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Redox- and enzyme-responsive fluorescent porous silica nanocarriers for drug delivery
Accepted Manuscript Title: Redox- and enzyme-responsive fluorescent porous silica nanocarriers for drug delivery Authors: Qianqian Zhang, Jia Guo, Xu Zhang, Yanbao Zhao, Liuqin Cao, Lei Sun PII: DOI: Reference:
S0925-4005(18)31556-9 https://doi.org/10.1016/j.snb.2018.08.118 SNB 25266
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
4-6-2018 16-8-2018 24-8-2018
Please cite this article as: Zhang Q, Guo J, Zhang X, Zhao Y, Cao L, Sun L, Redoxand enzyme-responsive fluorescent porous silica nanocarriers for drug delivery, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.08.118 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Redox- and enzyme-responsive fluorescent porous silica nanocarriers for drug delivery Qianqian Zhanga, Jia Guob, Xu Zhanga, Yanbao Zhao*a, Liuqin Caob, Lei Suna a Engineering Research Center for Nanomaterials, Henan University, Kaifeng 475004, China
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b College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, China
Highlights
Porous silica (pSiO2) nanoparticles were successfully prepared for drug release.
Both carbon dots (CDs) and hyaluronic acid (HA) are used to seal the loaded drug in
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pores.
pSiO2-ss-CDs/HA carriers exhibit redox/ enzyme-responsive release behavior.
The CDs could be used to trace the drug release.
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Abstract
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In this work, redox/enzyme-responsive fluorescent porous silica (pSiO2) nanoparticles (NPs) were constructed for drug delivery. The resultant pSiO2 NPs have large-pore core and mesoporous shell with size of 75 nm and present high specific surface area (605 m2·g-1).
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Oxidized glutathione (GSSG) as linker was conjugated on the surfaces of aminofunctionalized porous silica (pSiO2-NH2) NPs by the amide bonds for redox-responsive drug
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release. Dox is served as model drugs to evaluate the release performance of carriers. After loading Dox molecules, both carbon dots (CDs) as fluorescent label and hyaluronic acid (HA) as gatekeepers are capped on the surface of carries (pSiO2-ss-CDs/HA) so as to endow the
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delivery system with fluorescent monitoring and enzyme-responsive properties. The pSiO2-ssCDs/HA carriers displayed redox/enzyme-responsive and sustained release behavior. The controlled release of drug from the pSiO2-ss-CDs/HA delivery system was realized by the reduction of disulfide bonds in GSSG linker and degrading HA molecules. The incorporated
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Corresponding author. E-mail：[email protected] (Y Zhao); 1
CDs presented novel redox-dependent fluorescence, which could be used to trace the drug release. Keyword: Porous silica; carbon dots; redox/ enzyme-responsive; drug release.
1. Introduction
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In recent years, silica nanomaterials have attracted much attention as controlled drug delivery system [1,2]. Since 2001, when mesoporous silica (MS) nanoparticles (NPs) were reported as drug delivery firstly [3], a plenty of studies have been
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concentrated on their drug delivery properties and potential biomedical applications [4-6]. MS NPs have regular structural characteristics, large surface area and pore volume, tunable pore size, and ease surface modification, which make them ideal
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platforms to design multifunctional nanocarriers [7,8]. However, the limited loading
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capacity of conventional MS NPs cannot satisfy the biomedical application [9,10].
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Subsequently, many efforts have been devoted to the study of expanding the pore size
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of silica NPs. For instance, Dang et al. prepared the MS NPs with large radial pores of 17-78 nm and high surface area (1219 cm2 g-1), which exhibited high siRNA delivery
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capabilities [11]. Tu et al. synthesized cuboidal-like silica NPs with an average pore size of 9-11 nm and the carrier showed high encapsulation efficiency of proteins [12].
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Large-pore silica carrier has high loading capacity and can delivery large biomolecules, but it is easy to leak the loaded drug due to the large channels. In
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order to obtain the excellent performance of sustained release with high loading capacity, we propose the novel silica carrier with large-pore core and mesoporous shell to incorporate the advantages of MS and large pore silica into drug
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carriers.
Generally, the drug release from silica nanocarriers is the passive-diffusion
process, and the premature release is unavoidable in the delivery process, therefore stimuli-responsive release is necessary which could reduce premature release [13]. Doxorubicin (DOX) is a kind of anticancer drugs, which is often served as hydrophilic model drug to evaluate the responsive release of silica nanocarriers. 2
It is well documented that tumor tissues have unique physicochemical properties (compared to normal cell) with high levels of certain enzymes, redox, temperature and low pH, so the smart carriers can be tailored to be responsive to the pH variations [14], redox potential [15], enzymatic activation [16], light [17], hyperthermia [18] or magnetic field/ultrasound [19]. To further improve the release behavior, a variety of multifunctional silica nanocarriers have been intensively
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fabricated by incorporating diverse kinds of nanovalves utilized for controlled release of loaded drug by synthetic stimuli [20]. For example, Wu et al. designed a pH- and
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redox-responsive ZnO-gated hollow MS nanocarrier and ZnO quantum dots (QDs) were attached on the MS outer surface via disulfide bond. ZnO QDs that can be
dissolved in acidic environment and disulfide bond that can be broken by GSH [21].
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Furthermore, monitoring the drug release and on-demand release are both crucial
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for developing drug delivery systems. To date, various QDs and fluorescent dye are
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widely used in drug delivery to investigate the drug release process. Among them,
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carbon dots (CDs) have emerged as a new platform of QD-like fluorescent labels, which show good biocompatibility, tunable photoluminescent (PL) emission and
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excellent photostability [22-24]. Especially, both the optical absorption and fluorescent emissions in CDs are not band gap in origin, and their fluorescent
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performance can be tuned by doping other elements [25,26]. Here, we constructed a novel fluorescent dual-responsive drug delivery platform
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(pSiO2-ss-CDs/HA) by combining fluorescent CDs, oxidized glutathione (GSSG), hyaluronic acid (HA) and porous SiO2 (pSiO2) NPs. GSSG as linker was attached on the pSiO2 carrier through amido bonds and Dox was served as model drug to be
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encapsulated. After loading DOX, the CDs as fluorescent probe and hyaluronic acid (HA) as gatekeepers were coated on the surface of pSiO2 carrier (scheme 1). The pSiO2 carriers are composed of large pore core and mesoporous shell, which combine advantages of both mesopores and large pores. GSSG is a kind of biological peptide containing a disulfide bond, carboxyl groups and amino groups, which is used as linker to conjugate CDs and HA. HA is a naturally 3
occurring polysaccharide with nonimmunogenic, biocompatible, and biodegradable properties, which are used to cap the drug-loaded pores. In addition, HA possesses the active targeting toward tumor cells through selectively binding over-expressing CD44 receptors [27,28]. The disulfide bonds in GSSG linkers could be cleaved by redox, which would lead to the detachment of HA and redox-responsive release. HA could be degraded by Hyal-1, resulting in
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opening nanovalves and enzyme- responsive releasing drug. Due to high Hyal-1
and GSH concentration in tumor tissue, the drug release from the pSiO2 carrier could
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be triggered by the GSH and Hyal-1. In addition, CDs can be used to monitor the drug release by fluorescent signal.
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2. Experimental section
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2.1. Reagents
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Cetyltrimethylammonium bromide (CTAB) and citric acid were obtained from
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Tianjin Kermel Chemical Reagent Co. (Tianjin, China). Tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), polyethylenimine (PEI, 99%, Mw =
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600), n-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), oxidized glutathione (GSSG), hyaluronic acid (HA), reduced
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glutathione (GSH), hyaluronidase (Hyal-1) and doxorubicin (Dox) were purchased from Aldrich (Shanghai, China). Hydrochloric acid (HCl, 37%), ethanol and
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ammonium hydroxide (NH4OH, 30%) were provided by Luoyang Chemicals (Luoyang, China). Cyclohexane (C6H6) and toluene (C7H8) were from Tianjin Deen Chemical Reagent Co., Ltd. Distilled water with a resistivity of 18.2 MΩ cm was
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used in all experiment. All chemical reagents were of analytical grade and used as received without any further purification.
2.2. Preparation of N-doped CDs In a typical synthesis, 10 mL of PEI solution (0.1 mg/mL) was added into 20 mL of citric acid solution (0.1 g/mL) under stirring. Then, the above solution was 4
transferred to the Teflon-lined autoclave and heated to 150 ℃. After reacting for 2 h, the resulted bright brown solution was placed in a dialysis bag (Mw = 3500) for 24 h to purify the CDs solution.
2.3. Preparation and modification of pSiO2 NPs The porous SiO2 (pSiO2) NPs with core/shell structure were synthesized based
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on a previous report with some experimental modification [29]. Briefly, 0.25 g of
CTAB was dissolved in a mixed solution composed of 2.5 mL of ethanol, 35 mL
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of H2O, 7.5 mL of C6H6 and 0.4 mL of NH4OH (30%). The mixture was
vigorously stirred with a magnetic stirring rate of 1000 rpm for 0.5 h at 25 °C to form emulsion system.
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Then, a mixture of TEOS (1.25 mL), APTES (0.05 mL) and ethanol (1 mL) was
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dripped into the above solution and keep stirring for 3 h. Subsequently, 2 mL of
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TEOS/ethanol solution (1/1) was also added to the solution and reacting for another 3
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h to get core/shell pSiO2 sample. After that, the sample was centrifuged, washed for several times and then calcined by muffle furnace at 550 ℃ for 4 h to remove the
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template CTAB.
For amino-functionalization, 0.15 g of pSiO2 sample was dispersed in 60 mL of
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toluene under room temperature. After the mixture was mixed evenly, 1 mL of APTES was dropped into it and stirred for 24 h. The resulted amino-functionalized
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pSiO2 (pSiO2-NH2) was obtained after centrifuging and washing. For GSSG modification, GSSG molecule was attached to pSiO2-NH2 by amide
bonds. In brief, 100 mg of EDC and 100 mg of NHS were added to 15 mL of PBS
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(pH = 7.4) solution and stirring for 30 min at 25 ℃. Then, 50 mg of GSSG was added to above solution to active the carboxyl groups of GSSG. Afterwards, 50 mg of pSiO2-NH2 was added into the activated GSSG solution. After reacting for12 h at 25 ℃, the mixture was centrifuged, washed and dried to obtain pSiO2-GSSG.
2.4. Drug loading and capping 5
10 mg of pSiO2-GSSG was dispersed in 5 mL of PBS solution of Dox (1 mg/mL) by ultrasound and shaken at 37 ℃ for 24 h. Then Dox-loaded pSiO2-GSSG sample was collected via centrifugation and washing with water for several times. After that, the sample was dried in vacuum oven at 25℃.All the washing solution was collected and the loading capacity of Dox was calculated through detecting the
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UV absorbance of the washing solution and stock solutions. For CDs and HA capping, the Dox-loaded pSiO2-GSSG sample was dispersed in PBS solution containing 40 mg of NHS and 30 mg of EDC for 30 min. Then, 2 mL of
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CDs solution was added to the mixed solution and reacted for 12 h, followed by
addition of 2 mL of HA solution (1 mg/mL) which has been activated by EDC and NHS. After stirring for 10 h, the Dox-loaded CDs/HA capping silica carrier (pSiO2-
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ss-CDs/HA) was centrifuged, washed with PBS solution several times and dried in a
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vacuum oven at 25 ℃ for 24 h.
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2.5. Drug release
To investigate the drug release pattern under different microenvironments in
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vitro, a series of simulated release medium was prepared as follows: (1) PBS (pH=7.4) with different concentrations of GSH (0.1 mM, 1 mM and 5 mM). (2) PBS
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(pH=7.4) with different concentrations of Hyal-1 (0.1 mg/mL, 0.3 mg/mL, 0.5 mg/mL). (3) PBS (pH =7.4) with 5 mM GSH and 0.5 mg/mL Hyal-1.
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In vitro drug release experiment, 5 mL of PBS containing 5 mg of Dox-loaded
pSiO2-ss-CDs/HA was placed in dialysis bag, submersed in release medium under different conditions as above mentioned and shaken at 100 rpm and 36.8 ℃. After
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each fixed time intervals, 2 mL of release medium was taken out to measure UV absorbance. At the same time, an equivalent PBS was supplemented to release medium so as to maintain a constant total volume.
2.6. Characterization
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The morphology of the sample was observed at transmission electron microscopy (TEM, JEM-2100). N2 adsorption–desorption isotherm curves and pore size distribution were measured on a full-automatic specific surface and porosity analyzer (Quadrasorb SI, America). UV absorbable absorbance was obtained on a UV-2400 spectrophotometer. Fourier transform infrared (FTIR) spectra were performed on a Bruker VERTEX FTIR spectrometer. X-ray diffraction (XRD) was
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recorded on X-ray powder diffractometer ((D8-ADVANCE, German). Thermo gravimetric analysis (TGA) was carried out on a thermo-gravimetric analyser
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(TAQ600, America). Photoluminescence (PL) spectra was investigated by
fluorescence spectrophotometer (JY-HORIBA, France). X-ray photoelectron
spectroscopy (XPS) analysis was from a X-Ray Photoelectron Spectroscopy（AXIS
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ULTRA，Britain）
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3. Results and discussion
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3.1. Characterization
Fig. 1 (a) shows the TEM, HRTEM and size distribution of CDs. It is apparent
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that the CDs are of excellent dispersion and the average particle size is 3.7 nm. The HRTEM image of CDs reveals the lattice spacing of 0.21 nm, which is quite close to
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(100) diffraction facet of graphite [30, 31]. Fig. 1 b-f show the TEM images of pSiO2, pSiO2-NH2, pSiO2-GSSG, pSiO2-ss-CDs and pSiO2-ss-CDs/HA samples. The pSiO2
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sample presents uniform spheres with size of 70-80 nm and the sample presents distinct core/shell structure with large pores core and mesopore shell (Fig. 1b). After amino-modification, there is no change in the particle size and morphology
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compared to pSiO2 sample, which indicated that amino-modified layer is thin (Fig. 1c). After grafting GSSG, the morphology of the silica particles does not change significantly (Fig. 1d). When the CDs are attached, small agglomerated particles can be observed on the surface of the silica particles (Fig. 1e), which indicates that CDs are conjugated on the surface of pSiO2-GSSG. Obviously, the surface of silica
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particles turns to blurred after capping HA molecules and the pSiO2-ss-CDs/HA still remains porous structure (Fig. 1f). To investigate the chemical composition of CDs, XPS was performed as shown in Fig. 2. Fig. 2a shows the survey spectrum of the CDs that indicates the existence of C, N and O. The binding energies of C1s are found to be at 283.8, 284.5, 285.1, 285.7 and
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287.2 eV, suggesting the existence of C=C, C-C, C-N, C-O and C=N/C=O bond. The peak of N1s can be divided into three diff erent peaks at 398.3, 399.1 and 399.8 eV,
respectively, which are ascribed to C-N-C, N-(C)3 and N-H bonds. Meanwhile, after
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deconvoluting O 1s peak, it is found to exhibit two peaks at 530.3 and 531.3 eV, which indicates the presence of C=O and C-O bond respectively. These results reasonably
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demonstrate the formation of carbon dots from the carbon source of PEI and citric acid. The porous information was examined by N2 sorption. Fig. 3 gives the N2 adsorption-
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desorption isotherms and corresponding pore size distributions of the pSiO2，pSiO2-NH2
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，pSiO2-GSSG and pSiO2-ss-CDs/HA. Undoubtedly, all samples present IV isotherm
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typically with evident hysteresis loop (Fig. 3a), indicating the existence of mesopores [32]. The specific surface area (SSA) and total pore volume (Vp) of pSiO2 sample are
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605.87 m2g-1 and 0.92 cm3g-1. The high SSA and Vp would provide suitable conditions for the surface modification and drug loading. With each step of modification, the SSA
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and Vp display a decreasing trend. After amino-functionalization, the SSA and Vp decrease to 383.85 m2g-1 and 0.55 cm3g-1, which provide the proof of the successful
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grafting of amino groups. Similarly, the SSA and Vp of pSiO2-GSSG and pSiO2-ssCDs/HA are decreased to 226.55 m2g-1, 0.35 cm3g-1 and 96.00 m2g-1, 0.13 cm3g-1, respectively. The change in SSA and Vp of the prepared samples would provide
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evidences directly for drug-loading and each successful modification. The corresponding pore size distribution curves were obtained from the adsorption
branch by BJH method (Fig. 3b). For pSiO2 sample, there appears a narrow and strong peak centered at 7.5 nm, while pSiO2-NH2 sample presents a peak position at 6.4 nm. The slight decrease in pore size revealed that the amino group was not only grafted on the surface of the silica but also on the internal pore surface. Compared with pSiO2-NH2, the 8
pore size of pSiO2-GSSG and pSiO2-ss-CDs/HA are slightly increased, which may be attributed to that part of smaller channels of the silica are completely covered by GSSG (or Dox) molecules, resulting in an increasing in the average pore size. FTIR was used to identify the successful modification of pSiO2 NPs (Fig. 4a). For CDs, the absorption bands at 1645 and 1566 cm-1 are attributed to C=O and N-H
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stretching vibration, respectively [25]. The weak peaks at 2836 and 2963 cm-1 belong to characteristic absorption -CH2- chains of PEI, demonstrating the existence of PEI on CDs [33, 34]. For pSiO2-NH2, the peak at 1463 cm-1is assigned to N–H bending
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vibration, indicating the successful grafting of amino groups [35]. After connection with GSSG, the bands at 1643 and 1535 cm-1 are related to amide I and amide II absorption, which identifies the interaction between carboxyl groups of GSSG and
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amino groups of silica [21]. The peaks at 1405 and 2965 cm-1 correspond to C=O
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stretching vibration of HA and -CH2- chains stretching vibration in CDs, respectively,
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confirming the successful covering of CDs and HA [36].
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Fig. 4b shows the typical UV-Vis absorption, excitation and emission spectra of CDs. Two noticeable absorption peaks located at 255 and 368 nm may be attributed to
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the π-π* transitions of C=C bond and n-π* transitions of C=O bond [37, 38]. When CDs are excited at 370 nm, the maximum emission peak at 440 nm can be observed.
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Moreover, the synthesized CDs is quite well of water solubility and the aqueous solution of CDs presents pale yellow and displays bright blue fluorescence under UV
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light at 365 nm.
PL is one of most fascinated features of CDs. Fig. 5a shows a detailed PL
investigation with different excitation wave lengths. When the excitation wavelength
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is changed from 320 to 400 nm, the PL emission peak at 440 nm remains unchanged. This behavior of excitation-independent PL is considered to be associated with less surface defects and more uniform size of CDs, which is beneficial to avoid unwanted autofluorescence for down-conversion and up-conversion cell imaging [37, 39]. In addition, the PL intensity increases first and then decreases with the increase of excitation wavelength from 320 nm to 390 nm. When the excitation wavelength is 9
370 nm, the intensity of PL emission is maximum as shown in Fig. 5a. The excitation-dependent PL intensity may be related to the simultaneous absorption of CDs and the maximum absorption appears at 370 nm.
Fig. 5b shows the PL spectra of CDs, pSiO2-ss-CDs and pSiO2-ss-CDs/HA. In aqueous solution, the CDs exhibited blue emission peak at 440 nm. Under the
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excitation light of 370 nm, both pSiO2-ss-CDs and pSiO2-ss-CDs/HA also present remarkable emission peak at the same position of 440 nm with different PL intensity, indicating the successful of capping CDs. Compared with pSiO2-ss-CDs, the PL
interactions between CDs and HA molecules [40,41].
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3.2. In vitro release behavior
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intensity of pSiO2-SS-CDs/HA is weakened a lot, which may be related to electronic
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The pSiO2-ss-CDs/HA carrier with large inner channel is beneficial for the drug
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diffusion and loading, and its loading capacity of Dox is up to 20.5%. The drug
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release would be triggered by GSH and Hyal-1 in the way of cleaving disulfide bonds of GSSG and decomposing hyaluronic acid, respectively. Fig. 6 illustrates the
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cumulative release of Dox from pSiO2-ss-CDs/HA under different release medium. The drug release profiles in PBS solution (pH=7.4) with different GSH concentrations
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are shown in Fig. 6a. It can be seen that the pSiO2-ss-CDs/HA carrier displays an excellent GSH-responsive release manner. In the absence of GSH, only small amount
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of Dox was released into the PBS solution within 10 h and the release rate is 16.4 %, signifying the efficient confinement of the drug in the pores of the pSiO2-ss-CDs/HA. The release efficiency is gradually enhanced as the concentration of GSH increased
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from 0.1 to 10 mM. Under GSH concentration of 10 mM, the drug release rate is accumulated to 52.9% within 6 h. The GSH-triggered rapid release is related to the detachment of CDs or HA from pSiO2 carriers via the cleavage of disulfide bonds. In the case of redox, the disulfide bonds in GSSG may be not completely broken due to the hindrance of the coating layer HA or the cleaved CDs and large HA
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moiety may be not completely detached from on the surface of pSiO2 carriers, causing the drug to not be completely released. Fig. 6b depicts the cumulative drug release from pSiO2-ss-CDs/HA in PBS solution with different Hyal-1 concentration. Obviously, the pSiO2-ss-CDs/HA also displays Hyal-1-responsive sustained-release property without burst phenomenon. As
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the concentration of Hyal-1 increases, the drug release efficiency is enhanced gradually. When the concentration of Hyal-1 reaches 0.5 mg/mL, the drug release rate is accumulated to 62.2% in 6 h. This fact demonstrates the pSiO2 carriers with CDs
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and HA capping are sensitive to Hyal-1 and cause enzyme-responsive release.
Due to the high GSH concentration and over-expressed enzyme of tumor cells,
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the release efficiency of drug would be increased when pSiO2-ss-CDs/HA carriers were delivered under the tumor environment. The release behavior in buffer solution
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with Hyal-1 and GSH was also investigated (in Fig. 6b). The faster Dox release is
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observed at 10 mM GSH and 0.5 mg/mL Hyal-1, and the released Dox reaches at
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87% within 5 h, much high than that at 10 mM GSH or 0.5 mg/mL Hyal-1, showing that pSiO2-ss-CDs-HA carrier exhibits excellent GSH/Hyal-1 synergic responsive
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drug release. In the presence of Hyal-1 and GSH, the degradation of HA would help to completely cleave the disulfide bonds in GSSG, leading to more complete
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opening nanovalves and promoting the release of drug. Therefore, the release rate could be enhanced in the dual-responsive case. In addition, the CD44 receptor
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over-expressed on the surface of tumor can bind to hyaluronic acid specifically, which is promising to achieve carrier-targeted drug release. To understand the release mechanism of Dox from pSiO2-ss-CDs/HA carriers,
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the corresponding release kinetics was also studied. In this study, the experimental release data were fit using the first-order kinetic model involving an exponential decay. Q = Qmax (1 − exp (−kt)).
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where, Q represents the amount of drug released at time t, Qmax represents the maximum amount of drug released and k is the first-order constant. The graphical representations and the corresponding parameters calculated from the kinetics model are shown in Fig. 6c and d, respectively. The Dox release from pSiO2-ss-CDs/HA carrier follows the first order kinetics model with high correlation coefficient.
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3.3. Fluorescent behavior As a fluorescent label of drug carriers, CDs can be used not only to track the
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position of the carrier in real time, but also to monitor the process of drug release
based on changes in PL intensity. Fig. 7a gives the PL spectra of pSiO2-ss-CDs/HA with release time in the presence of 10 mM GSH and 0.5 mg/mL Hyal-1. It can be
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seen that the PL intensity of the pSiO2-ss-CDs/HA is reduced with increasing release
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time. The phenomenon can account for that the GSH reduction of disulfide bonds in
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GSSG, which lead to the CDs falling off from the pSiO2 carriers. In the meantime, the CDs detached from the carriers would be dispersed in release medium and cause a
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change of PL intensity of the release medium. To illustrate that the detached CDs
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were dispersed in the release medium, the PL intensity of the release medium was detected (Fig. 7b). As expected, the PL intensity of the release medium increases simultaneously as increasing release time, suggesting that the CDs were detached
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from carriers and dispersed in release medium under the action of GSH. To
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understand the relationship between detached CDs and drug release, we also compared the drug release rate and PL intensity of the release medium over time (Fig.7c). It can be seen that the curve of PL intensity of the release medium is similar to that of the accumulative drug release rate. The results indicate that the
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drug release can be monitored by the change in PL intensity of the drug delivery system.
Conclusions
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In this paper, we designed and prepared a novel drug delivery system based on fluorescent pSiO2 NPs. In this system, GSSG as linker was modified on the surface of pSiO2 NPs and both CDs and HA are used to seal the drug-loaded pores. The pSiO2ss-CDs/HA carrier presents high loading capacity and the drug release would be triggered by cleaving disulfide bonds of GSSG or decomposing HA. The Dox release
allows real-time monitoring of the release of drug from the pores.
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Acknowledgement
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followed the first order kinetics model very well. The PL characteristic of the CDs
Financial support of this work from National Natural Science Foundation of China (21271062) is gratefully acknowledged.
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Biographies Qianqian Zhang studied at Henan Normal University, and obtained her BSc in chemistry in 2016. She joined Professor Zhao’s research group at Henan University after her graduation.
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Her researches focus on fluorescent porous silica nanospheres for drug delivery. Jia Guo studied at Henan University, and obtained her MSc in chemistry in 2014. Her
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research focuses on drug delivery.
Xu Zhang studied at Huazhong Agricultural University, and obtained her Master degree in 2005. She joined Professor Zhao’s research group at Henan University in 2014. Her research
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focuses on porous silica nanospheres for drug delivery.
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Yanbao Zhao studied physical chemistry in Lanzhou Institute of Chemical Physics, Chinese
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Academy of Sciences, and received his PhD in 2004. He started his academic career at Henan
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University, and promoted as Professor in 2005. He engages in designing and preparation of functional nanomaterials and their applications and has published more than 80 scientific
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papers.
Liuqin Cao works at Henan University as an experimentalist of chemistry, and her research
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interests concentrate in preparation and modification of nanoparticles.
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Lei Sun studied physical chemistry in Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, and received his PhD in 2005. He is a professor of Chemistry at Henan University. He engages in designing and preparation of inorganic antibacterial material, and
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has published more than 60 scientific papers
Figure captions 18
Scheme 1 The preparation and release of pSiO2-ss-CDs/HA nanocarriers. Fig. 1. TEM image (a) with HRTEM and size distribution (insert) of CDs. TEM images of pSiO2 (b), pSiO2-NH2 (c), pSiO2-GSSG (d), pSiO2-ss-CDs (e) and pSiO2-ss-CDs/HA (f) samples.
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Fig. 2. XPS of the CDs (a) and C1s, N1s, O1s XPS spectra of CDs (b-d). Fig. 3. N2 sorption isotherms and the corresponding pore size distribution curves of samples.
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Fig. 4. FTIR spectra of pSiO2, pSiO2-NH2, pSiO2-GSSG and pSiO2-ss-CDs/HA samples (a) and UV/vis absorption, PL excitation, and emission spectra of CDs in aqueous solution (b).
Fig. 5. PL spectra of CDs excited from 320 to 400 nm (a). PL spectra of CDs, pSiO2-ss-CDs
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and pSiO2-ss-CDs/HA excited at 370 nm.
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Fig. 6. The cumulative release of Dox from pSiO2-ss-CDs/HA in PBS (pH=7.4) under
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different GSH concentration (a), different Hyal-1 concentration with or without GSH (b) and
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the corresponding release kinetics from pSiO2-ss-CDs/HA (c and d). Fig. 7. PL spectra of pSiO2-ss-CDs/HA carriers (a) and PBS solution (10 mM GSH and 0.5
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Scheme 1
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release medium (c).
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mg/mL Hyal-1) (b) with release time. Relationship between release rate and PL intensity of
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Figure 1
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Figure 2
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Total Fitting Background 283.8 sp2 C 284.5 sp3 C 285.1 C-N 285.7 C-O 287.2 C=N/C=O
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Total Fitting Background 398.3 C-N-C 399.1 N-(C)3
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Figure 3
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a
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pSiO2 pSiO2-NH2 pSiO2-GSSG pSiO2-ss-CDs/HA
SBET=605.87m
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SBET=383.85m
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Figure 6
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90 80 70
Q=53.36(1-e-0.6625t,R2=0.99)
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Q=63.15(1-e-0.726t,R2=0.99)
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S0925-4005(18)31556-9 https://doi.org/10.1016/j.snb.2018.08.118 SNB 25266
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
4-6-2018 16-8-2018 24-8-2018
Please cite this article as: Zhang Q, Guo J, Zhang X, Zhao Y, Cao L, Sun L, Redoxand enzyme-responsive fluorescent porous silica nanocarriers for drug delivery, Sensors and amp; Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.08.118 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Redox- and enzyme-responsive fluorescent porous silica nanocarriers for drug delivery Qianqian Zhanga, Jia Guob, Xu Zhanga, Yanbao Zhao*a, Liuqin Caob, Lei Suna a Engineering Research Center for Nanomaterials, Henan University, Kaifeng 475004, China
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b College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, China
Highlights
Porous silica (pSiO2) nanoparticles were successfully prepared for drug release.
Both carbon dots (CDs) and hyaluronic acid (HA) are used to seal the loaded drug in
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pores.
pSiO2-ss-CDs/HA carriers exhibit redox/ enzyme-responsive release behavior.
The CDs could be used to trace the drug release.
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Abstract
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In this work, redox/enzyme-responsive fluorescent porous silica (pSiO2) nanoparticles (NPs) were constructed for drug delivery. The resultant pSiO2 NPs have large-pore core and mesoporous shell with size of 75 nm and present high specific surface area (605 m2·g-1).
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Oxidized glutathione (GSSG) as linker was conjugated on the surfaces of aminofunctionalized porous silica (pSiO2-NH2) NPs by the amide bonds for redox-responsive drug
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release. Dox is served as model drugs to evaluate the release performance of carriers. After loading Dox molecules, both carbon dots (CDs) as fluorescent label and hyaluronic acid (HA) as gatekeepers are capped on the surface of carries (pSiO2-ss-CDs/HA) so as to endow the
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delivery system with fluorescent monitoring and enzyme-responsive properties. The pSiO2-ssCDs/HA carriers displayed redox/enzyme-responsive and sustained release behavior. The controlled release of drug from the pSiO2-ss-CDs/HA delivery system was realized by the reduction of disulfide bonds in GSSG linker and degrading HA molecules. The incorporated
*
Corresponding author. E-mail：[email protected] (Y Zhao); 1
CDs presented novel redox-dependent fluorescence, which could be used to trace the drug release. Keyword: Porous silica; carbon dots; redox/ enzyme-responsive; drug release.
1. Introduction
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In recent years, silica nanomaterials have attracted much attention as controlled drug delivery system [1,2]. Since 2001, when mesoporous silica (MS) nanoparticles (NPs) were reported as drug delivery firstly [3], a plenty of studies have been
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concentrated on their drug delivery properties and potential biomedical applications [4-6]. MS NPs have regular structural characteristics, large surface area and pore volume, tunable pore size, and ease surface modification, which make them ideal
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platforms to design multifunctional nanocarriers [7,8]. However, the limited loading
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capacity of conventional MS NPs cannot satisfy the biomedical application [9,10].
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Subsequently, many efforts have been devoted to the study of expanding the pore size
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of silica NPs. For instance, Dang et al. prepared the MS NPs with large radial pores of 17-78 nm and high surface area (1219 cm2 g-1), which exhibited high siRNA delivery
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capabilities [11]. Tu et al. synthesized cuboidal-like silica NPs with an average pore size of 9-11 nm and the carrier showed high encapsulation efficiency of proteins [12].
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Large-pore silica carrier has high loading capacity and can delivery large biomolecules, but it is easy to leak the loaded drug due to the large channels. In
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order to obtain the excellent performance of sustained release with high loading capacity, we propose the novel silica carrier with large-pore core and mesoporous shell to incorporate the advantages of MS and large pore silica into drug
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carriers.
Generally, the drug release from silica nanocarriers is the passive-diffusion
process, and the premature release is unavoidable in the delivery process, therefore stimuli-responsive release is necessary which could reduce premature release [13]. Doxorubicin (DOX) is a kind of anticancer drugs, which is often served as hydrophilic model drug to evaluate the responsive release of silica nanocarriers. 2
It is well documented that tumor tissues have unique physicochemical properties (compared to normal cell) with high levels of certain enzymes, redox, temperature and low pH, so the smart carriers can be tailored to be responsive to the pH variations [14], redox potential [15], enzymatic activation [16], light [17], hyperthermia [18] or magnetic field/ultrasound [19]. To further improve the release behavior, a variety of multifunctional silica nanocarriers have been intensively
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fabricated by incorporating diverse kinds of nanovalves utilized for controlled release of loaded drug by synthetic stimuli [20]. For example, Wu et al. designed a pH- and
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redox-responsive ZnO-gated hollow MS nanocarrier and ZnO quantum dots (QDs) were attached on the MS outer surface via disulfide bond. ZnO QDs that can be
dissolved in acidic environment and disulfide bond that can be broken by GSH [21].
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Furthermore, monitoring the drug release and on-demand release are both crucial
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for developing drug delivery systems. To date, various QDs and fluorescent dye are
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widely used in drug delivery to investigate the drug release process. Among them,
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carbon dots (CDs) have emerged as a new platform of QD-like fluorescent labels, which show good biocompatibility, tunable photoluminescent (PL) emission and
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excellent photostability [22-24]. Especially, both the optical absorption and fluorescent emissions in CDs are not band gap in origin, and their fluorescent
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performance can be tuned by doping other elements [25,26]. Here, we constructed a novel fluorescent dual-responsive drug delivery platform
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(pSiO2-ss-CDs/HA) by combining fluorescent CDs, oxidized glutathione (GSSG), hyaluronic acid (HA) and porous SiO2 (pSiO2) NPs. GSSG as linker was attached on the pSiO2 carrier through amido bonds and Dox was served as model drug to be
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encapsulated. After loading DOX, the CDs as fluorescent probe and hyaluronic acid (HA) as gatekeepers were coated on the surface of pSiO2 carrier (scheme 1). The pSiO2 carriers are composed of large pore core and mesoporous shell, which combine advantages of both mesopores and large pores. GSSG is a kind of biological peptide containing a disulfide bond, carboxyl groups and amino groups, which is used as linker to conjugate CDs and HA. HA is a naturally 3
occurring polysaccharide with nonimmunogenic, biocompatible, and biodegradable properties, which are used to cap the drug-loaded pores. In addition, HA possesses the active targeting toward tumor cells through selectively binding over-expressing CD44 receptors [27,28]. The disulfide bonds in GSSG linkers could be cleaved by redox, which would lead to the detachment of HA and redox-responsive release. HA could be degraded by Hyal-1, resulting in
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opening nanovalves and enzyme- responsive releasing drug. Due to high Hyal-1
and GSH concentration in tumor tissue, the drug release from the pSiO2 carrier could
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be triggered by the GSH and Hyal-1. In addition, CDs can be used to monitor the drug release by fluorescent signal.
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2. Experimental section
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2.1. Reagents
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Cetyltrimethylammonium bromide (CTAB) and citric acid were obtained from
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Tianjin Kermel Chemical Reagent Co. (Tianjin, China). Tetraethyl orthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES), polyethylenimine (PEI, 99%, Mw =
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600), n-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), oxidized glutathione (GSSG), hyaluronic acid (HA), reduced
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glutathione (GSH), hyaluronidase (Hyal-1) and doxorubicin (Dox) were purchased from Aldrich (Shanghai, China). Hydrochloric acid (HCl, 37%), ethanol and
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ammonium hydroxide (NH4OH, 30%) were provided by Luoyang Chemicals (Luoyang, China). Cyclohexane (C6H6) and toluene (C7H8) were from Tianjin Deen Chemical Reagent Co., Ltd. Distilled water with a resistivity of 18.2 MΩ cm was
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used in all experiment. All chemical reagents were of analytical grade and used as received without any further purification.
2.2. Preparation of N-doped CDs In a typical synthesis, 10 mL of PEI solution (0.1 mg/mL) was added into 20 mL of citric acid solution (0.1 g/mL) under stirring. Then, the above solution was 4
transferred to the Teflon-lined autoclave and heated to 150 ℃. After reacting for 2 h, the resulted bright brown solution was placed in a dialysis bag (Mw = 3500) for 24 h to purify the CDs solution.
2.3. Preparation and modification of pSiO2 NPs The porous SiO2 (pSiO2) NPs with core/shell structure were synthesized based
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on a previous report with some experimental modification [29]. Briefly, 0.25 g of
CTAB was dissolved in a mixed solution composed of 2.5 mL of ethanol, 35 mL
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of H2O, 7.5 mL of C6H6 and 0.4 mL of NH4OH (30%). The mixture was
vigorously stirred with a magnetic stirring rate of 1000 rpm for 0.5 h at 25 °C to form emulsion system.
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Then, a mixture of TEOS (1.25 mL), APTES (0.05 mL) and ethanol (1 mL) was
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dripped into the above solution and keep stirring for 3 h. Subsequently, 2 mL of
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TEOS/ethanol solution (1/1) was also added to the solution and reacting for another 3
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h to get core/shell pSiO2 sample. After that, the sample was centrifuged, washed for several times and then calcined by muffle furnace at 550 ℃ for 4 h to remove the
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template CTAB.
For amino-functionalization, 0.15 g of pSiO2 sample was dispersed in 60 mL of
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toluene under room temperature. After the mixture was mixed evenly, 1 mL of APTES was dropped into it and stirred for 24 h. The resulted amino-functionalized
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pSiO2 (pSiO2-NH2) was obtained after centrifuging and washing. For GSSG modification, GSSG molecule was attached to pSiO2-NH2 by amide
bonds. In brief, 100 mg of EDC and 100 mg of NHS were added to 15 mL of PBS
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(pH = 7.4) solution and stirring for 30 min at 25 ℃. Then, 50 mg of GSSG was added to above solution to active the carboxyl groups of GSSG. Afterwards, 50 mg of pSiO2-NH2 was added into the activated GSSG solution. After reacting for12 h at 25 ℃, the mixture was centrifuged, washed and dried to obtain pSiO2-GSSG.
2.4. Drug loading and capping 5
10 mg of pSiO2-GSSG was dispersed in 5 mL of PBS solution of Dox (1 mg/mL) by ultrasound and shaken at 37 ℃ for 24 h. Then Dox-loaded pSiO2-GSSG sample was collected via centrifugation and washing with water for several times. After that, the sample was dried in vacuum oven at 25℃.All the washing solution was collected and the loading capacity of Dox was calculated through detecting the
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UV absorbance of the washing solution and stock solutions. For CDs and HA capping, the Dox-loaded pSiO2-GSSG sample was dispersed in PBS solution containing 40 mg of NHS and 30 mg of EDC for 30 min. Then, 2 mL of
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CDs solution was added to the mixed solution and reacted for 12 h, followed by
addition of 2 mL of HA solution (1 mg/mL) which has been activated by EDC and NHS. After stirring for 10 h, the Dox-loaded CDs/HA capping silica carrier (pSiO2-
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ss-CDs/HA) was centrifuged, washed with PBS solution several times and dried in a
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vacuum oven at 25 ℃ for 24 h.
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2.5. Drug release
To investigate the drug release pattern under different microenvironments in
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vitro, a series of simulated release medium was prepared as follows: (1) PBS (pH=7.4) with different concentrations of GSH (0.1 mM, 1 mM and 5 mM). (2) PBS
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(pH=7.4) with different concentrations of Hyal-1 (0.1 mg/mL, 0.3 mg/mL, 0.5 mg/mL). (3) PBS (pH =7.4) with 5 mM GSH and 0.5 mg/mL Hyal-1.
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In vitro drug release experiment, 5 mL of PBS containing 5 mg of Dox-loaded
pSiO2-ss-CDs/HA was placed in dialysis bag, submersed in release medium under different conditions as above mentioned and shaken at 100 rpm and 36.8 ℃. After
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each fixed time intervals, 2 mL of release medium was taken out to measure UV absorbance. At the same time, an equivalent PBS was supplemented to release medium so as to maintain a constant total volume.
2.6. Characterization
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The morphology of the sample was observed at transmission electron microscopy (TEM, JEM-2100). N2 adsorption–desorption isotherm curves and pore size distribution were measured on a full-automatic specific surface and porosity analyzer (Quadrasorb SI, America). UV absorbable absorbance was obtained on a UV-2400 spectrophotometer. Fourier transform infrared (FTIR) spectra were performed on a Bruker VERTEX FTIR spectrometer. X-ray diffraction (XRD) was
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recorded on X-ray powder diffractometer ((D8-ADVANCE, German). Thermo gravimetric analysis (TGA) was carried out on a thermo-gravimetric analyser
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(TAQ600, America). Photoluminescence (PL) spectra was investigated by
fluorescence spectrophotometer (JY-HORIBA, France). X-ray photoelectron
spectroscopy (XPS) analysis was from a X-Ray Photoelectron Spectroscopy（AXIS
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ULTRA，Britain）
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3. Results and discussion
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3.1. Characterization
Fig. 1 (a) shows the TEM, HRTEM and size distribution of CDs. It is apparent
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that the CDs are of excellent dispersion and the average particle size is 3.7 nm. The HRTEM image of CDs reveals the lattice spacing of 0.21 nm, which is quite close to
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(100) diffraction facet of graphite [30, 31]. Fig. 1 b-f show the TEM images of pSiO2, pSiO2-NH2, pSiO2-GSSG, pSiO2-ss-CDs and pSiO2-ss-CDs/HA samples. The pSiO2
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sample presents uniform spheres with size of 70-80 nm and the sample presents distinct core/shell structure with large pores core and mesopore shell (Fig. 1b). After amino-modification, there is no change in the particle size and morphology
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compared to pSiO2 sample, which indicated that amino-modified layer is thin (Fig. 1c). After grafting GSSG, the morphology of the silica particles does not change significantly (Fig. 1d). When the CDs are attached, small agglomerated particles can be observed on the surface of the silica particles (Fig. 1e), which indicates that CDs are conjugated on the surface of pSiO2-GSSG. Obviously, the surface of silica
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particles turns to blurred after capping HA molecules and the pSiO2-ss-CDs/HA still remains porous structure (Fig. 1f). To investigate the chemical composition of CDs, XPS was performed as shown in Fig. 2. Fig. 2a shows the survey spectrum of the CDs that indicates the existence of C, N and O. The binding energies of C1s are found to be at 283.8, 284.5, 285.1, 285.7 and
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287.2 eV, suggesting the existence of C=C, C-C, C-N, C-O and C=N/C=O bond. The peak of N1s can be divided into three diff erent peaks at 398.3, 399.1 and 399.8 eV,
respectively, which are ascribed to C-N-C, N-(C)3 and N-H bonds. Meanwhile, after
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deconvoluting O 1s peak, it is found to exhibit two peaks at 530.3 and 531.3 eV, which indicates the presence of C=O and C-O bond respectively. These results reasonably
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demonstrate the formation of carbon dots from the carbon source of PEI and citric acid. The porous information was examined by N2 sorption. Fig. 3 gives the N2 adsorption-
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desorption isotherms and corresponding pore size distributions of the pSiO2，pSiO2-NH2
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，pSiO2-GSSG and pSiO2-ss-CDs/HA. Undoubtedly, all samples present IV isotherm
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typically with evident hysteresis loop (Fig. 3a), indicating the existence of mesopores [32]. The specific surface area (SSA) and total pore volume (Vp) of pSiO2 sample are
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605.87 m2g-1 and 0.92 cm3g-1. The high SSA and Vp would provide suitable conditions for the surface modification and drug loading. With each step of modification, the SSA
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and Vp display a decreasing trend. After amino-functionalization, the SSA and Vp decrease to 383.85 m2g-1 and 0.55 cm3g-1, which provide the proof of the successful
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grafting of amino groups. Similarly, the SSA and Vp of pSiO2-GSSG and pSiO2-ssCDs/HA are decreased to 226.55 m2g-1, 0.35 cm3g-1 and 96.00 m2g-1, 0.13 cm3g-1, respectively. The change in SSA and Vp of the prepared samples would provide
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evidences directly for drug-loading and each successful modification. The corresponding pore size distribution curves were obtained from the adsorption
branch by BJH method (Fig. 3b). For pSiO2 sample, there appears a narrow and strong peak centered at 7.5 nm, while pSiO2-NH2 sample presents a peak position at 6.4 nm. The slight decrease in pore size revealed that the amino group was not only grafted on the surface of the silica but also on the internal pore surface. Compared with pSiO2-NH2, the 8
pore size of pSiO2-GSSG and pSiO2-ss-CDs/HA are slightly increased, which may be attributed to that part of smaller channels of the silica are completely covered by GSSG (or Dox) molecules, resulting in an increasing in the average pore size. FTIR was used to identify the successful modification of pSiO2 NPs (Fig. 4a). For CDs, the absorption bands at 1645 and 1566 cm-1 are attributed to C=O and N-H
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stretching vibration, respectively [25]. The weak peaks at 2836 and 2963 cm-1 belong to characteristic absorption -CH2- chains of PEI, demonstrating the existence of PEI on CDs [33, 34]. For pSiO2-NH2, the peak at 1463 cm-1is assigned to N–H bending
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vibration, indicating the successful grafting of amino groups [35]. After connection with GSSG, the bands at 1643 and 1535 cm-1 are related to amide I and amide II absorption, which identifies the interaction between carboxyl groups of GSSG and
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amino groups of silica [21]. The peaks at 1405 and 2965 cm-1 correspond to C=O
N
stretching vibration of HA and -CH2- chains stretching vibration in CDs, respectively,
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confirming the successful covering of CDs and HA [36].
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Fig. 4b shows the typical UV-Vis absorption, excitation and emission spectra of CDs. Two noticeable absorption peaks located at 255 and 368 nm may be attributed to
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the π-π* transitions of C=C bond and n-π* transitions of C=O bond [37, 38]. When CDs are excited at 370 nm, the maximum emission peak at 440 nm can be observed.
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Moreover, the synthesized CDs is quite well of water solubility and the aqueous solution of CDs presents pale yellow and displays bright blue fluorescence under UV
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light at 365 nm.
PL is one of most fascinated features of CDs. Fig. 5a shows a detailed PL
investigation with different excitation wave lengths. When the excitation wavelength
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is changed from 320 to 400 nm, the PL emission peak at 440 nm remains unchanged. This behavior of excitation-independent PL is considered to be associated with less surface defects and more uniform size of CDs, which is beneficial to avoid unwanted autofluorescence for down-conversion and up-conversion cell imaging [37, 39]. In addition, the PL intensity increases first and then decreases with the increase of excitation wavelength from 320 nm to 390 nm. When the excitation wavelength is 9
370 nm, the intensity of PL emission is maximum as shown in Fig. 5a. The excitation-dependent PL intensity may be related to the simultaneous absorption of CDs and the maximum absorption appears at 370 nm.
Fig. 5b shows the PL spectra of CDs, pSiO2-ss-CDs and pSiO2-ss-CDs/HA. In aqueous solution, the CDs exhibited blue emission peak at 440 nm. Under the
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excitation light of 370 nm, both pSiO2-ss-CDs and pSiO2-ss-CDs/HA also present remarkable emission peak at the same position of 440 nm with different PL intensity, indicating the successful of capping CDs. Compared with pSiO2-ss-CDs, the PL
interactions between CDs and HA molecules [40,41].
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3.2. In vitro release behavior
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intensity of pSiO2-SS-CDs/HA is weakened a lot, which may be related to electronic
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The pSiO2-ss-CDs/HA carrier with large inner channel is beneficial for the drug
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diffusion and loading, and its loading capacity of Dox is up to 20.5%. The drug
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release would be triggered by GSH and Hyal-1 in the way of cleaving disulfide bonds of GSSG and decomposing hyaluronic acid, respectively. Fig. 6 illustrates the
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cumulative release of Dox from pSiO2-ss-CDs/HA under different release medium. The drug release profiles in PBS solution (pH=7.4) with different GSH concentrations
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are shown in Fig. 6a. It can be seen that the pSiO2-ss-CDs/HA carrier displays an excellent GSH-responsive release manner. In the absence of GSH, only small amount
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of Dox was released into the PBS solution within 10 h and the release rate is 16.4 %, signifying the efficient confinement of the drug in the pores of the pSiO2-ss-CDs/HA. The release efficiency is gradually enhanced as the concentration of GSH increased
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from 0.1 to 10 mM. Under GSH concentration of 10 mM, the drug release rate is accumulated to 52.9% within 6 h. The GSH-triggered rapid release is related to the detachment of CDs or HA from pSiO2 carriers via the cleavage of disulfide bonds. In the case of redox, the disulfide bonds in GSSG may be not completely broken due to the hindrance of the coating layer HA or the cleaved CDs and large HA
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moiety may be not completely detached from on the surface of pSiO2 carriers, causing the drug to not be completely released. Fig. 6b depicts the cumulative drug release from pSiO2-ss-CDs/HA in PBS solution with different Hyal-1 concentration. Obviously, the pSiO2-ss-CDs/HA also displays Hyal-1-responsive sustained-release property without burst phenomenon. As
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the concentration of Hyal-1 increases, the drug release efficiency is enhanced gradually. When the concentration of Hyal-1 reaches 0.5 mg/mL, the drug release rate is accumulated to 62.2% in 6 h. This fact demonstrates the pSiO2 carriers with CDs
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and HA capping are sensitive to Hyal-1 and cause enzyme-responsive release.
Due to the high GSH concentration and over-expressed enzyme of tumor cells,
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the release efficiency of drug would be increased when pSiO2-ss-CDs/HA carriers were delivered under the tumor environment. The release behavior in buffer solution
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with Hyal-1 and GSH was also investigated (in Fig. 6b). The faster Dox release is
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observed at 10 mM GSH and 0.5 mg/mL Hyal-1, and the released Dox reaches at
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87% within 5 h, much high than that at 10 mM GSH or 0.5 mg/mL Hyal-1, showing that pSiO2-ss-CDs-HA carrier exhibits excellent GSH/Hyal-1 synergic responsive
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drug release. In the presence of Hyal-1 and GSH, the degradation of HA would help to completely cleave the disulfide bonds in GSSG, leading to more complete
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opening nanovalves and promoting the release of drug. Therefore, the release rate could be enhanced in the dual-responsive case. In addition, the CD44 receptor
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over-expressed on the surface of tumor can bind to hyaluronic acid specifically, which is promising to achieve carrier-targeted drug release. To understand the release mechanism of Dox from pSiO2-ss-CDs/HA carriers,
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the corresponding release kinetics was also studied. In this study, the experimental release data were fit using the first-order kinetic model involving an exponential decay. Q = Qmax (1 − exp (−kt)).
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where, Q represents the amount of drug released at time t, Qmax represents the maximum amount of drug released and k is the first-order constant. The graphical representations and the corresponding parameters calculated from the kinetics model are shown in Fig. 6c and d, respectively. The Dox release from pSiO2-ss-CDs/HA carrier follows the first order kinetics model with high correlation coefficient.
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3.3. Fluorescent behavior As a fluorescent label of drug carriers, CDs can be used not only to track the
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position of the carrier in real time, but also to monitor the process of drug release
based on changes in PL intensity. Fig. 7a gives the PL spectra of pSiO2-ss-CDs/HA with release time in the presence of 10 mM GSH and 0.5 mg/mL Hyal-1. It can be
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seen that the PL intensity of the pSiO2-ss-CDs/HA is reduced with increasing release
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time. The phenomenon can account for that the GSH reduction of disulfide bonds in
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GSSG, which lead to the CDs falling off from the pSiO2 carriers. In the meantime, the CDs detached from the carriers would be dispersed in release medium and cause a
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change of PL intensity of the release medium. To illustrate that the detached CDs
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were dispersed in the release medium, the PL intensity of the release medium was detected (Fig. 7b). As expected, the PL intensity of the release medium increases simultaneously as increasing release time, suggesting that the CDs were detached
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from carriers and dispersed in release medium under the action of GSH. To
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understand the relationship between detached CDs and drug release, we also compared the drug release rate and PL intensity of the release medium over time (Fig.7c). It can be seen that the curve of PL intensity of the release medium is similar to that of the accumulative drug release rate. The results indicate that the
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drug release can be monitored by the change in PL intensity of the drug delivery system.
Conclusions
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In this paper, we designed and prepared a novel drug delivery system based on fluorescent pSiO2 NPs. In this system, GSSG as linker was modified on the surface of pSiO2 NPs and both CDs and HA are used to seal the drug-loaded pores. The pSiO2ss-CDs/HA carrier presents high loading capacity and the drug release would be triggered by cleaving disulfide bonds of GSSG or decomposing HA. The Dox release
allows real-time monitoring of the release of drug from the pores.
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Acknowledgement
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followed the first order kinetics model very well. The PL characteristic of the CDs
Financial support of this work from National Natural Science Foundation of China (21271062) is gratefully acknowledged.
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Biographies Qianqian Zhang studied at Henan Normal University, and obtained her BSc in chemistry in 2016. She joined Professor Zhao’s research group at Henan University after her graduation.
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Her researches focus on fluorescent porous silica nanospheres for drug delivery. Jia Guo studied at Henan University, and obtained her MSc in chemistry in 2014. Her
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research focuses on drug delivery.
Xu Zhang studied at Huazhong Agricultural University, and obtained her Master degree in 2005. She joined Professor Zhao’s research group at Henan University in 2014. Her research
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focuses on porous silica nanospheres for drug delivery.
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Yanbao Zhao studied physical chemistry in Lanzhou Institute of Chemical Physics, Chinese
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Academy of Sciences, and received his PhD in 2004. He started his academic career at Henan
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University, and promoted as Professor in 2005. He engages in designing and preparation of functional nanomaterials and their applications and has published more than 80 scientific
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papers.
Liuqin Cao works at Henan University as an experimentalist of chemistry, and her research
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interests concentrate in preparation and modification of nanoparticles.
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Lei Sun studied physical chemistry in Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, and received his PhD in 2005. He is a professor of Chemistry at Henan University. He engages in designing and preparation of inorganic antibacterial material, and
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has published more than 60 scientific papers
Figure captions 18
Scheme 1 The preparation and release of pSiO2-ss-CDs/HA nanocarriers. Fig. 1. TEM image (a) with HRTEM and size distribution (insert) of CDs. TEM images of pSiO2 (b), pSiO2-NH2 (c), pSiO2-GSSG (d), pSiO2-ss-CDs (e) and pSiO2-ss-CDs/HA (f) samples.
IP T
Fig. 2. XPS of the CDs (a) and C1s, N1s, O1s XPS spectra of CDs (b-d). Fig. 3. N2 sorption isotherms and the corresponding pore size distribution curves of samples.
SC R
Fig. 4. FTIR spectra of pSiO2, pSiO2-NH2, pSiO2-GSSG and pSiO2-ss-CDs/HA samples (a) and UV/vis absorption, PL excitation, and emission spectra of CDs in aqueous solution (b).
Fig. 5. PL spectra of CDs excited from 320 to 400 nm (a). PL spectra of CDs, pSiO2-ss-CDs
U
and pSiO2-ss-CDs/HA excited at 370 nm.
N
Fig. 6. The cumulative release of Dox from pSiO2-ss-CDs/HA in PBS (pH=7.4) under
A
different GSH concentration (a), different Hyal-1 concentration with or without GSH (b) and
M
the corresponding release kinetics from pSiO2-ss-CDs/HA (c and d). Fig. 7. PL spectra of pSiO2-ss-CDs/HA carriers (a) and PBS solution (10 mM GSH and 0.5
A
CC E
Scheme 1
PT
release medium (c).
ED
mg/mL Hyal-1) (b) with release time. Relationship between release rate and PL intensity of
19
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figure 1
20
a
IP T
Figure 2
SC R
O
Counts (a.u.)
C
100
200
300
400
500
M
0
A
N
U
N
600
700
A
CC E
PT
ED
Binding Energy (eV)
21
800
900 1000
b
C1s
280
282
284
286
288
290
292
A
CC E
PT
ED
M
A
N
U
Binding Energy (eV)
22
294
IP T
278
SC R
Counts (a.u.)
Total Fitting Background 283.8 sp2 C 284.5 sp3 C 285.1 C-N 285.7 C-O 287.2 C=N/C=O
c N1s
Counts (a.u.)
Total Fitting Background 398.3 C-N-C 399.1 N-(C)3
390
392
394
396
398
400
402
404
406
408
410
A
CC E
PT
ED
M
A
N
U
SC R
Binding Energy (eV)
IP T
399.8 N-H
23
d O1s
524
526
528
530
532
534
536
538
540
A
CC E
PT
ED
M
A
N
U
SC R
Binding Energy (eV)
IP T
Counts (a.u.)
Total Fitting Background 530.3 C=O 531.3 C-O
Figure 3
24
a
1400
1000 800
pSiO2 pSiO2-NH2 pSiO2-GSSG pSiO2-ss-CDs/HA
SBET=605.87m
2 -1 g
600
SBET=383.85m
400
2 -1 g
SBET=226.55m
200
SBET
IP T
Volume (cm3g-1)
1200
2 -1 g
2 -1 =96.00m g
0 0.2
0.4
0.6
0.8
1.0
SC R
0.0
N
U
Relative Pressure (p/p0)
b
pSiO2
A
2.5
2.9-14.3nm
pSiO2-NH2
M
1.5
pSiO2-ss-CDs/HA
2.9-11.3nm
1.0
ED
d /d (cm g v (logD)
3 -1
)
pSiO2-GSSG
2.0
3.5-17.7nm
0.5
3.8-12.3nm
10
20
30
Pore Diameter (nm)
A
CC E
PT
0.0
25
40
50
Figure 4
a 2836
pSiO2-NH2
pSiO2-ss-CDs/HA
3000
1460
1643 1535
1405
2965
3500
1566
1645
pSiO2-GSSG
IP T
2963
2500
2000
1500
SC R
Transmittance (a. u.)
CDs
1000
-1
b
ED
M
A
N
U
Wavenumber (cm )
500
A
Absorbance (a. u.)
Em
Ex
1.2 1.0
PL intensity (a. u.)
CC E
PT
1.4
Abs
0.8 0.6 0.4 0.2
0.0 200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
26
Figure 5
a λex
5
1200000
1 2 3 4 5 6 7 8 9
4
7
3
1000000
2
800000 8
1
600000 400000
320nm 330nm 340nm 350nm 360nm 370nm 380nm 390nm 400nm
9
200000 0 425
450
475
500
525
550
575
CC E
PT
ED
M
A
N
Wavelength (nm)
U
400
IP T
6
SC R
PL intensity (a. u.)
1400000
PL intensity (a. u.)
A
b 1
200000
1----CDs 2----pSiO2-ss-CDs
2
3----pSiO2-ss-CDs/HA
150000 3 100000
50000
0 350
400
450
500
550
Wavelength (nm)
27
600
650
IP T SC R U N A 120
1 ---- 0 mM GSH 2 ---- 0.1 mM GSH 3 ---- 1.0 mM GSH 4 ---- 10 mM GSH
110
PT
a
ED
M
Figure 6
100
A
CC E
Release (%)
90 80 70 60
4
50
3
40 30
2
20
1
10 0 0
1
2
3
4
5
6
Time (h)
28
7
8
9
10
11
120
1 ---2 ---3 ---4 ---5 ----
110 100
Release (%)
90
0 mg/mL Hyal-1 0.1 mg/mL Hyal-1 0.25 mg/mL Hyal-1 0.5 mg/mL Hyal-1 GSH (10mM)+ Hyal-1(0.5 mg/mL)
5
80 70
4 3
60 50
2
40 30
1
20 10 0 0
1
2
3
4
5
6
7
8
9
10
11
c
ED
M
A
N
U
SC R
Time (h)
IP T
b
120
1----0 mM GSH 2----0.1 mM GSH 3----1.0 mM GSH 4----10 mM GSH
110
PT
100
A
Release (%)
CC E
90 80 70
Q=53.36(1-e-0.6625t,R2=0.99)
60
4
50
3
Q=47.57(1-e-0.6107t,R2=0.98)
30
2
Q=35.87(1-e-1.1824t,R2=0.99)
20
1
40
Q=16.45(1-e-1.7174t,R2=0.99)
10 0 0
1
2
3
4
5
6
Time (h)
29
7
8
9
10
11
120
1---- 0 mg/mL Hyal-1
110
2---- 0.1 mg/mL Hyal-1 3---- 0.25 mg/mL Hyal-1
100
Release (%)
Q=89.22(1-e-0.5562t,R2=0.99)
4---- 0.5 mg/mL Hyal-1
90
5---- GSH(10mM)+ Hyal-1(0.5 mg/mL)
80
5
Q=63.15(1-e-0.726t,R2=0.99)
70 4
60 50
3
Q=56.18(1-e-0.6803t,R2=0.99)
2
Q=39.35(1-e-0.4882t,R2=0.98)
40 30 20 1
10
Q=16.45(1-e-1.7174t,R2=0.99)
IP T
d
0 0
1
2
3
4
5
6
7
8
9
10
11
M
A
N
U
SC R
Time(h)
140000
1
120000
2
1----0h
2----2h 3----4h 4----8h
100000
3
80000
4
60000 40000 20000
0 350
400
450
500
550
Wavelength (nm)
A
CC E
PT
PL intensity (a. u.)
a
ED
Figure 7
30
600
650
IP T
b
40000 4 1----0h
30000 25000
SC R
2----2h 3----4h 4----8h
3
20000 2 15000
5000 0 350
1 400
450
500
550
U
10000
600
650
N
PL intensity (a. u.)
35000
A
CC E
PT
ED
M
A
Wavelength (nm)
31
c
40000
100
35000 2
1 20000
40
15000 20
10000 1 2
0
0
2
4
6
8
A
CC E
PT
ED
M
A
N
U
Time (h)
32
0
SC R
5000
PL intensity of release medium Release rate of Dox
IP T
60
25000
Release (%)
PL intensity (a. u.)
80 30000