Controllable Synthesis of a Smart Multifunctional Nanoscale Metal

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Controllable synthesis of smart multifunctional nanoscal metal-organic framework for magnetic resonance/optical imaging and targeted drug delivery Xuechuan Gao, Manjue Zhai, Weihua Guan, Jingjuan Liu, Zhiliang Liu, and Alatangaole Damirin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14795 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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ACS Applied Materials & Interfaces

Controllable Synthesis of Smart Multifunctional Nanoscale Metal-organic Framework for Magnetic Resonance/Optical Imaging and Targeted Drug Delivery Xuechuan Gao†, Manjue Zhai‡, Weihua Guan†, Jingjuan Liu†, Zhiliang Liu†* and Alatangaole Damirin ‡* †College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot, 010021, P. R. China. E-mail: [email protected] ‡ College of Life Sciences, Inner Mongolia University, Hohhot, 010021, P. R. China. E-mail: [email protected] KEYWORDS: nanoscale MOFs, fluorescence imaging, magnetic resonance imaging, targeted drug delivery, multifunctional MOFs ABSTRACT: As a result of their extraordinarily large surfaces and well-defined pores, the design of multifunctional metal-organic framework (MOF) is crucial for drug delivery while has been rarely reported. In this paper, a novel drug delivery system (DDS) based on nanoscale metal-organic framework (MOF) was developed for using in cancer diagnosis and therapy. This MOF based tumour targeting DDS was fabricated by a simple post-synthetic surface

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modification process. Firstly, magnetic mesoporous nanomaterial Fe-MIL-53-NH2 was devoted for encapsulating the drug and served as a magnetic resonance (MR) contrast agent. Moreover, Fe-MIL-53-NH2 nanomaterial exhibited a high loading capacity for the model anticancer drug 5fluorouracil (5-FU). Subsequently, the fluorescence imaging agent 5-carboxyfluorescein (5-FAM) and the targeting reagent folic acid (FA) were conjugated to the 5-FU-loaded Fe-MIL-53-NH2, resulting in an advanced DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU. Owing to the multifunctional surface modification, the obtained DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU shows good biocompatibility, tumour enhanced cellular uptake, strong cancer cell growth inhibitory effect, excellent fluorescence imaging and outstanding magnetic resonance imaging (MRI) capability. Taken together, this study integrates diagnostic section and treated section into a single platform by a simple and efficient strategy, aiming for facilitating new possibilities for the MOF using as a multifunctional drug delivery. 1. INTRODUCTION Different from most of the existing pure organic and inorganic carrier materials, Metal-organic frameworks (MOFs) as a new class of porous crystalline materials have high stability, high surface area, large pore volume, regular porosity and intriguing variety of architectures

1-4

and

have already emerged as a promising drug delivery platform.5-6 In addition, the variable pore size of MOFs can be achieved by altering the metal or ligand and the appeared active sites within the framework permit an easier transmission of guest molecules, 7-8 offering unexpected advantages in the areas of drug delivery. Starting from Fe-based MOFs as drug delivery carriers demonstrated by Férey and co-workers,9 inspiring efforts have been committed to explore these fields and several studies have been carried out. Anionic Zn-based MOF 10 was being used as a carrier of cationic drug and a biocompatible porous Mg-based MOF

11

was being used as an


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antioxidant carrier. While these MOFs exhibit ideal drug loading and excellent drug delivery ability, lack of tumor-targeting capability results in strong toxicity to normal cells, which is adverse to the drug efficacy and limits their biomedical applications. Although nanomaterials integrating magnetic imaging or fluorescence imaging and drug delivery have been reported a lot,12-16 the preparation methods are usually complex and little attempt has been made to incorporate three functionalities: magnetic / fluorescence imaging, cell-targeting and drug storage delivery in one single platform based on MOF. Considering above mentioned disadvantages, designing targeted drug delivery system (DDS) based on MOF to reduce secondary action of drugs during clinic treatment and integrating multimodal imaging capabilities is a long-standing challenge in drug development. It is generally known that iron is not only a widespread element in nature but also a essential element in human body, which makes it extremely appealing for the application of Fe-MOFs-NH2 in in diverse biomedical applications.17-18 With fascinating properties such as well-defined pores and low toxicity, nanoscale materials Fe-MIL-53-NH2 are ideal candidates for drug carriers.19-20 Besides, the Fe-MIL-53-NH2 materials already reported show intensive magnetism, which enable them to be effective magnetic resonance imaging (MRI) contrast agents.21 Furthermore, the abundant amino on external surface of Fe-MOFs-NH2 nanocrystals also offer the possibility to functionalize the Fe-based MOF for targeted imaging or therapy, which would have a great significance in further developments. Since the external carboxyl on 5-carboxylfluorescein (5FAM, Figure S1a) can be covalently connected to the NH2 groups on the surface of Fe-MOFsNH2 nanocrystals, 5-FAM is appropriate as a fluorescent probe for observing the drug delivery process and the real-time imaging in cells.22


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To reduce an anticancer drug’s the toxicity to normal cells and enhance anticancer effects to cancer cells, targeted molecules should be combined with the drug carrier. As we know, folic acid (FA, Figure S1b) receptors are usually overexpressed on the outside surface of a number of cancer cells and FA has excellent affinity with folate receptors (FR), leading to a selective uptake within FR-positive cancer cells and avoiding uptake by normal tissues that do not express FR.2324

Thus FA is widely chosen as a model targeting reagent for efficiency of delivering drugs into

tumor cells.

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Additionally, the stability and compatibility in physiological conditions

facilitate the employment of FA as an ideal targeting molecules for delivery systems. 5-Fluorouracil (5-FU), a pyrimidine analogue (Figure S1c), has been one of the major clinically applied anticancer agents for the treatment of a wide variety of tumors since it can be incorporated into DNA and RNA, leading to cytotoxicity and cancer cell death27-28. And the small molecular weight of 5-FU boosts the burgeoning biomedical applications of 5-FU as a model anticancer drug. Based on the above background, a multifunctional MOF based tumor targeting DDS (Fe-MIL53-NH2-FA-5-FAM/5-FU), shown in Figure 1, was developed in the present study. In this composite, the Fe-MIL-53-NH2 is devoted for encapsulating the drug and magnetic resonance imaging, whereas the FA serves as targeted reagent and 5-FAM is employed for fluorescent imaging. The Fe-MIL-53-NH2-FA-5-FAM/5-FU, obtained via a simple post-modification method, displays satisfying magnetic performance and excellent green fluorescence-emission. Meanwhile Fe-MIL-53-NH2-FA-5-FAM/5-FU can target MGC-803 cells and exhibits significant cell growth inhibitory effects on MGC-803 cells on account of the sustained release of 5-FU. All results demonstrate that the developed DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU possesses great potential as a novel theranostic agent for simultaneous fluorescence/magnetic resonance imaging


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and drug delivery in biomedicine. We envision that this new hybrid Fe-MIL-53-NH2-FA-5FAM/5-FU nanocomposite will be proved to be a novel drug delivery platform for cancer disease therapy in the near future.

Figure 1. 2D plane view of Fe-MIL-53-NH2 nanocrystalline (A, B), schematic of DDS Fe-MIL53-NH2-FA-5-FAM/5-FU (C), schematic illustrations of DDS Fe-MIL-53-NH2-FA-5-FAM/5FU for targeting drug delivery (D) 2. EXPERIMENTAL SECTION 2.1 Material and methods In this work, all the starting analytical grade reagents and solvents were acquired from commercial sources and utilized without further purification. A PANalytical empyrean sharp shadow system X-ray diffractometer was used to get the X-ray diffraction (XRD) patterns of the


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samples over the 2θ range of 5°-80°and Cu Karadiation (λ = 1.540598 Å) was used during the testing procedure. Scanning electron microscope (SEM) images with high quality of all samples were acquired using a HITACHI S-4800 scanning electron microscope and the accelerating voltage in the process of testing is 200 kV. A NEXUS-670 Fourier transform infrared spectrophotometer was utilized to assess the functional groups in all samples and photoluminescence spectra of fluorescent reagents and the obtained sample were characterized by taking advantage of a Hitachi F-7000 fluorescence spectrophotometer. Thermal gravity analysis (TGA) of the samples was obtained under an N2 atmosphere and carried out using a NETZSCH STA449F3 thermal analyser in the temperature range from 40°C to 600°C and the heating rate was 10°C/min. A laser scanning confocal microscope (OLYMPUS, IX81) was used to confirm the cell targeting process and cell imaging capacity. UV-Vis spectra of the samples were recorded on a TU-1901 diode UV-visible spectrophotometer. 2.2 Synthesis of Fe-MIL-53-NH2 Fe-MIL-53-NH2 nanocrystalline was prepared by a facile low-temperature synthesis route via the reaction of FeCl3·6H2O, and 2-amino terephthalic acid (NH2-H2BDC) in the ethanol solution.29 Typically, 0.1 mmol FeCl3·6H2O, 0.1 mmol NH2-H2BDC and 9 mL ethanol solution were mixed into a three-neck round bottom flask (25 ml) and transferred into a 40 °C oil bath as well as stirred for 2 h. The obtained reaction product was purified three times with ethanol and dried at 30 °C in a vacuum oven. To investigate the effect of reactant concentration on the crystal size, the amount of FeCl3·6H2O and the amount of NH2-H2BDC were tuned from 0.1 to 0.4 mmol. 2.3 Preparation of 5-FU loaded Fe-MIL-53-NH2


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A pure and dried sample of Fe-MIL-53-NH2 (0.1 g) was immersed in 50 ml of water solution containing 5-FU (0.1g) for 48 hours. After centrifuging, the obtained product was washed with some water for several times. The 5-FU loading efficiency on Fe-MIL-53-NH2 can be inferred from the formula as follow: loading efficiency (%) = (m1 − m2) / m. In this equation, m1 represents the initial weight of 5-FU; m2 refers to the weight of 5-FU present in the excess of solvent; m refers to the total weight of the solid obtained. After 2 days of soaking, the loading efficiency of 5-FU on Fe-MIL-53-NH2 was 28% according to the standard 5-FU concentration curve shown in Figures S2. 2.4 Synthesis of Fe-MIL-53-NH2-FA-5-FAM/5-FU At room temperature, 0.1 g 5-FU loaded Fe-MIL-53-NH2, 0.2 g FA, 0.2g 5-FAM were added to 50 mL saturated aqueous solution of 5-FU. After the solution was mixed uniformly, 0.1 g N(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) as dehydrating agent was added to the above mixture, which was subsequently stirred for 16 h in the dark. At the end of the reaction, the obtained Fe-MIL-53-NH2-FA-5-FAM/5-FU nanocomposite was isolated from solution through centrifugation, followed by washing with water and then dried under vacuum at 25 °C. Similar to the calculation method of loading efficiency of 5-FU on Fe-MIL-53-NH2, the loading efficiency of 5-FU on Fe-MIL-53-NH2-FA-5-FAM/5-FU nanocomposite is 23%. And the loading amounts of FA and 5-FAM on Fe-MIL-53-NH2-FA-5-FAM/5-FU nanocomposite are 2.6% and 1.5%, respectively. Meanwhile, the standard concentration curves of FA and 5-FAM were shown in Figures S3 and Figures S4. The aforementioned synthetic procedure was subsequently used to synthesize Fe-MIL-53NH2-FA-5-FAM and Fe-MIL-53-NH2-5-FAM/5-FU. Details of the procedure were provided in the supporting information.


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2.5 Cytotoxicity study MGC-803 cells and HASMC cells were used to evaluate the in vitro cytotoxicities and antitumor efficiencies of Fe-MIL-53-NH2-FA-5-FAM, Fe-MIL-53-NH2-FA-5-FAM/5-FU and 5-FU via the MTT method. Shortly, MGC-803 cells and HASMC cells were seeded onto 96-well plates and the culture density was 104 cells per well. After MGC-803 cells and HASMC cells were incubated for 24h, the initial culture medium was removed and replaced by the dispersion of Fe-MIL-53-NH2-FA-5-FAM, Fe-MIL-53-NH2-FA-5-FAM/5-FU and 5-FU with various concentrations (0, 6.25, 12.5, 25, 50, 100 and 200 µg/mL). After incubating for another 24 h, 20 µL of MTT solution was added to each well in which the previous culture medium has been removed and incubated for another 4 h. At last, all media were removed out from each well before 100 µL of DMSO was added to and the cell viability was obtained by monitoring the absorbance value of each sample at 570 nm using a microplate reader. The cell viability was expressed as a percentage of the absorbance value of the sample well to that of the controlled cell. All experiments were executed in triplicate, and the results were averaged. 2.6 Cellular uptake study The in vitro targeting ability of the FA to FR was evaluated by luminescence imaging in MGC-803 cells and HASMC cells. MGC-803 cells and HASMC cells were attached onto a 6well plate for 24 h. Afterwards, the MGC-803 cells were treated with Fe-MIL-53-NH2-FA-5FAM/5-FU and Fe-MIL-53-NH2-5-FAM/5-FU for 4 h and HASMC cells were incubated with Fe-MIL-53-NH2-FA-5-FAM/5-FU for 4 h (concentration was 0.1 µg/mL). Before observed by laser confocal microscope (OLYMPUS, IX81) under excitation wavelength of 473 nm, the MGC-803 cells and HASMC cells were were washed several times with phosphate-buffered saline (PBS).


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2.7 In vivo magnetic resonance imaging All animal experiments were conducted according to the guidelines approved by the Institutional Animal Care and Use Committee of Peking University. To carry out the in vivo imaging study, athymic nude mices bearing glioblastoma were injected with Fe-MIL-53-NH2FA-5-FAM/5-FU aqueous solution (5 mg/kg). At 1h after intratumoral injection, magnetic resonance imaging was taken by Siemens Prisma 3.0 T MR scanner (Erlangen, Germany) with gradient strength up to 80 mT/m. 2.8 In vivo biodistribution in different organs To investigate the biodistribution of Fe-MIL-53-NH2-FA-5-FAM/5-FU in different organs, athymic nude mice were administered via intraperitoneal injection (10mg/kg) and were sacrificed at the 24 h time point post-injection. The major organs were collected and the fluorescence signal intensity was recorded on IVIS imaging system (IuminaⅡ) at excitation wavelength of 495 nm. And after deducting autofluorescence, the biodistribution of different organs was concluded as the ratio of average fluorescence intensity of individual organ to average fluorescence intensity of all measured organs. 2.9 In vitro drug release study The sustained 5-FU release assays were carried out by soaking the samples in phosphatebuffered saline (PBS, pH 7.4 and pH 5). At 37 °C, a dialysis containing 0.05 g Fe-MIL-53-NH2FA-5-FAM/5-FU was put into 10 mL PBS solution in a 50 mL centrifuge tube. To real-time monitoring the release behavior of 5-FU, 2 mL solution was withdrawn from the centrifuge tube at appropriate time intervals and the UV-visible absorption values of the solutions were recorded at 265 nm. The cumulative release profiles were expressed by cumulative release percentages of 5-FU from Fe-MIL-53-NH2-FA-5-FAM/5-FU as a function of time, in which the cumulative


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release percentages of 5-FU from Fe-MIL-53-NH2-FA-5-FAM/5-FU were calculated as follows: Cumulative 5-FU release = amount of released 5-FU / amount of total 5-FU × 100%. 3. RESULTS AND DISCUSSION 3.1 Fabrication and Characterization of Fe-MIL-53-NH2-FA-5-FAM/5-FU

Figure 2. SEM images of Fe-MIL-53-NH2 nanocrystalline with different reactant concentrations (A, B, C, D). Different reactant concentration is applied to control over the size of Fe-MIL-53-NH2 and the results are shown in Figure 2. Figure 2A–D shows the SEM images of the Fe-MIL-53-NH2 nanocrystalline prepared by employing decreasing amounts of FeCl3·6H2O from 0.4 mmol to 0.1 mmol and BDC-NH2 from 0.4 mmol to 0.1 mmol. Interestingly, as the reactant concentrations decrease, the sizes of the nanoparticles change from 120 nm to 1 µm. Thus, tuning the amount of the reactant is a facile and robust way to control the particle size. To check the crystallinity, the materials are further investigated by XRD, which is depicted in Figure 3. The XRD reflections of all the obtained Fe-MIL-53-NH2 nanocrystallines correspond well with the simulated pattern, readily indexing high crystallinity of the obtained Fe-MIL-53-NH2 nanomaterials and the same crystal structures of all the obtained Fe-MIL-53-NH2 nanocrystalline. Due to the abundant NH2


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on the surface of Fe-MIL-53-NH2 nanocrystalline and small particle size, Fe-MIL-53-NH2 about 120nm is chosen to form the multifunctional drug carriers by conjugating with targeted agents (FA) and fluorescent reagent (5-FAM).

Figure 3. XRD patterns of simulated Fe-MIL-53-NH2 (A) and Fe-MIL-53-NH2 nanocrystalline prepared with different reactant concentrations (B, C, D, E)

Figure 4. (a) FTIR spectra of Fe-MIL-53-NH2 nanocrystalline (A), FA (B), 5-FAM (C) and FeMIL-53-NH2-FA-5-FAM nanocrystalline (D); (b) FTIR spectra of 5-FU (A), Fe-MIL-53-NH2FA-5-FAM/5-FU nanocomposite (B) and Fe-MIL-53-NH2-FA-5-FAM nanocomposite (C). To identify the successful synthesis of Fe-MIL-53-NH2-FA-5-FAM nanocomposite, the FTIR spectra of the Fe-MIL-53-NH2 (Figure 4aA), FA (Figure 4aB), 5-FAM (Figure 4aC) and Fe-


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MIL-53-NH2-FA-5-FAM (Figure 4aD) were recorded. Fe-MIL-53-NH2 exhibits the N-H symmetrical deformation mode at 1573cm-1, while the stretching vibrations of the C=O of carboxyl groups in FA and 5-FAM appear at 1696 cm-1 and 1693cm-1 respectively. For comparison, the FTIR spectrum of Fe-MIL-53-NH2-FA-5-FAM shows typical bands of Fe-MIL53-NH2, FA and 5-FAM, as revealed by Figure 4aD. However, no band around 1696 cm-1 that is representative of C=O of carboxyl groups is observed in Figure 4aD and the characteristic peak of C=O of amide groups appears at 1596 cm-1, suggesting the successful conjugation of FA and 5-FAM to NH2 on the surface of Fe-MIL-53-NH2. Besides, confocal laser scanning microscopy images of Fe-MIL-53-NH2-FA-5-FAM (Figure S5) show strong green luminescent signal, originated from the representative fluorescence of 5-FAM, confirming the successful conjugation of 5-FAM.. After loading with 5-FU, FTIR spectrum of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU is collected. The FTIR spectra further indicate the presence of 5-FU on the DDS (Figure 4b). The peak at 1655 cm−1 belongs to the vibrations of C=O in 5-FU (Figure 4bA). The broad band at 1596 cm−1 is attribute to C=O of amide groups in Fe-MIL-53-NH2-FA-5-FAM nanomaterial (Figure 4bC). Generally, Fe-MIL-53-NH2-FA-5-FAM/5-FU exhibits the typical absorption of 5FU and Fe-MIL-53-NH2-FA-5-FAM, which indicates that the loading of 5-FU with Fe-MIL-53NH2-FA-5-FAM is successful. Due to the formation of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU, TGA curve of Fe-MIL-53-NH2-FA-5-FAM/5-FU is extremely different from TGA curve of FeMIL-53-NH2, as shown in Figure S6. The DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU exhibits lager weight loss because of decomposition of the loaded 5-FU, FA and 5-FAM. Meanwhile the postmodification and 5-FU loaded cause the XRD peaks to broaden while the dominating peaks of the XRD spectrum of Fe-MIL-53-NH2-FA-5-FAM/5-FU correlates with the Fe-MIL-53-NH2


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pattern, as shown in Figure S7, suggesting the crystalline integrity of the Fe-MIL-53-NH2-FA-5FAM/5-FU. 3.2 Properties and Applications of Fe-MIL-53-NH2-FA-5-FAM/5-FU

Figure 5. Relaxation rate 1/T2 versus concentrations of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU using a 3T MRI scanner; T2-weighted MRI of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU with diverse Fe concentrations in vitro (the insert). The transverse relaxation times (T2) of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU with various concentrations were measured and the relaxivity values (r2) of DDS Fe-MIL-53-NH2-FA-5FAM/5-FU can be calculated by the slope of a plot of the relaxation rate 1/T2 versus Fe concentration. As we know, dipolar interaction between the magnetic moments of Fe-MIL-53NH2-FA-5-FAM/5-FU and the protons in the water can cut down T2. Thus 1/T2 increases linearly within the tested range of Fe concentration as revealed by Figure 5 and DDS Fe-MIL-53-NH2FA-5-FAM/5-FU exhibits high relaxivity values ( r2 =18.8 mM−1s−1 ) for potential clinical use as contrast agents for T2 -weighted MRI. The T2-weighted MRI of DDS Fe-MIL-53-NH2-FA-5FAM/5-FU are shown in the insert of Figure 5 and the iron concentrations in 2 to 7 are 0.115, 0.2308, 0.4617, 0.9235, 1.847 and 3.694 mM, respectively. It is visible that the signal intensity


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decreases (darkening) with the increase of the iron concentrations, suggesting that Fe-MIL-53NH2-FA-5-FAM/5-FU is a better candidate for T2-weighted MRI. Furthermore, the fluorescence testing of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU was also performed. Clearly, compared to fluorescence spectrum of 5-FAM, the excitation and emission spectra of Fe-MIL-53-NH2-FA-5FAM/5-FU are similar, suggesting that the luminescent property of Fe-MIL-53-NH2-FA-5FAM/5-FU is mostly derived from the 5-FAM. It is noted that the slight discrepancies in the position of excitation and emission wavelength are due to the combination of 5-FAM and FeMIL-53-NH2. While 5-FAM exhibits a broad emission band with a maximum at 530 nm (excited at 480 nm), DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU generate a broad band with a maximum at 560 nm (excited at 495 nm) in Figure 6, which belongs to green emission and fulfills the requirements of fluorescence imaging.

Figure 6. The excitation (A) and emission (B) spectra of 5-FAM and the excitation (C) and emission (D) spectra of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU


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Figure 7. Fluorescence imaging of live MGC-803 cells cultured with Fe-MIL-53-NH2-FA-5FAM/5-FU (A) and Fe-MIL-53-NH2-5-FAM/5-FU (B) as well as HASMC cells cultured with Fe-MIL-53-NH2-FA-5-FAM/5-FU (C) for 4 h. Left panels, middle panels and right panels are dark-field images, bright-field images and overlays, respectively. Scale bar: 100 µm. To gain more insight into the targeting ability of DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU, we carried out fluorescence imaging tests using FA-positive MGC-803 cells and FA-negative HASMC cells. And the results are shown in Figure 7. When MGC-803 cells are incubated with Fe-MIL-53-NH2-FA-5-FAM/5-FU, bright green fluorescence is observed in the cell. However, no obvious fluorescence contrast is obtained in MGC-803 cells incubated with Fe-MIL-53-NH25-FAM/5-FU and HASMC cells incubated with Fe-MIL-53-NH2-FA-5-FAM/5-FU. This observation

shows

that

only

the

FA-conjugated

Fe-MIL-53-NH2-FA-5-FAM/5-FU

nanocomposite shows a high-affinity to the cancer cell whereas has little interaction with normal cells, confirming the targeted fluorescence imaging ability of DDS Fe-MIL-53-NH2-FA-5-


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FAM/5-FU. We also investigated the magnetic resonance imaging capability of Fe-MIL-53NH2-FA-5-FAM/5-FU in vivo (Figure 8). Clearly, at 1h post-injection, the tumor becomes darker and the T2-weighted MR image exhibits intense contrast enhancement. In brief, Fe-MIL53-NH2-FA-5-FAM/5-FU displays good imaging capability similar to the fluorescence and magnetic resonance imaging systems reported.30-33 Besides, Fe-MIL-53-NH2-FA-5-FAM/5-FU shows stable drug loading and release behavior, making it more superior to be used in practical application.

Figure 8. T2-weighted MR images of tumor-bearing athymic nude mices without Fe-MIL-53NH2-FA-5-FAM/5-FU (A) and with Fe-MIL-53-NH2-FA-5-FAM/5-FU by intratumoral injection (B).


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Figure 9. Accumulation of Fe-MIL-53-NH2-FA-5-FAM/5-FU in major organs excised from athymic nude mices after 24 h post-injection; ex vivo fluorescence imaging of different organs at 24h time point post-injection (inset) To explore the dispersibility and stability of this DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU for in vivo application, the biodistribution of Fe-MIL-53-NH2-FA-5-FAM/5-FU has been analyzed by ex vivo fluorescence intensity of different organs at 24h time point after deducting autofluorescence and the result is shown in Figure 9. It can be found that DDS Fe-MIL-53-NH2FA-5-FAM/5-FU is able to transport to tissues in vivo and the intensity from the liver was highest among all measured organs. Additionally, the ex vivo fluorescence imaging of different organs at 24h time point post-injection has been shown in the inset of Figure 9. Apparently, strong fluorescence signals can still be detected in the liver and the XRD of Fe-MIL-53-NH2-FA5-FAM/5-FU after soaking for 7 days in PBS agrees well with the original Fe-MIL-53-NH2-FA5-FAM/5-FU (Figure S8), suggesting that this DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU is stable under physiological conditions.


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Figure 10. (a) Viabilities of HASMC cells cultured with Fe-MIL-53-NH2-FA-5-FAM (A), DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU (B) and 5-FU (C), evaluated by MTT; (b) Viabilities of MGC-803 cells cultured with Fe-MIL-53-NH2-FA-5-FAM (A), DDS Fe-MIL-53-NH2-FA-5FAM/5-FU (B) and 5-FU (C), evaluated by MTT. It is necessary to investigate the cytotoxicity of Fe-MIL-53-NH2-FA-5-FAM/5-FU for their potential application in bioimaging. Figure 10 shows the effects of Fe-MIL-53-NH2-FA-5-FAM, DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU and 5-FU on the viabilities of MGC-803 cells and HASMC cells by MTT method. After incubating MGC-803 cells and HASMC cells with the FeMIL-53-NH2-FA-5-FAM at various concentrations, little toxicity for the nanocomposite is observed at concentration up to 200 µg/mL and the cell viability decreases by 80%, as shown in Figure 10aA and Figure 10bA, indicating that Fe-MIL-53-NH2-FA-5-FAM nanocomposite displays excellent biocompatibility. Similarly, DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU and 5FU show little toxicity to HASMC cells and more than 80 % of cells survive at concentration of 200µg/mL (Figure 10aB and 10aC). In contrast, incubation of DDS Fe-MIL-53-NH2-FA-5FAM/5-FU with MGC-803 cells exhibits a dose-dependent toxicity that similar to 5-FU and induces 45% death of MGC-803 cells when the concentration of Fe-MIL-53-NH2-FA-5-FAM/5FU is 200 µg/mL (Figure 10bB and 10bC). These data demonstrate that DDS Fe-MIL-53-NH2-


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FA-5-FAM/5-FU gives highly efficient and selective treatment of tumor cells due to the targeting ability of FA and the release of 5-FU.

Figure 11. Drug release profile for DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU in PBS buffer solution at pH = 7.4 (A) and pH = 5 (B). Due to the stable and lager pore size, the Fe-MIL-53-NH2 nanocomposite displays BET surface area up to 198 m2/g (Figures S9) and the drug loading efficiency of Fe-MIL-53-NH2-FA5-FAM/5-FU is 23%. To validate sustained release property of 5-FU from Fe-MIL-53-NH2-FA5-FAM/5-FU system, release experiments were carried out in PBS buffer solutions (pH = 7.4 and 5) at 37°C and Figure 11 shows the drug delivery profile. Apparently, the release rates of 5FU both in pH = 7.4 and pH = 5 gradually decrease with time. During the first 5 hours, a distinctly rapid 5-FU release from DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU is detected, which is mostly on account of the fast release of 5-FU molecules on Fe-MIL-53-NH2-FA-5-FAM/5-FU surface. Subsequently, drug release slows down and the gentle release lasts for 25 hours in pH = 7.4 while the gentle release lasts for 20 hours in pH = 5. This prolonged 5-FU release is ascribed to the extended diffusion route of 5-FU from pores and cavities of Fe-MIL-53-NH2-FA-5FAM/5-FU to surrounding environment. Meanwhile the faster and more stable drug release in


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pH = 5 may be due to the slight dissolution of Fe-MIL-53-NH2 in the acidic environment. Clearly, DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU exhibits remarkable time-release properties, making their presence known in the area of targeted drug delivery. 4. CONCLUSION Tumor-targeting and programmable chemotherapeutic delivery are demonstrated by designing DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU. This smart system provides a unique MOF platform for fluorescence/magnetic resonance dual-mode imaging and targeted drug delivery. Fe-MIL-53NH2-FA-5-FAM/5-FU nanocomposite was prepared using a refluxe method at low temperature, followed by a simple post-modification method. In vitro fluorescence imaging verifies that DDS Fe-MIL-53-NH2-FA-5-FAM/5-FU exhibits excellent receptor-specific targeting fluorescence imaging for FA-positive MGC-803 cells and the high transverse relaxivity of the nanocomposites gives it potential as a contrast agent for MRI. Additionally, such DDS is also found to generate larger toxicity to cancer cells (MGC-803 cells) due to the targeted 5-FU release and release studies state that the 5-FU release behavior can last for more than 20h. In conclusion, this paper provides a brief and efficacious method to explore MOFs as a new targeted drug delivery system, which generate significantly enhanced antitumor efficacy. These results provide a proof of concept for a new mode of drug delivery and we fully expect to see this drug delivery system make their presence known in the area of biological applications ASSOCIATED CONTENT Supporting Information Chemical structure, fluorescence imaging, TGA curves, XRD patterns, N2 adsorption-desorption isotherms and the absorbance of 5-FU in PBS buffer solution are obtained in Supporting information. Supporting information for this article is available on the http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author Zhiliang Liu* E-mail: [email protected] College of Chemistry and Chemical Engineering, Inner Mongolia University, Hohhot, 010021, P. R. China Alatangaole Damirin* E-mail: [email protected] College of Life Sciences, Inner Mongolia University, Hohhot, 010021, P. R. China. Notes All authors have no conflicts of interest to declare for the work. The authors declare no competing financial interest ACKNOWLEDGMENT All authors received funding from Natural Science Foundation of China (21361016) and Inner Mongolia Autonomous Region Fund for Natural Science (2013ZD09). REFERENCES [1] Della Rocca, J.; Liu, D.; Lin, W. Nanoscale Metal–Organic Frameworks for Biomedical Imaging and Drug Delivery. Acc. Chem. Res. 2011, 44, 957–968. [2] Yoon, M.; Srirambalaji, R.; Kim, K. Homochiral Metal–Organic Frameworks for Asymmetric Heterogeneous Catalysis. Chem. Rev. 2012, 112, 1196–1231. [3] Keskin, S.; Kizilel, S. Biomedical Applications of Metal Organic Frameworks. Ind. Eng. Chem. Res. 2011, 50, 1799–1812.


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Table of Contents

A novel drug delivery system based on nanoscale metal-organic framework was developed for using in cancer diagnosis and therapy, exhibiting good biocompatibility, excellent fluorescence imaging, outstanding magnetic resonance imaging capability, targeted drug transportation, and localized drug sustained release.


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Figure 1 81x71mm (300 x 300 DPI)



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Figure 3 160x70mm (300 x 300 DPI)



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Figure 7 90x105mm (300 x 300 DPI)



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Figure 9 150x65mm (300 x 300 DPI)



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Figure 10 80x60mm (300 x 300 DPI)


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Controllable Synthesis of a Smart Multifunctional Nanoscale Metal

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