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Halloysite nanotube-based H2O2-responsive drug delivery system with a turn on effect on fluorescence for real-time monitoring
Chemical Engineering Journal 380 (2020) 122474
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Halloysite nanotube-based H2O2-responsive drug delivery system with a turn on effect on fluorescence for real-time monitoring ⁎
Cong Chenga, Yan Gaoa,b, , Weihua Songb, Qiang Zhaoa, Haisong Zhangb, Hailei Zhanga, a b
T
⁎
College of Chemistry & Environmental Science, Hebei University, Baoding 071002, PR China Affiliated Hospital of Hebei University, Baoding 071000, PR China
H I GH L IG H T S
G R A P H I C A L A B S T R A C T
halloysite-based hydrogel • Awasintelligent prepared with a H O -responsive 2
• •
2
release character. A coprecipitation method was proposed to load the model drug into the cavity of HNTs. The fluorescence intensity enhances with the increase of the drug release rate.
A R T I C LE I N FO
A B S T R A C T
Keywords: Halloysite nanotube Clay science H2O2-responsive Drug loading
In this paper, a novel halloysite-based hydrogel with a “turn-on” fluorescence character upon H2O2 was facilely prepared and used to construct the H2O2-responsive drug delivery system, in which a coprecipitation method was proposed to afford the drug-loaded halloysite nanotubes (DHNTs). DHNTs were carefully characterized by FTIR, TGA, XPS, XRD and TEM to demonstrate that the drugs were mainly loaded into the cavity rather than attached on the external surface of halloysite nanotubes (HNTs). The B-C linkage in the as-prepared hydrogel was broken in the presence of H2O2, resulting in the degradation and thereby a responsive release. The drug release was almost not occurred under the physiological concentration ([H2O2] = 0.02 μM), while a complete release (> 90%) can be achieved under pathological concentration ([H2O2] = 200 μM). Moreover, the broken of B-C linkage triggered a transformation from arylboronates to phenols, in which the formed fluorescein gave rise to the change from non-fluorescent to fluorescent of the hydrogels. The fluorescence intensity enhanced with the increase of release rate, in which a good linear relationship can be achieved. The attractive properties make the halloysite-based hydrogels a promising application in the field of biomedicine.
1. Introduction Hydrogen peroxide (H2O2), one of reactive oxygen species (ROS), plays a fundamental role in the regulation of many physiological processes in living organism, especially in cellular signal transduction,
differentiation and proliferation [1]. The overexpression of H2O2 is viewed as a marker for oxidative stress and damage events in vivo, and thereby associated with the diseases including inflammation, hemorrhage, Alzheimer's disease, renal hypofunction and tumors [2–4]. For example, the concentration of H2O2 in normal tissues maintains
⁎ Corresponding authors at: No.180 Wusidong Road, College of Chemistry & Environmental Science, Hebei University, Baoding, Hebei Province 071002, PR China (H. Zhang) and No. 212 Yuhua Road, Affiliated Hospital of Hebei University, Baoding, Hebei Province 071000, PR China (Y. Gao). E-mail addresses: [email protected] (Y. Gao), [email protected] (H. Zhang).
https://doi.org/10.1016/j.cej.2019.122474 Received 17 April 2019; Received in revised form 1 August 2019; Accepted 9 August 2019 Available online 09 August 2019 1385-8947/ © 2019 Elsevier B.V. All rights reserved.
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ca.10−8 M, while a thousand-fold increase can be found in inflammation tissues or tumors [5]. The development of new chemical tools that are sensitive enough to report H2O2 production at pathological changing level has raised a lot of interests [6–9]. In past decades, a library of H2O2-responsive materials has been developed, including catalyst [10], fluorescence probe [11,12], MRI contrast agent [13] and drug delivery system [14,15]. However, the limitations of reported H2O2-responsive drug delivery systems severely restricted its applications, such as serious initial burst effect, low compatibility, high cost and so on. Moreover, the lack of an indicator in the drug delivery system result in a difficulty to monitor the release behavior in real-time, which may bring some unexpected delays in practical clinical uses. Halloysite nanotube (HNT) is a natural aluminosilicate clay mineral, which has the same chemical composition as kaolinite (Al2Si2O5(OH)4 nH2O) [16–19]. The multilayers of hollow nanotubes are formed by curved and rolled up of the adjacent alumina and silica sheets along with their water of hydration [20,21]. Benefiting from the high dispersibility, the hollow tubular shape, the large cavity volume and the modifiable surface structure, HNTs can be used as the desirable natural nano-carriers for loaded active agents and even fabrication of nanocomposites [22–30]. The attractive properties including biocompatibility, nontoxicity, non-degradation, hydrophilic and low-cost also make HNTs promising materials in the field of biochemistry and biomedicine [31–35]. Lvov’s group pioneered the HNTs-based drug delivery system in which the sustained-release character can be facilely achieved [36,37]. Liu et al enhanced the transmembrane transport ability of HNTs-based drug delivery system by lower the size of HNTs [38]. Lazzara and coworkers developed a library of HNTs-based drug delivery systems with pH- or thermo- sensitive release behaviors [39,40]. Das’ group developed a library of clay-based carriers, including the HNTs-based drug loading system with fine tuning in drug release behaviors, in which the kinetics studies were emphasized [41–44]. Zhang et al explored the HNTs-based anti-bacteria materials [45]. Our groups also devoted our efforts to prepare HNTs-based H2O2-responsive fluorescence probes and drug delivery system, in which the “burst effect” can be effectively suppressed [46–48]. It will be very interesting to further develop a more intelligent H2O2-responsive materials that the drug release can be monitored in real-time by the fluorescence changes, which can effectively enhance the efficiency in diagnosis and treatment. Hydrogels emerged as excellent candidates for controlled release and stimulate-responsive drug delivery as they are able to encapsulate nanoparticles well as biomacromolecules and small molecule drugs [49–51]. The loading of guest molecule into the cavity of nanotubes ahead of the formation of hydrogels can effectively reduce the burst release effect, while the attachment of guest molecules on the exterior surface may result in a low controllability on the release behavior. In this study, a coprecipitation method was proposed to load the model drug into the cavity of HNTs facilely, where the adhesion of the drug on the external surface of HNTs can be effectively avoided. Our strategy for achieving the H2O2-response characters relies on the H2O2mediated transformation of arylboronates to phenols. Boronic acid groups were installed at the 3′ and 6′-positions of a xanthenone scaffold in fluorescein. The obtained fluorescein derivative (PA) bearing two arylboronic acid groups can rapidly react with diols to form boronic ester groups in mild conditions, which can serve as cross-linking agent to crosslink multi-hydroxyl polymers and thereby affording hydrogels with H2O2-sensitivity. The installation at the 3′ and 6′ positions of a xanthenone scaffold in fluorescein would force this platform to nonfluorescent lactone form, which gave rise to non-fluorescent hydrogels. After treating with H2O2, the transformation of arylboronates to phenols resulted in the formation of fluorescein which triggers a prompt fluorescence increase. Following this way, the intelligent hydrogels can be facilely afforded by the addition of PA into the above polyvinyl alcohol (PVA) solution containing drug-loaded HNTs (DHNTs). The H2O2-responsive cleavage of the formed B-C link in the hydrogels can be achieved in a high concentration of H2O2 and thereby result in a
H2O2-responsive drug release behavior. Little release can be detected when [H2O2] = 0.02 μM suggesting a desirable control on the initial burst effect, while a rapid and complete release can be achieved in a concentration of H2O2 at 200 μM. The fluorescence intensity enhanced with the increase of release rate, which shows a good linear relationship to the drug release behavior. 2. Material and methods 2.1. Materials HNTs were obtained from GuangZhouShinshi Metallurgy and Chemical Company Ltd (Guangzhou, China) and purified according to our previous work [52]. Fluorescein and other reactants were obtained from J&K Scientific Ltd. Pentoxifylline (PTX) was obtained from TCI Development Co., Ltd. PVA and 30% H2O2 solution were purchased from Kemiou Chemical Reagent Co., Ltd. Dimethyl sulfoxide (DMSO) was dried and distilled from CaH2 under vacuum before use. Distilled water was used throughout the study. High-purity argon was used for degassing procedures. PF1 was synthesized according to the reference [53]. 2.2. Syntheses and preparations 2.2.1. Synthesis of (3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-3′,6′diyl) diboronic acid (PA) PF1 (940 mg, 1.7 mmol) and diethanolamine (400 mg, 3.8 mmol) were dissolved into an ethyl ether solution and then stirred at room temperature for about 50 min. The formed precipitate was filtered and washed with ethyl ether. After vacuum drying, the obtained brown powders were immersed in 0.1 M HCl. After ultrasound about 30 min, the yellow precipitation was formed by centrifugation and washed by 0.1 M HCl to give the product PA (435 mg, 66%) (Scheme 1). 1H NMR (600 MHz, (CD3)2SO) δ 8.06 (d, J = 7.8 Hz, 1H), 7.87 – 7.76 (m, 3H), 7.75 (t, J = 7.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.28 (d, J = 7.8 Hz, 1H), 6.80 (d, J = 7.8 Hz, 2H). 13C NMR (100 MHz, (CD3)2SO) δ 169.21, 153.43, 150.38, 138.34, 136.34, 130.81, 129.78, 127.19, 125.58, 125.41, 124.38, 122.88, 120.35, 81.98. FTIR (KBr): 3416, 1740, 1412, 1342, 1075, 925, 704 . 2.2.2. Preparation of drug loaded HNTs (DHNTs) 100 mg of HNTs was dispersed into a saturated solution of PTX in DMSO. The suspension was placed in a vacuum chamber by pumping and filling in with N2 for several times. The product was precipitated into excess amount of acetone washed for three times. The residue was collected by a fast certification process and then dried in vacuum to give DHNTs as white powders (drug loading degree: 17.0%). 2.2.3. Preparation of the physical mixture of HNTs and PTX (HNTs@PTX) 17 mg of PTX was dissolved in 10 mL anhydrous ethyl alcohol followed by 100 mg of HNTs were added. The mixture was stirred to achieve a uniform suspension and the evaporated under atmosphere pressure to afforded a solid residue. The residue was collected, grinded, and dried to give HNTs@PTX as faint yellow powders (drug loading degree: 17.0%).
Scheme 1. Synthesis of PA. 2
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Scheme 2. Preparation of PTX@PVA@PA hydrogel.
instrument (PerkinElmer, USA) operated under 20 mL/min nitrogen flow rate in which the heating rate was 10 °C/min. UV–Vis absorption and fluorescence spectra were obtained on a UV-2550 UV–visible spectrometer (Shimadzu, Japan) and a RF-5301PC fluorospectro photometer (Shimadzu, Japan). X-ray diffraction (XRD) patterns were performed at room temperature with a D8 ADVANCE X-ray powder diffractometer system (Bruker, Faellanden, Switzerland). The patterns were recorded on a quartz plate at a tube voltage of 40 kV and a current of 40 mA in a 2θ range of 10-90° using a step size 0.06° at a scan speed of 1 s/step.
2.2.4. Preparation of PTX@PVA@PA hydrogels Polyvinyl alcohol (200 mg) was dissolved in 2.0 mL of distilled water at 80 °C. 5.0 mg of PTX was dispersed in 2.0 mL of aqueous solution of PA, and then added carefully into the above aqueous solution. The system was stirring for about 3 min and incubated at 80 °C for 0.5 h to afford a unique hydrogel (PTX@PVA@PA) with yellow colour. The obtained hydrogel was stored at −20 °C before use (drug loading degree: 0.2%) (see Scheme 2). 2.2.5. Preparation of DHNTs@PVA@PA hydrogels Polyvinyl alcohol (200 mg) was dissolved in 2.0 mL distilled water at 80 °C. A certain amount of DHNTs containing 5 mg PTX was dispersed in 2.0 mL of aqueous solution of PA, and then quickly added into the above aqueous solution. The system was stirring for about 3 min and incubated at 80 °C for 0.5 h to afford a unique faint yellow hydrogel (DHNTs@PVA@PA). The obtained hydrogel was stored at −20 °C before use (drug loading degree: 0.2%) (see Scheme 3)
2.4. In vitro drug delivery Drug delivery studies were carried out using a ZRS-8G dissolution tester (Haiyida, China). The paddle rotation speed was set as 50 rpm at 37 ± 0.5 °C. The drug release behaviors were evaluated by immersing the samples into pH 7.4 phosphate buffer solution (PBS) with H2O2 in different concertation ([H2O2] = 200 μM and 0.02 μM) H2O2. The samples were filtered through a membrane filter (pore size 0.45 μm) before monitored. The filtrates were collected for further test.
2.3. Characterizations The Tecnai G2 F20 S-TWIN transmission electron microscope (TEM) was used to characterize the morphology of HNTs and DHNTs with an accelerating voltage of 200 kV. The morphological characterisations of the hydrogels were conducted on a JEOL Ltd. JSM-7500F Cryo field emission scanning electron microscopy (SEM). The lyophilized samples were placed on flat substrates and coated with gold for SEM observations. FTIR spectra were performed in the region of 4000–400 cm−1 for each sample on a Varian-640 spectrophotometer. Samples were previously ground and mixed thoroughly with spectrum pure KBr. The spectrum for each sample was obtained from averaging 32 scans over the selected wave number range. The scan rate is 5 kHz. 1H and 13C NMR spectra were recorded on a Bruker AVANCE-600 600 MHz spectrometer (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) was conducted on a Thermo Scientific ESCALab 250Xi using 200 W monochromated Al Ka radiation, in which 500 μm X-ray spot and 3 × 10−10 mbar base pressure were used. The hydrocarbon C1s line at 284.8 eV from adventitious carbon was used for energy referencing. Thermogravimetric analysis (TGA) was recorded by a Pyris1 TGA
3. Results and discussion 3.1. Drug loading in HNTs A coprecipitation method was used to load the model drug (PTX) into the cavity of HNTs. Briefly, the purified HNTs were immersed into saturated aqueous solution of PTX. The suspension was carefully degassed to fill the drug solution into the cavity of the tubes and then precipitated into acetone which can satisfy the solubility of PTX. It should be noted that vacuum pumping step is essential for achieving a desirable loading rate. The vapor pressure may be beneficial to evaporate the inherent water and empty the gas inside the lumen of HNTs, in which the molecules dissolved in the fresh aqueous solution can migrate to the halloysite lumen [54]. Owing to the poor dispersibility of HNTs in acetone, the drug loaded in the lumen was coprecipitated accompanying with the nanotubes. Precipitates were collected by centrifugation in a very short time in case the drug released from the
Scheme 3. Preparation of DHNTs@PVA@PA hydrogel. 3
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Fig. 1. Characterizations of HNTs, PTX, DHNTs, PTX@PVA@PA, DHNTs@PVA@PA (A: FTIR spectra; B: TGA and normalized differential curves; C: XRD patterns; D: XPS spectra).
determined based on Lambert-Beer laws. The calculated drug loading degree is in accordance with that given by TG analysis. A section of the medium was collected after certifying and then freeze-dried for 1H NMR analysis (Supporting Information). The 1H NMR spectrum shows a high purity and the results match well with the structure information of PTX, implying the drug retained a high chemical stability in the drug loading process based on the coprecipitation method. We have tried to increase the drug loading degree by changing the conditions, including extending the time in low pressure and increasing the temperature. We find that 17% is the highest drug loading degree for this approach. This value is slightly lower than those in literatures [55,56], which may be due to that part of the loaded molecules located near the opening of the tubes were dissolved in the precipitation process. Nevertheless, we focus on developing a facile loading approach to avoid the absorption of external surface rather than increasing the loading degree. The drug loading degree of 17% is sufficient for us to process the latter investigations. The XRD and XPS characterizations were carefully investigated to reveal that the drug was loaded in the lumen, rather than attached on the external surface. In addition, a physical mixture of HNTs and PTX (HNTs@PTX) was prepared by a simple grinding method. The XRD patterns of HNTs, DHNTs and HNTs@PTX were shown in Fig. 1C. The XRD pattern of HNTs features a diffraction peaks at ca. 11.9°, corresponding to a basal spacing of layer spaces. Other diffraction signals at ca 20.0°, 24.6°, 35.1°, 38.2°, 54.9° and 62.3° can also be observed, which are in good agreement with the literatures [57–59]. The diffraction peak of PTX can be clearly seen at 15°, 24.1° and 27.9° in the XRD pattern of HNTs@PTX, while the characteristic diffraction peaks assigned to PTX are difficult to be detected in the pattern of DHNTs. The wall thickness of HNTs is estimated as 20 nm, which well exceeds the penetration depth of X-ray (10 nm) [60,61]. Thus, it is difficult to disclose the chemical composition in the cavity of HNTs. The XRD results suggest that the PTX should be loaded in the lumen of the nanotubes.
cavity. The drug-loaded HNTs (DHNTs) were obtained after drying in vacuum. FTIR and TGA were performed to reveal the composition of the obtained composites. The FTIR spectrum of original HNTs depicted on Fig. 1A features two distinct peaks at 3717 and 3618 cm−1. Inner hydroxyl groups, lying between the tetrahedral and octahedral sheets, give the absorption near 3618 cm−1. The other strong band at 3717 cm−1 is associated with surface hydroxyl groups in the lumen of HNTs. Characteristic band of in-plane Si–O–Si stretching vibration is observed around 1030 cm−1. The bands < 1000 cm−1 can be assigned to symmetric or perpendicular stretching vibrations of SieO or AleO groups. The FTIR spectrum of DHNTs inherits the all characteristic peaks of HNTs, while the newly emerged peaks at 3023, 2934, 1705 and 1415 cm−1 can be attributed to the AreH, CeH, C]O and CeC stretching in the structure of PTX, respectively. The FTIR results indicate that the DHNTs should be a composite made by HNTs and PTX, which match well with the expected composition. TG curves of original HNTs and DHNTs are illustrated in Fig. 1B. For HNTs, the slight mass loss before 200 °C can be attributed to the adsorbed water existing in the surface, cavity or inter-layers of HNTs. The distinct mass loss ranging from 400 to 550 °C can be attributed to the presence of a small number of organic components and the dehydration effect. The TG curve of DHNTs shows a distinct difference from HNTs, in which a dramatically decrease can be clearly observed from 100 to 300 °C. The differential curves are also plotted based on the TG curves. The peak at 210.9 °C on the differential curve of DHNTs is in consistent with the inherent characteristic of PTX. The result matches well with the expected composition and FTIR analyses. The drug loading degree (the mass ratio of loaded PTX on the pure nanotubes) was calculated as 17.0% based on the residual masses at 800 °C of HNTs and DHNTs. To verify the accuracy of the calculation method based on the TG analysis, the drug loading degree was also measured by immersing DHNTs in aqueous medium for 48 h followed by the concentration was 4
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Fig. 2. TEM images of HNTs (A, B & C) and DHNTs (D, E & F).
non-HNTs contained drug-loaded hydrogel was prepared by adding PA into the PVA solution containing equivalent PTX to give a transparent yellow hydrogel (PTX@PVA@PA) (shown in Fig. 3D). The rheological characterizations were conducted and the detailed results were shown in supporting information. The storage modulus (G') is obviously larger than the loss modulus (G'') for PTX@PVA@PA and DHNTs@PVA@PA, which proves that the gels have been successfully prepared. The G' of DHNTs@PVA@PA is greater than that of PTX@PVA@PA, suggesting the addition of HNTs in the three-dimensional network can effectively improve the strength of hydrogels. Then the products were characterized by FTIR and SEM after freezedrying. The FTIR spectrum of PTX@PVA@PA depicted on Fig. S5 in Supporting Information features three distinct peaks at 3400, 2917 and 1450 cm−1. The vibrations centred at 3400 cm−1 can be attributed the presence of hydroxyl groups in the PVA. The peak located at 2917 and 1450 cm−1 is corresponding to the and CeC stretching. As for DHNTs@ PVA@PA, the aboved mentioned vibrations can also be detected, while the distinct peaks at 3701 and 3624 cm−1 demonstrate the presence of HNTs in the network. The SEM observation at the magnification of 1 × 104 times shows the typical morphology of the prepared hydrogels (Fig. 3A & B). The polymer fibres are found to possess curly morphology and arc-like endings in Fig. 3A. As for the SEM image of DHNTs@PVA@PA depicted in Fig. 3B, some tubular substances can be clearly found with high density, which show straight morphology and sharp edges. The tubular substances show obvious different morphologies from those of the polymer fibres in Fig. 3A. In addition, the lengths of some tubular substances are measured and given in Fig. 3B, the lengths and morphologies of the tubular substances matches well with those of HNTs. Therefore, tubular substances might be ascribed to the DHNTs (Fig. 3C) inserted in the hydrogel matrix. The SEM images of PTX@ PVA@PA and DHNTs@PVA@PA at low magnification (×25) were shown in Supporting Information. The rough surface can be found in both cases. In as-prepared hydrogels, the installation of boronic ester groups at the 3′ and 6′ positions of the xanthenone scaffold would force this platform to adopt a closed, colorless, and non-fluorescent lactone form. As a result, PA unit shows a very low fluorescence quantum yield (Φ < 0.10). The addition of H2O2 would break the B-C link and trigger the conversion of boronates to phenols and thereby result in the formation of fluorescein which exhibit a much higher fluorescence quantum yield (Φ = ca.0.94, Fig. 4A). The chemospecific boronate-tophenol switch triggers a dramatically enhancement of the fluorescence intensity as the addition of H2O2 to the as-prepared hydrogel (Fig. 4B). This phenomenon may be quite helpful to trace the H2O2-responsive
The XPS spectra (Fig. 1D) of original HNTs, HNTs@PTX and DHNTs show the presence of aluminum (Al 2s and Al 2p) and silicon (Si 3p), in accordance with the composition of aluminosilicate clay. The weak signal in the curve of original HNTs suggests that some organic component exists in the multilayer nanotubes. As for HNTs@PTX, it should be noted that the weak peak of PTX assigned to N 1s can be clearly observed at 400.5 eV with the atomic % of 11.7%, while it is difficult to find the absence of N 1s peak in the curve of DHNTs. The difference can be owing to the penetration depth of X-ray (10 nm), which is lower than that of the thickness of tube wall. When the drug loaded in the cavity of HNTs, the associated signals assigned to the drug are difficult to be detected. The XPS results, accompanying with the XRD patterns, suggest that the drug may be mainly loaded into the cavity rather than attached on the external surface of HNTs. TEM images of original HNTs and DHNTs are shown in Fig. 2. The images in Fig. 2A, B & C reveal that the original HNTs are cylindricalshaped with an open-ended lumen along the nanotubes. The nanotubes have an outer diameter of ca.100 nm and a lumen diameter of ca.30 nm, while the wall thickness is about 20 nm. The distinct borders in the lumen can be clearly seen from the images, which matches well with the inherent characteristic of original HNTs. As for the images shown in Fig. 2D, E & F, the external surface of DHNTs retains the smooth morphology, while the borders became indistinct. This phonomenon might be attributed to the fillness of the drug into the lumen of the HNTs. The coprecipitation method proposed in this study can feasibly load the model drug into the cavity of HNTs in a green and low-cost manner. Following this way, the adhesion of the drug on the external surface of HNTs can be effectively avoided, which is quite important for achieving controllable drug delivery behavior and reducing burst release. In addition, the adhesion of the drug on the external surface will change the surface charge and thereby damage the stable dispersity in aqueous medium. Moreover, the prepared DHNTs also shows a good dispersity in water (details shown in Supporting Information) which is quite helpful to construct uniform hydrogels. 3.2. Preparation of hydrogels A fluorescein derivative (PA) bearing two arylboronic acid groups was synthesized by a simple deprotection reaction. Arylboronic acid is able to react with diols promptly, which makes PA a potential crosslinking agent to prepare chemical hydrogels. As PA added into the PVA solution containing certain amount of DHNTs, the solution mixture became stiff rapidly, and the self-standing hydrogel (DHNTs@PVA@ PA) was formed within ca.30 min as depicted in Fig. 3D. In addition, a 5
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Fig. 3. Morphology characterizations: A) SEM image of PTX@PVA@PA; B) SEM image of DHNTs@PVA@PA (“D” is abbreviated for diameter); C) general view of DHNTs; D) photographs of prepared hydrogels (i: DHNTs@PVA@PA; ii: PTX@PVA@PA).
matrix of PTX@PVA@PA directly without the using of HNTs, the drugs dispersing in the surface of hydrogel are capable of releasing into the medium directly resulting in a serious initial burst effect. For DHNTs@ PVA@PA, the drugs were loaded into the cavity of HNTs before the formation of hydrogels, which can effectively suppress the initial burst effect. The little burst release of DHNTs@PVA@PA may be owing to that some drug might escape from the lumen before the formation of gels. The release behavior of DHNTs@PVA@PA and PTX@PVA@PA with H2O2 in high level ([H2O2] = 200 μM) was then evaluated in PBS solution as shown in Fig. 4C. The significant increase of the release rates can be observed as compared to those in the medium with H2O2 in low level, which can be owing to the degradation of the hydrogels triggered by H2O2. We have used the Korsmeyer–Peppas model to evaluate the release data between 0 and 60% for DHNTs@PVA@PA, in which the
drug release behavior by visual sense.
3.3. In vitro drug release behavior As the concentration of H2O2 in normal tissues maintains ca.10−8 μM, while a thousand-fold increase can be found in inflammation tissues or tumors. The drug release behavior was measured in PBS solution with different concentration of H2O2 (0.02 μM and 200 μM) to investigate the H2O2-responsive release characters. The release behaviors were summarized in Fig. 4C. A very small amount of PTX was released (< 5%) from DHNTs@ PVA@PA under the medium with H2O2 in low level, while the 4-fold increased initial-burst amount can be tracked for PTX@PVA@PA. The distinct difference for the initial burst effect can be owing to the introduction of HNTs in the matrix. When drugs were loaded in the
Fig. 4. A) The transformation mechanism from arylboronates to phenols to afford fluorescein with high fluorescence in the presence of H2O2; B) the changes of DHNTs@PVA@PA from non-fluorescent to fluorescent; C) the drug release profiles in different concertation of H2O2; D) the fluorescence spectra of the release medium (H2O2 = 200 μM) after addition of DHNTs@PVA@PA; E) plots of fluorescence intensity vs. the drug release rate. 6
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Fig. 5. H2O2-responsive release mechanism of DHNTs@PVA@PA.
Acknowledgements
correlation coefficient was calculated as R = 0.992. The diffusion component (n = 0.756) was found in the range of 0.45 ≤ n ≤ 0.89 (for cylinder shape geometry), meaning that the release of the drug from the hydrogel matched well with the non Fickian type (the synergistic effect of swelling control and diffusion control) [62]. The B–C linkage in the obtained hydrogels can be degraded into B–OH and C–OH groups in the presence of H2O2 and thereby cause the degradation of the hydrogels. The detailed mechanism is summarized in Fig. 5. The relationship between fluorescence intensity with the drug release behavior of DHNTs@PVA@PA is carefully investigated. Fig. 4D shows the changes in fluorescence intensity of the release medium over time. A remarkable increase of the intensity is observed from 5 to 40 min which can be owing to the transformation from arylboronates to phenols. The continuously generated fluorescein triggers the significant enhancement on fluorescence intensity. Fig. 4E shows the relationship between the release rate and fluorescence intensity. The plots were fitted linearly, in which the correlation index (R) was calculated as 0.983. The good relationship may be quite useful to trace the pathological changes and drug release behavior invisibly. Moreover, the visible release behavior can also be used to remind the patient to exchange the hydrogels in time when DHNTs@PVA@PA serving as external preparations.
This work was supported by the Foundation of Hebei Education Department (No. QN2018052) and the Natural Science Foundation of Hebei Province (No. B2019201138 and H2018201289). The authors also thank the Post-Graduate’s Innovation Fund Project of Hebei Province (No. CXZZSS2019003). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cej.2019.122474. References [1] S. Rhee, H2O2, a necessary evil for cell signaling, Science 312 (2006) 1882–1883. [2] H.J. Kwon, D. Kim, K. Seo, Y.G. Kim, S.I. Han, T. Kang, M. Soh, T. Hyeon, Ceria nanoparticle systems for selective scavenging of mitochondrial, intracellular, and extracellular reactive oxygen species in Parkinson's disease, Angew. Chem. Int. Ed. 57 (2018) 9408–9412. [3] Y.Q. Wang, L.L. Li, W.B. Zhao, Y. Dou, H.J. An, H. Tao, X.Q. Xu, Y. Jia, S. Lu, J.X. Zhang, H.Y. Hu, Targeted therapy of atherosclerosis by a broad-spectrum reactive oxygen species scavenging nanoparticle with intrinsic anti-inflammatory activity, ACS Nano 12 (2018) 8943–8960. [4] X. Bao, J. Zhao, J. Sun, M. Hu, X. Yang, Polydopamine nanoparticles as efficient scavengers for reactive oxygen species in periodontal disease, ACS Nano 12 (2018) 8882–8892. [5] H. Sies, Role of metabolic h2o2 generation: Redox signaling and oxidative stress, J. Biol. Chem. 289 (2014) 8735–8741. [6] C.R. Powell, K.M. Dillon, Y. Wang, R.J. Carrazzone, J.B. Matson, A persulfide donor responsive to reactive oxygen species: Insights into reactivity and therapeutic potential, Angew. Chem. Int. Ed. 57 (2018) 6324–6328. [7] L. Xu, Y. Yang, M. Zhao, W. Gao, H. Zhang, S. Li, B. He, Y. Pu, A reactive oxygen species-responsive prodrug micelle with efficient cellular uptake and excellent bioavailability, J. Mater. Chem. B 6 (2018) 1076–1084. [8] Y. Li, H. Bai, H. Wang, Y. Shen, G. Tang, Y. Ping, Reactive oxygen species (ros)responsive nanomedicine for rnai-based cancer therapy, Nanoscale 10 (2018) 203–214. [9] G. Chen, H. Deng, X. Song, M. Lu, L. Zhao, S. Xia, G. You, J. Zhao, Y. Zhang, A. Dong, H. Zhou, Reactive oxygen species-responsive polymeric nanoparticles for alleviating sepsis-induced acute liver injury in mice, Biomaterials 144 (2017) 30–41. [10] H. Ding, Y. Cai, L. Gao, M. Liang, B. Miao, H. Wu, Y. Liu, N. Xie, A. Tang, K. Fan, X. Yan, G. Nie, Exosome-like nanozyme vesicles for h2o2-responsive catalytic photoacoustic imaging of xenograft nasopharyngeal carcinoma, Nano Lett. 19 (2019) 203–209. [11] Y.X. Nan, W.J. Zhao, N.B. Li, Z.W. Liang, X.H. Xu, Chemiluminescence-triggered fluorophore release: approach for in vivo fluorescence imaging of hydrogen peroxide, Sens. Actuator B-Chem. 281 (2019) 296–302. [12] Y. Cheng, J. Dai, C.L. Sun, R. Liu, T.Y. Zhai, X.D. Lou, F. Xia, An intracellular h2o2responsive aiegen for the peroxidase-mediated selective imaging and inhibition of inflammatory cells, Angew. Chem. Int. Ed. 57 (2018) 3123–3127. [13] Y.X. Liu, Q. Jia, Q.W. Guo, W. Wei, J. Zhou, Simultaneously activating highly
4. Conclusion In summary, the halloysite-based hydrogel with a “turn-on” fluorescence character upon H2O2 was facilely prepared to construct the H2O2-responsive drug delivery system by the introduction of the fluorescein derivative bearing two arylboronic acid groups, in which a coprecipitation method was proposed to afford the drug-loading HNTs. The as-formed hydrogel can be degraded in the presence of H2O2 at pathological concertation, in which the “initial burst effect” was effectively suppressed. Moreover, a good linear relationship was achieved between the release rate and fluorescence intensity. This approach provides a promising opportunity to achieve a new generation of H2O2responsive preparations with visible fluorescence changes to trace the inherent release behavior.
Declaration of Competing Interest There are no conflicts to declare.
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Halloysite nanotube-based H2O2-responsive drug delivery system with a turn on effect on fluorescence for real-time monitoring ⁎
Cong Chenga, Yan Gaoa,b, , Weihua Songb, Qiang Zhaoa, Haisong Zhangb, Hailei Zhanga, a b
T
⁎
College of Chemistry & Environmental Science, Hebei University, Baoding 071002, PR China Affiliated Hospital of Hebei University, Baoding 071000, PR China
H I GH L IG H T S
G R A P H I C A L A B S T R A C T
halloysite-based hydrogel • Awasintelligent prepared with a H O -responsive 2
• •
2
release character. A coprecipitation method was proposed to load the model drug into the cavity of HNTs. The fluorescence intensity enhances with the increase of the drug release rate.
A R T I C LE I N FO
A B S T R A C T
Keywords: Halloysite nanotube Clay science H2O2-responsive Drug loading
In this paper, a novel halloysite-based hydrogel with a “turn-on” fluorescence character upon H2O2 was facilely prepared and used to construct the H2O2-responsive drug delivery system, in which a coprecipitation method was proposed to afford the drug-loaded halloysite nanotubes (DHNTs). DHNTs were carefully characterized by FTIR, TGA, XPS, XRD and TEM to demonstrate that the drugs were mainly loaded into the cavity rather than attached on the external surface of halloysite nanotubes (HNTs). The B-C linkage in the as-prepared hydrogel was broken in the presence of H2O2, resulting in the degradation and thereby a responsive release. The drug release was almost not occurred under the physiological concentration ([H2O2] = 0.02 μM), while a complete release (> 90%) can be achieved under pathological concentration ([H2O2] = 200 μM). Moreover, the broken of B-C linkage triggered a transformation from arylboronates to phenols, in which the formed fluorescein gave rise to the change from non-fluorescent to fluorescent of the hydrogels. The fluorescence intensity enhanced with the increase of release rate, in which a good linear relationship can be achieved. The attractive properties make the halloysite-based hydrogels a promising application in the field of biomedicine.
1. Introduction Hydrogen peroxide (H2O2), one of reactive oxygen species (ROS), plays a fundamental role in the regulation of many physiological processes in living organism, especially in cellular signal transduction,
differentiation and proliferation [1]. The overexpression of H2O2 is viewed as a marker for oxidative stress and damage events in vivo, and thereby associated with the diseases including inflammation, hemorrhage, Alzheimer's disease, renal hypofunction and tumors [2–4]. For example, the concentration of H2O2 in normal tissues maintains
⁎ Corresponding authors at: No.180 Wusidong Road, College of Chemistry & Environmental Science, Hebei University, Baoding, Hebei Province 071002, PR China (H. Zhang) and No. 212 Yuhua Road, Affiliated Hospital of Hebei University, Baoding, Hebei Province 071000, PR China (Y. Gao). E-mail addresses: [email protected] (Y. Gao), [email protected] (H. Zhang).
https://doi.org/10.1016/j.cej.2019.122474 Received 17 April 2019; Received in revised form 1 August 2019; Accepted 9 August 2019 Available online 09 August 2019 1385-8947/ © 2019 Elsevier B.V. All rights reserved.
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ca.10−8 M, while a thousand-fold increase can be found in inflammation tissues or tumors [5]. The development of new chemical tools that are sensitive enough to report H2O2 production at pathological changing level has raised a lot of interests [6–9]. In past decades, a library of H2O2-responsive materials has been developed, including catalyst [10], fluorescence probe [11,12], MRI contrast agent [13] and drug delivery system [14,15]. However, the limitations of reported H2O2-responsive drug delivery systems severely restricted its applications, such as serious initial burst effect, low compatibility, high cost and so on. Moreover, the lack of an indicator in the drug delivery system result in a difficulty to monitor the release behavior in real-time, which may bring some unexpected delays in practical clinical uses. Halloysite nanotube (HNT) is a natural aluminosilicate clay mineral, which has the same chemical composition as kaolinite (Al2Si2O5(OH)4 nH2O) [16–19]. The multilayers of hollow nanotubes are formed by curved and rolled up of the adjacent alumina and silica sheets along with their water of hydration [20,21]. Benefiting from the high dispersibility, the hollow tubular shape, the large cavity volume and the modifiable surface structure, HNTs can be used as the desirable natural nano-carriers for loaded active agents and even fabrication of nanocomposites [22–30]. The attractive properties including biocompatibility, nontoxicity, non-degradation, hydrophilic and low-cost also make HNTs promising materials in the field of biochemistry and biomedicine [31–35]. Lvov’s group pioneered the HNTs-based drug delivery system in which the sustained-release character can be facilely achieved [36,37]. Liu et al enhanced the transmembrane transport ability of HNTs-based drug delivery system by lower the size of HNTs [38]. Lazzara and coworkers developed a library of HNTs-based drug delivery systems with pH- or thermo- sensitive release behaviors [39,40]. Das’ group developed a library of clay-based carriers, including the HNTs-based drug loading system with fine tuning in drug release behaviors, in which the kinetics studies were emphasized [41–44]. Zhang et al explored the HNTs-based anti-bacteria materials [45]. Our groups also devoted our efforts to prepare HNTs-based H2O2-responsive fluorescence probes and drug delivery system, in which the “burst effect” can be effectively suppressed [46–48]. It will be very interesting to further develop a more intelligent H2O2-responsive materials that the drug release can be monitored in real-time by the fluorescence changes, which can effectively enhance the efficiency in diagnosis and treatment. Hydrogels emerged as excellent candidates for controlled release and stimulate-responsive drug delivery as they are able to encapsulate nanoparticles well as biomacromolecules and small molecule drugs [49–51]. The loading of guest molecule into the cavity of nanotubes ahead of the formation of hydrogels can effectively reduce the burst release effect, while the attachment of guest molecules on the exterior surface may result in a low controllability on the release behavior. In this study, a coprecipitation method was proposed to load the model drug into the cavity of HNTs facilely, where the adhesion of the drug on the external surface of HNTs can be effectively avoided. Our strategy for achieving the H2O2-response characters relies on the H2O2mediated transformation of arylboronates to phenols. Boronic acid groups were installed at the 3′ and 6′-positions of a xanthenone scaffold in fluorescein. The obtained fluorescein derivative (PA) bearing two arylboronic acid groups can rapidly react with diols to form boronic ester groups in mild conditions, which can serve as cross-linking agent to crosslink multi-hydroxyl polymers and thereby affording hydrogels with H2O2-sensitivity. The installation at the 3′ and 6′ positions of a xanthenone scaffold in fluorescein would force this platform to nonfluorescent lactone form, which gave rise to non-fluorescent hydrogels. After treating with H2O2, the transformation of arylboronates to phenols resulted in the formation of fluorescein which triggers a prompt fluorescence increase. Following this way, the intelligent hydrogels can be facilely afforded by the addition of PA into the above polyvinyl alcohol (PVA) solution containing drug-loaded HNTs (DHNTs). The H2O2-responsive cleavage of the formed B-C link in the hydrogels can be achieved in a high concentration of H2O2 and thereby result in a
H2O2-responsive drug release behavior. Little release can be detected when [H2O2] = 0.02 μM suggesting a desirable control on the initial burst effect, while a rapid and complete release can be achieved in a concentration of H2O2 at 200 μM. The fluorescence intensity enhanced with the increase of release rate, which shows a good linear relationship to the drug release behavior. 2. Material and methods 2.1. Materials HNTs were obtained from GuangZhouShinshi Metallurgy and Chemical Company Ltd (Guangzhou, China) and purified according to our previous work [52]. Fluorescein and other reactants were obtained from J&K Scientific Ltd. Pentoxifylline (PTX) was obtained from TCI Development Co., Ltd. PVA and 30% H2O2 solution were purchased from Kemiou Chemical Reagent Co., Ltd. Dimethyl sulfoxide (DMSO) was dried and distilled from CaH2 under vacuum before use. Distilled water was used throughout the study. High-purity argon was used for degassing procedures. PF1 was synthesized according to the reference [53]. 2.2. Syntheses and preparations 2.2.1. Synthesis of (3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-3′,6′diyl) diboronic acid (PA) PF1 (940 mg, 1.7 mmol) and diethanolamine (400 mg, 3.8 mmol) were dissolved into an ethyl ether solution and then stirred at room temperature for about 50 min. The formed precipitate was filtered and washed with ethyl ether. After vacuum drying, the obtained brown powders were immersed in 0.1 M HCl. After ultrasound about 30 min, the yellow precipitation was formed by centrifugation and washed by 0.1 M HCl to give the product PA (435 mg, 66%) (Scheme 1). 1H NMR (600 MHz, (CD3)2SO) δ 8.06 (d, J = 7.8 Hz, 1H), 7.87 – 7.76 (m, 3H), 7.75 (t, J = 7.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.28 (d, J = 7.8 Hz, 1H), 6.80 (d, J = 7.8 Hz, 2H). 13C NMR (100 MHz, (CD3)2SO) δ 169.21, 153.43, 150.38, 138.34, 136.34, 130.81, 129.78, 127.19, 125.58, 125.41, 124.38, 122.88, 120.35, 81.98. FTIR (KBr): 3416, 1740, 1412, 1342, 1075, 925, 704 . 2.2.2. Preparation of drug loaded HNTs (DHNTs) 100 mg of HNTs was dispersed into a saturated solution of PTX in DMSO. The suspension was placed in a vacuum chamber by pumping and filling in with N2 for several times. The product was precipitated into excess amount of acetone washed for three times. The residue was collected by a fast certification process and then dried in vacuum to give DHNTs as white powders (drug loading degree: 17.0%). 2.2.3. Preparation of the physical mixture of HNTs and PTX (HNTs@PTX) 17 mg of PTX was dissolved in 10 mL anhydrous ethyl alcohol followed by 100 mg of HNTs were added. The mixture was stirred to achieve a uniform suspension and the evaporated under atmosphere pressure to afforded a solid residue. The residue was collected, grinded, and dried to give HNTs@PTX as faint yellow powders (drug loading degree: 17.0%).
Scheme 1. Synthesis of PA. 2
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Scheme 2. Preparation of PTX@PVA@PA hydrogel.
instrument (PerkinElmer, USA) operated under 20 mL/min nitrogen flow rate in which the heating rate was 10 °C/min. UV–Vis absorption and fluorescence spectra were obtained on a UV-2550 UV–visible spectrometer (Shimadzu, Japan) and a RF-5301PC fluorospectro photometer (Shimadzu, Japan). X-ray diffraction (XRD) patterns were performed at room temperature with a D8 ADVANCE X-ray powder diffractometer system (Bruker, Faellanden, Switzerland). The patterns were recorded on a quartz plate at a tube voltage of 40 kV and a current of 40 mA in a 2θ range of 10-90° using a step size 0.06° at a scan speed of 1 s/step.
2.2.4. Preparation of PTX@PVA@PA hydrogels Polyvinyl alcohol (200 mg) was dissolved in 2.0 mL of distilled water at 80 °C. 5.0 mg of PTX was dispersed in 2.0 mL of aqueous solution of PA, and then added carefully into the above aqueous solution. The system was stirring for about 3 min and incubated at 80 °C for 0.5 h to afford a unique hydrogel (PTX@PVA@PA) with yellow colour. The obtained hydrogel was stored at −20 °C before use (drug loading degree: 0.2%) (see Scheme 2). 2.2.5. Preparation of DHNTs@PVA@PA hydrogels Polyvinyl alcohol (200 mg) was dissolved in 2.0 mL distilled water at 80 °C. A certain amount of DHNTs containing 5 mg PTX was dispersed in 2.0 mL of aqueous solution of PA, and then quickly added into the above aqueous solution. The system was stirring for about 3 min and incubated at 80 °C for 0.5 h to afford a unique faint yellow hydrogel (DHNTs@PVA@PA). The obtained hydrogel was stored at −20 °C before use (drug loading degree: 0.2%) (see Scheme 3)
2.4. In vitro drug delivery Drug delivery studies were carried out using a ZRS-8G dissolution tester (Haiyida, China). The paddle rotation speed was set as 50 rpm at 37 ± 0.5 °C. The drug release behaviors were evaluated by immersing the samples into pH 7.4 phosphate buffer solution (PBS) with H2O2 in different concertation ([H2O2] = 200 μM and 0.02 μM) H2O2. The samples were filtered through a membrane filter (pore size 0.45 μm) before monitored. The filtrates were collected for further test.
2.3. Characterizations The Tecnai G2 F20 S-TWIN transmission electron microscope (TEM) was used to characterize the morphology of HNTs and DHNTs with an accelerating voltage of 200 kV. The morphological characterisations of the hydrogels were conducted on a JEOL Ltd. JSM-7500F Cryo field emission scanning electron microscopy (SEM). The lyophilized samples were placed on flat substrates and coated with gold for SEM observations. FTIR spectra were performed in the region of 4000–400 cm−1 for each sample on a Varian-640 spectrophotometer. Samples were previously ground and mixed thoroughly with spectrum pure KBr. The spectrum for each sample was obtained from averaging 32 scans over the selected wave number range. The scan rate is 5 kHz. 1H and 13C NMR spectra were recorded on a Bruker AVANCE-600 600 MHz spectrometer (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) was conducted on a Thermo Scientific ESCALab 250Xi using 200 W monochromated Al Ka radiation, in which 500 μm X-ray spot and 3 × 10−10 mbar base pressure were used. The hydrocarbon C1s line at 284.8 eV from adventitious carbon was used for energy referencing. Thermogravimetric analysis (TGA) was recorded by a Pyris1 TGA
3. Results and discussion 3.1. Drug loading in HNTs A coprecipitation method was used to load the model drug (PTX) into the cavity of HNTs. Briefly, the purified HNTs were immersed into saturated aqueous solution of PTX. The suspension was carefully degassed to fill the drug solution into the cavity of the tubes and then precipitated into acetone which can satisfy the solubility of PTX. It should be noted that vacuum pumping step is essential for achieving a desirable loading rate. The vapor pressure may be beneficial to evaporate the inherent water and empty the gas inside the lumen of HNTs, in which the molecules dissolved in the fresh aqueous solution can migrate to the halloysite lumen [54]. Owing to the poor dispersibility of HNTs in acetone, the drug loaded in the lumen was coprecipitated accompanying with the nanotubes. Precipitates were collected by centrifugation in a very short time in case the drug released from the
Scheme 3. Preparation of DHNTs@PVA@PA hydrogel. 3
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Fig. 1. Characterizations of HNTs, PTX, DHNTs, PTX@PVA@PA, DHNTs@PVA@PA (A: FTIR spectra; B: TGA and normalized differential curves; C: XRD patterns; D: XPS spectra).
determined based on Lambert-Beer laws. The calculated drug loading degree is in accordance with that given by TG analysis. A section of the medium was collected after certifying and then freeze-dried for 1H NMR analysis (Supporting Information). The 1H NMR spectrum shows a high purity and the results match well with the structure information of PTX, implying the drug retained a high chemical stability in the drug loading process based on the coprecipitation method. We have tried to increase the drug loading degree by changing the conditions, including extending the time in low pressure and increasing the temperature. We find that 17% is the highest drug loading degree for this approach. This value is slightly lower than those in literatures [55,56], which may be due to that part of the loaded molecules located near the opening of the tubes were dissolved in the precipitation process. Nevertheless, we focus on developing a facile loading approach to avoid the absorption of external surface rather than increasing the loading degree. The drug loading degree of 17% is sufficient for us to process the latter investigations. The XRD and XPS characterizations were carefully investigated to reveal that the drug was loaded in the lumen, rather than attached on the external surface. In addition, a physical mixture of HNTs and PTX (HNTs@PTX) was prepared by a simple grinding method. The XRD patterns of HNTs, DHNTs and HNTs@PTX were shown in Fig. 1C. The XRD pattern of HNTs features a diffraction peaks at ca. 11.9°, corresponding to a basal spacing of layer spaces. Other diffraction signals at ca 20.0°, 24.6°, 35.1°, 38.2°, 54.9° and 62.3° can also be observed, which are in good agreement with the literatures [57–59]. The diffraction peak of PTX can be clearly seen at 15°, 24.1° and 27.9° in the XRD pattern of HNTs@PTX, while the characteristic diffraction peaks assigned to PTX are difficult to be detected in the pattern of DHNTs. The wall thickness of HNTs is estimated as 20 nm, which well exceeds the penetration depth of X-ray (10 nm) [60,61]. Thus, it is difficult to disclose the chemical composition in the cavity of HNTs. The XRD results suggest that the PTX should be loaded in the lumen of the nanotubes.
cavity. The drug-loaded HNTs (DHNTs) were obtained after drying in vacuum. FTIR and TGA were performed to reveal the composition of the obtained composites. The FTIR spectrum of original HNTs depicted on Fig. 1A features two distinct peaks at 3717 and 3618 cm−1. Inner hydroxyl groups, lying between the tetrahedral and octahedral sheets, give the absorption near 3618 cm−1. The other strong band at 3717 cm−1 is associated with surface hydroxyl groups in the lumen of HNTs. Characteristic band of in-plane Si–O–Si stretching vibration is observed around 1030 cm−1. The bands < 1000 cm−1 can be assigned to symmetric or perpendicular stretching vibrations of SieO or AleO groups. The FTIR spectrum of DHNTs inherits the all characteristic peaks of HNTs, while the newly emerged peaks at 3023, 2934, 1705 and 1415 cm−1 can be attributed to the AreH, CeH, C]O and CeC stretching in the structure of PTX, respectively. The FTIR results indicate that the DHNTs should be a composite made by HNTs and PTX, which match well with the expected composition. TG curves of original HNTs and DHNTs are illustrated in Fig. 1B. For HNTs, the slight mass loss before 200 °C can be attributed to the adsorbed water existing in the surface, cavity or inter-layers of HNTs. The distinct mass loss ranging from 400 to 550 °C can be attributed to the presence of a small number of organic components and the dehydration effect. The TG curve of DHNTs shows a distinct difference from HNTs, in which a dramatically decrease can be clearly observed from 100 to 300 °C. The differential curves are also plotted based on the TG curves. The peak at 210.9 °C on the differential curve of DHNTs is in consistent with the inherent characteristic of PTX. The result matches well with the expected composition and FTIR analyses. The drug loading degree (the mass ratio of loaded PTX on the pure nanotubes) was calculated as 17.0% based on the residual masses at 800 °C of HNTs and DHNTs. To verify the accuracy of the calculation method based on the TG analysis, the drug loading degree was also measured by immersing DHNTs in aqueous medium for 48 h followed by the concentration was 4
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Fig. 2. TEM images of HNTs (A, B & C) and DHNTs (D, E & F).
non-HNTs contained drug-loaded hydrogel was prepared by adding PA into the PVA solution containing equivalent PTX to give a transparent yellow hydrogel (PTX@PVA@PA) (shown in Fig. 3D). The rheological characterizations were conducted and the detailed results were shown in supporting information. The storage modulus (G') is obviously larger than the loss modulus (G'') for PTX@PVA@PA and DHNTs@PVA@PA, which proves that the gels have been successfully prepared. The G' of DHNTs@PVA@PA is greater than that of PTX@PVA@PA, suggesting the addition of HNTs in the three-dimensional network can effectively improve the strength of hydrogels. Then the products were characterized by FTIR and SEM after freezedrying. The FTIR spectrum of PTX@PVA@PA depicted on Fig. S5 in Supporting Information features three distinct peaks at 3400, 2917 and 1450 cm−1. The vibrations centred at 3400 cm−1 can be attributed the presence of hydroxyl groups in the PVA. The peak located at 2917 and 1450 cm−1 is corresponding to the and CeC stretching. As for DHNTs@ PVA@PA, the aboved mentioned vibrations can also be detected, while the distinct peaks at 3701 and 3624 cm−1 demonstrate the presence of HNTs in the network. The SEM observation at the magnification of 1 × 104 times shows the typical morphology of the prepared hydrogels (Fig. 3A & B). The polymer fibres are found to possess curly morphology and arc-like endings in Fig. 3A. As for the SEM image of DHNTs@PVA@PA depicted in Fig. 3B, some tubular substances can be clearly found with high density, which show straight morphology and sharp edges. The tubular substances show obvious different morphologies from those of the polymer fibres in Fig. 3A. In addition, the lengths of some tubular substances are measured and given in Fig. 3B, the lengths and morphologies of the tubular substances matches well with those of HNTs. Therefore, tubular substances might be ascribed to the DHNTs (Fig. 3C) inserted in the hydrogel matrix. The SEM images of PTX@ PVA@PA and DHNTs@PVA@PA at low magnification (×25) were shown in Supporting Information. The rough surface can be found in both cases. In as-prepared hydrogels, the installation of boronic ester groups at the 3′ and 6′ positions of the xanthenone scaffold would force this platform to adopt a closed, colorless, and non-fluorescent lactone form. As a result, PA unit shows a very low fluorescence quantum yield (Φ < 0.10). The addition of H2O2 would break the B-C link and trigger the conversion of boronates to phenols and thereby result in the formation of fluorescein which exhibit a much higher fluorescence quantum yield (Φ = ca.0.94, Fig. 4A). The chemospecific boronate-tophenol switch triggers a dramatically enhancement of the fluorescence intensity as the addition of H2O2 to the as-prepared hydrogel (Fig. 4B). This phenomenon may be quite helpful to trace the H2O2-responsive
The XPS spectra (Fig. 1D) of original HNTs, HNTs@PTX and DHNTs show the presence of aluminum (Al 2s and Al 2p) and silicon (Si 3p), in accordance with the composition of aluminosilicate clay. The weak signal in the curve of original HNTs suggests that some organic component exists in the multilayer nanotubes. As for HNTs@PTX, it should be noted that the weak peak of PTX assigned to N 1s can be clearly observed at 400.5 eV with the atomic % of 11.7%, while it is difficult to find the absence of N 1s peak in the curve of DHNTs. The difference can be owing to the penetration depth of X-ray (10 nm), which is lower than that of the thickness of tube wall. When the drug loaded in the cavity of HNTs, the associated signals assigned to the drug are difficult to be detected. The XPS results, accompanying with the XRD patterns, suggest that the drug may be mainly loaded into the cavity rather than attached on the external surface of HNTs. TEM images of original HNTs and DHNTs are shown in Fig. 2. The images in Fig. 2A, B & C reveal that the original HNTs are cylindricalshaped with an open-ended lumen along the nanotubes. The nanotubes have an outer diameter of ca.100 nm and a lumen diameter of ca.30 nm, while the wall thickness is about 20 nm. The distinct borders in the lumen can be clearly seen from the images, which matches well with the inherent characteristic of original HNTs. As for the images shown in Fig. 2D, E & F, the external surface of DHNTs retains the smooth morphology, while the borders became indistinct. This phonomenon might be attributed to the fillness of the drug into the lumen of the HNTs. The coprecipitation method proposed in this study can feasibly load the model drug into the cavity of HNTs in a green and low-cost manner. Following this way, the adhesion of the drug on the external surface of HNTs can be effectively avoided, which is quite important for achieving controllable drug delivery behavior and reducing burst release. In addition, the adhesion of the drug on the external surface will change the surface charge and thereby damage the stable dispersity in aqueous medium. Moreover, the prepared DHNTs also shows a good dispersity in water (details shown in Supporting Information) which is quite helpful to construct uniform hydrogels. 3.2. Preparation of hydrogels A fluorescein derivative (PA) bearing two arylboronic acid groups was synthesized by a simple deprotection reaction. Arylboronic acid is able to react with diols promptly, which makes PA a potential crosslinking agent to prepare chemical hydrogels. As PA added into the PVA solution containing certain amount of DHNTs, the solution mixture became stiff rapidly, and the self-standing hydrogel (DHNTs@PVA@ PA) was formed within ca.30 min as depicted in Fig. 3D. In addition, a 5
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Fig. 3. Morphology characterizations: A) SEM image of PTX@PVA@PA; B) SEM image of DHNTs@PVA@PA (“D” is abbreviated for diameter); C) general view of DHNTs; D) photographs of prepared hydrogels (i: DHNTs@PVA@PA; ii: PTX@PVA@PA).
matrix of PTX@PVA@PA directly without the using of HNTs, the drugs dispersing in the surface of hydrogel are capable of releasing into the medium directly resulting in a serious initial burst effect. For DHNTs@ PVA@PA, the drugs were loaded into the cavity of HNTs before the formation of hydrogels, which can effectively suppress the initial burst effect. The little burst release of DHNTs@PVA@PA may be owing to that some drug might escape from the lumen before the formation of gels. The release behavior of DHNTs@PVA@PA and PTX@PVA@PA with H2O2 in high level ([H2O2] = 200 μM) was then evaluated in PBS solution as shown in Fig. 4C. The significant increase of the release rates can be observed as compared to those in the medium with H2O2 in low level, which can be owing to the degradation of the hydrogels triggered by H2O2. We have used the Korsmeyer–Peppas model to evaluate the release data between 0 and 60% for DHNTs@PVA@PA, in which the
drug release behavior by visual sense.
3.3. In vitro drug release behavior As the concentration of H2O2 in normal tissues maintains ca.10−8 μM, while a thousand-fold increase can be found in inflammation tissues or tumors. The drug release behavior was measured in PBS solution with different concentration of H2O2 (0.02 μM and 200 μM) to investigate the H2O2-responsive release characters. The release behaviors were summarized in Fig. 4C. A very small amount of PTX was released (< 5%) from DHNTs@ PVA@PA under the medium with H2O2 in low level, while the 4-fold increased initial-burst amount can be tracked for PTX@PVA@PA. The distinct difference for the initial burst effect can be owing to the introduction of HNTs in the matrix. When drugs were loaded in the
Fig. 4. A) The transformation mechanism from arylboronates to phenols to afford fluorescein with high fluorescence in the presence of H2O2; B) the changes of DHNTs@PVA@PA from non-fluorescent to fluorescent; C) the drug release profiles in different concertation of H2O2; D) the fluorescence spectra of the release medium (H2O2 = 200 μM) after addition of DHNTs@PVA@PA; E) plots of fluorescence intensity vs. the drug release rate. 6
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Fig. 5. H2O2-responsive release mechanism of DHNTs@PVA@PA.
Acknowledgements
correlation coefficient was calculated as R = 0.992. The diffusion component (n = 0.756) was found in the range of 0.45 ≤ n ≤ 0.89 (for cylinder shape geometry), meaning that the release of the drug from the hydrogel matched well with the non Fickian type (the synergistic effect of swelling control and diffusion control) [62]. The B–C linkage in the obtained hydrogels can be degraded into B–OH and C–OH groups in the presence of H2O2 and thereby cause the degradation of the hydrogels. The detailed mechanism is summarized in Fig. 5. The relationship between fluorescence intensity with the drug release behavior of DHNTs@PVA@PA is carefully investigated. Fig. 4D shows the changes in fluorescence intensity of the release medium over time. A remarkable increase of the intensity is observed from 5 to 40 min which can be owing to the transformation from arylboronates to phenols. The continuously generated fluorescein triggers the significant enhancement on fluorescence intensity. Fig. 4E shows the relationship between the release rate and fluorescence intensity. The plots were fitted linearly, in which the correlation index (R) was calculated as 0.983. The good relationship may be quite useful to trace the pathological changes and drug release behavior invisibly. Moreover, the visible release behavior can also be used to remind the patient to exchange the hydrogels in time when DHNTs@PVA@PA serving as external preparations.
This work was supported by the Foundation of Hebei Education Department (No. QN2018052) and the Natural Science Foundation of Hebei Province (No. B2019201138 and H2018201289). The authors also thank the Post-Graduate’s Innovation Fund Project of Hebei Province (No. CXZZSS2019003). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cej.2019.122474. References [1] S. Rhee, H2O2, a necessary evil for cell signaling, Science 312 (2006) 1882–1883. [2] H.J. Kwon, D. Kim, K. Seo, Y.G. Kim, S.I. Han, T. Kang, M. Soh, T. Hyeon, Ceria nanoparticle systems for selective scavenging of mitochondrial, intracellular, and extracellular reactive oxygen species in Parkinson's disease, Angew. Chem. Int. Ed. 57 (2018) 9408–9412. [3] Y.Q. Wang, L.L. Li, W.B. Zhao, Y. Dou, H.J. An, H. Tao, X.Q. Xu, Y. Jia, S. Lu, J.X. Zhang, H.Y. Hu, Targeted therapy of atherosclerosis by a broad-spectrum reactive oxygen species scavenging nanoparticle with intrinsic anti-inflammatory activity, ACS Nano 12 (2018) 8943–8960. [4] X. Bao, J. Zhao, J. Sun, M. Hu, X. Yang, Polydopamine nanoparticles as efficient scavengers for reactive oxygen species in periodontal disease, ACS Nano 12 (2018) 8882–8892. [5] H. Sies, Role of metabolic h2o2 generation: Redox signaling and oxidative stress, J. Biol. Chem. 289 (2014) 8735–8741. [6] C.R. Powell, K.M. Dillon, Y. Wang, R.J. Carrazzone, J.B. Matson, A persulfide donor responsive to reactive oxygen species: Insights into reactivity and therapeutic potential, Angew. Chem. Int. Ed. 57 (2018) 6324–6328. [7] L. Xu, Y. Yang, M. Zhao, W. Gao, H. Zhang, S. Li, B. He, Y. Pu, A reactive oxygen species-responsive prodrug micelle with efficient cellular uptake and excellent bioavailability, J. Mater. Chem. B 6 (2018) 1076–1084. [8] Y. Li, H. Bai, H. Wang, Y. Shen, G. Tang, Y. Ping, Reactive oxygen species (ros)responsive nanomedicine for rnai-based cancer therapy, Nanoscale 10 (2018) 203–214. [9] G. Chen, H. Deng, X. Song, M. Lu, L. Zhao, S. Xia, G. You, J. Zhao, Y. Zhang, A. Dong, H. Zhou, Reactive oxygen species-responsive polymeric nanoparticles for alleviating sepsis-induced acute liver injury in mice, Biomaterials 144 (2017) 30–41. [10] H. Ding, Y. Cai, L. Gao, M. Liang, B. Miao, H. Wu, Y. Liu, N. Xie, A. Tang, K. Fan, X. Yan, G. Nie, Exosome-like nanozyme vesicles for h2o2-responsive catalytic photoacoustic imaging of xenograft nasopharyngeal carcinoma, Nano Lett. 19 (2019) 203–209. [11] Y.X. Nan, W.J. Zhao, N.B. Li, Z.W. Liang, X.H. Xu, Chemiluminescence-triggered fluorophore release: approach for in vivo fluorescence imaging of hydrogen peroxide, Sens. Actuator B-Chem. 281 (2019) 296–302. [12] Y. Cheng, J. Dai, C.L. Sun, R. Liu, T.Y. Zhai, X.D. Lou, F. Xia, An intracellular h2o2responsive aiegen for the peroxidase-mediated selective imaging and inhibition of inflammatory cells, Angew. Chem. Int. Ed. 57 (2018) 3123–3127. [13] Y.X. Liu, Q. Jia, Q.W. Guo, W. Wei, J. Zhou, Simultaneously activating highly
4. Conclusion In summary, the halloysite-based hydrogel with a “turn-on” fluorescence character upon H2O2 was facilely prepared to construct the H2O2-responsive drug delivery system by the introduction of the fluorescein derivative bearing two arylboronic acid groups, in which a coprecipitation method was proposed to afford the drug-loading HNTs. The as-formed hydrogel can be degraded in the presence of H2O2 at pathological concertation, in which the “initial burst effect” was effectively suppressed. Moreover, a good linear relationship was achieved between the release rate and fluorescence intensity. This approach provides a promising opportunity to achieve a new generation of H2O2responsive preparations with visible fluorescence changes to trace the inherent release behavior.
Declaration of Competing Interest There are no conflicts to declare.
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