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O2-Loaded pH-responsive Multifunctional Nanodrug Carrier for Overcoming Hypoxia and Highly Efficient Chemo-photodynamic Cancer Therapy Zhongxi Xie, Xuechao Cai, Chunqiang Sun, Shuang Liang, Shuai Shao, Shanshan Huang, Ziyong Cheng, Maolin Pang, Bengang Xing, Abdulaziz A. Al Kheraif, and Jun Lin Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b04321 • Publication Date (Web): 16 Dec 2018 Downloaded from http://pubs.acs.org on December 17, 2018
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Chemistry of Materials
O2-loaded pH-responsive Multifunctional Nanodrug Carrier for Overcoming Hypoxia and Highly Efficient Chemophotodynamic Cancer Therapy Zhongxi Xie,†, ‡ Xuechao Cai,† Chunqiang Sun,† Shuang Liang,†, ‡ Shuai Shao,† Shanshan Huang,† Ziyong Cheng,*, †, ‡ Maolin Pang,*, †, ‡ Bengang Xing,§ Abdulaziz A. Al Kheraif,# Jun Lin,*, †, ‡, £ State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China. ‡ University of Science and Technology of China, Hefei, 230026, P. R. China. § School of Physical & Mathematical Sciences, Nanyang Technological University, Singapore. # Dental Health department College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia £ School of Applied Physics and Materials, Wuyi University, Jiangmen, Guangdong 529020, P. R. China †
ABSTRACT: Tumor therapy is facing great challenges in improving drug efficiency while reducing side effects. Herein, a novel multifunctional nanodrug carrier UC@mSiO2-RB@ZIF-O2-DOX-PEGFA (URODF) that combines oxygen (O2)-enhanced photodynamic therapy (PDT) with pH-responsive chemotherapy is presented. 808-nm NIR light irradiated NaYF4:Yb/Er@NaYbF4:Nd@NaGdF4 nanoparticles (UC) were employed as both of the upconversion/MR imaging matrix and the motivator for photosensitizer in PDT with deep penetration depth. Mesoporous silica shell (mSiO2) was used as the carrier for photosensitizer Rose Bengal (RB). Zeolitic imidazolate framework-90 (ZIF-90) was coated outside of mSiO2 as O2 reservoir to quickly release O2 in tumor microenvironment and alleviate tumor hypoxia for enhanced PDT. Doxorubicin (DOX) and NH2-PEG modified folic acid (PEGFA) were covalently conjugated on the surface of nanoparticles for synergetic therapy. The drug loading capacity reaches 5.6% and 6.0% for RB and DOX, respectively. In vitro and in vivo experiments demonstrate the great therapeutic effect of URODF. This work presents for the first time a multifunctional nanodrug carrier that combines upconversion nanoparticles with MOF structure for O2-loaded combination therapy, which might open a promising way in enhancing tumor therapeutic efficacy.
Migone et al. reported the O2 adsorption properties for ZIF8.15 Very recently, Fairen-Jimenez et al. employed highthroughput screening (HTS) techniques through GCMC simulations to explore O2 storage in a database of 2,932 existing MOFs.16 Zhang and coworkers reported the enhanced antitumor efficacy by using zirconium (IV)-based UiO-66 as an O2 carrier.17 Although most MOFs are capable of storing O2, the heavy metal ions in MOFs, such as Cu2+, Co2+ etc., largely restrict their applications in the biological field. ZIF90, which belongs to MOF family, is composed of imidazolate-2- carboxyaldehyde (ICA) ligands and tetrahedral zinc(II) centers.18 Caro and coworkers have systematically studied the gas adsorption and separation properties of ZIF-90 in detail.19-21 Attributed to the highly thermal and chemical stability, easy scale-up production and post-synthetic modification as well as the excellent biocompatibility, ZIF-90 is more suitable for biomedical applications.4, 22-24 Moreover, it is reported that ZIF-90 could decompose quickly under acidic conditions while keep stable in physiological conditions.25 Therefore, employing ZIF-90 as the drug loading system is of great interest for tumor therapy. However, there is no report about using ZIF-90 as O2 storage material for PDT until now. During the past decades, a number of photosensitizers, such as porphyrins, chlorins, bacteriochlorins and phthalocyanines, have been explored for PDT.26-29 Most of them were activated by UV or visible light (typically below 700 nm). The
INTRODUCTION In recent years, photodynamic therapy (PDT) has received considerable attention in tumor treatment due to its high selectivity, minimal invasiveness and repeatability without inducing sever toxicity.1 It involves irradiation source with specific wavelength, O2 and photosensitizers to generate cytotoxic reactive oxygen species (ROS). However, the hypoxia environment caused by the enhanced tumor proliferative activity and the abnormal structure of tumor vessels in solid tumors increases the difficulty of PDT.2 Besides, the consumption of O2 during PDT would further deteriorate hypoxia. To overcome this problem, several strategies have been employed by using O2-generated materials, such as MnO2,3 catalase4 or O2-loaded materials like perfluorocarbon.5 However, the amount of O2 generated by MnO2 and catalase are highly dependent on catalyzing of the limited endogenous H2O2. For perfluorocarbon-based O2 carriers, only a small amount of O2 could be transported to the tumor site, which leads to the restricted therapeutic efficacy.6 Metal-organic frameworks (MOFs) are constructed by coordinating metal ions with organic linkers. They have been widely studied for chemical sensors, luminescent materials, catalysis, drug delivery and so on.7-13 Moreover, MOFs are good candidates for gas storage and separation due to the large surface area and uniform pore size. Farha and co-workers presented a systematic study of MOFs for the storage of O2.14
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Scheme 1. Schematic illustration of the preparation of UC@mSiO2-RB@ZIF-O2-DOX/PEGFA (URODF) nanoparticles. The meanings of symbols in URODF are as follows: U - UC@mSiO2@ZIF, R - Rose Bengal, O - oxygen, D - DOX, F PEGFA.
synergetic therapy. Finally, the resultant nanodrug carrier with remarkably enhanced tumor inhibition effect was demonstrated in vitro and in vivo. This novel multifunctional nanodrug system that combines upconversion nanoparticles with MOF structure for O2 enhanced synergetic therapy might provide new directions for improving tumor therapeutic efficiency.
insufficient ability to penetrate tissues and potential photodamage to living organisms greatly restrict their bioapplications. Recently, rare earth doped upconversion nanoparticles (UCNPs) have been considered as a promising matrix to increase penetration depth for PDT treatment by tuning the excitation wavelength to the “therapeutic window” (650 - 1100 nm).30-33 Additionally, they also have low background fluorescence interference and high chemical and physical stability. These advantages promote them as good candidates for multi-modal imaging and labeling, which are of great importance for the detection and diagnosis of cancer.34-37 By introducing tumor targeting agents like folic acid, hyaluronic acid, RGD peptide and DNA aptamer on the surface of nanoparticles, the therapeutic efficiency could be greatly enhanced.38-40 In this report, an O2-loaded pH-responsive multifunctional nanodrug carrier UC@mSiO2-RB@ZIF-O2-DOX-PEGFA (URODF) with enhanced chemo-photodynamic therapeutic effect was fabricated (Scheme 1, Figure 1a). NaYF4:Yb/Er@ NaYbF4:Nd@NaGdF4 (UC) nanoparticles were employed for dual modal upconversion/MR imaging. The core-shell structure allows UC nanoparticles to harvest 808-nm photons and achieve green emission through multi-photon process, which indicates that photosensitizer can be activated in the “therapeutic window”. Mesoporous silica (mSiO2) was coated outside of UC to encapsulate photosensitizer Rose Bengal (RB). The outmost shell was constructed with ZIF-90 as oxygen reservoir. It would decompose under acidic conditions allowing quickly release of O2 at low pH tumor microenvironment and therefore improve the PDT efficiency. Besides, the organic ligand imidazol-2-carbox-aldehyde (ICA) of ZIF-90 contains free aldehyde groups, which permits covalent conjugation with chemotherapy drug DOX and tumor targeted molecule PEGFA via Schiff base reaction to achieve
RESULTS AND DISCUSSIONS Fabrication and Characterization of UC@mSiO2-RB@ ZIF-DOX/PEGFA (URODF) Nanoparticles. Scheme 1 shows the synthetic protocol of URODF nanoparticles. First of all, NaYF4:Yb/Er@NaYbF4:Nd@NaGdF4 was fabricated via a modified one-pot hot injection protocol.41, 42 Figure 1b presents the monodispersed hexagonal phase UC nanoparticles with average size about 50 nm. The high-resolution TEM image indicates the crystal lattice fringes of UC with interplanar distance of 0.530 nm, which is accordance with that of (100) plane in β-NaGdF4 crystals (Figure 1c). Then, mSiO2 was decorated on the UC by the well-established solgel method. Figure 1d shows that the particle size increases to around 120 nm after coating with mSiO2. Next, photosensitizer RB was encapsulated into the mesoporous pores of mSiO2 and validated through photoluminescence (PL) and UV-vis spectra (Figure 1j-1n, Figure S1). The emission peaks between 500 and 600 nm decrease sharply after RB conjugation. This could be ascribed to the fluorescence resonance energy transfer (FRET) process occurred between UCNPs and RB.43 Besides, the maximum absorption value (𝜆max) for RB at 549 nm red-shift to 556 nm for UC@mSiO2RB, indicating the successful conjugation of RB into the pores of mSiO2.44 In addition, ZIF-90 was coated outside of UC@mSiO2-RB with the assistance of
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Chemistry of Materials
Figure 1. (a) Schematic illustration of URODF nanoparticle as pH sensitive double drug loaded nanodrug carrier for O2 enhanced PDT and chemotherapy. TEM images of (b) NaYF4:Yb/Er@NaYbF4:Nd@NaGdF4 (UC), (c) high-resolution TEM image of UC nanoparticles, (d) UC@mSiO2-RB and (e) UC@mSiO2-RB@ZIF (UR) nanoparticles. EDS elemental mapping of (f) UR, (g) Y, (h) Si and (i) Zn. (j) Emission spectra of UC@mSiO2, UC@mSiO2-RB and UV-Vis absorption spectrum of RB. White field photographs of (k) UC@mSiO2, (l) UC@mSiO2-RB and dark field photographs of (m) UC@mSiO2, (n) UC@mSiO2-RB. Dark field photographs were obtained under 808 nm excitation with power of 4 W/cm2. The nanoparticle concentration was 0.25 mmol/L. (o) Thermogravimetry (TG) analysis of UC, UC@mSiO2-RB and UC@mSiO2-RB@ZIF-O2-DOX/PEGFA (URODF) nanoparticles, respectively. (p) N2 adsorption (solid symbols)/desorption (hollow symbols) isotherms for ZIF-90, UC@mSiO2 and UC@mSiO2@ZIF nanoparticles, respectively. compounds and drug molecules. For URODF nanoparticles, a rapid weight loss appears at around 450 °C. This should be affected by the surface PEG macromolecules. The weight loss above 600 °C is corresponded to the decomposition of ZIF-90 frameworks.18, 45 The fabricated nanocomposites are also confirmed via fourier-transform infrared (FT-IR) spectra. As shown in Figure S6a, UC nanoparticles exhibit strong adsorption peaks at 2925 and 2854 cm-1, which belong to the stretching vibration of methylene (CH2) in oleic acid. After coating with mSiO2, a broad adsorption peak around 3433 and 1086 cm-1 appears. These two peak are due to the vibrations of O-H and Si-O-Si bonds, respectively (Figure S6b). For UC@mSiO2RB, C-N stretching mode at 1385 cm-1 arises, suggesting the conjugation of RB molecules (Figure S6c).46 Figure S6d shows a typical peak at 1661 cm-1 which can be attributed to the C=O vibration in aldehyde of ZIF-90 shell.47 Finally, after connecting with of PEGFA and DOX, the FT-IR spectrum shows increasing peaks at 1094, 1615 and 1733 cm-1 which are generated from the C-O-C vibration in PEG and the -COOH and phenyl ring in folic acid, respectively (Figure S6d). Besides, the band around 2886 cm−1 that corresponds to the asymmetric and symmetric C-H stretch vibrations of folic acid and PEG also increases in intensity.48 Nitrogen Adsorption/Desorption Isotherms. The porosity of ZIF-90, UC@mSiO2 and UC@mSiO2@ZIF nanoparticles
triehylamine.18 Owning to the weak absorbance in the region of 400 - 1200 nm, ZIF-90 shell will not interrupt the FRET process from UC to RB (Figure S2). Figure 1e demonstrates that the final nanoparticles have about 140 nm size and rougher surface after coating with ZIF-90. The energy dispersive X-ray spectroscopy (EDX) results are shown in Figure 1f-1i. The evenly distribution of Y, Si and Zn element signals suggest the successful fabrication of UC@mSiO2RB@ZIF (UR). In order to increase the biocompatibility, tumor targeting effect and enhance chemotherapy, folic acid (FA) modified PEG (PEGFA, Mw = 3400 Da) and DOX were anchored on UR through Schiff base reaction. UV-vis spectra results reveal that the DOX loading capacity is about 6.0% in weight (Figure S3). Dynamic light scattering (DLS) analysis indicates that the particle size increases by about 10 nm after modification with DOX and PEGFA (Table S1 and Figure S4). Figure S5 shows the X-ray powder diffraction (XRD) pattern of URODF nanoparticles, which indicates the presence of the three components including β-NaYF4 (JCPDS 16-0334), silica and ZIF-90 phase, respectively. Thermogravimetry (TG) analysis shows the weight lost trends of nanoparticles below 800 °C (Figure 1o). All samples have a little weight loss below 280 °C. This is attributed to the absorbed water and surfactants coated around nanoparticles.44 The second weight loss existing in the blue and red curve between 350 °C and 500 °C should be ascribed to the decomposition of small organic
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Figure 2. O2 concentration of 20 ml deoxygenated PBS solution after adding 25 mg of (a) UC@mSiO2-N2, (b)UC@mSiO2@ZIFN2, (c) UC@mSiO2-O2 and (d) UC@mSiO2@ZIF-O2 nanoparticles, respectively. Black lines were obtained from pure PBS solution (pH = 7.4) without addition of any nanoparticles. Inverted fluorescence microscope images of HeLa cells incubated with (e) UROD and (f) URODF nanoparticles at different time point. All images share the same scale bar of 150 μm. The absorbance changes of DPBF treated with (g) URDF and (h) URODF in deoxygenated PBS buffer (pH = 5.5) after 808 nm laser irradiation for different times. Linear fit of the peak intensity at 412 nm for DPBF in (i) URDF and (j) URODF treated group. are confirmed via nitrogen adsorption/desorption isotherms. As shown in Figure 1p, pure ZIF-90 crystals exhibits type I isotherms, which indicate the existence of microporosity. For UC@mSiO2 nanoparticles, the isotherm presents a hysteresis loop in desorption process, suggesting the typical type IV classification of mesoporous structure. Interestingly, type I and type IV curves are both observed for the isotherm of UC@mSiO2@ZIF composites, implying the coexistence of microporous and mesoporous structures in UC@mSiO2@ZIF. In addition, the nitrogen adsorption increases sharply at low pressure (P/P0 < 0.02) after ZIF-90 coating, which also demonstrates the presence of micropore in UC@mSiO2@ZIF structure. Table S2 summarizes the Brunauer-Emmett- Teller (BET) surface area (SBET) and total pore volume value (Vtotal) for ZIF-90, UC@mSiO2 and UC@mSiO2@ZIF nanoparticles, respectively. The SBET increases from 508 m2/g for UC@mSiO2 to 556.2 m2/g for UC@mSiO2@ZIF. Besides, the Vtotal decreases from 0.71 to 0.68 cm3g-1 after coating with ZIF-90. pH-responsive O2 and DOX Release. It is well known that ZIF-90 is stable under physiological conditions and will decompose under acidic conditions.25 Figure S7 shows the SEM images of ZIF-90 crystal after immersing in PBS buffer solutions (pH = 7.3, 5.5 and 2.0) for 10 min. For the group treated with PBS solution at pH = 7.3, crystals remain the
dodecahedron morphology very well. As the pH value decreases to 5.5, which is similar to that of the microenvironment around endosome and lysosome in cancer cells, ZIF-90 crystals partially collapse. To further demonstrate the pH-dependent morphology evolution for ZIF90, the crystals were also immersed in PBS buffer solutions at pH = 2.0 for 10 min. As shown in Figure S7c, the crystals are totally decomposed, confirming the pH sensitivity of ZIF-90. In order to test the oxygen uptake capacity and the pHresponsive O2 release behavior, UC@mSiO2-N2, UC@mSiO2@ZIF-N2, UC@mSiO2-O2 and UC@mSiO2@ ZIF-O2 nanoparticles were dispersed in deoxygenated PBS buffer solutions with different pH values, respectively. The dissolved O2 was in situ monitored by the O2 detector every 3 s. Nitrogen treated nanoparticles were used as the control groups. As shown in Figure 2a-2b, the dissolved O2 concentration changes a little for N2 containing groups, indicating the negligible influence of environment on the results. For UC@mSiO2-O2 treated group, only a small amount of O2 is detected (
