Micelles with Sheddable Dendritic Polyglycerol Sulfate Shells Show

Sep 27, 2016 - Cancer nanomedicines are typically stealthed by a poly(ethylene glycol) layer that is important to obtain extended blood circulation an...
2 downloads 6 Views 995KB Size
Subscriber access provided by Heriot-Watt | University Library

Article

Micelles with Sheddable Dendritic Polyglycerol Sulfate Shells Show Extraordinary Tumor Targetability and Chemotherapy In Vivo Yinan Zhong, Mathias Dimde, Daniel Stöbener, Fenghua Meng, Chao Deng, Zhiyuan Zhong, and Rainer Haag ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09204 • Publication Date (Web): 27 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces 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.

Page 1 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Micelles with Sheddable Dendritic Polyglycerol Sulfate Shells Show Extraordinary Tumor Targetability and Chemotherapy In Vivo

Yinan Zhong a, Mathias Dimde b, Daniel Stöbener b, Fenghua Meng a,*, Chao Deng a, Zhiyuan Zhong a,*, and Rainer Haag b

a

Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional

Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, People’s Republic of China.

b

Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, 14195, Germany

ABSTRACT: Cancer nanomedicines are typically stealthed by poly(ethylene glycol) layer that is important to obtain extended blood circulation and elevated tumor accumulation. PEG stealth, however, also leads to poor tumor cell selectivity and uptake thereby reducing treatment efficacy. Here, we report that biodegradable micelles with sheddable dendritic polyglycerol sulfate (dPGS) shells show an unusual tumor targetability and chemotherapy in vivo. The self-assembly of dPGS-SS-poly(-caprolactone) amphiphilic block copolymer with an Mn of 4.8-3.7 kg mol-1 affords negatively charged and small sized micelles (dPGS-SS-PCL Ms). dPGS-SS-PCL Ms reveal a low cytotoxicity, decent doxorubicin (DOX) loading and accelerated drug release under a reductive condition. Notably, DOX-loaded dPGS-SS-PCL

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ms exhibit a high tolerable dosage of more than 40 mg kg-1, a long plasma half-life of ca. 2.8 h, and an extraordinary tumor accumulation. Intriguingly, therapeutic results demonstrate that DOX-loaded dPGS-SS-PCL Ms induce complete tumor suppression, significantly improved survival rate, and diminishing adverse effects as compared to free drug (DOXHCl) in MCF-7 human mammary carcinoma models. Dendritic polyglycerol sulfate with a superior tumor homing ability appears to be an attractive alternative to PEG in formulating targeted cancer nanomedicines. KEYWORDS: polyglycerol, reduction-sensitive, biodegradable micelles, tumor-targeting, cancer chemotherapy

1. INTRODUCTION Advances in nanomedicine have promoted the quick development and commercialization of novel treatments for malignant tumors.1-3 In particular, micellar anticancer drugs derived from biodegradable polyesters like poly(-caprolactone) (PCL), with a reliable safety profile, have attracted the most attention.4-6 Notably, several nanomedicines like Genexol-PM®, paclitaxel-loaded poly(ethylene glycol)-polylactide micelles7 and BIND-014, nanoparticulate docetaxel derived from PEG-poly(lactide-co-glycolide) homing to prostate-specific membrane antigen (PSMA),8 are used in the clinics or under clinical trials for treating various cancers.9,

10

However, the treatment efficacy of these micellar anticancer drugs remains

suboptimal partly due to problems of low tumor targetability and limited drug release in cancer cells.11, 12 To obtain a long blood circulation, nanotherapeutics are usually protected by a stealth polymer such as PEG, which intends to reduce protein opsonization and phagocytosis by the nonparenchymal cells of the liver.13, 14 The dense layer of stealth polymers, however, induces modest tumor targeting and retention (so-called passive targeting) as well as poor tumor cell 2

ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

uptake.15,

16

In the past years, polyglycerol that is water-soluble, biocompatible, and

multi-functional has emerged as an appealing alternative to PEG.17-21 Interestingly, dendritic polyglycerol sulfate (dPGS) has shown an intrinsic targetability to inflammation and high affinity to L-selectin.22 It is known that development of tumor is usually accompanied with inflammation.23-26 In many aspects, tumor and inflammation share similar biological properties.27 Here we hypothesized that micelles with dPGS shells would effectively target to tumor in vivo. To verify our hypothesis, we designed reduction-sensitive dPGS-SS-PCL block copolymer micelles (dPGS-SS-PCL Ms) and investigated their doxorubicin (DOX) delivery to MCF-7 human mammary carcinoma xenografts in nude mice (Scheme 1). The presence of a disulfide linkage between dPGS and PCL would further promote drug release within tumor cells, 28-31 given that there presents an abundant glutathione (GSH) in the cytosol of cancerous cells.32-34 The reduction-sensitive strategy was widely used in the tumoral delivery of therapeutic agents including drugs,35-38 genes,39-42 siRNA,43 peptides,41 antigens,44 and NIR dyes.45 Our results show that DOX-loaded dPGS-SS-PCL Ms exhibit extraordinary targetability and chemotherapy toward MCF-7 human mammary carcinoma in vivo. This represents, to our knowledge, the first proof-of-concept study that dendritic polyglycerol sulfate can be used as an alternative to PEG for targeted cancer therapy.

3

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme

1.

Illustration

of

dendritic

polyglycerol

sulfate-SS-poly(-caprolactone)

(dPGS-SS-PCL) micelles for active targeting to inflammation-related tumor tissues and triggered cytoplasmic drug release within tumor cells.

2. EXPERIMENTAL SECTION 2.1. Synthesis of dPGS-SS-PCL and dPGS-PCL Conjugates. As shown in Scheme 2, dPGS-SS-PCL block polymer was obtained via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) between PCL-SS-cyclooctyne (Mn = 3.7 kg mol-1) and azide-dPGS (dPGS-N3, Mn = 4.8 kg mol-1, sulfation: 88%). Typically, a solution of dPGS-N3 (189 mg, 0.038 mmol) in water (5.0 mL) was added to a stirred THF solution (1.0 mL) of PCL-SS-cyclooctyne (100 mg, 0.027 mmol) under nitrogen. The reaction proceeded at r.t. for 24 h. The target compound dPGS-SS-PCL was isolated by rotary evaporation to remove THF and water, extensive washing by cold methanol to get rid of unreacted dPGS, and drying in vacuo. Yield: 52%. 1H NMR (400 MHz, DMSO-d6): δ 1.28, 1.51, 2.26, and 4.00 (PCL); δ 2.78, and 3.55 (cystamine); δ 0.65-0.88, and 1.98-2.20 (cyclooctyne); δ 3.15-3.7, 4.20, and 4

ACS Paragon Plus Environment

Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

4.54-4.85 (dPGS). Through contrasting the integrals of signals at δ 4.20, 4.54-4.85 (the methyl and methine protons next to -OSO3Na of dPGS) and 4.00 (the methyl protons next to –OC(O) of PCL), we could conclude an equal conjugation of dPGS-N3 and PCL-SS-cyclooctyne. Mn (1H NMR) = 8.8 kg mol-1. Reduction-insensitive dPGS-PCL was synthesized by a similar way. Yield: 47%. 1H NMR (400 MHz, DMSO-d6): δ 1.28, 1.51, 2.26, and 4.00 (PCL); δ 1.55, and 3.25 (butanediamine); δ 0.65-0.88, and 1.98-2.20 (cyclooctyne); δ 3.15-3.7, 4.20, and 4.54-4.85 (dPGS). Mn (1H NMR) = 8.6 kg mol-1.

2.2. Cytotoxicity Studies. The cytotoxicity of blank micelles was evaluated in MCF-7 human mammary carcinoma cells. The cells were seeded in a 96-well plate (1×104 cells well-1) using RPMI 1640 media supplemented with 10% fetal bovine serum, 1% L-glutamine, antibiotics penicillin (100 IU mL-1) and streptomycin (100 μg mL-1) at 5% CO2 and 37 oC. 24 h later, a prescribed amount of dPGS-SS-PCL or dPGS-PCL micellar dispersions (with final micellar concentrations of 0.1, 0.5 and 1.0 mg mL-1) was added and cultured with the cells for 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assays were performed according to a previous report.47 Also, the anticancer activities of DOX-encapsulated micelles and free DOX were evaluated as above except that DOX-encapsulated micelles or free DOX instead of blank micelles were added.

2.3. Blood Circulation. The protocols for animal experiments were approved by Soochow University Laboratory Animal Center and the Animal Care and Use Committee of Soochow University. The blood circulation experiments were performed in healthy nude mice (~ 8 week, ~ 20 g). DOX-encapsulated micelles or free DOX (10 mg DOX equiv. kg-1) were intravenously administrated into the mice. At different time intervals post injection, ~10 L of

5

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

blood samples were collected and dissolved in 0.3 mL of lysis buffer (1% Triton X-100) with intense sonification. The samples were exposed to 20 mM DTT solution of HCl-isopropanol at -20 oC overnight. After centrifugation (14.8 krpm, 30 min), the supernatant was collected for fluorescence spectrophotometry to determine DOX level. The pharmacokinetic parameters were obtained by fitting a typical two-compartment model. The half-lives of two phases (t1 and t2) were obtained based upon the following equation: y = A1×exp(-x / t1) + A2×exp(-x / t2) + y0

2.4. Maximum Tolerated Dose (MTD). 7~8 week old nude mice were randomly divided into five groups (n = 4) to evaluate the MTD of dPGS-SS-PCL/DOX Ms and free DOX at a single dose for a period of 10 days. The mice in each group were administrated intravenously with dPGS-SS-PCL/DOX Ms at a dose of 20 or 40 mg DOX equiv. kg-1, and free DOX at a dose of 5 or 7.5 mg DOX kg-1, respectively. The mice administrated with saline only were served as controls. MTD was defined as a maximal dose of DOX that causes neither body weight loss greater than 15% nor death or other significant changes in the general signs within the whole experimental period.

2.5. In Vivo and Ex Vivo Imaging Studies. The biodistribution of micelles in mice was studied by real-time near-infrared (NIR) fluorescence imaging using hydrophobic DIR as a fluorescence probe. MCF-7 human mammary carcinoma xenografted mice were obtained by injecting 1 × 106 MCF-7 cells into the mouse hind flank. As the tumor volumes reached about 150 mm3, DIR-loaded micelles were injected into the tumor-bearing mice intravenously. At scheduled time points (1, 4, 10 and 24 h) post intravenous injection, the mice were scanned using the Maestro in vivo fluorescence imaging system (CRi Inc.). Ex vivo fluorescence imaging was conducted using DOX-encapsulated micelles and free

6

ACS Paragon Plus Environment

Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

DOX. Tumor-bearing mice were killed 10 h after intravenous administration of DOX-encapsulated micelles or free DOX. The tumor burden and main organs were excised, washed, dried, and analyzed using the same fluorescence imaging system as described above.

2.6. In Vivo Therapeutic Efficiency. The therapeutic efficiency of dPGS-SS-PCL/DOX Ms, dPGS-PCL/DOX Ms, and free DOX were studied in MCF-7 human mammary carcinoma-bearing mice (7.5 mg DOX equiv. kg-1). The tumor-bearing mice were assigned to five groups (n = 6) at random, and each group was intravenously injected with the above drugs every 3 days. The tumor volumes measured using calipers was calculated based on the formula V = 0.5 × L × W × H, wherein L, W and H are the tumor size at the longest, widest and highest point, respectively. Relative tumor volumes were presented as the tumor volume at a scheduled time point divided by the tumor volume when the treatment was initiated. Additionally, the body weights of mice in each group were also measured, and the relative body weights were normalized to their initial weights. Mice were considered to be dead when the tumor volume exceeded 1000 mm3.

3. RESULTS AND DISCUSSION 3.1. Synthesis of dPGS-SS-PCL and dPGS-PCL Conjugates. dPGS-SS-PCL block polymer was obtained via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) between PCL-SS-cyclooctyne and azide-dPGS (Scheme 2). SPAAC shows a superior specificity, high coupling efficiency, and low cytotoxicity.48-52 PCL-SS-cyclooctyne was prepared via activating the hydroxyl terminal group of PCL with p-nitrophenyl chloroformate, reacting with cystamine, and coupling with bicyclo (6.1.0) non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (BCN). 1H NMR showed that PCL-SS-cyclooctyne had a nearly 100% functionality of terminal group and an Mn of 3.7 kg mol-1 (Figure S1). Gel-permeation

7

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 24

chromatography (GPC) displayed a low Mw/Mn of 1.12. SPAAC reaction between PCL-SS-cyclooctyne and dPGS-azide (Mn = 4.8 kg mol-1, sulfation: 88%) was carried out at a molar ratio of 1:1.4 in tetrahydrofuran-water solvent at r.t. for 24 h. dPGS-SS-PCL was purified via rotary evaporation to remove tetrahydrofuran (THF) and water, washing by cold methanol to get rid of unreacted dPGS, and vacuum drying. 1H NMR revealed an equal conjugation of dPGS and PCL (Figure S2). In a similar way, non-reductive dPGS-PCL copolymer with the same constituents was synthesized using butanediamine instead of cystamine (Figure S3 and S4).

OSO3OSO3-

O3SO

O O O

-

O3SO

HO

O

OSO3O

OSO3-

O

-

O

O

OH

OSO3-

OH

O

O3SO O

O

O O

O -

OSO3O O

O3SO

O -

O3SO

OSO3- OSO3O

O

O OH

OSO3-

O OH

OSO3-

O3SO

O -

O -

OSO3

OSO3

-

HO

S

S

O

O O

N H

O

O m

O

OO3SO

O O

O

H N

O

N3

(Cyclooctyne-SS-PCL)

O

O

OSO3-

-

O3SO

O

OSO3-

OSO3-

(dPGS-N3)

OSO3-

OSO3OSO3- HO

SPAAC -

O3SO

O O O

-

O3SO

OSO3

O

OSO3-

-

O

OSO3O

OSO3-

O -

O

O

OH

OSO3-

OH

-

O

O

O

O3SO

O3SO O O O

O -

-

O3SO

OSO3O O O

-

O3SO

OSO3 OSO3 O

O

O OH

OSO3O HO

O-

O

N

O

O3SO

O

O OH

-

OSO3-

O

N

H N

O

N

S

S

O

O N H

O

O O m

O -

O3SO

(dPGS-SS-PCL)

OSO3-

O

OSO3-

OSO3OSO3-

Scheme

2.

Synthesis

of

dPGS-SS-PCL

from

dPGS-azide

PCL-SS-cyclooctyne via SPAAC reaction. Condition: THF/H2O, r.t., 24 h. 8

ACS Paragon Plus Environment

(dPGS-N3)

and

Page 9 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

3.2. Characterization of Micelles and DOX-Encapsulated Micelles. dPGS-SS-PCL block polymer formed small-sized monodisperse micelles with a hydrodynamic diameter of about 60 nm as revealed by dynamic light scattering (Figure 1A). TEM measurement showed a mean diameter of ~ 45 nm with a spherical shape (Figure 1A). dPGS-SS-PCL Ms displayed a zeta potential of -11 mV and a low critical micelle concentration of ~ 5.4 mg L-1 (Table S1). Notably, DLS showed that dPGS-SS-PCL Ms swelled from ca. 60 nm to over 500 nm within 24 h in the exsitence of 10 mM GSH (an intracellular-mimicking reductive condition), while hardly any size variation was observed for non-reductive dPGS-PCL Ms under otherwise the same condition (Figure S5), confirming a high reduction-responsivity of dPGS-SS-PCL Ms. We studied the drug loading and release properties of dPGS-SS-PCL Ms using DOX, a widely used clinical anticancer agent,53,

54

as a model drug. The results showed that

dPGS-SS-PCL Ms had a decent DOX loading capacity of 13.4 wt.% (Table 1). DOX-loaded dPGS-SS-PCL Ms (dPGS-SS-PCL/DOX Ms) maintained a small size and a low PDI (Table 1). The release studies revealed ~ 79% drug release from dPGS-SS-PCL Ms in 24 h under 10 mM GSH condition, while significantly less drug release (< 30%) was detected for non-reductive dPGS-PCL counterparts under the same condition and dPGS-SS-PCL Ms under a non-reductive condition (Figure 1B), similar to previous reports for PEG-SS-PCL micelles.47, 55 MTT studies indicated that dPGS-SS-PCL/DOX Ms were highly effective against MCF-7 human mammary carcinoma cells, with an IC50 of 2.0 g mL-1, which was over 10 times better than non-reductive dPGS-PCL counterparts (Figure 1C). Confocal observation revealed a much stronger nucleic DOX fluorescent intensity in dPGS-SS-PCL/DOX Ms-treated MCF-7 cells than in dPGS-PCL/DOX Ms-treated ones (Figure S6A). Flow cytometry analysis indicated that dPGS-SS-PCL/DOX Ms-treated MCF-7 cells had a 7-fold higher DOX level than dPGS-PCL/DOX Ms-treated ones (Figure S6B). dPGS-SS-PCL/DOX 9

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

Ms afford enhanced internalization and cytoplasmic drug release. Importantly, blank dPGS-SS-PCL Ms possessed low cytotoxicity in MCF-7 cells (> 95% cell viability) at 1.0 mg mL-1 micelle concentration (Figure 1D), supporting their excellent biocompatibility.

dPGS-SS-PCL

Intensity (%)

20 15 10

50nm

5 0

C

100

Cumulative Release (%)

25

10

100 Size (nm)

1000

D

60 40 20 0

0

100 120 80 100 60 80 40 60 20 40 0 20

Cell Viability (%)

100 80 60 40 20 0

80

5

10 15 Time (h)

20

25

120

dPGS-SS-PCL/DOX dPGS-PCL/DOX Free DOX

120

dPGS-SS-PCL, 10 mM GSH dPGS-SS-PCL, No GSH dPGS-PCL, 10 mM GSH

B

dPGS-SS-PCL dPGS-PCL

***

A

Cell Viability (%)

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

0

0.01 0.1 1 10 DOX Concentration (g/mL)

dPGS-SS-PCL dPGS-PCL

0.01

0.1

1

10

0.1 0.5 1 Micelle Concentration (mg/mL)

Figure 1. Characterization of dPGS-SS-PCL and dPGS-PCL polymeric micelles. (A) The size determined by DLS (Inset: TEM image of dPGS-SS-PCL Ms); (B) In vitro DOX release in PB at 37 oC (n = 3). ***p < 0.001 (Student’s t test); (C) MTT assays of dPGS-SS-PCL/DOX Ms, dPGS-PCL/DOX Ms, and free DOX in MCF-7 human mammary carcinoma cells for 48 h (n = 4); (D) Cytotoxicities of empty dPGS-SS-PCL and dPGS-PCL Ms in MCF-7 cells.

Table 1. Characteristics of DOX-loaded micelles. Micelles

dPGS-SS-PCL/DOX

Size (nm)

a

PDI

a

DLC (wt.%) b Theory

Determined

DLE (%) b

76

0.14

10.0

7.1

68.9

80

0.13

20.0

12.4

56.4

10

ACS Paragon Plus Environment

Page 11 of 24

dPGS-PCL/DOX

68

0.16

10.0

7.3

70.8

75

0.12

20.0

13.4

61.7

a

Determined by DLS analysis at 25 ºC in PB (pH 7.4, 10 mM);

b

Determined by fluorescence measurement.

3.3. In Vivo Pharmacokinetics and Tolerability Studies. To verify the stealth effect of dPGS, the pharmacokinetic studies in nude mice were conducted. Interestingly, both dPGS-SS-PCL/DOX and dPGS-PCL/DOX Ms exhibited extended plasma circulation with elimination phase half-lives of 2.83 h and 2.88 h respectively, which were ten-fold higher than free DOX (Figure 2A). The total area under the curve (AUC) was calculated to be ca. 100 mgh L-1 for both dPGS-SS-PCL/DOX and dPGS-PCL/DOX Ms, 17-fold exceeding that for free

DOX.

Notably,

the

maximum-tolerated

dose

(MTD)

studies

showed

that

dPGS-SS-PCL/DOX Ms were well tolerated with an MTD of more than 40 mg DOX equiv. kg-1 (Figure 2B). In contrast, free DOX exhibited significant systemic toxicity at a dose of 7.5 mg kg-1 (Figure 2B). The occurrence of dose-limiting side effects is a major challenge for most clinical chemotherapeutics including DOX.56, 57 The high MTD of dPGS-SS-PCL/DOX Ms provides a broad therapeutic window for effective cancer therapy without causing adverse effects.

Figure

B 20

dPGS-SS-PCL/DOX dPGS-PCL/DOX Free DOX

15 10 5 0

2.

0

5

In

vivo

10 15 Time (h)

20

Relat. Body Weight (%)

A DOX Uptake (% ID/g)

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

25

pharmacokinetics

and

120 100 80

dPGS-SS-PCL/DOX, 20 mg/kg dPGS-SS-PCL/DOX, 40 mg/kg Free DOX, 5 mg/kg Free DOX, 7.5 mg/kg PBS

60 40 20 0

0

2

tolerability.

4 6 Time (d)

(A)

8

10

Pharmacokinetics

of

dPGS-SS-PCL/DOX Ms and dPGS-PCL/DOX Ms in healthy nude mice. DOX level was 11

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

measured by fluorescence spectrometer. DOX uptake is presented as %ID/g (n = 3); (B) Maximum tolerated dosage (MTD) measurements in healthy nude mice. The body weight of mice was monitored after intravenous injection of dPGS-SS-PCL/DOX Ms or free DOX (n = 3).

3.4. In Vivo and Ex Vivo Fluorescence Imaging. Based on the facts that tumor is usually accompanied by inflammation and dPGS can efficiently target to the inflammation site,22, 58 we hypothesized that micelles with dPGS shells may effectively target to solid tumors. In order to verify this assumption, we have adopted MCF-7 human mammary carcinoma xenografts in mice as a model, and monitored DIR-loaded dPGS-SS-PCL Ms in vivo following intravenous injection using a Maestro EX in vivo fluorescence imaging system (CRi, Inc.). Intriguingly, the results revealed a significant DIR accumulation in tumor at 1 h after injection (Figure 3A). Tumor DIR fluorescence increased in time and reached the maximum at 10 h (Figure 3A), confirming effective tumor targeting and retention of dPGS-SS-PCL Ms. We and others have shown that block copolymer micelles based on PEG typically lead to low tumor accumulation and retention.59-61 Notably, DIR-encapsulated PEG-SS-PCL micelles exhibited a weak fluorescence in subcutaneous glioblastoma U87MG tumor models.59 DIR-loaded dPGS-PCL Ms displayed also obvious tumor accumulation though with comparably weaker fluorescence (Figure 3A), as a result of self-quenching of DIR fluorescence when kept inside the micelles. The DOX fluorescence images of the major tissues harvested from mice 10 h after intravenous injection of dPGS-SS-PCL/DOX Ms revealed a significantly higher DOX fluorescence intensity in the tumor than in normal tissues including heart, spleen, lung, kidney and liver (Figure 3B). By comparison, mice treated with dPGS-PCL/DOX Ms showed weaker tumor DOX fluorescence likely due to poor drug release within the tumor (Figure 3B). DOX fluorescence would be quenched when located in the micellar core.62 On the contrary, free DOX-treated mice showed the highest fluorescence intensity in the liver (Figure 3B). The semi-quantitative analysis of DOX fluorescence in the 12

ACS Paragon Plus Environment

Page 13 of 24

tissues showed that the fluorescence intensity in the tumor of mice treated with dPGS-SS-PCL/DOX Ms was about 3-fold that with free DOX, further verifying the tumor targetability of dPGS-SS-PCL/DOX Ms (Figure S7). These results suggest that dPGS-SS-PCL Ms can efficaciously target and release payloads to human MCF-7 tumor in mice. A

dPGS-SS-PCL

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

4h

1h

10h

24h

high

low

B dPGS-SS-PCL/DOX

dPGS-PCL/DOX

high

Free DOX

1

2

3

1

2

3

1

2

3

4

5

6

4

5

6

4

5

6

low

Figure 3. In vivo and ex vivo fluorescence imaging studies. (A) In vivo NIR fluorescence images of MCF-7 human mammary carcinoma xenografted nude mice at indicated time points (1, 4, 10, and 24 h) post administration of DIR-loaded micelles; (B) DOX fluorescence images of the major tissues excised from tumor-bearing mice 10 h post i.v. injection of different formulations (1: heart, 2: liver, 3: spleen, 4: lung, 5: kidney, and 6: tumor).

3.5. In Vivo Antitumor Performance of dPGS-SS-PCL/DOX Ms. We further evaluated the therapeutic performance of dPGS-SS-PCL/DOX Ms using MCF-7 human mammary carcinoma in nude mice. When tumor volume reached ca. 50 mm3, the mice were intravenously injected with dPGS-SS-PCL/DOX Ms, dPGS-PCL/DOX Ms or free DOX (7.5 mg DOX equiv. kg-1) every three days. Strikingly, dPGS-SS-PCL/DOX Ms showed complete inhibition of tumor progression (Figure 4A). dPGS-PCL/DOX Ms could also significantly

13

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 24

delay tumor growth, though less effectively than dPGS-SS-PCL/DOX Ms (Figure 4A). Notably, both dPGS-SS-PCL/DOX and dPGS-PCL/DOX Ms did not show any influence on mice body weights (Figure 4B), further corroborating that they have little adverse effects. It is worth noting that free DOX at the same dosage though effectively inhibited tumor growth resulted in severe side effects (Figure 4A, B). Remarkably, Kaplan-Meier survival curves showed a 100% survival rate for mice treated with dPGS-SS-PCL/DOX Ms within an experimental period of 76 days (Figure 4C). dPGS-PCL/DOX Ms could also significantly improve the survival rate, with a median survival time of 60 days (Figure 4C). In comparison, PBS and free DOX treated groups showed median survival times of 36 and 16 days, respectively (Figure 4C). It is evident, therefore, that dPGS-SS-PCL/DOX Ms exhibit excellent targetability to and effective treatment of MCF-7 human mammary carcinoma in vivo. It should be noted that dPGS shows intrinsic anti-inflammatory effect,22 which would possibly also result in change of tumor microenvironment. The alternation of tumor microenvironment could be another factor for high antitumor effects of dPGS-SS-PCL/DOX Ms.

14

ACS Paragon Plus Environment

Relat. Body Weight (%)

dPGS-SS-PCL/DOX dPGS-PCL/DOX Free DOX PBS

12 A 10 8

***

6 4

**

2 0

Morbility Free Survival (%)

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Relat. Tumor Vol. (%)

Page 15 of 24

0

5

10 15 20 Time (d)

25

30

10 15 20 Time (d)

25

30

120 B 100 80 60 40 20

0

5

120 C 100 80 60 40 20 0

0 10 20 30 40 50 60 70 80 Time (d)

Figure 4. In vivo antitumor performances of dPGS-SS-PCL/DOX Ms in MCF-7 human mammary carcinoma xenografted nude mice. Tumor growth curves (A) and body weight changes

(B) of xenografted mice after

treatments of dPGS-SS-PCL/DOX Ms,

dPGS-PCL/DOX Ms, free DOX, or PBS. The drugs were administrated on day 1, 4, 7, 10, 13, and 16 (7.5 mg DOX equiv. kg-1) (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 (Student’s t test); (C) Survival data for mice in different groups within a period of 76 d.

The histological analyses showed that both dPGS-SS-PCL/DOX Ms and dPGS-PCL/DOX Ms caused little damage or inflammatory lesion of liver, heart, and kidney (Figure 5). dPGS-SS-PCL/DOX Ms induced significantly more tumor necrosis than dPGS-PCL/DOX Ms 15

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

(Figure 5). In contrast, free DOX resulted in obvious liver and heart damage (Figure 5), in line with their serious side effects. All of the results support dPGS-SS-PCL Ms can effectively deliver and release pharmaceuticals to target MCF-7 human mammary carcinoma, leading to

Heart

Liver

Tumor

high treatment efficacy with significantly reduced systemic toxicity.

Kidney

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 24

Figure 5. H&E-staining of tumor, liver, heart, and kidney slices from MCF-7 human mammary carcinoma xenografted nude mice on day 30 after different treatments. The images were recorded by Leica microscope at high magnification (400×). The scale bars represent 50 m.

4. CONCLUSIONS We show that biodegradable micelles with sheddable dendritic polyglycerol sulfate (dPGS) shells show an extraordinary tumor targetability and chemotherapy in MCF-7 human mammary carcinoma-bearing mice. This work provides a proof-of-concept that dPGS-shelled 16

ACS Paragon Plus Environment

Page 17 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

micelles can effectively home to tumor and dPGS is a better alternative to PEG in formulating tumor-targeting nanomedicines. Micellar doxorubicin based on dPGS-SS-PCL block copolymer possesses the following unique features: (i) they have a high drug loading, low drug leakage, while fast drug release inside the tumor cells; (ii) they show a remarkably high tolerated dose and therapeutic index; (iii) they exhibit a long circulation time and intrinsic tumor targetability; (iv) they display complete inhibition of tumor growth and markedly improved survival time; and (v) they cause little adverse effects. Dendritic polyglycerol sulfate with a superior tumor homing ability to PEG has opened a new avenue to advanced cancer nanomedicines.

ASSOCIATED CONTENT Supporting Information is available free of charge on the ACS Publications website. Synthesis of dPGS-azide, PCL-cyclooctyne and PCL-SS-cyclooctyne;

1

H NMR spectra of

PCL-cystamine, PCL-SS-cyclooctyne, dPGS-SS-PCL, PCL-NH2, PCL-cyclooctyne, and dPGS-PCL polymers; Characteristics of dPGS-SS-PCL and dPGS-PCL Ms; Size and size distribution of dPGS-SS-PCL Ms and dPGS-PCL Ms in response to 10 mM GSH; CLSM and flow cytometry analyses; ROI analysis of the ex vivo fluorescence imaging.

AUTHOR INFORMATION Corresponding Authors *Tel: +86-512-65880098. E-mail: [email protected] (F. Meng); [email protected] (Z. Zhong).

ACKNOWLEDGMENTS

17

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

This work is financially supported by the National Natural Science Foundation of China (51473111, 51225302), and a project funded by China Postdoctoral Science Foundation (7131705616). R.H. and M.D. are grateful to the SFB 1112 from the German Science Foundation for financial support.

REFERENCES (1)

Torchilin, V. P. Multifunctional, Stimuli-Sensitive Nanoparticulate Systems for Drug Delivery. Nature Rev. Drug Discov. 2014, 13 (11), 813-827.

(2)

Chauhan, V. P.; Jain, R. K. Strategies for Advancing Cancer Nanomedicine. Nature Mater. 2013, 12 (11), 958-962.

(3)

Cabral, H.; Kataoka, K. Progress of Drug-Loaded Polymeric Micelles into Clinical Studies. J. Control. Release 2014, 190, 465-476.

(4)

Gaucher, G.; Marchessault, R. H.; Leroux, J.-C. Polyester-Based Micelles and Nanoparticles for the Parenteral Delivery of Taxanes. J. Control. Release 2010, 143 (1), 2-12.

(5)

Seyednejad, H.; Ghassemi, A. H.; van Nostrum, C. F.; Vermonden, T.; Hennink, W. E. Functional Aliphatic Polyesters for Biomedical and Pharmaceutical Applications. J. Control. Release 2011, 152 (1), 168-176.

(6)

Chen, Y.; Li, Y.; Gao, J.; Cao, Z.; Jiang, Q.; Liu, J.; Jiang, Z. Enzymatic PEGylated Poly (Lactone-Co-β-Amino Ester) Nanoparticles as Biodegradable, Biocompatible and Stable Vectors for Gene Delivery. ACS Appl. Mater. Interfaces 2015, 8 (1), 490-501.

(7)

Gong, J.; Chen, M.; Zheng, Y.; Wang, S.; Wang, Y. Polymeric Micelles Drug Delivery System in Oncology. J. Control. Release 2012, 159 (3), 312-323.

(8)

Hrkach, J.; Von Hoff, D.; Ali, M. M.; Andrianova, E.; Auer, J.; Campbell, T.; De Witt, D.; Figa, M.; Figueiredo, M.; Horhota, A. Preclinical Development and Clinical Translation of A PSMA-Targeted Docetaxel Nanoparticle with A Differentiated Pharmacological Profile. Sci. Transl. Med. 2012, 4 (128), 128ra39-128ra39.

(9)

Min, Y.; Caster, J. M.; Eblan, M. J.; Wang, A. Z. Clinical Translation of Nanomedicine. Chem. Rev. 2015, 115 (19), 11147-11190.

(10) Wicki, A.; Witzigmann, D.; Balasubramanian, V.; Huwyler, J. Nanomedicine in Cancer Therapy: 18

ACS Paragon Plus Environment

Page 19 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Challenges, Opportunities, and Clinical Applications. J. Control. Release 2015, 200, 138-157. (11) Srinivasarao, M.; Galliford, C. V.; Low, P. S. Principles in the Design of Ligand-Targeted Cancer Therapeutics and Imaging Agents. Nature Rev. Drug Discov. 2015, 14, 203-219. (12) Cheng, R.; Meng, F.; Deng, C.; Zhong, Z. Bioresponsive Polymeric Nanotherapeutics for Targeted Cancer Chemotherapy. Nano Today 2015, 10 (5), 656-670. (13) Li, S.-D.; Huang, L. Stealth Nanoparticles: High Density but Sheddable PEG Is A Key for Tumor Targeting. J. Control. Release 2010, 145 (3), 178. (14) Romberg, B.; Hennink, W. E.; Storm, G. Sheddable Coatings for Long-Circulating Nanoparticles. Pharm. Res. 2008, 25 (1), 55-71. (15) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Poly (Ethylene Glycol) in Drug Delivery: Pros and Cons as well as Potential Alternatives. Angew. Chem. Int. Ed. 2010, 49 (36), 6288-6308. (16) Barz, M.; Luxenhofer, R.; Zentel, R.; Vicent, M. J. Overcoming the PEG-Addiction: Well-Defined Alternatives to PEG, from Structure–Property Relationships to Better Defined Therapeutics. Polym. Chem. 2011, 2 (9), 1900-1918. (17) Frey, H. Dendritic Polymers: Universal Glue for Cells. Nature Mater. 2012, 11 (5), 359-360. (18) Thomas, A.; Müller, S. S.; Frey, H. Beyond Poly (Ethylene Glycol): Linear Polyglycerol as A Multifunctional Polyether for Biomedical and Pharmaceutical Applications. Biomacromolecules 2014, 15 (6), 1935-1954. (19) Tekade, R. K.; Kumar, P. V.; Jain, N. K. Dendrimers in Oncology: An Expanding Horizon. Chem. Rev. 2008, 109 (1), 49-87. (20) Thota, B. N.; Urner, L. H.; Haag, R. Supramolecular Architectures of Dendritic Amphiphiles in Water. Chem. Rev. 2016, 116 (4), 2079-2102. (21) Wilms, D.; Stiriba, S.-E.; Frey, H. Hyperbranched Polyglycerols: from the Controlled Synthesis of Biocompatible Polyether Polyols to Multipurpose Applications. Acc. Chem. Res. 2009, 43 (1), 129-141. (22) Dernedde, J.; Rausch, A.; Weinhart, M.; Enders, S.; Tauber, R.; Licha, K.; Schirner, M.; Zügel, U.; von Bonin, A.; Haag, R. Dendritic Polyglycerol Sulfates as Multivalent Inhibitors of Inflammation. Proc. Natl. Acad. Sci. USA 2010, 107 (46), 19679-19684. (23) Mantovani, A.; Allavena, P.; Sica, A.; Balkwill, F. Cancer-Related Inflammation. Nature 2008, 19

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

454 (7203), 436-444. (24) Grivennikov, S. I.; Greten, F. R.; Karin, M. Immunity, Inflammation, and Cancer. Cell 2010, 140 (6), 883-899. (25) Lin, W.-W.; Karin, M. A Cytokine-Mediated Link between Innate Immunity, Inflammation, and Cancer. J. Clin. Invest. 2007, 117 (5), 1175-1183. (26) Karin, M.; Greten, F. R. NF-κB: Linking Inflammation and Immunity to Cancer Development and Progression. Nat. Rev. Immunol. 2005, 5 (10), 749-759. (27) Schäfer, M.; Werner, S. Cancer as An Overhealing Wound: An Old Hypothesis Revisited. Nat. Rev. Mol. Cell Biol. 2008, 9 (8), 628-638. (28) Wang, Y.; Zhang, L.; Zhang, X.; Wei, X.; Tang, Z.; Zhou, S. Precise Polymerization of A Highly Tumor Microenvironment-Responsive Nanoplatform for Strongly Enhanced Intracellular Drug Release. ACS Appl. Mater. Interfaces 2016, 8 (9), 5833-5846. (29) Ko, N. R.; Oh, J. K. Glutathione-Triggered Disassembly of Dual Disulfide Located Degradable Nanocarriers

of

Polylactide-Based

Block

Copolymers

for

Rapid

Drug

Release.

Biomacromolecules 2014, 15 (8), 3180-3189. (30) Wang, Y.-C.; Wang, F.; Sun, T.-M.; Wang, J. Redox-Responsive Nanoparticles from the Single Disulfide Bond-Bridged Block Copolymer as Drug Carriers for Overcoming Multidrug Resistance in Cancer Cells. Bioconjugate Chem. 2011, 22 (10), 1939-1945. (31) Gao, W.; Langer, R.; Farokhzad, O. C. Poly (Ethylene Glycol) with Observable Shedding. Angew. Chem. Int. Ed. 2010, 49 (37), 6567-6571. (32) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nature Mater. 2013, 12 (11), 991-1003. (33) Sun, H.; Meng, F.; Cheng, R.; Deng, C.; Zhong, Z. Reduction-Sensitive Degradable Micellar Nanoparticles as Smart and Intuitive Delivery Systems for Cancer Chemotherapy. Expert Opin. Drug Deliv. 2013, 10 (8), 1109-1122. (34) Wei, H.; Zhuo, R.-X.; Zhang, X.-Z. Design and Development of Polymeric Micelles with Cleavable links for Intracellular Drug Delivery. Prog. Polym. Sci. 2013, 38 (3), 503-535. (35) Ma, Y.-C.; Wang, J.-X.; Tao, W.; Sun, C.-Y.; Wang, Y.-C.; Li, D.-D.; Fan, F.; Qian, H.-S.; Yang, X.-Z. Redox-Responsive Polyphosphoester-Based Micellar Nanomedicines for Overriding Chemoresistance in Breast Cancer Cells. ACS Appl. Mater. Interfaces 2015, 7 (47), 20

ACS Paragon Plus Environment

Page 21 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

26315-26325. (36) Xu, P.; Meng, Q.; Sun, H.; Yin, Q.; Yu, H.; Zhang, Z.; Cao, M.; Zhang, Y.; Li, Y. Shrapnel Nanoparticles Loading Docetaxel Inhibit Metastasis and Growth of Breast Cancer. Biomaterials 2015, 64, 10-20. (37) Han, H.; Wang, H.; Chen, Y.; Li, Z.; Wang, Y.; Jin, Q.; Ji, J. Theranostic Reduction-Sensitive Gemcitabine Prodrug Micelles for Near-Infrared Imaging and Pancreatic Cancer Therapy. Nanoscale 2016, 8 (1), 283-291. (38) Wu, J.; Zhao, L.; Xu, X.; Bertrand, N.; Choi, W. I.; Yameen, B.; Shi, J.; Shah, V.; Mulvale, M.; MacLean, J. L. Hydrophobic Cysteine Poly (Disulfide) ‐ Based Redox ‐ Hypersensitive Nanoparticle Platform for Cancer Theranostics. Angew. Chem. Int. Ed. 2015, 127 (32), 9350-9355. (39) Hu, Y.; Zhu, Y.; Yang, W.; Xu, F. New Star-Shaped Carriers Composed of β-Cyclodextrin Cores and Disulfide-Linked Poly (Glycidyl Methacrylate) Derivative Arms with Plentiful Flanking Secondary Amine and Hydroxyl Groups for Highly Efficient Gene Delivery. ACS Appl. Mater. Interfaces 2013, 5 (3), 703-712. (40) He, Y.; Cheng, G.; Xie, L.; Nie, Y.; He, B.; Gu, Z. Polyethyleneimine/DNA Polyplexes with Reduction-Sensitive Hyaluronic Acid Derivatives Shielding for Targeted Gene Delivery. Biomaterials 2013, 34 (4), 1235-1245. (41) Chen, S.; Rong, L.; Lei, Q.; Cao, P.-X.; Qin, S.-Y.; Zheng, D.-W.; Jia, H.-Z.; Zhu, J.-Y.; Cheng, S.-X.; Zhuo, R.-X. A Surface Charge-Switchable and Folate Modified System for Co-Delivery of Proapoptosis Peptide and p53 Plasmid in Cancer Therapy. Biomaterials 2016, 77, 149-163. (42) Hu, Y.; Yuan, W.; Zhao, N.-N.; Ma, J.; Yang, W.-T.; Xu, F.-J. Supramolecular Pseudo-Block Gene Carriers Based On Bioreducible Star Polycations. Biomaterials 2013, 34, 5411-5422. (43) Chen, W.; Yuan, Y.; Cheng, D.; Chen, J.; Wang, L.; Shuai, X. Co‐Delivery of Doxorubicin and siRNA with Reduction and pH Dually Sensitive Nanocarrier for Synergistic Cancer Therapy. Small 2014, 10 (13), 2678-2687. (44) Li, D.; Kordalivand, N.; Fransen, M. F.; Ossendorp, F.; Raemdonck, K.; Vermonden, T.; Hennink, W. E.; Van Nostrum, C. F. Reduction‐Sensitive Dextran Nanogels Aimed for Intracellular Delivery of Antigens. Adv. Func. Mater. 2015, 25 (20), 2993-3003. (45) Lin, W.; Sun, T.; Xie, Z.; Gu, J.; Jing, X. A Dual-Responsive Nanocapsule via Disulfide-Induced 21

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

Self-Assembly for Therapeutic Agent Delivery. Chem. Sci. 2016, 7, 1846-1852. (46) Türk, H.; Haag, R.; Alban, S. Dendritic Polyglycerol Sulfates as New Heparin Analogues and Potent Inhibitors of the Complement System. Bioconjugate Chem. 2004, 15 (1), 162-167. (47) Zhong, Y.; Yang, W.; Sun, H.; Cheng, R.; Meng, F.; Deng, C.; Zhong, Z. Ligand-Directed Reduction-Sensitive Shell-Sheddable Biodegradable Micelles Actively Deliver Doxorubicin into the Nuclei of Target Cancer Cells. Biomacromolecules 2013, 14 (10), 3723-3730. (48) Steinhilber, D.; Rossow, T.; Wedepohl, S.; Paulus, F.; Seiffert, S.; Haag, R. A Microgel Construction Kit for Bioorthogonal Encapsulation and pH‐Controlled Release of Living Cells. Angew. Chem. Int. Ed. 2013, 52 (51), 13538-13543. (49) DeForest, C. A.; Polizzotti, B. D.; Anseth, K. S. Sequential Click Reactions for Synthesizing and Patterning Three-Dimensional Cell Microenvironments. Nature Mater. 2009, 8 (8), 659-664. (50) Dommerholt, J.; Schmidt, S.; Temming, R.; Hendriks, L. J.; Rutjes, F. P.; van Hest, J.; Lefeber, D. J.; Friedl, P.; van Delft, F. L. Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three‐Dimensional Imaging of Living Cells. Angew. Chem. Int. Ed. 2010, 49 (49), 9422-9425. (51) Canalle, L. A.; van Berkel, S. S.; de Haan, L. T.; van Hest, J. Copper‐Free Clickable Coatings. Adv. Func. Mater. 2009, 19 (21), 3464-3470. (52) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Floris, P.; van Hest, J. C.; van Delft, F. L. Aza-Dibenzocyclooctynes for Fast and Efficient Enzyme PEGylation via Copper-Free (3 + 2) Cycloaddition. Chem. Commun. 2010, 46 (1), 97-99. (53) Matsumura, Y.; Kataoka, K. Preclinical and Clinical Studies of Anticancer Agent ‐ Incorporating Polymer Micelles. Cancer Sci. 2009, 100 (4), 572-579. (54) Lammers, T.; Kiessling, F.; Hennink, W. E.; Storm, G. Drug Targeting to Tumors: Principles, Pitfalls and (Pre-) Clinical Progress. J. Control. Release 2012, 161 (2), 175-187. (55) Sun, H.; Guo, B.; Cheng, R.; Meng, F.; Liu, H.; Zhong, Z. Biodegradable Micelles with Sheddable Poly (Ethylene Glycol) Shells for Triggered Intracellular Release of Doxorubicin. Biomaterials 2009, 30 (31), 6358-6366. (56) Wang, A. Z.; Langer, R.; Farokhzad, O. C. Nanoparticle Delivery of Cancer Drugs. Annu. Rev. Med. 2012, 63, 185-198. 22

ACS Paragon Plus Environment

Page 23 of 24

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

(57) Caron, W. P.; Morgan, K. P.; Zamboni, B. A.; Zamboni, W. C. A Review of Study Designs and Outcomes of Phase I Clinical Studies of Nanoparticle Agents Compared with Small-Molecule Anticancer Agents. Clin. Cancer Res. 2013, 19 (12), 3309-3315. (58) Ji, T.; Zhao, Y.; Ding, Y.; Nie, G. Using Functional Nanomaterials to Target and Regulate the Tumor Microenvironment: Diagnostic and Therapeutic Applications. Adv. Mater. 2013, 25 (26), 3508-3525. (59) Zhu, Y.; Zhang, J.; Meng, F.; Deng, C.; Cheng, R.; Feijen, J.; Zhong, Z. cRGD-Functionalized Reduction-Sensitive Shell-Sheddable Biodegradable Micelles Mediate Enhanced Doxorubicin Delivery to Human Glioma Xenografts in vivo. J. Control. Release 2016, 233, 29-38. (60) Dai, J.; Lin, S.; Cheng, D.; Zou, S.; Shuai, X. Interlayer‐Crosslinked Micelle with Partially Hydrated Core Showing Reduction and pH Dual Sensitivity for Pinpointed Intracellular Drug Release. Angew. Chem. Int. Ed. 2011, 50 (40), 9404-9408. (61) Zou, Y.; Song, Y.; Yang, W.; Meng, F.; Liu, H.; Zhong, Z. Galactose-Installed Photo-Crosslinked pH-Sensitive Degradable Micelles for Active Targeting Chemotherapy of Hepatocellular Carcinoma in Mice. J. Control. Release 2014, 193, 154-161. (62) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Design of Environment ‐ Sensitive Supramolecular Assemblies for Intracellular Drug Delivery: Polymeric Micelles that Are Responsive to Intracellular pH Change. Angew. Chem. Int. Ed. 2003, 42 (38), 4640-4643.

23

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

1 2 3 4 5 6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic

24

ACS Paragon Plus Environment

Page 24 of 24