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Photo-induced surface grafting of phosphorylcholine containing copolymers onto mesoporous silica nanoparticles for controlled drug delivery
Materials Science and Engineering C 79 (2017) 596–604
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Photo-induced surface grafting of phosphorylcholine containing copolymers onto mesoporous silica nanoparticles for controlled drug delivery Liucheng Mao a,1, Meiying Liu a,1, Long Huang a, Dazhuang Xu a, Qing Wan a, Guangjian Zeng a, Yanfeng Dai a, Yuanqing Wen a,⁎, Xiaoyong Zhang a,⁎, Yen Wei b,⁎ a b
Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China Department of Chemistry, Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, PR China
a r t i c l e
i n f o
Article history: Received 9 April 2017 Received in revised form 15 May 2017 Accepted 16 May 2017 Available online 17 May 2017 Keywords: Mesoporous silica nanoparticles Surface-initiated polymerization Photo-induced ATRP Controlled drug release Zwitterionic phosphorylcholine
a b s t r a c t Surface modification of mesoporous silica nanoparticles (MSNs) with functional polymers has become one of the most interest topics over the last decade. Among various surface modification strategies, surface-initiated atom transfer radical polymerization (ATRP) has been regarded as one of the most effective methods. However, the typical ATRP strategy is relied on the transition metal ions and their organic ligands as the polymerization catalyst systems. In this work, a novel surface-initiated ATRP method was established for surface functionalization of MSNs using 10-Phenylphenothiazine (PTH) as the catalyst, 2-methacryloyloxyethyl phosphorylcholine (MPC) and itaconic acid (IA) as the monomers. We demonstrated that photo-induced ATRP is very effective for preparation of polymer functionalized MSNs (MSNs-NH2-poly(IA-co-MPC)). More importantly, MSNs-NH2-poly(IAco-MPC) displayed well water dispersity, low cytotoxicity, high loading capability and controlled release behavior towards cisplatin. Furthermore, the method based on photo-induced surface-initiated ATRP could effectively overcome the drawbacks of conventional ATRP, which may involve in the residue of transition metal ions, high polymerization temperature, long polymerization term and complex experimental procedure. Therefore, this strategy described above is of great interest for fabrication of multifunctional polymer composites for various applications. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hybrid materials and inorganic nanomaterials have tremendous promise for a number of biotechnological and information technology applications such as biological imaging, sensor technology, photodynamic therapy, gene delivery, microarrays and optical computing [1– 12]. Among them, the development of inorganic mesoporous silicon materials is particularly rapid. Since Kresge reported the preparation of mesoporous silica nanoparticles (MSNs) using cationic surfactants as template in 1992, MSNs have attracted intensive attention of scientists over the past few decades [13–18]. Up to now, various types of MSNs and their composites have been widely exploited for different applications in the fields of separation, catalysis, optoelectronic devices, micro-electronic technology, chemical sensors, nonlinear optical materials and biological medicine because of its characteristics of high ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Wen), [email protected] (X. Zhang), [email protected] (Y. Wei). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.msec.2017.05.107 0928-4931/© 2017 Elsevier B.V. All rights reserved.
specific surface areas, regular pore structure, narrow pore size distribution and the size of the continuous adjustable [19–34]. To achieve better performance and endow novel properties, the integration of MSNs with other functional components such as metal nanoparticles, metal oxides, semiconductor quantum dots and polymers is necessary [15,19,35–46]. Among them, the fabrication of MSNs based polymer nanocomposites has raised the most interest due to the combination advantages from both of polymers and MSNs [19,35,36]. Surface modification of MSNs with polymers through different surface-initiated polymerization strategies has demonstrated to be the effective routes for preparation of MSNs based polymer nanocomposites [47–52]. Over the past few decades, many polymerization methods have been developed and the resultant MSNs based polymer nanocomposites have shown great potential for different applications especially in the biomedical fields. The commonly utilized methods for surface modification of MSNs are atom transfer radical polymerization (ATRP), stable free radical polymerization (SFRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization [53–65]. These polymerization methods have been used for decades, the technology has been very mature. But they still have their own shortcomings,
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and there is an urgent need for them to make further improvements. For example, the typical ATRP is relied on metal ion catalysts and their organic ligands to initiate the polymerization under inert atmosphere and at elevated temperature. It is well known that the metal ion catalysts are difficult to be removed from the polymerization system and they are toxic towards living organisms or impeded the performance of resultant polymeric composites. On the other hand, the typical ATRP are required at high temperature to active the initiator for the polymerization procedure [65–70]. Therefore, it is highly desirable to develop novel ATRP methods to overcome these issues of traditional ATRP. Metal free ATRP is a recently emerged ATRP method that relied on the organic catalysts under light irradiation to initiate the polymerization [71–73]. In this work, we reported a photo-induced metal free ATRP for surface modification of MSNs using 10-Phenylphenothiazine (PTH) as the organic catalyst. The detailed procedure for the fabrication of MSNs based polymer nanocomposites was shown in Scheme 1. MSNs were prepared using cetyltrimethylammonium bromide (CTAB) as template agent and tetraethyl ortho-rthosilicate (TEOS) as silicon source. Then the amino groups were immobilized onto MSNs to obtain MSNs-NH2 through hydrolysis of (3-aminopropyl)triethoxysilane (APTES), which was used to react with α-Bromoisobutyryl bromide through amidation reaction and removed CTAB to generate Br-containing initiator MSNsBr. The synthetic MSNs-Br can be further used for surface polymerization with itaconic acid (IA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) monomer using PTH as the catalyst. The obtained MSNs-NH2-poly(IA-co-MPC) showed significantly improved dispersibility in various solvents. Furthermore, this facile and efficient method described in this work is also suitable for the surface modification of other materials. 2. Experiments
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2.2. Characterization Fourier transform infrared (FT-IR) spectra were performed on a Nicolet 380 Fourier transform spectrometer with a resolution of 2 cm−1. UV-Visible absorption spectra were obtained by Perkin Elmer LAMBDA35 UV/Vis spectroscopy. Transmission electron microscopy (TEM) images were recorded on a Hitachi 7650B microscope operated at 80 kV, the tested specimens were made by placing a drop of the nanoparticles suspension on a carbon-coated copper grid. X-ray photoelectron spectra (XPS) were measured on a VGESCALAB 220-IXL spectrometer using an AlKa X-ray source (1486.6 eV). thermogravimetric analysis (TGA) curves were recorded on a Q50 thermo gravimetric analyzer under N2 flow and the samples were heated from 25 to 600 °C with the weight of 5–10 mg. The nitrogen sorption characterization on the samples before and after nitridation was conducted on a Micromeritics ASAP2020 surface area and pore size analyzer at −196 °C (liquid nitrogen temperature) using accompanying software from Micromeritics, Inc. The surface area was calculated by using the conventional BET method. The pore parameters were calculated from the adsorption branches of these isotherms using BJH methods. 2.3. Synthesis of MSNs MSNs were synthesized through the hydrolysis of TEOS in the mixed solution that contained CTAB, NaOH according to our previous report [24]. In brief, CTAB (500 mg, 1.37 mmol) and NaOH (150 mg, 3.75 mmol) were dissolved in 250 mL water. The mixture solution was stirred appropriately 0.5 h at 80 °C in an oil bath. Then TEOS (5.00 mL, 22.4 mmol) was added dropwise to the mixture solution under vigorous stirring. After 4 h, the white precipitation was separated through centrifugation and washed with deionized water and methanol for three times. The MSNs were dried under vacuum oven drying at 40 °C to get dry MSNs.
2.1. Materials 2.4. Synthesis of MSNs-NH2 CTAB and TEOS were supplied by Alfa Aesar. APTES as silane coupling agent was supplied by Aldrich.2-Methacryloyloxy ethyl phosphorylcholine (MPC, Mw: 295.27 Da, 98%), IA (Mw: 130.10 Da, 99%), α-bromoisobutyryl bromide (Mw: 229.90 Da, 98%) were purchased from Aladdin (Shanghai, China). PTH was synthesized by phenothiazine and iodobenzene according to previous report. Acetone and toluene were obtained by Heowns (Tianjin, China). cisdichlorodiamineplatinum(II) (CDDP) was purchased from huaxia (Chengdu, China). All chemical agents were of analytical grade and were used directly without any purification.
The MSNs (1.5 g) and toluene (50 mL) were added into a round bottom flask of 100 mL. The mixture was stirred at room temperature about 20 min to make a well dispersed MSNs toluene solution. Then the APTES (0.75 mL) was added and refluxed at 110 °C for 3 h. After that, the white precipitate (MSNs-NH2-1) was separated by centrifugation at 8000 rpm for 3 min. MSNs-NH2-1 was further washed with toluene and isopropanol three times and dried at 40 °C in the vacuum oven. In order to remove the excess surfactant CTAB, 1.00 g of MSNs-NH2-1 was refluxed for 24 h in a methanolic solution that contains 6.00 mL
Scheme 1. Schematic showing the synthesis of MSNs-NH2-poly(IA-co-MPC).
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of HCl (37.4%) in 100.00 mL of methanol. The white precipitate (MSNsNH2) was separated by centrifugation at 8000 rpm for 3 min. The MSNsNH2 was washed with distilled water and methanol for several times and dried in vacuum oven at 35 °C.
and test concentrations per microplate, and the experiment was repeated three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as mean ± standard deviation (SD).
2.5. Synthesis of MSNs-NH2-Br
3. Results and discussion
The amidation reaction between MSNs-NH2 and 2-bromo-2methylpropionyl bromide was used to prepare the Br-containing initiator. MSNs-NH2 (300 mg) and triethylamine (60 mg) were added to toluene (40 mL) under N2. When the temperature of system achieved to 5 °C, a toluene solution of 30 μL of 2-bromo-2-methylpropionyl bromide in 10 mL of toluene was put dropwise into the reaction system. Mixture solution was stirred violently in ice water bath for 4 h. The solid product was separated by centrifuging at 8000 rpm for 3 min. The centrifugal product was washed with water and acetone three times and dried in vacuum oven at 40 °C to get MSNs-NH2-Br.
The successful preparations of MSNs and MSNs-NH2 were first confirmed by TEM. It can be seen that MSNs are spherical morphology with size about 120–180 nm (Fig. 1A). The high-magnification TEM image of MSNs vaguely shows the ordered and uniform pore size in the interior of MSNs. It indicates that taking a clear mesostructure in a TEM image is very difficult before removal of the template (Fig. 1B). After removal of the template, the highly ordered and unified mesostructure can be observed clearly in the TEM image of MSNs-NH2 (Fig.1C). When the scale is 20 nm, the mesoporous structure is more clearly displayed in the Fig. 1D. On the other hand, the regular pores with diameter about 4 nm were also distinguished through high resolution TEM image. However, the pores had disappeared on the edge of silica spheres, which may be because of the fact the continuous bombardment of high energy electron beams leads to the collapse of the pore structure. The pore structure of MSNs, MSNs-NH2 and MSNs-NH2-poly(IA-coMPC) materials is also evaluated by N2 sorption studies. The N2 sorption isotherms of these materials are presented in Fig. 2(A) and the corresponding textural properties are summarized in Table 1. MSNs exhibit type IV isotherms with a typical capillary condensation step into uniform pores without hysteresis. Compare with the N2 sorption isotherms of MSNs and MSNs-NH2, the adsorption capacity of MSNs-NH2 becomes larger, which can be attributed to the removal of template. The amount of adsorbed N2 of MSNs-NH2-poly(IA-co-MPC) decreased sharply compare with the amount of adsorbed N2 of MSNs-NH2, which can be attributed to the grafting of monomers. As shown in Table 1 and Fig. 3(B) the pore size increase obviously after removing the template. The aperture of MSNs after removing the template is about 4 nm, This is consistent with the previously mentioned TEM results. The grafting of polymers reduced BET surface area and BJH volume to 308.85 m2/g and 0.4012 cm3/g. 1 H NMR spectrum of MSNs-NH2-poly(IA-co-MPC) was shown in Fig. 3. A lot of important information can be directly perceived from the 1H NMR spectrum. The detailed information of 1H NMR spectrum in DMSO was listed below. For example, -NH-CO- (d, 7.92), -CH2-NH- (c, 2.85) indicted that the amino groups are successfully bound to the surface of MSNs by hydrolysis between APTES and the hydroxyl groups on the surface of the silica sphere. Subsequently, the amidation reaction occurred between the amino group and α-Bromoisobutyryl bromide. That was apparent from -NH-CO- (d, 7.92), -C-CH3 (e, 1.21). -CH2- (f, 1.91), -CH2-COOH (g, 2.73) can be attributed to the monomer of IA. -CH2- (h, 2.73), -CH3 (i, 1.91), -CH2- (j,k,l,m, 3.42) can be attributed to the monomer of MPC. The above results from 1H NMR spectrum indicated that we have successfully synthesized MSNs-NH2-poly(IA-co-MPC) via the photo-induced surface-initiated ATRP. The chemical information and functional groups of samples were characterized by FT-IR spectroscopy and FT-IR spectra of MSNs, MSNsNH2 and MSNs-NH2-poly(IA-co-MPC) were shown in Fig. 4. All these spectra display an obvious absorption band at 1070 cm−1. This is no doubt that these are silicon-oxygen bond (Si\\O). On the other hand, the C\\H stretching vibration that normally distributed between 2800 and 3000 cm−1 was found in the sample of MSNs. It can evidence the existence of CTAB. As compared with MSNs, the two peaks at 2800– 3000 cm−1 were disappeared in the sample of MSNs-NH2. Moreover, a characteristic absorbance band at 3485 cm− 1 was appeared in the spectrum of MSNs-NH2. This implied that amino groups have been successfully immobilized on the surface of MSNs. Based on the above comparison, we can conclude that CTAB has been removed from MSNs and amino groups have introduced on MSNs. However, in the spectrum of MSNs-NH2-poly(IA-co-MPC), the absorption band at 2920 cm−1 was
2.6. Preparation of MSNs-NH2-poly(IA-co-MPC) Grafting polymer is one of the most important steps in the surface modification of MSNs. MSNs-NH2-Br (150 mg), MPC (567 mg, 1.92 mmol), IA (250 mg, 1.92 mmol) and PTH (3 mg) were dispersed in 3 mL THF in the schlenk flask under N2. Then the schlenk flask was irradiated with LEDs at 380 nm under room temperature for 7 h. The MSNs-NH2-poly(IA-co-MPC) was separated by centrifugation and washing with distilled water and THF several times. The resultant solid was dried overnight in a vacuum at 40 °C. 2.7. CDDP loading and release behavior MSNs-NH2-poly(IA-co-MPC) (20 mg) and 10 mg cis-dichlorodiam mineplatinum(II) (CDDP) was dispersed in 100 mL PBS (pH = 7.4). The above solution was stirred under dark at 37 °C for 48 h. After that, the drug loading complexes MSNs-NH2-poly(IA-co-MPC)-CDDP were separated by centrifuging at 8000 rpm for 3 min. The concentration of CDDP in the supernatant was measured and calculated based on the absorption of UV-Visible spectrometer at 705 nm according to previous report. To determine their release behavior, MSNs-NH2-poly(IA-co-MPC)CDDP was divided into two portions and put into the dialysis bag (3500 Da). The pH values are (pH = 5.6 and 7.4). At different time points, the concentration in PBS was measured and the content of CDDP in PBS was calculated based on the standard curve of CDDP. In order to confirm the loading position and release behavior of CDDP, the amount of CDDP adsorbed into the MSNs-NH2 was measured by the same method. 2.8. Cytotoxicity of MSNs-NH2-poly(IA-co-MPC) To evaluate the potential biomedical applications of MSNs-NH2poly(IA-co-MPC), the cell viability of MSNs-NH2-poly(IA-co-MPC) towards A549 cells was examined using cell counting kit-8 (CCK-8) [74– 76]. In brief, A549 Cells were seeded in 96-well microplates at a density of 5 × 104 cells per mL in 160 μL of the respective media containing 10% fetal bovine serum (FBS). After 24 h of cell attachment, A549 cells were incubated with 10–120 μg mL−1 of MSNs-NH2-poly(IA-co-MPC) for 12 and 24 h. Then the cells were washed with PBS for three times to remove the uninternalized nanoparticles. After that, 10 μL of CCK-8 dye and 100 μL of Dulbecco's Modified Eagle's Medium (DMEM) cell culture media was added to each well and incubated for 2 h at 37 °C. Plates were then analyzed with a microplate reader (VictorIII, Perkin-Elmer). Measurements of dye absorbance were carried out at 450 nm, with the reference wavelength at 620 nm. The values were proportional to the number of live cells. The percent reduction of WST was compared to the control (cells not exposed to nanoparticles), which represented 100% WST reduction. Three replicate wells were used for each control
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Fig. 1. TEM images of MSNs. (A) Scale bar = 200 nm, (B) Scale bar = 100 nm. TEM images of MSNs-NH2. (C) Scale bar = 50 nm, (D) Scale bar = 20 nm.
emerged again. This suggested that the MPC have been grafted on the sample of MSNs through photo-induced surface-initiated ATRP. On the other hand, the some other characteristic bonds such as 1710 cm− 1 (C_O), 1660 cm−1 (C\\O) and 1410 cm−1 (C\\H) were also found in the sample of MSNs-NH2-poly(IA-co-MPC). The above results further evidenced indicated that the monomers of IA and MPC have been introduced on the surface of MSNs. Fig. 5 shows the TGA curves of MSNs, MSNs-NH2, and MSNs-NH2poly(IA-co-MPC). It clearly shows that the weight loss of MSNs-NH2 and MSNs-NH2-poly(IA-co-MPC) are much larger than that of MSNs. Significant weight loss at the temperature 100–200 °C was found in the curve of MSNs. The weight loss should be ascribed to the decomposition of CTAB. However, no obvious weight loss was occurred, suggesting that MSNs possess good thermal stability. On the contrary, there is a uniform weight loss (12%) between 100 and 600 °C from the TGA curve of MSNs-NH2. This implied that APTES was immobilized on MSNs through hydrolysis reaction. Furthermore, more significant weight loss was found in the sample of MSNs-NH2-poly(IA-co-MPC). The total weight loss percentage was achieved about 30% between 100 and 600 °C. As compared to MSN-NH2, the weight loss of MSNs-NH2-poly(IAco-MPC) increased by 17%, which means that the weight of polymer in the total weight is about 17%. The significant weight loss given
powerful evidence about the successful preparation of monomers MSNs-NH2-poly(IA-co-MPC). XPS is one of the most useful techniques for determination of chemical compositions of materials and surface. The survey XPS spectra ranging from 0 to 1400 eV were examined and results were displayed in Fig. 6. Elements of Si, O, C and N were observed in the sample of MSNs (Fig. 6A). The elements such Si and O are come from the hydrolysis of TEOS, while the C and N should be derived from CTAB. In Fig. 6B, the peak intensity of Si and O are significantly reduced, while the peak intensity of C was increased. This suggested that APTES has been successfully attached on the MSNs. As compared with the spectrum of MSNsNH2, the new element Br was emerged in the sample of MSNs-NH2-Br, suggesting the bromine-containing initiator has immobilized on the surface of MSNs-NH2 through amidation reaction (Fig. 6C). Moreover, the element of P as a unique element of MPC was appeared in the sample of MSNs-NH2-poly(IA-co-MPC), giving the powerful evidence for the surface modification of MSNs with MPC. On the other hand, the intensity of Si was further decreased after surface modification with copolymers while the intensity of C, N and O was relatively enhanced in the sample of MSNs-NH2-poly(IA-co-MPC), further confirming the successful grafting of MPC and IA on the surface of MSNs-NH2-poly(IA-coMPC). All of the above XPS results indicated that we have successfully
Fig. 2. (A) Adsorption–desorption isotherms of some samples at 77 K; (B) BJH pore size distribution of some samples calculated from the adsorption branch of the isotherm.
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Table 1 Textural properties of MSNs, MSNs-NH2 and MSNs-NH2-poly(IA-co-MPC). Sample
ABET (m2/g)
DBJH (nm)
VBJH (cm3/g)
MSNs MSNs-NH2 MSNs-NH2-poly(IA-co-MPC)
47.79 642.16 308.85
22.88 3.95 4.4905
0.08 0.7987 0.4012
ABET, BET surface area; DBJH, BJH pore diameter; VBJH, BJH volume.
modified MSNs with poly(IA-co-MPC) through photo-induced surfaceinitiated ATRP. The high resolution XPS spectra of C1s, N1s, O1s, Si2p, P2p and Br3d were shown in Fig. 7. In the spectra of C1s, the peak intensity of C1s in MSNs-NH2 and MSNs-NH2-poly(IA-co-MPC) is increased as compared with MSNs. More importantly, the peak position of C1s in MSNs-NH2poly(IA-co-MPC) has divided into three peaks, suggesting that C were bonded with different atoms (Fig. 7A). The C1s spectrum of MSNsNH2-poly(IA-co-MPC) with binding energy at 288.6 eV should be attributed to the \\O\\C_O, which was derived from IA and MPC. The N1s spectra were displayed in Fig. 7B, the intensity of N1s in the sample of MSNs–NH2 and MSNs-NH2-poly(IA-co-MPC) was also increased as compared with MSNs. Especially, the N1s spectrum in the sample MSNs-NH2-poly(IA-co-MPC) shows a sharp peak in Fig. 7B. The significant increase of N in sample of MSNs-NH2-poly(IA-co-MPC) suggested the MPC that contains -N(CH3)3 has been introduced onto MSNs successfully. Contrast with N, the peak intensity of O1s and Si2p was simultaneously decreased after their surface was modified with APTES and polymers (Fig. 7C and D). The above results clearly evidence that the MSNs were functionalized with poly(IA-co-MPC) through the photo-induced ATRP. The P2p spectra were shown in Fig. 7E. It can be seen that only the sample MSNs-NH2-poly(IA-co-MPC) found the P signal. The P signal should be derived from the MPC and further demonstrated the surface grafting of MSNs with polymers. Moreover, the successful immobilization of ATRP initiator on the surface MSNs and surface grafting of MSNs could also be evidenced by the Br3d spectra (Fig. 7F). For example, we can find intensive Br3d signal in the sample of MSNs-Br, but it was absent in the sample of MSNs and MSNs-NH2. This suggests that
Fig. 4. FTIR spectra of MSNs, MSNs-NH2, and MSNs-NH2-poly(IA-co-MPC).
the ATRP initiator was immobilized on the surface of MSNs-NH2 through amidation reaction. Furthermore, the intensity of Br3d is decreased in the sample of MSNs-NH2-poly(IA-co-MPC) as compared with the sample of MSNs-Br. This further implied that the monomers IA and MPC were grafted on MSNs. The percentages of different elements were calculated by XPS spectra. Table 2 shows the percentages of main elements in MSNs, MSNsNH2 and MSNs-NH2-poly(IA-co-MPC). The percentages of Si, C, N and O in MSNs are 22.42%, 24.94%, 1.56% and 50.85%, respectively. It is worth to mentioning that the percentages of Si and O were 22.65% and 50.85%, respectively. It is close to the expected ratio of Si/O (1:2). Compared with MSNs, the percentages of Si, C, N and O in the samples of MSNs-NH2 are changed to 17.2%, 41.14%, 4.13% and 37.36%, respectively. The increase of N content suggests clearly indicated that APTES has immobilized on the surface of MSNs through hydrolysis reaction. Moreover, the element Br with 1.35% was emerged in the sample of
Fig. 3. 1H NMR spectrum of MSNs-NH2-poly(IA-co-MPC). The characteristic signals suggest that MPC and IA were grafted onto MSNs through photo-induced surface-initiated ATRP.
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Fig. 5. TGA curves of MSNs, MSNs-NH2, and MSNs-NH2-poly(IA-co-MPC).
MSNs-Br. This gives powerful evidence for the linkage of ATRP initiator on MSNs through amidation reaction. However, the contents of Si and O in the sample of MSNs-NH2-poly(IA-co-MPC) was further decreased, while the percentage of P was obviously increased. The above changes suggested that MPC and IA were successfully introduced onto MSNs through the metal-free surface-initiated ATRP.
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Due to the introduction of IA onto the surface of MSNs, the loading behavior of MSNs-NH2-poly(IA-co-MPC) towards to CDDP was further examined. It is well known that the CDDP can coordinate with carboxyl groups. Therefore, CDDP can be loaded onto MSNs-NH2-poly(IA-coMPC) and the drug loading capability might be adjusted through the feed ratio of monomer IA and MPC. In this work, the concentrations of CDDP are determined using the UV-Visible spectroscopy. The CDDP and 1, 2-diaminobenzene can form complex and shows maximum absorption wavelength at 705 nm. The concentrations of CDDP are positively related with the absorption of CDDP-1, 2-diaminobenzene complexes at 705 nm and can be calculated using the standard curve (Fig. S1). Based on the determination, the amount of CDDP loaded onto MSNs-NH2-poly(IA-co-MPC) was calculated to be 213 mg/g. The amount of CDDP loaded into MSNs-NH2 was calculated to be 43 mg/g. It indicated that MSNs-NH2 which was not grafted with itaconic acid can also load CDDP with its mesoporous structure. Compare with the amounts of CDDP adsorbed in the MSNs-NH2-poly(IA-co-MPC) and MSNs-NH2, it is no doubt that the loading capacity of the drug in the modified MSNs via metal-free ATRP greatly enhanced. On the other hand, the release behaviors of MSNs-NH2-poly(IA-co-MPC)-CDDP and MSNs-NH2-CDDP were also determined when MSNs-NH2-poly(IA-coMPC)-CDDP and MSNs-NH2-CDDP complexes were dissolved in PBS solution (pH = 7.4, pH = 5.6) (Fig. 8). The results show that the release ratio of MSNs-NH2-poly(IA-co-MPC)-CDDP is significant different under pH = 7.4 and 5.6. For example, the percentage of CDDP release from MSNs-NH2-poly(IA-co-MPC)-CDDP can reach 59% at the solution of pH = 5.6. However, the percentage value is only 18% in PBS solution
Fig. 6. Survey XPS spectra of MSNs, MSNs-NH2, MSNs-Br and MSNs-NH2-poly(IA-co-MPC). The characteristic elements such as Br and P were found in the samples of MSNs-Br and MSNsNH2-poly(IA-co-MPC) confirmed the successful preparation of MSNs-NH2-poly(IA-co-MPC) through photo-induced metal free ATRP.
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Fig. 7. High resolution XPS spectra of MSNs, MSNs-NH2, MSNs-Br and MSNs-NH2-poly(IA-co-MPC). (A) The region of C1s, (B) The region of N1s, (C) The region of O1s, (D) The region of Si2p, (E) The region of P2p, (F) The region of Br3d.
with pH = 7.4 (Fig. 8A). This property provides the potential for targeted cancer therapy. In contrast, the drug release curves of MSNsNH2 were also drawn (Fig. 8B). The release behavior is almost the same under pH = 7.4 and 5.6, which proves MSNs-NH2 have no ability to control-release CDDP through different pH condition. More importantly, because the content of IA in final polymer composites can be tuned through the polymerization procedure, therefore the drug loading capability of the final MSNs polymer composites can also be Table 2 Percentages of Si, C, N and O based on XPS results.
MSNs MSNs-NH2 MSNs-Br MSNs-NH2-poly(IA-co-MPC)
Si(%)
C(%)
N(%)
O(%)
Br(%)
P(%)
22.42 17.2 16.6 2.91
24.94 41.14 44.2 55.82
1.56 4.13 3.63 4.18
50.85 37.36 33.96 33.41
0 0 1.35 0.35
0.23 0.17 0.26 3.33
adjusted. It is therefore, therapeutic effects of the final MSNs-NH2poly(IA-co-MPC)-CDDP would thus be regulated through design of polymerization parameters. Moreover, this method could also use for fabrication of many other functional polymer composites using different monomers. Therefore, it should be a general tool for preparation of MSNs based functional materials with various properties and prospective application potential in different fields. Furthermore, as compared with conventional ATRP that is relied on the transition metal ions and organic ligands as catalyst systems, the method described above provides an alternative and more attractive choice for surface-initiated controlled living polymerization. The cell viability of MSNs-NH2-poly(IA-co-MPC) towards A549 cells were determined by CCK-8 assay. It is well known that MPC is a biocompatible monomer which has been extensively for surface modification of materials for biomedical applications [77]. The poly(MPC) modified materials will simultaneously enhanced water dispersity and biocompatibility [78]. In this work, the cell viability values of MSNs-NH2-
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Fig. 8. The release behaviors of CDDP from MSNs-NH2-poly(IA-co-MPC)-CDDP (A) and MSNs-NH2-CDDP (B) complexes under different pH values (pH = 7.4 and 5.6). The results show that CDDP could be released from MSNs-NH2-poly(IA-co-MPC)-CDDP complexes faster under acidic solution. This implied that MSNs-NH2-poly(IA-co-MPC) can be acted as carriers for controlled delivery of CDDP.
poly(IA-co-MPC) was calculated based on CCK-8 data and results were shown in Fig. 9. It can be seen that the cell viability values were gradually decreased as the increasing of MSNs-NH2-poly(IA-co-MPC) concentrations. However, it is worth to noting that the cell viability values are still maintained at high levels. For example, the cell viability value of MSNs-NH2-poly(IA-co-MPC) at 120 μg mL−1 is as high as 87.9% when the incubation time is 24 h. The preliminary results suggest that MSNs-NH2-poly(IA-co-MPC) possess good biocompatibility and are promising for biomedical applications. Furthermore, the half maximal inhibitory concentration (IC50) values were also calculated. Results suggested that the IC50 values at12 and 24 h are 231.7 and 190.6 μg mL−1, respectively. Combined the high loading capability of CDDP on MSNsNH2-poly(IA-co-MPC), we can speculate that MSNs-NH2-poly(IA-coMPC) can used as promising candidates for pH controlled intracellular delivery of CDDP for cancer treatment. 4. Conclusions In summary, surface modification of MSNs with biocompatible and zwitterionic phosphorylcholine containing copolymers was reported through photo-induced surface-initiated ATRP for the first time. Various characterization techniques demonstrated that MSNs-NH2-poly(IA-coMPC) can be facilely fabricated via the novel ATRP strategy. As compared with traditional ATRP, the photo-induced surface-initiated ATRP can occur under visible light irradiation and room temperature, which can avoid the using transition metal ions/organic ligands catalyst systems and high polymerization temperature. More importantly, the
Fig. 9. Cell viability of MSNs-NH2-poly(IA-co-MPC) with A549 cells for 12 and 24 h. The cells were incubated with different concentrations (10–120 μg mL−1) of MSNs-NH2poly(IA-co-MPC). The cell viability values of MSNs-NH2-poly(IA-co-MPC) was calculated based on CCK-8 assay.
anticancer agent CDDP could be loaded onto MSNs-NH2-poly(IA-coMPC) with high efficiency. The MSNs-NH2-poly(IA-co-MPC) display good water dispersity, high drug-loading capability, pH controlled drug release behavior and desirable biocompatibility. These remarkable properties make MSNs-NH2-poly(IA-co-MPC) promising candidates for pH controlled intracellular drug delivery. Moreover, owing to the universality of photo-induced surface-initiated ATRP, many other functional polymeric composites can be also fabricated. Therefore, the novel ATRP should be an effective and general strategy for preparation of multifunctional biomaterials. Acknowledgements This research was supported by the National Natural Science Foundation of China (Nos. 51363016, 21474057, 21564006, 21561022, 21644014), Natural Science Foundation of Jiangxi Province in China (Nos. 20161BAB203072, 20161BAB213066). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2017.05.107. References [1] R. Lakshminarayanan, E.O. Chi-Jin, X.J. Loh, R.M. Kini, S. Valiyaveettil, Biomacromolecules 6 (2005) 1429–1437. [2] Q. Dou, X. Fang, S. Jiang, P.L. Chee, T.-C. Lee, X.J. Loh, RSC Adv. 5 (2015) 46817–46822. [3] E. Ye, M.D. Regulacio, M.S. Bharathi, H. Pan, M. Lin, M. Bosman, K.Y. Win, H. Ramanarayan, S.Y. Zhang, X.J. Loh, Y.W. Zhang, M.Y. Han, Nanoscale 8 (2016) 543–552. [4] B.M. Teo, D.J. Young, X.J. Loh, Part. Part. Syst. Charact. 33 (2016) 709–728. [5] R. Lakshminarayanan, X.J. Loh, S. Gayathri, S. Sindhu, Y. Banerjee, R.M. Kini, S. Valiyaveettil, Biomacromolecules 7 (2006) 3202–3209. [6] C. Dhand, N. Dwivedi, X.J. Loh, A.N. Jie Ying, N.K. Verma, R.W. Beuerman, R. Lakshminarayanan, S. Ramakrishna, RSC Adv. 5 (2015) 105003–105037. [7] Q.Q. Dou, C.P. Teng, E. Ye, X.J. Loh, Int. J. Nanomedicine 10 (2015) 419–432. [8] X.J. Loh, T.C. Lee, Q. Dou, G.R. Deen, Biomater. Sci. 4 (2016) 70–86. [9] Z. Li, E. Ye, R. Lakshminarayanan David, X.J. Loh, Small 12 (2016) 4782–4806. [10] H.C. Guo, E. Ye, Z. Li, M.Y. Han, X.J. Loh, Mater. Sci. Eng. C 70 (2017) 1182–1191. [11] K. Huang, Q. Dou, X.J. Loh, RSC Adv. 6 (2016) 60896–60906. [12] E. Ye, X.J. Loh, Aust. J. Chem. 66 (2013) 997. [13] C. Kresge, M. Leonowicz, W. Roth, J. Vartuli, J. Beck, Nature 359 (1992) 710–712. [14] Z. Li, J.C. Barnes, A. Bosoy, J.F. Stoddart, J.I. Zink, Chem. Soc. Rev. 41 (2012) 2590–2605. [15] F. Muhammad, M. Guo, W. Qi, F. Sun, A. Wang, Y. Guo, G. Zhu, J. Am, Chem. Soc. 133 (2011) 8778–8781. [16] F. Zhang, G.B. Braun, Y. Shi, Y. Zhang, X. Sun, N.O. Reich, D. Zhao, G. Stucky, J. Am. Chem. Soc. 132 (2010) 2850–2851. [17] N. Shadjou, M. Hasanzadeh, Mater. Sci. Eng. C 55 (2015) 401–409. [18] M. Moritz, M. Geszke-Moritz, Mater. Sci. Eng. C 49 (2015) 114–151. [19] X. Xu, S. Lü, C. Gao, X. Wang, X. Bai, H. Duan, N. Gao, C. Feng, M. Liu, Chem. Eng. J. 279 (2015) 851–860. [20] D. Dutta, D. Thakur, D. Bahadur, Chem. Eng. J. 281 (2015) 482–490. [21] S. Wang, K. Wang, C. Dai, H. Shi, J. Li, Chem. Eng. J. 262 (2015) 897–903.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Photo-induced surface grafting of phosphorylcholine containing copolymers onto mesoporous silica nanoparticles for controlled drug delivery Liucheng Mao a,1, Meiying Liu a,1, Long Huang a, Dazhuang Xu a, Qing Wan a, Guangjian Zeng a, Yanfeng Dai a, Yuanqing Wen a,⁎, Xiaoyong Zhang a,⁎, Yen Wei b,⁎ a b
Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031, China Department of Chemistry, Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing, 100084, PR China
a r t i c l e
i n f o
Article history: Received 9 April 2017 Received in revised form 15 May 2017 Accepted 16 May 2017 Available online 17 May 2017 Keywords: Mesoporous silica nanoparticles Surface-initiated polymerization Photo-induced ATRP Controlled drug release Zwitterionic phosphorylcholine
a b s t r a c t Surface modification of mesoporous silica nanoparticles (MSNs) with functional polymers has become one of the most interest topics over the last decade. Among various surface modification strategies, surface-initiated atom transfer radical polymerization (ATRP) has been regarded as one of the most effective methods. However, the typical ATRP strategy is relied on the transition metal ions and their organic ligands as the polymerization catalyst systems. In this work, a novel surface-initiated ATRP method was established for surface functionalization of MSNs using 10-Phenylphenothiazine (PTH) as the catalyst, 2-methacryloyloxyethyl phosphorylcholine (MPC) and itaconic acid (IA) as the monomers. We demonstrated that photo-induced ATRP is very effective for preparation of polymer functionalized MSNs (MSNs-NH2-poly(IA-co-MPC)). More importantly, MSNs-NH2-poly(IAco-MPC) displayed well water dispersity, low cytotoxicity, high loading capability and controlled release behavior towards cisplatin. Furthermore, the method based on photo-induced surface-initiated ATRP could effectively overcome the drawbacks of conventional ATRP, which may involve in the residue of transition metal ions, high polymerization temperature, long polymerization term and complex experimental procedure. Therefore, this strategy described above is of great interest for fabrication of multifunctional polymer composites for various applications. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hybrid materials and inorganic nanomaterials have tremendous promise for a number of biotechnological and information technology applications such as biological imaging, sensor technology, photodynamic therapy, gene delivery, microarrays and optical computing [1– 12]. Among them, the development of inorganic mesoporous silicon materials is particularly rapid. Since Kresge reported the preparation of mesoporous silica nanoparticles (MSNs) using cationic surfactants as template in 1992, MSNs have attracted intensive attention of scientists over the past few decades [13–18]. Up to now, various types of MSNs and their composites have been widely exploited for different applications in the fields of separation, catalysis, optoelectronic devices, micro-electronic technology, chemical sensors, nonlinear optical materials and biological medicine because of its characteristics of high ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Wen), [email protected] (X. Zhang), [email protected] (Y. Wei). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.msec.2017.05.107 0928-4931/© 2017 Elsevier B.V. All rights reserved.
specific surface areas, regular pore structure, narrow pore size distribution and the size of the continuous adjustable [19–34]. To achieve better performance and endow novel properties, the integration of MSNs with other functional components such as metal nanoparticles, metal oxides, semiconductor quantum dots and polymers is necessary [15,19,35–46]. Among them, the fabrication of MSNs based polymer nanocomposites has raised the most interest due to the combination advantages from both of polymers and MSNs [19,35,36]. Surface modification of MSNs with polymers through different surface-initiated polymerization strategies has demonstrated to be the effective routes for preparation of MSNs based polymer nanocomposites [47–52]. Over the past few decades, many polymerization methods have been developed and the resultant MSNs based polymer nanocomposites have shown great potential for different applications especially in the biomedical fields. The commonly utilized methods for surface modification of MSNs are atom transfer radical polymerization (ATRP), stable free radical polymerization (SFRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization [53–65]. These polymerization methods have been used for decades, the technology has been very mature. But they still have their own shortcomings,
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and there is an urgent need for them to make further improvements. For example, the typical ATRP is relied on metal ion catalysts and their organic ligands to initiate the polymerization under inert atmosphere and at elevated temperature. It is well known that the metal ion catalysts are difficult to be removed from the polymerization system and they are toxic towards living organisms or impeded the performance of resultant polymeric composites. On the other hand, the typical ATRP are required at high temperature to active the initiator for the polymerization procedure [65–70]. Therefore, it is highly desirable to develop novel ATRP methods to overcome these issues of traditional ATRP. Metal free ATRP is a recently emerged ATRP method that relied on the organic catalysts under light irradiation to initiate the polymerization [71–73]. In this work, we reported a photo-induced metal free ATRP for surface modification of MSNs using 10-Phenylphenothiazine (PTH) as the organic catalyst. The detailed procedure for the fabrication of MSNs based polymer nanocomposites was shown in Scheme 1. MSNs were prepared using cetyltrimethylammonium bromide (CTAB) as template agent and tetraethyl ortho-rthosilicate (TEOS) as silicon source. Then the amino groups were immobilized onto MSNs to obtain MSNs-NH2 through hydrolysis of (3-aminopropyl)triethoxysilane (APTES), which was used to react with α-Bromoisobutyryl bromide through amidation reaction and removed CTAB to generate Br-containing initiator MSNsBr. The synthetic MSNs-Br can be further used for surface polymerization with itaconic acid (IA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) monomer using PTH as the catalyst. The obtained MSNs-NH2-poly(IA-co-MPC) showed significantly improved dispersibility in various solvents. Furthermore, this facile and efficient method described in this work is also suitable for the surface modification of other materials. 2. Experiments
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2.2. Characterization Fourier transform infrared (FT-IR) spectra were performed on a Nicolet 380 Fourier transform spectrometer with a resolution of 2 cm−1. UV-Visible absorption spectra were obtained by Perkin Elmer LAMBDA35 UV/Vis spectroscopy. Transmission electron microscopy (TEM) images were recorded on a Hitachi 7650B microscope operated at 80 kV, the tested specimens were made by placing a drop of the nanoparticles suspension on a carbon-coated copper grid. X-ray photoelectron spectra (XPS) were measured on a VGESCALAB 220-IXL spectrometer using an AlKa X-ray source (1486.6 eV). thermogravimetric analysis (TGA) curves were recorded on a Q50 thermo gravimetric analyzer under N2 flow and the samples were heated from 25 to 600 °C with the weight of 5–10 mg. The nitrogen sorption characterization on the samples before and after nitridation was conducted on a Micromeritics ASAP2020 surface area and pore size analyzer at −196 °C (liquid nitrogen temperature) using accompanying software from Micromeritics, Inc. The surface area was calculated by using the conventional BET method. The pore parameters were calculated from the adsorption branches of these isotherms using BJH methods. 2.3. Synthesis of MSNs MSNs were synthesized through the hydrolysis of TEOS in the mixed solution that contained CTAB, NaOH according to our previous report [24]. In brief, CTAB (500 mg, 1.37 mmol) and NaOH (150 mg, 3.75 mmol) were dissolved in 250 mL water. The mixture solution was stirred appropriately 0.5 h at 80 °C in an oil bath. Then TEOS (5.00 mL, 22.4 mmol) was added dropwise to the mixture solution under vigorous stirring. After 4 h, the white precipitation was separated through centrifugation and washed with deionized water and methanol for three times. The MSNs were dried under vacuum oven drying at 40 °C to get dry MSNs.
2.1. Materials 2.4. Synthesis of MSNs-NH2 CTAB and TEOS were supplied by Alfa Aesar. APTES as silane coupling agent was supplied by Aldrich.2-Methacryloyloxy ethyl phosphorylcholine (MPC, Mw: 295.27 Da, 98%), IA (Mw: 130.10 Da, 99%), α-bromoisobutyryl bromide (Mw: 229.90 Da, 98%) were purchased from Aladdin (Shanghai, China). PTH was synthesized by phenothiazine and iodobenzene according to previous report. Acetone and toluene were obtained by Heowns (Tianjin, China). cisdichlorodiamineplatinum(II) (CDDP) was purchased from huaxia (Chengdu, China). All chemical agents were of analytical grade and were used directly without any purification.
The MSNs (1.5 g) and toluene (50 mL) were added into a round bottom flask of 100 mL. The mixture was stirred at room temperature about 20 min to make a well dispersed MSNs toluene solution. Then the APTES (0.75 mL) was added and refluxed at 110 °C for 3 h. After that, the white precipitate (MSNs-NH2-1) was separated by centrifugation at 8000 rpm for 3 min. MSNs-NH2-1 was further washed with toluene and isopropanol three times and dried at 40 °C in the vacuum oven. In order to remove the excess surfactant CTAB, 1.00 g of MSNs-NH2-1 was refluxed for 24 h in a methanolic solution that contains 6.00 mL
Scheme 1. Schematic showing the synthesis of MSNs-NH2-poly(IA-co-MPC).
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of HCl (37.4%) in 100.00 mL of methanol. The white precipitate (MSNsNH2) was separated by centrifugation at 8000 rpm for 3 min. The MSNsNH2 was washed with distilled water and methanol for several times and dried in vacuum oven at 35 °C.
and test concentrations per microplate, and the experiment was repeated three times. Cell survival was expressed as absorbance relative to that of untreated controls. Results are presented as mean ± standard deviation (SD).
2.5. Synthesis of MSNs-NH2-Br
3. Results and discussion
The amidation reaction between MSNs-NH2 and 2-bromo-2methylpropionyl bromide was used to prepare the Br-containing initiator. MSNs-NH2 (300 mg) and triethylamine (60 mg) were added to toluene (40 mL) under N2. When the temperature of system achieved to 5 °C, a toluene solution of 30 μL of 2-bromo-2-methylpropionyl bromide in 10 mL of toluene was put dropwise into the reaction system. Mixture solution was stirred violently in ice water bath for 4 h. The solid product was separated by centrifuging at 8000 rpm for 3 min. The centrifugal product was washed with water and acetone three times and dried in vacuum oven at 40 °C to get MSNs-NH2-Br.
The successful preparations of MSNs and MSNs-NH2 were first confirmed by TEM. It can be seen that MSNs are spherical morphology with size about 120–180 nm (Fig. 1A). The high-magnification TEM image of MSNs vaguely shows the ordered and uniform pore size in the interior of MSNs. It indicates that taking a clear mesostructure in a TEM image is very difficult before removal of the template (Fig. 1B). After removal of the template, the highly ordered and unified mesostructure can be observed clearly in the TEM image of MSNs-NH2 (Fig.1C). When the scale is 20 nm, the mesoporous structure is more clearly displayed in the Fig. 1D. On the other hand, the regular pores with diameter about 4 nm were also distinguished through high resolution TEM image. However, the pores had disappeared on the edge of silica spheres, which may be because of the fact the continuous bombardment of high energy electron beams leads to the collapse of the pore structure. The pore structure of MSNs, MSNs-NH2 and MSNs-NH2-poly(IA-coMPC) materials is also evaluated by N2 sorption studies. The N2 sorption isotherms of these materials are presented in Fig. 2(A) and the corresponding textural properties are summarized in Table 1. MSNs exhibit type IV isotherms with a typical capillary condensation step into uniform pores without hysteresis. Compare with the N2 sorption isotherms of MSNs and MSNs-NH2, the adsorption capacity of MSNs-NH2 becomes larger, which can be attributed to the removal of template. The amount of adsorbed N2 of MSNs-NH2-poly(IA-co-MPC) decreased sharply compare with the amount of adsorbed N2 of MSNs-NH2, which can be attributed to the grafting of monomers. As shown in Table 1 and Fig. 3(B) the pore size increase obviously after removing the template. The aperture of MSNs after removing the template is about 4 nm, This is consistent with the previously mentioned TEM results. The grafting of polymers reduced BET surface area and BJH volume to 308.85 m2/g and 0.4012 cm3/g. 1 H NMR spectrum of MSNs-NH2-poly(IA-co-MPC) was shown in Fig. 3. A lot of important information can be directly perceived from the 1H NMR spectrum. The detailed information of 1H NMR spectrum in DMSO was listed below. For example, -NH-CO- (d, 7.92), -CH2-NH- (c, 2.85) indicted that the amino groups are successfully bound to the surface of MSNs by hydrolysis between APTES and the hydroxyl groups on the surface of the silica sphere. Subsequently, the amidation reaction occurred between the amino group and α-Bromoisobutyryl bromide. That was apparent from -NH-CO- (d, 7.92), -C-CH3 (e, 1.21). -CH2- (f, 1.91), -CH2-COOH (g, 2.73) can be attributed to the monomer of IA. -CH2- (h, 2.73), -CH3 (i, 1.91), -CH2- (j,k,l,m, 3.42) can be attributed to the monomer of MPC. The above results from 1H NMR spectrum indicated that we have successfully synthesized MSNs-NH2-poly(IA-co-MPC) via the photo-induced surface-initiated ATRP. The chemical information and functional groups of samples were characterized by FT-IR spectroscopy and FT-IR spectra of MSNs, MSNsNH2 and MSNs-NH2-poly(IA-co-MPC) were shown in Fig. 4. All these spectra display an obvious absorption band at 1070 cm−1. This is no doubt that these are silicon-oxygen bond (Si\\O). On the other hand, the C\\H stretching vibration that normally distributed between 2800 and 3000 cm−1 was found in the sample of MSNs. It can evidence the existence of CTAB. As compared with MSNs, the two peaks at 2800– 3000 cm−1 were disappeared in the sample of MSNs-NH2. Moreover, a characteristic absorbance band at 3485 cm− 1 was appeared in the spectrum of MSNs-NH2. This implied that amino groups have been successfully immobilized on the surface of MSNs. Based on the above comparison, we can conclude that CTAB has been removed from MSNs and amino groups have introduced on MSNs. However, in the spectrum of MSNs-NH2-poly(IA-co-MPC), the absorption band at 2920 cm−1 was
2.6. Preparation of MSNs-NH2-poly(IA-co-MPC) Grafting polymer is one of the most important steps in the surface modification of MSNs. MSNs-NH2-Br (150 mg), MPC (567 mg, 1.92 mmol), IA (250 mg, 1.92 mmol) and PTH (3 mg) were dispersed in 3 mL THF in the schlenk flask under N2. Then the schlenk flask was irradiated with LEDs at 380 nm under room temperature for 7 h. The MSNs-NH2-poly(IA-co-MPC) was separated by centrifugation and washing with distilled water and THF several times. The resultant solid was dried overnight in a vacuum at 40 °C. 2.7. CDDP loading and release behavior MSNs-NH2-poly(IA-co-MPC) (20 mg) and 10 mg cis-dichlorodiam mineplatinum(II) (CDDP) was dispersed in 100 mL PBS (pH = 7.4). The above solution was stirred under dark at 37 °C for 48 h. After that, the drug loading complexes MSNs-NH2-poly(IA-co-MPC)-CDDP were separated by centrifuging at 8000 rpm for 3 min. The concentration of CDDP in the supernatant was measured and calculated based on the absorption of UV-Visible spectrometer at 705 nm according to previous report. To determine their release behavior, MSNs-NH2-poly(IA-co-MPC)CDDP was divided into two portions and put into the dialysis bag (3500 Da). The pH values are (pH = 5.6 and 7.4). At different time points, the concentration in PBS was measured and the content of CDDP in PBS was calculated based on the standard curve of CDDP. In order to confirm the loading position and release behavior of CDDP, the amount of CDDP adsorbed into the MSNs-NH2 was measured by the same method. 2.8. Cytotoxicity of MSNs-NH2-poly(IA-co-MPC) To evaluate the potential biomedical applications of MSNs-NH2poly(IA-co-MPC), the cell viability of MSNs-NH2-poly(IA-co-MPC) towards A549 cells was examined using cell counting kit-8 (CCK-8) [74– 76]. In brief, A549 Cells were seeded in 96-well microplates at a density of 5 × 104 cells per mL in 160 μL of the respective media containing 10% fetal bovine serum (FBS). After 24 h of cell attachment, A549 cells were incubated with 10–120 μg mL−1 of MSNs-NH2-poly(IA-co-MPC) for 12 and 24 h. Then the cells were washed with PBS for three times to remove the uninternalized nanoparticles. After that, 10 μL of CCK-8 dye and 100 μL of Dulbecco's Modified Eagle's Medium (DMEM) cell culture media was added to each well and incubated for 2 h at 37 °C. Plates were then analyzed with a microplate reader (VictorIII, Perkin-Elmer). Measurements of dye absorbance were carried out at 450 nm, with the reference wavelength at 620 nm. The values were proportional to the number of live cells. The percent reduction of WST was compared to the control (cells not exposed to nanoparticles), which represented 100% WST reduction. Three replicate wells were used for each control
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Fig. 1. TEM images of MSNs. (A) Scale bar = 200 nm, (B) Scale bar = 100 nm. TEM images of MSNs-NH2. (C) Scale bar = 50 nm, (D) Scale bar = 20 nm.
emerged again. This suggested that the MPC have been grafted on the sample of MSNs through photo-induced surface-initiated ATRP. On the other hand, the some other characteristic bonds such as 1710 cm− 1 (C_O), 1660 cm−1 (C\\O) and 1410 cm−1 (C\\H) were also found in the sample of MSNs-NH2-poly(IA-co-MPC). The above results further evidenced indicated that the monomers of IA and MPC have been introduced on the surface of MSNs. Fig. 5 shows the TGA curves of MSNs, MSNs-NH2, and MSNs-NH2poly(IA-co-MPC). It clearly shows that the weight loss of MSNs-NH2 and MSNs-NH2-poly(IA-co-MPC) are much larger than that of MSNs. Significant weight loss at the temperature 100–200 °C was found in the curve of MSNs. The weight loss should be ascribed to the decomposition of CTAB. However, no obvious weight loss was occurred, suggesting that MSNs possess good thermal stability. On the contrary, there is a uniform weight loss (12%) between 100 and 600 °C from the TGA curve of MSNs-NH2. This implied that APTES was immobilized on MSNs through hydrolysis reaction. Furthermore, more significant weight loss was found in the sample of MSNs-NH2-poly(IA-co-MPC). The total weight loss percentage was achieved about 30% between 100 and 600 °C. As compared to MSN-NH2, the weight loss of MSNs-NH2-poly(IAco-MPC) increased by 17%, which means that the weight of polymer in the total weight is about 17%. The significant weight loss given
powerful evidence about the successful preparation of monomers MSNs-NH2-poly(IA-co-MPC). XPS is one of the most useful techniques for determination of chemical compositions of materials and surface. The survey XPS spectra ranging from 0 to 1400 eV were examined and results were displayed in Fig. 6. Elements of Si, O, C and N were observed in the sample of MSNs (Fig. 6A). The elements such Si and O are come from the hydrolysis of TEOS, while the C and N should be derived from CTAB. In Fig. 6B, the peak intensity of Si and O are significantly reduced, while the peak intensity of C was increased. This suggested that APTES has been successfully attached on the MSNs. As compared with the spectrum of MSNsNH2, the new element Br was emerged in the sample of MSNs-NH2-Br, suggesting the bromine-containing initiator has immobilized on the surface of MSNs-NH2 through amidation reaction (Fig. 6C). Moreover, the element of P as a unique element of MPC was appeared in the sample of MSNs-NH2-poly(IA-co-MPC), giving the powerful evidence for the surface modification of MSNs with MPC. On the other hand, the intensity of Si was further decreased after surface modification with copolymers while the intensity of C, N and O was relatively enhanced in the sample of MSNs-NH2-poly(IA-co-MPC), further confirming the successful grafting of MPC and IA on the surface of MSNs-NH2-poly(IA-coMPC). All of the above XPS results indicated that we have successfully
Fig. 2. (A) Adsorption–desorption isotherms of some samples at 77 K; (B) BJH pore size distribution of some samples calculated from the adsorption branch of the isotherm.
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Table 1 Textural properties of MSNs, MSNs-NH2 and MSNs-NH2-poly(IA-co-MPC). Sample
ABET (m2/g)
DBJH (nm)
VBJH (cm3/g)
MSNs MSNs-NH2 MSNs-NH2-poly(IA-co-MPC)
47.79 642.16 308.85
22.88 3.95 4.4905
0.08 0.7987 0.4012
ABET, BET surface area; DBJH, BJH pore diameter; VBJH, BJH volume.
modified MSNs with poly(IA-co-MPC) through photo-induced surfaceinitiated ATRP. The high resolution XPS spectra of C1s, N1s, O1s, Si2p, P2p and Br3d were shown in Fig. 7. In the spectra of C1s, the peak intensity of C1s in MSNs-NH2 and MSNs-NH2-poly(IA-co-MPC) is increased as compared with MSNs. More importantly, the peak position of C1s in MSNs-NH2poly(IA-co-MPC) has divided into three peaks, suggesting that C were bonded with different atoms (Fig. 7A). The C1s spectrum of MSNsNH2-poly(IA-co-MPC) with binding energy at 288.6 eV should be attributed to the \\O\\C_O, which was derived from IA and MPC. The N1s spectra were displayed in Fig. 7B, the intensity of N1s in the sample of MSNs–NH2 and MSNs-NH2-poly(IA-co-MPC) was also increased as compared with MSNs. Especially, the N1s spectrum in the sample MSNs-NH2-poly(IA-co-MPC) shows a sharp peak in Fig. 7B. The significant increase of N in sample of MSNs-NH2-poly(IA-co-MPC) suggested the MPC that contains -N(CH3)3 has been introduced onto MSNs successfully. Contrast with N, the peak intensity of O1s and Si2p was simultaneously decreased after their surface was modified with APTES and polymers (Fig. 7C and D). The above results clearly evidence that the MSNs were functionalized with poly(IA-co-MPC) through the photo-induced ATRP. The P2p spectra were shown in Fig. 7E. It can be seen that only the sample MSNs-NH2-poly(IA-co-MPC) found the P signal. The P signal should be derived from the MPC and further demonstrated the surface grafting of MSNs with polymers. Moreover, the successful immobilization of ATRP initiator on the surface MSNs and surface grafting of MSNs could also be evidenced by the Br3d spectra (Fig. 7F). For example, we can find intensive Br3d signal in the sample of MSNs-Br, but it was absent in the sample of MSNs and MSNs-NH2. This suggests that
Fig. 4. FTIR spectra of MSNs, MSNs-NH2, and MSNs-NH2-poly(IA-co-MPC).
the ATRP initiator was immobilized on the surface of MSNs-NH2 through amidation reaction. Furthermore, the intensity of Br3d is decreased in the sample of MSNs-NH2-poly(IA-co-MPC) as compared with the sample of MSNs-Br. This further implied that the monomers IA and MPC were grafted on MSNs. The percentages of different elements were calculated by XPS spectra. Table 2 shows the percentages of main elements in MSNs, MSNsNH2 and MSNs-NH2-poly(IA-co-MPC). The percentages of Si, C, N and O in MSNs are 22.42%, 24.94%, 1.56% and 50.85%, respectively. It is worth to mentioning that the percentages of Si and O were 22.65% and 50.85%, respectively. It is close to the expected ratio of Si/O (1:2). Compared with MSNs, the percentages of Si, C, N and O in the samples of MSNs-NH2 are changed to 17.2%, 41.14%, 4.13% and 37.36%, respectively. The increase of N content suggests clearly indicated that APTES has immobilized on the surface of MSNs through hydrolysis reaction. Moreover, the element Br with 1.35% was emerged in the sample of
Fig. 3. 1H NMR spectrum of MSNs-NH2-poly(IA-co-MPC). The characteristic signals suggest that MPC and IA were grafted onto MSNs through photo-induced surface-initiated ATRP.
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Fig. 5. TGA curves of MSNs, MSNs-NH2, and MSNs-NH2-poly(IA-co-MPC).
MSNs-Br. This gives powerful evidence for the linkage of ATRP initiator on MSNs through amidation reaction. However, the contents of Si and O in the sample of MSNs-NH2-poly(IA-co-MPC) was further decreased, while the percentage of P was obviously increased. The above changes suggested that MPC and IA were successfully introduced onto MSNs through the metal-free surface-initiated ATRP.
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Due to the introduction of IA onto the surface of MSNs, the loading behavior of MSNs-NH2-poly(IA-co-MPC) towards to CDDP was further examined. It is well known that the CDDP can coordinate with carboxyl groups. Therefore, CDDP can be loaded onto MSNs-NH2-poly(IA-coMPC) and the drug loading capability might be adjusted through the feed ratio of monomer IA and MPC. In this work, the concentrations of CDDP are determined using the UV-Visible spectroscopy. The CDDP and 1, 2-diaminobenzene can form complex and shows maximum absorption wavelength at 705 nm. The concentrations of CDDP are positively related with the absorption of CDDP-1, 2-diaminobenzene complexes at 705 nm and can be calculated using the standard curve (Fig. S1). Based on the determination, the amount of CDDP loaded onto MSNs-NH2-poly(IA-co-MPC) was calculated to be 213 mg/g. The amount of CDDP loaded into MSNs-NH2 was calculated to be 43 mg/g. It indicated that MSNs-NH2 which was not grafted with itaconic acid can also load CDDP with its mesoporous structure. Compare with the amounts of CDDP adsorbed in the MSNs-NH2-poly(IA-co-MPC) and MSNs-NH2, it is no doubt that the loading capacity of the drug in the modified MSNs via metal-free ATRP greatly enhanced. On the other hand, the release behaviors of MSNs-NH2-poly(IA-co-MPC)-CDDP and MSNs-NH2-CDDP were also determined when MSNs-NH2-poly(IA-coMPC)-CDDP and MSNs-NH2-CDDP complexes were dissolved in PBS solution (pH = 7.4, pH = 5.6) (Fig. 8). The results show that the release ratio of MSNs-NH2-poly(IA-co-MPC)-CDDP is significant different under pH = 7.4 and 5.6. For example, the percentage of CDDP release from MSNs-NH2-poly(IA-co-MPC)-CDDP can reach 59% at the solution of pH = 5.6. However, the percentage value is only 18% in PBS solution
Fig. 6. Survey XPS spectra of MSNs, MSNs-NH2, MSNs-Br and MSNs-NH2-poly(IA-co-MPC). The characteristic elements such as Br and P were found in the samples of MSNs-Br and MSNsNH2-poly(IA-co-MPC) confirmed the successful preparation of MSNs-NH2-poly(IA-co-MPC) through photo-induced metal free ATRP.
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Fig. 7. High resolution XPS spectra of MSNs, MSNs-NH2, MSNs-Br and MSNs-NH2-poly(IA-co-MPC). (A) The region of C1s, (B) The region of N1s, (C) The region of O1s, (D) The region of Si2p, (E) The region of P2p, (F) The region of Br3d.
with pH = 7.4 (Fig. 8A). This property provides the potential for targeted cancer therapy. In contrast, the drug release curves of MSNsNH2 were also drawn (Fig. 8B). The release behavior is almost the same under pH = 7.4 and 5.6, which proves MSNs-NH2 have no ability to control-release CDDP through different pH condition. More importantly, because the content of IA in final polymer composites can be tuned through the polymerization procedure, therefore the drug loading capability of the final MSNs polymer composites can also be Table 2 Percentages of Si, C, N and O based on XPS results.
MSNs MSNs-NH2 MSNs-Br MSNs-NH2-poly(IA-co-MPC)
Si(%)
C(%)
N(%)
O(%)
Br(%)
P(%)
22.42 17.2 16.6 2.91
24.94 41.14 44.2 55.82
1.56 4.13 3.63 4.18
50.85 37.36 33.96 33.41
0 0 1.35 0.35
0.23 0.17 0.26 3.33
adjusted. It is therefore, therapeutic effects of the final MSNs-NH2poly(IA-co-MPC)-CDDP would thus be regulated through design of polymerization parameters. Moreover, this method could also use for fabrication of many other functional polymer composites using different monomers. Therefore, it should be a general tool for preparation of MSNs based functional materials with various properties and prospective application potential in different fields. Furthermore, as compared with conventional ATRP that is relied on the transition metal ions and organic ligands as catalyst systems, the method described above provides an alternative and more attractive choice for surface-initiated controlled living polymerization. The cell viability of MSNs-NH2-poly(IA-co-MPC) towards A549 cells were determined by CCK-8 assay. It is well known that MPC is a biocompatible monomer which has been extensively for surface modification of materials for biomedical applications [77]. The poly(MPC) modified materials will simultaneously enhanced water dispersity and biocompatibility [78]. In this work, the cell viability values of MSNs-NH2-
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Fig. 8. The release behaviors of CDDP from MSNs-NH2-poly(IA-co-MPC)-CDDP (A) and MSNs-NH2-CDDP (B) complexes under different pH values (pH = 7.4 and 5.6). The results show that CDDP could be released from MSNs-NH2-poly(IA-co-MPC)-CDDP complexes faster under acidic solution. This implied that MSNs-NH2-poly(IA-co-MPC) can be acted as carriers for controlled delivery of CDDP.
poly(IA-co-MPC) was calculated based on CCK-8 data and results were shown in Fig. 9. It can be seen that the cell viability values were gradually decreased as the increasing of MSNs-NH2-poly(IA-co-MPC) concentrations. However, it is worth to noting that the cell viability values are still maintained at high levels. For example, the cell viability value of MSNs-NH2-poly(IA-co-MPC) at 120 μg mL−1 is as high as 87.9% when the incubation time is 24 h. The preliminary results suggest that MSNs-NH2-poly(IA-co-MPC) possess good biocompatibility and are promising for biomedical applications. Furthermore, the half maximal inhibitory concentration (IC50) values were also calculated. Results suggested that the IC50 values at12 and 24 h are 231.7 and 190.6 μg mL−1, respectively. Combined the high loading capability of CDDP on MSNsNH2-poly(IA-co-MPC), we can speculate that MSNs-NH2-poly(IA-coMPC) can used as promising candidates for pH controlled intracellular delivery of CDDP for cancer treatment. 4. Conclusions In summary, surface modification of MSNs with biocompatible and zwitterionic phosphorylcholine containing copolymers was reported through photo-induced surface-initiated ATRP for the first time. Various characterization techniques demonstrated that MSNs-NH2-poly(IA-coMPC) can be facilely fabricated via the novel ATRP strategy. As compared with traditional ATRP, the photo-induced surface-initiated ATRP can occur under visible light irradiation and room temperature, which can avoid the using transition metal ions/organic ligands catalyst systems and high polymerization temperature. More importantly, the
Fig. 9. Cell viability of MSNs-NH2-poly(IA-co-MPC) with A549 cells for 12 and 24 h. The cells were incubated with different concentrations (10–120 μg mL−1) of MSNs-NH2poly(IA-co-MPC). The cell viability values of MSNs-NH2-poly(IA-co-MPC) was calculated based on CCK-8 assay.
anticancer agent CDDP could be loaded onto MSNs-NH2-poly(IA-coMPC) with high efficiency. The MSNs-NH2-poly(IA-co-MPC) display good water dispersity, high drug-loading capability, pH controlled drug release behavior and desirable biocompatibility. These remarkable properties make MSNs-NH2-poly(IA-co-MPC) promising candidates for pH controlled intracellular drug delivery. Moreover, owing to the universality of photo-induced surface-initiated ATRP, many other functional polymeric composites can be also fabricated. Therefore, the novel ATRP should be an effective and general strategy for preparation of multifunctional biomaterials. Acknowledgements This research was supported by the National Natural Science Foundation of China (Nos. 51363016, 21474057, 21564006, 21561022, 21644014), Natural Science Foundation of Jiangxi Province in China (Nos. 20161BAB203072, 20161BAB213066). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2017.05.107. References [1] R. Lakshminarayanan, E.O. Chi-Jin, X.J. Loh, R.M. Kini, S. Valiyaveettil, Biomacromolecules 6 (2005) 1429–1437. [2] Q. Dou, X. Fang, S. Jiang, P.L. Chee, T.-C. Lee, X.J. Loh, RSC Adv. 5 (2015) 46817–46822. [3] E. Ye, M.D. Regulacio, M.S. Bharathi, H. Pan, M. Lin, M. Bosman, K.Y. Win, H. Ramanarayan, S.Y. Zhang, X.J. Loh, Y.W. Zhang, M.Y. Han, Nanoscale 8 (2016) 543–552. [4] B.M. Teo, D.J. Young, X.J. Loh, Part. Part. Syst. Charact. 33 (2016) 709–728. [5] R. Lakshminarayanan, X.J. Loh, S. Gayathri, S. Sindhu, Y. Banerjee, R.M. Kini, S. Valiyaveettil, Biomacromolecules 7 (2006) 3202–3209. [6] C. Dhand, N. Dwivedi, X.J. Loh, A.N. Jie Ying, N.K. Verma, R.W. Beuerman, R. Lakshminarayanan, S. Ramakrishna, RSC Adv. 5 (2015) 105003–105037. [7] Q.Q. Dou, C.P. Teng, E. Ye, X.J. Loh, Int. J. Nanomedicine 10 (2015) 419–432. [8] X.J. Loh, T.C. Lee, Q. Dou, G.R. Deen, Biomater. Sci. 4 (2016) 70–86. [9] Z. Li, E. Ye, R. Lakshminarayanan David, X.J. Loh, Small 12 (2016) 4782–4806. [10] H.C. Guo, E. Ye, Z. Li, M.Y. Han, X.J. Loh, Mater. Sci. Eng. C 70 (2017) 1182–1191. [11] K. Huang, Q. Dou, X.J. Loh, RSC Adv. 6 (2016) 60896–60906. [12] E. Ye, X.J. Loh, Aust. J. Chem. 66 (2013) 997. [13] C. Kresge, M. Leonowicz, W. Roth, J. Vartuli, J. Beck, Nature 359 (1992) 710–712. [14] Z. Li, J.C. Barnes, A. Bosoy, J.F. Stoddart, J.I. Zink, Chem. Soc. Rev. 41 (2012) 2590–2605. [15] F. Muhammad, M. Guo, W. Qi, F. Sun, A. Wang, Y. Guo, G. Zhu, J. Am, Chem. Soc. 133 (2011) 8778–8781. [16] F. Zhang, G.B. Braun, Y. Shi, Y. Zhang, X. Sun, N.O. Reich, D. Zhao, G. Stucky, J. Am. Chem. Soc. 132 (2010) 2850–2851. [17] N. Shadjou, M. Hasanzadeh, Mater. Sci. Eng. C 55 (2015) 401–409. [18] M. Moritz, M. Geszke-Moritz, Mater. Sci. Eng. C 49 (2015) 114–151. [19] X. Xu, S. Lü, C. Gao, X. Wang, X. Bai, H. Duan, N. Gao, C. Feng, M. Liu, Chem. Eng. J. 279 (2015) 851–860. [20] D. Dutta, D. Thakur, D. Bahadur, Chem. Eng. J. 281 (2015) 482–490. [21] S. Wang, K. Wang, C. Dai, H. Shi, J. Li, Chem. Eng. J. 262 (2015) 897–903.
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