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Intracellular delivery colloidally-stable core-crosslinked triblock copolymer micelles with glutathioneresponsive enhanced drug release for cancer therapy Jung Kwon Oh, Depannita Biswas, So Young An, Yijing Li, and Xiangtao Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01146 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 17, 2017

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Molecular Pharmaceutics

Intracellular delivery colloidally-stable core-crosslinked triblock copolymer micelles with glutathione-responsive enhanced drug release for cancer therapy Depannita Biswas,+# So Young An,+# Yijing Li,§ Xiangtao Wang,§ Jung Kwon Oh+* + Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada H4B 1R6 § Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences &Peking Union Medical College, No. 151, Malianwa North Road, Haidian district, Beijing 100193, China. Corresponding author: J.K.Oh ([email protected]) # Two authors are equally contributed

Abstract Design and development of amphiphilic block copolymer-based nanocarriers exhibiting enhanced colloidal stability upon dilution in the blood and cellular glutathione-responsive rapid drug release is highly desired for tumor-targeting chemotherapy. Herein, we report a novel ABA-type triblock copolymer consisting of a hydrophilic central poly(ethylene glycol) block and two terminal hydrophobic blocks of a polymethacrylate having pendant disulfides (PHMssEt), thus PHMssEt-bPEG-b-PHMssEt (ssTP). Aqueous self-assembly and following disulfide-exchange reaction of the resulting ssTP allows for formation of core-crosslinked micelles (CCMs) through the formation of new disulfide linkages, retaining enhanced colloidal stability in physiological condition and in the presence of proteins. Further, they exhibit reduction-responsive enhanced release of encapsulated drugs in response to cellular concentrations of glutathione in cancer cells, confirmed by dynamic light scattering and spectroscopic analysis. Combined with these results, in vitro (cells) and in vivo (mouse model) biological results suggest that ssTP-based CCMs are effective candidates as intracellular nanocarriers targeting tumors for cancer therapy.

Keywords: triblock copolymer • drug delivery • crosslinked micelles • glutathione-responsive degradation • enhanced release

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Introduction Nanoscale platforms (or nanocarriers) have been developed to improve delivery and biodistribution of small drug therapeutics.1-3 In particular, the development of smart nanocarriers self-assembled from well-defined amphiphilic block copolymers exhibiting stimuli-responsive degradation (SRD) in response to cellular or tumor environments have drawn significant attention as promising candidates for tumor-targeting drug delivery applications.4-7 Upon intravenous injection, SRD (environment)exhibiting drug-loaded nanocarriers designed with 50-150 nm in diameter, similar to conventionallydesigned nanocarriers, continue through stages of circulation, extravasation, accumulation, distribution, endocytosis, endosomal escape, intracellular localization, and action.8-11 After extravasation into tumor tissues through the process called “Enhanced Permeation and Retention (EPR)” and further internalization to cancer cells through endocytosis,12-14 the SRD-exhibiting nanocarriers can be advantageous in that they can be degraded or disintegrated upon the cleavage of labile linkages in response to cellular components. This process enables the enhanced release of encapsulated drugs in tumor sites, offering maximized drug efficacy and minimal side effects.15-20 However, a challenge of SRD-exhibiting self-assembled nanocarriers to be addressed toward successful tumor-targeting chemotherapy involves the physically-driven self-assembly of block copolymer chains in aqueous solution. After intravenous injection, they undergo significant dilution in the blood by several orders of magnitude. Such a large dilution causes their destabilization or dissociation, thus leading to the premature release of drugs during blood circulation. A potential strategy to maintain colloidal stability upon dilution in blood is to synthesize core-crosslinked micelles (CCMs).21, 22 However, conventional methods utilizing permanent covalent crosslinkages could hamper the controlled release of encapsulated drugs inside cells due to their non-degradability. A promising solution is the design of well-defined CCMs with labile (or dynamic) crosslinkages that can be cleaved in response to cellular components; particularly disulfide linkages, which can be cleaved to the corresponding thiols in reducing environments.23, 24 It is known that glutathione (GSH, a tripeptide having cysteine group) can be found at millimolar concentration (2-10 mM) in intracellular compartments (mostly cytosol) and at elevated concentrations in cancer cells (4-5 times higher than in normal cells).25, 26 Given this feature, the disulfide-crosslinked CCMs endow colloidal stability during blood circulation, and then exhibit enhanced therapeutic release in response to cellular GSH inside tumor tissues and cancer cells after extravasation from blood streams.27, 28 A number of reports have described novel disulfide-based CCMs;23, 29-33 particularly including the systems fabricated through ACS Paragon Plus Environment

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disulfide exchange reaction of pendant disulfide linkages in the presence of a catalytic amount of a reducing agent.24, 34-39 Despite these advances, however, most of the systems with pendant disulfides have been designed with AB-type diblock copolymers.34-37 Herein, we report novel reduction-responsive, flower-like CCMs based on an ABA-type triblock copolymer designed with a hydrophilic central block of poly(ethylene glycol) (PEG) and two terminal hydrophobic blocks of a polymethacrylate having disulfide pendants (PHMssEt), thus PHMssEt-bPEG-b-PHMssEt, hereafter ssTP. The resulting CCMs had excellent colloidal stability in physiological conditions, and reduction-responsive enhanced/controlled release of encapsulated drugs in cancer cells. As illustrated in Scheme 1, the ssTP copolymer self-assembled in aqueous solution to form noncrosslinked micelles (NCMs) with pendant disulfide-containing hydrophobic PHMssEt cores and PEG coronas as flower petals. Being treated with a catalytic amount of a reducing agent, the flower-like micelles were converted to disulfide-CCMs through thiol-disulfide exchange reaction. These colloids were turned to be stable in organic solvents, confirming the occurrence of new disulfide crosslinking, and in the presence of proteins, ensuring colloidal stability during blood circulation. After being loaded with nile-red (NR, a fluorescent dye) or doxorubicin (Dox, a clinically-used anticancer drug), they were examined for reduction-responsive degradation and enhanced drug release in the presence of 10 mM GSH, compared with the corresponding NCMs. Their intracellular trafficking was evaluated in cellular environments using flow cytometry and confocal laser scanning microscopy (CLSM) for cellular uptake as well as a colorimetric assay for cell viability. Further to in vitro studies, the reduction-responsive Dox-loaded CCMs were evaluated for in vivo mouse models bearing 4T1 solid tumor.


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Endocytosis

SH

SH SH

SH

SH

GSH triggered degradation

cytoplasm

nucleus

Scheme 1. Schematic illustration of synthesis and intracellular drug delivery of reduction-responsive CCMs based on well-defined ssTP triblock copolymer having pendant disulfide linkages for cancer therapy.


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Experimental Instrumentation. 1H-NMR spectra were recorded using a 500 MHz Varian spectrometer. The CDCl3 singlet at 7.26 ppm was selected as the reference standard. Molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC). An Agilent GPC was equipped with a 1260 Infinity Isocratic Pump and a RI detector. Two Agilent PLgel mixed-C and mixed-D columns were used with DMF containing 0.1 mol% LiBr at 50 °C at a flow rate of 1.0 mL/min. Linear poly(methyl methacrylate) standards from Fluka were used for calibration. Aliquots of the polymer samples were dissolved in DMF/LiBr. The clear solutions were filtered using a 0.25 µm PTFE filter to remove any DMF-insoluble species. A drop of anisole was added as a flow rate marker. The size of micelles in hydrodynamic diameter by volume was measured by dynamic light scattering (DLS) at a fixed scattering angle of 175° at 25 °C with a Malvern Instruments Nano S ZEN1600 equipped with a 633 nm He-Ne gas laser. Fluorescence spectra on a Varian Cary Eclipse Fluorescence spectrometer and UV/Vis spectra on an Agilent Cary 60 UV/Vis spectrometer were recorded using a 1 cm wide quartz cuvette. Transmission electron microscopy (TEM) images were obtained using a Philips Tecnai 12 TEM, operated at 80kV and equipped with a thermionic LaB6 filament. An AMT V601 DVC camera with point to point resolution and line resolution of 0.34 nm and 0.20 nm respectively was used to capture images at 2048 by 2048 pixels. To prepare specimens, the micellar dispersions were dropped onto copper TEM grids (400 mesh, carbon coated), blotted and then allowed to air dry at room temperature. Materials. Poly(ethylene glycol) (HO-PEG-OH) with #EO = 240, α-bromoisobutyryl bromide (Br-iBuBr), triethylamine (Et3N), copper(I) bromide (CuBr, >99.99%), N,N,N′,N′′,N′′pentamethyldiethylenetriamine (PMDETA, >98%), glutathione (GSH, a reduced form), Nile Red (NR), doxorubicin hydrochloride (Dox, -NH3+Cl- forms, >98%), and immunoglobulin G from human serum (IgG, reagent grade, >95%, essentially salt-free, lyophilized powder) from Sigma Aldrich, Pierce BCA protein assay kit from Bio-Rad, DL-dithiothreitol (DTT, 99%) from Acros Organics, and dialysis tubing with MWCO = 12 kDa from Spectrum Labs were purchased and used as received. A methacrylate having pendant a disulfide linkage (HMssEt) was synthesized as described in our previous publication.37 Synthesis of Br-PEG-Br. Br-iBuBr (4.6 g, 20 mmol) dissolved in dichloromethane (20 mL) was added dropwise to a solution of OH-PEG-OH and Et3N (3.5 mL, 25 mmol) dissolved in dichloromethane in an ice bath at 0 °C for 15 min. The resulting mixture was stirred for 18 hrs at room temperature and washed with aqueous acidic and basic solution three times. The residues were then ACS Paragon Plus Environment

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dried over anhydrous Na2SO4 and precipitated from hexane. The resulting Br-PEG-Br was then isolated by vacuum filtration and further dried in a vacuum oven at room temperature for 18 hrs, resulting in white solids. Yield = 80% Synthesis of ssTPs by ATRP. Br-PEG-Br (0.34 g, 0.033 mmol), HMssEt (1.5 g, 4.3 mmol), PMDETA (6.7 µL, 0.032 mmol), and anisole (4.7 mL) were added to a 25 mL Schlenk flask. The resulting mixture was deoxygenated by three freeze-pump-thaw cycles. The reaction flask was filled with nitrogen and CuBr (3.8 mg, 0.026 mmol) was added to the frozen solution. The flask was sealed, purged with vacuum and backfilled with nitrogen once. The mixture was thawed and the flask was then immersed in an oil bath preheated at 50 °C to start the polymerization. After 6 hrs, the polymerization was stopped by cooling and exposing the reaction mixture to air. For purification, the as-prepared polymer solution was diluted with dichloromethane and passed through a basic alumina column to remove residual copper species. The solvent was removed under rotary evaporation at room temperature, and the polymer was isolated by precipitation from hexane, then dried under vacuum at room temperature for 15 hrs. Determination of critical micellar concentration using a NR probe. A stock solution of NR in THF at 1 mg/mL and a stock solution of ssTP in THF at 1 mg/mL and 5 µg/mL were prepared. Different amounts of the ssTP stock solution were mixed with the same amount of the NR stock solution (0.5 mL) in THF to prepare polymer and NR mixtures with increasing concentrations of ssTP. Total volume of the solution was adjusted to 2 mL by adding THF. After the drop-wise addition of water (10 mL), the resulting mixtures were stirred for 48 hrs to remove THF. They were then filtered through 0.45 µm PES filter to remove free NR, yielding a series of NR-loaded micelles with various concentrations of ssTP from 5x10-6 to 0.1 mg/mL. Their fluorescence spectra were recorded with λex = 480 nm to monitor the fluorescence intensity at maximum λem=620 nm. Aqueous micellization using dialysis method. Water (10 mL) was added drop-wise to an organic solution of ssTP (10 mg) in THF (2 mL) using a syringe pump equipped with a plastic syringe (20 mL volume, 20 mm diameter) at an addition rate of 0.2 mL/min. The resulting dispersion was dialyzed against deionized water (1 L) twice for 24 hrs, yielding aqueous micellar aggregates (NCMs) at 1 mg/mL concentration. In situ disulfide-crosslinking to core-crosslinked micelles. To aliquots of aqueous NCM dispersion (2.5 mL), an aqueous solution of DTT (177 µL, 0.2 mol equivalent to disulfide linkages in the micelles) was added using a syringe pump equipped with a plastic syringe (3 mL, 10 mm diameter) ACS Paragon Plus Environment

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at an addition rate of 0.1 mL/hr. The resulting mixture was stirred at room temperature for 2 days and dialyzed over water for 24 hrs to remove excess DTT to yield CCMs. To examine the occurrence of crosslinking through disulfide-thiol exchange reactions, aliquots of the aqueous CCM dispersion (0.1 mL) was mixed with DMF (1.9 mL) under stirring for 2 days. The resulting mixtures were analyzed using DLS to monitor any changes in size and size distribution. As controls, the similar procedures were conducted with aqueous NCM dispersions (with no DTT). Reduction-responsive degradation of aqueous CCMs in the presence of GSH. First, aliquots of aqueous micellar dispersion (1 mg/mL, 2 mL) were mixed with GSH (6.1 mg, 20 µmol, 10 mM) under stirring at room temperature. Samples were taken periodically to analyze the change in size distribution using DLS. In another set, NR-loaded NCM dispersion was prepared as described above, with water (8.3 mL), NR (0.83 mg), ssTP (8.3 mg), and THF (2 mL). Similarly, aqueous NR-loaded CCM dispersion was prepared by mixing aqueous NR-loaded NCM dispersion with catalytic amount of DTT as described above. Their fluorescence spectra (λex = 480 nm) were recorded at different time intervals to monitor fluorescence intensity at λem = 585-587 nm. Preparation of aqueous Dox-loaded CCM dispersions. Water (10 mL) was added drop-wise to the organic solution consisting of ssTP (20 mg), Dox (2 mg), and Et3N (2.0 µL) in DMF (1.5 mL). The resulting dispersion was stirred for 1 hr, and then dialyzed over water (1 L) for 24 hrs, yielding Doxloaded NCM dispersion at 1.5 mg/mL. To determine the loading level of Dox using UV/Vis spectroscopy, an aliquot of aqueous Dox-loaded micellar dispersion (1 mL) was mixed with DMF (5 mL) to form a clear solution of DMF/water (5/1 v/v) and its UV/Vis spectrum was recorded. The loading level was determined by the weight ratio of loaded Dox to dried ssTP. Next, Dox-loaded NCMs was mixed with catalytic amount of DTT to form Dox-loaded CCMs at 1.5 mg/mL (hereafter, Dox-CCM1.5). Similar procedure was used to prepare aqueous Dox-loaded CCM dispersions at 10 mg/mL (hereafter, Dox-CCM10), with the use of ssTP (107.5 mg), Dox (10 mg), Et3N (7.2 µL), DMF (2 mL), and water (8 mL). Note that Dox-CCM10 dispersion was used only for in vivo biodistribution studies. Colloidal stability of Dox-loaded micelles in the presence of proteins. Aliquots of Dox-loaded NCM and CCM1.5 dispersion (1 mL, 1.5 mg/mL) were mixed with aliquots of aqueous PBS solutions of BSA (1 mL, 80 mg/mL) and IgG (1 mL, 16 mg/mL). The resulting mixtures were incubated at 37 °C for 48 hrs. Aliquot of the aqueous mixtures were then subjected to centrifugation (10,000 rpm x 20 min) to precipitate any undesirably-formed aggregates. Quantitative analysis was conducted using


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bicinchoninic acid (BCA) assay (Pierce® BCA Assay Kit) according to our report40 based on manufacturer’s instruction. GSH-triggered release of Dox from Dox-loaded micelles. Aliquots of aqueous Dox-loaded NCM and CCM1.5 dispersion (3 mL, 1.5 mg/mL) were mixed with 10 mM GSH and transferred into dialysis tubing (MWCO = 12,000 g/mol) and immersed in PBS (40 mL) solution under stirring. An aliquot without GSH was used as control. Fluorescence spectra of outer solutions were measured over 40 hrs at λex = 470 nm. For quantitative analysis of %Dox release, the amount of Dox equivalent to that encapsulated in Dox-CCM1.5 (3 mL) was dissolved in water (40 mL, equivalent to outer water above) for fluorescence measurement. Cell culture. Human embryonic kidney (HEK 293T) and human breast adenocarcinoma (MCF-7) cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (fetal bovine serum) and 1% antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Flow cytometry. MCF-7 cells were plated at 5×105 cells/well into a 6-well plate and incubated in DMEM (2 mL) at 37 °C. After 48 hrs, cells were treated with Dox-CCM1.5 (300 µL, Dox = 4.7 µg/mL) and free Dox (Dox = 4.7 µg/mL) at 37 °C for 12 hrs. After the culture medium was removed, the cells were washed with PBS and treated with trypsin. The cells were suspended in DMEM (500 µL) for flow cytometry measurements. Data analysis was performed by means of a BD FACSCANTO II flow cytometer and BD FACSDiva software. Confocal laser scanning microscopy (CLSM). MCF-7 cells plated at 2×105 cells/well into a 6well plate and incubated for 48 hrs in DMEM (2 mL) were treated with Dox-CCM1.5 (Dox = 4.7 µg/mL) at 37 °C for 12 hrs. After culture medium was removed, cells were washed with PBS three times. After the removal of supernatants, the cells were fixed with cold methanol (-20 °C) for 20 min at 4 °C. The slides were rinsed five times with PBS and three times with TBST (tris-buffered saline Tween-20). Cells were stained with 2-(4-amidinophenl)-6-indolecarbamidine (DAPI). The fluorescence images were obtained using a LSM 510 Meta/Axiovert 200 (Carl Zeiss, Jena, Germany). Cell viability using MTT assay. HEK 293T normal and MCF-7 breast cancer cells were plated at 5 x 105 cells/well into a 96-well plate and incubated for 24 hrs in DMEM (100 µL) containing 10 % FBS and 1% antibiotics. They were then incubated with various concentrations of empty (Dox-free) and Dox-CCM1.5 as well as free Dox for another 48 hrs. Blank controls without micelles (cells only) were run simultaneously as control. Cell viability was measured using CellTiter 96 Non-Radioactive ACS Paragon Plus Environment

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Cell Proliferation Assay kit (MTT, Promega) according to the manufacturer’s protocol. Briefly, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (15 µL) was added into each well. After 4 hrs of incubation, the medium containing unreacted MTT was carefully removed. DMSO (100 µL) was added into each well in order to dissolve the formed formazan purple-blue crystals, and then the absorbance at λ = 570 nm was recorded using Powerwave HT Microplate Reader (Bio-Tek). Each concentration was 12-replicated. Cell viability was calculated as the percent ratio of absorbance of mixtures with micelles to control (cells only). In vivo bio-distribution. The amounts of Dox distributed in various tissues were investigated by fluorescence measurements. First, tumor models were established as 4T1 (2×106) was injected to right armpit of female BALB/c mice (20 ±2 g). After the sizes of tumors reached to 100 mm3 in volumes, 4T1-bearing mice were randomly divided into three groups (n = 5). They were then administrated via lateral tail veins with normal saline, free Dox, and Dox-CCM10 at a dosage of 2 mg equivalent Dox/kg. After 12 hrs, the mice were sacrificed. Tissues including liver, heart, spleen, lung, and kidney, as well as tumor were collected. They were then washed with saline and weighed after drying. Then each tissue was placed in dimethyl sulfoxide (DMSO, 2 mL), homogenized, vortexed for 15 min, and centrifuged for 5 min at 3,500 rpm. The resulting supernatants were analyzed for fluorescence spectroscopy with maximum fluorescence intensity. For quantification, different amounts of Dox were dissolved in DMSO to prepare a series of solutions of Dox at 5-2000 ng/mL in DMSO. Their fluorescence spectra were recorded to construct a calibration plot of fluorescence intensity of Dox over the concentration. Statistical analysis. Statistical analysis among experimental groups was performed using independent-samples t-test and IBM SPSS Statistics software, Version 19 (IBM Corporation, Armonk, NY, USA). P-value 350 nm. Due to the creation of disulfide inter-chain crosslinkages, the CCMs were swollen, not dissociated, in DMF/dispersion mixtures (95/5 v/v).


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a)

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b) Dav= 33 nm

ssTP Catalytic DTT

NCMs

10

100

1000

Dav= 28 nm

New disulfide crosslinks

CCMs

10

disulfide-thiol exchange

Dav < 200 nm (95% both proteins (BSA and IgG) remaining in the supernatants of the mixtures after centrifugation. These results suggest that hydrophilic PEG coronas with stealth effect43 provide Dox-loaded CCMs with excellent colloidal stability in the presence of serum proteins in pseudo-physiological conditions.


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GSH-triggered DOX release. Reduction-responsive enhanced release of Dox from Dox-loaded CCMs in the presence of glutathione was examined using a fluorescence spectroscopy. An aliquot of Dox-CCM1.5 was mixed with 10 mM GSH and the resulting mixture was placed in a dialysis tubing (MWCO = 12 kDa). Its Dox release kinetics was compared with an aliquot of NCMs mixed with and without 10 mM GSH as controls. Fluorescence spectra of the three systems in outer water was recorded over time upon excitation at λex = 470 nm (Figure S7). Then, the fluorescent intensity at λmax = 497 nm was followed to investigate %Dox release. Figure 6 shows the enhanced release of Dox from both NCMs and CCMs in the presence of 10 mM GSH, compared with no GSH. In response to GSH (a cellular reducing agent), the disulfide linkages and crosslinkages in the CCM cores can be cleaved. Such a reductive cleavage causes the disintegration of CCMs, resulting in the enhanced release of Dox. The released Dox molecules diffuse through the dialysis tubing into the outer water and thus fluorescent intensity of Dox in outer water increases. Note that Dox release was faster for NCMs, compared with CCMs in the presence of 10 mM GSH. 80 NCMs

70

10 mM GSH

60

Dox release (%)

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50 CCMs

40 30 20 NCMs/ No GSH

10 0 0

10

20

30

40

50

60

Time (Hrs) Figure 6. Release profile of Dox from Dox-loaded micelles (1.5 mg/mL) in the absence and presence of 10 mM GSH at pH = 7.4.

Antitumor activity and intracellular release. Given the promising results, the ssTP-based Doxloaded CCMs were evaluated as effective intracellular drug delivery nanocarriers for cancer therapy. ACS Paragon Plus Environment

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First, in vitro cytotoxicity were examined using a MTT colorimetric assay (Figure 7). The empty CCMs (Dox free) incubated with HEK 293T cells exhibit >80% viability up to 200 µg/mL concentration. For Dox-CCM1.5 incubated with MCF-7 adenocarcinoma cells, their viability gradually decreased to 70% when the amount of encapsulated Dox increased to 1.7 µg/mL. Upon further increase in the amount of encapsulated Dox to 2.4 µg/mL, the viability significantly decreased to 30%. These results suggest the inhibition of proliferation of MCF-7 cells in the presence of Dox-loaded CCMs. In parallel experiment with Dox only, the viability rapidly decreased to 30% with free Dox = 1.0 µg/mL and then gradually decreased to below 20% with an increasing amount of free Dox. Intracellular trafficking of Dox from Dox-loaded CCMs were examined using flow cytometry and CLSM. Figure 8 shows the flow cytometric histogram of MCF-7 cells incubated with Dox-CCM1.5 and free Dox. Note that the amount of free Dox was designed to be the same as that encapsulated in Dox-CCM1.5. Compared with MCF-7 cells only as a control, the histogram for Dox-CCM1.5 was shifted to high fluorescence intensity, even though the shift of the histogram for free Dox was more obvious. Figure 9 shows CLSM images of MCF-7 cells incubated with and without Dox-CCM1.5 and free Dox for 12 hrs. MCF-7 cells incubated with Dox-CCM1.5 show Dox fluorescence in their nuclei. Similar to flow cytometry, the signal for free Dox was more intense. Compared with free Dox, the Dox-loaded CCMs exhibit less cytotoxicity and less fluorescence intensities in flow cytometry and CLSM. The plausible reason could be free Dox being directly accumulation in cells, whereas, Doxloaded CCMs being required for the degradation that is essential for the release of the encapsulated cargoes.


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Viability of MCF-7 cells (%)

120

100 Dox-loaded CCMs

80

60

40

20 Free Dox

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Dox (µ µg/mL)

Figure 7. Viability of MCF-7 cells incubated with various amounts of free Dox and Dox-CCM1.5 for 48 hours determined by a MTT assay.

cells only

cells + Doxloaded CCMs

cells + Dox

Count

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Fluorescence intensity (a.u.)

Figure 8. Flow cytometric histogram of MCF-7 cells only and incubated with Dox-CCM1.5 and free Dox for 9 hrs.


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a)


DAPI

Dox

Merged

b)

c)

Figure 9. CLSM images of MCF-7 cells only (A) and incubated with Dox-CCM1.5 (B) and free Dox (C) for 12 hrs. Scale bar = 20 µm.

In vivo biodistribution studies. For preliminary in vivo studies of Dox-CCM10 with Dox loading level = 4% and diameter = 40 nm, their in vivo bio-distribution was further studied on a mouse model bearing 4T1 solid tumor (breast cancer cell lines). Fluorescence spectroscopy has been used for Dox as fluorescent maker.44, 45 In our experiment, a calibration plot of fluorescent intensity over concentration of Dox in DMSO was constructed (Figure S8). One group of the mice was intravenously injected through tail veins with Dox-CCM10 at a concentration of 3 mg/kg equivalent to Dox. The other two groups were i.v. administrated with free Dox and normal saline respectively as control. As seen in Figure 10, the accumulation of Dox in tumor sites was greater for Dox-CCM10 than free Dox (P

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