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Positively Charged Combinatory Drug Delivery Systems against Multidrug Resistant Breast Cancer: Beyond the Drug Combination Xu Yan, Qingsong Yu, Linyi Guo, Wenxuan Guo, Shuli Guan, Hao Tang, Shanshan Lin, and Zhihua Gan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14244 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 10, 2017

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ACS Applied Materials & Interfaces

Positively Charged Combinatory Drug Delivery Systems against Multidrug Resistant Breast Cancer: Beyond the Drug Combination

Xu Yan, Qingsong Yu*, Linyi Guo, Wenxuan Guo, Shuli Guan, Hao Tang, Shanshan Lin, and Zhihua Gan*

The State Key Laboratory of Organic-inorganic Composites, Beijing Laboratory of Biomedical Materials, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China

KEYWORDS: multidrug resistance, tumor vasculature targeting, combination therapy, drug delivery, positive charge.

ABSTRACT: The formation and development of cancer is usually accompanied with angiogenesis and related to multiple pathways. The inhibition of one pathway by monotherapy might result in the occurrence of drug resistance, tumor relapse or metastasis. Thus a combinatory therapeutic system that targets several independent pathways simultaneously is preferred for the treatment. To this end, we prepared combinatory drug delivery systems consist of cytotoxic drug SN38, pro-apoptotic 1

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KLAK peptide and survivin siRNA with high drug loading capacity and reductive responsiveness for the treatment of multidrug resistant (MDR) cancer. With the help of positive charge and the synergistic effect of different drug, the combinatory systems inhibited the growth of doxorubicin resistant breast cancer cells (MCF-7/ADR) efficiently. Interestingly, the systems without siRNA showed more superior in vivo anticancer efficacy than those with siRNA which exhibited enhanced in vitro cytotoxicity and pro-apoptotic ability. This phenomenon could be attributed to the preferential tumor accumulation, strong tumor penetration and excellent tumor vasculature targeting ability of the combinatory micelles of SN38 and KLAK. As a result, a combinatory multi-target therapeutic system with positive charge induced tumor accumulation and vasculature targeting which can simultaneously inhibit the growth of both tumor cell and tumor vasculature was established. This work also enlightened us that the design of combinatory drug delivery systems is not just a matter of simple drug combination. Besides the cytotoxicity and pro-apoptotic ability, the tumor accumulation, tumor penetration or vascular targeting may also influence the eventual antitumor effect of the combinatory system.

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1. INTRODUCTION Cancer, as one of the most notorious human diseases,1,2 is usually accompanied with neovascularization. Therefore, to suppress tumor progression thoroughly, the synergistic strategies against angiogenesis and tumor cell growth have attracted lots of attentions. Although some free drugs such as curcumin3 and azaindole derivatives4 have shown potential anticancer and anti-angiogenesis activities, the inhibition of one pathway by monotherapy may result in the emergence of drug resistance or tumor relapse, largely because of pathway redundancy, cross-talk, compensatory and neutralizing actions, or antitarget activities.5-8 It is broadly accepted that combination therapy which targets and inhibits multiple essential pathways of tumor growth, invasiveness and/or metastasis should be performed in order to enhance therapeutic efficiency, lower system toxicity, and overcome multi-drug resistance (MDR).9-11 For the simultaneous inhibition of tumor vasculature and tumor cell growth, some combinatory strategies which involved the co-administration or co-delivery of cytotoxic drugs or nucleic acid and anti-angiogenesis agents have been developed.12-15 However, in most of these combinatory systems, the inhibition of angiogenesis and cancer cells growth relied on different agents that act on neovasculature or tumor cells separately. In some other cases, researchers deliver combination of drugs and anti-angiogenesis siRNAs against complementary target inside tumor cells. But there is still only one active agent could act on tumor vasculatures.16 This may leave the backdoor for the potential drug resistance of cancer cells17,18 or the endothelial cells.19,20 Therefore, it is of great significance to establish multi-target therapeutic

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systems21 which could simultaneously overcome the MDR of cancer cells and weed out tumor vasculatures. In light of simultaneous targeting against tumor cells and tumor vasculatures, previous findings about the tumor accumulation enhancement and tumor vasculature colocalization by PEGylated positively charged micelles might provide potential options.22,23 Herein, we reported a positively charged combinatory drug delivery system with the ability to simultaneously suppress tumor cells and tumor vasculatures. To construct multi-target therapeutics, we introduced an integration of chemotherapeutic agents and pro-apoptotic peptides. 7-ethyl-10-hydroxycamptothecin (SN38), a metabolite of 7-ethyl-10-[4-(l-piperidino)-l-piperidino] carbonyloxy camptothecin (CPT-11, also known as irinotecan) which can inhibit the activity of topoisomerase I,24,25 was introduced in the form of PEGylated polymeric prodrugs to overcome its poor water solubility and form stable micelle core with the help of the strong intermolecular

π-π

stacking.26

The

cationic

pro-apoptotic

peptide,

KLAK

(CGG(KLAKLAK)2), which itself shows no obvious toxicity towards eukaryotic cells before endocytosis and can initiate caspase-dependent and/or caspase-independent cell apoptosis through the targeting damage of eukaryotic mitochondrial membranes after endocytosis,27-29 was introduced through a disulfide bond to facilitate its intracellular release.30 Therefore, compared with the conventional polymer-SN38 conjugates which rely on the neclear topoisomerase inhibition,31,32 the combinatory system may offer us more pathways to induce apoptosis.

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Since the combination of chemotherapeutic agents and nucleic acid such as small interfering RNA (siRNA) has exhibited great potential in reversing the drug resistance of tumor cells,33-35 we integrated survivin siRNA into this system to silence its target gene which is related to anti-apoptosis,36,37 and explored the influence of drug combination on the biological properties (similar combinations of chemotherapeutics and survivin siRNA have been proved to be efficient in the treatment of drug resistant cancer38,39). Meanwhile, simultaneous targeting towards tumor vasculature and tumor cells could be expected for the combinatory systems with positive charge. After the endocytosis by endothelial or cancer cells, SN38, survivin siRNA and KLAK peptide were expected to be efficiently released in acidic condition or in the presence of excess glutathione (GSH) to attack different intracellular target so as to activate the apoptosis and cell death (Figure 1A).

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Figure 1. A) Schematic illustration of transportation, vascular targeting, cellular uptake and intracellular drug delivery of the combinatory micelles. The cationic micelles can target tumor vasculature and inhibit angiogenesis; a preferable particle charge and size allows the micelles to penetrate and accumulate inside the tumor. Most of the micelles are endocytosized through both lysosomal and non-lysosomal pathways. The release of KLAK and siRNA mainly depend on the reductive environment inside cells. The combination of the multi-target therapeutics can efficiently overcome the multidrug resistance of tumor cells. B) Structure of PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38 copolymers. C, D) Size of 3Mf, 4Mf and 9Mf micelles measured by dynamic light scattering (DLS) (C) and Transmission Electron Microscopy (TEM) (D, 3Mf only). 6


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2. MATERIALS AND METHODS 2.1. Materials 2-Aminoethyl methacrylate hydrochloride (AEMA) and methoxy poly(ethylene glycol) (mPEG, Mw = 5/10 kDa) was purchased from Sigma-Aldrich. 2-hydroxy-1-ethanethiol, 2-hydroxyethyl aldrithiol-2,

2,2-dipyridyl

methacrylate

disulfide,

(HEMA),

mercaptoethanol,

2,

methacryloyl

2’-azobisisobutyronitrile

4-dimethylaminopyridine

chloride, (AIBN), (DMAP),

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and succinic anhydride was purchased from J&K chemical Inc. SN38 and irinotecan were purchased from Beijing Huafenginfo Co., Ltd. KLAK peptide (sequence: CGGKLAKLAKKLAKLAK) was purchased from China Peptide Co., Ltd. siRNA (sequence: Scrambled siRNA: sense 5’-CAGUCAGGAGGAUCCAAAGdTdG-3’, anti-sense 5’-CUUUGGAUCCUCCUGACUGdTdT-3’; GL3-FFL(luciferase) siRNA: sense

5’-

CUUACGCUGAGUACUUCGAdTdT-3’,

5’-UCGAAGUACUCAGCGUAAGdTdT-3’

;

survivin

5’-CGUACGCGGAAUACUUCGAdTdT-3’,

anti-sense siRNA:

sense anti-sense

5’-UCGAAGUAUUCCGCGUACGdTdT-3’) was purchased from Shanghai Gene Pharma Co., Ltd. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratory (Kumamoto, Japan). FBS (Fetal bovine serum), DMEM (Dulbecco’s Modified Eagle Medium), RPMI 1640 (Roswell Park Memorial Institute 1640 medium), DMEM/F12 (Dulbecco’s Modified Eagle Medium/Ham’s F12 50/50 Mix)

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and penicillin/streptomycin solution were obtained from Wisent (Canada). Chain transfer agent 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) and PEG macro chain transfer agent PEG-DDMAT,40 2-(pyridin-2-yldisulfanyl)ethyl methacrylate

(DSEMA)41

pentafluorophenyl

methacrylate

(MAPFP)42

and

HEMASN3832 were synthesized according to the literature procedure. The other chemicals (reagent grade) in this work were purchased from Sigma-Aldrich and used without further purification. 2.2.

Synthesis

of

Poly(ethylene

glycol)-b-poly(S-((2-(methacryloyloxy)ethyl)-S`-CGG(KLAKLAK)2-disulfide)-bpoly(HEMASN38) (PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38). The

block

copolymer

PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38

was

synthesized in two steps: Firstly, PEG-b-PDSEMA-b-PHEMASN38 was obtained through reversible addition-fragmentation chain transfer (RAFT) polymerization. Secondly, PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38 was synthesized with the conjugation of KLAK to the PDSEMA block. In the first step, PEG-DDMAT (0.2 g, 40 µmol), AIBN (0.4 mg, 2.4 µmol) and different amount of DSEMA were dissolved in 1.5 mL N, N-Dimethylformamide (DMF) and added into schlenck tubes. After three freeze-evacuate-thaw cycles, the tubes were sealed and the polymerization was conducted at 70 ℃ for 16 h. The reaction was halted by cooling with liquid nitrogen and the reaction mixture was precipitated into diethyl ether for three times. PEG-b-PDSEMA was collected as yellow powder, dried in vacuum overnight. Then 0.2 g PEG-b-PDSEMA, 0.4 mg

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AIBN and different amount of HEMASN38 were dissolved in 1.5 mL DMF and added into schlenck tubes. Same reaction and purification procedures were carried out to obtain PEG-b-PDSEMA-b-PHEMASN38. After the polymerization, the end capped chain transfer agent was removed by reaction with 2-fold excess AIBN at 70 ℃ for an additional 2 h. The molecular weight and chemical structure of all polymer precursors were analyzed by GPC (Waters 1515 GPC setup equipped with a Waters 2414 differential refractive index detector in DMF containing 20 mM lithium bromide at 50 ℃ with a flow rate of 1.0 mL min-1. A column set of a Styragel HR 1 DMF column, a Styragel HR 3 DMF column, and a Styragel HR 4 DMF column was employed. Narrowly distributed polystyrene standards in the range of 0.5~1000 kDa (Mainz, Germany) were used for calibration.) and 1H NMR (400MHz, Avance 400, Bruker, Germany), respectively. After the PEG-b-PDSEMA-b-PHEMASN38 was obtained, the KLAK peptide was conjugated to the PDSEMA block via sulfide exchange reaction. Briefly, 0.15 g PEG-b-PDSEMA-b-PHEMASN38, KLAK (0.1 g, 57 µmol) and glacial acetic acid (7 µL) were dissolved in 700 µL DMF and added to a glass bottle. After 6 h continuous stirring at room temperature, the product was dialyzed to remove DMF and excess KLAK, and lyophilized by freeze dryer (LABCONCO FreeZone®). The structure and molecular weight (Mn) of PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38 were characterized with 1H NMR. ℃ The

control

polymer

poly(ethylene

glycol)-b-poly(N-(3-(3-CGG(KLAKLAK)2-thio)-2-methylpropanamido)propyl)

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methacrylamide)-b-poly(HEMASN38) (PEG-b-P(MA-AEMA-KLAK)-b-PHEMASN38) was synthesized to investigate the influence of the disulfide linkage. Briefly, PEG-DDMAT (0.2 g, 40 µmol), AIBN (0.4 mg, 2.4 µmol) and MAPFP (0.08 g, 0.32 mmol) were dissolved in 1.5 mL DMF. The reaction was carried out following the same procedure as that of PEG-b-PDSEMA. After the polymer was dried in vacuum, PEG-b-PMAPFP (0.2 g, 40 µmol), AIBN (0.4 mg, 2.4 µmol) and HEMASN38 (0.15 g, 0.25 mmol) was dissolved in 1.5 mL DMF,

reacted

and

purified

under

the

same

condition

to

obtain

PEG-b-PMAPFP-b-PHEMASN38. Then 0.2 g PEG-b-PMAPFP-b-PHEMASN38 was reacted with AEMA (55 mg, 0.43 mmol) and triethylamine (65 mg, 0.64 mmol) in 2 mL DMF under room temperature for 24 h. The resultant solution was dialyzed and lyophilized to yield the PEG-b-P(MA-AEMA)-b-PHEMASN38. KLAK peptide was linked to the P(MA-AEMA) block through thiol-ene click reaction. Briefly, 100 mg PEG-b-P(MA-AEMA)-b-PHEMASN38 was reacted with 100 mg KLAK in 700 µL DMF in the presence of 20 mg triethylamine for 24 h. The mixture was then dialyzed and lyophilized to yield the final PEG-b-P(MA-AEMA-KLAK)-b-PHEMASN38. 2.3. Preparation and Characterization of Micelles Micelles were prepared via nanoprecipitation method. Briefly, 30 mg polymer was dissolved in 1 mL DMF, and slowly injected into 10 mL water with a Longerpump® LSP01-1A micro-injection pump under continuous stirring. The mixture solution was dialyzed in 10 mM sodium phosphate buffer (pH 7.4) to remove DMF.

For

the

ease

of

nomenclature,

micelles

10


made

from

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PEG114-b-P(DSEMA-SS-KLAK)5-b-PHEMASN3820, PEG114-b-P(DSEMA-SS-KLAK)12-b-PHEMASN385, PEG228-b-P(DSEMA-SS-KLAK)12-b-PHEMASN385, PEG114-b-PDSEMA5-b-PHEMASN3820,

and

PEG114-b-P(MA-AEMA-KLAK)6-b-PHEMASN3818 copolymers were abbreviated as 3M, 4M, 9M, PSN and NC, respectively. The sizes and zeta potentials of these micelles were determined at 25 ˚C by dynamic light scattering (DLS) using a zetasizer equipped with a 633 nm laser (Zeta-Sizer Nano Series; Malvern, Worcestershire, United Kingdom). Transmission electron microscopic (TEM) analysis was carried out on a JEM-2100F apparatus (JEOL, Japan) with 200 kV accelerating voltage. To test the stability of as-prepared micelles, micelles was added into 10% serum to reach a concentration of 1.5 mg mL-1 and incubated at 37 ˚C. The micelles sizes were monitored with DLS at 24 h, 48 h and 72 h, respectively. To determine the critical micelle concentration (CMC) of KLAK conjugated micelles, a known amount of Nile Red dissolved in dichloromethane (DCM) was added to a series of vials. When DCM was evaporated, a measured amount of PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38 micelles were added to each vial to obtain a final concentration of 1 µM Nile Red and stirred overnight in dark. The fluorescence emission intensity was measured at the wavelength of 620 nm (excited at 543 nm) using an F-4600 fluorescence spectrometer (Hitachi, Japan). CMC was determined at the intersection of the tangents to the two linear portions of the curve of the fluorescence intensity as a function of polymer concentration.43

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2.4. Preparation and Characterization of siRNA-Loaded Micelles The siRNA loaded micelles were fabricated by the electrostatic interaction between micelles and siRNA. Briefly, micelles and different amount of siRNA with N/P ratios of 1/1, 2/1, 4/1, 8/1, and 16/1 were mixed in UltraPure™ DEPC (diethylpyrocarbonate)-Treated Water (Thermo Fisher Scientific) by gentle pipetting. The mixtures were then maintained at 25 ℃ for 20 min to yield target micelles. During the fabrication process, different siRNA such as scrambled siRNA and survivin siRNA were introduced. For the ease of nomenclature, the micelles without siRNA were abbreviated as xMf; the micelles loading survivin siRNA were abbreviated as xMsur; and the micelles loading scrambled siRNA were abbreviated as xMs, i.e. 3Mf, 3Msur and 3Ms for 3M micelles without siRNA, 3M micelles with survivin siRNA and 3M micelles with scrambled siRNA, respectively. The siRNA loading ability of micelles was analyzed by electrophoresis with 1% agarose gel and a voltage of 200 V for 8 min in TAE buffer solution (40 mM Tris-HCl, 1 v/v% acetic acid, and 1 mM EDTA). The retardation of the complexes was visualized by staining with ethidium bromide. The sizes and zeta potentials of siRNA-loaded micelles with different N/P ratios were determined by DLS method at 25 ℃. The protection of siRNA by micelles was also tested with electrophoresis. Briefly, siRNA was firstly complexed with 3Mf at N/P ratio of 4/1, and then treated with 10% serum at 37 ˚C. After that, 10-fold excess of heparin sodium was added to the micelle/siRNA complex to completely replace the siRNA before electrophoresis.

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A control test of naked siRNA in 10% serum was also carried out at the same condition. 2.5. Cell Line and Cell Culture The murine breast cancer cell line (4T1) and the human breast cancer cell line (MCF-7) were obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China), 4T1-Luc cells stably expressing firefly luciferase was purchased from Cobioer Inc. (Nanjing, China), DOX resistant MCF-7/ADR cell line was purchased from Cellbio Inc. (Shanghai, China). 4T1 cells, 4T1-Luc cells and MCF-7 or MCF-7/ADR cells were grown in RPMI 1640 medium, DMEM/F12 medium and DMEM medium, respectively. All mediums were supplemented with 1% penicillin-streptomycin mixture and 10% fetal bovine serum (FBS). All cells were maintained at 37 ℃ in a humidified atmosphere of 5 % carbon dioxide and 95 % air. 2.6. Animal Models All animal experiments were performed in accordance with guidelines of Peking University Health Science Center Animal Care and Use Committee under the protocols approved by the Institutional Animal Care and Use Committee at Peking University (Beijing, China). 4T1 murine tumor model was established by subcutaneously injecting 4T1 cells (0.1 mL, 1×106) into the right backside of female BALB/c mice (18±2 g, 5-6 weeks old). Similarly, drug resistant tumor model was established by subcutaneously injecting DOX resistant MCF-7/ADR cells (0.1 mL, 1×106) into the right backside of female athymic nude mice (18±2 g, 5-6 weeks old). Tumors were allowed to grow up to 50 mm3 before the subsequent experiments.

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The other experimental procedures related to this work could be found in the supporting information.

3. RESULTS AND DISCUSSION 3.1. Fabrication and Characterization of Polymers and Micelles Table 1. Physicochemical Characterization of Polymers and Free Micelles Zeta potential Sample

CMC

SN38

KLAK

DLC

a)

Polymer composition

Size[nm]

[mV]a)

[µg mL-1] [wt%/mol%]b) [wt%/mol%]b) [wt%/mol%]b)

3M

PEG114-P(MEMA-SS-KLAK)5-PHEMASN3820

64.8±1.2

11.8±0.7

1.7

28.90/14.30

32.20/3.60

61.10/17.90

4M


89.6±0.9

1.83±1.8

2.4

6.25/3.82

65.2/9.16

71.50/12.98

9M


95.8±1.7

0.83±1.4

2.9

5.41/2.04

56.4/4.89

61.80/6.93

PSN

PEG114-PDSEMA5-PHEMASN3820

49.6±0.5

-3.43±0.9

1.3

42.80/14.30

-

42.80/14.30

NC

PEG114-P(MA-AEMA-KLAK)6-PHEMASN3818

92.1±2.4

4.63±0.3

1.9

25.6/13.00

38.10/4.30

63.70/17.30

a)

Size and zeta potential were obtained from DLS measurements in 10 mM PBS

buffer (pH 7.4). Micelle concentration was set as 1.0 mg mL-1;

b)

wt% and mol%

represents the weight percentage and molar percentage of SN38 and KLAK in the block copolymers measured by 1H NMR, respectively. DLC which stands for the drug loading content was calculated as the sum of SN38 and KLAK by weight or by molar amount.

For the synthesis of PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38 (Figure 1B, Figure S1), polymer precursors PEG-b-PDSEMA-b-PHEMASN38, was prepared 14


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through the sequential RAFT polymerization of DSEMA and HEMASN38 monomers with PEG-DDMAT. Cationic peptide KLAK was then conjugated to the polymer backbone via thiol exchange reaction. A series of block copolymers (1M—9M) which could be divided into three groups according to their composition (i.e. 1M—3M, 4M

— 6M and 7M — 9M) have been synthesized to show the influence of polymer composition on the in vivo biodistribution (Table S1). In order to evaluate the influence

of

disulfide

bond

on

the

drug

release,

PEG-b-P(MA-AEMA-KLAK)-b-PHEMASN38 without disulfide linkage was also synthesized (Figure S3). The chemical composition of the copolymers was determined by 1H NMR. As shown in Figure S2 and S4, the composition of DSEMA, HEMASN38 and KLAK could be determined by the integral area ratio between 8.2-8.3 ppm, 5.25-5.52 ppm, 1.4-1.7 ppm and 3.3-3.6 ppm (-CH2CH2- from PEG block), respectively. The disappearance of pyridine group (8.2-8.3 ppm) and the double bond peak (5.2-5.5 ppm) in PEG-b-P(MEMA-SS-KLAK)-b-PHEMASN38 and PEG-b-P(MA-AEMA-KLAK)-b-PHEMASN38 (Figure S4) indicated that the thiol-exchange reaction and the thiol-ene click reaction between KLAK peptide and corresponding polymers were quantitatively. The molecular weight and molecular weight distribution of as-prepared polymers were measured by GPC. The representative GPC elution curves of PEG114-b-PDSEMA5-b-PHEMASN3820 and the intermediate polymers are showed in Figure S5.

The weight averaged molecular

weight, number averaged molecular weight and molecular weight distribution of various PEG-b-PDSEMA-b-PHEMASN38 precursors were summarized in Table S1.

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Micelles of polymers with different composition were prepared via nanoprecipitation method. The physico-chemical characteristics of micelles could be found in Table 1 and Table S1. The size of micelles from 1M—9M ranged from 33 nm to 96 nm with relatively narrow distribution (Figure 1C&D) and remained stable after incubation with 10% serum for 72 h (Figure S8), indicating the efficient protection by PEG shell from serum proteins. Overall, higher SN38 content, higher KLAK content and longer PEG block all led to larger micelle size. The positive surface charge of all micelles could be attributed to the lysine residue on the KLAK peptide. In order to investigate the influence of polymer composition on the biodistribution, a hydrophobic near infrared dye Cy 7.5 was loaded into 1M—9M micelles which were then subjected to biodistribution study via fluorescent imaging techniques. As shown in Figure S6 & S7, the biodistribution of different micelles varied a lot from each other. For 1M—3M micelles with same hydrophilic block, 3M with longer hydrophobic block showed more specific tumor accumulation, probably due to its larger size. However, for the rest two groups such as 4M—6M and 7M— 9M, micelles with longer hydrophilic block and shorter hydrophobic block showed more specific tumor accumulation. For more in-depth study of the in vitro and in vivo biological properties after loading siRNA, micelles with most specific tumor accumulation were selected from each group, i.e. 3M, 4M and 9M Because the membrane-disrupting activity of the KLAK peptide mainly depends on its α-helical conformation,44 we then examined the secondary structure of KLAK-containing micelles. The circular dichroism spectrum of micelles containing

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KLAK peptide showed clear α-helical structure, whereas free KLAK peptide itself was predominantly random coil (Figure 2A). This indicated that the linkage of peptide to the polymer backbone may help the formation of α-helical structure.29 Accordingly, more KLAK peptide in the polymer chain (4Mf or 9Mf) caused stronger α-helical signals. In contrast to the increased amount of α-helix, the increase of KLAK content caused the decrease of zeta potential (Table 1). Compared with free micelles, the loading of siRNA which itself showed weak helical structure caused the significant increase of α-helical signals. The sizes and zeta potentials of micelles loaded with different amount of siRNA could be found in Figure 2C. For 3M, the zeta potential values underwent no significant change before N/P ratios decreased from 2/1 to 1/1 which caused the charge reversal of RNA loaded micelles. Similar charge reversal properties could also be observed for 4M and 9M which contained higher KLAK content. Besides the charge reversal, sudden zeta potential increase also occurred at N/P = 16/1 for 4M and 9M. As reported in the literature, KLAK peptide can form α-helical structure with hydrophobic residues distributed on one side of the helical axis and cationic residues on the other.44 Therefore, because of the rigidity of α-helical structure, only part of the positive charge could be exposed in water. For micelles with higher amount of KLAK peptide, the strong inter- or intra- molecular hydrogen bond (this might be proved by the gel formation in DMF solvent during the conjugation process of KLAK peptide, data not shown) might lead to more compact stacking of α-helix and cause the exposure of less positive charge. However, the loading of siRNA could occupy the spaces between KLAK and cause the exposure of

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more α-helical structure and positive charge as indicated by the stable size and increased zeta potentials (Figure 2C). Only with excess siRNA (N/P=1/1) can the positive surface be completely covered by negative charge. This might explain the zeta potential alteration of micelles with different amount of siRNA. It is noteworthy that the size of micelles loaded with various amount of siRNA maintained the same, possibly due to the strong π-π stacking interaction between SN38 molecules.32, 43 The stable size might facilitate the subsequent investigation about the influence of siRNA content. To investigate the siRNA loading capacity of the above micelles, electrophoresis analysis was carried out. As shown in Figure 2B and Figure S8, the maximum loading capacity of siRNA for 3M and micelles with higher KLAK content (4M and 9M) was 2/1 and 4/1, respectively. Besides the clear siRNA strip, another blurry strip in the middle of the gel could also be observed for responsive micelles other than NC micelles (Figure 2B and Figure S12), which might be caused by the breakage of the disulfide linkage during the electrophoresis.45 Moreover, the stability assay showed that the surface PEG layer could efficiently protect the siRNA from enzyme degradation (Figure S9).

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Figure 2. A) The secondary structure of free KLAK, free siRNA, free micelles and RNA-loaded micelles measured by circular dichroism, a positive peak at 192 nm and two negative peaks at 208 nm/222 nm indicates the α-helix structure in KLAK-conjugated micelles. B) Electrophoresis of siRNA-loaded micelles, a blurry strip (marked with red rectangle) above the strip of siRNA indicated the breakage of disulfide bond during electrophoresis. C) Size and zeta-potential alteration of micelles loaded with different amount of siRNA. Data are presented as mean ± SD (n = 3).

3.2. In Vitro Micelle-cell Interaction and Intracellular Release of siRNA To explore the interaction between micelles and tumor cells, the cellular uptake and the endocytic pathways of free micelles, KLAK induced mitochondrial injury, and the intracellular transportation of siRNA have been investigated. As shown in Figure 3A, because of the positive charge of KLAK peptide, the Nile Red loaded 3Mf micelles could be efficiently endocytosed by MCF-7/ADR cells. Dispersive red fluorescence was observed at the early stage of endocytosis and showed only partial

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colocalization with lysosome and no obvious colocalization with mitochondria (Figure S15), indicating that the micelles can either bypass the lysosomal pathway or escape from early endosomes which was beneficial for siRNA release. However, with the extended incubation time, the colocalization between micelles and lysosome increased. For the investigation of the endocytic pathways, cells were pretreated by chloropromazine,46 hypertonic sucrose,47,48 nystatin,49 mβCD,50 and cytochalasin D,51 respectively. The concentration of various inhibitors was set in the safe region according to the cytotoxicity assay (data not shown). The cells were also incubated at 4 ℃ to detect any energy-dependent endocytic process. As shown in Figure 3B, the cellular uptake of 3Mf could be inhibited by cytochalasin D, mβCD, nystatin and 4 ℃ incubation. Among these inhibitors, cytochalasin D and 4 ℃ incubation inhibited more efficiently than mβCD or nystatin. This indicated that the endocytosis of cationic

micelles

was

mainly

mediated

by

macropinocytosis.

But

the

caveolae-mediated endocytosis also contributed to the uptake of micelles.52 Accordingly, these results might explain the different lysosome colocalization level at different incubation time. The cellular uptake of 3M micelle loaded with different amount of siRNA was also characterized by CLSM and flow cytometer. As shown in Figure S13 and S14, 3Mf, 3Ms (4/1) showed higher uptake level than 3Ms (1/1). This was in accordance with the zeta potentials of these micelles. Compared with 3Ms (4/1), 4Ms (4/1) and 9Ms (4/1) showed similar endocytosis level.

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Figure 3. A) Endocytosis of Nile red (red) loaded 3Mf micelles in MCF-7/ADR cells, lysosome was stained with LysoTracker® Green DND-26. The micelles could be efficiently endocytosed by MCF-7/ADR cells, but only partial colocalization with lysosome could be observed. B) Endocytosis inhibition of 3Mf micelles after pretreated with different inhibitors, endocytosis rate was calculated through fluorescence intensity of Nile red. C, D) Detection of mitochondrial membrane potential change (∆ψm) by JC-1 in MCF-7/ADR cells, micelles with cleavable KLAK could destroy most of the mitochondria and result in the reduce of red fluorescence intensity. No obvious red fluorescence could be detected with CLSM (D) in 3Mf, 4Mf and 9Mf groups at 6h, which was consistent with the flow cytometry result (C). E) In 21



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vitro delivery of siRNA in MCF-7/ADR cells. FAM labelled siRNA (green) could be delivered into cells efficiently. The N/P ratios for 3Ms, 4Ms and 9Ms were 4/1. Only partial of colocolization with lysosome (stained with LysoTracker® Blue DND-22) could be observed. Data are presented as mean ± SD (n = 6). *p < 0.001; n.s. means no significant difference.

To evaluate the activity of KLAK peptide, mitochondrial membrane potential was determined by JC-1 assay (Figure 3C&D).28 For 3Mf micelles, the mitochondrial membrane potential which is adverse to the fluorescence ratio of JC-1 monomer (green) and JC-1 aggregates (red) was at the same level as the positive control, sodium azide (Figure S16).53,54 In contrast to this, for micelles with more cleavable KLAK such as 4Mf and 9Mf, the membrane potential declined significantly to be even lower than sodium azide. With the increase of incubation time, JC-1 fluorescence became dispersive, indicating the disruption of mitochondrial membrane. However, for free KLAK peptide which can hardly enter cells and NC micelles which cannot release KLAK peptide inside cells, the mitochondrial membrane potential was at the same level as normal cells. These results proved that KLAK peptide could be efficiently released from the micelles inside cells to attack mitochondrial membrane and that the reversible linkage well maintained the membrane disrupting activity of KLAK peptide.29 In order to study the intracellular transportation of siRNA in reduction sensitive micelles, FAM labeled siRNA was loaded into various micelles and co-incubated with

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MCF-7/ADR cells. The intracellular distribution of micelles loaded with FAM-labeled siRNA (N/P = 4/1) (Figure 3E) indicated that the siRNA loaded by micelles other than free siRNA could be efficiently endocytosed by MCF-7/ADR cells. The partial colocalization between siRNA and punctate stained lysosome for 3Ms could be attributed to the efficient escape from early endosome or the bypassing of lysosomal pathway which prevent the siRNA and KLAK peptide from lysosomal enzyme degradation.55 After longer incubation time, both dispersive blue and green fluorescence could be observed, indicating the disintegration of lysosome and lysosomal escape of siRNA. This lysosomal escape was observed earlier for 4Mf and 9Mf with more KLAK, probably due to the strong membrane disrupting capability of KLAK peptide.29 In contrast to the efficient intracellular release of siRNA in responsive micelles, siRNA loaded by NC micelles were mostly compartmentalized near the cell membrane even at 6 h. This indicated that the reduction sensitive cleavage of KLAK peptide (Figure S10) did help to release the siRNA.

3.3. Cytotoxicity, Apoptosis, and Gene Silencing Caused by Combinatory Micelles

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Figure 4. A) IC50 values of free drugs and micelles incubated with MCF-7/ADR cells for 24h as measured by CCK-8 Kit and calculated with Graph Pad Prism 5. *p < 0.001. B) Apoptosis of MCF-7/ADR cells induced by micelles and free drugs. C, D) Transfection of survivin siRNA into MCF-7/ADR cells, the amount of survivin mRNA and survivin protein was measured by RT-PCR and western blot (C and D), respectively. N/P ratios for all micelles are 4/1. Data are presented as mean ± SD (n = 6). *p < 0.001.

The cytotoxicity of free drugs, free micelles and survivin-loaded micelles against MCF-7 and MCF-7/ADR cells was evaluated by CCK-8 assay (Figure 4A, Figure S11). The drug resistant property of MCF-7/ADR could be proved by the different 24


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DOX cytotoxicity against the two cell lines (p < 0.001). In contrast to this, SN38 showed indiscriminate toxicity in both cell lines, indicating that SN38 might be an option to overcome the MDR of cancer cells.56 No noticeable toxicity could be observed for free survivin siRNA in the range of tested concentration (1000 µg mL-1) for both cell lines. Since the synergistic effect of different therapeutics which was affected by the polymer composition might play important role in cytotoxicity, the total drug concentration of each micelle was used for a better comparison of the results of the synergistic effect. The IC50 values of free KLAK were 74.83 µg mL-1 and 213.32 µg mL-1 towards MCF-7 and MCF-7/ADR cells, respectively, possibly due to the low internalization and the lack of helical structures.28 Considering the higher cytotoxicity of free SN38 (IC50 = 10.73 µg mL-1) than PEG-PSN38 micelles32 and the slow release profile of SN38 (Figure S11), the higher toxicity of 3Mf micelles (IC50 = 4.82 µg mL-1) compared with free SN38 and KLAK should be attributed to conjugated KLAK peptides. This could also be proved by the higher cytotoxicity of 3Mf compared with PSN micelle without KLAK. Similarly, the higher cytotoxicity of 3Msur micelle compared with NCsur micelle without disulfide bonds indicated that the intracellular release of KLAK played important roles in suppressing tumor cell growth. The higher cytotoxicity for 4Mf (IC50 = 2.24 µg mL-1) and 9Mf (IC50 = 3.15 µg mL-1) with higher KLAK content also led to the same conclusion. Besides KLAK peptide, the loading of survivin siRNA in each micelle also led to enhanced cytotoxicity, indicating the silencing of survivin mRNA was conducive to cell death.36,37

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The gene silencing efficiency of siRNA loaded micelles was determined in MCF-7/ADR cells with survivin siRNA. Since the observed IC50 values of MCF-7/ADR cells for 3Msur, 4Msur and 9Msur micelles were 2.17, 0.81 and 0.72 µg mL-1, respectively, the results might be influenced by the nonspecific effect such as the toxicity of micelles. This issue could be addressed by siRNA treatments over a concentration range and decisions were made based on non-toxic concentrations.57 However, the above method may not be applicable in this work because of the low IC50 values. In order to exclude the effect of cytotoxicity, 3Mf and NCsur micelles was introduced. Micelles with 100 ng mL-1 siRNA and N/P ratio of 4/1 was co-incubated with MCF-7/ADR cells, mRNA was extracted at 24 h after incubation and quantified by qRT-PCR. As shown in Figure 4C, free survivin siRNA showed no silencing effect on the mRNA while 3Mf and 3M with scrambled siRNA (3Ms) significantly down-regulated the amount of survivin mRNA (p < 0.001), probably due to the cytotoxicity of the micelles.58 Survivin siRNA-loaded 3M (3Msur) group had stronger silencing effect on survivin mRNA than micelles loaded scrambled siRNA, indicating the specificity of survivin siRNA. The silencing effect of 3Msur, 4Msur and 9Msur micelles with same siRNA concentration was similar to each other. The survivin mRNA level of NCsur micelle treated cells was similar to control group and significantly higher than 3Mf and 3Msur, indicating that the release of KLAK was crucial for gene silencing. Besides, the survivin expression level was further characterized with western blots (Figure 4D). The results were consistent with that of qRT-PCR except that the expression level of survivin in 4Msur group and 9Msur

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group was lower than that in 3Msur group. This can be interpreted by the different siRNA delivery efficiency of 3Msur, 4Msur and 9Msur micelles as indicated in Figure 3E. Since siRNA in 4Msur and 9Msur micelles could be delivered more rapidly than 3Msur, the efficient gene silencing would occur earlier for 4Msur and 9Msur, and the total inhibition effect of 4Msur and 9Msur within 36 h would be stronger than that of 3Msur. However, at 24 h, the siRNA in all micelles had been efficiently delivered into cells. Therefore, the survivin mRNA level of all the three groups at 24 h remained the same. The pro-apoptotic ability of micelles and free drugs (Figure 4B, Figure S18) was in good agreement with the drug delivery and gene silencing data. Both free micelles and siRNA-loaded micelles treated groups showed higher apoptotic rate than free SN38 of the same concentration, while free survivin siRNA and KLAK treated cells showed no obvious apoptosis. Notably, the late apoptotic ratio for 3Msur, 4Msur and 9Msur with siRNA was 92.05, 82.89 and 93.9%, respectively. These values were significantly higher than their RNA free counterpart whose apoptotic ratios were 85.66, 58.34 and 48%, respectively. Collectively, the above results proved that the combinatory systems of multi-target therapeutics were able to cause efficient apoptosis of MDR cells by the synergistic effect of mitochondrial membrane disrupting, survivin mRNA silencing and topoisomerase I inhibition.

3.4. In Vivo Biodistribution and Vasculature Targeting

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As shown in Figure 2C, different N/P ratios of micelles resulted in different surface charge. To investigate the influence of siRNA content on the biodistribution patterns of micelles, the in vivo and ex vivo distribution of 3M, 4M and 9M micelles with different N/P ratios were investigated (Figure 5A & B, Figure S14 & S15). Cy 7.5 and Nile Red loaded micelles were used for the biodistribution and intratumoral distribution study, respectively. According to the in vitro release study of these two fluorophores (data not shown), less than 2% was released from the diluted micelles during the 48 h incubation which allowed the further distribution study with these micelles. In order to study the biodistribution of micelles in both immunocompetent and immunodeficient animals, two different kinds of tumor models, 4T1 and MCF-7/ADR models on BALB/c and BALB/c nude mice, respectively, were used in the biodistribution experiments. As shown in Figure 5A which presents the biodistribution of 3Msur micelles with different N/P ratios in both 4T1 and MCF-7/ADR models, PEG outer shell provided efficient protection for positively charged micelles so that they could accumulate in tumor through the EPR effect.59 The loading of siRNA caused the increase of zeta potential which may increase the reticuloendothelial (RES) uptake. Therefore, for 3Msur, the liver to tumor (L/T) and spleen to tumor (S/T) ratios increased with the siRNA content from 0 to 2/1 (Figure 5B). However, for 4Msur and 9Msur, no strict dependence could be observed for the L/T or S/T ratios and the N/P ratios of micelles. This might be influenced by the different size, composition and the complicated surface properties of 3M micelles compared with micelles with higher KLAK content (4M or 9M). Overall, 3Mf, 4Msur

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(8/1), and 9Msur (8/1) showed the most optimal biodistribution patterns in 3Msur, 4Msur and 9Msur groups, respectively. The difference for same micelle with different siRNA loading content must be arributed to the different surface properties caused by loaded siRNA. Compared to micelles with N/P ratio of 1/1 which exhibited negative surface charge, micelles with positive charge were more prone to accumulate in tumor. As indicated in Figure 2, the absorption of siRNA should happen in the spaces between the rigid α-helix. Therefore, excess siRNA would inevitably cause the expansion of KLAK layer. As a result, the negatively charged surface lost the sufficient protection of PEG layer so as to be captured by the RES system. The superiority in tumor accumulation of positively charged micelles might be caused either by their stronger vasculature targeting ability22 or by their stronger tumor penetrating ability.23 Among all micelles, 3Mf and 3Msur (N/P = 16/1) with the smallest size (65 nm) and appropriate positive charge had the most favorable tumor accumulation and the least normal organ uptake (Figure 5B). In comparison with the biodistribution in 4T1 tumor bearing BALB/c mice, similar distribution patterns could also be observed in MCF-7/ADR tumor bearing female BALB/c athymic nude mice (Figure 5A & Figure S21). To further investigate the tumor penetrating and vasculature targeting ability of these micelles, tumor slices treated with Nile Red loaded 3Mf and 3Msur (N/P = 16/1) were stained with CD31-FITC antibody. Figure 5C shows the intratumoral distribution of Nile red-loaded micelles. Both tumor penetration and vascular colocalization (R = 0.821) could be observed for 3Mf micelles. After the loading of siRNA, the micelles showed similar penetrating ability,

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but the colocolization level with blood vessel (R = 0.400) decreased significantly even though they had similar size and zeta potential. The reason for this phenomenon was still unclear, we assumed that it might be due to the adsorption of siRNA which may change the surface properties and influence the interaction between micelles and endothelial cells.

Figure 5. A) Biodistribution of 3M micelles (labeled with cy7.5) loaded with different amount of siRNA in 4T1 tumor-bearing BALB/c mice and MCF-7/ADR

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tumor-bearing BALB/c nude mice. Optical imaging was conducted on an IVIS® Spectrum in vivo imaging system using 745/800 filter combination at 48h after administration. Main organs were presented in each group in a sequence of heart, lung, liver, spleen, kidney and tumor. B) The relative accumulation of micelles in liver and spleen calculated from the fluorescent quantitative analysis. The fluorescent intensity of tumor was set as 1.0. C) The intratumoral distribution and vascular targeting ability of 3Mf and 3Msur (16/1) micelles were evaluated at 24h after injection. For both groups, a large amount of the micelles could be observed to efficiently penetrate the tumor tissue, however, the colocalization index (R) of 3Mf was higher than 3Msur. Data are presented as mean ± SD (n = 5).

3.5. Tumor Growth Inhibition, Anti-Angiogenesis Ability and Histological Analysis To evaluate the anticancer efficacy, free drugs and micelles were given intravenously every other day for 8 days to female athymic nude mice bearing subcutaneous drug resistant breast cancer (MCF-7/ADR) at a dose of 5 mg kg-1 starting after the tumor volume reached 50~100 mm3. The siRNA loaded micelles which showed strong cytotoxicity or pro-apoptotic ability and the same cellular uptake level, i.e. 4Msur (4/1) and 9Msur (4/1), were chose to evaluate the effect of the synergisticity of three components and the biodistribution on the eventual anticancer efficacy. Besides, 3Mf and 3Msur with N/P of 16/1 was also administered to evaluate the role of vasculature targeting. The tumor growth inhibition was evaluated by the

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tumor volume change (Figure 6A) and the final tumor weight (Figure 6C). The angiogenesis ability of tumors treated by free drugs and micelles was preliminarily evaluated by CD31 immunostaining. Because of its poor solubility in most physiologically compatible and pharmaceutically acceptable solvents, SN38 was replaced with commercially available irinotecan

during the treatment even though

the latter drug showed 100~1000 times weaker cytotoxicity.32, 43 From Figure 6, the tumor growth inhibition ability of free drugs and micelles ranked as follows: 3Mf (86.3%) > 3Msur (72.4%) > irinotecan (65.9%) > 4Msur (50.2%) > 9Msur (29.9%) ≈ KLAK (25%) > PBS. No obvious tumor growth inhibition could be observed for free KLAK peptide, consistent with its in vitro cytotoxicity. Similarly, despite the strongest cytotoxicity and pro-apoptotic ability, 9Msur (4/1) showed comparable tumor growth inhibition as free KLAK peptide due to its poor tumor accumulation (Figure 6 & Figure S19). In contrast to 9Msur (4/1), 4Msur (4/1) which showed higher tumor accumulation (Figure S19) exhibited stronger tumor growth inhibition. Compared with free irinotecan which showed obvious tumor growth inhibition, both 3Mf and 3Msur showed significantly higher suppressing ability. Moreover, 3Mf showed much more superior tumor growth inhibition ability than all siRNA loaded micelles even though it possessed weaker in vitro cytotoxicity and pro-apoptotic ability (Figure 6C&F). For 3Mf and 3Msur, the difference in tumor growth at the initial 4 days was indistinguishable from each other. But after 6 days the superiority of 3Mf began to emerge, probably due to its better targeting ability against tumor vasculature. This could also be proved by the vasculature staining of tumor slices

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treated by free drugs or micelles. The preliminary CD31 staining (Figure 6D and Figure S22) and the relevant quantitative analysis (Figure 6E) of tumor slices showed that all positively charged micelles except for 9Msur showed more efficient tumor vasculature inhibition activity than free drugs. More intriguingly, even though free irinotecan showed more efficient tumor growth inhibition than 4Msur, the micelle treated tumor slice showed lower vascularization level, which further proved the tumor vasculature targeting ability of positively charged micelles. From Figure 6A, 6D and 6E, it could be found that 3Mf had the highest inhibition efficacy against tumor cells and tumor vasculatures than other micelles. This might be explained by its more optimal biodistribution pattern and vasculature targeting ability.

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Figure 6. In vivo antitumor efficacy of free drugs and micelles. Each female BALB/c nude mouse bearing MCF-7/ADR tumor received five intravenous injections (5 mg kg-1 for irinotecan, KLAK and micelles) at every other day. Tumor volume (A) and body weight (B) was measured every other day. All of the mice were pictured (F) and sacrificed at Day 10, tumors were collected and weighted (C). D, E) Tumors blood vessels were marked with CD31-TRITC (red) for the evaluation of micelles’ anti-angiogenesis ability (E), cell nuclei were stained with DAPI (blue). The fluorescence density of TRITC in the whole tumor section was calculated with Image-Pro plus 6.0 software (D). Tumor slices were stained with H&E and TUNEL 34


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(G). Data are presented as mean ± SD (n = 5). *p < 0.001; n.s. means no significant difference.

The TdT-mediated dUTP nick end labeling (TUNEL) and hematoxylin and eosin (H&E) staining of tumor slices are showed in Figure 6G. Most of tumor cells treated with 3Mf, 3Msur and irinotecan were TUNEL-positive (brown staining) and scored as apoptotic, but fewer apoptotic cells were found in tumors treated with other micelles or KLAK peptide. H&E staining showed that tumor treated with saline, KLAK as well as 9Msur consisted of typical, tightly packed cells, whereas massive necrosis was observed for those groups treated with irinotecan or the other micelles. For safety issues, the body weight was measured every other day since the first injection (Figure 6C). As shown in Figure 6C, no significant weight loss could be observed for all groups. Besides, no significant difference could be observed between the normal organs except for lung of control group and the drug administered groups (Figure S23). The difference between the lung slice of irinotecan and saline group might be caused by the potential irinotecan-associated pulmonary toxicity as reported in the literature.60,61 Above all, the aim of this work was to construct multi-target combinatory systems to overcome the drug resistance of breast cancer cells. Indeed, the three drug combination which contained SN38, KLAK and survivin siRNA exhibited extraordinary in vitro cytotoxicity and pro-apoptotic ability against MCF-7/ADR cells. The two drug combination of SN38 and KLAK possessed more superior tumor

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accumulation, stronger vascular targeting ability and more efficient tumor growth inhibition in the in vivo evaluation. Therefore, the final antitumor efficacy depends not just on the cytotoxicity but also on the in vivo pharmacological properties of the combinatory systems.

4. CONCLUSIONS In this work, we fabricated combinatory drug delivery systems consist of cytotoxic drug SN38 and pro-apoptotic KLAK peptide with high drug loading capacity for the simultaneous destruction of MDR cells and tumor vasculatures. Survivin siRNA was loaded into the combinatory micelles for more thorough inhibition of tumor cell proliferation, and its influence on the biological properties was evaluated. The conjugation of KLAK peptide via reduction sensitive linkage restored its α-helical structure and facilitated the destruction of mitochondrial membrane. The loaded survivin siRNA could also be efficiently released inside cancer cells to selectively silence its target gene due to the cleavage of disulfide bonds. Therefore, the combination of SN38, KLAK and survivin siRNA exhibited excellent in vitro cytotoxicity and pro-apoptotic ability against MCF-7/ADR cells. However, the loading of siRNA altered the surface properties of the combinatory micelles, i.e. the zeta potentials, which led to higher RES uptake. As a result, those micelles with poor tumor accumulation could not efficiently suppress the tumor growth, despite their excellent in vitro cytotoxicity against tumor cells. In contrast to this, positively charged 3Mf micelles with the combination of SN38 and KLAK exhibited the most

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favorable anticancer efficacy and vasculature disrupting capability due to its efficient tumor accumulation and tumor vasculature targeting ability. This work proved that the combination therapy was an efficient way to overcome the multidrug resistance of breast cancer. Meanwhile, it also enlightened us that some other aspects beyond the drug combination such as biodistribution and intratumoral distribution also need more attentions.

ASSOCIATED CONTENT

Supporting Information Supplementary data related to this article can be found at http:// dx.doi.org/XXXXX Some detailed experimental procedures, 1H NMR, drug release, cytotoxicity, apoptosis, images of electrophoresis, CLSM images, in vivo biodistribution data and histological analysis of normal organs and tissues. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Qingsong Yu); *E-mail: [email protected] (Zhihua Gan) Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS 37



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This work was financially supported by the National Natural Science Foundation of China (Grant no. 51503013, 51390481 and 81472412) and China Postdoctoral Science Foundation (Grant no. 2015M570919). This work was also supported by the Fundamental Research Funds for the Central Universities (ZY1519) and by the long-term subsidy mechanism from the Ministry of Finance and the Ministry of Education of PRC for BUCT.

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Graphical Abstract

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Positively Charged Combinatory Drug Delivery ... - ACS Publications

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