Enzyme Degradable Hyperbranched Polyphosphoester Micellar

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Enzyme Degradable Hyperbranched Polyphosphoester Micellar Nanomedicines for NIR Imaging-Guided ChemoPhotothermal Therapy of Drug Resistant Cancers Mengqun Yao, Yinchu Ma, Hang Liu, Malik Ihsanullah Khan, Song Shen, Shuya Li, Yangyang Zhao, Yi Liu, Guoqing Zhang, Xiaoqiu Li, Fei Zhong, Wei Jiang, and Yucai Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01793 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Biomacromolecules

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Enzyme Degradable Hyperbranched Polyphosphoester Micellar Nanomedicines for NIR Imaging-Guided Chemo-Photothermal Therapy of Drug Resistant Cancers

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Mengqun Yao,†,§,⊥ Yinchu Ma, ‡,⊥ Hang Liu, ‡,⊥ Malik Ihsanullah Khan,# Song Shen,# Shuya Li,# Yangyang Zhao,# Yi Liu,# Guoqing Zhang,ǁ Xiaoqiu Li,*, † Fei Zhong,*, †, § Wei Jiang,*, ǁ and Yucai Wang#

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8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

†

Department of Oncology, the First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P. R. China. § Department of Oncology, Fuyang Hospital of Anhui Medical University, Fuyang, Anhui 236000, P. R. China ‡ Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230027, China # School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China ǁ Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei 230027, P. R. China Xiaoqiu Li, email: [email protected]; Fei Zhong, email: [email protected]; Wei Jiang, email: [email protected]; ⊥ These authors contributed equally to this work.

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ABSTRACT

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Multidrug resistance (MDR) is the major cause for chemotherapy failure, which

3

constitutes a formidable challenge in the field of cancer therapy. The synergistic

4

chemo-photothermal treatment has been reported to be a potential strategy to

5

overcome MDR. In this work, rationally designed enzyme-degradable, hyperbranched

6

polyphosphoester nanomedicines were developed for reversing MDR via the

7

co-delivery

8

chemo-photothermal therapy. The amphiphilic hyperbranched polyphosphoesters with

9

phosphate bond as the branching point were synthesized via a simple but robust

10

one-step polycondensation reaction. The self-assembled hPPEDOX&IR exhibited good

11

serum stability, sustained release, preferable tumor accumulation, and enhanced drug

12

influx of doxorubicin in resistant MCF-7/ADR cells. Moreover, the degradation of

13

hPPEDOX&IR was accelerated in the presence of alkaline phosphatase which was

14

over-expressed in various cancers, resulting in the fast release of encapsulated

15

doxorubicin.

16

chemo-photothermal cyototxicity against MCF-7/ADR cells, and thus the efficient

17

ablation of DOX-resistant tumor without regrowth. This delivery system may open a

18

new avenue for co-delivery of chemo and photothermal therapeutics for MDR tumor

19

therapy.

20 21 22 23

KEYWORDS: hyperbranched polyphosphoester; chemo-photothermal therapy; enzyme degradable; synergistic effect; multidrug resistance; drug delivery;

of

doxorubicin

The

and

IR-780

enzyme-degradable

(hPPEDOX&IR)

polymer

2


as

generated

combined

synergistic

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Biomacromolecules

1

Introduction

2

Multidrug resistance (MDR) is one critical cause for chemotherapy failure and

3

constitutes a formidable challenge in cancer therapy.1-2 The mechanisms of MDR

4

include reduced cellular uptake, increased drug efflux, altered DNA repair, as well as

5

increased drug inactivation, resulting from unregulation of membrane protein

6

transporters and cytoplasmic enzymes and leading to insufficient accumulation of

7

active drug in cells.2-4 To achieve therapeutical cellular concentrations, higher drug

8

dosage should be given, which leads to greater toxic side effects in normal tissues.

9

Therefore, considerable efforts have been undertaken to overcome the drug resistance

10

of cancer cells. Combination therapy using different drugs (e.g., chemotherapeutics,

11

proteins, DNAs, or siRNAs) and therapeutic approaches (e.g., radiation,

12

photo-dynamic, photothermal therapy, ultrasound et al.) have been given particular

13

attentions in that they can cooperatively reverse tumor resistance with different

14

mechanisms and thus minimize side effects.5-7 Among them, there has been a great

15

interest in developing combined treatment of chemotherapy and near-infrared (NIR)

16

photothermal treatment to augment the cytotoxicity of chemotherapeutic agents.8-10

17

One mechanism underlying the synergistic chemo-photothermal therapy is that cancer

18

cells are more sensitive to heat in the hypoxic and acidic tumor microenvironment.11

19

Furthermore, several reports have demonstrated that DNA-damaging agents, such as

20

doxorubicin (DOX), are more effective in combination with hyperthermia, since high

21

temperature can inhibit DNA repair processes.12-13 From this point of view, to

22

establish maximal cooperation effect between photothermal treatment and 3



1

chemotherapy, it is expected that accurate doses of chemotherapeutic drugs and

2

photothermal agents should be simultaneously delivered to the same tumor cell

3

following systemic administration.

4

In the past few decades, nanomedicines (NMs) have been demonstrated

5

promising in cancer therapy, propelled by multiple advantages including sustained

6

drug release, prolonged blood circulation, enhanced permeation and retention (EPR)

7

effect, and superior cellular uptake.14-16 Moreover, co-delivery of therapeutics through

8

nanoformulation is one of the best approaches to achieve the optimal synergistic

9

effects and overcome the hurdles of MDR in cancer. Previous efforts made in this

10

regard involve the design of various co-delivery NMs including liposomes17,

11

polymers,18-19 metallic nanoparticles,20-21 and carbon-based materials.22 More recently,

12

to obtain better controlled release of chemoterapeutic or photothermal agents at the

13

targeted cancer cells, components that can respond to tumoral or cellular

14

microenvironments such as pH,23-24, redox25, hypoxia26 and emzyme27 have been

15

incorporated to the design of co-delivery NMs.28 These responsive NMs allow more

16

acute and specific release of chemo- photothermal reagents and represent superior

17

therapeutic effects. However, among these strategies, release systems triggered by the

18

biocatalytic action of enzymes have seldomly been developed. A number of enzymes

19

are over-expressed in tumors as compared to normal tissues.29 Therefore, exploiting

20

enzymes as a trigger would allow exceptionally selective degradation of their

21

substrates and consequently highly and biologically specific release of cargos.27, 30

22

Polyphosphoesters (PPEs) are a series of functional and biocompatible polymers 4


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Biomacromolecules

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with phosphate bonds as repeating units. The penvalent nautre of phosphorus atom

2

has brought them structural versatility, biodegrability, and various bioapplications,

3

including drug delivery.31-32 More importantly, the degradation of PPEs can be

4

catalyzed by phosphoesterase or alkaline phosphatase (ALP), which are over

5

expressed in various tumors.33-34 More recently, PEGylate and PPEylated in addition

6

to reducing protein adsorption, can affect the composition of the protein corona that

7

forms around nanocarriers, and the presence of distinct proteins is necessary to

8

prevent non-specific cellular uptake.35-36 Inspired by these features of PPEs, we

9

developed

enzyme

degradable

micellar

NMs

based

on

hyperbranched

10

polyphosphoesters (hPPE) for the co-delivery of chemo and photothermal

11

therapeutics for MDR cancer therapy (Figure 1). The hydrophobic segment of hPPE

12

forms the core of NMs, which can encapsulate DOX and photothermal agent IR-780.

13

The core undergoing a viscous-to-flow transition may exhibit temperature-sensitive

14

drug release under photothermal conversion according to our recent results.18

15

Moreover, the degradation of phosphate bonds in hPPE was accelerated by ALP,

16

which subsequently triggered the release of encapsulated DOX. The NMs as an

17

efficient vehicle exhibited enhanced cellular uptake and tumor accumulation in DOX

18

resistant MCF-7/ADR tumors. Consequently, these NMs were capable of generating

19

synergistic chemo-photothermal cytotoxicity against MCF-7/ADR cells, and thus

20

resulted in efficient ablation of MDR tumors. The above results suggested that these

21

hyperbranched polymer systems have great potential as excellent carriers for

22

co-delivery of therapeutic agents for cancer therapy. 5



1

6


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Biomacromolecules

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Materials and Methods

2

Materials. 1, 6-Hexanediol, phosphoryl chloride, triethylamine (TEA) and

3

magnesium sulfate (MgSO4) were purchased from Sinopharm Chemical Reagent

4

(Shanghai, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide

5

(MTT), methoxy polyethylene glycol (mPEG44-OH, Mn=2000 Da), IR-780 iodide,

6

alkaline phosphatase from bovine intestinal mucosa and bovine serum albumin (BSA)

7

were purchased from Sigma-Aldrich (St. Louis, MO). Doxorubicin hydrochloride

8

(DOX·HCl) was purchased from Dalian Mellon biological technology Co., Ltd.

9

(Dalian, China). Hydrophobic DOX was obtained according to previous reports.18

10

Milli-Q water was prepared using a Milli-Q Synthesis System (Millipore, Bedford,

11

MA).

12

Synthesis of PEGylated Hyperbranched Polyphosphoesters (hPPE). hPPEs were

13

synthesized via condensation polymerization with phosphoryl chloride, 1,

14

6-hexanediol, and mPEG44-OH through a two-step reaction. We fixed the amount of

15

the mPEG44-OH and adjusted the proportion of the 1, 6-hexanediol and phosphoryl

16

chloride. The proportion of the active chlorine of phosphoryl chloride and the

17

hydroxyl of 1, 6-hexanediol were 1: 1.2 (hPPE1) and 1: 0.85 (hPPE2). For example, 8

18

mL of anhydrous chloroform solution of mPEG44-OH (0.50 g, 0.25 mmol) was added

19

dropwise to phosphoryl chloride (0.43 g, 2.78 mmol) in anhydrous chloroform (5.0

20

mL) under stirring. After reacting at room temperature for 2 h, the former solution

21

was added dropwise to a three-necked flask under nitrogen with 1, 6-hexanediol (0.59

22

g, 5.00 mmol), TEA (0.84 g, 8.33 mmol) and chloroform (20 mL) at -5 °C for 24 h.

23

The by-product of triethylamine hydrochloride salt was removed by filtration. The

24

obtained filtrate was twice extracted by HCl (1 M), NaHCO3 (10%) and NaCl

25

(saturated) aqueous solution, respectively. After drying over anhydrous MgSO4, the

26

solvent was removed under vacuum, obtaining a solid product (yield 73%). H NMR

27

(300 MHz, CDCl3, δ ppm): 4.25-4.00 (m, 2H, PO-CH2-CH2-), 3.65 (s, 4H,

28

-O-CH2-CH2-O), 3.35 (s, 3H, CH3-O-CH2-CH2-O), 1.85-1.65 (m, 4H, PO-CH2-CH2-),

29

1.50-1.20 (m, 4H, PO-CH2-CH2-).

30

(O-CH2-CH2-O), 62.65 (P-OCH2CH2CH2-), 32.75 (P-OCH2CH2CH2), 25.2 (P-O

31

CH2CH2CH2).

32

Preparation of hPPEDOX&IR. The NMs were prepared by the nanoprecipitation

1

13

C NMR (75 MHz, CDCl3, δ ppm): 70.63

7



1

method. For DOX and IR-780 co-loaded NMs preparation, hPPE polymer (10.0 mg),

2

hydrophobic DOX (1.0 mg) and IR-780 (0.4 mg) were dissolved in 1.0 mL DMSO,

3

and then ultrapure water (10 mL) was dropwise added into the solution under stirring

4

for 2 h. The final solution was transferred to a dialysis tube (MWCO: 14000), and

5

dialyzed against ultrapure water for overnight to remove DMSO. The NMs were

6

centrifuged to remove free DOX and IR-780. The hPPEDOX and hPPEIR were prepared

7

using a similar method as described above when the IR-780 or DOX was absent,

8

respectively.

9

Characterization of hPPEDOX&IR. The size and zeta potential were measured by

10

dynamic light scattering (DLS) using a Malvern Zetasizer (Nano-ZS, Malvern

11

instruments, UK). All the DLS measurements were performed at 25 °C. Drug loading

12

content (DLC) and encapsulation efficiencies (EE) of DOX and IR-780 were

13

measured by high-performance liquid chromatography (HPLC) and fluorescence

14

spectrophotometer (RF-5301PC, Shimadzu, Kyoto, Japan), respectively. The

15

encapsulation efficiency (EE) and drug-loading (DLC) were calculated by the

16

following equations: DLC (%) = amount of drug in NMs/amount of hPPE

17

added×100%

18

EE (%) = amount of drug in NMs/amount of drug added×100%

19

In Vitro DOX Release. 1 mL of hPPEDOX&IR was transferred to a dialysis tube

20

(MWCO 14 kDa), which was immersed in 20 mL of phosphate buffer (PB, 0.02 M,

21

pH 7.4) with 0.1% Tween 80 (Sinopharm Chemical Reagent Co., Ltd., Shanghai,

22

China). The release experiment was executed at 37 °C in a culture incubator stirred at

23

the speed of 100 rpm. 10 mL of external buffer was collected and equal volume of

24

fresh PB buffer was immediately supplemented at preset time intervals. The

25

concentration of DOX in the collected solution was determined by HPLC.

26

Enzymatic Degradation of hPPEDOX&IR. To test the NMs degradation by enzyme,

27

200 µL hPPEDOX&IR (1.67 mg/mL, hPPE polymer) dispersion incubated with 1 µL

28

alkaline phosphatase (2.0 × 103 unit/mL, Sigma-Aldrich, St. Louis, MO) in cuvette,

29

and another cuvette with 1 µL PBS as control incubated at 37 °C. At predetermined

30

time, the size of NMs and fluorescence intensity of DOX were measured by DLS and 8


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Biomacromolecules

1

fluorescence spectrophotometer, respectively.

2

In Vitro DOX Release: The aqueous solution of hPPE1DOX&IR or hPPE2DOX&IR

3

([IR-780] = 3.5 µg mL−1, [DOX] = 5.0 µg/mL) were exposed to different laser power

4

densities of 0.5, and 1.0 W/cm2 for 5 s (laser on) and were then incubated for 5 min

5

(laser off). The laser on and laser off cycles were repeated several times. The release

6

of DOX at each laser on and laser off cycle was collected by ultrafiltration (3000 g,

7

10 min, Amicon Ultra-4 centrifugal filter), and the resulting DOX concentrations

8

were determined by HPLC analyses.

9

Cell Culture. The human breast cancer cell line MCF-7 and MCF-7/ADR (DOX

10

resident human breast cancer cell line with P-gp overexpression) were kindly

11

provided by Prof. Tao Zhu from the University of Science and Technology of China.

12

MCF-7 and MCF-7/ADR cells were respectively cultured in DMEM medium (Gibco,

13

Grand Island, UK) and RPMI 1640 medium (Gibco, Grand Island, UK) along with 10%

14

FBS (HyClone, Logan, UT) and 1% penicillin/streptomycin (Sigma-Aldrich, St.

15

Louis, MO) in a humidified atmosphere containing 5% CO2 at 37 °C. The DOX

16

resistance of MCF-7/ADR cells was maintained with 5.0 µg/mL DOX concentration

17

and cultured without DOX for 2 weeks before experiments.

18

Cellular Uptake and Intracellular Distribution. To analyze cellular uptake of NMs,

19

the cellular DOX fluorescence was measured by flow cytometry (BD Biosciences,

20

San Jose, CA). MCF-7 and MCF-7/ADR cells were seeded in 24-well plates (1.0 ×105

21

cells per well) and cultured for overnight. Then the culture medium was replaced by

22

fresh medium containing free DOX&IR, hPPE1DOX&IR and hPPE2DOX&IR at an

23

equivalent DOX concentration of 5.0 µg/mL for 2 or 4 h, respectively. Cells were

24

rinsed twice times with PBS, treated with trypsin (0.25%, with 0.02% ethylene

25

diamine tetracetic acid, Gibco, Canada) and resuspended 300 µL PBS for flow

26

cytometric analysis using an FACS Calibur flow cytometer (BD Biosciences, Bedford,

27

MA). The data were analyzed using FlowJo software.

28

Cellular uptake and intracellular distribution were observed by a confocal laser

29

scanning microscope. MCF-7 and MCF-7/ADR cells were seeded on coverslips in a

30

24-well plate at a density of 5 × 104 cells per well. After culturing overnight, the cells 9



1

were treated with these nanomedicines as mentioned above. After incubation for 2 h

2

and 24 h, the cells were washed three times with PBS, and then the cytoskeleton and

3

cell nuclei were stained by Alexa Fluor 488 Phalloidin (green) and DAPI (blue)

4

according to the manufacturer’s instructions. Briefly, the cells fixed with 4.0%

5

formaldehyde solution in 1×PBS for 10 min at room temperature. After wash three

6

times with 1×PBS, 0.1% Triton X-100 were added for 5 min at room temperature.

7

After wash three times with PBS. Alexa Fluor™ 488 Phalloidin (1:200 diluted) was

8

add into cells for 20 min at room temperature. Coverslips were mounted on glass

9

microscope slides with a drop of anti-fade mounting media (Sigma-Aldrich, St. Louis,

10

MO) to reduce fluorescence photobleaching, and then visualized by confocal laser

11

scanning microscopy (CLSM, LSM 710, Carl Zeiss Inc., Germany). For the detection

12

of DOX, the excitation wavelength was 530 nm and emission wavelength was

13

540-650 nm. As the excitation wavelength of NIR was 780 nm, they are not supposed

14

to interfere with each other.

15

Cytotoxicity of hPPEDOX&IR In Vitro. The cytotoxicity of drug-loaded NMs and free

16

DOX against MCF-7 and MCF-7/ADR cells was determined by a MTT assay. MCF-7

17

and MCF-7/ADR cells were seeded in 96-well plates at 5 × 103 cells per well. After

18

culturing overnight, the culture medium was replaced by 100 µL fresh medium

19

containing different concentrations of DOX (free DOX&IR and hPPE1DOX&IR) for

20

another 24 h. Thereafter, MTT stock solution (25 µL) was added to the wells to a final

21

concentration of 1.0 mg/mL. After incubation for 4 h, 100 µL of extraction buffer (20%

22

SDS in 50% DMF, pH 4.7, prepared at 37 °C) was added and incubation for another 4

23

h. The absorbance was measured at 570 nm using ELx800™ absorbance microplate

24

readers (Bio-Rad 680, Winooski, VT). Cell viabilities were normalized to that of cells

25

cultured in medium with PBS. IC50 value was calculated by GraphPad PRISM

26

(GraphPad Software, Inc., San Diego, CA).

27

To test the efficacy of chemo-photothermal treatment, MCF-7 and MCF-7/ADR

28

cells were seeded in 96-well plates at 5 × 103 cells per well. After culturing overnight,

29

the culture medium was replaced by 100 µL fresh medium containing different

30

concentration of free DOX&IR, hPPE1DOX, hPPE1IR, and hPPE1DOX&IR, respectively. 10


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Biomacromolecules

1

After 4 h incubation, the culture medium was replaced by fresh medium, followed by

2

irradiated with a 1 W/cm2 808-nm laser for 5 min with photothermal treatments. After

3

24 h incubation, cell viability of irradiated cells was evaluated by MTT assay.

4

Animals and Xenograft Tumor Model. Female Nonobese diabetic/severe combined

5

immunodeficiency/γc null (NOG) mice (6 weeks) were obtained from the Vital River

6

Laboratory Animal Technology Co. Ltd (Beijing, China). All animals were treated by

7

obeying the guidelines outlined in the Guide for the Care and Use of Laboratory

8

Animals and all procedures were approved by the Animal Care and Use Committee of

9

University of Science and Technology of China. To construct the MCF-7/ADR tumor

10

xenograft model, approximately 1 × 107 cells with 20% Matrigel (BD Biosciences,

11

Franklin Lakes, NJ) in 100 µL PBS was injected into the mammary fat pat of female

12

NOG mice. Tumor-bearing mice were used when the volume of tumors reached to 80

13

mm3.

14

Pharmacokinetics Studies and Biodistribution. Female NOG mice bearing

15

MCF-7/ADR tumor (tumor volume = 150 mm3) were randomly divided into three

16

groups (n = 4 per group). Free DOX&IR, hPPE1DOX&IR and hPPE2DOX&IR were

17

injected into NOG mice via tail vein at an equivalent dose of 3.5 mg IR-780 per kg

18

mouse weight. Blood samples were collected from the retro-orbital plexus of eyes at

19

different time intervals. The plasma was obtained by blood centrifuged (5000 rpm, 10

20

min, 4 °C), and the supernatant of samples (50 µL) and the major organs, including

21

the brain, heart, liver, spleen, lung, and kidney tissue were harvested for ex vivo

22

imaging via Xenogen IVIS Lumina system (Caliper Life Sciences, Alameda, CA).

23

In Vivo Distribution and Ex Vivo Tumor Accumulation of hPPE1DOX&IR and

24

hPPE2DOX&IR. Female NOG mice bearing MCF-7/ADR xenografts were i.v. injected

25

with hPPE1DOX&IR and hPPE2DOX&IR, respectively. At the preset times, in vivo

26

fluorescence images were acquired on the Xenogen IVIS Lumina system (Caliper

27

Life Sciences, Alameda, CA). Moreover, after 48 h post-injection, the organs of mice

28

including heart, lung, liver, spleen, kidney, and tumor were collected. The quantitative

29

content of IR-780 in tumors were detected by a Xenogen IVIS Lumina system.

30

Temperature Evolution on tumors and IR thermal images In Vivo. Temperature 11



Page 12 of 38

1

evolution on tumors under NIR irradiation was determined by infrared thermal

2

imaging camera (Ti27, Fluke, USA). The mice were divided into groups of 3 mice

3

each that were treated with the PBS, free DOX&IR, hPPE1DOX&IR, and hPPE2DOX&IR

4

via tail vein injections at an equivalent dose of 5 mg/kg DOX and 3.5 mg/kg IR-780.

5

After 12 h, the tumors were exposed to the NIR laser with 1.0 W/cm2 for 5 min and

6

tumor tissue temperature evolution was measured.

7

Tumor Suppression Study. When the tumor volume of the MCF-7/ADR xenograft

8

reached to 80 mm3, the mice were randomly divided into 6 groups (five mice per

9

group) and treated with PBS, free DOX&IR with irradiation (+NIR), hPPE1DOX,

10

hPPE1IR +NIR, hPPE1DOX&IR without irradiation and hPPE1DOX&IR +NIR at an

11

equivalent dose of 5 mg/kg DOX and 3.5 mg/kg IR-780 by intravenous injection. The

12

mice received injections two times at day 1 and 7. After injection 12 h, free DOX&IR,

13

hPPE1IR and hPPE1DOX&IR were given the NIR exposure using 808-nm laser at a

14

power density of 1.0 W/cm2 for 5 min. Tumor growths were monitored by measuring

15

the perpendicular diameter of the tumor using calipers every two days. The estimated

16

volume was calculated according to the formula: Tumor volume (mm3) = 0.5 × length

17

× width2. Weight of each mouse was also measured every two days.

18

Immunohistochemical and Immunofluorescence Staining. At the end of the above

19

tumor growth inhibition experiment, the mice were sacrificed. The tumor tissues were

20

excised, fixed in 4% formaldehyde and embedded in paraffin. The tumor tissues were

21

analyzed by hematoxylin–eosin (H&E) staining assay and immunofluorescence

22

analysis.

23

immune-histochemical analysis. The proliferating cell nuclear antigen (PCNA) and

24

the terminal transferase dUTP nick-end labeling (TUNEL) assay kit (Roche

25

Diagnostics, Indianapolis, IN) were used as per the manufacturer’s protocol.

26

Statistical Analysis. The statistical significance of treatment outcomes was assessed

27

using Student’s t-test (two-tailed); p < 0.05 was considered statistically significant in

28

all analyses (95% confidence level).

29

Results and Discussion

Paraffin-embedded

5

tumor

µm

sections

12


were

prepared

for

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Biomacromolecules

1

Synthesis and Characterization of hPPEs.

2

hPPEs were synthesized by the condensation reaction between phosphoryl chloride

3

(A3), 1, 6-hexanediol (B2), and linear heterobifunctional polyethylene glycol with a

4

methoxy and a hydroxyl group (mPEG44-OH, Mn = 2000 g mol-1, C1) using an A3 +

5

B2 + C1 method (Figure 1A). The pentavalent phosphorus atom served as a branching

6

point during the polymer growth. mPEG44-OH was used as a capping agent (i.e., C1

7

monomer). The reaction was carried at low temperature (i.e., -5 °C) using

8

triethylamine to neutralize the liberated hydrogen chloride. hPPEs were obtained after

9

overnight reaction followed by repeated precipitation. The products were denoted as

10

hPPE1 (for A3: B2: C1 = 1: 0.8: 0.03) and hPPE2 (for A3: B2: C1 = 1: 1.2: 0.03),

11

respectively. Both polymers showed good solubility in common organic solvents such

12

as tetrahydrogen furan, chloroform, and toluene. 1H and

13

and hPPE2 are shown in Figure S1 and S2, respectively. In Figure S2, the

14

resonances at 0.48, -0.80, and -13.0 ppm, corresponded to the dendritic, linear, and

15

terminal units, respectively. Degree of branching is a structural property that is

16

determined by the concentration of linear (L), terminal (T) and dendritic (D) units

17

within the polymer matrix. Hyperbranched polymers are characterized by a degree of

18

branching (DB) which represents the percentage of dendritic and terminal monomers

19

among the total monomers in the polymer, i.e., DB=(D+T)/(D+T+L).35 The degrees of

20

branching (DBs) of hPPE1 and hPPE2 were calculated to be 0.54 and 0.47,

21

respectively, using

22

pentavalent nature of the phosphorus atom can also allow the conjugation of other

31

31

P NMR spectra of hPPE1 31

P

P NMR spectrum analyses. It is worth mentioning that the

13



1

bioactive molecules as pendant groups. Furthermore, elemental analyses of the

2

obtained hPPE1 showed that the content of C, H, O, and P was 47.38%, 8.20%, 31.19%

3

and 13.12%, respectively, and hPPE2 the showed similar elemental contents. The

4

absorptions of FT-IR spectrum at 1260, 1111, and 1026 cm-1 are attributed to the

5

asymmetrical, symmetrical stretching of P=O and P-O-C, respectively. The peak at

6

3485 cm-1, is assigned to the large number of terminal hydroxyl groups (Figure S3).

7

The DSC curve showed a single glass transition temperature (Tg) of hPPE of ~-25 oC

8

(Figure S4), indicating that the polymers were not composed of mixed species.

9

Moreover, the weight-average molecular weight of hPPE characterized by GPC is

10

92000 g mol-1 with the polydispersity index of 1.75 (Table S1). These results well

11

demonstrate the successful synthesis of the single-component hPPE.

12

Preparation and Characterization of DOX and IR-780 co-Loaded hPPEDOX&IR.

13

Hydrophobic 1, 6-hexanediol and hydrophilic mPEG44-OH in the hPPE1 and hPPE2

14

polymers render the resultant hPPEs amphiphilic, and capable of encapsulating

15

hydrophobic cargos through hydrophobic interactions. As a proof-of-demonstration,

16

we used DOX as a chemotherapeutic model and IR-780 as a photothermal plus NIR

17

imaging agent, respectively. DOX and IR-780 were loaded into the hydrophobic core

18

of hPPEs-based NMs by a co-precipitation method.18 After removal of excess DOX

19

and IR-780 by centrifugation and dialysis, the purified NMs were obtained and

20

denoted as hPPEDOX&IR. NMs encapsulating DOX or IR-780 alone were denoted as

21

hPPEDOX or hPPEIR, respectively. For these DOX and IR-780 loaded NMs, the drug

22

loading content (DLC) of DOX and IR-780 was ca. 4.2% ± 0.4 wt% and 8.2% ± 1.3 14


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Biomacromolecules

1

wt%, respectively, when the feeding ratio of DOX or IR-780 to polymers was 1:10 by

2

weight (Table S2). Although hPPEDOX&IR could offer higher DLC as an increase of

3

drug feeding concentrations, the inferior stability in aqueous solutions occurred as

4

evident precipitation was observed after storage for a few days. The average size of

5

hPPE1DOX&IR and hPPE2DOX&IR as measured with dynamic light scattering (DLS)

6

were all ~150 nm (Figure 2A and Figure S5), indicating that they are suitable for

7

passive tumor targeting via EPR effect.37 The zeta potentials of hPPE1DOX&IR and

8

hPPE2DOX&IR were measured to be -19.2 ± 0.8 mV and -21.3 ± 0.3 mV, respectively

9

(Figure 2B), due to the PEG coating.36. In addition, hPPE1DOX&IR and hPPE2DOX&IR

10

exhibited high stability without aggregation and obvious drug leakages within 24 h in

11

10% FBS or 5% glucose at 37 °C (Figure 2C and Figure S6), which could be due to

12

the steric effect of the PEG corona.38 The release of the DOX from hPPE1DOX&IR as

13

well as hPPE2DOX&IR was then further examined. Both hPPE1DOX&IR and

14

hPPE2DOX&IR showed a sustained release of DOX over 72 h (Figure 2D). In particular,

15

approximately 30% of DOX loaded in hPPEDOX&IR was released within 12 h followed

16

by sustained released of ~62% (~46%) of total DOX within 72 h for hPPE1DOX&IR

17

(hPPE2DOX&IR). To study the photothermal transduction efficiency of hPPEDOX&IR, its

18

aqueous solution was irradiated with an 808-nm continuous laser with an intensity of

19

1.0 W/cm2. The change in temperature under irradiation was recorded via an infrared

20

thermal imaging camera. The temperatures for hPPE1DOX&IR and hPPE2DOX&IR with

21

20 µg/mL of IR-780 increased by up to 15.5 °C and 16.1 °C, respectively, after an

22

irradiation for 5 min, while the PBS control only increased by 1.35 °C (Figure 2E and 15



1

F). The results showed that IR-780 but not DOX produced significant temperature

2

elevation under laser irradiation, giving hPPEDOX&IR the photothermal therapy

3

potentials to produce irreversible damage to tumor cells.

4

Enzymatic Degradation of hPPE NMs.

5

ALP is an enzyme that is responsible for cleaving phosphate bonds and its levels are

6

frequently elevated in patients with various cancers.39-41 We next studied the

7

enzymatic degradation of hPPEs catalyzed by ALP. Considering the structure of the

8

degradation the polymer, we investigated ALP-triggered degradation using gel

9

permeation chromatography (GPC) after incubation with ALP. As shown in Figure S7,

10

there were increasing homopolymers with a lower molecular weight observed at an

11

elution volume of 28 mL, which was consistent with the molecular weight of PEG.

12

The size changes of hPPE1DOX&IR were monitored using DLS after incubated with

13

ALP (2.0 × 103 unit/mL) at 37 °C. As shown in Figure 3A, negligible size changes

14

occurred for hPPE1DOX&IR in the absence of ALP, while the size of hPPE1DOX&IR

15

increased rapidly upon enzyme treatment, as evidence by obvious precipitation. This

16

indicated that the phosphate bonds in the polymers were degraded, causing the

17

detachment of PEG and precipitation of the hydrophobic core. To further confirm our

18

results, we measured the fluorescence intensity of DOX in hPPE1DOX&IR at

19

predetermined time points. Fluorescence quenching occured once DOX molecules

20

were encapsulated in the NM’s core, due to homofluorescence resonance energy

21

transfer (homo FRET).42 The DOX fluorescence recovered gradually as time of

22

incubation with ALP (2.0 × 103 unit/mL) increased, indicating the release of DOX 16


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Biomacromolecules

1

was as a result of hPPE1DOX&IR degradation by ALP enzyme (Figure 3B). The

2

recovery of fluorescence intensity of DOX released from hPPE1DOX&IR (Figure 3C)

3

was further corroborated by the change in size of hPPE1DOX&IR (Figure 3A). The size

4

change and recovery of fluorescence intensity of DOX released from hPPE2DOX&IR

5

(data not shown) confirmed similar degradation trends of hPPE1DOX&IR. We further

6

explored the thermosensitive release of DOX from hPPEDOX&IR. As shown in Figure

7

3D, the DOX release from hPPE1DOX&IR and hPPE2DOX&IR exhibited a

8

thermo-triggered, stepwise, and intensity-dependent manner with response to pulsed

9

NIR irradiation. Specially, the NIR-activated drug release was observed when the

10

laser was on while stopped when the laser was switched off.

11

Intracellular Uptake of hPPE NMs.

12

Enhanced cellular internalization and increased intracellular drug concentration of the

13

hPPEDOX&IR is an essential factor to overcome drug resistance. We next investigated the

14

capability of cellular uptake of DOX for free DOX&IR and hPPEDOX&IR after incubation

15

with DOX-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells for 2 h and 4 h,

16

respectively. The intracellular fluorescence of DOX was determined by flow cytometric

17

analysis. In MCF-7 cells, the free DOX&IR group exhibited stronger cellular

18

fluorescent intensity compared with hPPEDOX&IR treated groups (Figure 4A and B),

19

which was attributed to efficient passive diffusion of free DOX.43 However, free

20

DOX&IR exhibited much weaker intracellular fluorescence presumably due to rapid

21

efflux by efflux pumps on the membrane of MCF-7/ADR cells (Figure 4C and D),

22

which eliminates most drug molecules. For hPPEDOX&IR, gradually enhanced 17



1

intracellular DOX content was observed with prolonged incubation time, suggesting

2

efficient internalization of hPPEDOX&IR. Specifically, the cellular fluorescence

3

intensities of DOX in MCF-7/ADR cell after treatment with hPPE1DOX&IR, were

4

3.0-fold and 3.5-fold more than those of free DOX&IR at 2, and 4 h, respectively. The

5

results indicated that hPPEDOX&IR as a vehicle could enter tumor cells by endocytosis

6

before releasing their drug into cytoplasm efficiently, and bypass the P-gp efflux

7

pump.44

8

The enhanced cellular uptake of hPPEDOX&IR in MCF-7 and MCF-7/ADR cell line

9

was further confirmed by confocal laser scanning microscopy (CLSM). After 4 h

10

incubation with free DOX&IR or hPPE1DOX&IR in MCF-7 cells, the cellular

11

fluorescence intensity of DOX was mainly localized within cell nucleus (Figure 5A),

12

indicating rapid cellular nucleus translocation of DOX.45 However, no obvious

13

cellular fluorescence intensity of DOX was observed in the nucleus of DOX-resistant

14

MCF-7/ADR cells under the same incubation condition, owing to decreased drug

15

uptake and enhanced drug efflux mediated by P-gp transporters over-expressed in

16

MDR cells.46 In contrast, DOX of hPPE1DOX&IR entered the MCF-7/ADR cells within

17

4 h and efficiently translocated into the nucleus at 24 h. Furthermore, to precisely

18

quantify the localization of DOX, intranuclear fluorescence intensity and the total

19

intracellular fluorescence intensity were denoted as I1 and I2, respectively. The value

20

obtained from dividing I1 by I2 was used to evaluate the relative fluorescence intensity

21

of the intranuclear layers (Figure 5B). Relative fluorescence intensity of DOX in

22

hPPE1DOX&IR-treated cells was 2.3-fold higher than that of free DOX&IR after 24 h 18


Page 18 of 38

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Biomacromolecules

1

incubation in MCF-7/ADR cells (Figure 5C and D), indicating hPPE NMs could

2

efficiently accumulate DOX into the nucleus, where DOX molecules interact with

3

DNA and thereby inhibit cell growth. hPPE2DOX&IR showed comparable cell uptake

4

with hPPE1DOX&IR (Figure S8). These results revealed that hPPEDOX&IR had the

5

potential to overcome drug resistance via enhanced nuclear distribution of DOX in

6

cells due to the passive diffusion of the internalized drugs.

7

Synergistic Chemo-Photothermal therapy of hPPE NMs In Vitro.

8

The enhanced cellular uptake of DOX in hPPEs is expected to be accompanied by

9

increased binding of DOX with DNA to exert its anticancer activity. We further

10

evaluated the synergistic chemo-photothermal therapeutic effect on MCF-7/ADR cells

11

in vitro. To demonstrate, free DOX&IR or hPPEDOX&IR was incubated with

12

DOX-resistant MCF-7/ADR cells at a series of DOX concentrations for 24 h

13

incubation, and the cell viabilities were measured. The resistance index (RI) of

14

MCF-7/ADR cells was determined to be 174.4 compared to MCF-7 cells (data not

15

shown). As shown in Figure S9, the IC50 values of the hPPE1DOX&IR and hPPE2DOX&IR

16

treatment in MCF-7/ADR cells were 13.9 µg/mL and 13.4 µg/mL, respectively, which

17

were both significantly lower than that of free DOX&IR treatment (IC50 = 37.4

18

µg/mL).

19

NMs containing photothermal agent has the ability to disrupt the endo/lysosomal

20

membrane and damage efflux effect by producing localized heat shock.47 To evaluate

21

whether the synergistic chemo-photothermal treatment with hPPE NMs can further

22

overcome the drug resistance, MCF-7/ADR cells were treated with hPPE1DOX, 19



1

hPPE1IR, and hPPE1DOX&IR at varying concentration gradient of DOX (0.6~10.0

2

µg/mL) and IR-780 (0.4~7.0 µg/mL) for 4 h, respectively. The procedure was

3

followed by 808-nm laser irradiation (1.0 W/cm2, 5 min) or no irradiation and then

4

further incubation for 24 h. As shown in Figure 6, increased cytotoxicity in

5

MCF-7/ADR cells was followed by gradually increased concentration of DOX and

6

IR-780. The IC50 value of the hPPE1DOX&IR with laser irradiation (+NIR) treatment

7

was 3.3 µg/mL, which was significantly lower than that of other treatments, i.e.,

8

0.24-fold, 0.32-fold, and 0.26-fold that of hPPE1DOX (13.6 µg/mL), hPPE1IR+NIR

9

(10.9 µg/mL) and hPPE1DOX&IR without NIR laser irradiation (12.6 µg/mL),

10

respectively. In addition, hPPE1DOX&IR without NIR laser irradiation showed lower

11

cytotoxicity compared to hPPE1DOX&IR with NIR laser irradiation, suggesting that

12

chemotherapy combined with photothermal therapy could significantly enhance the

13

cytotoxicity and could overcome the drug resistance in MCF-7/ADR cells.

14

hPPE2DOX&IR showed similar cytotoxicity under same treatment (data not shown). Our

15

results endorse the previous findings that photothermal therapy increased the

16

nanoparticles cellular uptake and enhanced DOX antitumor efficacy.48

17

Pharmacokinetics and Biodistribution of hPPE NMs.

18

The pharmacokinetics and biodistribution of hPPE NMs in mice were further

19

examined. Nonobese diabetic/severe combined immunodeficiency/γc null (NOG)

20

mice bearing MCF-7/ADR tumors (volume ~150 mm3) were intravenously injected

21

with free DOX&IR, hPPE1DOX&IR, and hPPE2DOX&IR at an equivalent dose of 5.0

22

mg/kg of DOX and 3.5 mg/kg of IR-780. Blood was collected at pre-determined times 20


Page 20 of 38

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Biomacromolecules

1

and fluorescence of IR-780 was quantified using Xenogen IVIS® Lumina system. As

2

shown in Figure 7A, hPPE1DOX&IR circulated in blood for more than 24 h, attributing

3

to the densely PEGylated surface layer which can resist protein absorption and

4

subsequent reticuloendothelial system (RES) clearance.49 hPPE2DOX&IR exhibited

5

shorter circulation half-life in contrast to hPPE1DOX&IR, owing to the low ratio of PEG

6

in hPPE2DOX&IR.

7

The biodistribution of hPPE1DOX&IR and hPPE2DOX&IR were further studied in

8

vivo. Following i.v. injection, the fluorescence intensity of IR-780 was found

9

intuitively in MCF-7/ADR tumor tissues, indicating the outstanding capacity of

10

hPPEDOX&IR to enhance tumoral accumulation (Figure 7B). The fluorescence intensity

11

of IR-780 continuously increased at the tumor site over the first 12 h, and then slowly

12

decayed (Figure 7C). In addition, at 48 h post-injection of the hPPEDOX&IR, the mice

13

were sacrificed and major organs and tumor tissues were collected for analysis with a

14

Xenogen IVIS® Lumina system (Figure 7D). For both NMs, most of fluorescence

15

intensities were accumulated in liver and spleen, which were mainly responsible for

16

clearing, processing, and degrading foreign materials from blood circulation.50 For

17

hPPE2DOX&IR, its clearance by liver was much more obvious than for hPPE1DOX&IR.

18

While lower tumoral accumulation than hPPE1DOX&IR, consistent with the better

19

pharmacokinetics of hPPE1DOX&IR (Figure 7E).

20

NIR Phothermal Properties of hPPE NMs.

21

We expected that the increased tumor accumulation of hPPE1DOX&IR caused more

22

efficient temperature elevation during photothermal therapy in tumors. To verify this, 21



1

the NIR-triggered temperature change of tumors was investigated in NOG mice

2

bearing MCF-7/ADR tumors. At 12 h post injection of free DOX&IR, hPPE1DOX&IR,

3

and hPPE2DOX&IR, the tumor regions were irradiated with an 808-nm laser at power

4

density of 1.0 W/cm2 for 5 min. Thermal images recorded at different time points

5

showed the temperature of the tumor region increased rapidly upon irradiation (Figure

6

8A and B). For the PBS control group or free DOX&IR injected group, the increase

7

of temperature was less than 6 °C. hPPE1DOX&IR exhibited the highest increase in

8

temperature of 21.5 °C, which is sufficient for tumor cell damage.37 The temperature

9

increase of hPPE2DOX&IR was much lower than hPPE1DOX&IR, since hPPE2DOX&IR had

10

inferior pharmacokinetics and tumor accumulation than hPPE1DOX&IR.

11

Synergistic Chemo-Photothermal therapy In Vivo.

12

Encouraged by the synergistic chemo- photothermal capacity of hPPE1DOX&IR to

13

circumvent DOX-resistance in vitro and its efficient tumoral accumulation, we then

14

performed animal experiments to further test its in vivo therapeutic potential. NOG

15

mice with MCF-7/ADR xenograft tumors (80 mm3 in size) were intravenously

16

injected with free DOX&IR, hPPE1DOX, hPPE1IR, or hPPE1DOX&IR at an equivalent

17

dose of 5.0 mg/kg of DOX and/or 3.5 mg/kg of IR-780. Mice injected with PBS were

18

used as a negative control. At 12 h post injection, the mice were irradiated with or

19

without a NIR laser (808-nm, 1 W/cm2) for 5 min as indicated in Figure 9. As shown

20

in Figure 9A, administration of free DOX&IR without NIR treatment only resulted in

21

~41% inhibition of tumor growth, because of the poor accumulation of both DOX and

22

IR-780 without being encapsulated in NMs. DOX encapsulated in hPPE1, i.e., 22


Page 22 of 38

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Biomacromolecules

1

hPPE1DOX, resulted in 64% of growth inhibition, attributing to the better tumoral

2

uptake of PEGylated NMs. hPPE1DOX&IR with additional IR-780 encapsulation

3

without NIR irradiation did not make an extra therapeutic benefit, as compared to

4

hPPE1DOX, which demonstrated the biocompatibility of IR-780. Similarly, hPPE1IR

5

with NIR treatment (+NIR) significantly inhibited the growth of tumors by ~60%.

6

Treatment with the synergistic chemo-thermotherapy using hPPE1DOX&IR with laser

7

irradiation showed the best growth inhibition and eventually inhibited the tumor

8

volume by 94% compared that PBS treatment. On day 15 after the treatment, all the

9

tumors were harvested, weighed, and photographed. Tumor weight measurement and

10

direct observation of tumors further proved that the antitumor efficacy was improved

11

markedly for hPPE1DOX&IR group with NIR treatment (Figure 9B). It is worth noting

12

that there was not significant body weight loss found in any of treatment group,

13

suggesting that the treatment had no observable adverse effect on mice health (Figure

14

9C), which made the hPPE NMs potentially safe vehicles for therapeutic and

15

photothermal reagents.

16

To

further

evaluate

the

antitumor

effect

of

hPPE1DOX&IR

mediated

17

chemo-photothermal therapy, tumor slices were investigated by pathological

18

examination using hematoxylin and eosin (H&E) staining. H&E staining of tumor

19

slices showed that tumor cells with PBS treatment largely retained their normal

20

morphology

21

chemo-thermotherapy showed a better remission than other treatment groups. This

22

further suggested that the synergistic therapy was superior to chemotherapy or

with

distinctive

nuclear

structures

23


and

membrane.

The


1

photothermal therapy alone (Figure 9D). Furthermore, the antitumor effect was

2

analyzed by immunohistochemical staining of the TUNEL and the Ki-67 assay at the

3

end of treatment. Meanwhile, immunohistochemical studies also confirmed improved

4

therapeutic effect of hPPE1DOX&IR with NIR irradiation, showing a significant

5

reduction in proliferation while an increase in the apoptosis of tumor cells.

6

Conclusion

7

In summary, enzyme-degradable NMs hPPEDOX&IR based on hyperbranched

8

polyphosphoester were constructed for efficient chemo-photothermal ablation of drug

9

resistant tumors. hPPEDOX&IR exhibited enhanced physicochemical and biological

10

features including enhanced cellular internalization, more efficient nucleus

11

translocation, and better tumoral accumulation of encapsulated DOX. The enzymatic

12

degradation of hPPE led to a rapid release of DOX with response to ALP, an enzyme

13

abundant in various cancers. NIR triggered hyperthermia resulted in the synergistic

14

cytotoxicity against both MCF-7 and MCF-7/ADR cells in vitro and efficient ablation

15

of DOX-resistant tumors in vivo. Our strategy provides an innovative approach in the

16

design of enzyme-responsive NMs for cancer therapy.

17

24


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Biomacromolecules

1

2 3 4 5 6

Figure 1. Illustration of (A) structure of DOX- and IR-780 co-loaded hyperbranched polyphosphoester nanomedicines (hPPEDOX&IR) and (B) the rationale of combined chemo-photothermal treatment of drug resistant cancer cells.

25



1 2 3 4 5 6 7 8 9

Figure 2. (A, B) Size distribution (A) and zeta potential (B) of hPPEDOX&IR. (C) The stability of hPPEDOX&IR in 10% FBS and 5% glucose aqueous solutions. (D) Percentile release of DOX from hPPEDOX&IR as a function of time in vitro. (E) Photothermal images of 200 µL of hPPEDOX&IR with different concentrations of IR-780 in 1.5 mL plastic tubes under NIR laser irradiation at a power intensity of 1.0 W/cm2 for 5 min. (F) Temperature changes of hPPEDOX&IR under irradiation as indicated in (E).

26


Page 26 of 38

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Biomacromolecules

1 2 3 4 5 6 7 8

Figure 3. (A) Size change of hPPEDOX&IR by dynamic light scattering (DLS) after incubation with or without alkaline phosphatase for various times. (B) Fluorescence spectra of DOX in hPPEDOX&IR after various time incubation with alkaline phosphatase. (C) Fluorescence intensity at 560 nm of hPPEDOX&IR at the same concentration of DOX after various time incubation with or without alkaline phosphatase enzyme. (D) DOX release profiles from hPPEDOX&IR with six laser off/laser on cycles of different NIR irradiation power levels (laser on time: 5 s)

27



1 2 3 4 5 6 7 8 9 10

Figure 4. (A) Flow cytometric analyses of cellular internalization of free DOX&IR and hPPEDOX&IR in MCF-7 cells after incubation for 2 h. (B) Flow cytometric analyses of cellular internalization of free DOX&IR and hPPEDOX&IR in MCF-7 cells after incubation for 2 h and 4 h, respectively. (C) Flow cytometric analyses of cellular internalization of free DOX&IR and hPPEDOX&IR in MCF-7/ADR cells after incubation for 2 h, respectively. (D) Flow cytometric analyses of cellular internalization of free DOX&IR and hPPEDOX&IR in MCF-7/ADR cells after incubation for 2 h and 4 h, respectively. *p

Enzyme Degradable Hyperbranched Polyphosphoester Micellar

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