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Article Cite This: ACS Omega 2019, 4, 7391−7399
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Coating Magnetic Nanospheres with PEG To Reduce Nonspecific Adsorption on Cells Xiao-Juan Dong,† Zhi-Ling Zhang,† Ling-Ling Wu,† Xu-Yan Ma,† Chun-Miao Xu,† and Dai-Wen Pang*,†,‡ †
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Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P. R. China ‡ College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China S Supporting Information *
ABSTRACT: With the rapid advancement of nanobioscience, the interaction between nanomaterials and cells has become increasingly important, among which, understanding the nonspecific adsorption (NSA) between nanomaterials and cells is beneficial to the comprehension of nano−bio interactions. Herein, magnetic nanospheres (MNs) modified with different types of poly(ethylene glycol) (MNsPEG) were used as models to systematically study the NSA between nanomaterials and cells. By exploring different surface properties of MNs modified with PEG, we found that the NSA between MNs and cells was greatly reduced after the modification of MNs-COOH with a long-chain (MW: 10 000), brush-conformational PEG, the extremely low NSA rate of which (less than 5%) was nearly one-fifth that of unmodified ones. Additionally, PEG modification improved the colloidal stability and adsorption uniformity of MNs to cells and remarkably reduced the number of adsorbed white blood cells (WBCs) in circulating tumor cell detection from complex blood samples. Surprisingly, the number of adsorbed WBCs by immunomagnetic nanospheres (IMNs) was 17 times more than that of PEGylated IMNs (IMNs-PEG). In a word, this work provides some perspective for the study on nanobio interactions and will be helpful to the construction and surface modification of low-adsorption nanomaterials.
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INTRODUCTION Nanomaterials have been widely used in life science over the past decades, such as cell imaging, drug delivery, fluorescence labeling, molecular recognition, and so forth.1 With the increasing advancement of nanobiomedicine, the interaction between nanomaterials and cells, tissues, or organisms, called nanobio interaction, is emerging as a rapidly growing scientific frontier.2,3 Among them, exploring the nonspecific adsorption (NSA) between nanomaterials and cells is beneficial to the comprehension of nanobio interaction and contributes to the biological application of nanomaterials.4 However, research studies on NSA to date have commonly concentrated on proteins adsorbed on substrates or nanomaterials called “protein conara”,5,6 and few of them focused on NSA between nanomaterials and cells. Hence, the fundamental understanding of kinetics, thermodynamics, or molecular mechanism of NSA between nanomaterials and cells, which is needed for the next decade, is yet to be achieved.7 Magnetic nanomaterial is one of the most commonly used materials in nanobiomedicine. Because of the unique magnetic properties, it has been widely used in targeted drug delivery, magnetic resonance imaging, magnetic hyperthermia therapy and biosensing, and so forth.8,9 Especially, as a rapid biological separation tool, magnetic nanomaterial has been widely used to © 2019 American Chemical Society
enrich tumor cells, bacteria, viruses, proteins, nucleic acids, and so on.10−12 Among them, one of the distinctive applications we have studied for years is magnetic nanosphere (MN)-based circulating tumor cell (CTC) detection.13−16 In our previous works, MNs were fabricated, modified, and then applied to CTCs enrichment because of their excellent properties such as faster magnetic response, stronger stability, and easier biofunctionality than magnetic nanoparticles (MNPs). However, when magnetic separation is performed to enrich CTCs, which are extremely rare (1−100/mL) in the blood of early cancer patients, it is inevitable for MNs to adsorb white blood cells (WBCs), which are abundantly present (106 to 107/mL). As a result, the purity of CTCs enriched by magnetic separation is greatly reduced undesirably, and the ubiquitous NSA of nontarget cells will greatly impact the sensitivity and accuracy for biosensing.17 On the one hand, literatures18 have reported that physicochemical properties of nanomaterials such as shape, size, surface charge, and hydrophilicity affected their protein adsorption performance. On the other hand, modifications Received: January 27, 2019 Accepted: March 22, 2019 Published: April 24, 2019 7391
DOI: 10.1021/acsomega.9b00245 ACS Omega 2019, 4, 7391−7399
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Scheme 1. Schematic Diagram for the NSA between MNs and Cells before or after PEG Modification
Figure 1. (A−C) NSA rates of MNs and MNs-PEG to Jurkat T cells. (A) MNs-COOH modified with NH2−PEG5000−NH2 and MNs-NH2 modified with HOOC−PEG5000−COOH. (B) MNs-COOH modified with NH2−PEG2000−NH2 and NH2−PEG2000−COOH. (C) MNsCOOH modified with NH2−PEG3−OH and NH2−PEG3−OCH3. (D) Mass of PEG immobilized on MNs at different reaction concentrations (0.5, 2.5, 10, 25 mg/mL) of NH2−PEG2000−NH2. (E) NSA rates of MNs-COOH treated with different concentrations of NH2−PEG2000−NH2. (F) MNs-COOH modified with different chain lengths (MW: 2000, 5000, 10 000) of NH2−PEG−NH2. (The reaction concentration of PEG unmarked was 10 mg/mL. Compared with MNs-COOH, *P < 0.05, **P < 0.01.)
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RESULTS AND DISCUSSION Surface Properties of MNs to Cell Adsorption. According to our previous work,23 MNs-COOH were fabricated by assembling five layers of MNPs on poly(styrene/acrylamide) copolymer nanospheres followed by coating with a silica layer outside and further modified with succinic anhydride to fortify their biocompatibility and stability in complex matrices. PEG-modified MNs (MNs-PEG) were all prepared by carbodiimide chemistry, and their characterizations are presented in Figures S1 and S2. To investigate how the surface properties of MNs affect their NSA to cells, the NSA rates of MNs modified with different types of PEG were obtained. Here, we explored the effect of surface charge of MNs, hydrophobicity of the PEG terminal group, surface coverage, and chain length of modified PEG on cell adsorption of MNs. The statistical data for cell adsorption are provided in Figure 1. Surface Charge of MNs. MNs-COOH were prepared with a remarkably negatively charged surface (−49.1 mV), and MNs-NH2 were obtained with an obviously positively charged surface (+47.2 mV) by coating MNs-COOH with PEI-750k
have been carried out to repel the nonspecific protein adsorption on substrates,19,20 especially the “gold-standard” poly(ethylene glycol) (PEG) modification because of chain flexibility and high hydrophilicity.21,22 However, to date, how to effectively resist the NSA between nanomaterials and cells especially in magnetic separation still remains to be tackled and how the surface properties of nanomaterials affect their cell adsorption performance is still unclarified. In this work, MNs were modified with different types of PEG to explore how surface properties affect their NSA to cells, such as the surface charge of MNs, hydrophilicity of the PEG terminal group, chain length, and surface coverage of PEG on MNs. The NSA between MNs and cells was quantitatively described with the NSA rate to cells in magnetic separation and the average number of MNs adsorbed on per cell, and the interaction between MNs and cells before or after PEG modification are depicted in Scheme 1. Additionally, the thermodynamics of NSA between MNs and the Jurkat T cell was studied. Finally, we applied the unmodified and modified MNs to complex blood samples to evaluate the NSA of WBCs in magnetic separation-based CTC detection. 7392
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Figure 2. (A) UV probes of PEG and EDC. (B) Proportional relationship of absorbance at 563 nm vs the concentration of RB−PEG10000−NH2. (C) Mass of RB−PEG10000−NH2 immobilized on MNs at different reaction concentrations of PEG. (D,E) TEM image (D) and size distribution histogram (E) of MNs. (F) Cartoon representative PEG in brush (a) and mushroom (b) conformation on MNs.
lated by determining the mass of PEG immobilized on MNs (Figure 1D) according to Supporting Information S1. As a result, the surface coverage at four different reaction concentrations of PEG on MNs were calculated as 0.58 × 104, 1.96 × 104, 6.12 × 104, and 7.04 × 104 PEG chains per square nanometer (PEG/nm2). Notably, the more dosage of PEG was used, the larger surface coverage of PEG on MNs was achieved, consequently, the lower NSA rate of MNs to cells was realized (Figure 1E). As described by the steric effect,29−31 when high coverage of PEG was achieved on MNs, compression of the chain destroyed its free extension state and thus created steric repulsion to resist NSA. Chain Length of PEG. MNs-COOH were modified with different chain lengths (MW: 2000, 5000, 10 000) of NH2− PEG−NH2 to gain MNs-PEG2000, MNs-PEG5000, and MNsPEG10000. The NSA rates of MNs and MNs-PEG are demonstrated in Figure 1F, from which the longer chain NH2− PEG−NH2 was modified to MNs, the lower NSA rate was achieved (MNs-PEG10000 < MNs-PEG5000 < MNsPEG2000). Moreover, the lowest NSA rate (less than 5%) was realized when modifying MNs with NH2−PEG10000− NH2. The effect of the PEG chain length32 on cell adsorption was related to the “water layer” theory and steric effect, that is, the long PEG chain on MNs form a thick hydrophilic layer and large steric repulsion on their surface to repel adsorption. To sum up, the NSA between MNs and cells was influenced by the surface charge of MNs, hydrophilicity of the PEG terminal group, surface coverage, and chain length of PEG on MNs. Furthermore, MNs for low NSA to cells were negatively charged, coated with the hydrophilic terminal, and long-chain PEG with high coverage on their surface. Among them, MNsPEG10000 presented an extremely low NSA rate, which were subsequently used for low cell adsorption in CTC detection. PEG Quantification and Conformation on MNs. PEG chains on a surface can acquire “mushroom” or “brush” conformation,33 which is based on the comparison of the Flory radius (RF) and the distance between PEG grafts (D). Mushroom conformation refers to a low-density PEG coverage (D RF), while brush conformation refers to densely packed PEG chains (D RF). RF and D were calculated according to Supporting Information S2,S3. As shown in Figure 2A,B, RB−
(Figure S1A), while MNs maintained the same kind of surface charge before or after PEGylation. As shown in Figure 1A, the NSA rates of negatively charged MNs were much lower than those of positively charged ones. Most surprisingly, when the surface was negatively charged, PEG modification was helpful to reduce cell adsorption of MNs, but no reduction was achieved to positively charged ones. According to the electrostatic interaction theory,24,25 MNs with the negatively charged surface tend to present low cell adsorption because of the electrostatic repulsion between both negatively charged MNs and cells. Even if MNs-COOH were modified with different surface-charged PEG (NH2−PEG−NH2 or NH2− PEG−COOH), their final surface charges were still negative (Figure S1B), while the NSA rates of both MNs-PEG were much less than the unmodified ones and the two differed little (Figure 1B). Thus, the surface charge of MNs was vital to cell adsorption, instead of the surface charge of the modified PEG terminal group. Hydrophilicity of the PEG Terminal Group. To avoid the influence of the long hydrophilic region of the PEG chain, NH2−PEG3−OH and NH2−PEG3−OCH3 were used to investigate the effect of hydrophilicity of the PEG terminal group on cell adsorption performance of MNs.26 As shown in Figure 1C, the NSA rate of MNs-COOH modified with hydrophilic terminal NH2−PEG3−OH (MNs-PEG3−OH) was greatly reduced, but no reduction was realized when they were modified with hydrophobic terminal NH2−PEG3− OCH3 (MNs-PEG3−OCH3). According to the “water barrier” theory27,28 (as well as hydrophobic interaction theory), MNsPEG3−OH with a hydrophilic surface generated a water layer around them in aqueous solution and formed an energetic and physical barrier to prevent adsorption. On the contrary, MNsPEG3−OCH3 with a more hydrophobic surface exhibited larger surface energy in aqueous solution and tended to be adsorbed to reduce the Gibbs free energy on their surface. Therefore, the modified PEG with a hydrophilic terminal group was beneficial to resist cell adsorption of MNs. Surface Coverage of PEG on MNs. The reaction concentration of PEG directly affects its coverage on MNs. The surface coverage of PEG chains on MNs at different reaction concentrations of NH2−PEG2000−NH2 was calcu7393
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Figure 3. (A) Schematic diagram for the quantification of MNs adsorbed on the cell. (B) Proportional relationship of absorbance at 400 nm vs the concentration of MNs. (C) Number of MNs and MNs-PEG10000 absorbed on per cell. (D) Thermodynamics of NSA between MNs and the Jurkat T cell at 25 °C (green), 37 °C (blue), and 50 °C (red) simulated by the Langmuir isotherm equation. (E) Linear fitting of ΔG to temperatures.
Figure 4. (A) Schematic diagram for NSA by magnetic separation. (B) NSA rates of Jurkat T cells by six batches of MNs and MNs-PEG10000.
CV = 2.7%). A3−A4 was the absorbance of MNs adsorbed on cells, which was used to calculate the number of MNs adsorbed on the cell. As a result, 0.504 μg of MNs was adsorbed on 5 × 104 cells, which is 192 MNs adsorbed on per cell on average. By the same method, the number of MNs-PEG10000 on per cell was estimated as 40, which was nearly one-fifth that of MNs (Figure 3C). In conclusion, PEGylation can effectively reduce the adsorption of MNs to the cell. Adsorption Thermodynamics. The thermodynamics of NSA between MNs and cells was studied in Figure 3D, where the equilibrium adsorption amount (Qe) and equilibrium concentration (Ce) of MNs fitted well with the Langmuir equation35,36 at different temperatures (25, 37 and 50 °C). Furthermore, the parameters of the Langmuir equation at different temperatures are listed in Table S1, suggesting that the higher the incubation temperature, the more the MNs adsorbed on the cell. Because of the extremely low nonspecific cell adsorption, the change in absorbance, before and after MNs-PEG were treated with cells, was undetectable, so we did not find out any law of MNs-PEG. Moreover, the Gibbs free energy change (ΔG), enthalpy change (ΔH), and entropy change (ΔS) were verified according to the van’t Hoff equation in the Supporting Information (Figure 3E), indicating adsorption between MNs and cells was spontaneous, exothermic, and entropy-increasing. Cell Adsorption Performance of MNs and MNs-PEG. The cell adsorption performance of six batches of MNs and MNs-PEG10000 was evaluated by NSA rates as illustrated in Figure 4A. The NSA rates of MNs prepared with the same
PEG10000−NH2 displayed a high absorption peak at 563 nm, and the absorbance here exhibited excellent linear relationship versus the concentration of RB−PEG10000−NH2 (R2 > 0.9999). Based on this, the PEG immobilized on MNs was quantified by comparing the absorbance of RB−PEG untreated and treated with MNs after magnetic separation. The optimal reaction concentration of PEG was 4 mg/mL (Figure 2C) and the diameter of MNs was 340 ± 20 nm (Figure 2D,E). The mass of PEG immobilized was 0.069 mg, thus, 1.16 × 106 PEG chains were immobilized on each MN. RF and D were eventually determined as 90.7 and 6.31 ± 0.37 Å respectively (D < RF), where PEG chains were brush conformation aligning on MNs visually illustrated in Figure 2F(a). As reported, the beneficial effects of PEG modification such as protein and cell resistance occur when brush conformation was achieved on a surface rather than mushroom conformation.34 Number of MNs and MNs-PEG Adsorbed on Cells. The method of quantifying the number of MNs adsorbed on cells is illustrated in Figure 3A based on the excellent linear relationship of absorbance at 400 nm versus the concentration of MNs (Figure 3B). Briefly, the absorbance of 5 × 104 Jurkat T cells in 200 μL 1× PBS at 400 nm was first measured (A1 = 0.143), followed by the absorbance of cell suspension after passing through a filter with a pore size of 0.8 μm (A2 = −0.004), indicating cells could hardly pass through the filter. Afterward, the absorbance of MNs (0.01 mg) passed through the filter was monitored (A3 = 1.096 ± 0.012, CV = 1.6%), then the absorbance of MNs treated with cells followed by passing through the filter was measured (A4 = 1.057 ± 0.015, 7394
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Table 1. Cell Adsorption Uniformity Analysis of MNs and MNs-PEG10000 group MNs MNs-PEG
NSA rates of six batches of MNs and MNs-PEG10000 (%) 34.2 6.9
52.6 4.0
21.9 3.6
44.4 8.6
32.8 0.7
18.0 4.1
mean
range
standard deviation
mean deviation
34.0 4.6
34.6 7.9
13.1 2.8
9.7 2.1
Figure 5. (A−F) Hydrodynamic sizes (A−C) and PDI (D−F) of MNs and MNs-PEG10000 in FBS, plasma, and blood. (G,H) Microscopic images of RMNs (G) and RMNs-PEG10000 (H) in the human whole blood.
and the more complex the sample was, the worse the colloidal stability of MNs was monitored. Additionally, the microscopic images of red fluorescent MNs (RMNs) and PEG-modified RMNs (RMNs-PEG) in blood also implied that RMNs aggregated seriously, but RMNs-PEG monodispersed well (Figure 5G−H). Therefore, PEG modification improved the colloidal stability of MNs and RMNs in complex samples. Cell Adsorption of MNs. To investigate the cell adsorption performance of MNs with different modifications, the NSA studies of MNs-COOH, MNs-PEG10000, MNs-IgG, and MNs-PEG-IgG were performed in 1× PBS, and their zeta potentials were presented in Figure S3. As shown in Figure 6A, the NSA rate of MNs-COOH was nearly 25%, which was decreased (less than 10%) after PEG or IgG modification, and further reduced after both PEG and IgG modification (nearly 3%), indicating it was possible to greatly reduce cell adsorption of MNs by PEGylation and biofunctionalization with the antibody. It is known that CTCs identification in the peripheral blood is significant for the diagnosis and treatment of early cancer,
method were dramatically different, but the difference was greatly decreased after the modification of MNs with NH2− PEG10000−NH2 (Figure 4B). The adsorption uniformity of MNs and MNs-PEG10000 to cells was further discussed in Table 1. Notably, the mean, range, standard deviation, and mean deviation of NSA rates of MNs-PEG10000 were much less than those of MNs. These results demonstrated that PEG modification can not only reduce cell adsorption of MNs but also improve their adsorption uniformity by unifying the surface chemistry. Colloidal Stability of MNs and MNs-PEG in Complex Matrices. Aggregation in the matrix limits the biological applications of nanomaterials, thus, it is crucial to improve their colloidal stability and maintain the monodispersity in complex matrices. Our MNs exhibited high uniformity and good monodispersity in water but not in complex matrices such as fetal bovine serum (FBS), plasma, and blood as shown in Figure 5A−F. The hydrodynamic sizes and polydispersity index (PDI) indicated that MNs-PEG10000 maintained better monodispersity and stability in complex matrices than MNs, 7395
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CTC detection. As shown in Figure 6D, the number of adsorbed WBCs by IMNs-PEG was commonly less than IMNs. Even if IMNs showed high adsorption to WBCs (3#), the adsorbed WBCs could be greatly reduced by PEGylation, and the number of WBCs adsorbed by IMNs was 17 times more than that of IMNs. Besides, the number of spiked and captured MCF-7 cells with four batches of IMNs and IMNsPEG are listed in Table 2. It is noticed that whether IMNs were modified with PEG or not, and they could capture nearly all spiked CTCs by magnetic separation. These results demonstrated that when efficient capture of CTCs was ensured under magnetic separation, PEG modification of IMNs could effectively reduce the NSA of WBCs in the blood, which in other words, improved the purity of enriched CTCs. Therefore, PEG modification benefits a lot to reduce NSA of MNs in CTC enrichment from complex blood samples.
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CONCLUSIONS In summary, we had successfully modified PEG on MNs with brush conformation and systematically explored the effect of the surface properties such as surface charge of MNs, hydrophilicity of the PEG terminal group, the chain length, and surface coverage of modified PEG on cell adsorption by modifying different types of PEG on MNs. The results suggested that modifying MNs-COOH with the hydrophilic terminal, long-chain and brush conformational PEG was beneficial to low NSA, which is helpful to the construction or surface modification of low-adsorption nanomaterials. We evaluated the NSA of MNs and MNs-PEG by comparing the NSA rates and number of nanospheres adsorbed on per cell, which demonstrated that PEG modification greatly reduced the cell adsorption of MNs. Besides, the colloidal stability and adsorption uniformity of MNs were remarkably improved by PEGylation, and the NSA of WBCs by IMNs-PEG in CTC detection from complex blood samples was also greatly reduced. Overall, we expect this work does some contributions to the study on nanobio interaction and low NSA in CTC detection.
Figure 6. (A) NSA rates of MNs, MNs-PEG, MNs-IgG, and MNsPEG-IgG in PBS. (B) Number of absorbed WBCs by MNs and MNsPEG in the human whole blood. (C) Capture efficiencies of MCF-7 cells and adsorption rates of Jurkat T cells by IMNs and IMNs-PEG in 1× PBS. (D) Number of absorbed WBCs by IMNs and IMNs-PEG in mixed blood samples with spiked MCF-7 cells.
but it is inevitable to adsorb the abundant WBCs (106 to 107/ mL) when magnetic separation is performed to enrich the extremely rare CTCs (1−100/mL). Hence, to further explore the cell adsorption performance of MNs in the blood, the NSA studies of MNs and MNs-PEG were first performed in 1× PBS, 10% FBS, FBS, and plasma (Figure S5), which suggested that the protein adsorbed on MNs in complex matrices was beneficial to reduce cell adsorption, and MNs-PEG maintained lower adsorption to cells than MNs. Then, MNs and MNsPEG were treated with the human whole blood and the number of adsorbed WBCs was counted in Figure 6B. Consequently, the number of adsorbed WBCs by three batches of MNs-PEG was less than the unmodified ones. Even if the unmodified MNs exhibited high adsorption to WBCs (3#), the adsorbed WBCs could be greatly reduced by PEGylation, and the number of WBCs adsorbed by MNs was even 14.5 times more than that of MNs-PEG. Capture and Adsorption Study of Immunomagnetic Nanospheres. To ensure the efficient capture of target cells and low adsorption to nontarget cells, we compared the capture efficiencies and adsorption rates of immunomagnetic nanospheres (IMNs) and PEG-modified IMNs (IMNs-PEG) in 1× PBS. It could be seen in Figure 6C that IMNs and IMNs-PEG all presented high capture efficiencies (more than 90%) of MCF-7 cells, but IMNs-PEG exhibited even lower adsorption to Jurkat T cells than IMNs. The result suggested that PEGylation did not influence the efficient capture of target cells by IMNs, but it could further reduce the NSA to nontarget cells. Finally, IMNs and IMNs-PEG were treated with mixed blood samples to evaluate their cell adsorption performance in
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EXPERIMENTAL SECTION Reagents and Instruments. All types of PEG were purchased from Shanghai Pengshuo Biotechnology Co., Ltd. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and anti-EpCAM monoclonal antibody (mAb) were from Sigma-Aldrich. FBS, filter (polyether sulfone, 0.8 μm), Hoechst 33342, and magnetic scaffold (12320D) were bought from Invitrogen Corp. Mouse Immunoglobulin G (IgG) was obtained from Beijing Solarbio Technology Co., Ltd. All cell lines were obtained from China Center for Type Culture Collection. Human blood samples were supplied by Stomatological Hospital of Wuhan University. Dynamic light scattering data were monitored with a Zetasizer Nano ZS instrument (Malvern). UV−vis probes and absorbance were acquired on a UV−vis spectrophotometer (UV-2550). Transmission electron micros-
Table 2. Number of Spiked and Captured MCF-7 Cells in the Human Whole Blood 1#
2#
3#
4#
IMNs and IMNs-PEG
IMNs
IMNs-PEG
IMNs
IMNs-PEG
IMNs
IMNs-PEG
IMNs
IMNs-PEG
number of spiked MCF-7 number of captured MCF-7
19 18
20 20
21 20
22 22
18 17
10 9
15 15
19 18
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(Qe) of MNs adsorbed on per cell. Finally, the equilibrium concentration (Ce) of MNs was calculated. Performance of MNs and MNs-PEG10000. Six batches of MNs-COOH prepared with the same method were modified with NH 2 −PEG2000−COOH and NH 2 − PEG10000−NH2. Then, the NSA rates of MNs and MNsPEG to Jurkat T cells were acquired to evaluate the uniformity of their adsorption performance. Six batches of MNs-COOH and MNs-PEG10000 were introduced into FBS, plasma, and human whole blood for 30 min at room temperature on a shaker. Afterward, the mixtures were washed twice with water by magnetic separation and then dispersed in ultrapure water to measure their hydrodynamic size and PDI. In addition, RMNs-COOH were modified with NH2−PEG10000−NH2 to obtain RMNs-PEG. After they were treated with blood and washed twice, the bright-field and fluorescence-field images of RMNs and RMNs-PEG were recorded. Adsorption and Capture Study of IMNs. MNs-COOH were modified with IgG (MNs-IgG), mAb (IMNs), and PEG followed by IgG (MNs-PEG-IgG) by carbodiimide chemistry. Afterward, the NSA rates of MNs-COOH, MNs-PEG10000, MN-IgG, and MNs-PEG-IgG to Jurkat T cells in 1× PBS were obtained, as well as the NSA rates of MNs and MNs-PEG to cells in 10% FBS, FBS and plasma. To further investigate the adsorption performance of MNs and MNs-PEG10000 in blood, three batches of MNs and MNs-PEG10000 (0.2 mg) were incubated with 200 μL of the untreated human whole blood for 30 min and washed twice, and then the number of WBCs in the supernatant was counted with a hemocytometer. Magnetic capture experiments were performed on human breast cancer MCF-7 cells, which were cultured in Dulbecco’s modified Eagle’s medium at 37 °C in a 5% CO2 atmosphere. IMNs and IMNs-PEG10000 were respectively incubated with spiked MCF-7 cells and Jurkat T cells in 1× PBS, and then the capture efficiencies and adsorption rates were gained. Furthermore, 100 MCF-7 cells stained by Hoechst 33342 were spiked into 1 mL of the untreated human whole blood to prepare the mixed blood sample. Then, IMNs and IMNs-PEG (0.2 mg) were incubated with 200 μL of the sample at 25 °C for 30 min. After washing by magnetic separation, the number of absorbed WBCs and the captured and uncaptured MCF-7 cells were identified with the help of a hemocytometer and fluorescence microscope.
copy (TEM) images were recorded by a FEI Tecnai G2 20 TWIN electron microscope. Microscopic images were taken with a CCD camera (Nikon digital sight DS-U3) mounted on an inverted fluorescence microscope (Ti, Nikon), and magnetic separation was performed on a magnetic scaffold. Fabrication, Characterization, and Quantification of MNs-PEG. MNs and RMNs were fabricated according to our previous work 23,37 and were modified with PEG by carbodiimide chemistry. Briefly, 1 mg of MNs-COOH was washed with PBS (pH 7.2, 0.01 M) twice by magnetic separation, followed by dispersed in 100 μL of PBS containing 10 mg EDC, then introduced into 900 μL of PBS containing 10 mg PEG, and incubated with gentle shaking at room temperature for modifying PEG on MNs (MNs-PEG). After 4 h of incubation, MNs-PEG were washed with PBS five times by magnetic separation, which were then stored in PBS at 4 °C for use. The zeta potential and hydrodynamic size of MNs and MNsPEG dispersed in deionized water were measured. Besides, the bright-field and fluorescence-field images of MNs-COOH were taken, as well as MNs-COOH mixed with RB−PEG10000− NH2 and MNs-COOH modified with RB−PEG10000−NH2. The mass and number concentrations of MNs were monitored,38 and the information on their size and shape was gained from the TEM image. Besides, UV−vis spectra of EDC, NH2−PEG10000−NH2 and RB−PEG10000−NH2 were monitored. Then, the proportional relationship of absorbance at the maximum absorption wavelength versus the concentration of RB−PEG10000−NH2 was acquired to quantify the number of PEG10000 chains on each MN. NSA Rates of MNs and MNs-PEG to Cells. NSA studies were performed on Jurkat T cells, which were cultured in RPMI-1640 medium at 37 °C in a 5% CO2 atmosphere. 1 × 105 Jurkat T cells were spiked into MNs (0.2 mg) in 1× PBS (400 μL) and then incubated gently at room temperature for 30 min. After washing twice with 1× PBS by magnetic separation, the number of cells adsorbed (N0) and in the supernatant (N1) were counted to calculate the NSA rate according to eq 1 NSA rate = N0/(N0 + N1) × 100%
(1)
The NSA rates of MNs modified with different types and dosages of PEG were calculated, and each experiment was repeated for three times to ensure its reliability. Number of MNs and MNs-PEG10000 Adsorbed on the Cell. To identify the number of MNs and MNsPEG10000 adsorbed on per cell, 200 μL of the incubated mixture was passed through a filter with a pore size of 0.8 μm and rinsed with 800 μL of 1× PBS. Meanwhile, MNs and MNs-PEG10000 untreated with cells were passed through the filter as controls. Comparing the absorbance of the filtrates at 400 nm according to the proportional relationship of absorbance versus the concentration of MNs, the total number of MNs and MNs-PEG10000 adsorbed on cells were determined. Subsequently, the average number of MNs and MNs-PEG10000 absorbed on per cell were estimated. Adsorption Thermodynamics. Different concentrations of MNs were incubated gently with spiked Jurkat T cells at 25, 37, and 50 °C to explore the thermodynamics of NSA between MNs and cells. Then, the mixtures were passed through the filter followed by the absorbance of the filtrates at 400 nm, which was monitored to determine the equilibrium amount
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00245. Calculations of surface coverage, conformation identification, and thermodynamic constant; zeta potentials and NSA rates of MNs and MNs-PEG; characterization of MNs and MNs-PEG10000; and adsorption uniformity of MNs and MNs-PEG2000-COOH to cells (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 0086-27-68756759. Fax: 0086-27-68754067. 7397
DOI: 10.1021/acsomega.9b00245 ACS Omega 2019, 4, 7391−7399
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Zhi-Ling Zhang: 0000-0001-7807-2264 Dai-Wen Pang: 0000-0002-7017-5725 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21535005; 91859123), the National Science and Technology Major Project of China (2018ZX10301405), the 111 Project (no. 111-2-10), and Collaborative Innovation Center for Chemistry and Molecular Medicine.
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DOI: 10.1021/acsomega.9b00245 ACS Omega 2019, 4, 7391−7399
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DOI: 10.1021/acsomega.9b00245 ACS Omega 2019, 4, 7391−7399
