Biodegradability of Disulfide-Organosilica ... - ACS Publications

Article www.acsanm.org

Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Biodegradability of Disulfide-Organosilica Nanoparticles Evaluated by Soft X‑ray Photoelectron Spectroscopy: Cancer Therapy Implications Harutaka Mekaru,*,† Akitaka Yoshigoe,‡ Michihiro Nakamura,§ Tomohiro Doura,∥ and Fuyuhiko Tamanoi⊥,# †

National Institute of Advanced Industrial Science and Technology (AIST), 1-2-1 Namiki, Tsukuba, Ibaraki 305-8564, Japan Japan Atomic Energy Agency (JAEA), 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan § Yamaguchi University, 1-1-1 Minami-Koguchi, Ube, Yamaguchi 755-8505, Japan ∥ Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachio̅ji, Tokyo 192-0392, Japan ⊥ University of California, Los Angeles, 602 Molecular Sciences Building, Los Angeles, California 90095-1489, United States # Institute for Integrated Cell-Material Sciences, Institute for Advanced Study, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan

Downloaded via 193.56.74.208 on January 19, 2019 at 06:32:10 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Two kinds of organosilica nanoparticles (NPs) that were fabricated from thiol-containing precursors, (3mercaptopropyl)trimethoxysilane (MPMS) and (3mercaptopropyl)methyldimethoxysilane (MPDMS), are potential delivery vehicles of anticancer drugs. MPMS can form three siloxane bonds, but MPDMS forms two siloxane bonds as the maximum limit. Hence, disulfide bonds can be involved in the three-dimensional morphology of MPDMS NPs. In addition, NPs containing disulfide bonds are potentially degraded by a reduced form of glutathione (GSH). To examine reactions between the organosilica NPs and GSH, the NPs were incubated in 10 mM GSH aqueous solution at 37 °C for 7 d and the products were analyzed using field-emission scanning electron microscopy (FESEM), Raman spectroscopy, and soft X-ray photoelectron spectroscopy (XPS). The Raman spectra showed the presence of disulfide bonds in the MPDMS NPs and the absence of disulfide bonds in MPMS NPs. The results of XPS measurements suggested that the disulfide bonds in the outer layer of MPDMS NPs were reduced to thiol groups. FE-SEM observations of MPDMS NPs detected changes in NP morphology after the GSH incubation. These results support the idea that MPDMS NPs contain disulfide bonds and are degradable by GSH. Therefore, MPDMS NPs possess a biodegradable feature that is advantageous for clinical translation, that is, nanomedicine. KEYWORDS: (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)methyldimethoxysilane, nanoparticles, biodegradability, glutathione, X-ray photoelectron spectroscopy, Raman spectroscopy

■

INTRODUCTION Silica nanoparticles (NPs) have recently emerged as a promising nanomaterial for anticancer drug delivery that can supply the minimum required amount of drugs to the necessary place at appropriate times, decreasing the overdose risk and side effects of conventional drug therapy.1,2 Compared with other materials such as liposomes and polymers, silica NPs have high physical strength, enabling various surface modifications and attachment of nanomachines to enable controlled release of anticancer drugs in response to light and magnetic fields.3 Similar to other types of NPs, silica NPs can accumulate selectively in tumor tissue by the enhanced permeation and retention (EPR) effect (unlike normal blood vessels, there are fenestrations of approximately 200 nm size in the blood vessel wall in tumor tissue, so that NPs smaller than © XXXX American Chemical Society

the fenestration leak out from the blood vessel wall). Silica NPs are biocompatible and safe.4 One of the key issues regarding silica NPs is that, after release of the drug, it is preferable that the NPs are promptly degraded inside the cells to maximize their safety. A number of studies report degradation of inorganic mesoporous silica nanoparticles (MSNs).5−10 Degradation products of MSNs are nontoxic, and the decomposition time of MSNs in biorelevant media can be controlled in the range between several hours and several weeks.5 Decomposition of MSNs in simulated body fluid and in human embryonic kidney 293T cells was Received: November 9, 2018 Accepted: December 27, 2018 Published: December 27, 2018 A

DOI: 10.1021/acsanm.8b02023 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Article

ACS Applied Nano Materials

We characterized MPDMS NPs using field-emission scanning electron microscopy (FE-SEM) and Raman spectroscopy and analyzed the reaction between GSH and MPDMS NPs by using XPS and FE-SEM. Raman spectra provided information about the presence of disulfide bonds, and XPS analyses provided information on the bonding species around sulfur atoms on the surface of MPDMS NPs. These results demonstrated that MPDMS NPs have a number of disulfide bonds and the GSH-responsive degradable feature. We would also like to point out that our work first reported that XPS using soft X-ray has been applied to the study of silica-based NPs. This provided important insight as well as valuable lessons on how this technology can be applied.

examined by inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis.6 Degradation of MSNs in phosphate buffered saline was examined by transmission electron microscopy (TEM) observation and ICP-AES measurements.7 The degradation rate of hollow MSNs in human umbilical vein endothelial cells was examined.8 The degradation of colloidal MSNs was found to be independent of their diameter differences.9 From the viewpoint of medical safety, more thorough study of biochemical degradability is needed. The biodegradability needs to be improved so that biodegradable NPs can be more easily decomposed and the decomposition sites and decomposition time in the body and cells can be controlled.5,10 In recent years, biodegradable periodic mesoporous organosilica (PMOs) that contain biodegradable bonds such as disulfide bonds have been developed.11−13 Their degradability was evaluated by using the reduced form of glutathione (GSH).14,15 GSH is a natural tripeptide in almost all cells, and it maintains the cellular redox conditions.16 In cancer cells, elevated levels of GSH are involved in drug resistance.16 Therefore, the elevated level of GSH is an attractive target of cancer-selective nanomedicine. Destruction of solid PMO was examined by TEM images collected after stirring solid PMO in 10 mM GSH solution for 24 h.17 This confirmed decomposition of PMO into small pieces with a size of ∼35 nm. Biodegradation of mesoporous organosilica NPs containing disulfide bonds was examined by TEM observation.18−21 These studies confirmed that the nanospheres with a diameter of 40 nm were decomposed into small pieces with a size of 5 nm after incubation for 7 d. In this work, we have explored a different approach utilizing thio-organosillica NPs that are synthesized from a thiolcontaining precursor such as (3-mercaptopropyl)trimethoxysilane (MPMS) and (3-mercaptopropyl)methyldimethoxysilane (MPDMS).22−24 The thiols and alkyl chains of the NPs have been used to incorporate fluorescent dyes into the NPs through the electrostatic interactions and the hydrophobic interactions,25 and the fluorescent NPs facilitate bioimaging studies.26 MPMS can form three siloxane bonds; hence, MPMS can produce silsesquioxane cores. On the other hand, MPDMS can form two siloxane bonds at a maximum limit. Nevertheless, MPDMS can form threedimensional (3D) morphology, such as NPs. Therefore, MPDMS NPs are expected to have disulfide bonds. As described above, disulfide bonds of NPs are reduced by GSH, and the break of disulfide bonds of MPDMS NPs will lead to decisive degradation of the NPs. Because the reactions between GSH and MPDMS NPs proceed on the surface of MPDMS NPs, X-ray photoelectron spectroscopy (XPS) will be a powerful tool to clarify the reactions. XPS is a spectroscopic method used to measure the kinetic energy of photoelectrons emitted in a vacuum by exciting electrons with an energy corresponding to a suitable energy level in a solid by irradiating the solid with X-rays. The resulting photoelectron spectrum reflects the local electron state of an atom of interest and its surroundings. Using XPS, it is possible to identify the chemical bonding state of an element near the surface of nanoparticles. XPS spectra have been measured to confirm the composition of prepared NPs,27−30 and there are other cases where chemical shift was measured by focusing on a particular element.31−34 However, XPS has not been used to evaluate organosilica NPs because organic molecules are easily damaged and charged by the X-rays.

■

EXPERIMENTAL SECTION

Synthesis and Degradation of NPs. NPs composed of organic− inorganic hybrid materials containing thiols were synthesized by the sol−gel method using MPMS and MPDMS as a single silica source in NH4OH as a base catalyst. The synthesized thiol-organosilica NPs were then washed with distilled water three times. The prepared NPs were diffused in a 10 mM GSH aqueous solution and incubated in a microtube using a rotation mixer placed inside an incubator temperature-controlled at 37 °C for 7 d. The incubated samples were washed with Milli-Q water three times using a centrifuge. For details of the experiment procedures, refer to Figure S1 in the Supporting Information. FE-SEM Observation and Sample Preparation. FE-SEM images were obtained at a low acceleration voltage of 5 kV using an FE-SEM instrument (S4800, Hitachi, Japan). A dispersed sample was dropped onto a Au sputtered layer on a Si substrate with a Cr intermediate layer and allowed to dry naturally for several hours. However, because the MPDMS NPs after incubation could not be centrifuged, it was dropped onto the Au/Cr/Si substrate in the state of being dispersed in the glutathione solution as shown in Figure S2 in the Supporting Information. The substrate was fixed on a dedicated mounter by a conductive tape and then inserted into the FE-SEM system without coating the sample surface with a conductive material. Raman Spectroscopy. Raman spectroscopy measurements were conducted at room temperature using a Raman spectrophotometer (NRS3100, JASCO, Japan) equipped with a laser (λ = 532 nm). Each type of solid thiol-organosilica NP was placed in a holder. Polypropylene was used as a standard sample for calibration. The obtained Raman spectra were deconvoluted using a nonlinear leastsquares curve-fitting procedure to extract single bands. A single band was assumed to be represented as a pseudo-Voigt function, f v(v) = γf L(v) + (1 − γ)f G(v), where f L(v) and f G(v) represent the Lorentzian and Gaussian components, respectively, and the parameter γ (0 < γ < 1) represents the Lorentzian component. The intensity I of a single band was evaluated according to I = γIL + (1 − γ)IG, where IL and IG denote the integrated intensities of the Lorentzian and Gaussian components, respectively. XPS. XPS measurements were performed using the surface chemical experiment station35 of BL23SU36 at SPring-8. The samples of NP dispersion were dropped and dried on Au/Cr/Si substrates, and the substrates were put in a designated holder as shown in Figure S2(a-3) of the Supporting Information. In the case of powdered samples, the powders were pressed against the Au/Cr/Si substrate with a finger via a weighing paper to attach the powders to the substrate, as shown in Figure S2(b-1) of the Supporting Information. Each substrate fixed to the designed holder was individually transported into the main chamber via a load lock chamber of the surface chemical experiment station. In the analysis of spectra obtained by XPS measurements, peak decomposition was conducted using CasaXPS 2319 dev1−1b (Casa Software, U.K.). B


Article


■

RESULTS Synthesis of Nanoparticles and Their Characterization by FE-SEM and Raman Spectroscopy. Two kinds of thiol-organosilica NPs, MPMS NPs and MPDMS NPs, were synthesized from MPMS and MPDMS via sol−gel chemistry (Figure 1). The precursors are hydrolyzed in

is expected that oxidation of thiols derived from MPDMS proceeds with the formation of siloxane bonds. If the MPDMS NPs are composed of siloxane bonds and disulfide bonds, it is expected that the MPDMS NPs are degraded by GSH. Figure 2 illustrates the degradation process of the MPDMS NPs exposed to GSH. GSH can react with disulfide bonds of MPDMS NPs through the thiol−disulfide exchange reaction, and GSH is transformed to the oxidized glutathione (GSSG). As a result, the 3D network structure of the MPDMS NPs will collapse and eventually the NPs will be degraded. On the other hand, the structure of MPMS NPs is expected to be mainly formed by siloxane bonds (Figure 1). Thus, the MPMS NPs appear to exhibit a resistance to the GSH-responsive degradability. FE-SEM images of the prepared thiol-organosilica NPs are shown in Figure 3. An enlarged image [inset of Figure 3(a-1)] revealed that the MPMS NPs had a diameter of ca. 500−600 nm before incubation with 10 mM GSH. Figure 3(a-2) shows the MPMS NPs incubated in a 10 mM GSH aqueous solution for 7 d. The MPMS NPs were washed to remove glutathione. The diameter of the incubated MPMS NPs was estimated to be ca. 500−650 nm from the corresponding inset, and the spherical appearance of the NPs did not change following incubation. Therefore, it is considered that the MPMS NPs were not degraded by GSH, as was expected. The diameter of the MPDMS NPs shown in Figure 3(b-1) measured from the enlarged image was approximately 100 nm, which is about a fifth the size of the MPMS NPs. The particle size of these NPs also coincides well with the measurement results by dynamic light scattering shown in Figure S3 of the Supporting Information. When the MPDMS NPs were incubated in 10 mM GSH aqueous solution for 7 days, it appears that they

Figure 1. Fabrication of thiol-organosilica NPs: MPMS NPs (a) and MPDMS NPs (b).

concentrated NH3 aqueous solutions, and the hydrolysates polymerize through formation of siloxane bonds. As described before, the fabrication of MPDMS NPs needs to form disulfide bonds in addition to the formation of siloxane bonds. Hence, it

Figure 2. Degradation of MPDMS NPs through the thiol−disulfide exchange reaction with GSH. MPMS NPs without internal disulfide bonds are not affected by GSH. In contrast, MPDMS NPs containing disulfide bonds are susceptive to GSH, and GSH is transformed to GSSG through the thiol−disulfide exchange reaction. As a result, the 3D network structure of the MPDMS NPs collapses, and finally the nanoparticles themselves are decomposed and are not able to keep the spherical shape. C


Article


Figure 3. FE-SEM images of MPMS NPs (a-1) and MPDMS NPs (b-1) before incubation with 10 mM GSH. (a-2) FE-SEM image of the products from MPMS NPs after incubation with 10 mM GSH at 37 °C for 7 d. The products were washed three times with Mill-Q water using a centrifuge. (b-2) FE-SEM images of the products from 0.02 mg/mL MPDMS NPs incubated with 10 mM GSH at 37 °C for 7 d without washing.

Both disulfide bonds and thiols are Raman active and display high-intensity signals.37 Therefore, Raman spectroscopy is suitable to characterize the disulfide bonds and thiols in thiolorganosilica NPs. The Raman spectra of thiol-organosilica NPs are shown in Figure 5, and the analytical data are listed in Table 1. Disulfide stretching modes and thiol stretching modes of the thiol-organosilica NPs were located at approximately 480−550 and 2420−2620 cm−1, respectively.37 As shown in Figure 5 and Table 1, the MPMS NPs displayed strong Raman peaks derived from the thiols and weak Raman peaks derived from disulfide bonds, whereas the MPDMS NPs showed strong Raman peaks derived from the disulfide bonds and little Raman peaks derived from thiols. This result indicated that MPMS NPs have a lot of thiols and MPDMS NPs have a number of disulfide bonds, as was expected. The two disulfide stretching modes of thiol-organosilica NPs (approximately 509 and 524 cm−1) are derived from two torsional conformers of a disulfide bond.38 The two main thiol stretching modes of MPMS NPs at 2514 and 2566 cm−1 probably originated from SH···O and SH···S vibrations, respectively.39 Because MPMS NPs have a small quantity of disulfide bonds, the 3D structure must be mainly composed of siloxane bonds. On the other hand, the Raman spectra demonstrated that disulfide bonds are involved in the 3D structure of MPDMS NPs. XPS Measurements Using Synchrotron Radiation. Synchrotron Radiation Provides Good Signals and Minimum Radiation Damage during XPS Measurements. First, we optimized the XPS experimental conditions to analyze the surface of thiol-organosilica NPs. Typical results of the XPS measurements of MPMS NPs were obtained at a photon energy of 690 eV using the surface chemical experiment station of BL23SU at SPring-8. The four energy regionsS 2p (155−

were mostly degraded as we did not observe nanoparticles [Figure 3(b-2)]. To gain insight into the process of degradation of MPDMS NPs during the incubation with GSH, we carried out FE-SEM observation. The results of the observation for 3 days are shown in Figure 4. While MPDMS NPs were detected during

Figure 4. FE-SEM images of MPDMS NPs observed once a day to gain insight into the degradation process of NPs.

this incubation period, the shape of the NPs blurred each day (the line on the periphery of NPs became blurred). This appears to suggest that the degradation occurs from the NP surface. By day 4, NPs were degraded, but we observed some lumpy aggregation. Therefore, the degradation speed is slow and it takes 7 days of incubation time to complete degradation. D


Article


Figure 5. Raman spectra of (a-1) disulfide stretching modes of MPMS NPs in the frequency range of 480−550 cm−1, (a-2) thiol stretching modes of MPMS NPs in the frequency range of 2420−2620 cm−1, (b-1) disulfide stretching modes of MPDMS NPs in the frequency range of 480−550 cm−1, and (b-2) thiol stretching modes of MPDMS NPs in the frequency range of 2420−2620 cm−1.

strong SR is continuously irradiated on organosilica NPs for a long period of time, there is a risk of radiation damage such as breakage of molecular bonds and deterioration of the sample. Therefore, while continuing to irradiate the same part of the surface of the MPMS NPs with SR overnight, the four energy regions corresponding to each element were subjected to seven consecutive XPS measurements of about 57 min each, and the changes of spectral shapes and intensities were examined. In Figure 6, the S 2p, C 1s, O 1s, and Si 2p spectra are arranged in order of increasing SR irradiation time from the bottom to the top. While drastic changes such as disappearance of some peaks or appearance of new peaks did not happen, we did observe significant changes in the O 1s spectra in Figure 6c; the relative intensities of the two peaks reversed between the first and second scans. According to ref 40, these peaks are thought to originate from O−C and O−Si bonds, meaning that the SR with a photon energy of 690 eV decreases the relative amount of O−Si bonds and increases that of O−C bonds. This suggests that a part of the precursor remained unreacted in the synthesis process of the NPs because O−C bonds derived from CH3O groups should not exist in MPMS NPs in which the chemical reaction to form the silsesquioxane network was completed. The decreasing intensity of the O−Si peak indicates that O−Si bonds were broken by the SR and the network structure of silsesquioxane was destroyed. Furthermore, in Figure 6b, because the peak intensity of C 1s derived from the C−C/C−H bonds increased with the irradiation time, carbon contamination may occur. After the SR damage induced under various conditions was examined, it was decided that the optimum time to expose a single region of a sample to SR during an XPS measurement was 3 min. Also, to avoid overlap with the peak of Auger electrons, the SR energy was selected to hν = 1150 eV, so that the XPS measurements were

Table 1. Raman Modes of MPMS and MPDMS NPs NPs MPMS

MPDMS

−1

a

wavenumber (cm )

assignment

relative intensity

508 522 2485 2514 2566 509 524 2445 2495 2586

disulfide disulfide thiol thiol thiol disulfide disulfide thiol thiol thiol

1.2 1 1.8 13 6.5 144 84 1 1.4 1.2

a

The relative intensities of the peaks were calculated on the basis of the intensity of the weakest peak.

177 eV), C 1s (279−297 eV), O 1s (527−541 eV), and Si 2p (98−110 eV)were scanned using a hemispherical electron energy analyzer. We then compared two different radiation sources for XPS: synchrotron radiation (SR) or an Al/Mg twin-anode X-ray gun. The XPS results for MPMS NPs obtained with the Al Kα line of hν = 1486.6 eV are shown as the bottom spectrum in Figure 6. Although there is noise, no peak was observed in the S 2p energy region, a region that can provide information about disulfide bonds and thiols. Likewise, the Si 2p peak originating from the core Si atom of silsesquioxane did not appear. These contrast with the results obtained using SR; the noise level was clearly lowered compared with that of the spectrum obtained with the Al Kα line, and S 2p and Si 2p peaks were clearly observed. Therefore, SR provides better signals compared with XPS measurements using the Al Kα line. The above results suggest that the SR is a good source of radiation for characterizing thiol-organosilica NPs. However, if E


Article


Figure 6. XPS analyses of powder MPMS NPs using SR and an Al Kα line to reveal SR-induced damage: (a) S 2p, (b) C 1s, (c) O 1s, and (d) Si 2p regions.

Figure 7. XPS analyses of MPMS NPs and MPDMS NPs before and after incubation in 10 mM GSH solutions at 37 °C for 7 d: (a) S 2p (158− 174 eV), (b) C 1s (278−294 eV), (c) O 1s (527−541 eV), and (d) Si 2p (97−110 eV) regions.

C−H bonds in the C 1s spectra was adjusted to 284.2 eV, which is the value of the C 1s peak of L-cysteine on a gold surface measured in surface-sensitive mode,41 to calibrate the binding energy. Peaks derived from S−H, S 2p1/2, and S 2p3/2 of the S−C covalent bonds were observed in the S 2p spectra for all NPs in a relatively low binding energy range. In contrast, characteristic peaks were observed for each type of NP at binding energies of 166 eV or higher. The results of MPMS NPs measurement are shown first. These particles before incubation displayed a high-intensity peak at 168.4 eV. Although this peak can be decomposed into multiple peaks, it was identified as C−SOx (x = 1−4) according to ref 42. This means that the thiols on the surface of MPMS NPs were changed to groups like sulfene (SOH) and sulfinic (SOOH) by oxidation. Because the Milli-Q water used

executed focusing on the inner shell level of the NPs, such as S 2p, C 1s, O 1s, and Si 2p. XPS Characterization of Surface States of Thiol-Organosilica NPs. Figure 7 displays XPS measurement results obtained for the organosilica NPs shown in Figure 3. The S 2p, C 1s, O 1s, and Si 2p spectra of the MPMS NPs and MPDMS NPs before and after incubation with GSH are shown. A sample containing organic molecules is easy to be charged. In particular, the MPDMS NPs after incubation in which glutathione remained could be difficult to observe even by FE-SEM at a high acceleration voltage because of charge up. Because they are chemically stable, XPS curves are usually calibrated with reference to the Au 4f5/2 or 4f7/2 peak. However, considering the influence of the above-described charge up, the binding energy for the peak derived from C−C/ F


Article


Figure 8. XPS analyses of GSH and GSSG powders: (a) S 2p (158−174 eV), (b) C 1s (278−294 eV), (c) O 1s (527−541 eV), and (d) Si 2p (97− 110 eV) regions.

were inhibited by relatively large and highly electronegative O atoms. In addition, because the surface of MPDMS NPs after incubation is covered with GSSG, it is difficult to detect the Si 2p peaks of the surface layer of MPDMS NPs incubated with GSH. A peak in the S 2p spectrum derived from disulfide bonds is calculated to appear at a binding energy of 166.7 eV. This peak is close to the binding energy of disulfide bonds (166.2 eV) of dodecanethiol−Au NPs in a sol−gel measured by XPS.43 However, this peak was not detected with MPDMS before GSH incubation (Figure 7a). This is presumably because XPS is not capable of detecting disulfide bonds that are inside the NP, as XPS detects electrons that are directly ionized by Xrays. Thus, only electrons from atoms located up to a depth of several nanometers from the NP surface are detected. Interestingly, however, a peak at 166.7 eV was detected with MPDMS NPs after GSH incubation. We believe that this result can be explained by the appearance of GSSG on the surface of the NPs. In order to evaluate the above interpretation that the spectra of disulfide bonds detected after GSH incubation are due to GSH remaining on the NP surface, we compared the MPDMS spectra with those obtained when GSSG was subjected to XPS. Figure 8 shows S 2p, C 1s, O 1s, and Si 2p spectra of GSH and GSSG powders. Peaks derived from S−H and S−C S 2p3/2 bonds with similar binding energies were observed for GSH and GSSG powders. In addition, a peak due to S−C S 2p1/2 can be confirmed in the GSH powder. For the GSSG powder, a peak derived from disulfide bonds was observed. This peak matched that of the MPDMS NPs after GSH incubation in Figure 7a. Although no marked change was observed in the C 1s spectrum of GSH, peaks originating from O−C=O and π → π* excitation were detected for the GSSG powder. The O 1s spectra for the different samples were identical. No peak was observed in the Si 2p spectra because both GSH and GSSG do not contain Si atoms (Figure 2). This means that GSH behaved as a reductant toward the MPDMS NPs and itself was oxidized to GSSG. In these XPS measurements, although there was no direct evidence that the disulfide bonds inside MPDMS NPs were dissociated by the action of GSH to form thiols, the direct circumstantial evidence of the conversion of GSH to GSSG indicated that the disulfide bonds on the surface of MPDMS NPs were cleaved.

as a buffer for the NPs had not been degassed to remove oxygen, it is considered to contain many oxygen molecules. These residual oxygen molecules are believed to have oxidized the thiols on the surface during sample storage and transport. When the organosilica NPs were incubated in 10 mM GSH aqueous solution for 7 d, the peak intensity of C−SOx decreased remarkably. Therefore, GSH acted as a reducing agent and it is presumed that many C−SOx groups in the NPs were reduced to C-SH. Consistent with the Raman spectra in Figure 5, disulfide bonds were not detected in MPMS NPs by XPS measurements. In the O 1s spectrum of MPMS NPs before incubation, the binding energy is shifted by roughly 5 eV at the peak derived from O−C and O=C in the same direction. When the surface of multiwalled carbon nanotubes (MWCNTs) was modified with SO3H, a similar shift was observed at the 0.48 eV low binding energy side with a mixed O−C and O=S peak. Moreover, it was reported that the peak where O=C and O−S were mixed was shifted at the 0.45 eV low binding energy side.44 Thus, in the O 1s spectrum, it was also confirmed that SH groups of MPMS NPs before incubation were oxidized to C−SOx. We then examined MPDMS NPs by XPS. The C 1s spectra of the MPMS NPs before and after incubation and MPDMS NPs before incubation are similar. In the O 1s spectrum of MPMS NPs before incubation, the binding energy of Si−OH in tetraethyl orthosilicate (TEOS) was reported to be approximately 532.9 eV,45 and this value is almost the same as the binding energy of the single peak. Conversely, in the C 1s and O 1s spectra of the incubated MPDMS NPs, two peaks derived from O−C=O and a π → π* excitation (290.6 eV) of π electrons emitted from the C 1s orbital containing an unsaturated bond were independently observed.46 The binding energy of the latter peak is in good agreement with that observed for GSSG on gold (290 eV).47 This result is direct evidence of GSSG adsorbed on the surface of MPDMS NPs after incubation with GSH. The production of GSSG is important indirect evidence of the reaction between GSH and MPDMS NPs. The Si 2p spectra of the MPMS NPs after incubation and MPDMS NPs before incubation contained peaks derived from Si−C and Si−O. This phenomenon can be understood by considering the S 2p spectra described above. In the MPMS NPs before incubation, the Si−CH2CH2CH2SH terminal groups extending from the spherical surface are oxidized to C−SOx. There is a possibility that X-rays did not reach Si atoms present in the surface layer of the NPs or even if X-rays arrived, the photoelectrons escaping from the Si atoms G


Article


■

DISCUSSION Silica-based NPs with a biodegradable feature are of major interest, as they may provide promising material for delivery of anticancer drugs. In this paper, we have synthesized two kinds of thiol-organosilica NPs, MPMS NPs and MPDMS NPs that are synthesized from two different thiol-containing precursors. Their physicochemical evaluation before and after incubation with GSH was carried out by using Raman spectroscopy, FESEM and XPS. MPDMS is of particular interest, as this precursor forms a maximum two siloxane bonds and requires formation of disulfide linkages in order to form the 3D structure of the NPs. The Raman spectra clarified the presence of disulfide bonds in the MPDMS NPs. On the other hand, little Raman peaks of disulfide bonds were detected from MPMS NPs. Nakamura and Ishimura reported that the fabrication rate of MPDMS NPs is slower than that of MPMS NPs under the same conditions.22 Considering this result, the hydrolysis of MPDMS and the formation of siloxane bonds are faster than the formation of disulfide bonds. In the synthetic process of MPMS NPs, dense silsesquioxane cores are formed. Hence, the mobility of thiols is suppressed in MPMS NPs, and the repression of reactions between thiols leads to preservation of thiols in MPMS NPs. On the other hand, in the synthetic process of MPDMS NPs, dense silsesquioxane cores are not formed. Thus, the mobility of thiols is not repressed in the intermediates of MPDMS NPs, and disulfide bonds between intermediates derived from MPDMS are formed using oxygen dissolved in the reaction solutions. Because the formation of disulfide bonds leads to the production of MPDMS NPs, thiols on the surface of MPDMS NPs are likely to remain. This explanation is supported by the peak of thiols on the surface of MPDMS NPs before incubation with GSH shown in S 2p spectra of Figure 7. Incubation of MPDMS NPs with GSH resulted in degradation of the MPDMS NPs into smaller particles, while no changes were detected with MPMS NPs. Finally, XPS that probes the surface of organosilica NPs provided indirect evidence that MPDMS NPs react with GSH but MPMS NPs do not react with GSH. Our analyses showed that MPDMS NPs have a lot of disulfide bonds and represent the GSH-responsive biodegradable feature. The organosilica NPs may be promising as a delivery vehicle for anticancer drugs. In this paper, we report the characterization of organosilica NPs by XPS. This method uses monochromatic SR with a photon energy of 1150 eV as the X-ray source and can provide detailed information on the chemical state of sulfur in the NPs. In particular, the atomic state of NP surface can be probed with this method. This powerful method has not been employed to characterize NPs containing organic components in the past, as radiation damage causes problems with the experiment. We have shown here that it is possible to carry out this type of experiment. This is because our organosilica NPs are relatively stable and it is possible to define experimental conditions where radiation damage can be minimized (the damage could be minimized by using a short irradiation time of less than 3 min). Nuclear magnetic resonance (NMR) spectroscopy is also effective for elemental analysis of organic−inorganic hybrid materials;48,49 however, it is difficult to measure the 33S NMR spectra of organic sulfur compounds using conventional NMR spectrometers.50 Our XPS study revealed information regarding the chemical state of the NP surface before and after incubation with GSH.

Spectra due to S−C, S−H, C−S, C−C, O−C, and O−S were detected with both MPDMS NPs and MPMS NPs. Incubation with GSH decreased the S 2p peak intensity derived from C− SOx bonds of the MPMS NPs. Regarding the spectrum due to disulfide bonds, this signal was not detected with MPDMS NPs by an influence of inelastic scattering on the surface of NPs. However, it was surprising that we detected the disulfide peak with MPDMS NPs after GSH incubation. Examination of GSH and GSSG spectra by XPS suggested that the signal actually originated from GSSG. It is likely that GSH adsorbed on the surface of MPDMS NPs was converted to GSSG when the internal disulfide bonds of MPDMS NPs were cleaved. This indirectly suggests that the disulfide bonds in the MPDMS NPs were cleaved by the thiol−disulfide exchange reaction induced by GSH. FE-SEM observations showed the shape of MPDMS NPs dramatically changed after incubation with GSH. Most of the MPDMS NPs were degraded to small particles after GSH incubation. On the other hand, MPMS NPs did not change by the incubation. These results demonstrate that MPDMS NPs are biodegradable in response to GSH, and the results are in agreement with the XPS observations.

■

CONCLUSION The physicochemical evaluation of organosilica NPs after incubation with GSH was conducted using XPS measurements. We investigated the radiation damage of organic molecules induced by X-ray irradiation, finding that damage could be suppressed using a short irradiation time of less than 3 min. When MPMS NPs and MPDMS NPs were incubated in 10 mM GSH aqueous solutions at 37 °C for 7 d and then observed by FE-SEM, we found that the MPMS NPs were not degraded by GSH. Conversely, the MPDMS NPs were degraded in association with oxidation of GSH. We investigated the chemical state of the NP surface before and after incubation with GSH by XPS measurements using monochromatic SR with a photon energy of 1150 eV as the Xray source. Incubation with GSH decreased the S 2p peak intensity derived from C−SOx bonds of the MPMS NPs. For the MPDMS NPs, a peak derived from disulfide bonds appeared that originated from GSSG. This indirectly indicated that the disulfide bonds on the surface of the organosilica NPs were cleaved by the thiol−disulfide exchange reaction induced by GSH. The change of MPDMS NPs after incubation with GSH supported the degradation of MPDMS NPs induced by GSH. It is known that organosilica NPs that have disulfide bonds are reduced and degraded by GSH.11−13,17−21 However, the analyses are mainly dependent on the morphological analysis using electron microscopy. To our knowledge, there is no report of direct and detailed analyses about interface chemistry between GSH and organosilica NPs containing disulfide bonds, and this paper is the first report. This report revealed that MPDMS NPs are a kind of attractive GSHresponsive biodegradable organosilica NPs that are potential nanomaterials for antitumor nanomedicine.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b02023. Biodegradation testing of organosilica NPs in GSH solution (Figure S1), preparation of liquid and powder H



■

■

samples for FE-SEM observation and XPS measurements (Figure S2), particle size distributions of MPMS NPs before and after incubation and MPDMS NPs before incubation measured by dynamic light scattering (Figure S3), and Experimental Details (PDF)

Article

REFERENCES

(1) Thassu, D.; Pathak, Y. V.; Deleers, M.; Rabinow, B. E.; Chakrauarthi, S. S.; Robinson, D. F.; De, S.; Burke, M. D.; Luzhansky, D.; Möschwitzer, J.; Müller, R. H.; Wu, J.; Zhan, X.; Lee, R. J.; Jayagopal, A.; Shastri, V. P.; Raula, J.; Eerikäinen, H.; Lähde, A.; Kauppinen, E. I.; Bunjes, H.; Kuntsche, J.; Paciotti, G. F.; Tamarkin, L.; Chiang, J. F.; Jain, K. K.; Souto, E. B.; Cohen-Sela, E.; Elazar, V.; Epstein-Banash, H.; Golomb, G.; Ludwig, A.; Olivier, J.-C.; de Oliveira, M. P.; Vasir, J. K.; Labhaselwar, V.; Gasco, M. R.; Espuelas, S.; Gamazo, C.; Blanco-Prieto, M. J.; Irache, J. M.; Shim, J. Nanoparticulate Drug Delivery Systems. In Drugs and the Pharmaceutical Science; Thassu, D., Deleers, M., Pathak, Y. V., Eds.; CRC Press: Boca Raton, FL, 2007; Vol. 166, pp 1−352. (2) Yamamoto, E.; Kuroda, K. Colloidal Mesoporous Silica Nanoparticles. Bull. Chem. Soc. Jpn. 2016, 89, 501−539. (3) Mekaru, H.; Lu, J.; Tamanoi, F. Development of Mesoporous Silica-Based Nanoparticles with Controlled Release Capability for Cancer Therapy. Adv. Drug Delivery Rev. 2015, 95, 40−49. (4) Lu, J.; Liong, M.; Li, Z.; Zink, J. I.; Tamanoi, F. Biocompatibility, Biodistribution, and Drug-Delivery Efficiency of Mesoporous Silica Nanoparticles for Cancer Therapy in Animals. Small 2010, 6, 1794− 1805. (5) Croissant, J. G.; Fatieiev, Y.; Khashab, N. M. Degradability and Clearance of Silicon, Organosilica, Silsesquioxane, Silica Mixed Oxide, and Mesoporous Silica Nanoparticles. Adv. Mater. 2017, 29, 1604634. (6) Chen, G.; Teng, Z.; Su, X.; Liu, Y.; Lu, G. Unique Biological Degradation Behavior of Stöber Mesoporous Silica Nanoparticles from Their Interiors to Their Exteriors. J. Biomed. Nanotechnol. 2015, 11, 722. (7) Kempen, P. J.; Greasley, S.; Parker, K. A.; Campbell, J. L.; Chang, H.-Y.; Jones, J. R.; Sinclair, R.; Gambhir, S. S.; Jokerst, J. V. Theranostic Mesoporous Silica Nanoparticles Biodegrade after ProSurvival Drug Delivery and Ultrasound/Magnetic Resonance Imaging of Stem Cells. Theranostics 2015, 5, 631−642. (8) Zhai, W.; He, C.; Lei, W.; Zhou, Y.; Chen, H.; Chang, J.; Zhang, H. Degradation of Hollow Mesoporous Silica Nanoparticles in Human Umbilical Vein Endothelial Cells. J. Biomed. Mater. Res., Part B 2012, 100B, 1397−1403. (9) Yamada, H.; Urata, C.; Aoyama, Y.; Osada, S.; Yamauchi, Y.; Kuroda, K. Preparation of Colloidal Mesoporous Silica Nanoparticles with Different Diameters and Their Unique Degradation Behavior in Static Aqueous Systems. Chem. Mater. 2012, 24, 1462−1471. (10) Du, X.; Li, X.; Xiong, L.; Zhang, X.; Kleitz, F.; Qiao, S. Z. Mesoporous Silica Nanoparticles with Organo-Bridged Silsesquioxane Framework as Innovative Platforms for Bioimaging and Therapeutic Agent Delivery. Biomaterials 2016, 91, 90−127. (11) Croissant, J. G.; Mauriello-Jimenez, C.; Maynadier, M.; Cattoën, X.; Wong Chi Man, M.; Raehm, L.; Mongin, O.; Blanchard-Desce, M.; Garcia, M.; Gary-Bobo, M.; Maillard, P.; Durand, J.-O. Synthesis of Disulfide-Based Biodegradable Bridged Silsesquioxane Nanoparticles for Two-Photon Imaging and Therapy of Cancer Cells. Chem. Commun. 2015, 51, 12324−12327. (12) Croissant, J. G.; Cattoën, X.; Durand, J.-O.; Man, M. W. C.; Khashab, M. K. Organosilica Hybrid Nanomaterials with a High Organic Content: Syntheses and Applications of Silsesquioxanes. Nanoscale 2016, 8, 19945−19972. (13) Noureddine, A.; Brinker, J. C. Pendant/Bridged/Mesoporous Silsesquioxane Nanoparticles: Versatile and Biocompatible Platforms for Smart Delivery of Therapeutics. Chem. Eng. J. 2018, 340, 125− 147. (14) Croissant, J. G.; Cattoën, X.; Man, M. W. C.; Gallud, A.; Raehm, L.; Trens, P.; Maynadier, M.; Durand, J.-O. Biodegradable Ethylene-Bis(Propyl)Disulfide-Based Periodic Mesoporous Organosilica Nanorods and Nanospheres for Efficient In-Vitro Drug Delivery. Adv. Mater. 2014, 26, 6174−6180. (15) Du, X.; Kleitz, F.; Li, X.; Huang, H.; Zhang, X.; Qiao, S.-Z. Disulfide-Bridged Organosilica Frameworks: Desighed, Synthesis, Redox-Triggered Biodegradation, and Nanobiomedical Applications. Adv. Funct. Mater. 2018, 28, 1707325.

AUTHOR INFORMATION

Corresponding Author

*Research Center for Ubiquitous MEMS and Micro Engineering (UMEMSME), National Institute of Advanced Industrial Science and Technology (AIST), 1-2-1 Namiki, Tsukuba, Ibaraki 305-8564, Japan. Phone: 81-29-861-2431. Fax: +81-29861-7225. E-mail: [email protected]. ORCID

Harutaka Mekaru: 0000-0002-1120-6018 Michihiro Nakamura: 0000-0002-9216-3215 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank C. Tsukada and H. Yoshida of JAEA for providing technical support for the XPS experiments at BL23SU in the SPring-8. The XPS experiments were performed under the Shared Use Program of JAEA Facilities (Proposal No. 2016BE17) with the approval of Nanotechnology Platform project supported by the Ministry of Education, Culture, Sports, Science and Technology (Proposal No. A-16-AE-0027) and were performed at JAEA beamline BL23SU in SPring-8 (Proposal Nos. 2016A3801, 2016B3834, and 2017B3801). Part of this work was financially supported by the National Institute for Materials Sciences (NIMS) as a trial use program of the Nanotechnology Platform (Proposal No. NPS16064) and JSPS KAKENHI Grant Number JP15K21764. Thiolorganosilica NPs were provided as part of the Japan Society for the Promotion of Science (JSPS) Bilateral Joint Research Project (Proposal No. 16039901-000721) in 2016−2018. We received technical support from K. Fujii and T. Yanko of Yamaguchi University regarding the Raman spectroscopy measurements. We thank K. Kuroda of Waseda University for critically reading the paper and giving us comments on sol− gel chemistry.

■

ABBREVIATIONS NP = nanoparticle EPR = enhanced permeation and retention MSN = mesoporous silica nanoparticles ICP-AES = inductively coupled plasma−atomic emission spectroscopy TEM = transmission electron microscopy PMO = periodic mesoporous organosilica GSH = reduced glutathione MPMS = (3-mercaptopropyl)trimethoxysilane MPDMS = (3-mercaptopropyl)methyldimethoxysilane 3D = three-dimensional XPS = X-ray photoelectron spectroscopy FE-SEM = field-emission scanning electron microscopy GSSG = oxidized glutathione SR = synchrotron radiation MWCNT = multiwalled carbon nanotube TEOS = tetraethyl orthosilicate NMR = nuclear magnetic resonance I


Article

ACS Applied Nano Materials (16) Balendiran, G. K.; Dabur, R.; Fraser, D. The Role of Glutathione in Cancer. Cell Biochem. Funct. 2004, 22, 343−352. (17) Yang, Y.; Wan, J.; Niu, Y.; Gu, Z.; Zhang, J.; Yu, M.; Yu, C. Structure-Dependent and Glutathione-Responsive Biodegradable Dendritic Mesoporous Organosilica Nanoparticles for Safe Protein Delivery. Chem. Mater. 2016, 28, 9008−9016. (18) Maggini, L.; Cabrera, I.; Ruiz-Carretero, A.; Prasetyanto, R. A.; Robinet, E.; De Cola, L. Breakable Mesoporous Silica Nanoparticles for Targeted Drug Delivery. Nanoscale 2016, 8, 7240−7247. (19) Hadipour Moghaddam, S. P.; Saikia, J.; Yazdimamaghani, M.; Ghandehari, H. Redox-Responsive Polysulfide-Based Biodegradable Organosilica Nanoparticles for Delivery of Bioactive Agents. ACS Appl. Mater. Interfaces 2017, 9, 21133−21146. (20) Möller, K.; Bein, T. Talented Mesoporous Silica Nanoparticles. Chem. Mater. 2017, 29, 371−388. (21) Yu, L.; Chen, Y.; Lin, H.; Du, W.; Chen, H.; Shi, J. Utrasmall Mesoporous Organosilica Nanoparticles: Morphology Modulations and Redox-Responsive Biodegradability for Tumor-specific Drug Delivery. Biomaterials 2018, 161, 292−305. (22) Nakamura, M.; Ishimura, K. Synthesis and Characterization of Organosilica Nanoparticles Prepared from 3-Mercaptopropyltrimethoxysilane as the Single Silica Source. J. Phys. Chem. C 2007, 111, 18892−18898. (23) Nakamura, M.; Ishimura, K. One-Pot Synthesis and Characterization of three Kinds of Thiol-Organosilica Nanoparticles. Langmuir 2008, 24, 5099−5108. (24) Doura, T.; Tamanoi, F.; Nakamura, M. Miniaturization of Thiol-Organosilica Nanoparticles Induced by An Anionic Surfactant. J. Colloid Interface Sci. 2018, 526, 51−62. (25) Nakamura, M. Biomedical Applications of Organosilica Nanoparticles toward Theranostics. Nanotechnol. Rev. 2012, 1, 469− 491. (26) Nakamura, M. Chapter Eight - Organosilica Nanoparticles and Medical Imaging. In The Enzymes, Mesoporous Silica-based Nanomaterials and Biomedical Applications, Part B; Tamanoi, F., Ed.; Elsevier: Amsterdam, 2018; Vol. 44, pp 137−173. (27) Liu, P.; Liu, W.; Xue, Q. In Situ Chemical Oxidative Graft Polymerization of Aniline from Silica Nanoparticles. Mater. Chem. Phys. 2004, 87, 109−113. (28) Hakim, L. F.; Blackson, J.; George, S. M.; Weimer, A. W. Nanocoating Individual Silica Nanoparticles by Atomic Layer Deposition in a Fluidized Bed Reactor. Chem. Vap. Deposition 2005, 11, 420−425. (29) Hong, C.-Y.; Li, X.; Pan, C.-Y. Grafting Polymer Nanoshell onto the Exterior Surface of Mesoporous Silica Nanoparticles via Surface Reversible Addition-Fragmentation Chan Transfer Polymerization. Eur. Polym. J. 2007, 43, 4114−4122. (30) Mahtab, F.; Yu, Y.; Lam, J. W. Y.; Liu, J.; Zhang, B.; Lu, P.; Zhang, X.; Tang, B. Z. Fabrication of Silica Nanoparticles with Both Efficient Fluorescence and Strong Magnetization, and Exploration of Their Biological Applications. Adv. Funct. Mater. 2011, 21, 1733− 1740. (31) Gu, G.; Dang, H.; Zhang, Z.; Wu, Z. Fabrication and Characterization of Transparent Superhydrophobic Thin Films Based on Silica Nanoparticles. Appl. Phys. A: Mater. Sci. Process. 2006, 83, 131−132. (32) Liu, F.; Wen, L.-X.; Li, Z.-Z.; Yu, W.; Sun, H.-Y.; Chen, J.-F. Porous Hollo Silica Nanoparticles as Controlled Delivery System for Water-Soluble Pesticide. Mater. Res. Bull. 2006, 41, 2268−2275. (33) An, Y.; Chen, M.; Xue, Q.; Liu, W. Preparation and SelfAssembly of Carboxylic Acid-Functionalized Silica. J. Colloid Interface Sci. 2007, 311, 507−513. (34) Zhang, K.; Ma, J.; Zhang, B.; Zhao, S.; Li, Y.; Xu, Y.; Yu, W.; Wang, J. Synthesis of Thermoresponsive Silica Nanoparticle/PNIPAN Hybrids by Aqueous Surface-Initiated Atom Transfer Radical Polymerization. Mater. Lett. 2007, 61, 949−952. (35) Teraoka, Y.; Yoshigoe, T. Design of Surface Chemistry EndStation of BL23SU in SPring-8. Jpn. J. Appl. Phys. 1999, 38, 642−645.

(36) Saitoh, Y.; Nakatani, T.; Matsushita, T.; Agui, A.; Yoshigoe, A.; Teraoka, Y.; Yokoya, A. First Results from the Actinide Science Beamline BL23SU at SPring-8. Nucl. Instrum. Methods Phys. Res., Sect. A 2001, 474, 253−258. (37) Bazylewski, P.; Divigalpitiya, R.; Fanchini, G. In Situ Raman Spectroscopy Distinguishes Between Reversible and Irreversible Thiol Modifications in L-Cysteine. RSC Adv. 2017, 7, 2964−2970. (38) Van Wart, H. E.; Scheraga, H. A. Agreement with The Disulfide Stretching Frequency-Conformation Correlation of Sugeta, Go, and Miyazawa. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 3064−3067. (39) Pawlukojć, A.; Leciejewicz, J.; Ramirez-Cuesta, A. J.; NowickaScheibe, J. L-Cysteine: Neutron Spectroscopy, Raman, IR and ab Initio Study. Spectrochim. Acta, Part A 2005, 61, 2474−2481. (40) Qiu, X.; Li, N.; Yang, S.; Chen, D.; Xu, Q.; Li, H.; Lu, J. A New Magnetic Nanocomposite for Selective Detection and Removal of Trance Copper Ions from Water. J. Mater. Chem. A 2015, 3, 1265− 1271. (41) Uvdal, K.; Bodö, P.; Liedberg, B. L-Cysteine Adsorbed on Gold and Copper: An X-Ray Photoelectron Spectroscopy Study. J. Colloid Interface Sci. 1992, 149, 162−173. (42) Sun, C.; Zhang, Y.; Wang, P.; Yang, Y.; Wang, Y.; Xu, J.; Wang, Y.; Yu, W. W. Synthesis of Nitrogen and Sulfur Co-Doped Carbon Dots from Garlic for Selective Detection of Fe3+. Nanoscale Res. Lett. 2016, 11, 110. (43) Ferrara, M. C.; Mirenghi, L.; Mevoli, A.; Tapfer, L. Synthesis and Characterization of Sol-Gel Silica Films Doped with Size-Selected Gold Nanoparticles. Nanotechnology 2008, 19, 365706. (44) Wei, Y.; Ling, X.; Zou, L.; Lai, D.; Lu, H.; Xu, Y. A Facile Approach Toward Preparation of Sulfonated Multi-Walled Carbon Manotubes and Their Dispersibility in Various Solvents. Colloids Surf., A 2015, 482, 507−513. (45) Harb, S. V.; Cerrutti, B. M.; Pulcinelli, S. H.; Santilli, C. V.; Hammer, P. Siloxane-PMMA Hybrid Anti-Corrosion Coating Reinforced by Lignin. Surf. Coat. Technol. 2015, 275, 9−16. (46) Subramanian, A. S.; Tey, J. N.; Zhang, L.; Ng, B. H.; Roy, S.; Wei, J.; Hu, X. M. Synergistic Bond Strengthening in Epoxy Adhesives Using Polydopamine/MWCNT Hybrids. Polymer 2016, 82, 285− 294. (47) Calborean, A.; Martin, F.; Marconi, D.; Turcu, R.; Kacso, I. E.; Buimaga-Iarinca, L.; Graur, F.; Turcu, I. Adsorption Mechanisms of L-Glutathione on Au and Controlled Nano-Patterning Through Dip Pen Nanolithography. Mater. Sci. Eng., C 2015, 57, 171−180. (48) Dufaud, V.; Davis, M. E. Design of Heterogeneous Catalysts via Multiple Active Site Positioning in Organic-Inorganic Hybrid Materials. J. Am. Chem. Soc. 2003, 125, 9403−9413. (49) Lee, A. S. S.; Choi, S.-S.; Baek, K.-Y.; Hwang, S. S. Thiol-Ene Photopolymerization of Well-Defined Hybrid Graft Polymers from a Ladder-Like Polysilsesquioxane. Macromol. Res. 2015, 23, 60−66. (50) Yamada, K. Development of Solid-State NMR of Quadrupolar Spins with Variable Superconducting Magnets − Solid-State 33S NMR of Organic Solids−. Teion Kogaku 2016, 51, 64−70. [in Japanese]

J


Biodegradability of Disulfide-Organosilica ... - ACS Publications

Recommend Documents