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PEG-Fmoc-Ibuprofen Conjugate as a Dual Functional Nanomicellar Carrier for Paclitaxel Min Zhao, Yixian Huang, Yichao Chen, Jieni Xu, Song Li, and Xingjie Guo Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.6b00415 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

1

PEG-Fmoc-Ibuprofen Conjugate as a Dual Functional Nanomicellar

2

Carrier for Paclitaxel

3 1

4

Min Zhao, 2Yixian Huang, 2Yichao Chen, 2Jieni Xu, 2Song Li*, 1Xingjie Guo*

5 6

1

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China; 2Center for

7

Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of

8

Pittsburgh, Pittsburgh, PA 15261, USA

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

*Corresponding authors: Song Li, Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, 313 Salk Pavilion, 3501 Terrace Street, Pittsburgh, PA 15261, USA; Phone: 412-383-7976; Fax: 412-648-1664; Email: [email protected] Xingjie Guo, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China; Phone: +86 24 23986285; Fax: +86-24-23986285; Email: [email protected] 1

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1

ABSTRACT:

2

Ibuprofen is a kind of non-steroidal anti-inflammatory drugs (NSAIDs), and

3

considered to possess some antitumor effect. In this study, a novel nanomicellar carrier

4

based on PEG-derivatized ibuprofen, PEG2K-Fmoc-Ibuprofen (PEG2K-FIbu) was

5

developed, for delivery of anticancer agents such as paclitaxel (PTX). This conjugate

6

readily forms stable mixed micelles with PTX with a relatively high PTX loading

7

capacity of 67%. The release of PTX from PTX-loaded PEG2K-FIbu micelles was

8

significantly slower than that from Taxol formulation. PTX-loaded PEG2K-FIbu

9

micelles and Taxol showed a comparable in vitro cytotoxicity. Importantly,

10

PTX-loaded PEG2K-FIbu micelles demonstrated a much more pronounced in vivo

11

therapeutic efficacy compared with Taxol with respect to both inhibition of tumor

12

growth and animal survival. Our system may represent an attractive dual-functional

13

delivery system to achieve synergistic activity with PTX while minimizing the

14

carrier-associated toxicity.

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INTRODUCTION:

2

Paclitaxel (PTX) is one of significant anticancer agents against various solid tumors.1-4

3

However, low water solubility and high toxicity limited its clinical application in

4

cancer therapy.5 In order to solve these problems, a variety of nanocarriers have been

5

developed, such as liposomes, dendrimers, and micelles.6-8 Of all these delivery

6

systems, self-assembling polymeric micelles have been a promising delivery system

7

for many hydrophobic drugs, due to their technical ease, small size, high

8

biocompatibility, and high efficiency in drug delivery.9 However, most of the current

9

delivery systems utilize carrier materials that do not have therapeutic activity, which

10

not only increase the cost, but also impose additional safety issues.10 Our group has

11

recently developed a series of dual-functional micellar delivery systems that are based

12

on PEG-derivatized hydrophobic anticancer agents, such as PEG-vitamin E,

13

PEG-embelin and PEG-farnesylthiosalicylate conjugates.5,11-15 In this study, we report

14

another novel dual-functional carrier based on PEG-derivatized ibuprofen for delivery

15

of poorly water-soluble anticancer drugs.

16 17

Ibuprofen is a kind of non-steroidal anti-inflammatory drugs (NSAIDs), and

18

considered as an effective anti-inflammatory, antipyretic and analgesic drug. A large

19

number of epidemiological studies showed that long-term and regular use of NSAIDs

20

can reduce the incidence of several malignancies.16-18 The antitumor effect of NSAIDs

21

is mainly attributed to the inhibition of cyclooxygenase (COX) enzyme activity,

22

especially the inhibition of COX-2. COX-2 is overexpressed in many inflammatory 3

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and cancerous tissues and plays an important role in tumorigenesis.19,20

2 3

Recent studies have shown that ibuprofen also inhibits tumor growth via

4

COX-independent mechanisms.20 Ibuprofen inhibited the cell proliferation of

5

MKN-45 gastric adenocarcinoma cells in a time- and concentration-dependent manner,

6

and the major mechanisms of the antiproliferative effects are associated with the early

7

alteration of cell-cycle regulatory genes and the later induction of apoptosis.21

8

Andrews’ group evaluated the effectiveness of five kinds of NSAIDs on human

9

prostate cancer cells. They found that among the NSAIDs tested, ibuprofen is more

10

effective in suppressing tumor cell proliferation and inducing apoptosis at clinically

11

relevant concentrations.22 It is known that VEGF plays an important role in the

12

regulation of angiogenesis. Ibuprofen downregulated VEGF expression in human

13

gastric adenocarcinoma cell line (AGS) and inhibited angiogenesis.23 In addition to its

14

antitumor activity ibuprofen has the analgesic effect and can relieve pain for cancer

15

patients, which is one of its advantages in the clinical therapy. In this study, we use

16

ibuprofen as a hydrophobic domain of the micellar carrier, and we expect it to

17

synergize with co-delivered PTX in the overall antitumor activity.

18 19

We have previously discovered that 9-fluorenylmethoxycarbony (Fmoc), a functional

20

group often used for amino acid protection, is a highly effective drug-interactive motif.

21

Inclusion of an Fmoc motif at the interfacial region of micellar carriers led to

22

significant improvement in both drug loading capacity and formulation stability. In the 4

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present study we developed and characterized a new nanomicellar carrier,

2

PEG2K-Fmoc-Ibuprofen (PEG2K-FIbu), which consists of a PEG hydrophilic segment,

3

an Fmoc motif, and an ibuprofen-based hydrophobic domain. We systematically

4

characterized the biophysical properties of PTX-loaded PEG2K-FIbu micelles

5

including size, loading capacity, and in vitro drug release kinetics. We also

6

investigated their in vitro and in vivo antitumor activity.

7

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1

RESULTS

2

Combinational effect of PTX & Ibuprofen on cancer cell proliferation.

3

Many studies have demonstrated the antitumor effect of ibuprofen. To examine its

4

potential broad application, we firstly tested the combinational effect of PTX and

5

ibuprofen in 4T1.2 murine breast cancer cells. 4T1.2 cells were well responsive to

6

each single treatment by PTX or ibuprofen with an IC50 of 6.1×10-8 M and 1.8×10-3

7

M, respectively (Figure 1). However, combination of the two agents was more active

8

than each treatment alone in inhibiting the tumor cell growth (Figure 1). The

9

combination effect of the two agents was then further evaluated by combination index

10

(CI). A CI of greater than 1 is suggestive of antagonism; a CI of equal to 1 indicates

11

additivity; while a CI of less than 1 suggests synergy. The calculated CI based on the

12

data in Figure 1 was 0.63, suggesting a significant synergy between ibuprofen and

13

PTX in inhibiting the growth of 4T1.2 cancer cells. This has prompted us to develop a

14

dual functional PTX micellar carrier with a built-in ibuprofen motif to achieve a

15

combination therapy.

16

6

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Figure 1. Proliferation inhibition of 4T1.2 murine breast cancer cell line treated with combination of

3

PTX and ibuprofen for 72 h.

4 5

Characterization of drug-free and PTX-Loaded PEG2K-FIbu Micelles.

6

PEG2K-FIbu was synthesized via direct coupling of ibuprofen to PEG-Fmoc-Lys(Boc)

7

via an amide linkage. As shown in 1H NMR spectrum (Supporting Information Figure

8

S1), the signals at 3.63 ppm were attributed to the methylene protons of PEG, the

9

signals at 7.0-7.3 ppm were attributed to the benzene ring protons of Fmoc motif, the

10

ibuprofen signals were at 0.8 and 2.4 ppm, and the carbon chain singles were at 1.45

11

ppm. PEG2K-FIbu readily self-assembled to form micelles in aqueous solution with

12

small size around 12 nm (Figure 2a). Figure 2c shows the TEM images of PEG2K-FIbu

13

micelles. Homogeneously distributed spherical particles were observed under TEM

14

and the sizes of the micelles were consistent with those determined by DLS. Figure 2e

15

shows that the CMC value of PEG2K-FIbu conjugate was 0.197 μM. Loading of PTX 7

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into PEG2K-FIbu micelles led to a significant increase in particle size. However, the size of the PTX-loaded micelles remained in the nanosize range (~100nm), which is confirmed by both DLS and TEM (Figure 2b, d).

We then evaluated the DLC, DLE and stability of PTX-loaded PEG2K-FIbu micelles at various carrier/drug molar ratios. As shown in Table 1, PTX could be loaded in PEG2K-FIbu micelles at a molar ratio as low as 0.5:1 with a relatively high drug loading capacity (~67%), and the micelles were stable for 12h at room temperature. Increasing the carrier/drug molar ratio was associated with an improvement in both DLE and the colloidal stability of PTX-loaded micelles.

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Figure 2. Particle size distribution of drug-free (a) and PTX-Loaded PEG2K-FIbu micelles (b). TEM

3

images of drug-free (c) and PTX-Loaded PEG2K-FIbu micelles (PEG2K-FIbu to PTX=2.5:1, mol/mol)

4

(d). Critical micelle concentration (CMC) of PEG2K-FIbu micelles (e).

5 6 7 8

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Table 1. Physicochemical Characterization of Drug-free and PTX-Loaded PEG2K-FIbu Micelles Micelles

Molar ratio

Size (nm)

PDI

DLC (%)

DLE (%)

Stabilitya (h)

PEG2K-FIbu

-

11.7±0.1

0.32

-

-

-

PEG2K-FIbu/PTX

0.5:1

103.4±0.5

0.27

67.3

88.4

12

1:1

127.9±0.9

0.26

33.6

93.1

48

2.5:1

126.6±1.0

0.26

13.5

95.6

48

5:1

122.3±0.5

0.23

6.7

97.5

72

2

PTX concentration in micelles was 1 mg/mL. Blank micelles concentration was 20 mg/mL. Values reported are the

3

means ± SD for triplicate samples.

4

PDI = polydispersity index. DLC = drug loading capacity. DLE = drug loading efficiency.

5

a

Data means no noticeable drug precipitation or significant size change during the follow up time period.

6 7

In Vitro Drug Release Study. The kinetics of PTX release from PEG2K-FIbu/PTX

8

micelles was examined using a dialysis method and compared to that of Taxol. As

9

shown in Figure 3, after first 24 h, only 38.62% of formulated PTX was released from

10

PEG2K-FIbu/PTX micelles, while 55.21% of PTX was released from Taxol

11

formulation. PEG2K-FIbu/PTX micellar formulation exhibited a relatively slower rate

12

of PTX release compared to Taxol formulation. The T1/2 of PTX release is 18.8 h for

13

Taxol formulation, while only 44% of PTX was released from PEG2K-FIbu/PTX

14

micelles even after 48 h.

15

10

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Figure 3. Cumulative PTX release profile from Taxol and PEG2K-FIbu micelles.

3 4

In Vitro Cytotoxicity Study. Figure 4 shows the cytotoxicity of drug-free PEG2K-FIbu

5

micelles and ibuprofen in 4T1.2 and DU145 cancer cell lines. At the concentration of

6

ibuprofen ranging from 0 to 5.0×10-4 M, both PEG2K-FIbu and ibuprofen showed

7

significant cell growth inhibition effect, and PEG2K-FIbu was more active than

8

ibuprofen. The IC50 of PEG2K-FIbu was about 3.5×10-4 M in both of the two cancer

9

cell lines.

10 11

We then examined the cytotoxicity of PTX-loaded PEG2K-FIbu micelles in four tumor

12

cell lines including murine breast cancer cell line 4T1.2, human breast cancer cell line

13

MCF-7, and two human prostate cancer cell lines DU145 and PC-3. Taxol, a clinically

14

used formulation of PTX, was included as a control formulation. As shown in Figure 5,

15

Taxol inhibited the tumor cell growth in a concentration-dependent manner.

16

PTX-loaded PEG2K-FIbu had similar effect as Taxol. PEG2K-FIbu carrier alone did not 11

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show any significant cytotoxicity among all of the cell lines tested, due to its relatively

2

low concentrations used in this study.

3

4 5

Figure 4. Cytotoxicity of ibuprofen and drug-free PEG2K-FIbu micelles against murine breast cancer

6

cell line 4T1.2 (a) and human prostate cancer cell line DU145 (b). Cells were treated for 72 h and

7

cytotoxicity was determined by MTT assay.

8

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Figure 5. Cytotoxicity of Taxol, drug-free and PTX-loaded PEG2K-FIbu micelles against murine breast cancer cell line 4T1.2 (a), human breast cancer cell line MCF-7 (b), and two human prostate cancer cell lines DU145 (c) and PC-3 (d). Cells were treated for 72 h and cytotoxicity was determined by MTT assay.

Tissue Distribution study. Tissue distribution of PTX-loaded PEG2K-FIbu micelles was investigated using female BALB/c mice bearing 4T1.2 breast tumor. Taxol and PTX-loaded PEG2K-FIbu micelles were injected at the same dose of 10 mg PTX/kg. At 24h postinjection, major organs and tumors were excised for PTX determination. As shown in Figure 6, PTX-loaded PEG2K-FIbu micelles showed almost two-fold increase in the uptake of PTX in the tumor, and a reduced PTX accumulation in some 13

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normal organs such as liver and lung compared to Taxol, suggesting that PEG2K-FIbu micelles were more effective in mediating delivery of PTX to tumor tissues while minimizing the Taxol-associated toxicity in normal organs.

Figure 6. Tissue distribution of PTX 24h following the injection. Taxol and PTX-loaded PEG2K-FIbu micelles were injected into female BALB/c mice bearing 4T1.2 breast tumor at the dose of 10 mg PTX/kg, respectively. *P < 0.05 (vs Taxol). N=5.

In Vivo Therapeutic Study. Figure 7a shows the in vivo antitumor activity of drug-free and PTX-loaded PEG2K-FIbu micelles in a 4T1.2 murine breast cancer model. Taxol exhibited a modest tumor growth inhibition at a dose of 10 mg PTX/kg. PTX-loaded PEG2K-FIbu micelles was more effective than Taxol in inhibiting the 14

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tumor growth (p98%) was purchased from TCI

4

American (PA, USA). Dulbecco’s phosphate-buffered saline (DPBS) was purchased

5

from Lonza (MD, USA). Poly-(ethylene glycol) methyl ether (MeO-PEG-OH, MW =

6

2000 kDa), Fmoc-lys-(Boc)-OH, dimethyl sulfoxide (DMSO), Dulbecco’s Modified

7

Eagle’s

8

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were all

9

purchased from Sigma-Aldrich (MO, USA). Fetal bovine serum (FBS) and

Medium

(DMEM),

trypsin-EDTA

solution,

and

10

penicillin-streptomycin

11

N,N′-dicyclohexylcarbodiimide (DCC) was purchased from Alfa Aesar (MA, USA).

12

4-Dimethylaminopyridine (DMAP) was purchased from Calbiochem-Novabiochem

13

Corporation (CA, USA). Trifluoroacetic acid (TFA) and triethylamine (TEA) were

14

obtained from Acros Organic (NJ, USA). All solvents used in this study were HPLC

15

grade.

solution

were

from

Invitrogen

(NY,

USA).

16 17

Synthesis of PEG2K-FIbu. The synthesis scheme of PEG2K-FIbu conjugate is shown

18

in Figure 8. Firstly, MeO-PEG2K-OH (1 eq.) was mixed with Fmoc-Lys (Boc)-OH (4

19

eq.) and DCC (4 eq.) in CH2Cl2 with addition of DMAP (0.2 eq.). After stirring at

20

room temperature for 48 h, the mixture was filtered, then precipitated and washed with

21

cold ethanol and ether twice, respectively. Then the Boc groups were removed by

22

treatment with 50% (v/v) TFA in CH2Cl2, and the PEG derivative was precipitated and 20

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washed with cold ether twice. Ibuprofen (3 eq.) was coupled to the deprotected amino

2

groups of lysine with the assistance of DCC (3 eq.) and DMAP (0.2 eq.). The resulting

3

PEG2K-FIbu was then purified by cold ethanol and ether precipitation.

4

MeO

NHFmoc

O

OH

+ HO

MeO-PEG 2k-O

NHBoc

n

O

DCC, DMAP

NHFmoc

O

MeO-PEG2k-O

NH2 NHFmoc

O

MeO-PEG2k-O

Ibuprofen

O

TFA

H N

O

DCC, DMAP

NHBoc

O

NH O

5 6 7

Figure 8. Synthesis scheme of PEG2K-FIbu conjugate.

8 9

Preparation and Characterization of Drug-free and PTX-Loaded PEG2K-FIbu

10

Micelles. Drug-free and PTX-loaded PEG2K-FIbu micelles were prepared via a

11

thin-film hydration method. Briefly, PEG2K-FIbu conjugate (10 mM in chloroform)

12

and PTX (10 mM in chloroform) were mixed at various carrier/drug molar ratios. The

13

organic solvent was removed by nitrogen flow and a thin film of carrier/drug mixture

14

was formed. The trace amount of remaining solvent was further removed under

15

vacuum for 2 h. Then the thin film was hydrated by DPBS and micelles were formed

16

following gentle agitation. The unincorporated PTX was removed by filtrating the

17

samples through a filter of 220 nm. The size of drug-free and PTX-loaded PEG2K-FIbu

18

micelles was measured by dynamic light scattering (DLS) using a Zetasizer (Zetasizer 21

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1

Nano ZS instrument, Malvern, Worcestershire, UK). The PTX concentration was kept

2

at 1 mg/mL.

3 4

The morphology of the micelles was observed by transmission electron microscopy

5

(TEM). The samples were dropped on a copper grid covered with Formvar and stained

6

with 1% uranyl acetate. Imaging was performed at room temperature.

7 8

The critical micelle concentration (CMC) of PEG2K-FIbu micelles was determined

9

using pyrene as a fluorescence probe. Various amounts of PEG2K-FIbu were prepared

10

in chloroform and mixed with 10 μl of 6×10−5 M pyrene in chloroform. The organic

11

solvent was completely removed, and 1 mL of DPBS was added to each tube to form

12

the micelles with PEG2K-FIbu concentrations ranging from 1×10−4 to 0.5 mg/mL and

13

the final pyrene concentration was 6×10-7 M. The fluorescence intensity of each

14

sample was detected at wavelength of 334/390 nm (excitation/emission), using a

15

Synergy H1 Hybrid Multi-Mode Microplate Reader (Winooski, VT). And the CMC

16

value was determined from the threshold concentration, where the sharp increase in

17

pyrene fluorescence intensity is observed.

18 19

The PTX loading efficiency was determined by high performance liquid

20

chromatography (HPLC) (Alliance, 2695–2998 system), using a Lichrospher® 100

21

RP-18 reversed-phase column (5 μm), a mobile phase of methanol/water (70: 30 v/v)

22

at the flow rate of 1.0 mL/min, and UV-detection at 227 nm at room temperature. Drug 22

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loading capacity (DLC) and drug loading efficiency (DLE) were calculated according

2

to the following formula:

3

DLC (%) = [weight of drug loaded /(weight of polymer + drug used) ] × 100%

4

DLE (%) = (weight of loaded drug /weight of input drug) × 100%

5 6

In Vitro Drug Release Study. Two mL of PTX-loaded PEG2K-FIbu micelles (1 mg

7

PTX/mL) in DPBS (PH = 7.4) was placed in a dialysis bag (MWCO = 12 KDa,

8

Spectrum Laboratories) that was incubated in 200 mL of DPBS containing 0.5% (w/v)

9

Tween 80 under gentle shaking at 37 °C. Taxol formulation (6 mg of PTX/mL in

10

Cremophor EL/ethanol, 1:1, v/v) diluted with DPBS to a final PTX concentration of 1

11

mg/mL was employed as a control. The concentration of PTX remaining in the

12

dialysis bags at designated time points was measured by HPLC using the above

13

method. Values were reported as the means from triplicate samples.

14 15

Cell Culture. 4T1.2 is a mouse metastatic breast cancer cell line. MCF-7 is a human

16

breast cancer cell line. DU145 and PC-3 are two androgen-independent human

17

prostate cancer cell lines. All these cells were cultured at 37 °C in DMEM containing

18

10% FBS and 1% penicillin−streptomycin in a humidified environment with 5% CO2.

19 20

In Vitro Cytotoxicity Study. 4T1.2 (1000 cells/well), MCF-7 (5000 cells/well),

21

DU145 (2000 cells/well), or PC-3 (3000 cells/well) were seeded in 96-well plates and

22

incubated for 24 h. Then the cells were treated with various concentrations of 23

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drug-free PEG2K-FIbu micelles, PTX-loaded PEG2K-FIbu micelles (PEG2K-FIbu to

2

PTX=5:1, mol/mol) or Taxol (at the equivalent concentrations of PTX). The

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concentrations of drug-free PEG2K-FIbu were the same as those of carrier in the

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corresponding PTX-loaded PEG2K-FIbu micelles. After incubation for 72 h, 20 μL of

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MTT in DPBS (5 mg/mL) was added into each well and cells were further incubated

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for 4 h. The medium was then removed, and DMSO was added to solubilize the MTT

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formazan. The absorbance of each well was measured with a microplate reader at a

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wavelength of 550 nm and a reference wavelength of 630 nm. Untreated cells were

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used as a control. Cell viability was calculated as [(ODtreat − ODblank)/(ODcontrol −

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ODblank) × 100%]. The cytotoxicity of drug-free PEG2K-FIbu was also similarly

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examined as described above and compared to free ibuprofen.

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Animals. Female BALB/c mice (10 - 12 weeks) were purchased from Charles River

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(Davis, CA). All animals were housed under pathogen-free conditions according to

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Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)

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guidelines. All animal-related experiments were performed in full compliance with

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institutional guidelines and approved by the Animal Use and Care Administrative

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Advisory Committee at the University of Pittsburgh.

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Tissue Distribution Study. Tissue distribution of PTX-loaded PEG2K-FIbu micelles

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was investigated using female BALB/c mice bearing 4T1.2 breast tumor. When the

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tumor volume reached around 400–600 mm3, Taxol and PTX-loaded PEG2K-FIbu 24

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micelles (PEG2K-FIbu to PTX=5:1, mol/mol) were injected via tail vein injection at

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the same dose of 10 mg PTX/kg. Mice were sacrificed at 24h postinjection, major

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organs and tumors were excised for PTX determination. The tissues were weighed and

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homogenized with 2 mL solvent (acetonitrile to H2O=2:1, v/v). The samples were

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centrifuged (4°C, 4500 rpm, 10 min) and supernatants were transferred to clean tubes.

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The samples were dried by nitrogen flow, and then dissolved in 300 μL MeOH. The

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solutions were centrifuged at 4°C, 12,500 rpm for 15 min. The supernatants were

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collected, dried, redissolved in 200 μL MeOH and centrifuged again at 4°C, 12,500

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rpm for 15 min, and the clear supernatant was used for HPLC measurement.

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In Vivo Therapeutic Study. A syngeneic murine breast cancer model (4T1.2) was

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used to evaluate the therapeutic efficacy of PTX-loaded PEG2K-FIbu micelles. 4T1.2

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cells (2×105 in 200 μL PBS) were inoculated s.c. at the right flank of female BALB/c

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mice. When the tumor volume reached ~50 mm3 (day 1), mice were randomly divided

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into four groups (n=5) and received i.v. administration of PBS (control), free

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PEG2K-FIbu micelles (at equivalent dose as that in corresponding PTX-loaded

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micelles), PTX-loaded PEG2K-FIbu micelles (PEG2K-FIbu to PTX=5:1, mol/mol), and

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Taxol (all at 10 mg PTX/kg), respectively on days 1, 3, 5, 8, and 11. Tumor volumes

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were measured with digital caliper and calculated as V= (L×W2)/2, where L is the

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longest and W is the shortest tumor diameters (mm). Each group was compared by

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relative tumor volume (RTV=V/V0, V0 was the tumor volume prior to first treatment).

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Mice were sacrificed when the tumor volume reached ~2000 mm3.24 The survival rates 25

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are presented as Kaplan-Meier curves. The body weights were also monitored during

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the entire course of treatment to evaluate the potential toxicity.

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Histochemical Staining. After in vivo therapeutic study, tumor tissues were excised

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and preserved in 4% formaldehyde in PBS, followed by embedment in paraffin. The

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paraffin-embedded tumor samples were cut into thin slices of 5 µm with an HM 325

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Rotary Microtome. Then the slices were stained with hematoxylin and eosin (H&E)

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for histopathological examination under a Zeiss Axiostar plus Microscope (PA, USA).

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Statistical Analysis. All results were reported as the mean±standard deviation (SD)

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unless otherwise indicated. Statistical analysis was performed with Student's t-test for

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two groups, and one-way ANOVA for multiple groups, followed by Newman-Keuls

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test if P < 0.05. In all statistical analysis, P < 0.05 was considered statistically

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significant.

15

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ACKNOWLEDGEMENTS

This work was supported by NIH grants R01CA174305, R01GM102989, R01HL083365, and R21CA173887.

SUPPORTTING INFORMATION

Additional figures as discussed in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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