Article pubs.acs.org/Biomac
Nucleic Acid-Scavenging Electrospun Nanofibrous Meshes for Suppressing Inflammatory Responses Jihyun Kang† and Hyuk Sang Yoo*,†,‡ †
Department of Medical Biomaterials Engineering and ‡Institute of Bioscience and Bioengineering, Kangwon National University, Chuncheon 200-701, Republic of Korea S Supporting Information *
ABSTRACT: Fragmented nucleic acids are potent stimulators for inflammatory responses provoking pathological outcomes by activating adaptive immunity. In this study, highly cationic surfaces were prepared on electrospun nanofibrous meshes to scavenge nucleic acids to the surfaces. Poly(ε-caprolactone) [PCL]-poly(ethylenimine) [PEI] block copolymers were synthesized by coupling the carboxyl-terminated PCL to the primary amines of branched PEI. Polymeric solutions composed of PCL−PEI and PCL were electrospun to nanofibrous mats, and the surfaces were further methylated to prepare highly cationic surfaces on the mats. Raman spectroscopy revealed that the presence of increased methylated amines on the surfaces of the mats compared to unmodified mats. The methylated surfaces showed significant increases of wettability after methylation, suggesting highly charged surfaces caused by methylation of the primary amines. When the blend ratio of PCL−PEI was increased, the scavenged DNA was also increased, and the methylation further strengthened the scavenging ability of the mats. Fluorescently labeled oligodeoxynucleic acids were significantly adsorbed on the surface of the mats depending on the amounts of PCL−PEI and the degree of methylation. In the presence of the methylated nanofibrous mats, inflammatory responses induced by CpG oligonucleotides in murine macrophages were significantly reduced, which was confirmed by measuring inflammatory cytokine levels including TNF-α and IFN-γ.
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INTRODUCTION
suppress synthesis of nitric oxide and tumor necrosis factor-α (TNF-α) in macrophages.6 Nucleic acid delivery systems employing electrospun nanofibrous mats have recently been studied with the aim of accomplishing gene transfection in local areas.7−9 Due to the extremely high surface-to-volume ratios of the nanofibrous structures, released nucleic acids have increased chances of contacting targeted cells for further transfection due to the large surface areas available for contact between cells and nanofibers. Similar to the strategy of colloidal gene carriers, which employ electrostatic interactions between nucleic acids and cationic polymers, cationic polymers or their derivatives are primarily employed in the nanofibrous gene carriers for fabricating the transfection matrix.10,11 Negatively charged nucleic acids are electrostatically incorporated into the matrix when the surface is oppositely charged with cationic polymers. For this purpose, polyethylenimine (PEI) in particular has received much attention for its great affinity toward nucleic acids, because of the highly cationic charge density along the chains.12,13 Branched or linear PEI has been extensively employed for preparing colloidal carriers for gene transfection because it
Fragmented nucleic acids from apoptotic cell death or foreign organisms have been recognized as inflammatory inducers, provoking undesirable pathological responses by activating innate immune cells such as macrophages, dendritic cells and neutrophils.1,2 Once recognized by nucleic acid-sensing toll-like receptors (TLRs) on the immune cells, extracellular fragments, such as ssRNA, dsRNA, and unmethylated CpG-containing DNA, subsequently stimulate cytokine production pathways for TNF-α, chemokines, and interferon, to develop adaptive immunity.3 Thus, scavenging those nucleic acids has been considered an effective means of controlling immediate or prolonged hyper-inflammatory responses from bacterial or viral infection and autoimmune diseases.4 This scavenging has mostly been performed by cationic polymers or lipids such as cyclodextrin-containing polycation (CDP), hexadimethrine bromide (HDMBr), protamine sulfate (PAMAM), and dioleyoltrimethylammonium propane (DOTAP), which inhibit the ability of synthetic CpG DNAs to activate nucleic acidsensing TLRs. Such cationic polymers are specific for neutralizing the immune-stimulatory activity of TLR agonists in a variety of primary cells, including B lymphocytes, fibroblasts, and dendritic cells.5 Cationic liposomes composed of cationic lipids mixtures were shown to electrostatically scavenge intracellular oligonucleotides and consequently © 2014 American Chemical Society
Received: March 25, 2014 Revised: May 30, 2014 Published: June 2, 2014 2600
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Figure 1. Surface-methylation of PCL−PEI nanofibrous meshes for scavenging nucleic acids causing inflammatory responses. (A) Synthetic scheme of preparing amine-terminated poly(ε-caprolactone)-block-branched polyethylenimine (PCL−PEI). Ring-opening polymerization of ε-caprolactone and octanoic acid generated a carboxylic acid-terminated PCL, which was subsequently reacted with the primary amines of PEI in the presence of NHS and DCC. (B) Iodomethane (CH3I) reacted with the surface-exposed amine of PCL−PEI nanofibrous meshes to give methyl groups and the iodide counterions were subsequently replaced with chloride ions to prepare methylation of PCL−PEI nanofibrous meshes. (C) Highly cationic meshes scavenge negatively charged nucleic acids to reduce nucleic acid fragments-induced inflammatory response of immune cells. GACGTTCCTGATGCT-3′) were purchased from Bioneer (Seoul, Republic of Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Trevigen, Inc. (Gaithersburg, MD). Dulbecco’s modified Eagle’s medium (DMEM), streptomycin/penicillin, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Enzyme-linked immunosorbent assay (ELISA) kits for mouse TNF-α and IFN-γ were purchased from eBioscience (San Diego, CA). Synthesis of PCL−PEI Block Copolymers. A block copolymer composed of PCL and PEI was synthesized by a three-step reaction as shown in Figure 1A. Briefly, ε-caprolactone (180 mmol) and octanoic acid (18 mmol) were put into a flask in nitrogen atmosphere, and Sn(Oct)2 (0.12 mmol) was subsequently injected. The polymerization reaction was carried out at 130 °C for 24 h and the reaction mixture was diluted with dichloromethane, precipitated in cold diethyl ether, and completely dried in vacuum. The carboxyl-terminated PCL [PCLCOOH] (82 μmol) was activated with N-hydroxysuccinimide (NHS) and 1,3-dicyclohexylcarbodiimide (DCC) in dichloromethane (250 mL) and at room temperature for 12 h (molar ratio of PCLCOOH:NHS:DCC = 1:5:5). The reaction mixture was filtered with a paper filter (11 μm), and the filtrates were precipitated in an excess amount of cold diethyl ether. The activated PCL (PCL-COO-NHS, 74.10 μmol) in chloroform (50 mL) was slowly added to branched PEI (0.22 mmol) in chloroform (5 mL) with gentle stirring and reacted at room temperature for 24 h (molar ratio PEI:PCL−COO− NHS = 3:1). The reaction mixtures were precipitated in ice-chilled methanol and completely dried in vacuum. The conjugation amount of PEI to PCL was confirmed by 1H NMR spectroscopy in CDCl3 at the
spontaneously forms an electrostatic complex between the basic polymers and the acidic nucleic acids. In the current work, we prepared nucleic acid-scavenging nanofibrous mats to eliminate excess amounts of nucleic acid fragments, and thus to suppress hyperimmune responses. A nanofibrous mat composed of PCL−PEI block copolymers was fabricated by electrospinning and further methylated to induce a highly cationic charge density on the surface. Since CpG oligodeoxynucleotides (ODN) have been reported to stimulate dendritic cells and macrophages to secrete cytokines, we examined whether the methylated nanofibrous mats can suppress the release of TNF-α and interferon (IFN)-γ from the ODN-stimulated macrophages.14
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MATERIALS AND METHOD
Materials. ε-Caprolactone (ε-CL) was purchased from Alfa-aesar (Ward Hill, MA). Octanoic acid was purchased from Junsei Chemical Co. (Tokyo, Japan). Tin(II)-ethylhexanoate (Sn(Oct)2), N-hydroxysuccinimide (NHS), 1,3-dicyclohexylcarbodiimide (DCC), flourescamine, and deoxyribonucleic acid (DNA) from salmon testes were purchased from Sigma-Aldrich (St. Louis, MO). Branched polyethylenimine (PEI, Mw 1.8 kDa) was purchased from Polysciences Inc. (Warrignton, PA). Iodomethane (CH3I) was purchased from Daejung Chemical Co. (Cheongwon, Republic of Korea). Murine macrophage cell line RAW 264.7 was obtained from ATCC (Manassas, VA). CpG oligodeoxynucleotide (ODN) (5′-TCCATGACGTTCCTGATGCT3′), and Texas red-labeled ODN (Texas red-5′-TCCAT2601
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Central Laboratory of Kangwon National University (DPX 400, Bruker). Electrospinning and Methylation of PCL/PCL−PEI Nanofibrous Mats. The polymeric solution (12%, w/v) of PCL or PCL− PEI mixtures in methanol/chloroform mixture (1:3, v/v) was electrospun to the ground at +15 kV with a flow rate of 1 mL/h through a 25 G needle. The amount of PCL−PEI block copolymer in the polymeric solution was varied from 50 to 100% (w/w), and the ground-to-needle distance was 15 cm. The PCL/PCL−PEI nanofibrous mats (NFs) were soaked in methanol for 3 h and hydrated in phosphate buffered saline for 30 min (PBS, pH 7.4) (Figure 1B). One hundred microliters of iodomethane (70 mg/mL in methanol) was added to the hydrated nanofibers and reacted at 37 °C for 48 h. The reaction was quenched with 0.5 M NaCl solution, and the methylated NFs were washed with deionized water and dried under vacuum. The amount of the surface-exposed primary amines on the PCL/PCL−PEI NFs before and after methylation was determined by a fluorescamine assay as described previously.15 Briefly, the PCL/PCL−PEI NFs (1 mg) were sequentially soaked in 30% ethanol and PBS (pH 7.4) for 15 s and 30 min, respectively. Fluorescamine in acetone (0.3 mg/mL) was added to the hydrated NFs and then reacted at room temperature for 30 min. After washing with methanol, the reacted NFs were completely dissolved into 1,4-dioxane to measure the fluorescence intensity. The excitation wavelength and the emission wavelength were 390 and 475 nm, respectively, and ethylenediamine was employed as a standard for primary amines. Characterization of the Methylated PCL/PCL−PEI Nanofibers. Methylated PCL/PCL−PEI NFs were characterized by using Raman spectroscopy and water-contact angle measurements. For Raman spectroscopy, NFs (1 mg/device) were placed on a sample holder and excited with an air-cooled diode-pumped solid state laser at 633 nm (Horiba Jobin Yvon, France). The scattered Raman spectrum was detected in the range of 600−4000 cm−1 by a charge-coupled device (CCD) camera. Five collections of accumulation times of 10 s were employed with a spectral resolution of 1 cm−1. In order to determine the wettability of the NFs, the sample (1 mg/device) was placed on a sample stage, and water (10 μL) was dropped on the surface of the NFs. After 1 s, the image of the droplet on the NFs was obtained by a digital camera and analyzed by Image-Pro v6.0 software to obtain the contact angles between NFs and the water droplets. The measurements were repeated three times, and the averaged values were obtained. Nucleic Acid-Scavenging Efficacy of PCL/PCL−PEI NF. The nucleic-acid scavenging efficacy of the methylated NFs was determined by employing salmon testes DNA as a model nucleic acid for 12 h. PCL/PCL−PEI NFs (1 mg/device) were prewetted with 30% ethanol, and 50 μg of DNA was added to prehydrated NFs in 1 mL of PBS (pH 7.4). The mixture was incubated at 37 °C in an orbital shaker, and the supernatant was subjected to DNA quantification at 260 nm at different time intervals within 12 h. In order to visualize the scavenged nucleic acids on the NFs, fluorescently labeled ODN was incubated with the NF and visualized by In Vivo Imaging System (IVIS). Presoaked NF was attached to a 12-well plate, and Texas red-labeled ODN (0.5 μg) in PBS (0.5 mL) was added. After gentle shaking at 37 °C for 12 h, the NFs were thoroughly washed with PBS three times. The fluorescent images of the NFs were obtained by accumulating fluorescence signals for 15 s at 580 nm/630 nm of the excitation and the emission wavelength, respectively, at Korea Basic Science Institute (KBSI) (IVIS 200, PerkinElmer, Waltham, MA, USA). Cytotoxicity of PCL/PCL−PEI NFs. The cytotoxicity of the NFs with various blend ratios of PCL−PEI was determined by a MTTbased cytotoxicity assay against macrophages. RAW 264.7 cell lines were cultured in DMEM with 10% (v/v) FBS and streptomycin/ penicillin and maintained at a humidified atmosphere of 5% CO2 at 37 °C. PCL/PCL−PEI NFs (1 mg) were presoaked in 70% ethanol solution for 30 min and washed with PBS three times. Cells (1 × 105 cells/well) were seeded on PCL/PCL−PEI NFs placed in a 12-well plate and then incubated in the culture medium (1 mL/well) at 37 °C for 12 h. Ten microliters of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 3 h. After removing the cell
culture medium, formazan crystals were dissolved with DMSO (1 mL) for 1 h, and the absorbance was measured at 570 nm. Cell viability was determined by dividing the absorbance of the cells on PCL/PCL−PEI NFs by those on TCPS. Determination of Anti-inflammatory Effects. The antiinflammatory effects of the NFs were determined by measuring cytokine levels released from macrophages. The RAW 264.7 cell line (1 × 105 cells/well) was placed in a 12-well culture plate and cultured in DMEM (1 mL) supplemented by 10% FBS and streptomycin/ penicillin for 12 h. The cells were replenished with fresh serum-free DMEM containing CpG ODN (50 μg/mL), and the NFs (1 mg/well) were placed on the monolayer of cells. The medium was collected for detection of TNF-α or IFN-γ after 6 or 24 h, respectively. Cytokine levels were quantified by ELISA kits in accordance with the manufacturer’s instructions, and the sensitivity was 4pg/mL for all cytokines. Briefly, 50 μL of the media and 50 μL of the biotinconjugate were added to a 96-well plate coated with the antibody against either TNF-α or IFN-γ. After 2 h, the sample was washed with a wash solution two times, and 0.1 mL of a streptavidin-Horseradish peroxidase (HRP) solution was added to each well and incubated at room temperature for 2 h. After washing each well twice with a wash solution, yellow color was developed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate solution (0.1 mL) and a stop solution (0.1 mL). The absorbance at 450 nm was measured with a microplate reader, and the concentration of the cytokines was calculated by employing the cytokine solution in the kit as a standard (ELx800TM, BioTek Inc., Winnski, VT). Statistical Analysis. All data were evaluated with One Way Analysis of Variance (ANOVA) for the statistical significance, and the average values and standard deviations were also presented. P < 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION Cationic surfaces of nanofibrous meshes were generated by employing block copolymers composed of PCL and branched PEI and subsequent methylation upon electrospinning of the copolymers (Figure 1). In our previous works, we synthesized diblock copolymers composed of PCL and amine-terminated PEG and fabricated polymeric nanofibrous mats by electrospinning.15 Various molecules were chemically conjugated to the surface-exposed amine group of the mat in aqueous phase and shown to perform significant roles as drug and gene delivery carriers. However, the amount of surface-exposed amine groups was not high enough to sequester nucleic acids fragments by electrostatic interactions (0.7 to 2.1 nmol/mg nanofibrous mat). In the current work, we synthesized PCL− PEI block copolymer by conjugating the primary amines of PEI to the terminal carboxylic acid of PCL and confirmed the conjugate by 1H NMR spectrum (Figure 1 and Figure S1), where the peaks at 1.42, 1.62, 2.34, and 4.09 ppm correspond to the proton of −(CH2)3−, −OCHH2−, and −CH2OOC− in the PCL units, respectively. 1H NMR indicated that the proton peaks near the secondary amines of PEI appeared at 3.65 ppm. By comparing the peak area of the secondary amines of PEI (3.65 ppm) to −CH2OOC− of PCL (4.09 ppm), we speculated that one molecule of PCL had 0.5 molecule of PEI. Then, the block copolymer, at various blend ratios to PCL, was subsequently electrospun to fibrous mats, and the primary amine groups of the mats were methylated in aqueous phase to replace the primary amine with secondary or tertiary amines (Figure 1B). We expected that the methylation of the primary amine groups would strengthen the cationic charge density on the surface because the basicity of amines tends to be elevated in the order of primary, secondary, and tertiary amines.16,17 Thus, the methylated NFs can scavenge nucleic acid fragments because of stronger electrostatic interactions between the NF 2602
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surfaces and the nucleic acids, and the inflammation responses of the immune cells can be further attenuated (Figure 1C). The degree of methylation was determined by quantifying the residual primary amines of the methylated NFs (Figure 2).
Figure 3. Raman spectra of (A) PCL NF, (B) PCL−PEI NF (0/100,) and (C) methylated NFs (0/100). The significant peaks at 3000 cm−1 correspond to υCH2 at 2918, 2866 cm−1; CO at 1727, 1030 cm−1; δCH2 at 1421, 1444, 1469 cm−1, and ωCH2 at 1286, 1308 cm−1 of PCL.
Figure 2. Surface-exposed amine groups on PCL/PCL−PEI NFs before and after methylation (bars) at various blend ratios of PCL to PCL−PEI and the methylation efficiency (line). The blend ratio of PCL (x) to PCL−PEI (y) is presented in x/y (w/w) (n = 4). Asterisk (*) indicates statistical significance (p < 0.05). *ND: the primary amines were not determined because no PEI exists on the surface of PCL NF.
Among the primary, secondary, and tertiary amines of branched PEI, the secondary amines show higher basicity (Kb = 5.4 × 10−4 M) compared to the primary amines (Kb = 4.4 × 10−4 M) and consequently contribute strong electrostatic interactions, because of the effects of alkyl groups on the amine.21 Thus, electrostatic interactions between PEI and nucleic acids can be manipulated by introducing substitute groups of the amine. For example, plasmid DNA was electrostatically incorporated into a polymeric matrix composed of poly(ethylene glycol)−polyethyleneimine [PEG−PEI].22 While the incorporation of DNA by pure PCL fibers was less than 15%, the incorporation of DNA by PCL/PEG−PEI fibers reached the maximum (50%) within 30 min. In another study, DNA was electrostatically incorporated on the electrospun nanofibrous matrix at various charge ratios of DNA to the immobilized LPEI. The incorporation efficiency of the DNA increased significantly, from 17.4% to 79.5%, when N/P ratios were increased from 2 to 16.23 In order to confirm the effects of methylation on the surfacewettability of the NF, we determined the hydrophilicity of the methylated NF by measuring the water contact angles of PCL/ PCL−PEI NFs (Table 1). At the same blend ratio of PCL− PEI, the contact angles of the methylated NFs were dramatically decreased. Notably, PCL−PEI NF showed the most dramatic contact angle decrease, of 83.05°, from 93.35 ±
After methylation of the PCL/PCL−PEI NFs, the amount of primary amine groups decreased significantly, in accordance with the increased blend ratio. The primary amine groups of PCL/PCL−PEI NFs were expected to be methylated from 50.5 to 87.5% for all blend ratios. PCL−PEI NF with a blend ratio of 0/100 showed that the amount of primary amine groups was 1.96 ± 0.25 nmol per device, suggesting that 50.5 ± 1.80% of the surface-exposed primary amine groups were substituted with secondary or tertiary amines, compared to the initial amount on the same NFs (4.14 ± 0.41 nmol/device). The degree of substitution efficiency was gradually increased when the blend ratio reached 50/50. We speculate that steric hindrance of the bulky PEI chains can limit the reaction between the primary amines and methyl iodide and subsequently decrease the methylation efficiency. Thus, this result clearly suggests that an optimized amount of surfaceexposed PEI is required to obtain high degrees of substitution to methylated PEI moieties on the NFs. However, it should also be noticed that the 0/100 NF showed the highest degree of methylated amines because the amount of PCL−PEI in the NFs is the highest among the other NFs. We also surveyed the methylated surface of the NFs by Raman spectroscopy to observe methyl groups on the surface (Figure 3). After methylation of the NFs, a new absorption peak appeared at 3000 cm−1, which indicates the amine component of methyl groups, in comparison with the characteristic peaks of PCL, such as υCH2 at 2918, 2866 cm−1; υCO at 1727 cm−1; δCH2 at 1421, 1444, 1469 cm−1; ωCH2 at 1286, 1308 cm−1; and skeletal stretching at 1112 cm−1.18,19 Based on the Raman spectrum of N(CH3)3 in the methylated NFs, we can speculate that methylation occurred at the surface of the nanofibers because methylation of the NF was performed after electrospinning. However, we could not estimate the number of secondary or tertiary amines on the methylated NF because the spectrum is not quantitative.20
Table 1. Contact Angles of PCL/PCL−PEI NFs with Various Blend Ratios before and after Methylationa water contact angle (deg) PCL/PCL−PEI NF
before methylation
after methylation
PCL NF 50/50 25/75 0/100
139.74 ± 5.33 102.57 ± 1.50 102.11 ± 5.86 93.35 ± 1.46
106.10 ± 2.94 21.57 ± 4.04 13.00 ± 5.89 10.30 ± 6.22
The water contact angles were measured after 1sec of dropping 10 μL of water on the NFs (mean ± SD, n = 4).
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1.46° to 10.30 ± 6.22°. The extent of contact angle decrease gradually diminished as the blend ratio of PCL−PEI decreased from 50/50 to 0/100. In fact, the water droplet was instantly absorbed into the methylated NF after only 1s compared to the unmodified NFs (Figure S2, Supporting Information). Many studies have previously modified charge-densities on the surface to manipulate the hydrophobicity or hydrophilicity of polymeric surfaces.24,25 Thus, this result also suggests that the surface-exposed amines were successfully converted into electrostatically stronger cations such as secondary or tertiary amines. Figure 4 shows the nucleic acid-scavenging efficiency of the methylated NF in comparison to the unmethylated one. All
comparison to the unmethylated counterparts. The scavenging efficiency of the NF was gradually increased in all NFs containing PCL−PEI and became saturated after 12 h (data not shown). Thus, we concluded that the nucleic acids are scavenged by the NF in a time-dependent manner. We also fluorescently visualized the scavenged ODN on the NF with IVIS, and it indicated the high retention of ODN on the thoroughly washed surface of the NFs (Figure 4B). The fluorescence intensities on the NF gradually increased when the blend ratio of PCL−PEI increased, and the methylated NFs always showed higher intensities compared to their counterparts. This result accordingly suggests that the scavenging efficiency of DNA is proportional both to the amounts of PCL−PEI and the methylation, as shown in Figure 4A. Thus, we speculate that these meshes can be applied to any biological surfaces and will locally reduce excessive amounts of nucleic acids. We previously showed that the PCL NFs had the effect of producing well-contouring wound sites due to the high surface to volume ratios of the nanoporous meshes.26 Furthermore, considering that the topically administered NFs can be easily removed after scavenging unwanted nucleic acids on any biological surfaces, the NF can potentially be employed as a “molecular-sponge” to reduce inflammation responses in a limited area. In order to investigate the cytotoxicity of the methylated nanofibers on macrophage, the MTT assay was used (Figure S3). The cell viability of the methylated nanofibers was increased from 45.1% to 61.0% at 50/50 for 12h. We only monitored the viability for 12 h because most cytokines has been reported to be released within 12h.27 In fact, poly(εcaprolactone) (PCL) nanofibers have limited cellular affinity due to their high hydrophobicity. Surface modification by methylation can overcome that limitation because the increasing amount of PCL−PEI can increase cell adhesion and can reduce PCL hydrophobicity. However, the cytotoxicity of PCL−PEI NFs can be influenced, depending on the amount of PCL−PEI, by the strong cationic charge of PEI. The cytotoxicity of PEI depends on its molecular weight and it is accepted that PEI with a molecular weight of 1.8 kDa shows cytotoxicity.28 As shown in Figure 5, we investigated the anti-inflammation efficacy of the methylated NFs by quantifying major inflammatory cytokines such as TNF-α and IFN-γ in the macrophages in the presence of CpG ODN. Because CpG ODN has been considered to be a potent inflammatory stimulator against various immune cells such as macrophages, the in vivo condition for anti-inflammation efficacy can be simulated and tested with the ODN and macrophages mixtures in the presence of various NFs.29−31 Upon addition of various NFs to the ODN and macrophages mixtures, the cytokine releases were considerably attenuated, as shown in Figure 5. The most dramatic results were obtained from the methylated NF, where both TNF-α and IFN-γ were below detection limits by ELISA assay. Unmethylated NF also greatly suppressed TNF-α and IFN-γ release from the macrophages, by 8.42% and 0.50% for TNF-α and IFN-γ compared to the untreated group. Thus, we can speculate that the nucleic acids were electrostatically scavenged by the NFs, and the macrophages were not sufficiently stimulated by CpG ODN to show inflammatory responses. It should be of noticeable that the ELISA kit could not detect TNF-α and IFN-γ production for the macrophages without addition of nucleic acids for 12 h.
Figure 4. DNA scavenging efficacy of methylated or unmodified PCL/ PCL−PEI NFs. (A) DNA scavenging efficiency of the NFs in DNA solution (50 μg/mL) for 12h. The unincorporated DNA was quantified at 260 nm and calculated for the scavenging efficiency (the amounts of the scavenged DNA/total amounts of DNA) (mean ± SD, n = 4). All data in the figures have statistical significance (p < 0.05). (B) Scavenged nucleic acids on the NFs. Fluorescently labeled ODN was incubated with the PCL/PCL−PEI NF and visualized by In Vivo Imaging System (IVIS).
NFs exhibited increasing scavenging effects with increasing amounts of PCL−PEI in the NF from 50/50 to 0/100. The increased amounts of PEI obviously played a pivotal role in the scavenging of any nucleic acids onto the NFs; the higher densities of cationic PEI moieties on the surface of the NFs are electrostatically favorable for scavenging anionic nucleic acids. These cationic charges are dramatically strengthened by methylation and, in fact, the measured scavenging efficiency showed a several fold increase, ranging from 8% to 40% in 2604
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methylation. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]; tel: +82-33-250-6563; fax: +82-33-253-6560; website: http://nano-bio.kangwon.ac.kr. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant from the National Research Foundation (NRF) (NRF-2012R1A2A2A01005857) and partly by Kangwon National University.
Figure 5. Suppression of inflammatory responses of RAW 264.7 cells with PCL NF, PCL−PEI NF (0/100), and methylated NFs (0/100). RAW 264.7 cells (1 × 105) were stimulated with CpG oligonucleotides (50 μg/mL) in the presence of NFs, and the released amount of TNFα and IFN-γ in the culture supernatant was quantified by ELISA (n = 4). The black bars and the gray bars indicate the levels of TNF-α and the IFN-γ, respectively. Asterisk (*) indicates statistical significance (p < 0.05). *ND: TNF-α or IFN-γ was not determined because level of cytokine was below the detection limits.
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Additionally, it should be noted that PCL NF also suppressed TNF-α and IFN-γ release from the macrophages, because PCL NF only scavenged minimal amounts of the nucleic acids in Figure 4A. This can be attributed to the difference in incubation conditions: in the nucleic acidscavenging test, we employed vigorous shaking to minimize nonspecific binding of nucleic acids to the NFs. However, for the cell cultivation, to minimize cell detachment from the surfaces, we could not use such a vigorous condition. Thus, nonspecific interactions between NFs and nucleic acids could be further increased, and many more nucleic acids can be adsorbed on the surfaces of PCL NF in the cell culture experiments. In fact, the electrospun nanofibers feature a high surface-to-volume ratio, which can be advantageous for the surface-functionalization of biomolecules.32 In our group, we previously showed that electrospun fibrous meshes can be applied to potent wound dressing candidates in aims to reduce undesirable changes of cellular phenotypes. Surface-functionalized nanofibrous meshes showed high wound healing efficacy as well as superior prognosis in comparison to unmodified meshes. Thus, in combination with the current study, the cationic nanofibers are expected to contribute to controlling undesirable inflammatory responses, which leads to a faster and efficient wound healing process.
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CONCLUSION PCL/PCL−PEI NFs were methylated to increase cationic charges on their surfaces, and the methylated NF showed high wettability compared to unmethylated NFs. The methylated NFs showed high nucleic acid-scavenging efficacy and suppressed inflammatory responses of macrophages in CpGstimulation conditions.
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REFERENCES
(1) Pisetsky, D. S. Autoimmun. Rev. 2008, 8, 69−72. (2) Barbalast, R.; Ewald, S. E.; Mouchess, M. L.; Barton, G. M. Annu. Rev. Immunol. 2011, 29, 185−214. (3) Kawai, T.; Akira, S. Semin. Immunol. 2007, 19, 24−32. (4) Marshak-Rothstein, A.; Rifkin, I. R. Annu. Rev. Immunol. 2007, 25, 419−41. (5) Lee, J. W.; Sohn, J. W.; Zhang, Y.; Leong, K. W.; Pisetsky, D.; Sullenger, B. A. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 14055−14060. (6) Filion, M. C.; Phillips, N. C. Br. J. Pharmacol. 1997, 122, 551− 557. (7) Yoo, H. S.; Kim, T. G.; Park, T. G. Adv. Drug Deliver Rev. 2009, 61, 1033−1042. (8) Pham, Q. P.; Sharma, U.; Mikos, A. G. Tissue Eng. 2006, 12, 1197−1211. (9) Kim, T. G.; Park, T. G. Biotechnol. Prog. 2006, 22, 1108−1113. (10) Storrie, H.; Mooney, D. J. Adv. Drug Deliver Rev. 2006, 58, 500− 514. (11) De Smedt, S. C.; Demeester, J.; Hennink, W. E. Pharm. Res. 1999, 17, 113−126. (12) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (13) Xu, P.; Van Kirk, E. A.; Zhan, Y.; Murdoch, W. J.; Radaxa, M.; Shen, Y. Angew. Chem. 2007, 46, 4999−5002. (14) Shoda, L. K. M.; Keaerreis, K. A.; Suarez, C. E.; Mwangi, W.; Knowles, D. P.; Brown, W. C. J. Leukocyte Biol. 2001, 70, 103−112. (15) Choi, J. S.; Yoo, H. S. J. Bioactive Compatible Polym. 2007, 22, 508−524. (16) Lee, J. H.; Lim, Y. B.; Choi, H. S.; Lee, Y.; Kim, T. I.; Kim, H. J.; Yoon, J. K.; Kim, K.; Park, J. S. Bioconjugate Chem. 2003, 14, 1214− 1221. (17) Germershaus, O.; Mao, S.; Sitterberg, J.; Bakowsky, U.; Kissel, T. J. Controlled Release 2008, 125, 145−154. (18) Clippard, P. H.; Taylor, R. C. J. Chem. Phys. 1969, 50, 1472. (19) Ku, S. H.; Park, C. B. Biomaterials 2010, 31, 9431−9437. (20) Rao, A. M.; Richter, E.; Bandow, S.; Chase, B.; Eklund, P. C.; Williams, K. A.; Fang, S.; Subbaswamy, K. R.; Menon, M.; Thess, A.; Samlley, R. E.; Dresselhaus, G.; Dresselhaus, M. S. Science 1997, 275, 187−191. (21) Kean, T.; Roth, S.; Thanou, M. J. Controlled Release 2005, 103, 643−653. (22) Zhang, J.; Duan, Y.; Wei, D.; Wang, L.; Wang, H.; Gu, Z.; Kong, D. J. Biomed Mater. Res. A 2011, 96A, 212−220. (23) Kim, H. S.; Yoo, H. S. J. Controlled Release 2010, 146, 264−271. (24) Sanders, J. E.; Lamont, S. E.; Karchin, A.; Golledge, S. L.; Ratner, B. D. Biomaterials 2004, 26, 813−818. (25) Chen, J. P.; Su, C. H. Acta Biobater 2011, 7, 234−243. (26) Choi, J. S.; Leong, K. W.; Yoo, H. S. Biomaterials 2008, 29, 587−596.
ASSOCIATED CONTENT
S Supporting Information *
1
H NMR spectrum of PCL−PEI block copolymer (Figure S1), the water contact angles (Figure S2), and the cell viability (Figure S3) with various blending ratio before and after 2605
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(27) Anne, K.; Simon, R.; Veit, H.; Bernd; Susan, B.; Zuhair, K. B.; Stefan, E.; Arthur, M. K.; Gunther, H. Eur. J. Immunol. 2001, 31, 2154−5163. (28) Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Biomed Mater. Res. 1999, 45, 268−275. (29) Krieg, A. M. Annu. Rev. Immunol. 2002, 20, 709−760. (30) Puangpetch, A.; Anderson, R.; Huang, Y. Y.; Sermswan, R. W.; Chaicumpa, W.; Sirisinha, S.; Wongratanacheewin, S. Clin. Vaccine Immunol. 2012, 19, 675−683. (31) Bianco, A.; Hoebeke, J.; Godefroy, S.; Chaloin, O.; Pantarotto, D.; Briand, J. P.; Muller, S.; Prato, M.; Partidos, C. D. J. Am. Chem. Soc. 2005, 127, 58−59. (32) Lee, S. H.; Iten, R.; Muller, M.; Spencer, N. D. Macromolecules 2004, 37, 8349−8356.
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Nucleic Acid-Scavenging Electrospun Nanofibrous Meshes for Suppressing Inflammatory Responses Jihyun Kang† and Hyuk Sang Yoo*,†,‡ †
Department of Medical Biomaterials Engineering and ‡Institute of Bioscience and Bioengineering, Kangwon National University, Chuncheon 200-701, Republic of Korea S Supporting Information *
ABSTRACT: Fragmented nucleic acids are potent stimulators for inflammatory responses provoking pathological outcomes by activating adaptive immunity. In this study, highly cationic surfaces were prepared on electrospun nanofibrous meshes to scavenge nucleic acids to the surfaces. Poly(ε-caprolactone) [PCL]-poly(ethylenimine) [PEI] block copolymers were synthesized by coupling the carboxyl-terminated PCL to the primary amines of branched PEI. Polymeric solutions composed of PCL−PEI and PCL were electrospun to nanofibrous mats, and the surfaces were further methylated to prepare highly cationic surfaces on the mats. Raman spectroscopy revealed that the presence of increased methylated amines on the surfaces of the mats compared to unmodified mats. The methylated surfaces showed significant increases of wettability after methylation, suggesting highly charged surfaces caused by methylation of the primary amines. When the blend ratio of PCL−PEI was increased, the scavenged DNA was also increased, and the methylation further strengthened the scavenging ability of the mats. Fluorescently labeled oligodeoxynucleic acids were significantly adsorbed on the surface of the mats depending on the amounts of PCL−PEI and the degree of methylation. In the presence of the methylated nanofibrous mats, inflammatory responses induced by CpG oligonucleotides in murine macrophages were significantly reduced, which was confirmed by measuring inflammatory cytokine levels including TNF-α and IFN-γ.
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INTRODUCTION
suppress synthesis of nitric oxide and tumor necrosis factor-α (TNF-α) in macrophages.6 Nucleic acid delivery systems employing electrospun nanofibrous mats have recently been studied with the aim of accomplishing gene transfection in local areas.7−9 Due to the extremely high surface-to-volume ratios of the nanofibrous structures, released nucleic acids have increased chances of contacting targeted cells for further transfection due to the large surface areas available for contact between cells and nanofibers. Similar to the strategy of colloidal gene carriers, which employ electrostatic interactions between nucleic acids and cationic polymers, cationic polymers or their derivatives are primarily employed in the nanofibrous gene carriers for fabricating the transfection matrix.10,11 Negatively charged nucleic acids are electrostatically incorporated into the matrix when the surface is oppositely charged with cationic polymers. For this purpose, polyethylenimine (PEI) in particular has received much attention for its great affinity toward nucleic acids, because of the highly cationic charge density along the chains.12,13 Branched or linear PEI has been extensively employed for preparing colloidal carriers for gene transfection because it
Fragmented nucleic acids from apoptotic cell death or foreign organisms have been recognized as inflammatory inducers, provoking undesirable pathological responses by activating innate immune cells such as macrophages, dendritic cells and neutrophils.1,2 Once recognized by nucleic acid-sensing toll-like receptors (TLRs) on the immune cells, extracellular fragments, such as ssRNA, dsRNA, and unmethylated CpG-containing DNA, subsequently stimulate cytokine production pathways for TNF-α, chemokines, and interferon, to develop adaptive immunity.3 Thus, scavenging those nucleic acids has been considered an effective means of controlling immediate or prolonged hyper-inflammatory responses from bacterial or viral infection and autoimmune diseases.4 This scavenging has mostly been performed by cationic polymers or lipids such as cyclodextrin-containing polycation (CDP), hexadimethrine bromide (HDMBr), protamine sulfate (PAMAM), and dioleyoltrimethylammonium propane (DOTAP), which inhibit the ability of synthetic CpG DNAs to activate nucleic acidsensing TLRs. Such cationic polymers are specific for neutralizing the immune-stimulatory activity of TLR agonists in a variety of primary cells, including B lymphocytes, fibroblasts, and dendritic cells.5 Cationic liposomes composed of cationic lipids mixtures were shown to electrostatically scavenge intracellular oligonucleotides and consequently © 2014 American Chemical Society
Received: March 25, 2014 Revised: May 30, 2014 Published: June 2, 2014 2600
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Figure 1. Surface-methylation of PCL−PEI nanofibrous meshes for scavenging nucleic acids causing inflammatory responses. (A) Synthetic scheme of preparing amine-terminated poly(ε-caprolactone)-block-branched polyethylenimine (PCL−PEI). Ring-opening polymerization of ε-caprolactone and octanoic acid generated a carboxylic acid-terminated PCL, which was subsequently reacted with the primary amines of PEI in the presence of NHS and DCC. (B) Iodomethane (CH3I) reacted with the surface-exposed amine of PCL−PEI nanofibrous meshes to give methyl groups and the iodide counterions were subsequently replaced with chloride ions to prepare methylation of PCL−PEI nanofibrous meshes. (C) Highly cationic meshes scavenge negatively charged nucleic acids to reduce nucleic acid fragments-induced inflammatory response of immune cells. GACGTTCCTGATGCT-3′) were purchased from Bioneer (Seoul, Republic of Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Trevigen, Inc. (Gaithersburg, MD). Dulbecco’s modified Eagle’s medium (DMEM), streptomycin/penicillin, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Enzyme-linked immunosorbent assay (ELISA) kits for mouse TNF-α and IFN-γ were purchased from eBioscience (San Diego, CA). Synthesis of PCL−PEI Block Copolymers. A block copolymer composed of PCL and PEI was synthesized by a three-step reaction as shown in Figure 1A. Briefly, ε-caprolactone (180 mmol) and octanoic acid (18 mmol) were put into a flask in nitrogen atmosphere, and Sn(Oct)2 (0.12 mmol) was subsequently injected. The polymerization reaction was carried out at 130 °C for 24 h and the reaction mixture was diluted with dichloromethane, precipitated in cold diethyl ether, and completely dried in vacuum. The carboxyl-terminated PCL [PCLCOOH] (82 μmol) was activated with N-hydroxysuccinimide (NHS) and 1,3-dicyclohexylcarbodiimide (DCC) in dichloromethane (250 mL) and at room temperature for 12 h (molar ratio of PCLCOOH:NHS:DCC = 1:5:5). The reaction mixture was filtered with a paper filter (11 μm), and the filtrates were precipitated in an excess amount of cold diethyl ether. The activated PCL (PCL-COO-NHS, 74.10 μmol) in chloroform (50 mL) was slowly added to branched PEI (0.22 mmol) in chloroform (5 mL) with gentle stirring and reacted at room temperature for 24 h (molar ratio PEI:PCL−COO− NHS = 3:1). The reaction mixtures were precipitated in ice-chilled methanol and completely dried in vacuum. The conjugation amount of PEI to PCL was confirmed by 1H NMR spectroscopy in CDCl3 at the
spontaneously forms an electrostatic complex between the basic polymers and the acidic nucleic acids. In the current work, we prepared nucleic acid-scavenging nanofibrous mats to eliminate excess amounts of nucleic acid fragments, and thus to suppress hyperimmune responses. A nanofibrous mat composed of PCL−PEI block copolymers was fabricated by electrospinning and further methylated to induce a highly cationic charge density on the surface. Since CpG oligodeoxynucleotides (ODN) have been reported to stimulate dendritic cells and macrophages to secrete cytokines, we examined whether the methylated nanofibrous mats can suppress the release of TNF-α and interferon (IFN)-γ from the ODN-stimulated macrophages.14
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MATERIALS AND METHOD
Materials. ε-Caprolactone (ε-CL) was purchased from Alfa-aesar (Ward Hill, MA). Octanoic acid was purchased from Junsei Chemical Co. (Tokyo, Japan). Tin(II)-ethylhexanoate (Sn(Oct)2), N-hydroxysuccinimide (NHS), 1,3-dicyclohexylcarbodiimide (DCC), flourescamine, and deoxyribonucleic acid (DNA) from salmon testes were purchased from Sigma-Aldrich (St. Louis, MO). Branched polyethylenimine (PEI, Mw 1.8 kDa) was purchased from Polysciences Inc. (Warrignton, PA). Iodomethane (CH3I) was purchased from Daejung Chemical Co. (Cheongwon, Republic of Korea). Murine macrophage cell line RAW 264.7 was obtained from ATCC (Manassas, VA). CpG oligodeoxynucleotide (ODN) (5′-TCCATGACGTTCCTGATGCT3′), and Texas red-labeled ODN (Texas red-5′-TCCAT2601
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Central Laboratory of Kangwon National University (DPX 400, Bruker). Electrospinning and Methylation of PCL/PCL−PEI Nanofibrous Mats. The polymeric solution (12%, w/v) of PCL or PCL− PEI mixtures in methanol/chloroform mixture (1:3, v/v) was electrospun to the ground at +15 kV with a flow rate of 1 mL/h through a 25 G needle. The amount of PCL−PEI block copolymer in the polymeric solution was varied from 50 to 100% (w/w), and the ground-to-needle distance was 15 cm. The PCL/PCL−PEI nanofibrous mats (NFs) were soaked in methanol for 3 h and hydrated in phosphate buffered saline for 30 min (PBS, pH 7.4) (Figure 1B). One hundred microliters of iodomethane (70 mg/mL in methanol) was added to the hydrated nanofibers and reacted at 37 °C for 48 h. The reaction was quenched with 0.5 M NaCl solution, and the methylated NFs were washed with deionized water and dried under vacuum. The amount of the surface-exposed primary amines on the PCL/PCL−PEI NFs before and after methylation was determined by a fluorescamine assay as described previously.15 Briefly, the PCL/PCL−PEI NFs (1 mg) were sequentially soaked in 30% ethanol and PBS (pH 7.4) for 15 s and 30 min, respectively. Fluorescamine in acetone (0.3 mg/mL) was added to the hydrated NFs and then reacted at room temperature for 30 min. After washing with methanol, the reacted NFs were completely dissolved into 1,4-dioxane to measure the fluorescence intensity. The excitation wavelength and the emission wavelength were 390 and 475 nm, respectively, and ethylenediamine was employed as a standard for primary amines. Characterization of the Methylated PCL/PCL−PEI Nanofibers. Methylated PCL/PCL−PEI NFs were characterized by using Raman spectroscopy and water-contact angle measurements. For Raman spectroscopy, NFs (1 mg/device) were placed on a sample holder and excited with an air-cooled diode-pumped solid state laser at 633 nm (Horiba Jobin Yvon, France). The scattered Raman spectrum was detected in the range of 600−4000 cm−1 by a charge-coupled device (CCD) camera. Five collections of accumulation times of 10 s were employed with a spectral resolution of 1 cm−1. In order to determine the wettability of the NFs, the sample (1 mg/device) was placed on a sample stage, and water (10 μL) was dropped on the surface of the NFs. After 1 s, the image of the droplet on the NFs was obtained by a digital camera and analyzed by Image-Pro v6.0 software to obtain the contact angles between NFs and the water droplets. The measurements were repeated three times, and the averaged values were obtained. Nucleic Acid-Scavenging Efficacy of PCL/PCL−PEI NF. The nucleic-acid scavenging efficacy of the methylated NFs was determined by employing salmon testes DNA as a model nucleic acid for 12 h. PCL/PCL−PEI NFs (1 mg/device) were prewetted with 30% ethanol, and 50 μg of DNA was added to prehydrated NFs in 1 mL of PBS (pH 7.4). The mixture was incubated at 37 °C in an orbital shaker, and the supernatant was subjected to DNA quantification at 260 nm at different time intervals within 12 h. In order to visualize the scavenged nucleic acids on the NFs, fluorescently labeled ODN was incubated with the NF and visualized by In Vivo Imaging System (IVIS). Presoaked NF was attached to a 12-well plate, and Texas red-labeled ODN (0.5 μg) in PBS (0.5 mL) was added. After gentle shaking at 37 °C for 12 h, the NFs were thoroughly washed with PBS three times. The fluorescent images of the NFs were obtained by accumulating fluorescence signals for 15 s at 580 nm/630 nm of the excitation and the emission wavelength, respectively, at Korea Basic Science Institute (KBSI) (IVIS 200, PerkinElmer, Waltham, MA, USA). Cytotoxicity of PCL/PCL−PEI NFs. The cytotoxicity of the NFs with various blend ratios of PCL−PEI was determined by a MTTbased cytotoxicity assay against macrophages. RAW 264.7 cell lines were cultured in DMEM with 10% (v/v) FBS and streptomycin/ penicillin and maintained at a humidified atmosphere of 5% CO2 at 37 °C. PCL/PCL−PEI NFs (1 mg) were presoaked in 70% ethanol solution for 30 min and washed with PBS three times. Cells (1 × 105 cells/well) were seeded on PCL/PCL−PEI NFs placed in a 12-well plate and then incubated in the culture medium (1 mL/well) at 37 °C for 12 h. Ten microliters of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 3 h. After removing the cell
culture medium, formazan crystals were dissolved with DMSO (1 mL) for 1 h, and the absorbance was measured at 570 nm. Cell viability was determined by dividing the absorbance of the cells on PCL/PCL−PEI NFs by those on TCPS. Determination of Anti-inflammatory Effects. The antiinflammatory effects of the NFs were determined by measuring cytokine levels released from macrophages. The RAW 264.7 cell line (1 × 105 cells/well) was placed in a 12-well culture plate and cultured in DMEM (1 mL) supplemented by 10% FBS and streptomycin/ penicillin for 12 h. The cells were replenished with fresh serum-free DMEM containing CpG ODN (50 μg/mL), and the NFs (1 mg/well) were placed on the monolayer of cells. The medium was collected for detection of TNF-α or IFN-γ after 6 or 24 h, respectively. Cytokine levels were quantified by ELISA kits in accordance with the manufacturer’s instructions, and the sensitivity was 4pg/mL for all cytokines. Briefly, 50 μL of the media and 50 μL of the biotinconjugate were added to a 96-well plate coated with the antibody against either TNF-α or IFN-γ. After 2 h, the sample was washed with a wash solution two times, and 0.1 mL of a streptavidin-Horseradish peroxidase (HRP) solution was added to each well and incubated at room temperature for 2 h. After washing each well twice with a wash solution, yellow color was developed with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate solution (0.1 mL) and a stop solution (0.1 mL). The absorbance at 450 nm was measured with a microplate reader, and the concentration of the cytokines was calculated by employing the cytokine solution in the kit as a standard (ELx800TM, BioTek Inc., Winnski, VT). Statistical Analysis. All data were evaluated with One Way Analysis of Variance (ANOVA) for the statistical significance, and the average values and standard deviations were also presented. P < 0.05 was considered statistically significant.
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RESULTS AND DISCUSSION Cationic surfaces of nanofibrous meshes were generated by employing block copolymers composed of PCL and branched PEI and subsequent methylation upon electrospinning of the copolymers (Figure 1). In our previous works, we synthesized diblock copolymers composed of PCL and amine-terminated PEG and fabricated polymeric nanofibrous mats by electrospinning.15 Various molecules were chemically conjugated to the surface-exposed amine group of the mat in aqueous phase and shown to perform significant roles as drug and gene delivery carriers. However, the amount of surface-exposed amine groups was not high enough to sequester nucleic acids fragments by electrostatic interactions (0.7 to 2.1 nmol/mg nanofibrous mat). In the current work, we synthesized PCL− PEI block copolymer by conjugating the primary amines of PEI to the terminal carboxylic acid of PCL and confirmed the conjugate by 1H NMR spectrum (Figure 1 and Figure S1), where the peaks at 1.42, 1.62, 2.34, and 4.09 ppm correspond to the proton of −(CH2)3−, −OCHH2−, and −CH2OOC− in the PCL units, respectively. 1H NMR indicated that the proton peaks near the secondary amines of PEI appeared at 3.65 ppm. By comparing the peak area of the secondary amines of PEI (3.65 ppm) to −CH2OOC− of PCL (4.09 ppm), we speculated that one molecule of PCL had 0.5 molecule of PEI. Then, the block copolymer, at various blend ratios to PCL, was subsequently electrospun to fibrous mats, and the primary amine groups of the mats were methylated in aqueous phase to replace the primary amine with secondary or tertiary amines (Figure 1B). We expected that the methylation of the primary amine groups would strengthen the cationic charge density on the surface because the basicity of amines tends to be elevated in the order of primary, secondary, and tertiary amines.16,17 Thus, the methylated NFs can scavenge nucleic acid fragments because of stronger electrostatic interactions between the NF 2602
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surfaces and the nucleic acids, and the inflammation responses of the immune cells can be further attenuated (Figure 1C). The degree of methylation was determined by quantifying the residual primary amines of the methylated NFs (Figure 2).
Figure 3. Raman spectra of (A) PCL NF, (B) PCL−PEI NF (0/100,) and (C) methylated NFs (0/100). The significant peaks at 3000 cm−1 correspond to υCH2 at 2918, 2866 cm−1; CO at 1727, 1030 cm−1; δCH2 at 1421, 1444, 1469 cm−1, and ωCH2 at 1286, 1308 cm−1 of PCL.
Figure 2. Surface-exposed amine groups on PCL/PCL−PEI NFs before and after methylation (bars) at various blend ratios of PCL to PCL−PEI and the methylation efficiency (line). The blend ratio of PCL (x) to PCL−PEI (y) is presented in x/y (w/w) (n = 4). Asterisk (*) indicates statistical significance (p < 0.05). *ND: the primary amines were not determined because no PEI exists on the surface of PCL NF.
Among the primary, secondary, and tertiary amines of branched PEI, the secondary amines show higher basicity (Kb = 5.4 × 10−4 M) compared to the primary amines (Kb = 4.4 × 10−4 M) and consequently contribute strong electrostatic interactions, because of the effects of alkyl groups on the amine.21 Thus, electrostatic interactions between PEI and nucleic acids can be manipulated by introducing substitute groups of the amine. For example, plasmid DNA was electrostatically incorporated into a polymeric matrix composed of poly(ethylene glycol)−polyethyleneimine [PEG−PEI].22 While the incorporation of DNA by pure PCL fibers was less than 15%, the incorporation of DNA by PCL/PEG−PEI fibers reached the maximum (50%) within 30 min. In another study, DNA was electrostatically incorporated on the electrospun nanofibrous matrix at various charge ratios of DNA to the immobilized LPEI. The incorporation efficiency of the DNA increased significantly, from 17.4% to 79.5%, when N/P ratios were increased from 2 to 16.23 In order to confirm the effects of methylation on the surfacewettability of the NF, we determined the hydrophilicity of the methylated NF by measuring the water contact angles of PCL/ PCL−PEI NFs (Table 1). At the same blend ratio of PCL− PEI, the contact angles of the methylated NFs were dramatically decreased. Notably, PCL−PEI NF showed the most dramatic contact angle decrease, of 83.05°, from 93.35 ±
After methylation of the PCL/PCL−PEI NFs, the amount of primary amine groups decreased significantly, in accordance with the increased blend ratio. The primary amine groups of PCL/PCL−PEI NFs were expected to be methylated from 50.5 to 87.5% for all blend ratios. PCL−PEI NF with a blend ratio of 0/100 showed that the amount of primary amine groups was 1.96 ± 0.25 nmol per device, suggesting that 50.5 ± 1.80% of the surface-exposed primary amine groups were substituted with secondary or tertiary amines, compared to the initial amount on the same NFs (4.14 ± 0.41 nmol/device). The degree of substitution efficiency was gradually increased when the blend ratio reached 50/50. We speculate that steric hindrance of the bulky PEI chains can limit the reaction between the primary amines and methyl iodide and subsequently decrease the methylation efficiency. Thus, this result clearly suggests that an optimized amount of surfaceexposed PEI is required to obtain high degrees of substitution to methylated PEI moieties on the NFs. However, it should also be noticed that the 0/100 NF showed the highest degree of methylated amines because the amount of PCL−PEI in the NFs is the highest among the other NFs. We also surveyed the methylated surface of the NFs by Raman spectroscopy to observe methyl groups on the surface (Figure 3). After methylation of the NFs, a new absorption peak appeared at 3000 cm−1, which indicates the amine component of methyl groups, in comparison with the characteristic peaks of PCL, such as υCH2 at 2918, 2866 cm−1; υCO at 1727 cm−1; δCH2 at 1421, 1444, 1469 cm−1; ωCH2 at 1286, 1308 cm−1; and skeletal stretching at 1112 cm−1.18,19 Based on the Raman spectrum of N(CH3)3 in the methylated NFs, we can speculate that methylation occurred at the surface of the nanofibers because methylation of the NF was performed after electrospinning. However, we could not estimate the number of secondary or tertiary amines on the methylated NF because the spectrum is not quantitative.20
Table 1. Contact Angles of PCL/PCL−PEI NFs with Various Blend Ratios before and after Methylationa water contact angle (deg) PCL/PCL−PEI NF
before methylation
after methylation
PCL NF 50/50 25/75 0/100
139.74 ± 5.33 102.57 ± 1.50 102.11 ± 5.86 93.35 ± 1.46
106.10 ± 2.94 21.57 ± 4.04 13.00 ± 5.89 10.30 ± 6.22
The water contact angles were measured after 1sec of dropping 10 μL of water on the NFs (mean ± SD, n = 4).
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1.46° to 10.30 ± 6.22°. The extent of contact angle decrease gradually diminished as the blend ratio of PCL−PEI decreased from 50/50 to 0/100. In fact, the water droplet was instantly absorbed into the methylated NF after only 1s compared to the unmodified NFs (Figure S2, Supporting Information). Many studies have previously modified charge-densities on the surface to manipulate the hydrophobicity or hydrophilicity of polymeric surfaces.24,25 Thus, this result also suggests that the surface-exposed amines were successfully converted into electrostatically stronger cations such as secondary or tertiary amines. Figure 4 shows the nucleic acid-scavenging efficiency of the methylated NF in comparison to the unmethylated one. All
comparison to the unmethylated counterparts. The scavenging efficiency of the NF was gradually increased in all NFs containing PCL−PEI and became saturated after 12 h (data not shown). Thus, we concluded that the nucleic acids are scavenged by the NF in a time-dependent manner. We also fluorescently visualized the scavenged ODN on the NF with IVIS, and it indicated the high retention of ODN on the thoroughly washed surface of the NFs (Figure 4B). The fluorescence intensities on the NF gradually increased when the blend ratio of PCL−PEI increased, and the methylated NFs always showed higher intensities compared to their counterparts. This result accordingly suggests that the scavenging efficiency of DNA is proportional both to the amounts of PCL−PEI and the methylation, as shown in Figure 4A. Thus, we speculate that these meshes can be applied to any biological surfaces and will locally reduce excessive amounts of nucleic acids. We previously showed that the PCL NFs had the effect of producing well-contouring wound sites due to the high surface to volume ratios of the nanoporous meshes.26 Furthermore, considering that the topically administered NFs can be easily removed after scavenging unwanted nucleic acids on any biological surfaces, the NF can potentially be employed as a “molecular-sponge” to reduce inflammation responses in a limited area. In order to investigate the cytotoxicity of the methylated nanofibers on macrophage, the MTT assay was used (Figure S3). The cell viability of the methylated nanofibers was increased from 45.1% to 61.0% at 50/50 for 12h. We only monitored the viability for 12 h because most cytokines has been reported to be released within 12h.27 In fact, poly(εcaprolactone) (PCL) nanofibers have limited cellular affinity due to their high hydrophobicity. Surface modification by methylation can overcome that limitation because the increasing amount of PCL−PEI can increase cell adhesion and can reduce PCL hydrophobicity. However, the cytotoxicity of PCL−PEI NFs can be influenced, depending on the amount of PCL−PEI, by the strong cationic charge of PEI. The cytotoxicity of PEI depends on its molecular weight and it is accepted that PEI with a molecular weight of 1.8 kDa shows cytotoxicity.28 As shown in Figure 5, we investigated the anti-inflammation efficacy of the methylated NFs by quantifying major inflammatory cytokines such as TNF-α and IFN-γ in the macrophages in the presence of CpG ODN. Because CpG ODN has been considered to be a potent inflammatory stimulator against various immune cells such as macrophages, the in vivo condition for anti-inflammation efficacy can be simulated and tested with the ODN and macrophages mixtures in the presence of various NFs.29−31 Upon addition of various NFs to the ODN and macrophages mixtures, the cytokine releases were considerably attenuated, as shown in Figure 5. The most dramatic results were obtained from the methylated NF, where both TNF-α and IFN-γ were below detection limits by ELISA assay. Unmethylated NF also greatly suppressed TNF-α and IFN-γ release from the macrophages, by 8.42% and 0.50% for TNF-α and IFN-γ compared to the untreated group. Thus, we can speculate that the nucleic acids were electrostatically scavenged by the NFs, and the macrophages were not sufficiently stimulated by CpG ODN to show inflammatory responses. It should be of noticeable that the ELISA kit could not detect TNF-α and IFN-γ production for the macrophages without addition of nucleic acids for 12 h.
Figure 4. DNA scavenging efficacy of methylated or unmodified PCL/ PCL−PEI NFs. (A) DNA scavenging efficiency of the NFs in DNA solution (50 μg/mL) for 12h. The unincorporated DNA was quantified at 260 nm and calculated for the scavenging efficiency (the amounts of the scavenged DNA/total amounts of DNA) (mean ± SD, n = 4). All data in the figures have statistical significance (p < 0.05). (B) Scavenged nucleic acids on the NFs. Fluorescently labeled ODN was incubated with the PCL/PCL−PEI NF and visualized by In Vivo Imaging System (IVIS).
NFs exhibited increasing scavenging effects with increasing amounts of PCL−PEI in the NF from 50/50 to 0/100. The increased amounts of PEI obviously played a pivotal role in the scavenging of any nucleic acids onto the NFs; the higher densities of cationic PEI moieties on the surface of the NFs are electrostatically favorable for scavenging anionic nucleic acids. These cationic charges are dramatically strengthened by methylation and, in fact, the measured scavenging efficiency showed a several fold increase, ranging from 8% to 40% in 2604
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methylation. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]; tel: +82-33-250-6563; fax: +82-33-253-6560; website: http://nano-bio.kangwon.ac.kr. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a grant from the National Research Foundation (NRF) (NRF-2012R1A2A2A01005857) and partly by Kangwon National University.
Figure 5. Suppression of inflammatory responses of RAW 264.7 cells with PCL NF, PCL−PEI NF (0/100), and methylated NFs (0/100). RAW 264.7 cells (1 × 105) were stimulated with CpG oligonucleotides (50 μg/mL) in the presence of NFs, and the released amount of TNFα and IFN-γ in the culture supernatant was quantified by ELISA (n = 4). The black bars and the gray bars indicate the levels of TNF-α and the IFN-γ, respectively. Asterisk (*) indicates statistical significance (p < 0.05). *ND: TNF-α or IFN-γ was not determined because level of cytokine was below the detection limits.
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Additionally, it should be noted that PCL NF also suppressed TNF-α and IFN-γ release from the macrophages, because PCL NF only scavenged minimal amounts of the nucleic acids in Figure 4A. This can be attributed to the difference in incubation conditions: in the nucleic acidscavenging test, we employed vigorous shaking to minimize nonspecific binding of nucleic acids to the NFs. However, for the cell cultivation, to minimize cell detachment from the surfaces, we could not use such a vigorous condition. Thus, nonspecific interactions between NFs and nucleic acids could be further increased, and many more nucleic acids can be adsorbed on the surfaces of PCL NF in the cell culture experiments. In fact, the electrospun nanofibers feature a high surface-to-volume ratio, which can be advantageous for the surface-functionalization of biomolecules.32 In our group, we previously showed that electrospun fibrous meshes can be applied to potent wound dressing candidates in aims to reduce undesirable changes of cellular phenotypes. Surface-functionalized nanofibrous meshes showed high wound healing efficacy as well as superior prognosis in comparison to unmodified meshes. Thus, in combination with the current study, the cationic nanofibers are expected to contribute to controlling undesirable inflammatory responses, which leads to a faster and efficient wound healing process.
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CONCLUSION PCL/PCL−PEI NFs were methylated to increase cationic charges on their surfaces, and the methylated NF showed high wettability compared to unmethylated NFs. The methylated NFs showed high nucleic acid-scavenging efficacy and suppressed inflammatory responses of macrophages in CpGstimulation conditions.
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ASSOCIATED CONTENT
S Supporting Information *
1
H NMR spectrum of PCL−PEI block copolymer (Figure S1), the water contact angles (Figure S2), and the cell viability (Figure S3) with various blending ratio before and after 2605
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