Rational Design of Metal Organic Framework Nanocarrier-Based

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Rational Design of MOF Nanocarrier-Based Co-Delivery System of Doxorubicin Hydrochloride/Verapamil Hydrochloride for Overcoming Multidrug Resistance with Efficient Targeted Cancer Therapy Huiyuan Zhang, Wei Jiang, Ruiling Liu, Jing Zhang, Di Zhang, Zhonghao Li, and Yuxia Luan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 25, 2017

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ACS Applied Materials & Interfaces

Rational Design of MOF Nanocarrier-Based Co-Delivery System of Doxorubicin Hydrochloride/Verapamil Hydrochloride for Overcoming Multidrug Resistance with Efficient Targeted Cancer Therapy Huiyuan Zhang,a Wei Jiang,a Ruiling Liu,a Jing Zhang,a Di Zhang,a Zhonghao Li,b Yuxia Luan,a∗

a

School of Pharmaceutical Science, Key Laboratory of Chemical Biology, Ministry of Education, Shandong

University, Jinan, 250012, China. Fax: (86) 531-88382548; Tel: (86) 531-88382007; E-mail: [email protected]

b

Key Lab of Colloid & Interface Chemistry, Shandong University, Ministry of Education, Jinan, 250100,

China.

ABSTRACT Conventional organic and inorganic drug nanocarriers suffer from serious drawbacks, such as low drug-storage capacity and uncontrolled release. Moreover, multidrug resistance (MDR) has been one of the primary causes leading to chemotherapy failure for cancers. The main reason of MDR is the overexpressed active efflux transporters such as P-glycoprotein. Here, zeolitic imidazolate framework ZIF-8, as one of the biocompatible metal organic frameworks (MOFs), is reported for the first time as the multidrug carrier to realize the efficient co-delivery of verapamil hydrochloride (VER) as the P-glycoprotein inhibitor as well as doxorubicin hydrochloride (DOX) as an anticancer drug to overcome the MDR in addition to realizing the active targeted ability for an efficient anticancer effect. Uniform ZIF-8 nanoparticles encapsulating DOX and VER are 1

ACS Paragon Plus Environment


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achieved by a facile one-pot process, in which the VER is used to overcome the multidrug resistance. Furthermore, Methoxy poly (ethylene glycol)-folate (PEG-FA) is used to stabilize the (DOX+VER)@ZIF-8 to realize prolonged circulations and an active targeting drug delivery. In particular, the ZIF-8 exhibits high drug loading content up to ca. 40.9% with a pH-triggered release behavior. Importantly, the PEG-FA/ (DOX+VER)@ZIF-8 shows enhanced therapeutic efficiencies with much safety compared with the direct administration of free DOX both in vitro and in vivo. Near infrared fluorescent (NIRF) imaging indicates that the PEG-FA/ (DOX+VER)@ZIF-8 can increase the drug accumulations in tumors for targeted cancer therapy. Therefore, the PEG-FA/ (DOX+VER)@ZIF-8 multidrug delivery system can be used as a promising efficient formulation in reversing the multidrug resistance for targeted cancer therapy.

KEYWORDS: MOF, ZIF-8, drug co-delivery, cancer therapy, active targeting, multidrug resistance reversal

1.

INTRODUCTION The development of effective drug delivery systems to diminish the inherent adverse reactions

of chemotherapy medications and improve the therapeutic effects has been one of the key issues for the treatment of cancers.1 Currently, by virtue of the enhanced permeability and retention effect (EPR), various drug nanocarriers have been developed to improve the accumulations of drugs in the region of tumors, including polymersomes,2 micelles,3 polymeric nanoparticles,4 inorganic nanoparticles5 and hybrid porous solids.6 Among others, nanosized MOFs have 2


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recently attracted much attention as the drug nanocarriers with high drug loadings due to their high pore volume, large surface area and an easy modulation of the pore sizes with tunable organic groups within the frameworks.7 However, the previous reported works mostly focused on realizing one drug loaded into the MOF,8 which were ineffective on the treatment of cancers due to the multidrug resistance effect (MDR). It has been reported that combining the multiple drugs with different therapeutic effects could efficiently overcome MDR.9 Therefore, it was of great interest to construct the co-delivery system based on MOF, which had high drug loading content and simultaneously overcame MDR. Among the MOFs, zeolitic imidazolate framework ZIF-8 was particularly promising for the application as drug carrier as ZIF-8 was a nontoxic and biocompatible MOF built from zinc ions and 2-methylimidazolate, which was stable under physiological conditions while decomposed at the tumor sites with lower pH.10 Therefore, the rational design of pH-sensitive, multi-drugs co-delivered system based on ZIF-8 has great significance in achieving new efficient cancer therapy formulations. Doxorubicin hydrochloride (DOX) represented one of the first line chemotherapeutic drugs in the treatments of various human cancers such as breast cancer, ovarian cancer and acute lymphoblastic leukemia.11 However, the cytotoxic effects of DOX in tumor cells have been suppressed due to the MDR. The overexpressed P-glycoprotein (P-gp) as a typical drug efflux transport protein was thought as one of the major reasons for an efflux of anticancer drugs.12,13 Verapamil hydrochloride (VER) as P-gp inhibitor possesses the ability of reversing the drug resistance associated with P-gp when being used in combination with given anticancer drugs, resulting in increased local concentrations of drugs in MDR tumor cells for improved therapeutic 3



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efficacy.14 Therefore, the construction of new co-delivery system of DOX and VER is particularly important to overcome MDR in tumor cells for realizing efficient cancer therapy. Folate as a targeting ligand used for the delivery of pharmaceuticals has been extensively studied, which could be selectively recognized by their receptors (FRs) overexpressed on the cancer cell surfaces and stimulate the receptor mediated endocytosis thus to increase the tumor uptake of drugs while reduce the systemic toxicity.15,16 Polyethyleneglycol (PEG) was considered to be the best biocompatible material which was well known to prolong the circulation time.17 Therefore, PEG-FA was thought as one of the promising stabilizers to modify drug delivery systems to achieve the satisfied tumor-targeted therapy. Herein, we rationally designed a pH-responsive, PEG-FA functionalized multidrug delivery system (defined as PEG-FA/ (DOX+VER)@ZIF-8) to realize the active targeted delivery and overcome the MDR for an efficient anticancer ability. As revealed in Scheme 1, ZIF-8 drug co-delivery systems were prepared by an environmentally friendly one-pot process. Then, PEG-FA functionalized (DOX+VER)@ZIF-8 was constructed by coordination, which tended to accumulate at tumor tissues due to the EPR effect after intravenous injection. After that, the PEG-FA/ (DOX+VER)@ZIF-8 were internalized into tumor cells via FR-mediated endocytosis and played a toxic role toward cancer cells by the release of DOX. Meanwhile, the released VER blocked the efflux of DOX mediated by P-gp. The as-prepared PEG-FA/ (DOX+VER)@ZIF-8 with high drug loadings presented excellent stability and suitable particle size for EPR effect. The pH-sensitivity of ZIF-8 was validated and the results of in vitro cytotoxicity assay demonstrated that PEG-FA/ (DOX+VER)@ZIF-8 was highly cytotoxic towards B16F10 and 4


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multidrug resistant MCF-7 (MCF-7/A) cells. Furthermore, in vivo tumor inhibition efficacy and systemic toxicity of synthesized PEG-FA/ (DOX+VER)@ZIF-8 were evaluated via intravenous injection in mice bearing B16F10 melanoma. NIRF imaging with IR820 as a near-infrared probe indicated that the PEG-FA/ (DOX+VER)@ZIF-8 could increase the drug accumulations in tumors. Hence, this study offered a new path widening the applications of ZIF-8 for anticancer therapy with high anticancer effects and low side effects.

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Scheme 1. Schematic illustration of pH-responsive ZIF-8 as drug delivery vehicles: the synthesis of PEG-FA/ (DOX+VER)@ZIF-8; accumulation in tumors via the EPR effect; internalization by tumor cells via FR-mediated endocytosis; pH-sensitive drug release under the weak acidic environments of tumor cells; and multidrug resistant reversal mediated by VER.

2.

EXPERIMENTAL SECTION 6


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2.1.

Materials. 2-methylimidazole and Zn(NO3)2·6H2O of analytic grade were supplied by

Beijing J & K Technology Co., Ltd. PEG-FA (MW=2000 Da) was purchased from Shanghai Ponsure Biotech. Inc. Verapamil hydrochloride (VER, >99%) and doxorubicin hydrochloride (DOX, >98%) were provided by Dalian Mellon, Biological Technology Co., Ltd. 2.2.

Synthesis of (DOX+VER)@ZIF-8. The drug-loaded ZIF-8 was prepared based on

previous report with some modification.18 For the typical preparation of (DOX+VER)@ZIF-8, stock solutions of DOX (10 mg·mL−1) and VER (30 mg·mL−1) were prepared using deionized H2O. Firstly, 1 g of 2-methylimidazole (2-mim) was dissolved in 4 g of H2O, and 0.1 g of Zn(NO3)2·6H2O was dissolved in 0.4 g of H2O. Subsequently, the stock solution (2 mL) of DOX and VER was added to the Zn(NO3)2 solution. After this had been stirred for 5 min, the mixed solution was added dropwise to the 2-methylimidazole solution with stirring. After being stirred for 10 min, precipitation could be obtained utilizing a centrifuge, which was then being washed by deionized water for more than three times to completely get rid of the unreacted reactants. These operations were conducted under room temperature. Samples for dynamic light scattering (DLS) and transmission electron microscopy (TEM) examinations were prepared by re-dispersing some product in water. And then the product used for other characterization studies was dried thoroughly at room temperature under vacuum. Moreover, DOX@ZIF-8 and ZIF-8 were synthesized in a similar way for comparison, using 2 mL of DOX stock solutions or H2O. 2.3.

Preparation of PEG-FA/ (DOX+VER)@ZIF-8. The PEG-FA/ (DOX+VER)@ZIF-8

was prepared by the modification with PEG-FA on the surface of (DOX+VER)@ZIF-8 through the

formation

of

coordination

bonds

with

Zn2+.19-21

7


Briefly,

the

as-synthesized


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(DOX+VER)@ZIF-8 was dispersed in 10 mL of deionized H2O containing 100 mg of PEG-FA, which was firstly sonicated for 10 minutes and then agitated for 48 hours under room temperature. Afterwards, PEG-FA-capped (DOX+VER)@ZIF-8 could be obtained by the centrifuging of resulting solution. The precipitation was washed more than 3 times with deionized H2O to fully wash off unadsorbed PEG-FA. Samples for DLS and TEM characterizations were prepared by re-dispersing some product in water. And then the product used for other characterization studies was dried thoroughly at room temperature under vacuum. 2.4.

Characterization. The JEM-200CX transmission electron microscope was used for

TEM characterization. The average size and size distribution were detected using DLS (Brookhaven BI-200SM). The UV-vis absorption spectroscopy was conducted using a UV-vis spectrophotometer of TU-1810. Zeta potential was measured by the Zetasizer Nano ZS90 instrument (Malvern, Westborough, MA). Thermal gravimetric analysis (TGA) measurements were performed on the TGA/SDTA 851e equipment. X-ray powder diffraction (XRD) was conducted with the X'Pert3 Powder (PANalytical) using Cu Kα radiation over the 2θ range 5-50º. 2.5. In vitro pH-sensitive release of drugs from (DOX+VER)@ZIF-8. A typical release system was prepared to study the release behavior of (DOX+VER)@ZIF-8 in vitro. The obtained (DOX+VER)@ZIF-8 were directly dispersed into 5 mL of the phosphate buffer saline (PBS, pH = 7.4 and 5.0), which was then kept under stirring with the rate of 100 rpm at 37.0 °C. Supernatant (2 mL) was collected by centrifugation at each scheduled time and replaced by the same volume of fresh PBS. UV-vis spectrophotometry was used to determine the amount of 8


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DOX and VER that had been released. This release experiment of drugs was carried out in triplicate. 2.6.

Cell culture. The anticancer effects and cellular uptake of the prepared PEG-FA/

(DOX+VER)@ZIF-8 were evaluated on two kinds of cell lines including melanoma cells of mouse (B16F10) and breast cancer cells of human (MCF-7). The growth of these two kinds of cells was incubated with folate-free RPMI-1640 culture media containing fetal bovine serum (FBS, 10%). In addition, the medium with cells was kept in a humidified atmosphere of 37 °C with constant CO2 (5%). 2.7. In vitro cytotoxicity assay. The in vitro cytotoxicities of PEG-FA/ (DOX+VER)@ZIF-8, DOX@ZIF-8 and free DOX solution were conducted on FR-positive B16F10 and MCF-7/A cells using the MTT method. Briefly, cells (MCF-7/A and B16F10) were planted on the 96-well plates to modulate the cell count to be 5×103 per well. After being cultivated overnight, the culture media with cells were incubated with different concentrations of PEG-FA/ (DOX+VER)@ZIF-8, DOX@ZIF-8 or DOX solution for 24 h or 48 h, respectively. The concentration of DOX for all samples was prepared over the range of 0.01 µg·mL-1 to 50 µg·mL-1 (0.01, 0.1, 1, 10, 20 and 50 µg·mL-1).

Fresh

cell

medium

with

5

mg·mL-1

3-(4,

5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide (MTT) was applied to replace the medium after predetermined times. And then, additional incubation of 4 h was carried out on cells at the temperature of 37 °C. Thereafter, DMSO (100 µL) was added in each well of 96-well plate to make the purple formazan dissolve after the discard of supernatant. Microplate reader (ELIASA of Perkin Elmer) was used to read the absorbance of every well at 490 nm. For the research, the cytotoxicity 9



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experiment for all samples was repeated three times and cell inhibition rates presenting the cytotoxicity of PEG-FA/ (DOX+VER)@ZIF-8 were calculated as follows:

Cell inhibition rate (%) =

A positive - A sample × 100 % A positive - A blank

Where the culture media without drugs or cells represented the blank control, while the positive control group containing untreated cells displayed 100% viability. The IC50 values were then calculated, which were the concentrations of drugs inhibiting the viability of cells by 50%. 2.8. In vitro cellular uptake assay. MCF-7 and MCF-7/A cells (FR-upregulated) were studied to evaluate the cell uptake capacity for PEG-FA/ (DOX+VER)@ZIF-8. Cell uptake of the PEG-FA/ (DOX+VER)@ZIF-8 was assessed both qualitatively and quantitatively based on the inherent fluorescent property of DOX, compared with (DOX+VER)@ZIF-8, DOX@ZIF-8 and free DOX solution. Briefly, cells (MCF-7 and MCF-7/A) were cultured in 6-well microplates for two days with folate-free medium to the concentration of 2×105 cells per well. Afterwards, medium containing PEG-FA/ (DOX+VER)@ZIF-8, (DOX+VER)@ZIF-8, DOX@ZIF-8 or free DOX solution with the final concentration of DOX as 3 µg·mL-1 was used to replace the cell medium. After the incubation of 4 h at 37 °C, the cell medium was taken away. The cells were washed with fresh PBS for 3 times for the removal of the sample that had not been ingested by cells. Cells were imaged with a fluorescent inverted microscope (ECLIPSE-Ti, Nikon) to study the cell uptake ability qualitatively and the same parameters (the exposal time, intensity of exciting light, etc) were set for the microscopy of all samples. For the quantification of the cell 10


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uptake capacities of PEG-FA/ (DOX+VER)@ZIF-8, flow cytometer (BD FACSAria Ⅲ) was further used to measure the fluorescent signal intensity in cells quantitatively. All cells were harvested by the trypsinization after being rinsed with fresh PBS for 3 times, and then cells were dispersed in PBS for the examination of fluorescence signal intensity. 2.9.

Hemolysis

Assay.

To

determine

the

blood

compatibility

of

PEG-FA/

(DOX+VER)@ZIF-8 related to the feasibility for intravenous administration, a hemolysis study was carried out. The blood samples extracted from the New Zealand white rabbit were centrifuged and resuspended with saline solution to prepare the red blood cells suspension (RBC, 2%, v/v). Then, 0.15 mL PEG-FA/ (DOX+VER)@ZIF-8 with different concentrations (5, 10, 50, 100, 500, 1000 and 5000 µg·mL-1) were added into normal saline solution (1.1 mL) and incubated with 1.25 mL RBC suspension. The negative group contained 1.25 mL 2% RBC suspension and 1.25 mL saline which produced no hemolysis, while the positive control group was obtained by mixing 1.25 mL distilled water with 1.25 mL 2% RBC suspension which produced 100% hemolysis. All groups were incubated for 3 h under a constant temperature of 37.0 ± 1.0 °C, and then were centrifugated for 15 min with 1500 rpm. Afterwards, a UV-vis spectrophotometer was employed to measure the absorbance of the diluted supernatant at 576 nm known as the characteristic absorption wavelength of released haemoglobin from the hemolyzed RBC.22 All these tests were carried out for three times and the degree of hemolysis was obtained as follows:

Hemolysis (%) =

Asample-Anegative × 100% Apositive -Anegative 11



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Where Asample, Apositive and Anegative refered to the ultraviolet absorbancy of PEG-FA/ (DOX+VER)@ZIF-8 group, positive control group and negative control group, respectively. 2.10. Establishment of animal melanoma models. Healthy female Kunming mice with body weight of 18-22 g were purchased from Shandong University laboratory animal center (Jinan, China). All mice were under humane care throughout the studies. All experimental procedures were carried out on the basis of the requirements of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Animal Experiment Ethics Review of Shandong University. B16F10 cells were suspended in PBS to modulate the cell density at 1.0×107 cells per mL, which were kindly supplied from the Laboratory of Pharmacology, School of Pharmaceutical Science, Shandong University. The tumor model was established by subcutaneous injection of cell suspension (0.1 mL) with 1.0×106 B16F10 cells into the right anterior armpit of each mouse. Tumor progression was monitored by the measurements of the tumors along length with width using vernier calipers and the calculation equation of tumor volumes was as follows: 2 Tumor volume = L × W 2

Where the smallest and largest diameters of tumors were measured as W (mm) and L (mm), respectively.23 2.11. In vivo antitumor efficacy. Treatments were started after the volumes of tumors in mice reaching 50-100 mm3 (1 week after inoculation), and this day was designated as day 1. The antitumor efficacy of (a) normal saline, (b) free DOX, (c) (DOX+VER)@ZIF-8, and (d) PEG-FA/ (DOX+VER)@ZIF-8 was evaluated on the melanoma-bearing mice (n=5 per group). 12


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Each group was treated by tail vein injection once every 3 days until 13 days and the dose of DOX for all treatment groups was 2.0 mg per kg body weight.24 To study the treatment efficacy and safety of our formulations, the body weights and tumor sizes of mice of the four groups were monitored periodically during the treatment process. All the mice were euthanized two days after the last injection, and then the tumors were excised followed by the weighing. Photographs of the tumors from four groups’ mice were taken for a visual comparison. The antitumor efficacy of samples were displayed as the growth inhibition ratio of tumors, which was calculated as follows:25

Tumor inhibition ratio (%) =

(Wc - Wt) × 100% Wc

Where Wt and Wc represented the average tumor weight after the administration with treatment group and normal saline, respectively. 2.12. Histopathological evaluation. Mice were sacrificed at the end of chemotherapy treatment and tumors combined with individual organs (the kidney, heart, lung, liver, and spleen) were dissected to examine the in vivo systematic toxicity of our formulations. Formalin buffer solution (10%) was utilized for the fixation of freshly excised organs for 24 h immediately after those being rinsed with saline for further histological diagnosis. For the histopathological evaluation, hematoxylin and eosin (H&E) were employed to stain the paraffin-embedded tissue blocks that had been sliced. The NIKON Eclipse Ci light microscope was applied in pathologic observation and then photographs were taken. 2.13. In vivo and ex vivo NIRF imaging. Near-infrared optical imaging was carried out for 13



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the monitoring of the biological distribution of the PEG-FA modified ZIF-8 drug delivery system in vivo intuitively. IR820 was chosen as the indicator for in vivo imaging,26,27 and the PEG-FA/IR820@ZIF-8

was

prepared

through

the

similar

method

as

PEG-FA/

(DOX+VER)@ZIF-8. The biodistribution of the nano-scaled ZIF-8 was monitored after intravenous injection of PEG-FA/IR820@ZIF-8 into B16F10 inoculated mice melanoma models by the NIRF optical imaging method. Meanwhile, the NIRF signal of free IR820 (1.5 mg·kg-1) was also measured as control. When the tumor volumes reached approximately 150 mm3, the mice received tail vein injection of free IR820 and PEG-FA/IR820@ZIF-8. The mice were anesthetized with 10% chloral hydrate (i.p.), followed by the collection of fluorescent images using Xenogen IVIS Kinetic system at predetermined time post-injection (1, 2, 4, 8, 24 h), and then analyzed by IVIS Living Image 3.1 software. To compare the distributions of PEG-FA/IR820@ZIF-8 in tissues and tumors, the mice were sacrificed at the end of the imaging. Tumors combined with individual organs (the kidney, heart, lung, liver, and spleen) were carefully dissected and their fluorescence intensities were determined.

3.

RESULTS AND DISCUSSION TEM characterization (Figure 1a) was carried out for the as-prepared ZIF-8, DOX@ZIF-8,

(DOX+VER)@ZIF-8, and PEG-FA/ (DOX+VER)@ZIF-8 to study the morphology of our nanosized MOFs. The TEM images revealed that all of the samples exhibited uniform 14


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spherical-like morphology. Moreover, the PEG-FA/ (DOX+VER)@ZIF-8 particles were in well-dispersion, which demonstrated that the PEG-FA were capable of avoiding aggregation of (DOX+VER)@ZIF-8 particles effectively. The particle size and its’ distribution for all studied samples were further determined by means of the DLS. As shown in Figure 1b, the particle sizes were 147 nm for ZIF-8, 251 nm for DOX@ZIF-8, 138 nm for (DOX+VER)@ZIF-8 and 185 nm for PEG-FA/ (DOX+VER)@ZIF-8, respectively with narrow distributions (polydispersity index less than 0.1). It was clear that the particle size of PEG-FA/ (DOX+VER)@ZIF-8 was larger than that of (DOX+VER)@ZIF-8 due to the coating of PEG-FA. The zeta potential of ZIF-8, DOX@ZIF-8, (DOX+VER)@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8 was characterized to be 35.0 ± 6.22 mV, 18.7 ± 5.39 mV, 23.8 ± 4.69 mV and –34.5 ± 7.43 mV, respectively. The zeta potential of DOX@ZIF-8 and (DOX+VER)@ZIF-8 were lower than ZIF-8 because the surface of drug-loaded ZIF-8 were covered by a little DOX via strong adsorption.28 Compared with others, PEG-FA-coated nanosized MOFs showed a negative surface charge property, which resulted from the carboxylic ions of PEG-FA, confirming the successful adsorption of PEG-FA. Moreover, the PEG-FA/ (DOX+VER)@ZIF-8 exhibited high dispersibility and stability in water for several weeks, which could be attributed to its larger absolute value of zeta potential and the steric stabilization of PEG-FA molecules.

15



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Figure 1. (a) TEM images and (b) size distributions of (1) ZIF-8, (2) DOX@ZIF-8, (3) (DOX+VER)@ZIF-8, and (4) PEG-FA/ (DOX+VER)@ZIF-8. Figure S1 showed the XRD patterns of ZIF-8, DOX@ZIF-8, (DOX+VER)@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8. It demonstrated that all of the particles were of high crystallinity, confirming the formation of ZIF-8 according to the published crystal structure data.29 The XRD data of DOX@ZIF-8 and (DOX+VER)@ZIF-8 showed that the structural integrity of ZIF-8 was unaltered after the load of drugs. In contrast, the peak intensity at low angles of PEG-FA/ (DOX+VER)@ZIF-8 decreased because of the coating of amorphous PEG-FA. The thermal gravimetric analysis (TGA) was further studied for ZIF-8, DOX@ZIF-8, (DOX+VER)@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8 (Figure S2). The TGA results of ZIF-8 were similar to the previous reported data.30 The high thermal stabilities of the prepared MOF could be indicated by the long plateau over the temperature ranging from 200 °C to 365 °C. The thermogravimetric analysis of DOX@ZIF-8 showed three weight-loss stages. The weight loss of ca. 20% in the 16


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193-365 °C range originated from the loss of DOX molecules.31 As for (DOX+VER)@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8, the weight loss of VER and PEG-FA occurred combined with the decomposition of DOX and ZIF-8 proceeding in the temperature range of 193-365 °C and above 365 °C, respectively. The drug loading content of (DOX+VER)@ZIF-8 was evaluated utilizing UV-vis spectrophotometry after being fully disintegrated in hydrochloride solution. The drug loading contents of DOX and VER were determined to be approximately 8.9% and 32%, respectively. Such high loading capacity for the two drugs was superior to the traditional drug delivery system, suggesting ZIF-8 as a promising nano-carrier for drug co-delivery. In order to verify the pH-response release behavior of (DOX+VER)@ZIF-8, the in vitro release was performed in PBS with pH values of 5.0 and 7.4. PH 7.4 represented the physiological conditions with neutral pH, while pH 5.0 condition was chosen because it represented the acidic condition of endosome in tumor cell.32 The (DOX+VER)@ZIF-8 in pH 7.4 and pH 5.0 PBS both exhibited the obvious sustained release behaviors for DOX and VER (Figure S3).18 Furthermore, it could be seen that the (DOX+VER)@ZIF-8 displayed faster release in the PBS with pH value of 5.0 compared with that in PBS of pH 7.4 for both DOX and VER. For example, only (9.68 ± 1.25) % of loaded DOX and (18.18 ± 0.74) % of VER has been released from the drug carrier after being immersed in pH 7.4 PBS for 24 hours. In comparison, (27.37 ± 0.92) % of DOX and (76.48 ± 0.68) % of VER were released in the PBS of pH 5.0 over the identical time period. After 132 h, (41.61 ± 2.06) % of loaded DOX was released cumulatively in PBS of pH 5.0 while that in pH 7.4 PBS was only (29.81 ± 2.53) %. The pH-responsive release behavior was because of the dissolution in 17



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acid environment for ZIF-8,33 and the release rate of VER was higher than that of DOX in the same release condition due to the formation of coordination bonding Zn (II)-DOX.34 Therefore, this pH-responsive release profile for drugs enable (DOX+VER)@ZIF-8 to be used in clinic, leading to a controlled drug co-delivery behavior for enhanced cytotoxicity towards tumor cells as well as the lowest side effects in normal physiological conditions. In an effort to demonstrate the anticancer activity of PEG-FA/ (DOX+VER)@ZIF-8, the cytotoxicity assay in vitro was conducted on B16F10 and MCF-7/A cells. For comparison, free DOX and DOX@ZIF-8 were also studied. As shown in Figure 2, the inhibition rates of all samples against B16F10 and MCF-7/A cells increased as the DOX concentration raised from 0.01 to 50 µg·mL-1. The inhibition rates of nanoparticle formulations (DOX@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8) were higher than that of free DOX to both B16F10 and MCF-7/A cells. Free drugs are mainly taken in cells via passive diffusion. However, nano-drug delivery systems can be taken in by cells via endocytosis, resulting in higher cellular uptake of the drugs.35 Therefore, the drug-loaded carriers usually show higher inhibition rates than free drugs. Moreover, it was found that the inhibition rates of PEG-FA/ (DOX+VER)@ZIF-8 were higher than that of DOX@ZIF-8 towards both B16F10 and MCF-7/A cells. On one hand, the folate component in PEG-FA/ (DOX+VER)@ZIF-8 could bind efficiently with FRs markedly up-regulated in B16F10 and MCF-7/A cells, so as to largely increase the cellular uptake of anticancer agents into cancer cells. On the other hand, for MCF-7/A cells, VER was able to overcome the drug efflux mediated via the overexpressed membrane P-gp on MDR cancer cells, increasing intracellular concentrations of DOX. Therefore, PEG-FA/ (DOX+VER)@ZIF-8 18


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showed the best inhibition rates for MCF-7/A as well as B16F10 cells. To

compare

the

cytotoxicity

exhibited

by

DOX,

DOX@ZIF-8

and

PEG-FA/

(DOX+VER)@ZIF-8 on tumor cells intuitively, the half maximal inhibitory concentrations (IC50 values) were calculated and presented in Table 1. The IC50 values of DOX@ZIF-8 at 24 h and 48 h were calculated to be coincidently same (0.156 µg·mL-1) on B16F10 cells, which were much lower than those of free DOX (0.445 µg·mL-1 at 24 h and 0.270 µg·mL-1 at 48 h). The poor efficiency of nonspecific endocytosis,25 and the efflux of released DOX by the P-gp hampered the significant improvement of the cytotoxicity of DOX@ZIF-8 at 48 h compared with that at 24 h. The IC50 values of PEG-FA/ (DOX+VER)@ZIF-8 on B16F10 cells (0.036 µg·mL-1 at 24 h and 0.022 µg·mL-1 at 48 h) were much lower than those of DOX@ZIF-8. The PEG-FA/ (DOX+VER)@ZIF-8 also exhibited the lowest IC50 values on MCF-7/A cells among PEG-FA/ (DOX+VER)@ZIF-8, DOX@ZIF-8 and free DOX samples. In summary, it was indicated clearly by the results of cytotoxicity assay that the PEG-FA/ (DOX+VER)@ZIF-8 could suppress the cell viability effectively towards MCF-7/A as well as the B16F10 cells.

19



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Figure 2. The cell inhibition rates of DOX, DOX@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8 to B16F10 and MCF-7/A cells after the incubations of 24 h and 48 h (0.01 ≤ *p < 0.05, **p < 0.01, n=3).

Table 1. IC50 values of different samples against B16F10 and MCF-7/A cells after 24 h and 48 h incubations. IC50 (µg·mL-1) Formulation 24h B16F10 cells 20


48h

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Free DOX

0.445

0.270

DOX@ZIF-8

0.156

0.156

PEG-FA/ (DOX+VER)@ZIF-8

0.036

0.022

Free DOX

7.467

5.912

DOX@ZIF-8

5.014

4.512

PEG-FA/ (DOX+VER)@ZIF-8

2.187

0.949

MCF-7/A cells

Internalization of drug nanocarriers into cells was the critical process for chemotherapeutic agents to achieve the effective anticancer efficacy. The cellular uptake of PEG-FA/ (DOX+VER)@ZIF-8 on MCF-7 cells was evaluated. For comparison, free DOX and (DOX+VER)@ZIF-8 were also studied. Figure 3a showed images of the internalization of the studied samples by fluorescence inverted microscopy. It could be seen that PEG-FA/ (DOX+VER)@ZIF-8 showed higher intensity of red fluorescence than (DOX+VER)@ZIF-8 in MCF-7 cells. The remarkable enhancement of cellular uptake exhibited by PEG-FA/ (DOX+VER)@ZIF-8 was ascribed to the specific binding between folate and overexpressed FA-receptors on the cells. In contrast, the fluorescent intensity of PEG-FA/ (DOX+VER)@ZIF-8 in the presence of 1 mM free folate36 was notably weaker than that of PEG-FA/ (DOX+VER)@ZIF-8 without free folate. In this case, FR-mediated endocytosis of the PEG-FA functionalized nanoMOF was blocked efficiently by free folate, which was in competition with

21



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PEG-FA/ (DOX+VER)@ZIF-8 to bind with the FRs on cell surfaces, resulting in saturation of the receptors. The competitive binding with FRs proved that the enhanced cellular uptake of PEG-FA/ (DOX+VER)@ZIF-8 was directly associated with FR-mediated endocytosis. Flow cytometry analysis was further applied to confirm the accumulations of DOX quantitatively in MCF-7 cells incubated with different formulations. The results were shown in Figure 3a and were quite compatible with the consequence acquired by fluorescence microscope. The results could clearly evidence that the uptake of DOX was higher for cells (MCF-7) administrated with folate-targeted drug delivery systems compared with being administrated with non-folate pharmaceutical preparation, indicating the successful improvement of the uptake for drugs into tumor cells with overexpressed FR utilizing the folate-modified ZIF-8. Furthermore, the cell uptake was evaluated on MCF-7/A cells with overexpressed P-gp to investigate whether the VER added in PEG-FA/ (DOX+VER)@ZIF-8 contributed to the enhancement of cell uptake into MDR cells. Figure 3b displayed the drug accumulation results of DOX into MCF-7/A cells qualitatively and quantitatively. When treated with free DOX, almost none fluorescence could be observed, suggesting that DOX solution alone was difficult to penetrate the cell membrane and the overexpressed P-gp on MCF-7/A cells could cause the reflux of free drugs easily. By contrast, cells cultivated with (DOX+VER)@ZIF-8 and DOX@ZIF-8 emitted strong fluorescence, indicative of much more efficient cellular uptake of DOX. This result could be explained by the different cellular uptake routes for the studied samples. Passive diffusion mechanisms affected by efflux transport protein (P-gp) were the main path for free DOX to enter cytoplasm, yet nano-scaled carriers could benefit intracellular uptake through endocytotic pathways which was 22


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effective for drugs to circumvent the efflux associated with MDR. As a result, (DOX+VER)@ZIF-8 and DOX@ZIF-8 displayed more accumulation of drugs fast within tumor cells than that of free DOX. By comparison, the cells treated with (DOX+VER)@ZIF-8 emitted stronger fluorescence than that treated with DOX@ZIF-8 after being incubated for 4 h, which resulted from the VER-induced P-gp inhibition. As shown in Figure 3b, the quantitative result acquired by flow cytometry indicated that the cellular uptake of (DOX+VER)@ZIF-8 into MCF-7/A cells was 6.49-times and 2.69-times as high as that of DOX solution and DOX@ZIF-8, respectively. The study indicated that the accumulation of DOX in MCF-7/A cells was seriously inhibited by P-gp efflux when there was no VER in the formulation. However, the accumulation of DOX could be largely increased by the encapsulation of VER along with DOX in MOF due to the VER-induced P-gp inhibition. The cell uptake results agree well with the results of cytotoxicity in vitro.

23



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Figure 3. (a) Fluorescence microscopy images and flow cytometry results of MCF-7 cells after incubation with (A1) blank, (A2) DOX, (A3) (DOX+VER)@ZIF-8, (A4) PEG-FA/ (DOX+VER)@ZIF-8 with 1 mM free folate and (A5) PEG-FA/ (DOX+VER)@ZIF-8 without free folate for 4 h, (cDOX = 3 µg·mL-1). (b) Fluorescence microscopy images and flow cytometry results of MCF-7/A cells after incubation with (B1) blank, (B2) DOX, (B3) DOX@ZIF-8 and (B4) (DOX+VER)@ZIF-8 for 4 h, (cDOX = 3 µg·mL-1). Before the in vivo experiment, hemolytic study was performed to evaluate the biocompatibility and security of PEG-FA/ (DOX+VER)@ZIF-8 as formulations for intravenous administration. The result in Figure S4 revealed that the PEG-FA/ (DOX+VER)@ZIF-8 with the concentration ranging from 5 µg·mL-1 to 500 µg·mL-1showed negligible hemolytic potential (less than 1%) to RBCs after incubation of 3 h. The PEG-FA/ (DOX+VER)@ZIF-8 showed an increased 24


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hemolytic toxicity as the concentration exceeded 1000 µg·mL-1, while the vast majority of RBCs still maintained intact even at high concentration of 5000 µg·mL-1, resulting in low hemolysis ratios less than 5%. Pharmaceutical formulation with hemolysis ratio below 10% was viable to be

administrated

via

intravenous,37

demonstrating

the

feasibility

of

PEG-FA/

(DOX+VER)@ZIF-8 for intravenous injection. To verify the capability of PEG-FA/ (DOX+VER)@ZIF-8 as an efficient drug delivery system for in vivo tumor inhibition, therapeutic efficacy was evaluated on Kunming mice bearing melanoma. For comparison, the groups of normal saline, free DOX and (DOX+VER)@ZIF-8 were also studied. A series of researches on the tumor volume, tumor weight, the average tumor inhibition ratio and the body weight of mice were carried out (Figure 4). Figure 4a displayed the tumor volume monitored during the treatment with saline, DOX solution, (DOX+VER)@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8. It could be seen that all of these treatment groups suppressed the growth of tumors compared to normal saline group. Among them, mice of PEG-FA/ (DOX+VER)@ZIF-8 group exhibited the smallest tumor volumes. To further compare the antitumor effects of different samples, the tumors dissected after the last injection were photographed and weighed. Thus, the tumor inhibition ratios of the four groups could be calculated. The photograph of excised tumors from each group after treatment was shown in Figure 4b while the mean tumor weight and the average tumor inhibition ratios of each group were displayed as Figure 4c. Mice injected with PEG-FA/ (DOX+VER)@ZIF-8 showed obviously smaller tumor sizes than those treated with other samples (Figure 4b). Based on the tumor weight in Figure 4c, the tumor inhibition ratio of PEG-FA/ (DOX+VER)@ZIF-8 was 25



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Page 26 of 40

calculated to be 95.4% compared with 84.3% of (DOX+VER)@ZIF-8 groups and 56.8% of DOX group (Figure 4c). Therefore, it was obvious that the PEG-FA/ (DOX+VER)@ZIF-8 showed the best tumor inhibition ratio among the studied formulations. Free DOX expressed relatively low therapeutic efficacy in vivo as a result of the non-specific distribution and premature elimination by kidney.38 In contrast with other formulations, our prepared PEG-FA/ (DOX+VER)@ZIF-8 had the greatest in vivo therapeutic efficacy, ascribed to the EPR effects, pH-responsive drug release, the active targeting effect mediated by folate and the MDR reversal via VER. The safety of samples was studied in vivo by the monitoring of weight for mice during the experiment and the results were exhibited in Figure 4d. It was remarkable that severe weight loss took place for mice treated with DOX solution, while the (DOX+VER)@ZIF-8 group and PEG-FA/ (DOX+VER)@ZIF-8 group gained weight slightly after treatment like the normal saline group. This result demonstrated that our as-prepared formulations were well-tolerated at the tested dosage level, overcoming the inherent toxicity of DOX. Therefore, our drug co-delivery system of PEG-FA/ (DOX+VER)@ZIF-8 was more safe and more efficient in antitumor therapy compared with the direct administration of free DOX and the (DOX+VER)@ZIF-8.

26


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Figure 4. (a) Tumor volume after treatment with DOX, (DOX+VER)@ZIF-8, PEG-FA/ (DOX+VER)@ZIF-8, or normal saline. (b) Photograph of excised tumors from each group after treatment. (c) Mean tumor weight and the average tumor inhibition ratios of each group. (d) Changes in relative body weights of each group. To further investigate the systematic toxicity of PEG-FA/ (DOX+VER)@ZIF-8, histological assessments were performed. For comparison, the groups of normal saline, free DOX and (DOX+VER)@ZIF-8 were also studied. The morphology of sectioned organs from mice treated with the studied groups by staining with hematoxylin and eosin (H&E) was displayed in Figure 5. Obvious pathological damage could not be found at the tumor tissues of the normal saline group. 27



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However, aggravating necrosis of cells, detectable atypia of nucleus, and inferior dyeing of chromatin were shown at the tumor tissues treated with DOX, (DOX+VER)@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8 groups. While the PEG-FA/ (DOX+VER)@ZIF-8 group showed the most obvious atypia of the cancer cells, indicating its optimal efficacy. The main organs (kidney, heart, lung, liver and spleen) experienced negligible changes in cellular integrity and tissue morphology for (DOX+VER)@ZIF-8 and PEG-FA/ (DOX+VER)@ZIF-8 groups compared with the normal saline group. For the DOX group, abnormal morphology was observed in heart due to the intrinsic cardiotoxicity of free DOX.39 These results indicated the meaningful antitumor effects and reduced systematic toxicity of our prepared PEG-FA/ (DOX+VER)@ZIF-8. These results were mainly related to the sustained release and targeting effect of PEG-FA/ (DOX+VER)@ZIF-8, revealing that the typical biocompatible PEG-FA/ (DOX+VER)@ZIF-8 has the promise of becoming a multidrug co-delivery system in tumor treatment with minor side effects to normal tissues.

28


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Figure 5. H&E stained images of tumors and major organs after treatment with normal saline, DOX, (DOX+VER)@ZIF-8, PEG-FA/ (DOX+VER)@ZIF-8. The scale bar was 100 µm. To study the targeted behaviors of PEG-FA/ZIF-8 in vivo, the mice bearing tumors derived from B16F10 cells were administered with IR820 labeled PEG-FA/ZIF-8 and free IR820 intravenously, which were then monitored using a NIRF optical imaging system. The in vivo pseudocolor-adjusted fluorescence images of mice at 1, 2, 4, 8 and 24 h following the injection of free IR820 or PEG-FA/IR820@ZIF-8 were shown in Figure 6a. The mice injected with PEG-FA/IR820@ZIF-8 exhibited higher intensity of fluorescence at the tumor sites than that injected with free IR820 during the whole observation process, especially 4 h after injection. We noted that the fluorescent signal for free IR820 group decreased with time. Compared with free IR820, strong fluorescent signals could still be detected in mice especially at the area of tumors 24 h after the injection of PEG-FA/IR820@ZIF-8. This indicated the half-life time of PEG-FA/IR820@ZIF-8 was prolonged in the systemic circulation, which was attributed to the 29



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modification

with

PEG

chain

block.40,41

The

enhanced

Page 30 of 40

fluorescent

signal

of

PEG-FA/IR820@ZIF-8 at the tumor sites represented increased accumulations of PEG-FA/ZIF-8 at tumors, as a result of the EPR effect and folate mediated active targeting. Importantly, ex vivo NIRF

imaging

results

of

dissected

tumors

and

organs

for

IR820

group

and

PEG-FA/IR820@ZIF-8 group 24 h after the treatment were shown as Figure 6b, illustrating the stronger fluorescence of PEG-FA/IR820@ZIF-8 group in the tumor than that of free IR820 group. Liver, as the primary metabolic organ, exhibited the excellent accumulations of both IR820 as well as PEG-FA/IR820@ZIF-8. However, fluorescent intensities in liver were weaker in the cases of PEG-FA/IR820@ZIF-8 injected mice relative to the cases of free IR820 injected mice, which was due to the tumor-targeted effects for PEG-FA/IR820@ZIF-8. Thus, our designed drug co-delivery system of PEG-FA/ (DOX+VER)@ZIF-8 exhibited maximized therapeutic efficacy and minimized side effects such as toxicity to other normal organs.

30


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Figure 6. (a) In vivo fluorescence imaging of B16F10 bearing mice at 1, 2, 4, 8 and 24 h after the injection of free IR820 or PEG-FA/IR820@ZIF-8. The decrease of fluorescent intensities was indicated by the color bar from red to blue. (b) Fluorescence imaging of major organs and tumors taken from mice treated with IR820 or PEG-FA/IR820@ZIF-8 at 24 h post-injection, respectively.

4.

CONCLUSIONS In summary, a co-delivery of DOX/VER by PEG-FA functionalized ZIF-8 nanocarriers was

realized for the first time by a one-pot process for active targeted drug delivery in addition to the 31



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Page 32 of 40

overcoming of multidrug resistance, which led to an efficient anticancer effectiveness. Our PEG-FA/ (DOX+VER)@ZIF-8 showed uniform size and high stability. It exhibited excellent drug loading contents of DOX and VER with the pH-sensitive release characteristic. The PEG-FA/ (DOX+VER)@ZIF-8 showed the negligible hemolytic activity and minor toxicity on normal organs, demonstrating its excellent biocompatibility and safety as intravenous drug delivery system. PEG-FA/ (DOX+VER)@ZIF-8 showed remarkably enhanced therapeutic efficiencies compared with the direct administration of free DOX and other formulations such as DOX@ZIF-8 both in vivo and in vitro. The enhanced cytotoxicity for killing the cancer cells resulted from the improved internalizing of DOX caused by the FR-mediated endocytosis and VER-mediated multidrug resistance reversal. The cell uptake and NIRF imaging results indicated that the PEG-FA/ (DOX+VER)@ZIF-8 could increase the drug accumulations in tumors for targeted cancer therapy. Accordingly, the PEG-FA/ (DOX+VER)@ZIF-8 drug co-delivery system can be used as a promising efficient formulation in reversing the multidrug resistance for targeted cancer therapy. Therefore, our constructed pH-sensitive and tumor-targeting drug co-delivery system based on the MOF nanocarriers provides a promising platform in cancer treatment for reversing the multidrug resistance with enhanced therapeutic efficacy and reduced side effects, broadening the applications of MOF in biomedical field.

ASSOCIATED CONTENT Supporting Information XRD pattern of ZIF-8, DOX@ZIF-8, (DOX+VER)@ZIF-8, and PEG-FA/ (DOX+VER)@ZIF-8 32


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(Figure S1). TGA curves of ZIF-8, DOX@ZIF-8, (DOX+VER)@ZIF-8, and PEG-FA/ (DOX+VER)@ZIF-8 (Figure S2). The in vitro release profiles of DOX and VER from (DOX+VER)@ZIF-8 in different PBS (pH 7.4 and 5.0) (Figure S3). The hemolysis ratio of the PEG-FA/ (DOX+VER)@ZIF-8 at different concentrations (Figure S4).

AUTHOR INFORMATION Corresponding Authors ∗

E-mail: [email protected]. Tel: (86) 531-88382007. Fax: (86) 531-88382548.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (NSFC, No. 21373126 and No. 21673128).

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Rational Design of Metal Organic Framework Nanocarrier-Based

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