Degradation and Bio-Safety Evaluation of mPEG-PLGA-PLL

Article pubs.acs.org/JPCC

Degradation and Bio-Safety Evaluation of mPEG-PLGA-PLL Copolymer-Prepared Nanoparticles Zelai He,†,‡ Ying Sun,†,‡ Qi Wang,† Ming Shen,† Mingjie Zhu,† Fengqian Li,*,§ and Yourong Duan*,† †

State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, No 25, Lane 2200, Xietu Road, Xuhui District, Shanghai 200032, China § Dahua Hospital, Xuhui District, Shanghai 200237, China ABSTRACT: Studies have shown that monomethoxy(polyethylene glycol)−poly(D,Llactic-co-glycolic acid)−poly(L-lysine) (mPEG-PLGA-PLL)-prepared nanoparticles (NPs) are promising drugs carriers, with good drug loading and delivery performance. To further promote the use of this material in clinical applications, its degradation and biosafety were evaluated. This paper describes degradation studies and biosafety evaluations of different block composition ratios (LA/GA = 60/40, 70/30, and 80/20) of the main material, PLGA, for mPEG-PLGA-PLL (PEAL) NPs. The degradation of PEAL NPs was studied by characterizing the change in molecular weight, the chemical composition, and the degradation rate in addition to the pH value, the particle size, the zeta potential, and the lactic acid and lysine contents in degradation solutions by transmission electron microscopy (TEM), gel permeation chromatography (GPC), and 1H NMR. The results show that with prolonged degradation time, the pH, particle size, zeta potential, and molecular weight were reduced and that the lactic acid and lysine contents and the molecular weight distribution were increased. 1H NMR demonstrated that the hydrolysis rate for glycolic units was faster than those for lactic acid and lysine units. The degradation rate of NPs in pH 7.4 PBS was faster than that in pH 5.0 PBS. The degradation rate of PEAL NPs increased as the LA/GA increased from LA/GA = 60/40 to 80/20. Investigations of intracellular protein synthesis, lactate dehydrogenase (LDH) release, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining and reactive oxygen species (ROS) content in Huh7, L02, and RAW 264.7 cells showed that the PEAL NPs had no effect on protein synthesis or cell membrane integrity and did not induce chromatin agglutination. Although the ROS content was slightly concentration-dependent and time-dependent, the change in content was minimal and diffusely distributed within the cell. After THP-1 cells were induced to differentiate into macrophages, a subsequent incubation with 5 mM PEAL NPs for 24 h did not significantly induce the macrophage release of IL-1β, TNF-α, and TGF-β1 compared with the negative control. Embryos that had their chorion removed were coincubated with PEAL NPs to determine if there were any effects on embryonic development. It is known that zebrafish embryos at 10−24 h post-fertilization (hpf) are most sensitive to PEAL NPs. Zebrafish embryos treated with different concentrations of PEAL NPs within this sensitive time frame demonstrated that PEAL NPs have a high level of biosafety. Our work demonstrates that PEAL NPs are safe candidates for use as biodegradable carriers for drug and gene delivery.

1. INTRODUCTION

by hydrolytic or enzymatic degradation and further metabolized or excreted by renal clearance in humans. Therefore, PLGA has been widely studied in the area of sustained, targeted drug delivery systems. However, the hydrophobic nature and the negative potential of PLGA and the lack of modifiable functional groups complicate the grafting of reactive groups, which leads to difficulties in loading siRNA and penetrating tumor cell membranes. Therefore, there is a continuing need to promote research related to the development of drug carrier materials for the treatment of tumors. Poly(L-lysine) (PLL) is a flexible, stable material with a cationic charge and can be modified with a variety of functional groups. In addition, its degradation products are essential

Nanomaterials are typically defined as materials that are onedimensional or multidimensional and contain particles less than 100 nm in size. However, some researchers also classify materials composed of particles below 200 nm in size as nanomaterials.1 Nanomaterial technology emerged in the late 1980s and has developed rapidly over the past several decades. It has dramatically affected all fields of science, especially the life sciences and has become one of the most attractive and promising areas of research, opening a door for the development of drug and gene delivery systems. Nanomaterialprepared nanocarriers have been shown to allow the alteration of body distributions and selective retention at tumor sites due to enhanced permeability and retention (EPR) effects.2 Poly(D,L-lactic-co-glycolic acid) (PLGA) is an FDA-approved material with good biodegradable and biocompatible properties. It can be degraded into small molecules (e.g., lactic acid) © XXXX American Chemical Society

Received: October 9, 2014 Revised: January 19, 2015

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DOI: 10.1021/jp510183s J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C Scheme 1. Schematic Diagram of mPEG-PLGA-PLL (PEAL) Copolymer Synthesis

tion mechanisms. In addition to the biodegradability and capability of being metabolized, the nondegraded biomaterial should be safe and nontoxic to humans. Therefore, to further promote the use of PEAL in clinical applications, the health hazards of PEAL nanoparticles (NPs) in humans and other species must be assessed.7 In this paper, we report on the biosafety of PEAL using zebrafish as a model organism. Zebrafish have been widely used in metal nanomaterial biosafety studies and in cardiovascular disease research. However, there have been limited studies on the use of zebrafish for the biosafety studies of organic polymer nanomaterials. In the work reported here, zebrafish were used as model organisms for evaluating the biocompatibility of PEAL NPs.8 The physiochemical properties, degradation mechanisms, and biocompatibility of a biodegradable biomaterial can be changed by adjusting the chemical and stereochemical structures of its monomers. When using PEAL NPs as drug carriers, past studies have shown that the use of 10% mPEG with molecular weight (Mw) 2000 promoted long circulation times. Furthermore, when the molar ratio of mPEG-PLGA/ PLL was 1/15, the stability, loading drug efficacy and release properties of PEAL NPs were optimal.9−12 As a continuation of our previous studies, this work primarily studied the impact of different block ratios (LA/GA ratio) in the main body material, PLGA, on the degradation and biosafety of PEAL NPs.3,5,6,9−12

amino acids for the human body. Monomethoxy(polyethylene glycol) (mPEG) chains are hydrophilic and can substantially decrease the amount of plasma protein adsorbed onto the surface of materials; therefore, it can significantly reduce specific phagocytosis by macrophages and enhance the stability of particles. The feasibility of incorporating PLL and mPEG into the backbones of PLGA polymers to synthesize monomethoxy(polyethylene glycol)−poly(D,L-lactic-co-glycolic acid)−poly(Llysine) (mPEG-PLGA-PLL or PEAL) as a drug carrier nanomaterial has been studied. Various studies have confirmed its excellent ability to act as an anticancer drug and as an siRNA carrier with high drug loading and encapsulation efficiencies and excellent circulation and sustained drug release capabilities. These and related works have been published in the Journal of Biomaterials.3−6 These findings indicated that PEAL has potential clinical applications. However, if nanomaterials that are administered in vivo cannot degrade or metabolize under physiological conditions, repeated intravenous administration could lead to nanomaterial aggregation at the injection sites or elsewhere in the body, which could be toxic. Therefore, to avoid nanomaterial accumulation in vivo, nanomaterials must degrade into soluble oligomers and monomers able to be cleared from the injection site without severe inflammatory reactions or further degraded and metabolized. Due to these requirements, it is important to understand polymer degradaB


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The Journal of Physical Chemistry C Scheme 2. Possible Degradation Products and the Degradation Process of (A) mPEG-PLGA and (B) PEAL In Vitro

emulsified by ultrasonication (400 W, 2 s × 25 times; JY 92-II ultrasonic processor; Ningbo Scientz Biotechnology Co., Ltd., China). The resulting W/O emulsion was evaporated in a rotary evaporator (SHZ-DIII, Shanghai Qiangqiang Industrial Development Co., Ltd., China) at 37 °C and 0.05 MPa. After 1 h, the solution was centrifuged for 45 min at 16000 rpm to obtain the NPs and subsequently freeze-dried. 2.4. Characterization and Degradation. 2.4.1.. Particles Size, Zeta Potential, pH Value, and TEM Assay. The particle size and zeta potential of the NPs were characterized using a ZetaSizer Nano ZS (Malvern Instruments Ltd., U.K.). The average diameter and size distribution profile of the NPs were determined by dynamic light scattering (DLS). The zeta potential (ζ) was determined using a He−Ne laser beam at a wavelength of 633.8 nm at 25 °C. Three batches of each sample were analyzed. The pH was determined using an FE20 pH meter (FiveEasy Plus, Shanghai, China). The morphology of the particles was observed with a JEM-1400 transmission electron microscope (TEM; JEOL, Japan).13,14 2.4.2. Detection of Lactic Acid and Lysine in Degradation Medium. The samples were centrifuged, and the lactic acid and lysine contents were determined from the supernatant. Lactic acid was determined using an Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 μm) at 25 °C. The flow rate of the mobile phase (methanol/0.2% phosphoric acid = 5:95, v/v) was 1 mL/min, and the UV detector wavelength was 210 nm. A 20 μL aliquot of the supernatant was injected for each analysis. Lactic acid (500 μg/mL) was used as the standard. Lysine was determined using the Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 μm) at 25 °C. The flow rate of the mobile phase (20 mM KH2PO4 solution/acetonitrile = 99:1, v/ v) was 1.0 mL/min, and the UV detector wavelength was 203 nm. A 20 μL aliquot of the supernatant was injected for each analysis. Lysine (500 μg/mL) was used as the standard. 2.4.3. Molecular Weight Distribution and 1H NMR Assay. The molar masses and the molar mass distributions of the copolymers were measured by GPC and performed on a Polymer PL-GPC 50 GPC system with an RI detector (Polymer Laboratories, U.K.); the RI detector wavelength was 240 nm. Chloroform was used as the mobile phase at a flow rate of 1.0 mL/min, 20 μL of polymer solution were injected for each analysis. Polystyrene (molar mass range from 500 to 480000) was used as the standard. The composition of the copolymers was determined by 1H NMR, using a Bruker Avance 400 NMR spectrometer and CDCl3 as solvent. Tetramethylsilane was used as the internal reference to determine chemical shifts (δ) in ppm.15 2.4.4. Determination of the In Vitro Degradation of the NPs. Samples of PLGA (LA/GA = 80/20), mPEG-PLGA (LA/ GA = 80/20), and PEAL (LA/GA = 60/40, 70/30, 80/20) NPs were prepared at concentrations of 0.5% (w/v) in PBS solution

2. EXPERIMENTAL SECTION 2.1. Materials. PEG (Mw 2000) was obtained from SigmaAldrich (China) Co., Ltd. (Shanghai, China). L-lactide and glycolide were purchased from Yuanshengrong Co. (Beijing, China). Nε-carbobenzyloxy-L-lysine N-carboxyanhydride (NεCBZ-L-lysine NCA) was obtained from Shanghai Yuaniu Biotechnology Co. (Shanghai, China). Pluronic 188 was purchased from BASF (Ludwigshafen, Germany). Coomassie brilliant blue G250, 4′,6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA), an LDH kit, and phorbol-12myristate-13-acetate (PMA) were obtained from Beyotime Institute of Biotechnology (Nantong City, China). A reactive oxygen species (ROS) kit and Pronase were purchased from Qcbio Science and Technologies Co., Ltd. (Shanghai, China). All cell culture media and reagents were obtained from Gibco (Grand Island, NY, U.S.A.) unless otherwise specified. Acetone, ethyl acetate, and other reagents and solvents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). RAW 264.7 (murine macrophage), Huh7 (human hepatic carcinoma cells), and L02 (normal embryo liver cells) cell lines were grown in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/ mL streptomycin at 37 °C under 5% CO2. THP-1 cells (human monocytic leukemia cell line) were obtained from the Pasteur Institute of Algeria and cultured in RPMI supplemented with 10% FBS, 2 mM glutamine, 1.5 mg/mL glucose, 1% penicillin, and streptomycin. Adult zebrafish (AB strain of Danio rerio) were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and reared in fish water (FW) without chlorine gas under standard laboratory conditions of 28.5 °C on a 14 h light/10 h dark photoperiod, and tests were conducted in strict compliance with the “Guide for the Care and Use of Laboratory Animals”. 2.2. Synthesis of PEAL Copolymer. The synthesis of PEAL (Mw 12000, mPEG 10%) has been described in detail elsewhere and have a little improvement (Scheme 1).6 Briefly, (1) the mPEG-PLGA-OH diblock copolymer was prepared using zinc lactate as a catalyst; (2) its −OH end-group was converted into −NH2, and the synthesis of mPEG-PLGApoly(Nε-(Z)-L-lysine) occurred through the ring-opening polymerization (ROP) of the initiated NCA by the aminoterminated mPEG-PLGA; (3) the PEAL copolymer was obtained in an HBr/CH3COOH solution by removing the Nε-(carbonylbenzoxy) end-group of the mPEG-PLGA-poly(Nε-(Z)-L-lysine) block copolymer. 2.3. NP Preparation. The copolymer (2 μM) was dissolved in 250 μL of ethyl acetate and added to 1 mL of a 1% (w/w) Pluronic 188 solution. The mixture was then homogenized and C


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The Journal of Physical Chemistry C

solution. After the cells were adhered in 96-well plates, different concentrations (0, 10, 100, 500, and 1000 μM) of NPs were used to treat the cells for 24, 48, and 72 h. Cells treated with H2O2 (50 mM) were used as the positive control. After predetermined treatment times, the cells were coincubated for 30 min with 10 μM DCFH-DA at 37 °C in the dark. Next, the cells were washed three times with DMEM medium (without serum), and the fluorescent intensity of DCF was detected using a microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 528 nm. After the fluorescent intensity was detected, the intracellular fluorescence distribution was imaged using a fluorescence microscope. 2.9. Inflammatory Cytokine Release Assay. THP-1 cells and 25 ng/mL PMA in RPMI medium were dispensed at 200 μL/well into a 96-well flat-bottom plate (Nunc, U.S.A.) using a Matrix WellMate (Thermo Fisher Scientific, U.S.A.). The plate was incubated for 72 h in an incubator to allow the THP-1 cells to be induced by the PMA and differentiate into adherent macrophages. Then, RPMI medium was replaced with 5 mM PEAL NP medium. The 6.4% phenol solution in the medium and the medium were used as the positive and negative controls, respectively. After 24 h, the supernatant was collected for measuring the concentrations of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and transforming growth factor-β1 (TGF-β1) by ELISA according to kit instructions. 2.10. Sensitive Time Window and Dose Effects during the Sensitive Time Window for Zebrafish Embryos Development. Zebrafish embryos were tested in two studies. Study 1 was designed to broadly identify the sensitive time windows for the NP-induced embryo lethality and teratogenicity after coincubation with 3 × 103 μM NPs. Study 2 was conducted to explore the effects of different doses of NPs on zebrafish embryo development during the sensitive time window. 2.10.1. Exposure Protocol. Adult zebrafish were spawned (female/male = 1:2) and embryos were collected after 30 min. As described by Truong et al., to obtain a high bioavailability, the chorion surrounding the embryos was enzymatically removed with Pronase at 4 h post-fertilization (hpf). Briefly, embryos were placed in Petri glass dishes containing 25 mL of FW with 50 μL of 50 mg/mL Pronase for 4−5 min. Then, the water was decanted and replaced with fresh E3 medium (13.7 mmol/L NaCl, 0.54 mmol/L KCl, 0.025 mmol/L Na2HPO4, 0.044 mmol KH2PO4, 0.42 mmol/L NaHCO3, 1.3 mmol/L CaCl2, and 1.0 mmol/L MgSO4, and the pH was adjusted to 7.2 before use) for a total of 10 min. Next, the embryos were maintained at rest for at least 45 min.19 After the rest period, the dechorionated embryos were transferred to individual wells of 96-well plates with 100 μL of E3 medium (negative control) or E3 medium containing NPs/acetone, at one embryo per well. Nonexposed embryos (embryos raised in E3 medium with the chorion intact) were also incubated to monitor the quality of the inherent embryos. An exposure to E3 medium containing acetone was used as the positive control. The media were replaced every 24 h. The test was initiated when each group of at least 30−48 embryos were distributed in 96-wells plates in triplicate (for a total of at least 120 embryos). A total of 15 morphological malformations were evaluated at 96 hpf: yolk sac edema, bent body axis, eye, snout, jaw, otolith, pericardial edema, brain, somite, caudal fin, pectoral fin, heart, pigmentation, trunk length, and swim bladder. The representative images of malformation embryos were captured using an

(0.1 M, pH 5.0 and 7.4). The NP solutions were incubated at 37 °C, and at predetermined time intervals, samples were removed for DLS, TEM, pH value, lactic acid, and lysine measurements. Subsequently, the samples were lyophilized and analyzed using GPC and 1H NMR. Each group consisted of three parallel samples. For the elucidation of structural degradation, a few of the possible water-soluble products and the degradation process were proposed for the synthesized PEAL, as shown in Scheme 2. 2.5. Total Protein Synthesis. 2.5.1. Determination of Protein Content. Standard protein solutions of BSA (100, 200, 300, 400, 500, and 600 μg/mL) were prepared using normal saline. 2.5.2. Preparation of Coomassie Brilliant Blue G-250 Solution. Coomassie Brilliant Blue G-250 (50 mg) was dissolved in 25 mL of ethanol (90%). Then, 50 mL of 85% (w/v) phosphoric was added, and the solution was brought to 500 mL using distilled water. 2.5.3. OD Measurements. Sample and BSA solutions (20 μL) were added to the wells of a 96-well plate, followed by the addition of 180 μL of Coomassie Brilliant Blue G-250 solution. After 5−10 min, the OD was measured at 595 nm using a microplate reader (Synergy H4 Hybrid Microplate Reader, BioTek Instruments, Inc., U.S.A.). A blank was used as the baseline. A standard concentration curve was also constructed. 2.5.4. Preparation of Sample Solutions. Cells were seeded at 1−5 × l04 cells/well in 24-well plates. After adherence, the cells were treated with 1000 μM PEAL NPs. After 12, 24, 48, and 72 h, the NPs were aspirated and gently washed three times with HBSS solution at 4 °C. The cells were digested, washed with 500 μL of distilled water, and ultrasonicated in an ice bath with agitation. Finally, the protein content was measured as described in OD measurements.16 2.6. LDH Release Assay. Cells were cultured in 96-well plates in 200 μL of DMEM medium (1% FBS, 1000 μM NPs). DMEM medium with 1% FBS and 0.9% Triton X-100 served as the positive control, and DMEM medium with 1% FBS only served as the negative control. After 72 h, the samples were centrifuged for 10 min at 1200 rpm to remove the cells. The supernatant was then centrifuged for 30 min at 12000 rpm at 4 °C. The supernatant was analyzed for LDH with the LDH kit following the manufacturer instructions. 2.7. DAPI Staining for Chromatin Agglutination. To evaluate the effect of NPs on chromatin agglutination, DAPI staining was used. The NPs (0, 10, 100, 500, and 1000 μM) were coincubated with the cells. After 72 h, DAPI (1 μg/mL) solution was added for a 10 min coincubation with the cells in the dark at 37 °C, and the cells were then gently washed three times with PBS (5 min per wash). Next, the cells were fixed with 4% paraformaldehyde, and the nuclei were imaged using an Olympus IX51 fluorescence microscope (Tokyo, Japan) at 340−360 nm excitation and 450−480 nm emission wavelengths, with an exposure time of 1/30 to 1/100 s.17 2.8. Detection of Intracellular ROS. The nonfluorescent molecule 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) passes freely through a cell membrane into the intracellular region, where it can be hydrolyzed and oxidized to generate dichlorofluorescein (DCF), a fluorescent compound that cannot permeate the membranes of live cells. By measuring the fluorescent intensity of DCF, the intracellular levels of ROS can be determined.18 In this study, a 10 mM DCFH-DA stock solution was diluted in DMEM medium without FBS to yield a 10 μM working D


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decreasing of the size accelerated as the pH increased. Moreover, the erosion, cracks, and defect lobes of the NPs at 144 h were more obvious than those for the NPs at 48 h (Figure 2A1−A5 vs Figure 2B1−B5, Figure 2C1−C5 vs Figure 2D1−D5). The degradation of the polylactic acid NPs was likely affected by the hydrophobic and semicrystalline structures, which do not allow rapid water penetration, and the degradation rate was proportional to the ester concentration. Therefore, the lactate regions of the NP shells may be amorphous or nonperfect, partially oriented crystals that can rapidly degrade and permit the slow degradation of perfect crystalline regions.22 For the NPs in the pH 5.0 and 7.4 media, the zeta potential appeared negative. As the degradation time increased, the absolute value of the zeta potential increased (Figure 1C,D), because the lysine units likely degraded faster than the lactic acid units. The absolute value of the zeta potential in the pH 5.0 medium was less than that in the pH 7.4 medium, which may be due to the pH 7.4 medium having more OH− than the pH 5.0 medium. 3.2.2. Determination of pH, Lactic Acid, and Lysine Content. It can be seen from Figure 3 that the concentrations of lactic acid and lysine exhibited an increasing trend with prolonged degradation time. In the pH 7.4 medium, the concentrations of lactic acid and lysine were significantly higher than in the pH 5.0 medium (Figure 3C vs Figure 3D, Figure 3E vs Figure 3F), indicating that the degradation rate of the NPs in the pH 7.4 medium was faster than that in the pH 5.0 medium. Thus, pH has a significant impact on the degradation rate of the NPs. Furthermore, at a later stage of degradation, the lactic acid and lysine contents exhibited “burst” increments, and a greater number of NP fragments were observed by TEM (details not shown), implying that the NPs could rupture during the later stage and release the degradation products from inside the NPs. Before the rupture, the carboxylic end groups inside the NPs could not diffuse out and accumulate, which would generate an “autocatalytic effect” that would accelerate the hydrolysis of the internal remaining ester bonds, resulting in more oligomers being released and a broader molar mass distribution (Figure 4).23 If the contents of lactic acid and lysine at 0 h were set as the baseline values, then the lactic acid content in mPEG−PLGA (LA/GA = 80/20) medium at each predetermined interval was the highest, followed in turn by PLGA (LA/GA = 80/20) and PEAL (LA/GA = 80/20; Figure 3C,D). When the material weights were the same, the lactic acid content in the mPEG− PLGA (LA/GA = 80/20) polymer was less than that in the PLGA (LA/GA = 80/20) polymer. However, the lactic acid content in the mPEG−PLGA (LA/GA = 80/20) medium was higher than that in the PLGA (LA/GA = 80/20) medium, indicating that the degradation rate of the mPEG−PLGA (LA/ GA = 80/20) NPs was faster than that of the PLGA (LA/GA = 80/20) NPs. It is possible that the introduction of PEG into PLGA increased the hydrophilicity of PLGA and allowed water molecules to more easily enter the polymer chains and promote the degradation of the polymer.24 In the PEAL (LA/GA = 80/ 20) polymer, the molecular weight ratios of PEG, LA, GA, and lysine blocks were approximately 1:3.6:0.95:0.65 (Figure 5ii). By converting PEG and lysine into PLGA, the lactic acid content in the PEAL (LA/GA = 80/20) medium will increase approximately 36%, still far lower than the lactic acid content in the PLGA (LA/GA = 80/20) medium, indicating that the degradation rate of the PEAL (LA/GA = 80/20) NPs was

IX51 inverted microscope from OLYMPUS Corporation (Tokyo, Japan).20 2.10.2. Determination of Sensitive Time Windows during Zebrafish Embryos Development. To examine the effects of timing exposure on embryo development, several exposure durations were chosen. These selected time durations included 6−10 hpf (gastrula period), 6−14 hpf (for 14−24 h, important organ formation stage), 10−24 hpf (somite stage), 24−48 hpf (pharyngeal phase), and 48−72 hpf (the incubation period). At the corresponding time, stage embryos were exposed to 3 × 103 μM NPs solution or 2.5% acetone solution (corresponding to 34 × 104 μM). At 96 hpf, larvae/embryos were microscopically examined to determine the mortality and malformation rates.21 2.10.3. Dose Effect for Zebrafish Embryos Development during a Sensitive Time Window. Embryos at a sensitive time window were exposed to NP solutions at several different concentrations (0.0625, 0.125, 0.25, 0.5, 1, and 2 × 103 μM), acetone (0.125, 0.25, 0.5, 1, 2 and 4%, corresponding to 1.70, 3.40, 6.80, 13.6, 27.2, and 54.4 × 104 μM, respectively), or E3 medium (negative control or nonexposed group). Then, each embryo was scored for mortality and morphological malformations at 96 hpf. The percent mortality and total malformations were calculated and graphed. 2.11. Statistical Analysis. All analyses were compiled using SPSS 20.0 software. The results were expressed as the means ± standard deviation (SD) for the values obtained from at least three independent experiments. The statistical analyses were performed using one-way analysis of variance (ANOVA) and Tukey’s post hoc test. The half-positive response concentration (EC50) and the half-lethal concentration (LC50) were calculated using Microsoft Excel software. All tests were considered statistically significant when P < 0.05.

3. RESULTS AND DISCUSSION 3.1. Characterization of PEAL NPs. The mean diameters of PEAL (LA/GA = 60/40, 70/30, and 80/20) NPs were 144.4, 126.6, and 107.1 nm, and the zeta potentials were 15.9, 9.2, and 6.1 mV, respectively (Table 1). The mean sizes and polydispersity indices indicated that the NPs had narrow size distributions, homogeneous spatial distributions, and did not agglomerate. Table 1. Size distribution and zeta potential of PEAL NPs index

LA/GA = 60/40

LA/GA = 70/30

LA/GA = 80/20

size (nm) PDI ζ-potential (mV) ζ-deviation (mV)

141.8 ± 8.74 0.144 ± 0.08 17.6 ± 2.04 6.53 ± 1.77

120.9 ± 7.16 0.146 ± 0.07 10.9 ± 1.71 5.74 ± 1.45

100.6 ± 6.71 0.155 ± 0.05 5.6 ± 1.79 3.39 ± 1.52

3.2.. In Vitro Degradation Studies. 3.2.1. Size, Zeta Potential, and Morphology Changes of NPs during Degradation. Figure 1A,B shows that the sizes of NPs have a decreasing trend and that the NPs in the pH 7.4 medium were smaller than the NPs in the pH 5.0 medium during degradation. It can also be been seen that in both the pH 5.0 and 7.4 media, the NPs after 144 h of degradation were smaller than the NPs after 48 h of degradation (Figure 2A1−A5 vs Figure 2B1−B5, Figure 2C1−C5 vs Figure 2D1−D5). These results indicated that the sizes of the NPs decreased with prolonged degradation time. The sizes of the NPs in the pH 7.4 medium were slightly smaller than those of the NPs in the pH 5.0 medium (Figure 1A vs Figure 1B), indicating that the E


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Figure 1. Sizes of NPs in (A) pH 5.0 and (B) pH 7.4 medium decreased with prolonged degradation time, and the sizes of NPs in pH 7.4 medium were slightly smaller than NPs in pH 5.0 medium. The zeta potential of NPs in (C) pH 5.0 and (D) pH 7.4 media appeared negative, and the zeta potential of NPs in pH 5.0 medium was higher than NPs in pH 7.4 medium.

70/30) and PEAL (LA/GA = 80/20) NPs (Figure 3C,D). The lysine content in the PEAL medium also followed similar trends (Figure 3E,F). For prolonged degradation times, the pH values tended to decline due to the accumulation of acidic degradation products. Figure 3A,B shows that during the prophase degradation stage, the declining rate of the pH value was greater than that of the later period. This was likely due to the degradation rate in the beginning high pH value condition was faster than the degradation rate in the latter lower pH value condition. When the material weights were the same, the degradation products of the mPEG−PLGA (LA/GA = 80/20) NPs had a greater effect than those of the PLGA (LA/GA = 80/20) NPs for the same pH value (Figure 3A,B), and the degradation of PEG polymer fragments was inert (Figure 5i,ii). This indicated that the degradation rate of the mPEG−PLGA (LA/GA = 80/ 20) NPs was faster than that of the PLGA (LA/GA = 80/20) NPs. Similarly, when the PEAL copolymer weight was the same, the lactic acid unit content in the PEAL (LA/GA = 80/ 20) copolymer was the highest, followed in turn by those of the PEAL (LA/GA = 70/30) and PEAL (LA/GA = 60/40) copolymers. However, the greatest reduction of the pH value in the medium was with PEAL (LA/GA = 60/40), followed in turn by PEAL (LA/GA = 70/30) and PEAL (LA/GA = 80/20; Figure 3A,B). These results indicated that the acidic degradation product contents and degradation rates of the PEAL (LA/GA = 60/40) NPs were the highest, followed in turn by those of the PEAL (LA/GA = 70/30) and PEAL (LA/ GA = 80/20) NPs. 3.2.3. Changes in Molecular Weight and Molecular Weight Distribution. Changes in the weight-averaged molec-

Figure 2. Upper two rows show the morphology of NPs (pH 5.0) with degradation times of 48 h (A1−A5) and 144 h (B1−B5); the lower two rows show the morphology of NPs (pH 7.4) with degradation times of 48 h (C1−C5) and 144 h (D1−D5). Scale bar: 200 nm.

slower than that of the PLGA (LA/GA = 80/20) NPs. In the PEAL copolymer, the lactic acid units content of the PEAL (LA/GA = 80/20) copolymer was the highest, followed in turn by those of PEAL (LA/GA = 70/30) and PEAL (LA/GA = 60/ 40). However, the highest lactic acid content in the medium was PEAL (LA/GA = 60/40), then PEAL (LA/GA = 70/30) and PEAL (LA/GA = 80/20). This indicated that the degradation rate of the PEAL (LA/GA = 60/40) NPs was the fastest, followed in turn by those of the PEAL (LA/GA = F


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Figure 3. PH value decreased in (A) pH 5.0 and (B) pH 7.4 media with prolonged degradation time. The lactic acid content increased in (C) pH 5.0 and (D) pH 7.4 media with prolonged degradation time. The lysine content increased in (E) pH 5.0 and (F) pH 7.4 media with prolonged degradation time.

media, which indicated the generation of more oligomers in the pH 7.4 media. At different time points, small differences in the Mw changes of PEAL NPs indicated that the degradation patterns were very similar. At pH 5.0, the degradation time was between 48 and 96 h, and at pH 7.4, the degradation time was between 3 and 48 h. The Mw of PEAL (LA/GA = 60/40) was the highest. The Mw for PEAL (LA/GA = 80/20) to change into PEAL (LA/GA = 60/40) was the lowest and that to change into PEAL (LA/GA = 80/20) was the highest. These results indicated that the degradation rate of PEAL (LA/GA = 60/40) NPs was the highest, followed in turn by PEAL (LA/GA = 70/30) and PEAL (LA/GA = 80/20). The results also indicated that the higher the pH value, the faster the NP degradation, leading to a more rapid change in the Mw of the PEAL NPs in pH 7.4 media. Polylactic acid polymer degradation appeared to follow first order kinetics. When the linear portion of the Mw change was fit to a curve, the degradation rate constants were 0.0022 (R2 = 0.99), 0.0026 (R2 = 0.93), 0.0011 (R2 = 0.94), 0.0008 (R2 =

ular weight (Mw) and the number-averaged molecular weight (Mn) can affect many properties of biomaterials, such as crystallinity, viscosity, weight loss, and morphology. The Mw/ Mn is known as the molecular weight polydispersity index (PI), which is characteristic of the width of the polymer molecular weight distribution.25,26 GPC can detect changes in the Mw and PI of biomaterials during degradation. It can be seen in Figure 4 that the Mw of the remaining NPs decreased exponentially and that the PI increased with the degradation time. The GPC analysis indicated that the PI change for mPEG−PLGA (LA/GA = 80/20) was the widest, followed in turn by PLGA (LA/GA = 80/20), PEAL (LA/GA = 60/40), PEAL (LA/GA = 70/30), and PEAL (LA/GA = 80/20). After 144 h of degradation, the PI for mPEG−PLGA (LA/GA = 80/20), PLGA (LA/GA = 80/ 20), PEAL (LA/GA = 60/40), PEAL (LA/GA = 70/30), and PEAL (LA/GA = 80/20) in pH 5.0 media were 1.70, 1.49, 1.23, 1.21, and 1.18, respectively; and the PI in pH 7.4 media were 1.84, 1.60, 1.31, 1.27, and 1.20. Furthermore, the PI of the NPs in the pH 7.4 media were higher than those in the pH 5.0 G


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Figure 4. Mw decrease due to degradation of NPs in (A) pH 5.0 and (B) pH 7.4 media with prolonged degradation time; Mw/Mn increase due to degradation of NPs in (C) pH 5.0 and (D) pH 7.4 media with prolonged degradation time.

0.98), and 0.0006 (R2 = 0.85) day−1 in pH 5.0 media and 0.0025 (R2 = 0.89), 0.0032 (R2 = 0.95), 0.0017 (R2 = 0.97), 0.0013 (R2 = 0.97), and 0.0009 (R2 = 0.92) day−1 in pH 7.4 media for PLGA (LA/GA = 80/20), mPEG−PLGA (LA/GA = 80/20), PEAL (LA/GA = 60/40), PEAL (LA/GA = 70/30), and PEAL (LA/GA = 80/20), respectively.27 3.2.4. NMR Detection. The change in composition of the mPEG−PLGA (LA/GA = 80/20) and PEAL (LA/GA = 80/ 20) components during degradation in vitro were determined by 1H NMR measurements. For mPEG−PLGA (LA/GA = 80/ 20), it is known that the −CH− protons of hydroxylated lactic acid end units appear at 5.2 ppm and the CH3− protons of the methyl end units appear at 1.6 ppm, as shown at positions c and d in Figure 5i. Resonances at 5.2 ppm (−CH−) and at 1.6 ppm (CH3−) belong to lactic acid units, including both the PEGconnecting and main chain units. The signals detected at 3.6 ppm can be assigned to −CH2−, which belong to the PEG unit, as shown at position a. In addition, the signal detected at 4.8 ppm was −CH2− from the glycolic acid unit (unit b). Compared with mPEG−PLGA (LA/GA = 80/20), PEAL (LA/ GA = 80/20) has only a lysine unit peak, and the signals detected at 1.8−2.0 ppm can be assigned to both −CH− and −NH− from the lysine unit (unit e).23,28 Compared with the other units in mPEG−PLGA (LA/GA = 80/20) and PEAL (LA/GA = 80/20), the peak areas for the PEG unit (a) were 1.15, 4.71, 4.85, 4.93, and 5.39 for mPEG− PLGA (LA/GA = 80/20) and 1.15, 4.47, 4.99, 5.04, and 5.60 for PEAL (LA/GA = 80/20), respectively (Figure 5i,ii). The gradually increasing trend indicated that degradation of the other units in the material was more rapid than the PEG degradation, resulting in a relative increase of PEG. This is likely related to the inertness of PEG and may be used as an

imprecise standard to measure the degradation rate of other units. Using the calculated peak areas of the −CH2− signal of the glycolic acid unit (b) to the −CH− signal of the lactic acid unit (c + d), the lactic acid unit to glycolic acid unit proportion of the remaining mPEG−PLGA (LA/GA = 80/20) and PEAL (LA/GA = 80/20) was obtained (Figure 5i,ii).28,29 The proportions were 3.20, 3.66, 4.03, 4.39, and 4.82 for mPEG− PLGA (LA/GA = 80/20) and 3.824, 4.27, 4.46, 5.40, and 5.69 for PEAL (LA/GA = 80/20) at 0, 48 (pH 5.0), 48 (pH 7.4), 144 (pH 5.0), and 144 h (pH 7.4), respectively. It can be seen that the proportions increase with prolonged degradation time and rising pH. The greater hydrophilic nature of the GA−GA bonds compared to those of the GA−LA and LA−LA bonds causes the degradation of the GA−GA bonds to be faster than the GA−LA and LA−LA bonds. Furthermore, these results also indicated that, as the pH values increased, the extent of the hydrolysis of the LA−LA and LA−GA bonds was less than that for the GA−GA bonds. Related studies confirm that the degradation mechanisms of the poly(lactic acid) and poly(glycolic acid) units are the same, and the hydrolysis of their ester bonds leads to degradation. This is in agreement with the 1H NMR results for the mPEG− PLGA (LA/GA = 80/20) and PEAL (LA/GA = 80/20) NPs. The degradation of poly(lactic acid) and poly(glycolic acid) units may be divided into surface degradation and ontology degradation.23,25−28 For example, the hydrolysis of poly(lactic acid) initially generates small molecules of lactic acid, and then the lactic acid from the NP surfaces quickly spreads and leads to a slower degradation. The lactic acid in the interior of the NPs diffuse with difficulty, causing an accumulation of lactic acid within the NPs, which leads to autocatalytic ester bond H


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Figure 5. 1H NMR spectra of (i) mPEG−PLGA (LA/GA = 80/20) and (ii) PEAL (LA/GA = 80/20) in CDCl3.

hydrolysis, rapid degradation, and finally, NP splitting. A few studies have shown that the degradation rates of porous materials are obviously slower than those for compact materials, which also implicates an autocatalytic effect.23,25,27 Because of the autocatalytic effect, the increased rate of GA−GA bond hydrolysis is faster than the increased rate for LA−LA and GA− LA bond hydrolyses, and fewer glycolic acid units remain in the polymer relative to the lactic acid units in the remaining polymer. As a result, the lactic acid to glycolic acid unit proportion in the remaining polymer showed an increased trend. By calculating the signal peak areas of glycolic acid (b) to lysine units (e) in the PEAL (LA/GA = 80/20) spectrum (Figure 5ii), the glycolic acid to lysine unit proportions of the remaining PEAL (LA/GA = 80/20) may be obtained. They were 1.46, 0.63, and 0.49 at 0, 48 (pH 5.0), and 144 h (pH 5.0), respectively, and 1.46, 0.94, and 0.71 at 0, 48 (pH 7.4), and 144 h (pH 7.4), respectively. The proportions in the pH 5.0 and pH 7.4 media decreased with prolonged degradation time, which indicated that the hydrolysis rate of the GA−GA bonds of the glycolic acid units was faster than those for the −CH− and −NH− bonds of the lysine units. In addition, the proportion of

the glycolic acid to lysine units at 48 h (pH 5.0) and 48 h (pH 7.4) was 0.63 and 0.94, respectively, and at 144 h (pH 5.0) and 144 h (pH 7.4), the proportion was 0.49 and 0.71, respectively. The incremental increase of the proportion with an increased pH value implied that the accelerated degradation by pH of the lysine units was more significant than that for the glycolic acid units. 3.3. Total Protein Synthesis. Measurement of the intracellular protein content in the Huh7, L02, and RAW 264.7 cell lines and comparison with negative controls demonstrated that intracellular protein synthesis did not decrease for cells treated with 1000 μM PEAL NPs (Figure 6A−C). As the incubation time increased, the intracellular protein content also exponentially increased, as did the protein level of the negative control, indicating that the PEAL NPs and their degradation products did not adversely affect protein synthesis, and the cells continued to proliferate. These findings indirectly indicated the nontoxicity and safety of the PEAL NPs. 3.4. LDH Release Assay. LDH is present in the cytoplasm of living cells and is an important and stable enzyme related to cell metabolism (glycolysis). It can catalyze the conversion of I


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Figure 6. After cell treatment with 1000 μM PEAL NPs, the total protein synthesis and dynamic protein content changed with time. Changes in dynamic protein content in (A) Huh7 cells, (B) L02 cells, and (C) RAW 264.7 cells. (D) Huh7 cells, (E) L02 cells, and (F) RAW 264.7 cells were treated with 1000 μM PEAL NPs for 72 h; then, LDH leakage was quantitatively analyzed. The cells treated with PEAL NPs were compared with the negative control, and P > 0.05 in total protein synthesis and LDH release tests.

lactate to pyruvate and simultaneously produce ATP. Under normal circumstances, LDH cannot pass through the cell membrane; however, if the cell membrane is damaged, its permeability increases.1,30 Thus, detecting the activity of LDH in the medium can provide a quantitative indication of cytotoxicity. In this experiment, three types of cells were treated with 1000 μM NPs for 72 h. The LDH levels were not significantly higher than that of the negative control. The LDH levels in the medium of Triton X-100-treated cells were 900−1300 U/L, far higher than those of the negative control (200−600 U/L) and the NP-treated groups (200−500 U/L), and these differences were significant (P < 0.05; Figure 6D−F). These findings indicated that the 1000 μM PEAL NPs did not significantly affect membrane integrity. 3.5. Induction of Chromatin Agglutination. Intracellular DNA is mainly located in the chromatin, and its content is very stable. The chromatin agglutination test may reflect the cell nuclei toxicity of the nanomaterial.31 In cells treated with 10, 100, 500, and 1000 μM PEAL NPs for 72 h, the chromatin did not agglutinate and were uniformly stained, regularly shaped and similar to the control. Figure 7 shows the stained chromatin of cells treated with 1000 μM PEAL NPs. The corresponding images for the cells treated with 10, 100, and 500 μM PEAL NPs are not shown. 3.6. Detection of Intracellular ROS. The superoxide anion radical (O2•−), hydrogen peroxide (H2O2), and nitric oxide (NO) reactive oxygen species are natural byproducts of the normal metabolism of oxygen. However, excessive ROS can result in biomolecular damage, including lipid peroxidation and DNA damage.32 In the present study, slight concentration and time-dependent effects were observed. Compared with the negative control, the Huh7 and L02 cells treated with 10, 100, 500, and 1000 μM of the PEAL (LA/GA = 60/40, 70/30, and 80/20) NPs did not exhibit significant increases in ROS, suggesting that the Huh7 and L02 cells could safely be treated with up to 1000 μM of these NPs (Figure 8A,B). For the RAW

Figure 7. Chromatin of Huh7, L02, and RAW 264.7 cells treated with 1000 μM PEAL NPs were uniformly stained, regularly shaped and indistinguishable from the control (Figure A1 vs Figure A2−A4, Figure B1 vs Figure B2−B4, Figure C1 vs Figure C2−C4).

264.7 cells, 1000 μM NPs stimulated the cells to produce ROS, with the highest ROS content reaching 196.4%, which is significantly higher than those of the Huh7 (142%) and L02 cells (136.2%) at 72 h. The reason for this difference may be that RAW 264.7 cells are macrophages that can phagocytize more NPs. Furthermore, with prolonged incubations (24, 48, or 72 h), and as the LA/GA ratio decreased from 80/20 to 70/30 to 60/ 40, the accumulation of intracellular ROS also increased (data not shown). For example, the ROS content of the 1000 μM PEAL (LA/GA = 60/40, 70/30, and 80/20) NP-treated RAW 264.7 cells increased from 140.3, 138.2, and 120.1% at 24 h and J


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Figure 8. Intracellular ROS content (compared with negative control) of treated cells. (A) Huh7 cells and (B) L02 cells treated for 72 h. RAW 264.7 cells treated for (C) 48 h and (D) 72 h. Compared with the negative control, *P < 0.05, **P < 0.01, ***P < 0.001.

175.3, 160.2, and 138.7% at 48 h, to 196.4, 178.9, and 146.1% at 72 h, respectively. The incremental increases of the ROS content of the 1000 μM of the PEAL (LA/GA = 60/40 and 70/ 30) NP-treated RAW 264.7 cells were significant at 48 and 72 h (P < 0.05; Figure 8C,D). These findings indicated that the PEAL (LA/GA = 80/20) NPs had the lowest cytotoxicity. ROS generation was also confirmed in DCFH-DA-treated cells using fluorescence microscopy (Figure 9). The fluorescence was diffusely distributed in the cells, indicating that intracellular ROS could generate a diffuse distribution. Additionally, their generation may have occurred as a result of the direct interactions between the NPs and the biomolecules in the cells. 3.7. Inflammatory Cytokine Release Assay. 3.7.1. THP-1 Cells Induced to Differentiate into Macrophages. Before induction with PMA, THP-1 cells were in suspension and their morphology was round and smooth (Figure 10A. (i). At 24 h after PMA induction, the THP-1 cells were in an adherent state. The cell shapes turned from round to spindle-like, becoming oval or irregular, and showed the formation of lamellipodia and pseudopods (Figure 10A. (ii). At 48 h after PMA induction, almost all the cells were adherent and converted to a spindle or oval shape, indicating that the THP-1 cells differentiated into macrophages (Figure 10A. (iii). 3.7.2. Quantitative detection of THP-1 release of IL-1β, TNF-α and TGF-β1. IL-1β is a lymphocyte stimulating factor and is mainly produced by the activation of mononuclear macrophages. When the in vivo IL-1β concentration is low, its biological function is to primarily coordinate the stimulation of APC, T cell activation and to promote B cell proliferation and antibody production as an immune function. If the in vivo IL-1β concentration is high, the primary biological functions are endocrine in nature, which may induce acute phase protein synthesis in the liver and cause fever and cachexia. TNF-α can also have dual biological functions. On the one hand, if the in

Figure 9. ROS production sites in NP-treated cells (Figures A2−A4, B2−B4, C2−C4) were diffusely distributed in the cells, similar to the controls (Figures A1, B1, C1). As the LA/GA ratio increased from 60/ 40 to 70/30 to 80/20, the intracellular fluorescent intensity gradually decreased. The fluorescent intensity of PEAL (LA/GA = 80/20) NPtreated cells was the closest to that of the control (Figure A1 vs Figure A4; Figure B1 vs Figure B4; Figure C1 vs Figure C4).

vivo TNF-α concentration is low, it can play important roles in immune regulation, physiological functions and anti-infection. For example, TNF-α can stimulate T cells to produce a variety of inflammatory factors, then promote inflammatory reactions. On the other hand, if released or produced at a high concentration, TNF-α can cause fever, shock, and cachexia. K


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39 pg/mL, and the TGF-β1 contents were 18.51, 21.5, 20.5, and 16.43 pg/mL, respectively. These values were significantly lower than the IL-1β (647.53 pg/mL) (P < 0.001), the TNF-α (80.24 pg/mL) (P < 0.05 or 0.01) and the TGF-β1 (58.11 pg/ mL) (P < 0.05 or 0.01) values in the positive control. Compared with the negative control, the differences of the IL1β, TNF-α and TGF-β1 contents were not significant for the PEAL (LA/GA = 60/40, 70/30, 80/20) NP groups. These results indicated that the PEAL NPs have ideal immune compatibilities. 3.8. Sensitive Time Window and Dose Effects during the Sensitive Time Window for Zebrafish Embryo Development. 3.8.1. Sensitive Time Window. The sensitive time window is the most important period of zebrafish embryonic development. Many important systems and organs begin to develop and differentiate at this time, such as the central nervous system, circulatory system, digestive system, eyes and trunk. In this period, embryos are most sensitive to external stimuli and are vulnerable to and easily damaged by internal and external environmental factors, which may lead to death or serious body and organ malformation. Therefore, this period is known as the sensitive period. The mortality and malformation rates of zebrafish embryos at different stages of development exposed to PEAL NPs at 3 × 103 μM are shown in Figure 11A and Table 2. For the negative control and nonexposed embryos, the mortality rate was 0 at 96 hpf. This mortality rate is typical for zebrafish embryos under normal conditions and confirmed that there was nothing unusual about the rearing conditions in this study. The mortality rates with 2.5% acetone (positive control) were 100, 79.58, 66.67, 33.10, and 28.67% at 10−24, 6−14, 6−10, 24−48, and 48−72 hpf, respectively. The malformation rates with 2.5% acetone were 0, 12.68, 15.15, 21.83, and 16.17% at 10−24, 6− 14, 6−10, 24−48, and 48−72 hpf, respectively. The total positive response rates with 2.5% acetone were 100, 92.26, 81.82, 54.93, and 44.84% at 10−24, 6−14, 6−10, 24−48, and 48−72 hpf, respectively. Thus, the sensitive window was 10−24 hpf for acetone, followed by 6−14 hpf for the PEAL NPs. The highest mortality rate was approximately 10−16% during 10− 24 hpf (Figure 11A), and the highest malformation rate was also during 10−24 hpf (Table 2), followed by 24−48 hpf,

Figure 10. Inflammatory cytokine release assays. (A) Morphology of THP-1 cells induced by PMA after 0, 24, and 48 h. (B) Cells incubated with medium containing 6.4% phenol are used as positive controls, cells incubated with medium are used as negative controls. Cells incubated with medium containing 5 mM PEAL (LA/GA = 60/40, 70/30, 80/20) NPs are used as experimental groups. The cells treated with PEAL NPs are compared with the negative control, P > 0.05 for cytokine release.

TGF-β1 may also play important regulatory roles in inflammation, tissue repair, and embryonic development. However, an overexpression can suppress the proliferation of immune cells, inhibit the differentiation of lymphocytes, and inhibit cytokines production. The excessive expressions of these cytokines can cause an immune function disorder and a series of symptoms to occur. After PMA-induced differentiation, THP-1 monocytes become defensive secretory macrophages and can initiate immune inflammatory responsive processes in vivo. The release of IL-1β, TNF-α and TGF-β1 cytokines is important for investigating the immune compatibility of PEAL NPs. Figure 10B shows the IL-1β contents of the negative controls and the PEAL (LA/GA = 60/40, 70/30, and 80/20) NP groups, which were 72.41, 87.14, 94.6, and 75.5 pg/mL, respectively. The TNF-α contents were 42.24, 49.5, 46.5, and

Figure 11. (A) Sensitive time window test. The highest mortality rate is at 10−24 hpf and approximately 10−16% for PEAL NPs; thus, the sensitive time window is 10−24 hpf. (B) The mortality rate of different concentrations of PEAL NPs lead to the death of zebrafish embryos during the sensitive time window. 1: PEAL = 0.0625 × 103 μM; 2: PEAL = 0.125 × 103 μM; 3: PEAL = 0.25 × 103 μM; 4: PEAL = 0.5 × 103 μM; 5: PEAL = 1 × 103 μM; 6:2 × 103 μM. E3 medium and 4% acetone solution are used as negative and positive controls, respectively; 2 × 103 μM PEAL (LA/GA = 60/40, 70/30, 80/20) NPs and 1 × 103 μM PEAL (LA/GA = 60/40) NPs induced a mortality rate that is higher than the negative control (P < 0.05); when the PEAL NPs concentration is less than 1 × 103 μM or the PEAL (LA/GA = 70/30, 80/20) NPs concentration is 1 × 103 μM, the mortality rate is not significantly different; * indicated compared with negative control, the P < 0.05. L


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The Journal of Physical Chemistry C Table 2. Malformation Rate of Zebrafish Embryos at Different Stages Exposed to PEAL NPs at 3 × 103 μMa malformation types and proportion (%) LA/GA

time (hpf)

total malformation rate (%)

60/40

6−10 6−14 10−24 24−48 48−72 6−10 6−14 10−24 24−48 48−72 6−10 6−14 10−24 24−48 48−72

0±0 0.56 ± 0.96 11.68 ± 5.03* 4.32 ± 1.51* 0±0 0±0 0±0 5.34 ± 0.76* 2.74 ± 2.96 0±0 0±0 0±0 4.85 ± 1.62* 3.12 ± 1.07* 0±0

70/30

80/20

yolk sac edema 0±0 0±0 1.54 ± 1.34 0±0 0±0 0±0 0±0 0.90 ± 1.56 0±0 0±0 0±0 0±0 0±0 0±0 0±0

bent body axis 0±0 0.56 ± 4.68 ± 3.17 ± 0±0 0±0 0±0 3.43 ± 2.08 ± 0±0 0±0 0±0 2.26 ± 2.24 ± 0±0

0.96 1.95* 0.54*

0.99* 1.97

2.08 2.18

caudal fin abnormal

pericardial edema

0±0 0±0 0.72 ± 1.25 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0.90 ± 1.56 0±0 0±0

0±0 0±0 1.60 ± 1.41 0.62 ± 1.07 0±0 0±0 0±0 0.9 ± 1.56 0±0 0±0 0±0 0±0 0.68 ± 1.18 0±0 0±0

yolk sac absorption delay 0±0 0±0 2.26 ± 0.53 ± 0±0 0±0 0±0 1.01 ± 0.65 ± 0±0 0±0 0±0 1.01 ± 0.88 ± 0±0

2.18 0.92

1.74 1.13

1.75 1.52

yolk sac opaque 0±0 0±0 0.88 ± 1.52 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0

a The highest malformation rate was during 10−24 hpf, and the lowest malformation rate was 0% during 6−10 hpf and 48−72 hpf. During 6−14 hpf, PEAL (LA/GA = 60/40) NPs produces only 0.56% of the bent body axis, and PEAL (LA/GA = 70/30, 80/20) produce no malformations. The 10− 24 hpf corresponds to human pregnancy at 3−4 weeks. The low malformation rate at 6−14 hpf and 24−72 hpf, indicated that the PEAL NPs main impact is at the somite stage for embryonic growth and development, and has little effect at other phases of the entire developmental process, suggesting that clinical medication should be avoid during the sensitive somite stage.

rates were 9.7, 4.9, and 3.17%, which were all higher than that of the negative control (P < 0.05). When the concentration of the PEAL (LA/GA = 60/40, 70/30, and 80/20) NPs was 1 × 103 μM, the mortality rates were 3.71% (P < 0.05), 2.83% (P > 0.05), and 1.36% (P > 0.05), respectively, and the malformation rates were 5.16% (P < 0.05), 3.06% (P < 0.05), and 1.46% (P > 0.05; the P value was based on comparisons with the negative control). When the concentration of the PEAL (LA/GA = 60/ 40, 70/30, and 80/20) NPs was 500 μM, the mortality rates were 0.71, 0, and 0%, respectively, and the malformation rates were also 0.71, 0, and 0%, which were all lower than the negative control (P > 0.05). When the concentration of PEAL NPs was 250, 125, and 62.5 μM, the mortality and malformation rates were 0%. The main embryonic malformations observed include body axis bending, pericardial edema, yolk sac edema, yolk sac absorption delay and hypoevolutism, among others (Figure 12). Although there was a concentration dependence for toxicity with PEAL NPs for organs, the EC50 and LC50 for the PEAL NPs for zebrafish embryos were also very high (Table 4). These findings indicated that the toxicity of the PEAL NPs for biological bodies was very low, and its safe dosage range was wide. As the LA/GA ratio increased, the EC50 and LC50 also increased, indicating that the zebrafish embryos had a higher resistance for the PEAL (LA/GA = 80/20) NPs than for the PEAL (LA/GA = 60/40 and 70/30) NPs. The PEAL (LA/GA = 80/20) NPs demonstrated minimum side effects for biological bodies. This could be due to the following: first, the degradation rate of the PEAL (LA/GA = 80/20) NPs was the slowest. The lactic acid, glycolic acid and lysine degradation product contents were also low. Thus, the effect on the inner environment of the embryos during the critical stage would be minimal, and the mortality and malformation rates decreased. Second, according to the literature, within a certain range the larger NPs could lead to greater toxicity for the embryos.33−35 When LA/GA = 80/20, the sizes of the PEAL NPs were the smallest. Thus, the PEAL (LA/GA = 80/20) NPs have a

different from that of acetone. The highest total positive response rate with the PEAL NPs was during 10−24 hpf, followed in turn by 24−48, 6−14, 6−10, and 48−72 hpf. Thus, the sensitive window time for the PEAL NPs was 10−24 hpf. The primary malformations were body axis bend, yolk sac absorption delay, pericardial edema, and yolk sac edema. Representative images are shown in Figure 12.

Figure 12. Types of NP-induced malformations in zebrafish embryos at 96 hpf. (A) Normal development; (B) yolk sac edema; (C) pericardial edema; (D) yolk sac absorption delay; (E) hypoevolutism; (F) bent body axis. Arrows indicate the typical features of the malformation region.

3.8.2. Toxicity of PEAL NPs for Zebrafish Embryo Development. Few studies have used zebrafish embryos to investigate the toxicity of NPs prepared from organic polymer materials.8 The mortality and malformation rates obtained from the sensitive time window and dose effect experiments indicated that the positive response rate of the physical development of zebrafish embryos was correlated with NP concentrations (Figure 11B and Table 3). This indicated that the NP concentration is an important factor in the severity of the effect. When the concentration of the PEAL (LA/GA = 60/ 40, 70/30, and 80/20) NPs was 2 × 103 μM, the mortality rates were 6.51, 4.5, and 3.88%, respectively, and the malformation M


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The Journal of Physical Chemistry C Table 3. Zebrafish Embryo Malformation Types and Prevalences for Different Concentrations of PEAL NPsa malformation prevalence (%) material

concentration (×103 μM)

total malformation rate (%)

2 1 0.5 0.25 0.125 0.0625 2 1 0.5 0.25 0.125 0.0625 2 1 0.5 0.25 0.125 0.0625 544

9.70 ± 2.52*b 5.16 ± 1.15*b 0.71 ± 1.23 0±0 0±0 0±0 4.9 ± 2.07*b 3.06 ± 1.08*b 0±0 0±0 0±0 0±0 3.17 ± 1.44*b 1.46 ± 2.00 0±0 0±0 0±0 0±0 0±0 0±0

60/40

70/30

80/20

acetone E3

yolk sac edema 1.51 ± 0.76 ± 0±0 0±0 0±0 0±0 0.74 ± 0±0 0±0 0±0 0±0 0±0 0.79 ± 0±0 0±0 0±0 0±0 0±0 0±0 0±0

bent body axis

1.39 1.31

1.28

1.37

4.46 ± 2.15 ± 0.71 ± 0±0 0±0 0±0 2.05 ± 2.39 ± 0±0 0±0 0±0 0±0 1.59 ± 0.79 ± 0±0 0±0 0±0 0±0 0±0 0±0

2.29*b 1.13 1.23

1.19 1.27

1.17 1.21

pericardial edema 1.65 ± 0.78 ± 0±0 0±0 0±0 0±0 1.37 ± 0.67 ± 0±0 0±0 0±0 0±0 0.79 ± 0.67 ± 0±0 0±0 0±0 0±0 0±0 0±0

yolk sac absorption delay

hypoevolutism

1.37 ± 1.44 0.71 ± 1.23 0±0 0±0 0±0 0±0 0.74 ± 1.28 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0

0.71 ± 1.41 0.76 ± 1.31 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0 0±0

1.43 1.35

1.33 1.16

1.37 1.16

Compared with the negative control, the malformation rate with 2 × 103 μM PEAL NPs was significantly different (P < 0.05) from those with 1 × 103 μM PEAL (LA/GA = 60/40, 70/30) NPs (P < 0.05). The malformation rates with 0.5, 0.25, 0.125, and 0.0625 × 103 μM PEAL NPs and 1 × 103 μM PEAL (LA/GA = 80/20) NPs were not significantly different from the negative control. E3 medium containing acetone served as the positive control, in which all embryos died; therefore, the malformation rate was 0. E3 medium served as the negative control and the malformation rate was 0. b*P < 0.05 relative to the negative control. a

cell compatibility and a lower mortality rate among zebrafish embryos. In addition, it was demonstrated that the degradation rate for the glycolic acid units was faster than for the lactic acid and lysine units. The degradation products of lactic acid and lysine were nontoxic and metabolizable. However, the actual intermediate degradation products and the degradation mechanisms in vivo and in vitro require further exploration.

Table 4. EC50 and LC50 of PEAL NPs for Zebrafish Embryos; High EC50 and LC50 Values Indicate Good Biocompatibility index

LA/GA = 60/40

LA/GA = 70/30

LA/GA = 80/20

EC50 (×103 μM) LC50 (×103 μM)

5.75 14.01

9.55 19.75

13.38 24.42

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minimal impact on embryo growth and development. The safe dose in some cell experiments was higher than that for the embryo toxicity test, which may be due to the embryo toxicity by NPs being mainly based on the NPs generating oxidation emergency response. In cell experiments, the oxidation emergency response was lower than those of the surviving individuals.

AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86 21 64437139. E-mail: [email protected]. *Tel.: +86 21 64535555. E-mail: [email protected]. Author Contributions ‡

These authors contributed equally (Z.H. and Y.S.).

Notes

4. CONCLUSIONS Our experimental results showed that the low and routinely used concentrations (167 μM) of the PEAL NPs, and especially the PEAL (LA/GA = 80/20) NPs, exhibited good biosafety for Huh7, L02, and RAW 264.7 cells and zebrafish embryos.3,5,6 When the concentration was as high as 1000 μM, there was an effect on the biological development. Moreover, we studied the degradation of the PEAL NPs in vitro and demonstrated that the degradation rate increased with the pH of the medium, and the degradation rate of PEAL (LA/GA = 60/40) NPs was the fastest, followed in turn by the PEAL (LA/GA = 70/30) and PEAL (LA/GA = 80/20) NPs. Within a certain experimental time and concentration range, following an increase in LA/GA ratio from 60/40 and 70/30 to 80/20, both the degradation rate of PEAL NPs and the amount of degradation products decreased. This led to ever smaller internal environmental changes in cells and organisms, thus, in turn leading to better

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 81301973, 81172989, and 81472841), the Nature Science Foundation of Shanghai (No. 13ZR1440200), and the Science and Technology Commission foundation of Shanghai (No. 14JC1492500).

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Degradation and Bio-Safety Evaluation of mPEG-PLGA-PLL

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