Improving Tumor Specificity and Anticancer Activity of Dasatinib by

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Improving tumor specificity and anticancer activity of dasatinib by dual-targeted polymeric micelles Qing Yao, Jong Hoon Choi, Zhi Dai, Jiao Wang, Dongin Kim, Xing Tang, and Lin Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12233 • Publication Date (Web): 29 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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ACS Applied Materials & Interfaces

Improving Tumor Specificity and Anticancer Activity of Dasatinib by Dual-targeted Polymeric Micelles Qing Yao†, ‡, Jong Hoon Choi‡, Zhi Dai‡, Jiao Wang‡, Dongin Kim‡, Xing Tang†,* and Lin Zhu‡,* †

‡

DEPARTMENT OF PHARMACEUTICS, COLLEGE OF PHARMACY, SHENYANG PHARMACEUTICAL UNIVERSITY, SHENYANG, LIAONING, 110016, CHINA

DEPARTMENT

OF

PHARMACEUTICAL SCIENCES, IRMA LERMA RANGEL COLLEGE

OF

PHARMACY, TEXAS A&M UNIVERSITY HEALTH SCIENCE CENTER,

KINGSVILLE, TEXAS 78363, UNITED STATES

Keywords: multifunctional nanocarrier, polymeric micelle, tumor targeting, matrix metalloproteinase, folate receptor, dasatinib.

* Corresponding author: Xing Tang, Ph.D. Professor, Department of Pharmaceutical Sciences, Shenyang Pharmaceutical University Wen Hua Road, No. 103 Shenyang, Liaoning, 110016, China Tel: +86 24 23986343 Fax: +86 24 23911736. E-mail: [email protected] Lin Zhu, Ph.D. Assistant Professor, Department of Pharmaceutical Sciences, Irma Lerma Rangel College of Pharmacy, Texas A&M University Health Science Center 1010 West Ave. B, MSC 131, Kingsville, Texas 78363, USA Phone: (361)221-0757 Fax: (361)221-0793 Email: [email protected]

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Abstract To improve tumor targetability and drug efficacy and decrease drug resistance of dasatinib (DSB), the multifunctional micellar nanoparticles that combined the matrix metalloproteinase 2 (MMP2)-sensitive tumor (site) targeting with folate receptor-mediated tumor (cell) targeting were developed. Two major functional polymers, polyethylene glycol (5000 Da) - MMP2sensitive peptide - phosphoethanolamine (PEG5k-pp-PE) and folic acid - polyethylene glycol (2000 Da) - phosphoethanolamine (FA-PEG2k-PE), were synthesized to construct the dualtargeted micellar nanoparticles (MMP/FR micelles). In the absence of MMP2, the FA was shielded by PEG5k and the MMP/FR micelles showed low bioactivity. In the presence of MMP2, the nanoparticulate structure, stability, and cargo release profile of the MMP/FR micelles were not significantly affected, however, the MMP2-mediated PEG5k deshielding and FA exposure remarkably increased the cellular uptake and anticancer activity of the micelles in the MMP2 and FR expressing (MMP2+/FR+) cells, including multidrug resistant (MDR) cancer cells, rather than the MMP2- and/or FR- cells. In the 3D MDR tumor spheroids, the significant MMP2dependent tissue penetration, uptake and cytotoxicity of the MMP/FR micelles were also observed. Furthermore, in the in vivo biodistribution study, the MMP2 and FR dual targeting strategy could significantly prolong the systemic circulation, decrease the nonspecific distribution in non-tumor tissues, and increase the tumor accumulation of the polymeric micelles in a melanoma xenograft mouse model. The MMP2-sensitive FR-targeted micelles might have great potential as a tumor-targeted platform for delivery of molecular targeted therapeutics. Keywords: multifunctional nanocarrier, polymeric micelle, tumor targeting, matrix metalloproteinase, folate receptor, dasatinib.


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Introduction Cancer remains one of the major challenges although a lot of efforts have been made in past several decades, as evidenced by various clinical interventions and anticancer therapeutics. The unsatisfactory outcome of drug treatment is because of not only the drugs’ low efficacy to stop the tumor growth and progression, but also the serious side effects, drug resistance and cancer relapse, especially for chemotherapy drugs. Among various newly emerged cancer therapeutics, the molecularly targeted cancer therapy gradually replaces chemotherapy and becomes a major option for treating human cancers.1 The molecularly targeted therapy drugs that inhibit the tumor growth by interfering with various cancer-specific intracellular or membrane-anchored biomolecules and their signaling pathways, rather than exert cytotoxicity to all rapidly dividing cells, are considered to have higher cancer selectivity and better tolerance than traditional chemotherapy. However, the growing evidence shows that the molecularly targeted therapeutics may face the similar issues as other anticancer drugs, such as poor solubility, low bioavailability, insufficient tumor specificity, and drug resistance. 1-2 Nanoparticle-based drug delivery systems (NDDS) have shown various advantages over naked drugs in treating human cancers, including the increased drug solubility and stability, improved pharmacokinetics, enhanced permeability and retention (EPR) effect-mediated tumor targeting, and capability of further engineering to impart various functionalities. PEGylation is the most commonly used strategy for prolonging the residence time of drugs/nanoparticles in the bloodstream, so as to facilitate the EPR-mediated “passive” tumor targeting. In addition, further engineering of drug nanocarriers with the cancer-specific ligands, such as antibodies, peptides and aptamers, as well as small molecules, e.g. mannose and folic acid (FA), could impart the “active” tumor targeting mechanism to the nanoparticles for the improved tumor specificity.3 Folate receptors (FRs) are abundantly expressed on many cancer cells, including both solid tumors, e.g. ovarian cancer, breast cancer, etc., and hematological malignancies, e.g. multiple myeloma, etc., as one of the major mechanisms supportive of rapid cell growth 4. The FR overexpression provides the rationale for the development of FR-targeted therapeutics and various FR-mediated cancer-targeting strategies have been investigated in the preclinical and clinical studies. Currently, several FR-targeted therapeutics are under clinical investigation 5. FA or folate, as the native ligand of FRs, binds to FRs with high affinity 6. Obviously, compared to the antibody-based therapeutics, the small molecular size, simple chemistry, lack of immunogenicity, broader spectrum, and low costs, make folate an ideal molecule for the preparation of FR-targeted tumor-specific therapeutics 7. FA modified liposomes 8, polymeric micelles 9, albumin nanoparticles 10, conjugates11, and other nanoparticles12-13 have been developed for delivery of a wide range of cargo, including small moleucles, biologicals, and



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imaging agents. However, the FR-mediated tumor targeting approach is not satisfactory. The FA-modified nanoparticles often show the suboptimal tumor accumulation due to unintended uptake by normal tissues/cells expressing FRs. For instance, some normal tissues, such as lung and kidney, contain the FR-expressing cells, increasing the risk of off-target toxicity. The cancer targetability of FR-targeted therapeutics has to be further improved. Matrix metalloproteinase 2 (MMP2), a major enzyme responsible for cancer initiation, growth and metastasis, is up-regulated in many cancer tissues 14. It is secreted by cancer cells and cancer associated cells, e.g. stromal cells 15 or macrophages 16. In addition to residing in the tumor extracellular matrix, the secreted MMP2 binds to cancer cells 17 and endothelial cells of blood vessels 18 to facilitate cancer invasion and angiogenesis. MMP2 has been used as a biomarker for cancer diagnosis and prognosis and been also investigated as a therapeutic target 14. Recently, MMP2 has been used as a stimulus for tumor-targeted delivery of imaging agents and drugs by us 19-22 and others 23-24. In this study, we sought to design a dual-targeted multifunctional micellar nanocarrier by combining the MMP2-sensitive tumor site-targeting with FR-mediated cancer cell-targeting, to improve the tumor specificity and overcome the drug resistance of dasatinib (DSB), an FDAapproved anticancer tyrosine kinase inhibitor (TKI) with dual PDGFR and SFK inhibitory effects (Figure 1). Three polymers were used to build the micellar nanoparticles: the MMP2-sensitive polymer (PEG5k-pp-PE), FR-targeted polymer (FA-PEG2k-PE), and micelle building block (PEG2k-PE). Here, the synthesis of the functional polymers and preparation of the polymeric micelles were described. The physicochemical properties (particle size, zeta potential and morphology), micelle stability, MMP2 sensitivity, and drug release of the micelles were studied. The cellular uptake, tissue penetration, cytotoxicity and drug resistance of the micelles were examined in both cancer cell monolayers and 3D spheroids. Furthermore, the preliminary in vivo biodistribution study was performed in the tumor-bearing mice.


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Figure 1 MMP2-sensitive FR-targeted delivery of dasatinib to tumor cells for the improved tumor specificity and decreased drug resistance.



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1. Material and methods 1.1. Materials Polyethylene glycol 5000-succinimidyl valerate (PEG5k-SVA) and maleimide-PEG2k-DSPE (MAL-PEG2k-PE) were purchased from Laysan Bio, Inc. (Arab, AL). 1, 2-dioleoylsn- glycero3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE) and 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 1-Hydroxybenzotriazole hydrate (HoBt) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Chem-Impex International, Inc. (Wood Dale, IL). Chloroform, dichloromethane (DCM), dimethylformamide (DMF), acetonitrile (ACN), and methanol were purchased from Thermo Fisher Scientific (Rockford, IL). N, N'Dicyclohexylcarbodiimide (DCC), polysorbate 80 (Tween 80), L-Cysteine ethyl ester hydrochloride, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) were from Alfa Aesar (Ward Hill, MA). 1,1'-dioctadecyl-3,3,3',3'- tetramethylindotricarbocyanine iodide (DiR) were from Marker Gene Technologies, Inc (Eugene, OR). The MMP2-cleavable peptide, pp (PLGIAG), was synthesized by Sigma BioSciences Inc. (St. Louis, MO). Collagenase Type IV, molybdenum blue spray reagent, and folic acid (FA) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). Bovine serum albumin (BSA) and TLC plate (silica gel 60 F254) were from EMD Biosciences (La Jolla, CA). Dialysis tubings (MWCO 8000 and 12000-14000 Da) were purchased from Spectrum Laboratories, Inc. (Houston, TX). Fetal bovine serum (FBS) was purchased from VWR International (Radnor, PA). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute medium (RPMI)-1640, penicillin-streptomycin solution, and trypsin-EDTA were from Invitrogen Corporation (Carlsbad, CA). Hank’s Balanced Salt Solution (HBSS) was from Mediatech (Manassas, VA). Agarose was purchased from Amresco (Solon, OH). The human breast cancer (MCF-7 and MDA-MB-231) and MDR ovarian cancer (NCI/ADRRES) cells were grown in the DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS at 37 ºC in a 5% CO2. The human non-small cell lung cancer (A549), cervical cancer (HeLa) and murine melanoma (B16F10) cells were grown in the RPMI1640 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS at 37 ºC in a 5% CO2. 1.2. Synthesis of PEG5k-pp-PE and FA-PEG2k-PE The PEG5k-pp-PE was synthesized by a previously reported method.25 First, the PEG5k-SVA was reacted with the MMP2-cleavable peptide (pp) in the presence of a trace amount of triethylamine at room temperature overnight. The reaction mixture was purified by the dialysis (MWCO 3500 Da) against water for 48 h, followed by lyophilization. Then, the obtained PEG5k-pp was activated by the excess amount of the coupling reagents (DCC/HOBT) and conjugated with DOPE in DMF in the presence of triethylamine at room temperature overnight. The product PEG5k-pp-PE was purified by the preparative TLC (chloroform/methanol, 4:1, v/v).


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The final product PEG5k-pp-PE was lyophilized and characterized by the TLC and 1H NMR spectroscopy in deuterated chloroform (CDCl3). The FA-PEG2k-PE was synthesized according to the method 26 with modifications. Two steps were involved. First, the FA was activated by DCC, then reacted to the L-cysteine ethyl ester hydrochloride (Cys) in the DMSO/ pyridine mixture (5/2, v/v) at room temperature overnight. The FA-Cys was purified by the preparative TLC. Then, the FA-Cys was reacted with maleimide (Mal)-PEG2k-PE, followed by the dialysis (MWCO 8000 Da) against water. The FA-PEG2k-PE was lyophilized and characterized by the TLC and 1H NMR in deuterated DMSO (D6-DMSO). The ultraviolet–visible (UV-Vis) absorbance spectra of FA, PEG2k-PE and FA-PEG2k-PE were compared. 1.3. Preparation of polymeric micelles The DSB-loaded micelles were prepared by the modified film-hydration method. The formulations of the micellar nanoparticles were listed in Table 1. Briefly, DSB and polymers were dissolved in methanol and dried to form a drug-polymer film. The film was hydrated in HBSS by probe-sonication on ice for five minutes. The unentrapped DSB was removed by filtration through a 0.45 µm filter (GE Healthcare). The DSB in the filtrate was quantitated on a reversed-phase C18 column (250 mm×4.6 mm) using an isocratic mobile phase of acetonitrile and water (60:40, v/v) by a Waters HPLC system. The pH of the mobile phase was adjusted to 5.0 using triethylamine and ortho phosphoric acid. The flow rate was 1.0 mL/min and detection wavelength was 280 nm. The fluorescent dye (Rh-PE, DiI or DiR) -labeled micelles were prepared by the similar methods. The drug loading (DL) and encapsulation efficiency (EE) were calculated using the following equations. DL (%) =

×100 %

EE (%) =

×100 % Table 1 Formulations of the DSB-loaded micelles

Formulation

DSB (µg)

PEG5kPE (mg)

PEG2kPE (mg)

PEG5k-PE Mic MMP/FR Mic FR Mic

100 100 100

3 -

1 1.5

FAPEG2kPE (mg) 1 1.5

PEG5kpp-PE (mg) 1

DL (%)

EE (%)

2.47 2.58 2.42

74.10 77.40 72.60

1.4. Particle size and zeta potential The particle size of the polymeric micelles was measured by dynamic light scattering (DLS) on the NanoBrook 90Plus PALS Particle Size and Zeta Potential Analyzer (Brookhaven Instruments). The zeta potential was measured by the same instrument. The experiments were performed at 25 ºC.



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1.5. Morphological observations The morphology of the polymeric micelles was analyzed by the transmission electron microscopy (TEM). One drop of the polymer solution was placed on a 400-mesh carbon-coated copper grid (Ted Pella, USA). The grid was then dried at room temperature for several minutes. The TEM images were taken using JEOL JEM-2010 TEM (JEOL, Japan). 1.6. Micelle stability study The DiI (20μg/mL) -loaded micelles were incubated with PBS in the presence of different concentrations of collagenase IV (0, 10 or 50 μg/mL). The fluorescence spectra were monitored at 1 and 4 h (λex = 450 nm) on a Tecan Infinite M1000 Pro microplate reader. 1.7. In vitro drug release The DSB release from the polymeric micelles was studied by the dialysis method. Briefly, one milliliter of the DSB-loaded micelles was dialyzed (MWCO 12000-14000 Da) against 30 mL of PBS containing 0.5% Tween 80 to simulate the “sink condition” at 37 ºC. The released DSB in the outside medium was determined by HPLC over 48 h. To study the influence of the MMP2mediated cleavage on drug release, the DSB-loaded micelles were pre-incubated with the collagenase IV (50 µg/mL) at 37 ºC for 1 h. 1.8. Evaluation of FR expressions on cancer cells The cancer cell lines (HeLa, A549, MCF-7, MDA-MB231, NCI-ADR-RES and B16F10) were incubated in the folic acid-free culture medium overnight. The cells were treated with FITC or FA-FITC (10 µM) for 1 h and washed three times with PBS. Then, the cells were trypsinized and collected in 200 μL of PBS for fluorescence-activated cell sorting (FACS) analysis on a BD Accuri™ C6 flow cytometer. The cells were gated upon acquisition using forward vs. side scatter to exclude debris and dead cells. The cells (1.5×104 cell counts) were recorded and analyzed with CFlow Plus Software. 1.9. Cellular uptake of Rh-PE-labeled micelles The HeLa cells or B16F10 cells were seeded in 24-well plates at 1×105 cells/well 24 h before the experiment. The cells were washed with PBS and the culture medium was replaced with the folic acid-free and serum-free medium. The Rh-PE (2.5 μg/mL)-labeled micelles were incubated with the cells at 37 ºC for 2 h. Then, the medium was removed and the cells were washed with the PBS for three times, followed by fluorescence microscopy. The trypsinized cells were analyzed by FACS (same as section 2.8). To study the influence of MMP2 on cellular uptake, the micelles were pretreated with 50 μg/mL of collagenase IV at 37 ºC for 1h. For the FA competition study, 1mM FA was pre-added to the medium during the cellular uptake study. 1.10. Drug (DSB) uptake analysis


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The HeLa or NCI/ADR-RES cells were seeded in 12-well plates at 2×105 cells/well 24 h before experiments. Equivalent doses of free DSB or DSB-loaded micelles were added to the cells and incubated at 37°C for 4 h. The cells were washed with ice-cold PBS for four times to remove any extracellular DSB. Then, the cells were lysed by 1% Triton X-100 in PBS. The 150 μL of cell lysate was mixed with 300 μL of DMSO/ACN mixture (1/4, v/v) by ultrasonication for drug extraction and protein precipitation. The mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was analyzed by HPLC. To analyze the efflux-mediated drug resistance, verapamil (2.5 μM) or cyclosporine A (40 μM) was co-incubated with free DSB on NCI/ADRRES cells. 1.11. Tumor cell spheroid penetration study The NCI/ADR-RES multicellular spheroids were established by a liquid overlay method 27. Briefly, the 96-well plates were pre-coated with 1.5% (w/v) agarose in the culture medium. Then, the cell suspensions (9×103cells / 180μL per well) were transferred into the agarose-coated plates and maintained at 37 °C for spheroid formation. The spheroids were identified by their size and shape. The 4-5 day spheroids with a diameter of 400–600 μm were used in the study. To study the influence of the formulations on tumor penetration, the Rh-PE (25 μg/mL)-labeled micelles were incubated with the NCI/ADR-RES spheroids. After 2h incubation, the spheroids were removed by pipet and gently washed with PBS for 3 times. Then, the spheroids were imaged with a confocal microscope at 100×. Z-stack images were obtained at a fixed interval of 25 μm. The fluorescence intensity of each image was analyzed by ImageJ software. The mean fluorescence intensity was plotted against distance. To study the impact of the MMP2-mediated cleavage on tumor penetration, the MMP/FR micelles were pre-incubated with 50 µg/mL collagenase IV at 37 ºC for 1h. 1.12. Cytotoxicity study on cancer cell monolayers Cells were seeded into 96-well plates at 2×103 cells/well 24h before the treatment. The formulations were diluted with the medium and immediately added to the cells. After incubation for 72h, cell viability was determined by the MTT assay. Briefly, the culture medium was replaced with 200 µL of fresh medium containing 0.5 mg/mL MTT and incubated at 37 °C for 4h. Then, the medium was removed and 200 µL DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm on a microplate reader. 1.13. Cytotoxicity study on 3D tumor cell spheroids The 4-5 day NCI/ADR-RES spheroids with the similar size were treated with the formulations for 72h. The cytotoxicity in the spheroids was evaluated by CellTiter-Blue® Cell Viability Assay. Briefly, 20 µL of Cell Titer-Blue Reagent was added after treatment and incubated at 37 ºC for



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12 h.28 Then, the fluorescence intensity was measured at λex 560 nm and λ microplate reader.

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em

590 nm on a

1.14. In vivo biodistribution The murine melanoma cancer (B16F10) cells were cultured in RPMI 1640 media with 10 % FBS and 1× penicillin-streptomycin at 37°C in a 5% CO2. After reaching confluence, the cells were detached using trypsin-EDTA for in vivo tumor inoculation. The 6-8-week-old C57BL/6 mice purchased from Charles River Laboratories (Wilmington, MA) were housed in autoclaved microisolator cages that were placed in a positive pressure containment rack and maintained according to an approved protocol from the Texas A&M University Institutional Animal Care and Use Committee. The mice were randomly assigned to experimental and control groups of 4 animals each. The tumor xenografts were initiated by subcutaneously implanting 100 μL of B16F10 (5×106) cells in the right rear flank of the mice. The tumor volume was calculated by the following equation: V = (w2×l)/2, where w and l are the width and length of the tumor as measured by a caliper. When the tumor volume reached 100~300 mm3, each mouse was treated with 100 μL of DiR (25 μg)encapsulated nanoparticles by tail vein injection. A 100 μL of DiR dye (25 μg) itself served as a control. At each time point (1, 6, 12 and 24 h), the animals were sacrificed and the major organs, tumor and blood were harvested. The fluorescence intensity in the tissues was determined on an in vivo molecular imaging instrument (Kodak Imaging Station In-vivo F/FX, MA). 1.15. Statistical analysis All experiments were performed at least in triplicate and analyzed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA). The difference between two groups was analyzed using unpaired t-test analysis. P < 0.05 was considered to be statistically significant.

2. Results and discussion 2.1. Synthesis of PEG5k-pp-PE and FA-PEG2k-PE. In the previous work, we have successfully synthesized a series of PEG-pp-PE copolymers.25 Here, the same method was used for the synthesis of PEG5k-pp-PE. The final product was characterized by the TLC (Figure 2A) and 1H NMR (Figure 2B). Because the PEG5k-pp-COOH was attached to the NH3+ of DOPE to form the amide linker (-CONH-), the overall hydrophobicity of the product was increased, as evidenced by the spot far from the DOPE spot on the TLC plate.29 The spots were visualized by the phospholipid-staining reagent, molybdenum blue reagent. In the 1H NMR spectrum, the characteristic peaks of PEG5k-pp-PE (PE, 1.4 ppm;


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pp, 2.0 ppm; and PEG, 3.6 ppm) were clearly shown, indicating the successful conjugation of PEG5k-pp-PE.29 The peak integration result indicated that the ratio of PEG5k/PE was about 1:1. FA-PEG2k-PE was synthesized via the facile sulfhydryl-maleimide reaction. Three methods were used to characterize the product including, TLC (Figure 2C), 1H NMR (Figure 2D), and UV-Vis spectroscopy (Figure 2E). After conjugation with FA, the migration of PEG2k-PE was inhibited on TLC plates due to the increased polarity. The molybdenum blue reagent was used to stain the phospholipids of both compounds. In the 1H NMR spectrum, the proton peaks of 6.6– 8.8 ppm (FA), the peak at 3.50 ppm (PEG); as well as the peak at 1.3 ppm (PE), were all observed, confirming the successful conjugation. 29-30 The ratio of FA/PE was about 1:1 according to the peak integration. FA alone couldn’t be fully dissolved in methanol with the yellow-brown crystals settled down on the bottom (left bottle), while the conjugation to PEG2k-PE (highly soluble in methanol, right bottle) increased FA’s solubility in methanol (pale yellow solution, middle bottle) (Figure 2E). The UV-Vis spectra of these materials dissolved in DMSO were compared. PEG2k-PE had no significant absorbance in the range from 300 nm to 500 nm. In contrast, both FA and FAPEG2k-PE had a significant peak at around 360 nm. The conjugation didn’t significantly influence the optical property of FA.

Figure 2 Characterization of PEG5k-pp-PE and FA-PEG2k-PE. (A) TLC of PEG5k-pp-PE stained by molybdenum blue reagent (chloroform/methanol, 4/1, v/v); (B) 1H NMR of PEG5kpp-PE (in CDCl3); (C) TLC of FA-PEG2k-PE stained by molybdenum blue reagent



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(chloroform/methanol, 4/1, v/v); (D) 1H NMR of FA-PEG2k-PE (D6-DMSO); (E) UV-vis absorption spectra in DMSO and appearance in methanol. 2.2. Stability of PEG-PE-based micelles We have demonstrated that PEG-pp-PE polymers could be cleaved by human MMP2 21 and the PEG-pp-PE self-assembled micelles exhibited the MMP2-dependent particle size and drug release 25. The lipophilic membrane dye DiI was used as a probe since it shows strong fluorescence only when incorporated into the lipophilic core of micelles. Here, we studied the stability of the micelles formed by PEG5k-PE, PEG5k-pp-PE, and PEG5k-PE/ PEG5k-pp-PE (1:1, wt/wt). In all incubation media (PBS or collagenase IV), as shown in Figure 3, the PEG5kPE micelles were stable and there was no obvious leakage of the loaded DiI, suggesting that the collagenase didn’t have any effects on their stability. In contrast, the PEG5k-pp-PE micelles showed a significant dye leakage even after one-hour incubation with collagenase IV. The rate of cargo release was both dose- and time- dependent. The data indicated that the compact micelle structure was not a significant hindrance to MMP2 and the PEG5k-pp-PE micelles underwent dissociation after the MMP2-mediated cleavage, which confirmed our previous results 25. Surprisingly, the PEG5k-PE/PEG5k-pp-PE micelles were also stable and had no dye leakage even after collagenase incubation. The data suggested that the PEG5k-PE/PEG5k-pp-PE mixed micelles could hold the guest molecules tightly and the cleavage of PEG5k-pp-PE didn’t significantly influence the micelles’ stability/nanoparticulate structure. High stability of the mixed micelles made it possible to build the proposed dual-targeted micelles (PEG5k-pp-PE/FAPEG2k-PE/PEG2k-PE) which utilize the PEG shielding/deshielding to control the surface properties of nanoparticles for tumor targeting and cell internalization.


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Figure 3 Micelle stability study. The fluorescence of the DiI-loaded micelles was monitored after incubation with PBS or collagenase IV (10μg/mL and 50μg/mL) for 1 and 4 h. 2.3. Characterization of MMP/FR micelles It has been proven that the physicochemical characteristics of nanoparticles, i.e. particle size, surface charge and morphology, significantly influence the in vivo performance of the nanoparticles 31. Here, these properties of the MMP/FR micelles were studied. In the aqueous buffer, the MMP/FR micelles were a transparent yellow solution with a particle size of 186.17±5.56 nm measured by DLS (Figure 4A). Interestingly, the TEM results showed that the MMP/FR micelles were a spindle/oval shaped nanoparticle with a size of 100-200 nm, instead of smaller and near spherical-shaped micelles formed by PEG2k-PE or PEG5k-pp-PE (Figure 4B) and other PEG-PE-containing mixed micelles 20. Here, the FA is only slightly soluble in water and the conjugation of FA to PEG might decrease the hydrophilicity of the PEG chain, so as to decrease the hydrophilic-lipophilic balance (HLB). The decreased HLB might only allow the polymers to form a micelle with a relatively larger size and oval shape and might not be favorable for the formation of a tiny spherical micelle under the tested condition 25. To see if the MMP2-mediated cleavage influence the physicochemical characteristics of the MMP/FR micelles, the particle size and zeta potential of the micelles were determined (Figure 4C). The MMP/FR micelles were stable in the absence of MMP2 because the FA was shielded



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by high PEG density and long PEG chains (PEG5k), while after incubation with 50μg/mL collagenase IV, the long chain PEG (PEG5k) was deshielded and the FA was exposed. This change decreased the overall HLB and slightly increased the particle size of the MMP/FR micelles from 186.17±5.56 nm to 215.45±7.85 nm. However, compared with the MMP2mediated micelle dissociation (from 800 nm),25, 32 this slight change didn’t significantly influence the micelle stability (Figure 3) and drug release (Figure 4D). We also found that the zeta potential (surface charge) of MMP/FR Mic (with the hidden FA) was 15.05±1.84 mV in PBS, while the micelles with the fully-exposed FA on their surface (FR Mic) had the zeta potential of 4.25±1.89 mV (Figure 4C). High PEG density on the micelle surface, including both PEG5k and PEG2k, accounted for the negative charge of the micelles and, after the PEG5k deshielding, the zeta potential of MMP/FR Mic was moved up to -7.02 ±2.63 mV, indicating the exposure of FA. The zeta potential didn’t become positive, probably due to the incomplete PEG deshielding and the hindrance of the peptide residues after the cleavage. DSB, as a model drug, was loaded into the micelles, as shown in Table 1. To achieve the similar concentrations of drug and polymers, the same initial (drug/ total polymers) weight ratio was used to prepare the micelles. Three micellar nanoparticles showed similar drug loading (~2.5 wt%) capacity and encapsulation efficiency (~75%) (Table 1). The in vitro drug release profiles of the micelles were shown in Figure 4D. Free dasatinib was released rapidly and reached its plateau within 6 hours. In contrast, the drug release profiles from all PEG-PE-based micelles were pretty similar, as evidenced by a sustained release without the burst release. Compared to free DSB, at 6h, only around 40% of drugs were released from the micelles. It was worth noting that the MMP2 pretreatment didn’t significantly change the drug release pattern from the micelles. These data suggested that the slight expansion and the altered zeta potential of the micelles due to the MMP2-triggered PEG5k deshielding (Figure 4C) didn’t significantly influence the micelle stability (Figure 3) and the remaining micelles could tightly hold the loaded drugs and showed the sustained drug release rate as their counterparts in the absence of MMP2 (Figure 4D).


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Figure 4. (A) Particle size and size distribution of the MMP/FR micelles; (B) TEM micrographs of different micellar formulations (scale bar represents 200 nm); (C) Mean particle size and zeta potential of the micelles with/without the collagenase IV preincubation for 1h; (D) In vitro cumulative dasatinib release. 2.4. Evaluation of FR expression in the tested cell lines FA has been successfully exploited as a cancer-specific ligand for delivery of anticancer drugs, nanoparticles, photosensitizers and diagnostic probes.33 To evaluate the performance of the FAmodified nanoparticles, it is critical to know the cellular FR expressions. Although various methods have been used to determine the cellular FR levels 34, the FA-modified FITC (FA-FITC) was used as a fluorescent probe to detect the FRs and the FA-FR interaction-mediated endocytosis 35 in this study. Here, we tested the uptake of the FA-FITC and FITC on the nonsmall cell lung cancer (A549), cervical cancer (HeLa), breast cancer (MCF-7 and MDA-MB231), MDR ovarian cancer (NCI/ADR-RES), and murine melanoma (B16F10) cells by flow cytometry. After normalized by the fluorescence intensity of FITC (as an indicator of passive diffusion), we found that all tested cell lines showed the increased cellular uptake of FA-FITC (Figure 5A and S1A), confirming that the FRs are one of the major mechanisms supporting rapid cancer cell growth 4. Among them, the MCF-7 and A549 cells showed relatively low FA-FITC uptake (FA-FITC/FITC, 1.5 and 1.2 respectively) (Figure 5B), indicating that they could be the “FR-negative (FR-)” cancer cells for the study. In fact, they were widely used in the FR-targeting



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studies 36-37. In contrast, the FA-FITC/FITC ratios were around 4.6, 3.5 and 5.3 in HeLa, NCI/ADR-RES and MDA-MB-231, respectively, indicating the FR overexpression (FR+). The data were in consistent with previous reports 36, 38-39. Furthermore, the cellular uptake of FAFITC was significantly inhibited at 4 ºC (Figure S1B), suggesting the uptake process was an energy-dependent endocytosis.

Figure 5 (A) Flow cytometry analysis of the FR expression on the tested cell lines: MCF-7 (a), A549 (b), HeLa (c), NCI/ADR-RES (d) and MDA-MB-231 (e); (B) Mean fluorescence intensity (MFI) of FA-FITC over FITC obtained from flow cytometry data. 2.5. In vitro cellular uptake The cellular uptake of the Rh-PE-labeled dual-targeted micelles was first assessed on the HeLa cells (Figure 6A). We found that the cellular uptake of the MMP/FR micelles was much higher in the presence of MMP2 than that in the absence of MMP2 (~13000 vs. ~5000) and was similar to that of the FR micelles with the fully exposed FA (~14000). The PEG5k-PE micelles had low cellular uptake similar to the untreated cells (~2500). The addition of the excess amount FA to the culture medium significantly inhibited the uptake of the collagenase IV-pretreated micelles, indicating that the cell internalization was mediated mainly by FA. The data indicated that the PEGylation inhibited the nanoparticle internalization, in consistent with previous reports 20-22.


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For the MMP/FR micelles, the long chain PEG5k could efficiently shield the activity of FA residing at the distal end of short PEG2k chain. In the presence of MMP2, the PEG5k was deshielded and the previously hidden FA was exposed on the micelle surface as evidenced by the increased zeta potential (Figure 4B), resulting in the enhanced endocytosis. The rank of cellular uptake was summarized as: the FR micelles ˃ MMP/FR micelles (+ MMP2) ˃ MMP/FR micelles (+ MMP2, + FA) ˃ MMP/FR micelles (- MMP2) ˃ PEG5k-PE micelles. The MMP2triggered cellular uptake of the MMP/FR micelles was confirmed by the confocal microscopy images (Figure 6B). The microscopy data also demonstrated that the internalized micelles were mainly located in the cytoplasm, instead of attached to the cell surface. Since Rh-PE contains a phospholipid moiety and could be tightly held by the lipid-core nanoparticles, it was widely used as a fluorescent probe to visualize these nanoparticles. However, it might not represent the behavior of the loaded drug molecules. Here, the intracellular uptake of DSB was determined by HPLC on both drug sensitive and MDR cancer cells. On the sensitive (HeLa) cells, like the uptake of the micelles, the dense surface-anchored PEGs inhibited DSB uptake, while the MMP2-mediated PEG5k deshielding significantly increased the DSB uptake, whose extent was similar to that of the FR micelles (Figure 6C). The data was in consistent with the results in Figures 6A-B, indicating that high cellular uptake of the micelles facilitated the uptake of the micellar drugs. Drug resistance was widely reported among various anticancer therapeutics, including chemotherapy, targeted therapy and many other drugs. DSB was reported to be a substrate of Pglycoprotein (ABCB1, Pgp) and breast cancer resistance protein (ABCG2) and co-administration of the efflux inhibitors increased the DSB’s oral absorption/bioavailability 40. Here, free DSB could be easily pumped out by the Pgp, while the efflux could be effectively reversed by the well-known Pgp inhibitors, verapamil and cyclosporine A, as evidenced by the almost doubled intracellular drug concentrations (Figure 6D), which confirming that the efflux was a major factor that influenced the uptake of DSB. To evaluate if the dual-targeted micelles could overcome the efflux-mediated drug resistance, the MDR cells (NCI/ADR-RES) were as the model cells (Figure 6E). Unlike the results from the sensitive cells (Figure 6C), all PEG-PE micelles, including the conventional PEG5k-PE micelles, could significantly increase the DSB intracellular concentration, probably because that the nanoparticle-mediated non-specific endocytosis and delayed drug release bypassed the Pgp pathway 41. We also found that the MMP/FR micelles had the similar uptake as that of the PEG5k-PE micelles in the absence of MMP2, while the MMP2 pretreatment dramatically increased the DSB uptake from ~140% to ~180% compared to untreated cells, which was close to the FR micelles. All these data suggested that the MMP2 could be an “on/off” switch to control the PEG shielding /deshielding and FA exposure, resulting in the inhibition of drug uptake in the non-cancer tissues/cells with low MMP2 level but the enhancement of drug uptake in the MMP2-expressing cancer tissues/cells.



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Figure 6 (A) Flow cytometry of the Rh-PE labeled micelles. MFI, mean fluorescence intensity. (B) Confocal micrographs of the Rh-PE loaded MMP/FR micelles with/without MMP2 treatment. The scale bar represents 200 μm. (C) DSB uptake on HeLa cells. (D) Effect of the Pgp inhibitors on DSB uptake on NCI/ADR-RES cells. (E) DSB uptake on NCI-ADR/RES cells. Incubation time: 2h for micelle uptake and 4h for DSB uptake. (*p < 0.05, ***p < 0.001) 2.6. Penetration of polymeric micelles through tumor cell spheroids In addition to epigenetic and genetic alterations in cancer cells, the tumor architecture and microenvironment mediates responses of solid tumors to drug treatments 42. In addition, the cells distal from blood vessels are likely to be resistant to systemic therapy because of poor penetration-resulted low drug concentration, especially for large molecules or nanomedicine. The monolayer cell culture cannot fully represent the in vivo tumor in terms of architecture heterogeneity, nutrient and oxygen gradients, cell-cell interactions, matrix deposition, etc. 43. To better mimic the in vivo tumor condition, we developed the tumor cell spheroids in the previous study 20. Here, the similar protocol was used to generate the NCI/ADR-RES spheroids. The spheroids with the diameter of around 500μm made it a perfect in vitro cell model since it could better mimic the solid tumor tissues which had the distance between the blood capillaries similar to this size.42 After 2h incubation with the NCI/ADR-RES spheroids, the PEG5k-PE micelles were observed only at the periphery of the spheroids, as evidenced by the dark spheroid core and weak surrounding fluorescence, indicating their limited penetration capability, although their size was


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the smallest among the tested micellar formulations. The MMP/FR micelles (- MMP2) with the hidden FA also penetrated a short distance in the spheroids, while it was slightly deeper than that of PEG5k-PE micelles, probably because their rod shape facilitated their penetration and cellular uptake.44-45 These data suggested that the long chain PEG (5k Da) could inhibit the cellular uptake in not only the cell monolayers but also 3D spheroids. In contrast, the FR micelles and the MMP/FR micelles (+ MMP2) with the exposed FA penetrated much deeper and more extensive, as evidenced by stronger fluorescence even in the core of the spheroids, indicating their excellent capability to penetrate through the 3D tumor model (Figure 7A). The plots of the fluorescence intensity vs. the distance from the spheroid periphery (Figure 7B) also showed that both the overall uptake (area under curve) and penetration capability/depth [the height of peak and the distance from the periphery (0µm) to peak] were significantly greater in the FR Mic and MMP/FR Mic (+ MMP) groups than the MMP/FR Mic (- MMP) and PEG5k-PE groups. The data suggested that the MMP2-mediated PEG5k deshielding and exposure of FA could effectively improve the tumor penetration and cancer cell uptake of the micelles.

Figure 7 Penetration of the Rh-PE-labeled polymeric micelles through the NCI/ADR-RES spheroids after 2h incubation. (A) The representative Z-stack images taken by confocal microscope. The step size is 25 μm. The scale bar represents 200 μm. (B) The curves of the normalized mean fluorescence intensity vs. the distance from the spheroid periphery. 2.7. MMP2-sensitive FR-targeted cytotoxicity The anticancer activity of the DSB-loaded micelles was first tested on the drug-sensitive human cancer cell lines expressing different levels of MMP2 and FR, including the MMP2 and FR lowexpressing (MMP2-/FR-) breast cancer (MCF-7), MMP2 overexpressing but FR low-expressing



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(MMP2+/FR-) lung cancer (A549), and MMP2 and FR both overexpressing (MMP2+/FR+) cervical cancer (HeLa) cells. In the MCF-7 cells (a, Figure 8A), all micellar formulations (PEG5k-PE Mic, FR Mic and MMP/FR Mic) had the similar cytotoxicity which was lower than that of free DSB although the polymeric micelles showed similar or even higher cellular uptake efficiencies compared with free drug in the 2h uptake study (Figure 6), most likely because of slow drug release from the polymeric micelles (Figure 4D) 25. The data also indicated that the loaded drugs could not be benefited from the exposure of FA on the micelle surface when treating the FR- cells. In the A549 cells (b, Figure 8A), the MMP/FR Mic and FR Mic showed the similar cytotoxicity which was comparable to that of free DSB, while the PEG5k-PE micelles were still less potent than free DSB, suggesting that the MMP2-mediated PEG5k deshielding decreased the PEG density on the nanoparticles, leading to the increased “non-specific” cellular uptake 46 although the cells were FR-. In the HeLa cells (c, Figure 8A), the MMP/FR Mic and FR Mic showed significantly higher cytotoxicity than free DSB, while the PEG5k-PE micelles showed much less cytotoxicity, suggesting that the extracellular MMP2 deshielded the PEG5k and exposed FA on the MMP/FR Mic, facilitating the FR-mediated endocytosis in the MMP2+/FR+ cells. The data also indicated that the MMP2 sensitivity and FR targeting functions of the MMP/FR micelles could be fully activated only in the presence of both MMP2 and FR. The 50% inhibitory concentration (IC50) (Table 2) showed the similar trend as the drug response curves. Particularly, compared to the non-targeted micelles (PEG5k-PE micelles), the dualtargeted micelles (MMP/FR micelles) could bring down the IC50 of DSB for more than three-fold in the HeLa (MMP2+/FR+) cells. It is worth noting that the MMP/FR micelles and FR micelles also had lower drug release compared to free DSB (Figure 4D), however, unlike the PEG5k-PE micelles, they could be taken up by the MMP2+/FR+ cells more efficiently (Figure 6), resulting in high cytotoxicity. We already knew that the DSB’s uptake could be improved by the Pgp inhibitors in the MDR cancer cells (Figure 6D). However, the co-delivery of drugs and Pgp inhibitors which have different physicochemical and pharmacokinetic properties, might be complicated and cause additional undesired side effects 47. We demonstrated that the MMP/FR micelles could also significantly improve DSB’s cellular uptake and inhibit the drug efflux (Figure 6E). To test if the MMP/FR micelle-mediated efflux inhibition could sensitize the DSB treatment, two drugresistant MMP2+/FR+ cell lines, NCI/ADR-RES (ovarian cancer) and MDA-MB-231 (breast cancer), were used as the models. It was not surprising that free DSB had the lowest cytotoxicity in both cell lines among the treatments (d-e, Figure 8A), indicating that the drug efflux significantly decreased drug efficacy. In contrast, all the polymeric micelles showed the improved cytotoxicity compared to free drug, mainly due to the increased intracellular drug accumulation (Figure 6E). Among them, the MMP/FR micelles and FR micelles had the similar cytotoxicity which was much higher than that of the PEG5k-PE micelles, suggesting that the endogenous MMP2 level/activity was sufficient to trigger the PEG5k deshielding and exposure


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of FA, resulting in the improved endocytosis and anticancer activity of the MMP/FR micelles even in the drug-resistant cells. Though the shape might influence the performance of the micelles 48, our current data (Figure 6-8) indicated that it played a minor role in the MMP/FR Mic. In addition to the limited drug penetration through the tumor spheroids (Figure 7), the discrepancy in cytotoxicity between the 3D and monolayer cultures might be a result of drug resistance induced by the cell-cell/cell-matrix interaction in the spheroid microenvironment 49. To further challenge the prepared micelles, the drug efficacy was tested in the NCI/ADR-RES cell spheroids. We found that 2 μM of DSB had no effects on cell viability in the spheroids while it decreased the cell viability by around 75% in the cell monolayers (Figure 8B), confirming that the 3D structure of tumor cells was more resistant to the drug treatment than the monolayer cells 49 . In contrast, all polymeric micelles showed the improved cytotoxicity in the spheroids compared to free drugs. Similar to the monolayer cytotoxicity data, the MMP/FR micelles showed significantly higher cancer cell-killing capability than the PEG5k-PE micelles, mainly due to their high micelle uptake/penetration through the spheroids (Figure 7). The data suggested that the MMP/FR micelles could overcome not only the Pgp-mediated drug efflux but also the 3D tumor microenvironment-mediated drug resistance.



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Figure 8 (A) Cytotoxicity of the DSB-loaded micelles on MCF-7 cells (a), A549 cells (b), HeLa cells (c), NCI-ADR-RES cells (d) and MDA-MB-231 cells (e); (B) Comparison of cytotoxicity of the DSB-loaded micelles on NCI-ADR-RES cell monolayers and 3D spheroids. DSB dose: 2μM. (*p < 0.05, ***p < 0.001) Table 2 IC50 values on different cell lines IC50 （nM） MCF-7

A549

HeLa

NCI/ADR-RES

MDA-MB-231

Free DSB

311.4±77.6

103.6±10.8

70.3±1.9

11668.1±322.4

6501.3±337.6

PEG5k-PE Mic

1304.0±169.2

179.1±13.0

172.6±3.6

6397.3±435.4

3198.9±209.3

FR Mic

892.7±79.4

85.1±10.8

36.2±3.8

2192.8±107.0

879.02±37.1

MMP/ FR Mic

899.5±93.1

106.4±15.6

47.1±3.7

3013.0±152.0

1164.1±68.2

2.8. Pharmacokinetics and biodistribution As shown in Figure S1, the MMP/FR micelles achieved high cellular uptake on the FR+ murine B16F10 melanoma cells. To study the biodistribution and tumor targeting of the MMP/FR micelles, the B16F10 melanoma xenograft model was established. To make the in vivo study easy and accurate, we prepared the DiR-loaded micelles. After i.v. injection, the ex vivo fluorescence was measured at 1, 6, 12 and 24h. The fluorescence intensity-time profiles of DiR in the blood are shown in Figure 9A. A rapid and dramatic decrease in the intensity was observed after injection of free DiR, indicating the fast clearance of free DiR from the blood circulation. In contrast, the elimination half-life of all micellar formulations was significantly prolonged. No significant difference was observed among the tested three micellar systems. The results demonstrated the long circulation property and high stability of the polymeric micelles in the bloodstream, which made it possible to achieve the (EPR-mediated) tumor accumulation of the nanomicelles and FR-mediated tumor cell internalization of the micellar drugs. The long blood circulation could be attributed to the stealth behavior of the polymeric micelles induced by the protective hydrophilic PEG corona, which prevented the plasma protein adsorption (opsonization) to the surface of micelles and reduced their uptake by the mononuclear phagocyte system (MPS). After i.v. injection, the DiR accumulation (fluorescence intensity) in vital organs, including the heart, liver, lung, kidney and spleen (Figure 9B), and tumors (Figure 9C) were examined. The results showed that the free DiR was rapidly distributed to most tissues except tumors, probably


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due to its hydrophobicity-induced protein adsorption and phagocytic clearance 50. All polymeric micelles could distribute to all tissues, including tumors, due to the PEG-mediated long blood circulation. Among them, the PEG5k-PE micelles and FR micelles were preferentially accumulated in the lung and kidney (> 12500), while the mice administered with the MMP/FR micelles showed much lower DiR concentrations (< 7000) in the normal tissues compared with the FR micelles, PEG5k-PE micelles and free DiR solution. However, it was clearly shown that the MMP/FR dual-targeted micelles were able to deliver the loaded cargo to the tumor at a significantly higher level as compared with the plain PEG5k-PE micelles, FR targeting micelles, and free DiR (Figure 9C). The fluorescence in the tumor after injection of the MMP/FR micelles was initially ~1000/g tumor tissue at 1 h, and thereafter, the intensity gradually increased and reached ~5000 at 12h and ~7000 at 24h, which were almost double compared to the PEG5k-PE micelles and FR micelles at the same time points. In contrast, extremely low fluorescence intensity (~300) was detected in the tumor after injection of free DiR. We noticed that the FR micelles (active tumor targeting) failed to show the advantages compared with the plain PEG5kPE micelles (passive tumor targeting). It is probably because that (i) the significant EPR effect in the B16F10 solid tumor 51 increased the tumor accumulation of all nano-sized particles; and (ii) the FR expression in normal tissues 4 decreased the FR-mediated tumor targeting. For the MMP/FR micelles, their high tumor accumulation was most likely due to the cooperation of the micelles’ excellent physicochemical properties and stability, PEG’s stealth property, MMP2 sensitivity, and FR targetability. Though the MMP/FR micelles had similar or slightly lower cytotoxicity compared with the FR micelles in the in vitro experiments, we might speculate that the improved PK and tumor targetabiltiy of the MMP/FR micelles would result in the enhanced in vivo anticancer activity and decreased off-target toxicity.



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Figure 9. PK and biodistribution of the DiR-loaded micelles after i.v. injection to the B16F10 melanoma-bearing mice. (A) Mean fluorescence intensity (MFI)-time profiles of the DiR solution and DiR-loaded micelles in the blood. (B) In vivo distribution of the DiR solution and DiR-loaded micelles in the major organs. (C) Tumor accumulation of the DiR solution and DiRloaded micelles. (n=4, **p < 0.01)

3. Conclusions In summary, the novel MMP2-sensitive FR-targeted polymeric micelles were successfully prepared and characterized. The hydrophobic targeted therapy drug, dasatinib, could be easily loaded into the micelles and be released sustainedly. After the MMP2-triggered PEG5k deshielding, the nanoparticulate micelle structure was remained and the previously-hidden FA was exposed on the surface of the micelles, resulting in high cellular uptake, deep penetration as well as enhanced anticancer activity in both the cancer cells and their 3D spheroids. Furthermore, the dual-targeted micelles could efficiently overcome the drug resistance and sensitized the drug treatment in the drug-resistant cancer cells. The excellent physicochemical property, stability, MMP2 sensitivity and FR targetability significantly prolonged the blood circulation, decreased the distribution in the non-tumor tissues, and improved the tumor specificity of the dual-targeted


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micelles in the tumor-bearing mice. The MMP2 and FR dual-targeted micelles might have great potential for cancer-targeted intracellular delivery of anticancer drugs. Associated content Supporting Information Additional experimental results on B16F10 cells (FR expression and cellular uptake study). Acknowledgments Qing Yao thanks the China Scholarship Council (CSC) for the financial support (File No. CSC No.201508210197). Author contribution Lin Zhu conceived the idea, and designed and supervised the project. Qing Yao performed most research and analyzed data. Jong Hoon Choi and Dongin Kim performed the TEM and in vivo biodistribution study. Zhi Dai help interpret polymer structures. Jiao Wang help develop tumor cell spheroids. Xing Tang advised formulation characterization. Lin Zhu, Qing Yao, Dongin Kim, and Xing Tang wrote the paper.



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Improving Tumor Specificity and Anticancer Activity of Dasatinib by

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