- Email: [email protected]
Functional dendrimer modified ultra-hydrophilic trapping copolymer network towards highly efficient cell capture
Talanta 153 (2016) 366–371
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Functional dendrimer modified ultra-hydrophilic trapping copolymer network towards highly efficient cell capture Peiming Zhang, Mingxia Gao n, Xiangmin Zhang Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
art ic l e i nf o
a b s t r a c t
Article history: Received 17 January 2016 Received in revised form 10 March 2016 Accepted 12 March 2016 Available online 14 March 2016
Highly efficient isolation of living tumor cells possesses great significance in research of cancer. Hence, we have designed the 3-aminophenylboronic acid (APBA) derivative dendrimer-functionalized 3D network polyacrylamide/poly (methyl methacrylate) copolymer as capture substrate which is easily prepared, template free and low-cost. The structure of copolymer is compared to “fishing net” in order to increase the contact between cells and substrates. The application of poly (amidoamine) dendrimers provides abundant amino groups to react with APBA which is just like “baits” that can bond with sialic acid in the cytomembrane to realize cell capture. The 3D network structure trammels cancer cells, offers great reaction space and displays hydrophilic surface, which has immensely improved the contact probability of cells and materials. Due to the 3D network structure and dendrimer, this material can achieve a high capture efficiency of 87 75% in 45 min. The viability of captured cells is nearly 100%, as a result of the soft and hydrophilic surface and hypotoxicity of this copolymer. & 2016 Elsevier B.V. All rights reserved.
Keywords: Network structure Cell capture Functional copolymers Dendrimers Cell viability
1. Introduction Research on the isolation of tumor cells has obtained enormous attention in recent years. As it is reported, different types of tumor cells can be utilized as prognostic markers and its detection can be able to reflect the metastatic relapse and progression in other tumor entities as well [1–6]. Hence, it is pivotal and promising to develop highly efficient and sensitive methods to realize the isolation and detection of tumor cells, which provide a wide platform for the further research on the diagnosis, metastatic mechanisms and therapeutic methods of tumor [7]. In recent years, a diverse range of new techniques have been involved in the task to isolate and detect tumor cells. According to the separation mode, these new methods can be classified as physical isolation and biological affinity isolation [8]. The enrichment of tumor cells by physical properties takes full advantage of the difference in size, deformability and so on [9–11]. However, physical enrichment is short of sensitivity and specificity which limit its application, while biological enrichment often utilizes antibody [12–15], aptamer [16] and other specific markers [17–19] to recognize and capture tumor cells and possesses better efficiency and diversity than physical methods. In terms of biological isolation of tumor cells, microfluidic chips and functional materials such as magnetic beads [12,13], nanostructure array [14–16] and n
Corresponding author. E-mail address: [email protected] (M. Gao).
http://dx.doi.org/10.1016/j.talanta.2016.03.044 0039-9140/& 2016 Elsevier B.V. All rights reserved.
“smart particles” [20,21] are selected as substrates. Research on microfluidic devices has shown that this technique can achieve high capture efficiency by optimization of flow conditions and contact frequency [15,16,22]. Our research group has imitated the principle of microfluidic chip to design the nitrocellulose membrane substrate and also prepared aptamer conjugated magnetic beads [23] for efficient analysis of cancer cells and detecting the captured cells by surface-enhanced Raman scattering imaging [24]. Nevertheless, functional materials obtain unique superiorities in the following respects: high surface area to volume ratio, imitation of biomolecules, porosity [25] and so on, so that it is easier to be modified and contact with cells in rapid response for the functional materials. Especially, three-dimensional network materials display similar structure to the composition of cellular surface, such as microvilli and filopodia [17]. Besides, the polymer provides soft surface that is close to the extracellular matrix [26]. In total, 3D hierarchical polymer is able to offer large space for modification and cell incubation and the network structure can trap cells in the materials to enhance cell contact and capture efficiency of the polymer. Dendrimers are a series of synthetic macromolecules, which own abundant branches and monodispersity [27]. Nowadays, dendrimers are widely applied in modification of functional materials, for they possess excellent preponderance of biocompatibility, uniformity and a large number of functional ending groups [28]. In this way, functional molecules such as antibodies, aptamer, folic acid and phenylboronic acid can be bonded with dendrimer by conjugation for cell capture. Sialic acid (SA), which has been
P. Zhang et al. / Talanta 153 (2016) 366–371
clinically approved as one of the tumor markers, is an anionic monosaccharide that frequently appears at the terminal of glycan chains in eukaryotic cells [29]. Recently researches on sialic acid have manifested that there exists overexpression of SA on membrane glycoproteins and glycolipids in tumor cells of various origins [30]. As a result, overexpression of SA on the membrane has been selected as an implication of the malignant and metastatic phenotypes for several different types of cancers [29]. Phenylboronic is widely utilized as the select targets of SA [31], for it can form a stable complex with sialic acids [18,19,32], benefitting from the diols-containing targets existing in the sialic acids [33]. As it is reported, the polyacrylamide possesses network structure [34], biocompatibility [35], hydrophilicity [36] and this material is easy to obtain. The polyacrylamide has been widely used in gel electrophoresis, drug-release, oilfield chemical assistants and so on [37,38]. However, this polymer has been rarely applied in the isolation of tumor cells. Benefited from the characteristics of polyacrylamide, we designed functional polyacrylamide (PAM)/ poly (methyl methacrylate) (PMMA) copolymer as substrates, followed by the modification of dendrimers and 3-aminophenylboronic acid (APBA) in sequence. Through optimization of experimental conditions, the functional dendrimer modified 3D network copolymer could achieve a high capture efficiency and viability due to the soft and hydrophilic surface as well as the particular network structure which supported our idea and provided a good method to capture cells.
367
(0.1 g/mL). After this procedure, the 96-well plate was placed in a constant temperature vibrator (50 °C, 100 rpm) for 1 h. The products were washed with PBS for three times. The physical state of the copolymer changed from liquid to solid and the shape of this white PAM-PMMA copolymer was just fitted with the 96-well plate. 2.3. Synthesis of dendrimer-functionalized copolymer Before modification, the copolymer was exposed under UV for 10 min and washed with PBS. The copolymer was activated by EDC solution (14 mg/mL in PBS, pH 7.4) for half an hour, then added 7.5 μL PAMAM in it. During 2 h of reaction, the dendrimer-functionalized copolymer was generated and should be washed with PBS for three times. 2.4. Preparation of APBA modified dendrimer-copolymer Briefly, the dendrimer-copolymer was activated in glutaraldehyde solution (5%) for 2 h at first. Subsequently, the activated copolymer was added in APBA (10 mg/mL) and reacted for 3 h. After that, the copolymer was washed with PBS. Finally, the product was immersed in PBS which contained NaBH3CN (10 mg/mL) for 1 h to end the reaction. The APBA modified dendrimer-copolymer was synthesized and took on a claybank surface while the surface of non-modified material was white. 2.5. Capture assay towards cancer cells
2. Experimental section 2.1. Materials Acrylamide, ammonium persulfate (APS), Methyl methacrylate (MMA) and polyethylene glycol (PEG-6000) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tetramethylethylenediamine (TEMED) was acquired from Beijing Biodee Biotechnology Co., Ltd. Bis-Acrylamide was bought from SinoAmerican Biotechnology Co. The chemical reagents above were utilized to synthesize the copolymer. Dendrimer (PAMAM 5.0), 3-aminophenylboronic acid (APBA), N-(3-Dimethylaminopropyl)N-ethylcarbodiimide hydrochloride Crystalline (EDC), N-Hydroxysuccinimide (NHS), glutaraldehyde and sodium cyanoborohydride, which were applied in modification of copolymer, were obtained through Sigma-Aldrich Co., LLC. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), trypsin and phosphate buffer saline (PBS) were purchased from Shanghai solarbio Bioscience & Technology Co., LTD. Fluorescent dyes involved in our experiments were as follows: 4′,6-diamidino-2-phenylindole (DAPI), acridine orange (AO), prodium iodide (PI), and alizarin red S (ARS), they were provided by Sigma-Aldrich Co., LLC. U251 cancer cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). SEM images were taken by Philips XL30. FT-IR spectrum data was obtained from Nicolet Nexus 470. UV absorption spectra was measured with UV spectrophotometer (Agilent 8453). Fluorescence images of cells and materials were photographed by Leica DM4000 B LED.
To characterize the great potential of the materials in cell capture, we conducted several groups of parallel capture experiments to optimize the properties of materials and the capture conditions. In this task, U251 cell line was selected as targeted tumor cells for the reason that sialic acids overexpressed in its cytomembrane. The cells were incubated at 37 °C, 5% CO2 in DMEM which was supplemented with 10% FBS. As the information mentioned above, the APBA modified dendrimer-copolymer was synthesized in 96-well plate which was convenient for cell capture and image, so we injected 100 μL cell suspension at a density of 105 cells per mL in the 96-well plate and incubated at 37 °C, 5% CO2 for 45 min. After incubation, the supernate was sucked up and the materials bonded with U251 were rinsed by PBS (pH 7.4) for three times, all the washing solution were reserved for the calculation of cell capture efficiency. 2.6. Test of the viability of captured cells AO and PI [26] were used to evaluate the viability of captured U251 cells. The stock solution of AO/PI was prepared by using PBS (pH 7.4, without calcium and magnesium) to dilute the dye liquor from 1 mg/ml to 10 μg/mL, respectively. After incubation and rinse, the materials were added in equivalent amount of AO and PI stock solution and reacted for 15 min. The dyed cells were rinsed by PBS for several times, then observed by fluorescence microscope. 2.7. Characterization
2.2. Polymerization of polyacrylamide/poly (methyl methacrylate) copolymer Firstly, we chose 96-well plate as reactors to shape the copolymer. Then we prepared mixed solution as follows: 20 mg acrylamide, 20 mg bis-acrylamide and 30 mg PEG-6000 were dissolved in 600 μL PBS solution (pH 7.4) under ultrasonic treatment. Each well was added in 50 μL of the solution above, 1.068 μL methyl methacrylate, 0.6 μL TEMED and 2.5 μL APS solution
To describe the morphology of copolymer, we used scanning electron microscope (SEM). The components of copolymer were preliminarily examined by Fourier transform infrared spectroscopy (FTIR). The elemental analysis was conducted through energy dispersive spectrometer (EDS) to evaluate the APBA modified dendrimer-materials. To testify whether the dendrimer was successfully modified onto the copolymer, we applied ultraviolet absorption spectrum to
368
P. Zhang et al. / Talanta 153 (2016) 366–371
contrast the absorption intensity of the same amount of PAMAM in EDC solution before and after the reaction. To take one step further, alizarin red S (ARS) was chosen to prove that APBA was bonded with dendrimer. The APBA modified material was treated with ARS solution (0.05 mM pH 7.4) for 1 h and non-modified dendrimer-copolymer was acted as control group. After washing with PBS, experimental group and control group were observed under fluorescence microscope. To characterize the capture effect, SEM images were involved in displaying the combination of materials and U251. The captured U251 were fixed by 5% glutaraldehyde solution overnight and washed with PBS. Then the fixed cells were treated with different concentrations of ethanol and the concentration was 30%, 50%, 75%, 85%, 95%, 100% in sequence. Each concentration was treated twice and 15 min for once. The dehydrated samples were dried afterwards for SEM. We also introduced fluorescent staining method to further characterize the interaction between materials and cells. After capture, the cells were rinsed and fixed by 5% glutaraldehyde solution overnight. Then we chose DAPI as cell stain, the stock concentration should be 10 μg/mL and the staining time was 15 min. The well-disposed sample was viewed by fluorescence microscope. To estimate the capture efficiency, we combined direct method and indirect method together. Thus, we calculate the capture efficiency according to the equation: N − N′ cell capture efficiency (%)= N ×100% , N is represented as the total of cells that were added in 96-well plate, N′ is represented as the sum of cells that were not combined with materials and cells rinsed with PBS; and the fluorescence image can be also used to evaluate the efficiency by counting the fluorescence dots in the
whole area. It is the same way for testing the viability of captured cells.
3. Results and discussion 3.1. Design and characterization of the synthesized APBA modified dendrimer-copolymer To increase the contact probability of cells and materials, we designed the 3D network copolymer as substrate, which possessed porous structure and hydrophilic surface for further application. The SEM image (Fig. 1a) revealed the microstructure of the materials proving the net-like structure and hierarchical porous formation. This copolymer was compared to “fishing net” which possessed crosslinked network structure and large pore size so that it had great potential in cell capture. Besides, the SEM micrography of materials with captured U251 was shown in Fig. 1b which indicated the U251 was successfully targeted on the functional 3D network copolymer. In addition, the infrared spectrum showed basic components and structure of the network copolymer (Fig. S1). From this figure, we could conclude the characteristic absorption peak of 3411 cm 1 belonged to the stretching vibration of N–H and the peak of 1531 cm 1 belonged to the bending vibration of amide, the characteristic absorption peak of C¼ O was 1665 cm 1, as well as the asymmetrical stretching vibration (1221 cm 1) and symmetrical stretching vibration (1115 cm 1) of C–O manifested the existence of methyl methacrylate. The information mentioned in the Fig. S1 demonstrated
Fig. 1. (a) SEM micrography of copolymer showing the network structure of the substrate. (b) SEM image taken after cell capture, captured cells and materials were treated with fixation and dehydration. (c) EDS analysis of APBA modified dendrimer-copolymer testified the APBA was successfully immobilized on the substrate.
P. Zhang et al. / Talanta 153 (2016) 366–371
Fig. 2. The impact of incubation time on capture efficiency. Judged by the graph, capture efficiency could reach a higher level under the condition that the incubation time no less than 45 min.
the formation of PAM-PMMA copolymer. To prove the feasibility of the protocols, we arranged experiments to testify whether the copolymer was successfully modified with dendrimers and APBA. In order to inspect whether dendrimers were conjugated with the copolymer, we utilized the ultraviolet absorption spectrum to detect the variation of absorbance before and after this reaction (Fig. S2). Judged from it, we could distinctly observe the decrease of the absorption peak after the conjugation reaction, which manifested the amount of dendrimers in the supernate were fewer than in the stock solution, thus a number of dendrimers were bonded with the copolymer. Besides its hydrophilic characterization, the dendrimer had a rich amount of amino, so that the dendrimer was easily to react with other reactants that were able to capture cells. Giving to the properties of dendrimers, APBA which had boric acid group that could react with cis-dihydroxy of sialic acid in cell membrane was selected as the “baits” to target cells. Hence, the copolymer immobilized with dendrimer was reacted with APBA sequentially. To confirm the APBA was modified onto the materials, energy dispersive spectrometer (EDS) was conducted and the image (Fig. 1c) proved the feasibility. In this figure, the atomic percentage of boron was 20.9% that confirmed the APBA was tightly bonded with dendrimers and the large number of amino groups of the dendrimer had good modification performance in this reaction. Meanwhile, the boric acid group could bond with alizarin red S (ARS) [39] which was able to emit green fluorescence, while the group free of the modification of APBA could not emit green fluorescence as Fig. S3 showed. Therefore, all of these results mentioned above demonstrated that we successfully fabricated the 3D network dendrimer-copolymer which was modified with APBA. 3.2. Cell capture assay and optimization of experimental conditions As the APBA modified dendrimer-copolymer possessed the following abilities: soft surface, hydrophilic characterization, 3D network morphology and enhanced modification of APBA, it was easy to trap the cells in the pores and react with APBA to intensify the interaction between materials and target cells. Hence, we designed cell capture assays to evaluate its capture efficiency and optimize experimental conditions. As the foundation of polymerization, the optimal dosage of functional monomer methyl methacrylate (MMA) was one of the key points that we concerned about. By setting the usage of MMA as a single variable, we prepared copolymer of which the usage of MMA was 4.256 μL, 2.128 μL, 1.064 μL, 0.638 μL and 0.425 μL, respectively. After modification, the materials mentioned above were applied in cell capture assay and then calculated the capture
369
efficiency separately. As the result shown in Fig. S4, we could draw the conclusion that 1.064 μL MMA was the optimal volume in all the treatment groups for it might balance the extent of polymerization to form the cell-fitting pores and the quantity of modification sites which were from MMA hydrolysis. To increase the number of modification sites and keeping the hydrophilicity, we went on searching for the better modification amount of dendrimer. The copolymer was modified within different amount of dendrimer as follows: 0.75 μL, 1.5 μL, 2.5 μL, 5.0 μL, 7.5 μL and 10.0 μL, respectively. The result was also examined by cell capture tests and shown in Fig. S5. The figure revealed that the better usage of dendrimer was 7.5 μL, for the reason that less amount of dendrimer offered less amino groups for the reaction with APBA which would finally reduce the captured cells, while excess amount of dendrimer might lead to overload and instability that were adverse to capture efficiency as well. In order to improve the capture efficiency, the “bait”-APBA was the critical factor. When the modified quantity rose, the materials could provide more boric acid groups to target on the sialic acid in the cytomembrane of U251 cells. As a result, we designed six treatment groups with different usage of APBA and the concentrations were 2, 5, 10, 15, 20 and 25 mg/mL. After reaction and wash, the APBA modified copolymer was used in cell capture assay to characterize the capture ability. Last but not least, the operating conditions, temperature and reaction time should keep constant. As it was shown in Fig. S6, the material had a better capture capacity when the concentration of APBA was 10 mg/mL. To better investigate the relation between incubation time and cell capture efficiency, we prepared identical modified materials to conduct cell capture experiments and added the same amount of cells in 96-well plate, then incubated 5, 15, 30, 45, 60, 90 min, respectively. With incubation time extending, capture efficiency increased and when time went up to 45 min, 87 75% of cells were captured by the material (Fig. 2). Due to deficiently contact, the capture efficiency was low within short incubation time. As time extending, more and more cells trapped in the 3D network copolymer and reacted with APBA modified on the material that contributed to the high capture efficiency. Following by the optimization of synthetic procedures and conditions, the capture efficiency upgraded step by step from 21% up to surpassing 90%. This result firmly indicated that the functional copolymer substrate bonded the targeted tumor cells efficiently. Compared with the materials of phenylboronic acid-containing polymer brush [18] and the poly(APBA) nanostructures substrates [31], our APBA functionalized copolymer showed a higher efficiency under the same incubation conditions (the capture efficiency of polymer brush was 60% and of poly APBA substrates was 80%) and caused less damage to captured cells. The 3D network structure significantly enhanced the contact between cells and materials, and the illustration of this concept was concisely displayed in Fig. 3. Furthermore, dendrimers and APBA were modified surrounding the network structure, so the 3D structure could get pseudopod trapped in and let APBA easily react with sialic acid in this way. In virtue of the functional 3D network structure, the APBA modified dendrimer-copolymer exhibited great potential in cell isolation. To further investigate the function of 3D network copolymer in respect of structure and modification, we designed contrast experiments as follows: we prepared the APBA modified dendrimercopolymer and the APBA directly modified copolymer without the addition of dendrimer as capture substrates, meanwhile the synthesized PAM-PMMA copolymer exempted from the modification of dendrimer and APBA was individually used in cell capture assay as blank control group. Under the same operation and condition, the capture efficiency of APBA modified dendrimer-copolymer
370
P. Zhang et al. / Talanta 153 (2016) 366–371
Fig. 3. A simple illustration that showed how the U251 targeted on the functional 3D network copolymer.
Fig. 4. Experimental results that displayed the influence of 3D network structure and modification on the capture efficiency. (a) The column chart provided the capture efficiency of APBA modified dendrimer-copolymer, APBA modified copolymer without dendrimer and copolymer without any modification, which manifested the synergistic effect of dendrimer and 3D network copolymer. (b)–(d) Fluorescence images of the APBA modified group with dendrimer, APBA modified group without dendrimer and blank control group (copolymer without modification), respectively.
could reach nearly 90%, while the efficiency of APBA directly modified copolymer without dendrimer was 30.4 75% and the efficiency of the synthesized PAM-PMMA copolymer without modification was only 7 7 3% (Fig. 4a). This result was also confirmed by fluorescence image shown in Fig. 4b–d. The fluorescence images mentioned above demonstrated that the APBA modified dendrimer-copolymer had the higher density of fluorescent spots which meant more cells bonded with the material there. Judged by the results of control experiments, this 3D network copolymer offered large space for reaction and the low non-specific character was another superiority of this material. So that it could make cells easily caught in the material which provided great advantage in the cell capture assay. What's more, the use of dendrimers tremendously enhanced the volume of modification, so a great deal of APBA was utilized to react with sialic acid to increase the capture efficiency. To testify the influence of the copolymer to cell viability, we conducted AO/PI bi-label assay to test the viability of captured U251. After 2 h, the captured U251 on the copolymer was stained by AO/PI and washed with PBS for three times. The fluorescence
Fig. 5. Fluorescence image of AO/PI, which proved the capture U251 remained excellent viability.
P. Zhang et al. / Talanta 153 (2016) 366–371
image (Fig. 5) indicated almost all the cells kept alive and the viability nearly 100%. This result was attributed to that the copolymer and dendrimers were extremely hydrophilic and the surface of the material was soft and smooth. To sum up, our experimental results prove the functional 3D network copolymer have great potential in cells isolation and capture.
[4]
[5]
[6]
4. Conclusions We successfully synthesized the functional dendrimer modified 3D network copolymer as cell capture substrate in a simple and inexpensive way. The 3D network copolymer possessed hydrophilic surface and macroporous structure which enhanced the contact probability of cells and also induced the cells trapped in the pores for the consequent reaction. In addition, the usage of dendrimers kept the hydrophilic character of the copolymer and provided a great number of modification sites for the chemical conjugation with APBA that immensely improved the bonding quantity of APBA. The APBA modified copolymer was similar to the fishing net with baits and the dendrimer was acted as the bridge between the copolymer and functional group. As a result of the unique structure and functional modification, the capture efficiency of the material could reach 877 5% in 45 min. Moreover, the functional dendrimer modified copolymer was hypotoxicity and provided the soft and hydrophilic surface for the capture assay so that the viability of captured cells was nearly 100% after captured for 2 h. To sum up, the functional 3D network copolymer has a promising prospect of application in cell isolation.
Conflict interest The authors declare no competing financial interest.
[7] [8] [9] [10] [11]
[12] [13] [14]
[15] [16] [17] [18] [19] [20] [21] [22]
[23] [24] [25]
Acknowledgment [26]
This work was supported by National Basic Research Program of China (Project: 2012CB910604), the National High-Tech R&D Program (Project: 2012AA020202) and the National Natural Science Foundation of China (Project: 21275034 and 21475027).
[27] [28] [29] [30]
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2016.03. 044.
[31] [32]
[33] [34] [35]
References [1] S.M. Leong, K.M.L. Tan, H.W. Chua, D. Tan, D. Fareda, S. Osmany, M. Li, S. Tucker, E.S.C. Koay, J. Hematol. Oncol. 8 (2015) 75–82. [2] H.I. Scher, X. Jia, J.S. de Bono, M. Fleisher, K.J. Pienta, D. Raghavan, G. Heller, Lancet Oncol. 10 (2009) 233–239. [3] J. Hou, M.G. Krebs, L. Lancashire, R. Sloane, A. Backen, R.K. Swain, L.J.C. Priest,
[36] [37] [38] [39]
371
A. Greystoke, C. Zhou, K. Morris, T. Ward, F.H. Blackhall, C. Dive, J. Clin. Oncol. 30 (2012) 525–532. M.G. Krebs, R. Sloane, L. Priest, L. Lancashire, J. Hou, A. Greystoke, T.H. Ward, R. Ferraldeschi, A. Hughes, G. Clack, M. Ranson, C. Dive, F.H. Blackhal, J. Clin. Oncol. 29 (2011) 1556–1563. C. Aggarwal, N.J. Meropol, C.J. Punt, N. Lannotti, B.H. Saidman, K.D. Sabbath, N. Y. Gabrail, J. Picus, M.A. Morse, E. Mitchell, M.C. Miller, S.J. Cohen, Ann. Oncol. 24 (2013) 420–428. E. Denève, S. Riethdorf, J. Ramos, D. Nocca, A. Coffy, J. Daurès, T. Maudelonde, J. Fabre, K. Pantel, C. Alix-Panabières, Clin. Chem. 59 (2013) 1384–1392. J.M. Lang, B.P. Casavant, D.J. Beebe, Sci. Transl. Med. 4 (2012) 141–143. C. Alix-Panabières, K. Pantel, Nat. Rev. Cancer 14 (2014) 623–631. H.K. Lin, S. Zheng, A.J. Williams, M. Balic, S. Groshen, H.I. Scher, M. Fleisher, W. Stadler, R.H. Datar, Y. Tai, R.J. Cote, Clin. Cancer. Res. 16 (2010) 5011–5018. H.W. Hou, M.E. Warkiani, B.L. Khoo, Z.R. Li, R.A. Soo, D.S. Tan, W.T. Lim, J. Han, A.A. Bhagat, C.T. Lim, Sci. Rep. 3 (2013) 1259–1266. E. Sollier, D.E. Go, J. Che, D.R. Gossett, S. O'Byrne, W.M. Weaver, N. Kummer, M. Rettig, J. Goldman, N. Nickols, S. McCloskey, R.P. Kulkarni, D. Di Carlo, Lab Chip 14 (2014) 63–77. C. Wen, L. Wu, Z. Zhang, Y. Liu, S. Wei, J. Hu, M. Tang, E. Sun, Y. Gong, J. Yu, D. Pang, ACS Nano 8 (2014) 941–949. N. Lu, M. Xie, J. Wang, S. Lv, J. Yi, W. Dong, W. Huang, ACS Appl. Mater. Interfaces 7 (2015) 8817–8826. S. Wang, K. Liu, J. Liu, Z.T.-F. Yu, X. Xu, L. Zhao, T. Lee, E.K. Lee, J. Reiss, Y.K. Lee, L.W.K. Chung, J. Huang, M. Rettig, D. Seligson, K.N. Duraiswamy, C.K.-F. Shen, H. Tseng, Angew. Chem. Int. Ed. 123 (2011) 3140–3144. S. Wang, H. Wang, J. Jiao, K. Chen, G.E. Owens, K. Kamei, J. Sun, D.J. Sherman, C. P. Behrenbruch, H. Wu, H. Tseng, Angew. Chem. Int. Ed. 121 (2009) 9132–9135. L. Chen, X. Liu, B. Su, J. Li, L. Jiang, D. Han, S. Wang, Adv. Mater. 23 (2011) 4376–4380. Y. Zhao, X. Zhu, H. Liu, Y. Luo, S. Wang, M. Shen, M. Zhu, X. Shi, J. Mater. Chem. B 2 (2014) 7384–7393. H. Liu, Y. Li, K. Sun, J. Fan, P. Zhang, J. Meng, S. Wang, L. Jiang, J. Am. Chem. Soc. 135 (2013) 7603–7609. J. Lee, S. Chung, H. Cho, D. Kim, Adv. Funct. Mater. 25 (2015) 3705–3717. J. Meng, H. Liu, X. Liu, G. Yang, P. Zhang, S. Wang, L. Jiang, Small 10 (2014) 3735–3741. C. Huang, G. Yang, Q. Ha, J. Meng, S. Wang, Adv. Mater. 17 (2015) 310–313. S. Nagrath, L.V. Sequist, S. Maheswaran, D.W. Bell, D. Irimia, L. Ulkus, M. R. Smith, E.L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U.J. Balis, R. G. Tompkins, D.A. Haber, M. Toner, Nature 450 (2007) 20–27. C. Sun, R. Zhang, M. Gao, X. Zhang, Anal. Bioanal. Chem. 470 (2015) 8883–8892. P. Zhang, R. Zhang, M. Gao, X. Zhang, ACS Appl. Mater. Interfaces 6 (2014) 370–376. H. Yoon, T.H. Kim, Z. Zhang, E. Azizi, T.M. Pham, C. Paoletti, J. Lin, N. Ramnath, M.S. Wicha, D.F. Hayes, D.M. Simeone, S. Nagrath, Nat. Nanotechnol. 194 (2013) 735–742. L. Ma, G. Yang, N. Wang, P. Zhang, F. Guo, J. Meng, F. Zhang, Z. Hu, S. Wang, Y. Zhao, Adv. Healthc. Mater. 4 (2015) 838–843. D.A. Tomalia, J.M.J. Frechet, Dendrimers and Other Dendritic Polymers, John Wiley & Sons Ltd, New York, 2001. Y. Wang, J. Wang, M. Gao, X. Zhang, J. Mater. Chem. B 3 (2015) 8711–8716. A. Matsumoto, N. Sato, Ki Kataoka, Y. Miura, Y. Miyahara, J. Am. Chem. Soc. 131 (2009) 12022–12023. C. Bull, M.A. Stoel, M.H. den Brok, G.J. Adema, Cancer Res. 74 (2014) 3199–3204. J. Ouyang, M. Chen, W. Bao, Q. Zhang, K. Wang, X. Xia, Adv. Funct. Mater. 10 (2015) 6122–6130. S. Deshayes, H. Cabral, T. Ishii, S. Kobayashi, T. Yamashita, A. Matsumoto, Y. Miyahara, N. Nishiyama, K. Kataoka, J. Am. Chem. Soc. 135 (2013) 15501–15507. Q. Wu, L. Wang, H. Yu, J. Wang, Z. Chen, Chem. Rev. 111 (2011) 7855–7875. J. Gregory, J. Cannell, M. Kofron, L. Yeghiazarian, V. Nistor, J. Appl. Polym. Sci. 132 (2015) 41557–41563. P. Nghe, S. Boulineau, S. Gude, P. Recouvreux, J.S. van Zon, S.J. Tans, PloS One 8 (2013) 75537–75548. A.A. Adewunmi, S. Ismail, A.S. Sultan, J. Appl. Polym. Sci. 132 (2015) 42110–42118. W. Lee, Y. Fu, J. Appl. Polym. Sci. 89 (2003) 3652–3660. R. Zolfaghari, A.A. Katbab, J. Nabavizadeh, R.Y. Tabasi, M.H. Neyad, J. Appl. Polym. Sci. 100 (2006) 2096–2103. J. Liu, K. Yang, Y. Qu, S. Li, Q. Wu, Z. Liang, L. Zhang, Y. Zhang, Chem. Commun. 51 (2015) 3896–3898.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Functional dendrimer modified ultra-hydrophilic trapping copolymer network towards highly efficient cell capture Peiming Zhang, Mingxia Gao n, Xiangmin Zhang Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
art ic l e i nf o
a b s t r a c t
Article history: Received 17 January 2016 Received in revised form 10 March 2016 Accepted 12 March 2016 Available online 14 March 2016
Highly efficient isolation of living tumor cells possesses great significance in research of cancer. Hence, we have designed the 3-aminophenylboronic acid (APBA) derivative dendrimer-functionalized 3D network polyacrylamide/poly (methyl methacrylate) copolymer as capture substrate which is easily prepared, template free and low-cost. The structure of copolymer is compared to “fishing net” in order to increase the contact between cells and substrates. The application of poly (amidoamine) dendrimers provides abundant amino groups to react with APBA which is just like “baits” that can bond with sialic acid in the cytomembrane to realize cell capture. The 3D network structure trammels cancer cells, offers great reaction space and displays hydrophilic surface, which has immensely improved the contact probability of cells and materials. Due to the 3D network structure and dendrimer, this material can achieve a high capture efficiency of 87 75% in 45 min. The viability of captured cells is nearly 100%, as a result of the soft and hydrophilic surface and hypotoxicity of this copolymer. & 2016 Elsevier B.V. All rights reserved.
Keywords: Network structure Cell capture Functional copolymers Dendrimers Cell viability
1. Introduction Research on the isolation of tumor cells has obtained enormous attention in recent years. As it is reported, different types of tumor cells can be utilized as prognostic markers and its detection can be able to reflect the metastatic relapse and progression in other tumor entities as well [1–6]. Hence, it is pivotal and promising to develop highly efficient and sensitive methods to realize the isolation and detection of tumor cells, which provide a wide platform for the further research on the diagnosis, metastatic mechanisms and therapeutic methods of tumor [7]. In recent years, a diverse range of new techniques have been involved in the task to isolate and detect tumor cells. According to the separation mode, these new methods can be classified as physical isolation and biological affinity isolation [8]. The enrichment of tumor cells by physical properties takes full advantage of the difference in size, deformability and so on [9–11]. However, physical enrichment is short of sensitivity and specificity which limit its application, while biological enrichment often utilizes antibody [12–15], aptamer [16] and other specific markers [17–19] to recognize and capture tumor cells and possesses better efficiency and diversity than physical methods. In terms of biological isolation of tumor cells, microfluidic chips and functional materials such as magnetic beads [12,13], nanostructure array [14–16] and n
Corresponding author. E-mail address: [email protected] (M. Gao).
http://dx.doi.org/10.1016/j.talanta.2016.03.044 0039-9140/& 2016 Elsevier B.V. All rights reserved.
“smart particles” [20,21] are selected as substrates. Research on microfluidic devices has shown that this technique can achieve high capture efficiency by optimization of flow conditions and contact frequency [15,16,22]. Our research group has imitated the principle of microfluidic chip to design the nitrocellulose membrane substrate and also prepared aptamer conjugated magnetic beads [23] for efficient analysis of cancer cells and detecting the captured cells by surface-enhanced Raman scattering imaging [24]. Nevertheless, functional materials obtain unique superiorities in the following respects: high surface area to volume ratio, imitation of biomolecules, porosity [25] and so on, so that it is easier to be modified and contact with cells in rapid response for the functional materials. Especially, three-dimensional network materials display similar structure to the composition of cellular surface, such as microvilli and filopodia [17]. Besides, the polymer provides soft surface that is close to the extracellular matrix [26]. In total, 3D hierarchical polymer is able to offer large space for modification and cell incubation and the network structure can trap cells in the materials to enhance cell contact and capture efficiency of the polymer. Dendrimers are a series of synthetic macromolecules, which own abundant branches and monodispersity [27]. Nowadays, dendrimers are widely applied in modification of functional materials, for they possess excellent preponderance of biocompatibility, uniformity and a large number of functional ending groups [28]. In this way, functional molecules such as antibodies, aptamer, folic acid and phenylboronic acid can be bonded with dendrimer by conjugation for cell capture. Sialic acid (SA), which has been
P. Zhang et al. / Talanta 153 (2016) 366–371
clinically approved as one of the tumor markers, is an anionic monosaccharide that frequently appears at the terminal of glycan chains in eukaryotic cells [29]. Recently researches on sialic acid have manifested that there exists overexpression of SA on membrane glycoproteins and glycolipids in tumor cells of various origins [30]. As a result, overexpression of SA on the membrane has been selected as an implication of the malignant and metastatic phenotypes for several different types of cancers [29]. Phenylboronic is widely utilized as the select targets of SA [31], for it can form a stable complex with sialic acids [18,19,32], benefitting from the diols-containing targets existing in the sialic acids [33]. As it is reported, the polyacrylamide possesses network structure [34], biocompatibility [35], hydrophilicity [36] and this material is easy to obtain. The polyacrylamide has been widely used in gel electrophoresis, drug-release, oilfield chemical assistants and so on [37,38]. However, this polymer has been rarely applied in the isolation of tumor cells. Benefited from the characteristics of polyacrylamide, we designed functional polyacrylamide (PAM)/ poly (methyl methacrylate) (PMMA) copolymer as substrates, followed by the modification of dendrimers and 3-aminophenylboronic acid (APBA) in sequence. Through optimization of experimental conditions, the functional dendrimer modified 3D network copolymer could achieve a high capture efficiency and viability due to the soft and hydrophilic surface as well as the particular network structure which supported our idea and provided a good method to capture cells.
367
(0.1 g/mL). After this procedure, the 96-well plate was placed in a constant temperature vibrator (50 °C, 100 rpm) for 1 h. The products were washed with PBS for three times. The physical state of the copolymer changed from liquid to solid and the shape of this white PAM-PMMA copolymer was just fitted with the 96-well plate. 2.3. Synthesis of dendrimer-functionalized copolymer Before modification, the copolymer was exposed under UV for 10 min and washed with PBS. The copolymer was activated by EDC solution (14 mg/mL in PBS, pH 7.4) for half an hour, then added 7.5 μL PAMAM in it. During 2 h of reaction, the dendrimer-functionalized copolymer was generated and should be washed with PBS for three times. 2.4. Preparation of APBA modified dendrimer-copolymer Briefly, the dendrimer-copolymer was activated in glutaraldehyde solution (5%) for 2 h at first. Subsequently, the activated copolymer was added in APBA (10 mg/mL) and reacted for 3 h. After that, the copolymer was washed with PBS. Finally, the product was immersed in PBS which contained NaBH3CN (10 mg/mL) for 1 h to end the reaction. The APBA modified dendrimer-copolymer was synthesized and took on a claybank surface while the surface of non-modified material was white. 2.5. Capture assay towards cancer cells
2. Experimental section 2.1. Materials Acrylamide, ammonium persulfate (APS), Methyl methacrylate (MMA) and polyethylene glycol (PEG-6000) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tetramethylethylenediamine (TEMED) was acquired from Beijing Biodee Biotechnology Co., Ltd. Bis-Acrylamide was bought from SinoAmerican Biotechnology Co. The chemical reagents above were utilized to synthesize the copolymer. Dendrimer (PAMAM 5.0), 3-aminophenylboronic acid (APBA), N-(3-Dimethylaminopropyl)N-ethylcarbodiimide hydrochloride Crystalline (EDC), N-Hydroxysuccinimide (NHS), glutaraldehyde and sodium cyanoborohydride, which were applied in modification of copolymer, were obtained through Sigma-Aldrich Co., LLC. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), trypsin and phosphate buffer saline (PBS) were purchased from Shanghai solarbio Bioscience & Technology Co., LTD. Fluorescent dyes involved in our experiments were as follows: 4′,6-diamidino-2-phenylindole (DAPI), acridine orange (AO), prodium iodide (PI), and alizarin red S (ARS), they were provided by Sigma-Aldrich Co., LLC. U251 cancer cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). SEM images were taken by Philips XL30. FT-IR spectrum data was obtained from Nicolet Nexus 470. UV absorption spectra was measured with UV spectrophotometer (Agilent 8453). Fluorescence images of cells and materials were photographed by Leica DM4000 B LED.
To characterize the great potential of the materials in cell capture, we conducted several groups of parallel capture experiments to optimize the properties of materials and the capture conditions. In this task, U251 cell line was selected as targeted tumor cells for the reason that sialic acids overexpressed in its cytomembrane. The cells were incubated at 37 °C, 5% CO2 in DMEM which was supplemented with 10% FBS. As the information mentioned above, the APBA modified dendrimer-copolymer was synthesized in 96-well plate which was convenient for cell capture and image, so we injected 100 μL cell suspension at a density of 105 cells per mL in the 96-well plate and incubated at 37 °C, 5% CO2 for 45 min. After incubation, the supernate was sucked up and the materials bonded with U251 were rinsed by PBS (pH 7.4) for three times, all the washing solution were reserved for the calculation of cell capture efficiency. 2.6. Test of the viability of captured cells AO and PI [26] were used to evaluate the viability of captured U251 cells. The stock solution of AO/PI was prepared by using PBS (pH 7.4, without calcium and magnesium) to dilute the dye liquor from 1 mg/ml to 10 μg/mL, respectively. After incubation and rinse, the materials were added in equivalent amount of AO and PI stock solution and reacted for 15 min. The dyed cells were rinsed by PBS for several times, then observed by fluorescence microscope. 2.7. Characterization
2.2. Polymerization of polyacrylamide/poly (methyl methacrylate) copolymer Firstly, we chose 96-well plate as reactors to shape the copolymer. Then we prepared mixed solution as follows: 20 mg acrylamide, 20 mg bis-acrylamide and 30 mg PEG-6000 were dissolved in 600 μL PBS solution (pH 7.4) under ultrasonic treatment. Each well was added in 50 μL of the solution above, 1.068 μL methyl methacrylate, 0.6 μL TEMED and 2.5 μL APS solution
To describe the morphology of copolymer, we used scanning electron microscope (SEM). The components of copolymer were preliminarily examined by Fourier transform infrared spectroscopy (FTIR). The elemental analysis was conducted through energy dispersive spectrometer (EDS) to evaluate the APBA modified dendrimer-materials. To testify whether the dendrimer was successfully modified onto the copolymer, we applied ultraviolet absorption spectrum to
368
P. Zhang et al. / Talanta 153 (2016) 366–371
contrast the absorption intensity of the same amount of PAMAM in EDC solution before and after the reaction. To take one step further, alizarin red S (ARS) was chosen to prove that APBA was bonded with dendrimer. The APBA modified material was treated with ARS solution (0.05 mM pH 7.4) for 1 h and non-modified dendrimer-copolymer was acted as control group. After washing with PBS, experimental group and control group were observed under fluorescence microscope. To characterize the capture effect, SEM images were involved in displaying the combination of materials and U251. The captured U251 were fixed by 5% glutaraldehyde solution overnight and washed with PBS. Then the fixed cells were treated with different concentrations of ethanol and the concentration was 30%, 50%, 75%, 85%, 95%, 100% in sequence. Each concentration was treated twice and 15 min for once. The dehydrated samples were dried afterwards for SEM. We also introduced fluorescent staining method to further characterize the interaction between materials and cells. After capture, the cells were rinsed and fixed by 5% glutaraldehyde solution overnight. Then we chose DAPI as cell stain, the stock concentration should be 10 μg/mL and the staining time was 15 min. The well-disposed sample was viewed by fluorescence microscope. To estimate the capture efficiency, we combined direct method and indirect method together. Thus, we calculate the capture efficiency according to the equation: N − N′ cell capture efficiency (%)= N ×100% , N is represented as the total of cells that were added in 96-well plate, N′ is represented as the sum of cells that were not combined with materials and cells rinsed with PBS; and the fluorescence image can be also used to evaluate the efficiency by counting the fluorescence dots in the
whole area. It is the same way for testing the viability of captured cells.
3. Results and discussion 3.1. Design and characterization of the synthesized APBA modified dendrimer-copolymer To increase the contact probability of cells and materials, we designed the 3D network copolymer as substrate, which possessed porous structure and hydrophilic surface for further application. The SEM image (Fig. 1a) revealed the microstructure of the materials proving the net-like structure and hierarchical porous formation. This copolymer was compared to “fishing net” which possessed crosslinked network structure and large pore size so that it had great potential in cell capture. Besides, the SEM micrography of materials with captured U251 was shown in Fig. 1b which indicated the U251 was successfully targeted on the functional 3D network copolymer. In addition, the infrared spectrum showed basic components and structure of the network copolymer (Fig. S1). From this figure, we could conclude the characteristic absorption peak of 3411 cm 1 belonged to the stretching vibration of N–H and the peak of 1531 cm 1 belonged to the bending vibration of amide, the characteristic absorption peak of C¼ O was 1665 cm 1, as well as the asymmetrical stretching vibration (1221 cm 1) and symmetrical stretching vibration (1115 cm 1) of C–O manifested the existence of methyl methacrylate. The information mentioned in the Fig. S1 demonstrated
Fig. 1. (a) SEM micrography of copolymer showing the network structure of the substrate. (b) SEM image taken after cell capture, captured cells and materials were treated with fixation and dehydration. (c) EDS analysis of APBA modified dendrimer-copolymer testified the APBA was successfully immobilized on the substrate.
P. Zhang et al. / Talanta 153 (2016) 366–371
Fig. 2. The impact of incubation time on capture efficiency. Judged by the graph, capture efficiency could reach a higher level under the condition that the incubation time no less than 45 min.
the formation of PAM-PMMA copolymer. To prove the feasibility of the protocols, we arranged experiments to testify whether the copolymer was successfully modified with dendrimers and APBA. In order to inspect whether dendrimers were conjugated with the copolymer, we utilized the ultraviolet absorption spectrum to detect the variation of absorbance before and after this reaction (Fig. S2). Judged from it, we could distinctly observe the decrease of the absorption peak after the conjugation reaction, which manifested the amount of dendrimers in the supernate were fewer than in the stock solution, thus a number of dendrimers were bonded with the copolymer. Besides its hydrophilic characterization, the dendrimer had a rich amount of amino, so that the dendrimer was easily to react with other reactants that were able to capture cells. Giving to the properties of dendrimers, APBA which had boric acid group that could react with cis-dihydroxy of sialic acid in cell membrane was selected as the “baits” to target cells. Hence, the copolymer immobilized with dendrimer was reacted with APBA sequentially. To confirm the APBA was modified onto the materials, energy dispersive spectrometer (EDS) was conducted and the image (Fig. 1c) proved the feasibility. In this figure, the atomic percentage of boron was 20.9% that confirmed the APBA was tightly bonded with dendrimers and the large number of amino groups of the dendrimer had good modification performance in this reaction. Meanwhile, the boric acid group could bond with alizarin red S (ARS) [39] which was able to emit green fluorescence, while the group free of the modification of APBA could not emit green fluorescence as Fig. S3 showed. Therefore, all of these results mentioned above demonstrated that we successfully fabricated the 3D network dendrimer-copolymer which was modified with APBA. 3.2. Cell capture assay and optimization of experimental conditions As the APBA modified dendrimer-copolymer possessed the following abilities: soft surface, hydrophilic characterization, 3D network morphology and enhanced modification of APBA, it was easy to trap the cells in the pores and react with APBA to intensify the interaction between materials and target cells. Hence, we designed cell capture assays to evaluate its capture efficiency and optimize experimental conditions. As the foundation of polymerization, the optimal dosage of functional monomer methyl methacrylate (MMA) was one of the key points that we concerned about. By setting the usage of MMA as a single variable, we prepared copolymer of which the usage of MMA was 4.256 μL, 2.128 μL, 1.064 μL, 0.638 μL and 0.425 μL, respectively. After modification, the materials mentioned above were applied in cell capture assay and then calculated the capture
369
efficiency separately. As the result shown in Fig. S4, we could draw the conclusion that 1.064 μL MMA was the optimal volume in all the treatment groups for it might balance the extent of polymerization to form the cell-fitting pores and the quantity of modification sites which were from MMA hydrolysis. To increase the number of modification sites and keeping the hydrophilicity, we went on searching for the better modification amount of dendrimer. The copolymer was modified within different amount of dendrimer as follows: 0.75 μL, 1.5 μL, 2.5 μL, 5.0 μL, 7.5 μL and 10.0 μL, respectively. The result was also examined by cell capture tests and shown in Fig. S5. The figure revealed that the better usage of dendrimer was 7.5 μL, for the reason that less amount of dendrimer offered less amino groups for the reaction with APBA which would finally reduce the captured cells, while excess amount of dendrimer might lead to overload and instability that were adverse to capture efficiency as well. In order to improve the capture efficiency, the “bait”-APBA was the critical factor. When the modified quantity rose, the materials could provide more boric acid groups to target on the sialic acid in the cytomembrane of U251 cells. As a result, we designed six treatment groups with different usage of APBA and the concentrations were 2, 5, 10, 15, 20 and 25 mg/mL. After reaction and wash, the APBA modified copolymer was used in cell capture assay to characterize the capture ability. Last but not least, the operating conditions, temperature and reaction time should keep constant. As it was shown in Fig. S6, the material had a better capture capacity when the concentration of APBA was 10 mg/mL. To better investigate the relation between incubation time and cell capture efficiency, we prepared identical modified materials to conduct cell capture experiments and added the same amount of cells in 96-well plate, then incubated 5, 15, 30, 45, 60, 90 min, respectively. With incubation time extending, capture efficiency increased and when time went up to 45 min, 87 75% of cells were captured by the material (Fig. 2). Due to deficiently contact, the capture efficiency was low within short incubation time. As time extending, more and more cells trapped in the 3D network copolymer and reacted with APBA modified on the material that contributed to the high capture efficiency. Following by the optimization of synthetic procedures and conditions, the capture efficiency upgraded step by step from 21% up to surpassing 90%. This result firmly indicated that the functional copolymer substrate bonded the targeted tumor cells efficiently. Compared with the materials of phenylboronic acid-containing polymer brush [18] and the poly(APBA) nanostructures substrates [31], our APBA functionalized copolymer showed a higher efficiency under the same incubation conditions (the capture efficiency of polymer brush was 60% and of poly APBA substrates was 80%) and caused less damage to captured cells. The 3D network structure significantly enhanced the contact between cells and materials, and the illustration of this concept was concisely displayed in Fig. 3. Furthermore, dendrimers and APBA were modified surrounding the network structure, so the 3D structure could get pseudopod trapped in and let APBA easily react with sialic acid in this way. In virtue of the functional 3D network structure, the APBA modified dendrimer-copolymer exhibited great potential in cell isolation. To further investigate the function of 3D network copolymer in respect of structure and modification, we designed contrast experiments as follows: we prepared the APBA modified dendrimercopolymer and the APBA directly modified copolymer without the addition of dendrimer as capture substrates, meanwhile the synthesized PAM-PMMA copolymer exempted from the modification of dendrimer and APBA was individually used in cell capture assay as blank control group. Under the same operation and condition, the capture efficiency of APBA modified dendrimer-copolymer
370
P. Zhang et al. / Talanta 153 (2016) 366–371
Fig. 3. A simple illustration that showed how the U251 targeted on the functional 3D network copolymer.
Fig. 4. Experimental results that displayed the influence of 3D network structure and modification on the capture efficiency. (a) The column chart provided the capture efficiency of APBA modified dendrimer-copolymer, APBA modified copolymer without dendrimer and copolymer without any modification, which manifested the synergistic effect of dendrimer and 3D network copolymer. (b)–(d) Fluorescence images of the APBA modified group with dendrimer, APBA modified group without dendrimer and blank control group (copolymer without modification), respectively.
could reach nearly 90%, while the efficiency of APBA directly modified copolymer without dendrimer was 30.4 75% and the efficiency of the synthesized PAM-PMMA copolymer without modification was only 7 7 3% (Fig. 4a). This result was also confirmed by fluorescence image shown in Fig. 4b–d. The fluorescence images mentioned above demonstrated that the APBA modified dendrimer-copolymer had the higher density of fluorescent spots which meant more cells bonded with the material there. Judged by the results of control experiments, this 3D network copolymer offered large space for reaction and the low non-specific character was another superiority of this material. So that it could make cells easily caught in the material which provided great advantage in the cell capture assay. What's more, the use of dendrimers tremendously enhanced the volume of modification, so a great deal of APBA was utilized to react with sialic acid to increase the capture efficiency. To testify the influence of the copolymer to cell viability, we conducted AO/PI bi-label assay to test the viability of captured U251. After 2 h, the captured U251 on the copolymer was stained by AO/PI and washed with PBS for three times. The fluorescence
Fig. 5. Fluorescence image of AO/PI, which proved the capture U251 remained excellent viability.
P. Zhang et al. / Talanta 153 (2016) 366–371
image (Fig. 5) indicated almost all the cells kept alive and the viability nearly 100%. This result was attributed to that the copolymer and dendrimers were extremely hydrophilic and the surface of the material was soft and smooth. To sum up, our experimental results prove the functional 3D network copolymer have great potential in cells isolation and capture.
[4]
[5]
[6]
4. Conclusions We successfully synthesized the functional dendrimer modified 3D network copolymer as cell capture substrate in a simple and inexpensive way. The 3D network copolymer possessed hydrophilic surface and macroporous structure which enhanced the contact probability of cells and also induced the cells trapped in the pores for the consequent reaction. In addition, the usage of dendrimers kept the hydrophilic character of the copolymer and provided a great number of modification sites for the chemical conjugation with APBA that immensely improved the bonding quantity of APBA. The APBA modified copolymer was similar to the fishing net with baits and the dendrimer was acted as the bridge between the copolymer and functional group. As a result of the unique structure and functional modification, the capture efficiency of the material could reach 877 5% in 45 min. Moreover, the functional dendrimer modified copolymer was hypotoxicity and provided the soft and hydrophilic surface for the capture assay so that the viability of captured cells was nearly 100% after captured for 2 h. To sum up, the functional 3D network copolymer has a promising prospect of application in cell isolation.
Conflict interest The authors declare no competing financial interest.
[7] [8] [9] [10] [11]
[12] [13] [14]
[15] [16] [17] [18] [19] [20] [21] [22]
[23] [24] [25]
Acknowledgment [26]
This work was supported by National Basic Research Program of China (Project: 2012CB910604), the National High-Tech R&D Program (Project: 2012AA020202) and the National Natural Science Foundation of China (Project: 21275034 and 21475027).
[27] [28] [29] [30]
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2016.03. 044.
[31] [32]
[33] [34] [35]
References [1] S.M. Leong, K.M.L. Tan, H.W. Chua, D. Tan, D. Fareda, S. Osmany, M. Li, S. Tucker, E.S.C. Koay, J. Hematol. Oncol. 8 (2015) 75–82. [2] H.I. Scher, X. Jia, J.S. de Bono, M. Fleisher, K.J. Pienta, D. Raghavan, G. Heller, Lancet Oncol. 10 (2009) 233–239. [3] J. Hou, M.G. Krebs, L. Lancashire, R. Sloane, A. Backen, R.K. Swain, L.J.C. Priest,
[36] [37] [38] [39]
371
A. Greystoke, C. Zhou, K. Morris, T. Ward, F.H. Blackhall, C. Dive, J. Clin. Oncol. 30 (2012) 525–532. M.G. Krebs, R. Sloane, L. Priest, L. Lancashire, J. Hou, A. Greystoke, T.H. Ward, R. Ferraldeschi, A. Hughes, G. Clack, M. Ranson, C. Dive, F.H. Blackhal, J. Clin. Oncol. 29 (2011) 1556–1563. C. Aggarwal, N.J. Meropol, C.J. Punt, N. Lannotti, B.H. Saidman, K.D. Sabbath, N. Y. Gabrail, J. Picus, M.A. Morse, E. Mitchell, M.C. Miller, S.J. Cohen, Ann. Oncol. 24 (2013) 420–428. E. Denève, S. Riethdorf, J. Ramos, D. Nocca, A. Coffy, J. Daurès, T. Maudelonde, J. Fabre, K. Pantel, C. Alix-Panabières, Clin. Chem. 59 (2013) 1384–1392. J.M. Lang, B.P. Casavant, D.J. Beebe, Sci. Transl. Med. 4 (2012) 141–143. C. Alix-Panabières, K. Pantel, Nat. Rev. Cancer 14 (2014) 623–631. H.K. Lin, S. Zheng, A.J. Williams, M. Balic, S. Groshen, H.I. Scher, M. Fleisher, W. Stadler, R.H. Datar, Y. Tai, R.J. Cote, Clin. Cancer. Res. 16 (2010) 5011–5018. H.W. Hou, M.E. Warkiani, B.L. Khoo, Z.R. Li, R.A. Soo, D.S. Tan, W.T. Lim, J. Han, A.A. Bhagat, C.T. Lim, Sci. Rep. 3 (2013) 1259–1266. E. Sollier, D.E. Go, J. Che, D.R. Gossett, S. O'Byrne, W.M. Weaver, N. Kummer, M. Rettig, J. Goldman, N. Nickols, S. McCloskey, R.P. Kulkarni, D. Di Carlo, Lab Chip 14 (2014) 63–77. C. Wen, L. Wu, Z. Zhang, Y. Liu, S. Wei, J. Hu, M. Tang, E. Sun, Y. Gong, J. Yu, D. Pang, ACS Nano 8 (2014) 941–949. N. Lu, M. Xie, J. Wang, S. Lv, J. Yi, W. Dong, W. Huang, ACS Appl. Mater. Interfaces 7 (2015) 8817–8826. S. Wang, K. Liu, J. Liu, Z.T.-F. Yu, X. Xu, L. Zhao, T. Lee, E.K. Lee, J. Reiss, Y.K. Lee, L.W.K. Chung, J. Huang, M. Rettig, D. Seligson, K.N. Duraiswamy, C.K.-F. Shen, H. Tseng, Angew. Chem. Int. Ed. 123 (2011) 3140–3144. S. Wang, H. Wang, J. Jiao, K. Chen, G.E. Owens, K. Kamei, J. Sun, D.J. Sherman, C. P. Behrenbruch, H. Wu, H. Tseng, Angew. Chem. Int. Ed. 121 (2009) 9132–9135. L. Chen, X. Liu, B. Su, J. Li, L. Jiang, D. Han, S. Wang, Adv. Mater. 23 (2011) 4376–4380. Y. Zhao, X. Zhu, H. Liu, Y. Luo, S. Wang, M. Shen, M. Zhu, X. Shi, J. Mater. Chem. B 2 (2014) 7384–7393. H. Liu, Y. Li, K. Sun, J. Fan, P. Zhang, J. Meng, S. Wang, L. Jiang, J. Am. Chem. Soc. 135 (2013) 7603–7609. J. Lee, S. Chung, H. Cho, D. Kim, Adv. Funct. Mater. 25 (2015) 3705–3717. J. Meng, H. Liu, X. Liu, G. Yang, P. Zhang, S. Wang, L. Jiang, Small 10 (2014) 3735–3741. C. Huang, G. Yang, Q. Ha, J. Meng, S. Wang, Adv. Mater. 17 (2015) 310–313. S. Nagrath, L.V. Sequist, S. Maheswaran, D.W. Bell, D. Irimia, L. Ulkus, M. R. Smith, E.L. Kwak, S. Digumarthy, A. Muzikansky, P. Ryan, U.J. Balis, R. G. Tompkins, D.A. Haber, M. Toner, Nature 450 (2007) 20–27. C. Sun, R. Zhang, M. Gao, X. Zhang, Anal. Bioanal. Chem. 470 (2015) 8883–8892. P. Zhang, R. Zhang, M. Gao, X. Zhang, ACS Appl. Mater. Interfaces 6 (2014) 370–376. H. Yoon, T.H. Kim, Z. Zhang, E. Azizi, T.M. Pham, C. Paoletti, J. Lin, N. Ramnath, M.S. Wicha, D.F. Hayes, D.M. Simeone, S. Nagrath, Nat. Nanotechnol. 194 (2013) 735–742. L. Ma, G. Yang, N. Wang, P. Zhang, F. Guo, J. Meng, F. Zhang, Z. Hu, S. Wang, Y. Zhao, Adv. Healthc. Mater. 4 (2015) 838–843. D.A. Tomalia, J.M.J. Frechet, Dendrimers and Other Dendritic Polymers, John Wiley & Sons Ltd, New York, 2001. Y. Wang, J. Wang, M. Gao, X. Zhang, J. Mater. Chem. B 3 (2015) 8711–8716. A. Matsumoto, N. Sato, Ki Kataoka, Y. Miura, Y. Miyahara, J. Am. Chem. Soc. 131 (2009) 12022–12023. C. Bull, M.A. Stoel, M.H. den Brok, G.J. Adema, Cancer Res. 74 (2014) 3199–3204. J. Ouyang, M. Chen, W. Bao, Q. Zhang, K. Wang, X. Xia, Adv. Funct. Mater. 10 (2015) 6122–6130. S. Deshayes, H. Cabral, T. Ishii, S. Kobayashi, T. Yamashita, A. Matsumoto, Y. Miyahara, N. Nishiyama, K. Kataoka, J. Am. Chem. Soc. 135 (2013) 15501–15507. Q. Wu, L. Wang, H. Yu, J. Wang, Z. Chen, Chem. Rev. 111 (2011) 7855–7875. J. Gregory, J. Cannell, M. Kofron, L. Yeghiazarian, V. Nistor, J. Appl. Polym. Sci. 132 (2015) 41557–41563. P. Nghe, S. Boulineau, S. Gude, P. Recouvreux, J.S. van Zon, S.J. Tans, PloS One 8 (2013) 75537–75548. A.A. Adewunmi, S. Ismail, A.S. Sultan, J. Appl. Polym. Sci. 132 (2015) 42110–42118. W. Lee, Y. Fu, J. Appl. Polym. Sci. 89 (2003) 3652–3660. R. Zolfaghari, A.A. Katbab, J. Nabavizadeh, R.Y. Tabasi, M.H. Neyad, J. Appl. Polym. Sci. 100 (2006) 2096–2103. J. Liu, K. Yang, Y. Qu, S. Li, Q. Wu, Z. Liang, L. Zhang, Y. Zhang, Chem. Commun. 51 (2015) 3896–3898.