Inflammation-Responsive Nanovalves of Polymer-Conjugated Dextran on a Hole Array of Silicon Substrate for Controlled Antibiotic Release

Poly(methacrylic acid) (PMAA) brushes were tethered on a silicon surface possessing a 500-nm hole array via atom transfer radical polymerization after the modification of the halogen group. Dextran-biotin (DB) was sequentially immobilized on the PMAA chains to obtain a P(MAA-DB) brush surrounding the hole edges on the silicon surface. After loading antibiotics inside the holes, biphenyl-4,4′-diboronic acid (BDA) was used to cross-link the P(MAA-DB) chains through the formation of boronate esters to cap the hole and block the release of the antibiotics. The boronate esters were disassociated with reactive oxygen species (ROS) to open the holes and release the antibiotics, thus indicating a reversible association. The total amount of drug inside the chip was approximately 52.4 μg cm−2, which could be released at a rate of approximately 1.6 μg h−1 cm−2 at a ROS concentration of 10 nM. The P(MAA-DB) brush-modified chip was biocompatible without significant toxicity toward L929 cells during the antibiotic release. The inflammation-triggered antibiotic release system based on a subcutaneous implant chip not only exhibits excellent efficacy against bacteria but also excellent biocompatibility, recyclability, and sensitivity, which can be easily extended to other drug delivery systems for numerous biomedical applications without phagocytosis- and metabolism-related issues.


Introduction
Bacteria are currently one of the major causes of nosocomial infections, which may lead to many unattended infectious diseases that can progress to systemic inflammation and multiorgan dysfunction, threatening human beings in more severe cases [1]. These pathogenic bacteria include Gram-negative and Gram-positive bacteria responsible for many infections in human beings with severe and even fatal consequences [2]. Sepsis is a multiorgan dysfunction disease caused by bacteria that can present with a poor prognosis and potential long-term sequelae. Bacterial infection can induce the systemic inflammatory response syndrome of sepsis, which poses great threat to human life [3]. To improve the sepsis survival rates, preferable antimicrobial treatment is recommended within the first hour of suspecting sepsis [4]. However, antibiotic-resistant pathogens are still particularly problematic in the context of sepsis caused by bacterium infections, which cause poor outcomes. The inflammatory phase generally results in the increase of reactive oxygen species acid (BDA) to form a boronate ester bond that can cap the holes and block the release of the drug (Scheme 1). The cleavage of the bond formed by the binding between DB and BDA in the polymer-capped system is not stable under ROS conditions. The boronate esters disassociated in the presence of H2O2, leading to the opening of the holes and the release of the loaded drug. This strategy offers a convenient approach for an antibiotic delivery system with ROS-triggered valves that can be applied as an implanted chip for long-term antimicrobial treatment. Scheme 1. ROS-triggered valves of P(MAA-DB) brush cross-linked with BDA for the controlled release of FITC-vancomycin.

Developing Antibiotic Delivery System with ROS-Triggered Valves
Scheme 2 illustrates the construction of the antibiotic delivery system with ROS-triggered valves [45]. A and B: Photoresist was coated on silicon wafers to pattern hole array of 500-nm resolution by I-line lithography. C: Patterns of the samples were transferred to silicon wafer by inductive couple plasma etcher (Hakuto Corp, Tokyo, Japan) with SF6, and the residual photoresists were removed from the surfaces of silicon wafers, denoted as 500H.

Developing Antibiotic Delivery System with ROS-Triggered Valves
Scheme 2 illustrates the construction of the antibiotic delivery system with ROStriggered valves [45]. A and B: Photoresist was coated on silicon wafers to pattern hole array of 500-nm resolution by I-line lithography. C: Patterns of the samples were transferred to silicon wafer by inductive couple plasma etcher (Hakuto Corp, Tokyo, Japan) with SF 6 , and the residual photoresists were removed from the surfaces of silicon wafers, denoted as 500H. D: After oxygen plasma treatment for 30 s to generate the hydrophilic groups on the surface, the samples were treated with 3A solution, and 2B solution was used to immobilize halogen groups on the surface as SI-ATRP initiators. A mixture of MAA, Cu(I)Br, CuBr2, and PMDETA in methanol/water (1:1), denoted as 500H-PMAA, was employed to Scheme 2. Illustration of the preparation processes of a polymer-capped system for ROS-triggered drug release. A and B: Photoresist was coated on silicon wafers to pattern hole array of 500-nm resolution by I-line lithography. C: Patterns of the samples were transferred to silicon wafer by inductive couple plasma etcher with SF 6 , and the residual photoresists were removed from the surfaces of silicon wafers. D: After oxygen plasma treatment, the samples were treated with 3A solution, and 2B solution was used to immobilize halogen groups on the surface as SI-ATRP initiators. E: PMAA was grafted from the initiator-modified chip via ATRP. DB units were conjugated to the carboxylic acid functional groups of PMAA brush as pendant groups. E: The prepared sample was loaded the drug inside the holes. F: The P(MAA-DB) brushes surrounding the hole edges were cross-linked by BDA to cap the holes of the chip. A network of P(MAA-DB)-BDA could be cleaved and capped reversibly to release and load the antibiotic for recycling. D: After oxygen plasma treatment for 30 s to generate the hydrophilic groups on the surface, the samples were treated with 3A solution, and 2B solution was used to immobilize halogen groups on the surface as SI-ATRP initiators. A mixture of MAA, Cu(I)Br, CuBr 2 , and PMDETA in methanol/water (1:1), denoted as 500H-PMAA, was employed to synthesize PMAA brushes on the initiator-modified chip via ATRP following treatment for 8 h at 25 • C. DB units were conjugated to the carboxylic acid functional groups of PMAA brush as pendant groups by EDC/NHS reaction and denoted as 500H-P(MAA-DB) [46]. E: The prepared 500H-P(MAA-DB) was immersed in FITC-vancomycin solution and incubated for 24 h to load the drug inside the holes. F: The P(MAA-DB) brushes surrounding the hole edges, denoted as 500H-P(MAA-DB)-BDA [47], were crosslinked by BDA to cap the holes of the chip. A network of P(MAA-DB)-BDA was cleaved under a H 2 O 2 concentration range of 5-20 mM to release the antibiotic from the holes. As-prepared samples could be loaded the antibiotic and the holes capped reversibly for recycling. Surface components and functional groups of the samples for each stage were analyzed by X-ray photoelectron spectroscopy (XPS; Scientific Theta Probe, Delta-T Devices, Cambridge, UK) and Fourier transform infrared spectrometry (FTIR, Digilab, FTS-1000, Holliston, MA, USA), respectively. The morphologies of the samples in dry and liquid states for each stage were observed using a field emission scanning electron microscope (FESEM; JEOL 7900F, Tokyo, Japan) and atomic force microscope (AFM; Veeco Dimension 5000 scanning probe microscope, Plainview, NY, USA) equipped with temperature control and liquid modules, respectively.

Cell In Vitro Studies
The biocompatibility of the drug-loaded 500H-P(MAA-DB)-BDA chip was evaluated with the cell viabilities by seeding the L929 cells on the surfaces of chips. L929 cells were incubated in the medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin, refreshed ever two days. For the biocompatibility assays, the cells were seeded at 10 4 cells/mL in each 96-well plate that filled with 150 µL medium without cells as blank references. 10 4 cells were also seeded onto the sterilized chips with 150 µL medium for cell proliferation. The cell viabilities were evaluated by CCK-8 assay at 1, 3, 5 and 7 days. A toll-like receptor 4 agonist, lipopolysaccharide (LPS), was employed to induce the production of ROS and evaluate the behavior of inflammation-triggered release in vitro by activating macrophages [48], and pH decreased in the microenvironment during the macrophage activation. Accordingly, the enhancement of macrophage activity leads to both pH and ROS stimuli, which can simulate an inflammatory microenvironment for evaluating the drug release and anti-inflammatory efficacy of the chips. 20,000 RAW 264.7 murine macrophages were grown and stimulated with LPSs at a concentration of 5 µg/mL for 2 h before incubation with the chips [48]. After various incubation times with chips, the media were collected to measure the released drug by fluorescence spectrometry at 515 nm using a plate reader (Molecular Devices SpectraMax M5, Sunnyvale, CA, USA). The macrophages without LPS activation after various incubation times were employed to evaluate the stability of the polymer network cap. The loading capacity of the chips, calculated by gravimetric analysis for FITC-vancomycin (52.4 µg/cm 2 ) in the same medium was employed as a standard to normalize the collected media. In addition, fluorescence microscopy images of macrophages were also employed to verify the cellular uptake of drug.
To trigger the inflammation-targeting efficacy of the antibiotics released from the chips, Raw 264.7 murine macrophages were activated by LPS (100 ng/mL). Each square-cut sample (0.5 cm × 0.5 cm) was incubated with 0.5 mL of LPS-activated macrophages solution, completely immersing the substrate for 2 h, to trigger the drug release by dissociating the cap of polymer network. After the dissociation of the cap, the chips were taken from the solution and then placed horizontally inside a 24-well plate and coincubated with Staphylococcus aureus (SA) in 0.5 mL of the medium, cation-adjusted Mueller Hinton Broth II (CMHB) per well. Briefly, 0.50 mL of SA in the exponential growth phase at 10 5 CFU/mL in CMHB was injected in the well to submerge the chip. These 24-well plates including chip and SA were incubated at 37 • C under gentle shaking for various periods. SA density was obtained with a calibration curve of the optical intensity at 600 nm in a microplate spectrophotometer. For each set of assays, culture media were incubated with bacteria under the same conditions without the chips as the negative controls to normalize the bacteria density. These assays were administered from the time of seeding to data recording by fluorescence spectrometry. Assays of the inflammation-triggered efficacy of antibiotics were performed based on the optical density of the medium cultures at 600 nm. SA was also cultured in the medium with chips under inflammation-free condition to evaluate the stability of drug release.

Morphology of the Drug Delivery Chip
Nanoparticles are generally employed as carriers of inflammation-responsive systems of drug releases [7]. However, the issues of phagocytosis and metabolism in the human body may influence the efficiency of ROS-responsive drug delivery systems in clinical trials. The use of a subcutaneous implant chip may facilitate autonomously antimicrobial treatment without these issues. Figure 1a illustrates FESEM topographies of the silicon template with a hole array. The holes had a diameter and depth of 500 nm with a smooth surface, indicating the regularity of the designed textures. For 500H-P(MAA-DB), and a thin layer surrounding the hole edge was observed, suggesting that P(MAA-DB) grafts collapse forward into the hole edge in a dry state (Figure 1b). The hole diameter decreased from 500 to 232 nm, indicating that the length of P(MAA-DB) chains in a dry state was 134 nm. Although the holes were not capped completely, the P(MAA-DB) swelled and extended the polymer chain 2-3 times in aqueous solution to cap the holes. Thus, the length of P(MAA-DB) brush was sufficient to cross-link by BDA and form a solid cap upon the holes.  The oxygen plasma treatment facilitated the charge accumulation at the hole, resulting in higher initiator and P(MAA-DB) grafting densities. The P(MAA-DB) brush with high grafting density formed a protuberance surrounding the hole edges. After cross-linking P(MAA-DB) graft with BDA upon the holes, a thick layer upon the chip holes over a large area was observed (Figure 1c). The cross-linked P(MAA-DB) with BDA accumulated around the holes to cap the holes, as observed in the top-view FESEM image. (Figure 1c) After immersing the sample in 10 mM of H 2 O 2 solution, the surface morphology of the chip became similar to that shown in Figure 1b, indicating the ROS-responsive behavior of the cross-linked P(MAA-DB) brush. Figure 1d,e show FESEM cross-section profiles of the 500H-P(MAA-DB)-BDA before and after immersion in the H 2 O 2 solution. The hole chamber had a diameter and depth of 500 nm and 800 nm as drug tank, respectively. Based on the cross-sectional FESEM images, caps of P(MAA-DB)-BDA grafts was observed upon each hole but not grafted inside the holes, attributing that neither the ATRP initiator nor MAA penetrated the hole sufficiently. Because the caps of polymer network were dissociated after immersion in the H 2 O 2 solution, the holes appear to be opened in the cross-section image of holes. Figure 2a illustrates the 2D/3D AFM topographies and cross-section profiles of the hole array structure on silicon substrate. Morphology of the hole array was consistent with the FESEM image. For 500H-P(MAA-DB), the stretched polymer brush altered the surface textures slightly, indicating that the hole array with polymer brush did not completely match the original template. In comparison with morphology in the dry state (Figure 1b), the flower-like structure of 500H-P(MAA-DB)-BDA remained without significant changes ( Figure 2b).
The hole profile did not appear in the cross-section profile because of narrowing of the holes with high aspect ratio. For 500H-P(MAA-DB)-BDA, the polymer brush surrounding the hole edge collapsed forward into the holes to cross-link with BDA as solid caps upon the holes, resulting in a complete close state of holes (Figure 2c). From the cross-section profile, the solid cap upon the holes bulged upon the holes to form the regular 500 nm polymer network, confirming that the drug could be contain inside the holes. The polymeric network of P(MAA-DB)-BDA chains could be dissociated under a low concentration of H 2 O 2 resulting in the reopening of holes. The results suggest that the ROS-triggered valve is appropriate to apply in drug delivery systems as implant chips. Figure 3 shows the photographs of 500H, 500H-P(MAA-DB), 500H-P(MAA-DB)-BDA. Because of the regular hole array, the 500H exhibited blue color that observed along an invariable angle. (Figure 3a) For 500H-P(MAA-DB), the color turns obviously to yellow color instead of initial blue color. (Figure 3b) The results suggest that the P(MAA-DB) grafts varied the hole diameter substantially, resulting in the change of grating effect. The grating effect vanished after cross-linking P(MAA-DB) graft with BDA upon the holes, indicated the disappearance of hole array structure.       [49]. The broad peaks of stretching vibrations of hydroxyl (COH) group of PMAA appeared in the range of 3130-3690 cm −1 [50]. For the FTIR spectrum of P(MAA-DB), a decrease in the intensity of broad peaks of stretching vibrations of COH group of PMAA indicated binding between carboxyl groups of PMAA and amide groups of DB. A decrease in the intensity of the carboxyl stretching vibration at 1554 cm −1 (secondary amide C=O stretching) can be attributed to the DB grafts on the PMAA brush. Because boronic acid derivatives can bind to diol groups, the P(MAA-DB) could be cross-linked by diboronic acid derivatives. BDA was introduced as a cross-linker to cap the holes on the silicon substrate through cross-linking the P(MAA-DB) brush with the boronate esters. Thus, adsorption wavelengths at 1272 and 1354 cm −1 were identified as characteristic bands of B-O to confirm the cross-linking [51].

Loading and Releasing of FITC-Vancomycin
FITC-vancomycin as selected as a model drug to evaluate ROS-triggered release behavior from 500H-P(MAA-DB)-BDA. The FITC-vancomycin was loaded through the hydrophilic P(MAA-DB) brush layer on the substrate into the hole array. The cross-linker, BDA, was employed to cap the hole and preserve the FITC-vancomycin inside the holes. The encapsulating efficiency (E) was calculated using the following equation determined using the fluorescence emission spectra: substrate was observed (Figure 4c). For releasing FTIC-vancomycin from the holes for 18 h, the high fluorescent intensity indicated the long-term drug diffusion from the hole array to the liquid phase (Figure 4d). To evaluate the stability and ROS-responsive behavior of drug release, the drug-loaded 500H-P(MAA-DB)-BDA was immersed in PBS at pH 7.4 in the absence of ROS for 4 h and H2O2 was added to the solution at various concentrations. The solutions were withdrawn at various time intervals to measure the total amount of the loaded FITC-vancomycin released based on the fluorescence intensities.  The release rate of FITC-vancomycin with 10 mM of H2O2 was markedly reduced. After ROS-triggering, 74.8% of the drug was released gradually within 48 h, indicating that H2O2 concentration is markedly associated with the release rate of FITC-vancomycin. We observed a linear increase in FITC-vancomycin release from 0 to 38.9% at 5 mM H2O2 solution in real time from 4 to 48 h, implying the long-term drug release. In addition, the coordination between the functional groups of boronic acid and carboxyl groups on the PMAA chain also decreased the pKa, which may be influenced by pH values [51]. Figure (Figure 5b). These results suggest that FITC-vancomycin was also released from the chip in an acidic environment, consistent with the findings of previous studies [52].   (Figure 5b). These results suggest that FITC-vancomycin was also released from the chip in an acidic environment, consistent with the findings of previous studies [46,53].
The compatibility of an implant chip is an important factor for facilitating host cell adhesion, spreading, and permanent growth. Heparin L929 cells were seeded on the tissue culture plate and sample surfaces to examine the cell affinity with cell proliferation by CCK-8 assay at 1, 3, 5 and 7 days. Optical density at 600 nm after cell proliferation on the tissue culture plate at various periods was employed as a standard to calculate the cell viability ratio of sample surfaces. The compatibility of an implant chip is an important factor for facilitating host cell adhesion, spreading, and permanent growth. Heparin L929 cells were seeded on the tissue culture plate and sample surfaces to examine the cell affinity with cell proliferation by CCK-8 assay at 1, 3, 5 and 7 days. Optical density at 600 nm after cell proliferation on the tissue culture plate at various periods was employed as a standard to calculate the cell viability ratio of sample surfaces. Figure 6a shows cell viability ratio proliferated on the sample surface measured by CCK-8 assay within 7 days. The cell viability ratio of 500H surface ranged from 34.6 to 42.3% indicating poor ability of cell proliferation. The ability of cell proliferation, ranged from 82.3 to 86.6%, was enhanced significantly with the PMAA graft. Additionally, the cell viability ratio of 500H-P(MAA-DB) surface exhibited a tendency of increase, which indicated that DB molecules possessed better compatibility than PMAA. After cross-linking 500H-P(MAA-DB) with BDA, the cell viability ratio of the surface slightly decreased, which might be ascribed to the change in morphology. All the chips can support cell adhesion and proliferation without toxicity. To evaluate the reversibility of the polymercapped system, 500H-P(MAA-DB)-BDA, after the cleavage of the phenylboric acid ester bond following treatment with 10 mM of H2O2 for 48 h, was loaded the drug reversibly, reacted with BDA to cap the hole, and reused with immersion in 10 mM of H2O2 for 48 h. Figure 6b illustrates the amount of FITC-vancomycin that can be loaded inside the chip and released from the chip in five cycles. The ability of the chip to store FITC-vancomycin decreased slightly from 43.7-52.4 μg/cm 2 after the drug was reloaded reversibly for five cycles. FITC-vancomycin concentration inside the chip after ROS-triggered release for 48 h remained in the range of 3.3-4.5 μg/cm 2 . The excellent reversibility of FITC-vancomycin loading and release through the polymer-capped system suggests that the stable hole opening and closing behavior can be switched to release and block FITC-vancomycin reversibly using H2O2 and BDA, respectively. P(MAA-DB) cross-linked with BDA can satisfactorily gate the hole array with strong potential for application in antimicrobial treatment.  Figure 6a shows cell viability ratio proliferated on the sample surface measured by CCK-8 assay within 7 days. The cell viability ratio of 500H surface ranged from 34.6 to 42.3% indicating poor ability of cell proliferation. The ability of cell proliferation, ranged from 82.3 to 86.6%, was enhanced significantly with the PMAA graft. Additionally, the cell viability ratio of 500H-P(MAA-DB) surface exhibited a tendency of increase, which indicated that DB molecules possessed better compatibility than PMAA. After cross-linking 500H-P(MAA-DB) with BDA, the cell viability ratio of the surface slightly decreased, which might be ascribed to the change in morphology. All the chips can support cell adhesion and proliferation without toxicity. To evaluate the reversibility of the polymer-capped system, 500H-P(MAA-DB)-BDA, after the cleavage of the phenylboric acid ester bond following treatment with 10 mM of H 2 O 2 for 48 h, was loaded the drug reversibly, reacted with BDA to cap the hole, and reused with immersion in 10 mM of H 2 O 2 for 48 h. Figure 6b illustrates the amount of FITC-vancomycin that can be loaded inside the chip and released from the chip in five cycles. The ability of the chip to store FITC-vancomycin decreased slightly from 43.7-52.4 µg/cm 2 after the drug was reloaded reversibly for five cycles. FITC-vancomycin concentration inside the chip after ROS-triggered release for 48 h remained in the range of 3.3-4.5 µg/cm 2 . The excellent reversibility of FITC-vancomycin loading and release through the polymer-capped system suggests that the stable hole opening and closing behavior can be switched to release and block FITC-vancomycin reversibly using H 2 O 2 and BDA, respectively. P(MAA-DB) cross-linked with BDA can satisfactorily gate the hole array with strong potential for application in antimicrobial treatment.
LPS was employed to activate macrophages, resulting in both pH and ROS stimuli that can simulate inflammatory microenvironment in vitro. FITC-vancomycin was encapsulated into the 500H-P(MAA-DB) with seal of polymeric network as a model drug to investigate the behavior of inflammation-responsive drug release. Fluorescence of the drug did not be observed with seal of polymer network; however, it could be observed after release from the hole array and internalization in the cellular esterase. Macrophages with LPS activation were incubated on the drug-loaded chips to evaluate the performance of the drug release. In addition, the LPS-free macrophages with the chip in the media were considered as a control. LPS was employed to activate macrophages, resulting in both pH and ROS stimuli that can simulate inflammatory microenvironment in vitro. FITC-vancomycin was encapsulated into the 500H-P(MAA-DB) with seal of polymeric network as a model drug to investigate the behavior of inflammation-responsive drug release. Fluorescence of the drug did not be observed with seal of polymer network; however, it could be observed after release from the hole array and internalization in the cellular esterase. Macrophages with LPS activation were incubated on the drug-loaded chips to evaluate the performance of the drug release. In addition, the LPS-free macrophages with the chip in the media were considered as a control. Figure 7a,b shows the fluorescence microscopy images of in LPS-activated and LPSfree macrophages with the 500H-P(MAA-DB)-BDA, respectively. The macrophages without LPS activation did not exhibit significant fluorescence in the image. The obvious fluorescence of macrophages with LPS activation appeared in the image, indicating the excellent performance of inflammation-responsive drug release. Figure 7c shows that the normalized fluorescence of LPS-activated and LPS-free macrophages plotted as a function of time on the chips. The fluorescence intensity of macrophages with LPS activation increased gradually from 0 to 86.9% with time from 0 to 2 h and reached a plateau from 2 to 3 h, indicating that 2 h of the optimal incubation time of the inflammation-triggered drug release. While the fluorescence intensity of LPS-free macrophages did not increase significantly, suggested that the FITC-vancomycin was contained inside the hole array stably. The fluorescence intensity of macrophages with LPS activation for 4 h was 9.1 times higher than that without LPS activation, suggested that the pH/ROS dual-responsive chip release drug in an inflammatory environment. Furthermore, we evaluated inflammation-triggered efficacy of antibiotics released from the chips and exhibited the potential of the chip as drug delivery vehicles. SA, a common bacterium of biomedical implant infections, was employed in vitro assessments of the therapeutic activities. The drug-loaded 500H-P(MAA-DB)-BDA chip were incubated with LPS-activated and LPS-free macrophages for 2 h, and then coincubated with SA sample. Figure 7d shows the dependence of normalized bacteria density on the culture time. Normalized bacteria density (NBD) was calculated as follows:   Figure 7c shows that the normalized fluorescence of LPS-activated and LPS-free macrophages plotted as a function of time on the chips. The fluorescence intensity of macrophages with LPS activation increased gradually from 0 to 86.9% with time from 0 to 2 h and reached a plateau from 2 to 3 h, indicating that 2 h of the optimal incubation time of the inflammation-triggered drug release. While the fluorescence intensity of LPS-free macrophages did not increase significantly, suggested that the FITC-vancomycin was contained inside the hole array stably. The fluorescence intensity of macrophages with LPS activation for 4 h was 9.1 times higher than that without LPS activation, suggested that the pH/ROS dual-responsive chip release drug in an inflammatory environment. Furthermore, we evaluated inflammation-triggered efficacy of antibiotics released from the chips and exhibited the potential of the chip as drug delivery vehicles. SA, a common bacterium of biomedical implant infections, was employed in vitro assessments of the therapeutic activities. The drug-loaded 500H-P(MAA-DB)-BDA chip were incubated with LPS-activated and LPS-free macrophages for 2 h, and then coincubated with SA sample. Figure 7d shows the dependence of normalized bacteria density on the culture time. Normalized bacteria density (NBD) was calculated as follows: where OD chip and OD control represented the optical density of SA that cultured with and without the chips at 600 nm, respectively. While OD blank represented the optical density of blank medium. The SA density in the medium with the chip remained 100% within 6 h under inflammation-free condition, indicating that chip did not release antibiotics significantly under inflammation-free condition to suppress the SA growth. The SA density decreased markedly in the medium with the inflammation-triggered chip, suggesting that SA growth was suppressed obviously due to release of the antibiotic from the chip. These results verified the behavior of inflammation-triggered antibiotic release to inhibit SA growth for 500H-P(MAA-DB)-BDA chip.
blank medium. The SA density in the medium with the chip remained 100% within 6 h under inflammation-free condition, indicating that chip did not release antibiotics significantly under inflammation-free condition to suppress the SA growth. The SA density decreased markedly in the medium with the inflammation-triggered chip, suggesting that SA growth was suppressed obviously due to release of the antibiotic from the chip. These results verified the behavior of inflammation-triggered antibiotic release to inhibit SA growth for 500H-P(MAA-DB)-BDA chip.

Conclusions
PMAA brushes were grafted from a 500-nm hole array on a silicon wafer via SI-ATRP to immobilize with DB through an EDC/NHS reaction. The P(MAA-DB)-modified hole array loaded with FITC-vancomycin inside was capped by cross-linking between BDA and P(MAA-DB) to block the release of FITC-vancomycin. The polymer brush caps upon the 500-nm holes of the substrate were cleaved under the stimuli of ROS to release FITCvancomycin from the holes via diffusion. The dissociation of the polymer brush network

Conclusions
PMAA brushes were grafted from a 500-nm hole array on a silicon wafer via SI-ATRP to immobilize with DB through an EDC/NHS reaction. The P(MAA-DB)-modified hole array loaded with FITC-vancomycin inside was capped by cross-linking between BDA and P(MAA-DB) to block the release of FITC-vancomycin. The polymer brush caps upon the 500-nm holes of the substrate were cleaved under the stimuli of ROS to release FITC-vancomycin from the holes via diffusion. The dissociation of the polymer brush network was rebound by BDA reversibly to cap the hole for recycling. The reversible hole closing and opening with BDA and ROS could block and release the drug, respectively, which are appropriate for applications in antibiotic storage and delivery systems. The chips were also exhibited the excellent in vitro efficacy against bacteria growth without significant toxicity toward the cells. Our proposed chip could be more convenient than antibiotic-loaded nanoparticles for applications in clinical trials without phagocytosis-and metabolism-related issues. Although the amount of antibiotic loaded inside the hole array is not remarkable, the space of each hole can be extended using dry etching technology to enhance the storage capacity. The proposed antibiotic storage and delivery system implant chip not only exhibits smart polymer valves but also excellent biocompatibility, recyclability, and stability, which can be easily extended to other drug deliver system for numerous biomedical applications.

Institutional Review Board Statement:
This study has been officially approved by National Taiwan University of Science and Technology. The protocol was performed in accordance with the recommendations of the Guide of the Taichung Veterans General Hospital.

Informed Consent Statement: Not applicable.
Data Availability Statement: The authors declare that data related to this study are provided upon request.