA microspheres, and 10% Mg/ PLLA microspheres on 10 and 30 days postsurgery. (A) Images of IL-1β staining. (B) Images of TNF-α staining. Scale bar is 50 μm.

the 2.5% Mg/PLLA group. After 30 days of implantation, overexpression of IL-1β and TNF-α was still observed around the cotton and Mg-free PLLA microspheres, indicating the persistent transition from acute to chronic inflammation in these two groups. For the 10% Mg/PLLA microspheres, the expression levels of IL-1β and TNF-α decreased significantly, which were comparable to that of the 2.5% Mg/PLLA group, showing a negligible expression around the microspheres.

microspheres were associated with a milder tissue reaction. The microspheres in these two groups were surrounded by 6− 7 layers of cells. Minimal inflammatory reaction was seen in the 2.5% Mg/PLLA microspheres. Only 2−3 layers of cells were observed around the implants. After 30 days of implantation, the cell infiltration around the cotton and Mg-free PLLA microspheres was almost the same as that on day 10, showing the sustained presence of inflammation, whereas different degrees of decline in cell infiltration were observed in the 2.5% Mg/PLLA and 10% Mg/PLLA microspheres, resulting in only 1−2 layers of cells around the implants. To confirm the cell type presented in the local inflammatory response, some representative markers were stained. The results revealed that several inflammatory cells, especially macrophages, could be suppressed by the presence of Mg in the PLLA-based microspheres (Figures 6B and S3−S5 in the Supporting Information). In Figure 6B, after 10 days of implantation, significantly higher amounts of F4/80 positive cells were seen in the cotton, Mg-free PLLA, and 10% Mg/ PLLA groups as compared with that in the 2.5% Mg/PLLA group. Further to 30 days of implantation, obvious reducing cell count was observed in the 10% Mg/PLLA group, resulting in similar amounts of F4/80 positive cells to the 2.5% Mg/ PLLA group. However, the cotton and Mg-free PLLA groups still showed significantly higher amounts of F4/80 positive cells. Following the Mg-induced alleviation of macrophage infiltration around the implants, we need to further explore whether it can affect the secretion of inflammatory cytokines from the macrophages. Thus, two predominant inflammatory cytokines expressed by macrophages, interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α), were examined in this study (Figure 7). High levels of IL-1β and TNF-α were observed in the cotton, Mg-free PLLA, and 10% Mg/PLLA groups 10 days after implantation, whereas only a small amount of inflammatory cytokine expression was observed in

4. DISCUSSION In this study, we develop a kind of Mg/PLLA microspheres as a novel drug delivery system, in which Mg particle, as a basic agent, can not only suppress PLLA-induced inflammation by neutralizing the acidic byproducts but also tailor drug release profile of PLLA microspheres. It is well known that Mg is widely used as biodegradable implants in the biomaterial field. As a promising biodegradable material, the fast dissolution of Mg in aqueous medium limits its further application.35,36 To avoid this drawback, alloying and surface modification were traditionally concerned in the literature.37,38 Here, we try to turn this drawback into an advantageous feature for controlled drug release. Thus, the first objective of this study is to apply the fast dissolution character of Mg to regulate drug release kinetics. Several types of Mg/PLLA microspheres with different Mg contents and particle sizes are successfully synthesized using a typical double-emulsion technique. We hypothesize that Mg particles would play a function in tailoring drug release rates. It is known to us that drug release from microspheres depends both on drug diffusion through the polymer matrix and on polymer degradation.39−41 To eliminate the effect of polymer degradation on drug release rates, PLLA with high molecular weight is selected to fabricate the microspheres. During the whole degradation period in vitro, the microspheres show no obvious morphology change, except that some pores and cracks appear on the surface and inside of microspheres. Considering the negligible molecular 23553

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration of the Proposed Mechanism for the Release of BSA from Mg/PLLA Microspheresa

a

Stage I: microspheres before Mg dissolution and BSA release. Stage II: initial pore and channel formation with the dissolution of Mg particles distributed on the surface of microspheres. Water can penetrate the microspheres and initiate BSA release. Stage III: following the continuous dissolution of Mg particles contained inside, new cavities and channels appear in the deeper parts of microspheres, which further induce release of BSA into the aqueous medium through the network consisting of pores, cavities, and channels. Especially, for in vivo study, the slight PLLA degradation may also account for a small amount of BSA release from the microspheres (dotted box).

weight change of PLLA, the pores are believed to be caused by the dissolution of Mg particles exposed on the surface of microspheres. With the dissolution of Mg, the mechanical stability of microspheres also decreases, further resulting in the formation of cracks. Given that the slight degradation occurs in the PLLA matrix, we believe that the release of BSA from these microspheres is dominant by diffusion, which is regulated by the dissolution of Mg. The possible mechanism of BSA release from the Mg/PLLA microspheres is illustrated in Scheme 1. In the initial stage of the release experiment, the exposed Mg particles on the surface of microspheres would first contact with water and then the dissolution of Mg particles occurs. Mg dissolution brings about the formation of pores on the surface of microspheres, through which water can easily penetrate into the microspheres and bring out BSA encapsulated inside. The loss of Mg particles partly decreases the stability of the internal structure, resulting in the formation of cracks, that is, water channels, inside the microspheres. These channels allow water to penetrate into the inner parts of microspheres and dissolve the Mg particles contained inside, leading to the formation of new cavities and channels within the microspheres. Subsequently, water would fill these new cavities and enter into the deeper parts of microspheres through these new channels. Finally, a network consisting of numerous cavities and water channels is formed inside the microspheres, and BSA can diffuse out of the microspheres through this network. For in vitro release studies, the effects of both Mg content and its particle size on BSA release are investigated. First, we find that when the particle size is the same, BSA release rates increase with improving Mg content in the microspheres. Interestingly, the initial BSA release within 7 days in the 5% Mg/PLLA microspheres is similar to that in the 10% Mg/ PLLA group, whereas the following release gradually tends to the curve of the 2.5% Mg/PLLA group, which indicates that BSA release regulated by Mg content has a threshold. For the initial release as illustrated in stage II of Scheme 1, 5% Mg content is saturated, whereas it is insufficient for the following release in stage III. As an extension of these results, the 10% Mg/PLLA microspheres exhibit a four-phasic release pattern. Second, we measure BSA release profiles of 2.5% Mg(L)/ PLLA and 10% Mg(L)/PLLA microspheres and then compare them with the corresponding results of 2.5% Mg/PLLA and 10% Mg/PLLA groups. The results show that when the Mg

content is the same, microspheres with a larger Mg particle size exhibit a slower BSA release rate. Especially, the 2.5% Mg/ PLLA microspheres have a faster release rate than the 10% Mg(L)/PLLA group from 7 days of in vitro release experiment. Cross-sectional view of the Mg/PLLA microspheres reveals how Mg particles affect BSA release behavior. According to the mechanism illustrated in Scheme 1, we know that diffusion plays a dominant role in the release of BSA. Microspheres with good internal connectivity would allow BSA diffusion through the PLLA matrix and hence improve the release rate. The number of cavities and channels is positively related to the content of Mg particles, whereas it is negatively related to the Mg particle size. That is why the 2.5% Mg/PLLA and 10% Mg/PLLA microspheres exhibit faster BSA release than their counterparts. The in vivo release experiments exhibit different BSA release patterns from the in vitro studies. For the Mg-free PLLA microspheres, a consistently faster BSA release rate is observed over the 28 days of in vivo experiment as compared with that of in vitro release, which should attribute to the PLLA degradation in vivo (Table S2, Supporting Information). For the 2.5% Mg/PLLA and 10% Mg/PLLA microspheres, the in vivo BSA release rates are faster than the corresponding in vitro rates in the early 3 days. At this period of time, we believe that BSA release is dominant by diffusion. Afterward, the in vivo BSA release rates are gradually close to and further slower than their in vitro counterparts. We suspect that the microspheres are encapsulated by surrounding tissues at this stage, which limits the effect of diffusion on drug release and eventually leads to the decreasing BSA release rates. Inflammatory response induced by exogenous implants includes acute and chronic inflammation, and inflammatory cell infiltration and cytokines expression are the most direct standards to evaluate the degree of inflammation. The main infiltrating cells participating in foreign body reaction are macrophages, lymphocytes, and fibroblasts. Cytokines, including IL-1β and TNF-α, are two important proinflammatory mediators produced by macrophages, which can mediate the adhesion, migration, and activation of leukocytes during inflammation.42−44 PLLA is easy to bring about serious acidinduced inflammation due to its acidic degradation products, and this is the main reason for the failure of some PLLA-based biodegradable implants.45 Our second hypothesis is that the incorporation of proper amounts of Mg can produce a more 23554

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ACS Applied Materials & Interfaces favorable host response toward PLLA-based implants. We first conducted an in vitro cytotoxicity experiment. The results show that the low Mg dose group possesses favorable cell viability, which is comparable to the control group, whereas the Mg-free group and the high Mg dose group exhibit different degrees of decrease in cell viability. To further assess the longterm host response in vivo, microspheres were implanted into mice. According to the results of inflammatory cell infiltration and cytokine expression in vivo, we know that a low dose of Mg can significantly alleviate both acute and chronic inflammation, whereas a high dose of Mg is more favorable for long-term effects. We suspect that the reason for this is related to the variation of local pH values around the implants since the degradation products of PLLA are acidic, whereas those of Mg are basic. In the early stage of inflammation, inflammatory site exhibits an acidic environment because of the formation of insufficient oxidized intermediates and the generation of acid products by macrophages.46,47 The acidic environment can catalyze PLLA degradation, producing acidic byproducts. These acidic products overwhelm the body’s ability to flush them away. This leads to a buildup of acidic byproducts, resulting in acid-induced inflammation. Based on the media pH test in vitro, we believe that for the 2.5% Mg/ PLLA microspheres, the acid in the implant site achieves a balance with the base produced by Mg, resulting in a neutral local environment during the whole implantation period. While for the 10% Mg/PLLA microspheres, we suspect that the high amount of Mg particles not only neutralizes the acid but also increases the local pH to a value far higher than 7 in the early stage, resulting in a base-induced inflammation on day 10. With time, the release of Mg gradually decreases, which may just balance the acid and result in a neutral environment in the implant site. Hence, a favorable tissue response is observed in the 10% Mg/PLLA group on day 30.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-10-82338755. Fax: +86-10-82315554 (X.N.). *E-mail: [email protected]. Phone: +86-10-82339428. Fax: +86-10-82339428 (Y.F.). ORCID

Xufeng Niu: 0000-0003-2669-2028 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 11872097, 31470915, 31872735, 11421202, and 11827803), Beijing Natural Science Foundation (No. L182017), The National Key R&D Program of China (Nos. 2018YFC1106600, 2017YFC0108505, and 2017YFC0108500), Shenzhen Science and Technology Project (No. JCYJ20170817140537062), Young Elite Scientists Sponsorship Program By CAST (No. 2017QNRC001), the Fundamental Research Funds for the Central Universities (No. YWF-19-BJ-J-234), the 111 Project (No. B13003), and the International Joint Research Center of Aerospace Biotechnology and Medical Engineering, Ministry of Science and Technology of China.

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REFERENCES

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5. CONCLUSIONS The Mg/PLLA composite microspheres have been developed using a w/o/w double-emulsion method and used as a novel drug delivery system. Both in vitro and in vivo experiments have proved that this system has the ability of manipulating the drug release kinetics by regulating the contents or particle sizes of Mg contained inside the microspheres. The introduction of Mg into the microspheres can also alleviate PLLA-induced macrophage infiltration and inflammatory cytokine expression. The present Mg/PLLA microsphere-based delivery system has potential applications in various drug and protein-mediated tissue regeneration, especially in the growth factor-mediated bone regeneration field.

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10% Mg/PLLA microspheres on 10 and 30 days postsurgery (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b03766. Weight-averaged molecular weight (Mw) and polydispersity index (Mw/Mn) of PLLA before and after microsphere degradation in vitro and in vivo; size distribution of microspheres; SEM photograph of the cross-sectional view of Mg-free PLLA microspheres after 28 days of in vitro degradation; images of Ly6G, CD11b, and CD3 positive cell staining for the cotton, Mg-free PLLA microspheres, 2.5% Mg/PLLA microspheres, and 23555

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