Gated Mesoporous Silica Nanocarriers for Hypoxia

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Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24377−24385

Gated Mesoporous Silica Nanocarriers for Hypoxia-Responsive Cargo Release Qi Yan, Xuliang Guo, Xiaoli Huang, Xuan Meng, Fang Liu, Peipei Dai, Zheng Wang, and Yanjun Zhao* School of Pharmaceutical Science & Technology, Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China

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S Supporting Information *

ABSTRACT: Mesoporous silica nanocarriers (MSNs) are appealing in terms of their large cavity surface area and high loading capacity, but they have been suffering from premature cargo release. Herein, we report a gated smart MSN that is sensitive to low oxygen concentration (i.e., hypoxia) via taking advantage of the superior electron-accepting ability of the azobenzene moiety. The azobenzene polymer was employed as the responsive gate-keeper that was deposited on the MSN surface, followed by coating with amphiphilic Pluronic F68 for steric stabilization. The obtained nanocarriers were less than 200 nm. The in vitro polymer degradation was spectrophotometrically witnessed via the employment of a reducing agent, namely, sodium dithionite, with a strong electron-donating ability. The hypoxia-responsive cargo release from the gated MSN was quantitatively demonstrated in breast cancer cells (MCF-7) using the fluorescence resonance energy transfer (FRET) technique where coumarin 6 and rhodamine B was selected as the FRET donor and acceptor, respectively. The FRET ratio was used as the index and decreased linearly over time under hypoxia, whereas it almost remained steady under normoxia. In addition, a model photosensitizer, namely, chlorin e6, was also loaded in the gated MSN whose toxicity under hypoxia was verified. This study developed a hypoxia-responsive MSN with the azobenzene polymer as the removable gate-keeper, which would expand the application of MSNs in pharmaceutical and biomedical areas since the low oxygen concentration is a unique trigger in many pathological conditions. KEYWORDS: mesoporous silica nanocarriers, gate-keeper, hypoxia-responsive, azobenzene, controlled release adenosine triphosphate).15−20 Among these, hypoxia is a distinctive trigger as many pathological conditions (e.g., solid tumors) are well characterized with low oxygen levels.21−23 In terms of antitumor photodynamic therapy and sonodynamic therapy, the degree of hypoxia was further enhanced due to the consumption of molecular oxygen for singlet oxygen production.24−26 In addition, there is a rich collection of motifs (e.g., azobenzene and nitroimidazole) that are sensitive to hypoxia and can be used as the construction materials of hypoxia-responsive systems.27−29 However, to the best of our knowledge, the hypoxia-responsive MSN has been rarely investigated,30 which constrains its power in the area of triggered nanomaterials and nanomedicines. Among the two typical hypoxia-sensitive materials, the responsiveness of hydrophobic nitroimidazole was realized by its conversion to hydrophilic aminoimidazole, resulting in nanocarrier disassembly and payload liberation.31,32 The hypoxia sensitivity of azobenzene was less popular than its property of ultraviolet-light-induced conformational switch by trans−cis isomerization, but the azobenzene was broken into aniline via accepting four electrons.33 Such different mecha-

1. INTRODUCTION Mesoporous silica nanocarriers (MSNs) have been extensively employed in drug delivery, imaging, biosensor, diagnosis, and other pharmaceutical and biomedical fields.1−3 The MSN is a porous vehicle that is featured with a plethora of unique characteristics. The large specific surface area enables a high cargo loading as well as codelivery of multiple payloads in a single vehicle.4,5 This trait is also beneficial for encapsulating therapeutic macromolecules.6 The building block of the MSN is inert, which minimizes the undesirable carrier−cargo interaction to maintain the cargo integrity and activity.7 The degradation of MSN can be customized via the introduction of responsive linkers (e.g., disulfide bonds) to ensure the in vivo elimination.8 The surface of MSN can be tailored by different coatings to customize the surface charge and ligand density for enhanced targeting.9,10 Similar to other types of nanosystems, the MSN also faces the challenge of premature cargo release despite all these above advantages.11−13 Installing a responsive gate-keeper has been the routine approach to address this problem.14 Various external and internal stimuli can induce the nanocarrier disassembly and then cargo release, including light, ultrasound, magnetic field, temperature, pH shift, redox potential gradients, ionic strength, reactive oxygen species, oxygen depletion, and unique substrate (e.g., gases, sugars, and © 2019 American Chemical Society

Received: March 7, 2019 Accepted: June 14, 2019 Published: June 14, 2019 24377

DOI: 10.1021/acsami.9b04142 ACS Appl. Mater. Interfaces 2019, 11, 24377−24385

Research Article

ACS Applied Materials & Interfaces

Figure 1. Schematic illustration of hypoxia-responsive mesoporous silica nanocarriers (MSNs). The cargo (Ce6) was noncovalently loaded within the MSN that was gated by poly(4,4′-azodianiline) (pDAB) and further coated by Pluronic F68. Upon systemic circulation and endocytosis, the azo bonds (N=N) in the coating layer were broken under hypoxic conditions, which would aid Ce6 release and enhance the photodynamic effect upon laser irradiation (660 nm).

Figure 2. Physiochemical characterization of hypoxia-responsive mesoporous silica nanocarriers (MSNs). TEM images of (A) MSN and (B) Ce6@ MSN/pDAB/F68. (C) Hydrodynamic sizes of two nanocarriers. (D) Fourier transform infrared spectra of 4,4′-azodianiline (DAB) and poly(4,4′azodianiline) (pDAB). (E) Ultraviolet−visible absorption spectra of MSN, free Ce6, and Ce6@MSN/pDAB/F68 (in water) and pDAB (in THF). (F) Emission spectra of MSN/pDAB/F68 and Ce6@MSN/pDAB/F68 with the excitation wavelength at 404 nm (inset: the fluorescence image).

2. RESULTS AND DISCUSSION

nisms of azobenzene and nitroimidazole in response to low oxygen concentration indicate that the former is more efficient as a hypoxia-sensitive moiety. Therefore, the aim of this work was to engineer a novel MSN coated by the azobenzene polymer, that is, poly(4,4′-azodianiline) (pDAB), for hypoxiaresponsive cargo release. A standard photosensitizer, chlorin e6 (Ce6) was employed as the model cargo to be encapsulated within the MSN. As azobenzene was hydrophobic, an amphiphilic copolymer, namely, Pluronic F68 (F68), was further deposited on its exterior to ensure good affinity or dispersibility of the MSN in water (Figure 1).

2.1. Synthesis and Characterization of HypoxiaResponsive MSN. The hypoxia-sensitive, Ce6-loaded MSN was gated with the pDAB and further coated with the F68 polymer by a thin-film hydration method (i.e., Ce6@MSN/ pDAB/F68). The empty MSN without any surface modification was used as the control. Both types of nanocarriers displayed a spherical morphology and transmission electron microscope (TEM) sizes below 100 nm at 45.1 ± 4.8 (control) and 58.1 ± 6.0 nm (hypoxia-sensitive) (Figure 2A,B, respectively, Figures S1 and S2). The corresponding hydro24378


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ACS Applied Materials & Interfaces dynamic sizes were determined at 179.0 ± 12.3 (MSN) and 202.9 ± 9.1 nm (Ce6@MSN/pDAB/F68) (Figure 2C). It is a typical phenomenon that the hydrodynamic diameter of nanocarriers is larger than the TEM size, which was due to the presence of the hydrophilic poly(ethylene oxide) moiety in amphiphilic F68.34 The formation of loose aggregations is also a possible reason; such a phenomenon was consistent with previous publications.35 The cargo loading involves the mixing of the MSN core together with the drug, DAB monomer, and cross-linker (glutaraldehyde) in methanol. The polymerization of DAB relies on the presence of glutaraldehyde to react with the amino group of DAB, resulting in the formation of the C=N bond between each monomer. The chemical structure of the generated DAB polymer (i.e., pDAB) is shown in Figure S3. The polymer-gated MSN was purified by centrifugation and methanol washing prior to the surface adsorption of amphiphilic F68. Fourier transform infrared spectroscopy (FTIR) of pDAB was employed to verify the existence of such a characteristic band (1618 cm−1) that was attributed to the stretching vibration of the C=N bond (Figure 2D, Figure S4). The presence of this unique C=N vibration signal in the MSN/ pDAB nanocarrier further confirmed the successful coating of the gating polymer (Figure S5). In contrast to the control MSN, pDAB displayed a broad absorption peak from 400 to 600 nm that was clearly shown in the spectrum of Ce6@MSN/ pDAB/F68, indicating the successful capping of pDAB at the surface of the MSN (Figure 2E). The free cargo (Ce6) showed two characteristic absorption maxima at 404 and 660 nm. Both peaks were present in the hypoxia-sensitive MSN after cargo loading. The emission spectrum of Ce6@MSN/pDAB/F68 also confirmed the encapsulation of Ce6 in the nanocarrier (Figure 2F). The cargo loading was determined as 4.7 ± 0.2% (w/w) by a spectrophotometric approach. The deposition of hydrophobic pDAB would avoid or minimize the entry of water into the MSN cavity and prevent premature cargo release. The function of F68 was to form a steric layer to maintain the stability of the MSN in an aqueous medium after pDAB coating. 2.2. Degradation of pDAB under a Mimicked Hypoxic Environment in Vitro. The intracellular reduction of the azobenzene moiety under hypoxia requires the catalysis of azoreductase and the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as the reducing agent. To mimic such a reaction in vitro, we selected an alternative reductant, that is, sodium dithionite (Na2S2O4), as the electron donor to enable the reduction of azobenzene, which can avoid the use of enzyme.25,36 When the polymer (pDAB) was incubated with Na2S2O4, its absorption maximum dramatically decreased. The extent of the decrease was proportional to the concentration of Na2S2O4 and hence the degree of pDAB degradation (Figure 3A, Figure S6). The degradation mechanism of pDAB was thought to be due to a simple redox reaction with the polymer and Na2S2O4 as the electron acceptor and electron donor, respectively (Figure 3E). A unique probe, 4-(dimethylamino)benzaldehyde was used as a chromogenic moiety to monitor the breakdown of the azo bond (N=N) via the reaction of aldehyde and amine (Figure 3F). Upon treating pDAB with Na2S2O4 (0−20 mM) in the presence of the probe, the absorption maximum at approximately 500 nm dramatically increased, indicating a high degree of polymer degradation at the elevated reductant level (Figure 3B). When replacing pDAB with the gated

Figure 3. Degradation of pDAB polymer in response to a reducing agent. UV−vis spectra of pDAB (0.5 mg/mL) in water/THF mixture (1:1, v/v) (A) and aqueous MSN/pDAB/F68 (2 mg/mL) (C) upon incubation with sodium dithionite (0−30 mM) aqueous solution; UV−vis spectra of pDAB (0.5 mg/mL) in water/THF mixture (1:1, v/v) (B) and aqueous MSN/pDAB/F68 (2 mg/mL) (D) upon incubation with sodium dithionite (0−20 mM) aqueous solution in the presence of 4-(dimethylamino)benzaldehyde probe (130 mM); (E) proposed mechanism of sodium dithionite-induced pDAB degradation; (F) reaction between 4-(dimethylamino)benzaldehyde probe and pDAB degradation product.

nanocarrier (i.e., MSN/pDAB/F68), the same phenomenon was observed (Figure 3C,D, Figure S7). 2.3. FRET Analysis of Hypoxia-Responsive Gate Removal. We employed the fluorescence resonance energy transfer (FRET) technique to examine the pDAB degradationinduced removal of the MSN gate-keeper. FRET is a phenomenon in which an excited donor fluorophore transfers energy to an acceptor fluorophore via the nonradiative process that involves the intermolecular long-range dipole−dipole coupling.37 In the current work, coumarin 6 (Cou6) and rhodamine B (RhoB) were selected as the FRET donor and acceptor, respectively. The emission spectrum of Cou6 and excitation spectrum of RhoB well overlapped, indicating both as a good FRET pair (Figure 4A). When the excitation wavelength was fixed at 450 nm (i.e., the absorption maximum of Cou6), free Cou6 displayed a normal emission spectrum, whereas free RhoB almost had no emission (Figure 4B). However, when both fluorophores were coloaded within the nanocarrier, that is, Cou6 + RhoB@MSN/pDAB/F68 (Cou6 loading: 3.9 ± 0.3%, RhoB loading: 1.6 ± 0.1%, w/w), the excitation at 450 nm produced a strong emission (Figure 4B). This was because FRET was a distance-dependent mechanism. When both the donor and acceptor were encapsulated in the single nanocarrier, the distance between them was dramatically reduced to favor the intermolecular energy transfer and hence the generation of the FRET phenomenon. A robust washing procedure was employed to rule out the effect of Cou6/RhoB 24379


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Figure 4. FRET analysis of hypoxia-responsive pDAB gate removal from MSN. (A) Excitation and emission spectra of Cou6 (FRET donor) and RhoB (FRET acceptor) in methanol; (B) emission spectra of free Cou 6 (in methnol), free RhoB (in methanol), and Cou6 + RhoB@MSN/ pDAB/F68 nanocarrier (in water) with the excitation wavelength at 450 nm. (C) FRET ratios for Cou6 + RhoB@MSN/pDAB/F68 nanocarrier with or without Na2S2O4 treatment (n = 3). (D) Kinetic FRET images of Cou6 + RhoB@MSN/pDAB/F68 nanocarriers under normoxia and hypoxia. Scale bar: 20 μm. (Cou6: Ex = 488 nm, Em = 490−540 nm; RhoB: Ex = 488 nm, Em = 570−630 nm). (E) Hypoxia-responsive fluorophore release and FRET activation. (F) FRET ratios of Cou6 + RhoB@MSN/pDAB/F68 nanocarrier in MCF-7 cells under hypoxia or normoxia (n = 3).

the FRET mechanism (Figure 4E). Quantitative analysis of the FRET ratio further demonstrated that the pDAB degradationenabled FRET phenomenon diminished over time under hypoxia (Figure 4F). 2.4. In Vivo Cargo Release from Gated MSN under Hypoxia. To visualize the cargo release from gated MSN under hypoxia, we established a 4T1 tumor-bearing mice model. Cy5 and Cy7 were selected as the FRET donor and acceptor, respectively, because of their long-wavelength excitation and hence suitability for in vivo imaging. The individual loadings of FRET pairs were 3.4 ± 0.4 (Cy5) and 5.1 ± 0.1% (Cy7). The Cy5/Cy7@MSN/pDAB/F68 nanocarrier was intravenously administered, and the kinetic fluorescence of the tumor together with the major healthy organs was recorded from 4 to 24 h after dosing (Figure 5A,B). The emissive fluorescence intensities of both Cy5 and Cy7 in the tumor were quantified and compared (Figure 5C); both values reached the peak at 8 h after intravenous dosing due to the enhanced permeability and retention (EPR) effect.18,27 As an index of cargo release, the FRET ratio gradually decreased with time (Figure 5D), which signified that Cy5 and Cy7 were gradually released from the gated MSN under a hypoxic 4T1 tumor microenvironment. 2.5. Cytotoxicity of Hypoxia-Responsive Gated MSN. We selected a model photosensitizer, that is, chlorin e6 (Ce6), to examine the performance of the gated nanocarrier (i.e., Ce6@MSN/pDAB/F68) in MCF-7 cells. The cytotoxicity of Ce6 can only be activated when the light irradiation is applied to enable the production of highly toxic singlet oxygen.39 Therefore, regardless of oxygen concentration, all three

adsorption onto the MSN surface on the FRET performance (Figure S8). To assess the extent of FRET with or without the gate-keeper, we employed the index of FRET ratio that was defined as the ratio of the emissive maxima (584 nm) of the acceptor that was excited at 560 (acceptor excitation wavelength) and 450 nm (donor excitation wavelength).38 It was clear that the FRET ratio for nanocarriers without the reductant treatment remained almost constant, indicating that both fluorophores were safely locked within the MSN cavity. In contrast, in terms of Na2S2O4-treated nanocarriers, the FRET ratio proportionally decreased against incubation time, demonstrating the separation of the acceptor and donor fluorophores; that is, an evident fluorophore releases from the MSN due to the gate opening induced by pDAB degradation (Figure 4C). The same FRET technique was used in a model human breast adenocarcinoma cell line (MCF-7 cells) to verify the pDAB-mediated hypoxic-responsive cargo release. The cells were incubated with the Cou6 + RhoB@MSN/pDAB/F68 nanocarriers under both hypoxic and normoxic conditions for different times. Irrespective of the oxygen level, the kinetic fluorescence intensity of Cou6 (Ex = 488, Em = 490−540 nm) was similar (Figure 4D). In contrast, the fluorescence intensity of the acceptor fluorophore (RhoB, Ex = 488, Em = 570−630 nm) decreased over time under hypoxia, which was due to the release of both fluorophores from the MSN that was initiated by pDAB degradation with the aid of intracellular azoreductase and NADPH (Figure 4D). This phenomenon was not evident under normoxia due to the difficulty of cargo release from the gated MSN. These confocal images could be well explained by 24380


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7 cells at different intracellular oxygen levels with corresponding half maximal inhibitory concentrations (IC50) of 3.5 ± 0.4 (normoxia) and 5.2 ± 0.1 μg/mL (hypoxia). Such a discrepancy was because molecular oxygen was one of the key elements of photodynamic therapy and the generation of singlet oxygen was hindered at the low oxygen level.40 Under normoxia, the IC50 of the Ce6@MSN/pDAB/F68 nanocarrier was 6.4 ± 0.8 μg/mL, and the reduced cytotoxicity compared to free Ce6 was partly attributable to the short half-life and limited diffusion radius of singlet oxygen that necessitated rapid cargo release (Figure 6C).41 Under hypoxia, the IC50 of the Ce6-loaded gated nanocarrier was 8.2 ± 1.1 μg/mL; likewise, this value was also larger than that of free Ce6 (Figure 6D). The interplay of the oxygen level and cargo release rate from the MSN makes it hard to directly compare the cytotoxicity of the Ce6@MSN/pDAB/F68 nanocarrier under normoxia and hypoxia; the former favors singlet production and the latter aids cargo release. Hence, we selected a model drug (doxorubicin/Dox) whose activity is not dependent on the oxygen level, which would better demonstrate the superiority of hypoxia-responsive cargo release from the gated MSN. As expected, there was significant difference between the cytotoxicity of encapsulated Dox under hypoxia (3.7 ± 0.2 μg/mL) and that under normoxia (5.3 ± 0.5 μg/mL) (p < 0.01) (Figure 7). This was a direct consequence of rapid Dox

Figure 5. In vivo FRET analysis of Cy5 + Cy7@MSN/pDAB/F68 in 4T1 tumor-bearing BALB/c mice. (A,B) Fluorescence images of ex vivo tumor tissues; the excitation wavelength of Cy5 was 635 nm; the emission wavelengths were 670−700 (Cy5) and 700−800 nm (Cy7). (C) Fluorescence intensities of Cy5 and Cy7 in tumor with the excitation wavelength being fixed at 635 nm. (D) Kinetic FRET ratios of Cy5 + Cy7@MSN/pDAB/F68 in 4T1 tumor (n = 3).

samples (free Ce6, MSN/pDAB/F68 nanocarrier, and Ce6@ MSN/pDAB/F68 nanocarrier) did not induce significant toxicity in the absence of laser treatment for a Ce6 dose up to 20 μg/mL (Figure 6A,B). When the light irradiation was applied (i.e., laser on), the control nanocarrier (MSN/pDAB/ F68) did not cause any reduction of cell viability simply because no Ce6 was present and hence no singlet oxygen was produced (Figure 6C,D). Free Ce6 induced the death of MCF-

Figure 7. Cytotoxicity of Dox-loaded MSN (i.e., Dox@MSN/pDAB/ F68) in MCF-7 cells under normoxia (A) and hypoxia (B) (n = 4). Free Dox and placebo MSN/pDAB/F68 were used as controls.

release from the MSN under hypoxia. The release behavior of Dox under mimicked hypoxia conditions fully concurred with the cell viability data (Figure 8).

Figure 6. Cell viability of MCF-7 cells when incubated with free Ce6, MSN/pDAB/F68 nanocarrier, and Ce6@MSN/pDAB/F68 nanocarrier under four different conditions (n = 4). (A) Normoxia without laser treatment; (B) hypoxia without laser treatment; (C) normoxia with laser treatment; (D) hypoxia with laser treatment. “Laser on” represents the irradiation conditions of 660 nm, 100 mW/cm2, and 10 min.

Figure 8. Release profile of Dox from gated Dox@MSN/pDAB/F68 nanocarrier under mimicked hypoxic conditions with sodium dithionite concentrations ranging from 0 to 10 mM (n = 3). 24381


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4.3. Preparation of Ce6@MSN/pDAB/F68 Nanocarrier. The encapsulation of Ce6 in the gated MSN employed a similar approach. In brief, 20 mg MSN and 10 mg Ce6 were mixed in 4 mL, and the system was maintained at 60 °C for 12 h followed by the supplement of DAB, acetic acid, and glutaraldehyde. The procedure of DAB polymerization and F68 coating was the same as described in Section 4.2, and the obtained product was lyophilized to get the Ce6@MSN/ pDAB/F68 nanocarrier. The Ce6 loading was determined using a UV−vis spectrophotometer. Briefly, 1 mg of Ce6@MSN/pDAB/F68 was dispersed in 1 mL of tetrahydrofuran (THF) followed by ultrasonic treatment for 2 h to remove the gate. Then the samples were centrifuged at 10000 g for 10 min to collect the supernatant whose absorbance was recorded at 660 nm. The Ce6 loading was calculated by dividing the Ce6 mass in the nanocarriers and the total weight of the Ce6@MSN/pDAB/F68 nanocarrier (n = 3). 4.4. Characterization of Gated MSN Nanocarrier. The diameter and morphology of MSN and Ce6@MSN/pDAB/F68 nanocarriers were analyzed by a transmission electron microscope (TEM, Hitachi, HT7700). The hydrodynamic sizes of both nanocarriers were determined using a Zetasizer Nano (Malvern Instruments, ZS90). The FTIR spectra of DAB and pDAB were obtained by a Bruker Tensor 27 FTIR spectrophotometer. The pDAB polymer was obtained by dispersing 10 mg of MSN/pDAB in 10 mL of THF for 2 h followed by centrifugation (10000 g, 10 min) and solvent evaporation in the supernatant prior to vacuum-drying. The UV−vis absorption spectra of MSN aqueous dispersion, pDAB solution in THF, Ce6 aqueous water, and Ce6@MSN/pDAB/F68 aqueous dispersion were recorded using an Agilent Cary 60 UV−vis spectrophotometer. The concentration of all fours samples was fixed at 0.1 mg/mL. The emission spectra of MSN/pDAB/F68 and Ce6@ MSN/pDAB/F68 aqueous dispersion (both at 0.5 mg/mL) were obtained by a SpectraMax M2 microplate reader with the excitation wavelength at 404 nm. 4.5. Reductant-Induced Gate Removal. The pDAB polymer solution in THF (0.5 mg/mL, 1 mL) was mixed with a series of Na2S2O4 aqueous solutions (1 mL) at different concentrations (0, 2, 5, 10, 20, and 30 mM). After 12 h, the UV−vis spectra of the mixture were recorded. The MSN/pDAB/F68 nanocarrier aqueous solution (2 mg/mL) was mixed with solid Na2S2O4 to reach five target concentrations (0, 2, 5, 10, and 20 mM). To further verify the reduction of the pDAB, 4-(dimethylamino)benzaldehyde was used as the chromogenic probe to react with the amine-containing degradation products of pDAB. In detail, the probe (2 g) was dissolved in a mixed solvent containing 2 mL of sulfuric acid and 48 mL of ethanol. Then the probe solution and nanocarrier system were mixed at identical volumes prior to recording the UV−vis spectra. 4.6. FRET Analysis in Aqueous Medium. The FRET pair (Cou6 and RhoB) was loaded in the MSN using a similar approach to Ce6 encapsulation. The feeding ratio was set as 1:1:2 (w/w/w MSN/ Cou6/RhoB). The loading of both probes in the MSN was fluorescently determined (n = 3). The emission spectra were obtained with excitation wavelengths of 450 (Cou6) and 560 nm (RhoB). The excitation spectra were recorded with corresponding emission wavelengths at 500 (Cou6) and 584 nm (RhoB). The Cou6 + RhoB@MSN/pDAB/F68 nanocarrier aqueous dispersion (2.5 mg/ mL) was excited with a laser at 450 nm to get the emission spectrum. The Cou6 solution (0.5 mg/mL) and RhoB solution (0.5 mg/mL) were used as controls and excited under the same condition (450 nm). To test the hypoxia-dependent FRET phenomenon, the Cou6 + RhoB@MSN/pDAB/F68 aqueous nanosystem (0.5 mg/mL) was treated with Na2S2O4 (10 mM) followed by kinetic aliquot sampling (100 μL) at different time points (0, 20, 30, 60, 80, 100 min) after mixing. The nanocarrier without reductant treatment was used as the control. The sample’s fluorescence intensity at 584 nm was recorded using two different excitation wavelengths (450 and 560 nm). The FRET ratio is defined by eq 1:

3. CONCLUSIONS In summary, based on our previous report on azobenzenebased hypoxia-sensitive micelles,27 the current work expanded the application of the azobenzene polymer as the gate-keeper of mesoporous silica nanocarriers. The hypoxia-responsive gate removal was enabled by the polymer degradation in the presence of azoreductase and electron donors to enable rapid cargo release without premature dose dumping. Such a phenomenon was demonstrated in vitro using sodium dithionite as the model reductant. The hypoxia-responsive intracellular release of model fluorophores in MCF-7 cells was also proven by the employment of the FRET technique. Two model drugs, namely, Ce6 and Dox, were separately loaded in the gated MSN with established cytotoxicity under hypoxia. This work enriches the family of stimuli-responsive mesoporous silica nanocarriers, which could find application in the field of triggered release of various diagnostic and therapeutic payloads under hypoxic conditions. 4. EXPERIMENTAL SECTION 4.1. Materials. Chlorin e6 (Ce6) was obtained from Beijing JL Technology Co., Ltd. (Beijing, China). Hexadecyltrimethylammonium chloride (CTAB) and tetraethyl orthosilicate (TEOS) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). 4,4′-Azodianiline (DAB) was purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Pluronic F68 (F68) was obtained from Beijing Ouhe Technology Co., Ltd. (Beijing, China). 4-(Dimethylamino)benzaldehyde and rhodamine B (RhoB) were provided by Tianjin Heowns Biochem Co., Ltd. (Tianjin, China). Coumarin 6 (Cou6) was obtained from J&K Scientific Co., Ltd. (Beijing, China). Triethanolamine (TEOA), sodium dithionite, and all standard solvents were purchased from Tianjin Jiangtian Chemical Technology Co., Ltd. (Tianjin, China). Doxorubicin (Dox· HCl) was from Hvsf United Chemical Materials Co., Ltd. (Beijing, China). The FRET pairs (Cy5 and Cy7) were obtained from Dalian Meilun Biotechnology Co., Ltd. All chromatographic grade solvents came from Concord Tech Co., Ltd. (Tianjin, China). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were sourced from Tianjin Baibei Biotech Co., Ltd. (Tianjin, China). Female BALB/c mice (6 weeks, 18−22 g) were sourced from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). 4.2. Preparation of MSN/pDAB/F68. The synthesis of the mesoporous silica nanoparticle (MSN) was based on a previous report with minor modification.42 In brief, 0.64 g (1.76 mmol) of CTAB, 16.7 mL of water, and 2.3 mL of ethanol was mixed to form a stock solution. Then 0.92 mL (6.91 mmol) of triethanolamine (TEOA) was added to the stock, and the obtained solution was heated to 60 °C followed by the dropwise supplement of 1.541 mL (6.90 mmol) of TEOS within 3 min. After 2 h, the system was cooled down to ambient temperature, and the supernatant was removed after centrifugation (10000 g, 10 min). The sediment was then transferred to a mixed solvent containing 120 mL of ethanol and 15 mL of hydrochloric acid followed by reflux for 24 h, supernatant removal by centrifugation (10000 g, 10 min), and extensive washing by deionized water. Then the MSN was obtained via vacuum-drying overnight. The MSN core (20 mg) was dispersed in 4 mL of methanol followed by adding DAB (2 mg, 9.42 μmol), acetic acid (4 μL), and 25% (w/v) aqueous glutaraldehyde (4 μL, 9.42 μmol). After 1 h, the supernatant was removed after centrifugation (10000g, 10 min), and the sediment (MSN/pDAB) was washed by methanol. Thereafter, F68 (100 mg) was mixed with the sediment in 10 mL of methanol in a roundbottom flask. After 30 min, the solvent was evaporated to generate a thin film followed by hydration by deionized water. The excess F68 was removed by ultrafiltration, and the samples (MSN/pDAB/F68) were lyophilized ready for use.

FRET ratio =

24382

intensity(Ex = 450nm) intensity(Ex = 560nm)

(1)


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gated MSNs.27 The in vivo FRET analysis was carried out in accordance with the guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals with the approval of the Animal Ethics Committee of Tianjin University. Cy5/Cy7 was selected as the in vivo FRET pair. Both were coloaded in the gated MSN using a similar approach described in Section 4.3; the feeding ratio was set at 1:1:4 (Cy5/Cy7/MSN, w/w/w). The loadings of both cargos were fluorescently quantified after nanocarrier gate removal (n = 3). When the tumor size reached 50−100 mm3, the Cy5 + Cy7@MSN/pDAB/ F68 nanocarriers (200 μL, 1 mg/mL) were intravenously injected into the mice via the tail vein. At predefined time points after nanocarrier dosing (4, 8, and 24 h), the tumor and other major organs were collected and scanned using the CRi Maestro in vivo imaging instrument (Cambridge Research & Instrumentation, Inc., MA, USA). The excitation/emission wavelengths were fixed at 635/670−700 (Cy5) and 635/700−800 nm (Cy7). The kinetic FRET ratio in terms of the hypoxic tumor was calculated using eq 3:

4.7. Intracellular FRET Analysis. MCF-7 cells were seeded at a density of 1 × 104 cells in the 20 mm glass bottom cell culture dish (NEST Biotechnology Co., Ltd.). The culture medium was Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotics of penicillin/streptomycin that was maintained at 37 °C. The cells were divided into two groups; one group at the standard normoxic condition (5% CO2) and the other at the hypoxic condition (1% O2, 5% CO2, and 94% N2) using a BillupsRothenberg incubator chamber.27 The MCF-7 cells at both conditions were treated with Cou6 + RhoB@MSN/pDAB/F68 nanocarriers (0.5 mg/mL) for 2 h followed by PBS washing in triplicate and continued culturing under the same conditions for up to 4 h. At predefined time points after washing (0, 2, and 4 h), the cells were fixed using 1 mL of paraformaldehyde (4%, w/v) for 20 min and washed by PBS three times. Afterward, the nuclei were stained with 1 mL of DAPI (1 μg/mL) for 10 min. The FRET analysis employed an excitation wavelength of 450 nm for all samples, and the emission wavelength was set at either 490−540 or 570−630 nm. The intracellular FRET ratio was calculated using eq 2. FRET ratio =

FRET ratio =

intensity(Em = 570 − 630 nm) intensity(Em = 490 − 540 nm)

intensity(Em = 700 − 800 nm) intensity(Em = 670 − 700 nm)

(3)

4.11. Statistical Analysis. The Student t test was used to compare the average values of different samples. The p value was set at 0.05 for the threshold of significant difference.

(2)

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4.8. Cytotoxicity Analysis of Ce6 Nanocarrier. The typical MTT cell viability assay was used to investigate the cytotoxicity of Ce6@MSN/pDAB/F68 under four conditions: (A) normoxia and laser off; (B) hypoxia and laser off; (C) normoxia and laser on; and (D) hypoxia and laser on. Briefly, MCF-7 cells were seeded into each well of 96-well plates at a density of 4 × 103 cells/well followed by 24 h of culture. The cells were cultured in a humidified incubator containing 5% CO2 at 37 °C. The hypoxic condition was realized by using the same Billups-Rothenberg incubator chamber. After 12 h, the cells were treated with the following samples including free Ce6, MSN/pDAB/F68, and Ce6@MSN/pDAB/F68. The Ce6 concentration was varied (0, 2, 4, 6, 8, 10, and 20 μg/mL) for all drugcontaining samples. The concentration of the MSN/pDAB/F68 nanocarrier was maintained to be identical to that in Ce6@MSN/ pDAB/F68. The sample incubation under either hypoxia or normoxia was maintained for 4 h followed by PBS washing and the supplement of fresh medium. For the conditions of laser on, the samples were irradiated by 660 nm light (100 mW/cm2) for 10 min. For those with laser off, no light irradiation was applied. Afterward, the cells were further cultured for 24 h under normoxia or hypoxia. Then the medium was discarded, and the MTT solution (100 μL, 0.5 mg/mL) was added followed by incubating for 4 h in the dark and replacing the old medium with DMSO (100 μL). The obtained purple solutions were analyzed using a SpectraMax M2 microplate reader to get the absorbance at 490 nm followed by plotting the cell viability curve and IC50 calculation. 4.9. Cytotoxicity Analysis of Dox Nanocarrier. Likewise, Dox was encapsulated in the MSN (i.e., Dox@MSN/pDAB/F68) as an oxygen-independent active agent. In brief, 20 mg of MSN and 10 mg of Dox·HCl were mixed in 10 mL of PBS (pH = 7.4). After 24 h, 4 mL of methanol was used to replace PBS followed by the supplement of DAB, acetic acid, and glutaraldehyde. The procedure of DAB polymerization and F68 coating was the same as described in Section 4.2. The Dox loading was determined by high-performance liquid chromatography.43 The Dox release behavior was investigated as follows. The Dox@MSN/pDAB/F68 aqueous dispersion (2 mg/mL) was mixed with sodium dithionite solution differing in concentration (0, 10, and 20 mM) (n = 3). The mixture was stirred at 37 °C, and the amount of releasing Dox was determined by a UV−vis spectrophotometer after the removal of the intact nanocarrier by centrifugation (10000 g, 10 min) at 0.5, 1, 2, 4, 6, 8, 12, and 24 h. The cytotoxicity of Dox@MSN/pDAB/F68 together with free Dox and placebo nanocarrier (MSN/pDAB/F68) was assessed in MCF-7 cells using the same protocol for Ce6@MSN/pDAB/F68. 4.10. FRET Analysis in Vivo. Because hypoxia is well known as one of the hallmarks of solid tumors, we chose murine breast 4T1 tumor-bearing BALB/c mice for in vivo analysis of cargo release from

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b04142. TEM size distribution of nanocarriers; chemical structure of pDAB; FTIR spectra of DAB, pDAB, MSN, and MSN/pDAB; optical images of pDAB and MSN/pDAB/F68 upon sodium dithionite treatment; flowchart for producing FRET pair-loaded nanocarrier (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-22-2740 7882. Fax: +8622-2740 4018. ORCID

Yanjun Zhao: 0000-0001-5739-1960 Author Contributions

All authors have contributed to the manuscript writing and have approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Basic Research Program of China (2015CB856500) and the Tianjin Research Program of Application Foundation and Advanced Technology (18JCZDJC35700).

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