Highly Stable Fluorinated Nanocarriers with iRGD for Overcoming the

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Highly stable fluorinated nanocarriers with iRGD for overcoming the stability dilemma and enhancing tumor penetration in an orthotopic breast cancer Shengnan Ma, Jie Zhou, Yuxin Zhang, Yiyan He, Qian Jiang, Dong Yue, Xianghui Xu, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09633 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 13, 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 34

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

Highly stable fluorinated nanocarriers with iRGD for overcoming the stability dilemma and enhancing tumor penetration in an orthotopic breast cancer

Shengnan Ma†, Jie Zhou†, Yuxin Zhang†, Yiyan He*†, Qian Jiang†, Dong Yue†, Xianghui Xu†, and Zhongwei Gu*†‡ †

National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610065, P. R. China. ‡

College of Materials Science and Engineering, Nanjing Tech University, 30 South Puzhu Road, Nanjing 211816, P. R. China. *E-mail: [email protected] (Dr. Yiyan He), [email protected] & [email protected] (Prof. Zhongwei Gu)

KEYWORDS: drug delivery, self-assembly nanocarriers, stability dilemma, tumor penetration, orthotopic breast cancer

1 ACS Paragon Plus Environment


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

ABSTRACT The stability dilemma and limited tumor penetration of nanocarriers in cancer chemotherapy remain two predominant challenges for their successful clinical translation. Herein, the pHsensitive fluorocarbon functionalized nanocarriers (SFNs) with a tumor homing and penetrating peptide iRGD are reported to overcome the stability dilemma and enhance tumor accumulation and penetration in an orthotopic breast cancer. The highly stable SFNs with a low critical association concentration provide a safe and spacious harbor for hydrophobic drugs. Furthermore, the stimulus-responsive evaluation and in vitro drug release study show that the SFNs can balance the intracellular dissociation for drug release and the extracellular stability in the blood circulation. Additionally, the tumor penetration capacity has been dramatically enhanced in 3D multicellular spheroids, effectively affecting cells far from the periphery. This can be ascribed to the co-administration of iRGD having the tumor penetrating ability and fluorocarbon chains having the good cell membrane permeability. The combination of SFNs and iRGD is a viable approach to assist drugs effectively accumulation in primary and metastasized tumor sites, significantly inhibiting the breast tumor growth and curbing lung and liver metastases in an orthotopic tumor bearing mouse model. Taken together, this pH-sensitive fluorinated nanosystem having the excellent stability and tumor accumulation and penetration properties paves the way to combat cancer.


Page 2 of 34

Page 3 of 34

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


1. INTRODUCTION Breast cancer is the most commonly diagnosed disease in middle-aged women in China,1 which is a primary cause of female cancer death, and usually accompanies with distant organ metastasis.2 Systemic chemotherapy based on nanocarriers with antineoplastic agents plays an increasingly important role in the treatment of both primary breast cancer and metastasis. Although considerable achievements have been gained, the therapeutic outcomes are still unable to meet the expectations of clinicians.3 One of the predominant challenges in the successful systemic chemotherapy is that the low stability4-6 of nanocarriers to resist the extensive dilution in the blood circulation,7, 8 which always gives rise to the dissociation of nanocarriers and the premature leakage of the encapsulated drugs. Moreover, the limited tumor penetration and distribution of traditional nanocarriers are another well-known hurdles for effective chemotherapy of many solid tumors. It can be attributable to the high interstitial fluid pressure, the heterogeneous vasculature and the dense interstitial matrix of tumors,9, 10 which make drugs more tough to penetrate into tumors than normal tissues, and lead to insufficient dose of anticancer drugs in the whole tumor.

Desirable nanocarriers encapsulated anticancer drugs should keep stable and avoid nonspecific interaction in the circulation, reach to the tumor site and deeply penetrate into the entire tumor, arousing a prospective biological response to the signals from target tumor cells and releasing their loads inside cells.11, 12 Prevalent attentions have been paid to address the issue of the stability dilemma by virtue of stimuli responsive strategy. Stimuli responsive designed nanocarriers can effectively balance the intracellular dissociation for drug release and the extracellular stability in the blood circulation.13-17 Moreover, the adjustment of driving forces, especially the hydrophobic effect of amphiphilic molecules,18 for the self-assembly nanocarriers is always used to improve the extracellular stability. Nevertheless, the hydrophobic forces from the hydrophobic hydrocarbon segments of the amphiphile remain 3 ACS Paragon Plus Environment


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 4 of 34

insufficient for the stability,19 thus these types of self-assembly nanocarriers are unable to withstand the extensive dilution in the blood circulation.

In addition, the tumor penetration is another precondition to achieve satisfying chemotherapy. Approaches on remodeling of the tumor microenvironment have been developed to cope with the tumor penetration problem, including the normalization of abnormal vessels,20 the decompression of blood vessels,21 and the degradation of the extracellular matrix.22 There are also some favorable solutions to combat the poor tumor penetration and diffusion by modulating the nature of the delivery systems.23-25 One promising strategy is to use multistage delivery systems based on size-controlled nanoparticles,26-29 which can switch their sizes from 100 or so nanometers to about 10 nm at tumor sites by stimuli, then penetrate deeply into the tumor. Another is the intra-intercellular transportation strategy,30,

31

which is that

nanoparticles released from dead tumor cells are able to infect neighboring cells. Although the reconstitution of tumor microenvironment and modulation the nature of delivery systems are both promising strategies for the improvement of tumor penetration ability, it remains a long and winding road to their clinical translation.

Perfluorinated carbon molecules have recently attracted considerable interest by virtue of their super-hydrophobicity or “fluorous effect”, a strong propensity to self-association, and the good membrane permeability as compared with hydrocarbon analogues.32,

33

Herein, we

report the pH-sensitive fluorocarbon functionalized nanocarriers (SFNs) generated from the peptide dendrons end-capped with fluorocarbon chains (FPD), which are attached to the hydrophilic dextran via an intracellularly cleavable hydrazone linkage. The inner cores of the self-assembly nanocarriers with characteristic properties, such as highly-branched architectures, the fluorous effect, and amplification effects of dendrons, provide a safe and spacious harbor for the hydrophobic anticancer agents. It is very interesting to note that 4 ACS Paragon Plus Environment

Page 5 of 34

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


fluorocarbons have poor miscibility with hydrocarbons, thus the highly stable self-assembly with the water repellent fluorocarbon hydrophobic layer and a low critical association concentration (CAC) are expected to endure the extensive dilution of the blood. In addition, because iRGD contains a tissue penetration element named CendR having the ability to mediate an active transport channel through tumor vessels even continue within the extravascular tumor tissue in a αvβ3 integrin and neuropilin-1 dependent manner,34,

35

the

predicament of insufficient tumor penetration can be addressed by co-administrating iRGD to the highly stable SFNs (Scheme 1). The stability and stimulus responsive properties of SFNs were systematically investigated, as compared with the pH-sensitive hydrocarbon functionalized nanocarriers (SHNs) and the insensitive fluorocarbon functionalized nanocarriers (IFNs). The accumulation and penetration abilities and therapeutic impact on the primary tumor growth and metastasis of SFNs were assessed in 3D multicellular spheroids and the orthotopic breast cancer.

2. MATERIALS AND METHODS 2.1. Materials Anhydrous methanol, dimethyl sulfoxide (DMSO) and hydrazine hydrate (NH2–NH2·H2O) were obtained from Asta Tech Pharmaceutical (Chengdu, China). Butyric anhydride and sodium borohydride were ordered from Aladdin® Ltd. The generation 2 poly (L-lysine) dendron (MeO-PD) and aldehyde-functionalized dextran (Dex-CHO) were synthesized as previously reported.36 iRGD was purchased from GL Biochem Ltd (Shanghai, China). The molecular structure, mass spectra and HPLC analysis of iRGD can be found in Figure S1. Doxorubicin hydrochloride (DOX·HCl) was purchased from Zhejiang Hisun Pharmaceutical. Dialysis membrane (MWCO = 100–500, 1000 and 3500 Da) were commercial available from Spectrum/Por (Houston, USA). Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) were from Life Technologies Corporation (Gibco®, USA). The 4T1 5 ACS Paragon Plus Environment


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

cells were obtained from Shanghai Institutes for Biological Sciences (China). A CellCounting Kit-8 (CCK-8) was commercially purchased from Dojindo Laboratories (Tokyo, Japan). PBS buffer and other buffers were prepared in Milli-Q® ultrapure water, and all the other chemicals were purchased from Aldrich and used without purification. 2.2. Synthesis of hydrazine modified peptide dendron The generation 2 poly (L-lysine) dendron (MeO-PD, 2 mmol) was dissolved in 30 mL methanol under nitrogen atmosphere. Then, the excess amount of TEA (17 mL) and butyric anhydride (1.6 mL) were added under magnetic stirring and reacted for 48 h at 30 °C. The yellow liquid (MeO-HPD) was obtained after removing the solvents. The product was confirmed using Mass Spectrum (MS). 5 mL methanol solution of MeO-HPD (0.2 mmol) and hydrazine hydrate (6 mmol, 500 µL) were added into a glass flask under nitrogen atmosphere, and the solution was reacted for 2 days at 40 °C. To remove the excess hydrazine hydrate, the solution was packaged into a dialysis bag (MWCO = 100-500 Da) and dialyzed exhaustively against water. After centrifuging and drying, the white solid of hydrazine modified alkylated peptide dendron (Hydrazine-HPD) was obtained and further confirmed using Mass Spectrum (MS). The synthesis of hydrazine modified fluorinated peptide dendron (Hydrazine-FPD) was similar to that of Hydrazine-HPD. 2.3. Synthesis of pH-sensitive peptide dendron functionalized dextran The aldehyde-functionalized dextran (Dex-CHO) with different oxidation degree was synthesized according to our previously reported method. For the synthesis of the pH sensitive fluorinated or alkylated peptide dendron functionalized dextran (FPD-HZN-Dex or HPD-HZN-Dex), the Hydrazine-FPD or the Hydrazine-HPD was conjugated to the Dex-CHO via hydrazone bonds, respectively. In detail, the molar ratio of hydrazine groups in Hydrazine-FPD or Hydrazine-HPD to the aldehyde groups of oxidized dextran (oxidization 67.2%) was 1:1, and both chemicals were dissolved in 10 mL DMSO under a nitrogen environment. Then, the solution was stirred at 40 °C for 48 h in the dark and further purified 6 ACS Paragon Plus Environment

Page 6 of 34

Page 7 of 34

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


using

dialysis

method.

The

obtained

product

was

confirmed

using

Differential Scanning Calorimeter (DSC) and Fourier Transform Infrared Spectroscopy (FTIR). 2.4. Synthesis of pH-insensitive peptide dendron functionalized dextran The pH-insensitive fluorinated peptide dendron functionalized dextran (FPD-Dex) was obtained through the reduction of hydrazone bonds in FPD-HZN-Dex. FPD-HZN-Dex was dissolved in 10 mL DMSO, then NaBH4 (18 mg) was added to the DMSO solution. The reduction reaction was performed for 24 h at room temperature. The product was then purified via dialysis and confirmed using DSC and FTIR. 2.5. Preparation of DOX-encapsulated nanocarriers The SFNs, SHNs and IFNs are self-assembled from the amphiphilic FPD-HZN-Dex, HPDHZN-Dex and FPD-Dex, respectively. The blank nanocarriers were prepared using dialysis method. Briefly, 10 mg freeze-dried amphiphilic FPD-HZN-Dex, HPD-HZN-Dex and FPDDex were dissolved in 1.5 mL DMSO, and dispersed into 10 mL deionized water under ultrasound, respectively. After stirred for 2 h, the mixture solution was dialyzed using dialysis tube (MWCO = 1000 Da) against deionized water for 1 day. The drug-loaded nanoparticles were prepared in the similar method. 10 mg FPD-HZN-Dex, HPD-HZN-Dex and FPD-Dex and 2.5 mg DOX co-dissolved in 2 mL DMSO were dispersed slowly into 10 mL water under ultrasound in the dark, which was stirred for another 2 h. Then, they were dialyzed against deionized water at 4 °C for 24 h and freeze-dried subsequently. The drug loading content (LC) and encapsulation efficiency (EE) of obtained SFNs/DOX, SHNs/DOX and IFNs/DOX were calculated according to the following equations: LC (%) = (weight of loaded DOX /weight of drug loaded nanoparticles) × 100% EE (%) = (weight of loaded DOX/weight of DOX in feeding) × 100%



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

2.6. Determination of CAC The self-assembly of amphiphilic FPD-HZN-Dex and HPD-HZN-Dex were investigated in aqueous medium. The CAC of FPD-HZN-Dex and HPD-HZN-Dex were estimated through fluorescence spectroscopy using pyrene as a probe. The pyrene solutions in acetone (1.2 × 106

M, 1 mL) were added to 10 mL volumetric flasks, and the acetone was allowed to evaporate

under vacuum. Then, 2 mL of FPD-HZN-Dex or HPD-HZN-Dex solutions with different concentrations ranging from 0.1 to 0.0001 mg/mL were added to the vials, and the final pyrene concentration is 6 × 10-7 M. The solutions were equilibrated at 60 °C for 1 h and kept in the dark place for 1 day at room temperature. The excitation spectra were recorded from 300 to 360 nm with an emission wavelength of 390 nm, and both excitation and emission bandwidths were set at 5 nm. The intensity ratios of I338 to I334 were plotted as a function of logarithm of FPD-HZN-Dex or HPD-HZN-Dex concentration. 2.7. Physicochemical characterization of nanocarriers In order to demonstrate the superiority of SFNs/DOX in storage stability, the size distribution of SFNs/DOX and SHNs/DOX were measured using dynamic light scattering (DLS). After the nanocarriers were stored for 1 week at 4 °C, DLS measurements were performed at 25 °C to record their size distributions, which was used to compare the storage stability of SFNs/DOX and SHNs/DOX. To investigate the responsiveness of SFNs/DOX in acidic lysosomal condition, DLS and TEM were performed to confirm the size and morphology of SFNs/DOX and IFNs/DOX in the physiological condition (pH 7.4) and in acidic lysosomal environment (pH 5.0), respectively. Moreover, the dilution stability of DOX loaded SFNs and SHNs was investigated through incubating them in PBS buffer (pH 7.4) in 1 to 1000 folds dilutions at room temperature. Changes in particle size were recorded. 2.8. In vitro drug release The release profile of DOX-encapsulated nanocarriers at different pH (pH 5.0, 6.8 and 7.4) was carried out using dialysis method. The freeze-dried SFNs/DOX and SFNs/DOX/iRGD 8 ACS Paragon Plus Environment

Page 8 of 34

Page 9 of 34

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


were immersed into 25 mL of PBS buffer at a final concentration of 1 mg/mL, then the solutions were poured into dialysis tubes (MWCO = 1000 Da) and maintained at 37.0 ± 0.5 °C in a shaking bed at 120 rpm. The medium (1 mL) was taken out and replaced with an equal quantity of fresh corresponding PBS medium at designated intervals. The amount of released DOX was determined using fluorescence measurement (F-7000) with 480 nm excitation and 557 nm emission. 2.9. In vitro cytotoxicity and antitumor efficacy The biocompatibility of blank SHNs, IFNs, and SFNs, as well as SFNs/iRGD was evaluated using CCK-8 assays. In brief, 4T1 cells were seeded in 96-well plates at 5× 103 cells per well in 100 µL of culture medium and allowed to attach for 24 h. Then blank SHNs, IFNs and SFNs as well as SFNs/iRGD with 0.5 mM iRGD were added with different concentrations from 0.01 to 200 µg/mL, and incubated for another 24 h. Then, the culture medium was removed and the wells were washed with PBS (pH 7.4) for thrice. Subsequently, 100 µL of the fresh media containing 10 µL CCK-8 was added to the wells, and the plates were incubated for an additional 2 h in the dark. Finally, the absorbance was measured at 450 nm using a microplate reader. In order to study the antitumor efficacy of DOX encapsulated nanocarriers, the nanocarriers (SFNs/DOX, SFNs/DOX/iRGD, SHNs/DOX and IFNs/DOX) were dissolved in culture medium with the final DOX concentrations from 0.0001 to 100 µg/mL. After the cells (5 × 103 per well) were seeded in the 96-well plates, the culture medium was replaced with 100 µL of medium containing the drug encapsulated nanocarriers. The cancer cells were incubated for 24 h and measured by CCK-8 assays. 2.10. Cellular uptake 4T1 cells were seeded in 35 mm confocal dishes (Φ = 12 mm) at a density of 1× 104 cells per well and incubated for 24 h. The medium was then replaced with fresh culture media containing DOX encapsulated nanocarriers (DOX concentration: 10 µg/mL). The medium of 9 ACS Paragon Plus Environment


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

SFNs/DOX was added 0.5 mM free iRGD additionally. After being incubated for 0.5 hour, the medium was removed and washed thrice with ice-cold PBS. The cells were observed using confocal laser scanning microscopy. To quantitatively analyze the uptake of nanocarriers, cells were harvested through trypsin treatment, centrifuged at 1000 rpm for 3 min and washed with PBS for another time. Finally, the cells were re-suspended in 0.5 mL PBS and analyzed using flow cytometry. 2.11. DOX penetration into multicellular 4T1 tumor spheroids The agarose solution (1%, w/v) was prepared in PBS and sterilized for 20 min. Then, coating the plate well with 1.5 mL agarose solution for every well, 4T1 cells (2× 105 cells/well) were seeded in 6-well plates in 1.5 mL of culture medium with 10% FBS. After cultivation for 4 days in humidified incubator, the diameter of tumor spheroids was about 150 µm, which were transferred to the glass bottom plate and treated with DOX and DOX encapsulated nanocarriers respectively (DOX: 10 µg/mL). After incubation for 3 h in humidified incubator, the culture medium with drug solution was taken out and washed with PBS twice, which was suspended in 100 µL PBS at last. Confocal laser scanning microscopy was used to observe the penetration of DOX encapsulated nanocarriers into 4T1 tumor spheroids by serial bright field and fluorescence sections. 2.12. In vivo study Female BALB/c nude mice weighting 16~20 g were obtained from Chengdu Dashuo Biological Technology Co. Ltd. (China). Murine mammary carcinoma 4T1 cells were used for the establishment of orthotopic breast cancer models.37 The orthotopic cancer models were established by injecting 1 × 106 4T1 cells into the right mammary fat pad of the female BALB/c nude mice. All animal procedures and experiments were conformed to the ethics of our institutional and NIH guidelines for care and use of research animals. To evaluate the in vivo biodistribution and tumor accumulation, a near-infrared fluorescence dye, the hydrophobic DiR encapsulated nanocarriers (SHNs/DiR, SFNs/DiR and 10 ACS Paragon Plus Environment

Page 10 of 34

Page 11 of 34

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


SFNs/DiR/iRGD) were used. Mice bearing an orthotopic model of breast cancer metastasis to lungs were intravenously injected with saline, free DiR, SHNs/DiR, SFNs/DiR and SFNs/DiR/iRGD at a dose of 40 µg DiR per kg body weight. Fluorescent signals were recorded at 0.5 h and 2 h post-administration using an in vivo fluorescence imaging system (CRi Maestro EX, USA) with excitation at 745 nm and emission at 780 nm. Mice were sacrificed at 2 h post-injection and the hearts, lungs and tumors were excised to evaluate the tissue distribution. When the tumor volume reached about 80 mm3, tumor-bearing BALB/c nude mice were randomly divided into five groups with 8 mice in each group. Mice were intravenously injected with saline, DOX.HCl, blank SFNs,SFNs/DOX and SFNs/DOX/iRGD (4 µmol/kg iRGD) respectively at a dose of 5 mg DOX per kilogram body weight every 3 days for 4 treatments. During the treatment, the tumor volume (length × width2 × 0.5) and body weight were measured every two days for 14 days. After treatment for 14 days, all mice were sacrificed. Tumors and major organs were dissected from the mice and fixed in 4% (v/v) formalin saline for 1 day. Tissues were embedded in paraffin and cut at 5 µm thickness. The sections were stained using hematoxylin and eosin (H&E) for histopathological evaluation. Platelet endothelial cell adhesion molecule1 (CD31) test was carried out to research vessels in the tumor tissues. Monoclonal antibody Ki-67 response was used to investigate the proliferation activity of tumor cells. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay was conducted to examine the apoptosis level of tumor cells. 2.13. Statistical analysis All experiments were repeated at least six independent samples and each measurement were performed in triplicates. Average results are expressed as mean values ± standard deviation. The statistical differences between treatments groups were determined using two-way analysis of variance. In all tests, P values ≤ 0.05 were considered statistically significant. 11 ACS Paragon Plus Environment


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

3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of SFNs The pH-sensitive fluorocarbon functionalized nanocarriers (SFNs) are self-assembled from amphiphilic FPD-HZN-Dex polymer. The generation 2 poly (L-lysine) dendron (MeO-PD) and the aldehyde-functionalized dextran (Dex-CHO) were prepared as previously reported.36 The pH-sensitive amphiphilic FPD-HZN-Dex polymer bears hydrophilic dextran and four super-hydrophobic fluorocarbon chains functionalized peptide dendrons bridged via intracellularly cleavable hydrazone linkages (Scheme 1). For comparison, we also prepared the insensitive fluorocarbon functionalized nanocarriers (IFNs) and the pH-sensitive hydrocarbon functionalized nanocarriers (SHNs) as negative controls. The insensitive amphiphilic FPD-Dex and the pH-sensitive amphiphilic HPD-HZN-Dex containing dendrons end-capped with hydrocarbon analogues were synthesized using similar methods as illustrated in Figure 1.

Scheme 1. The highly stable SFNs combined with iRGD for overcoming the stability dilemma and enhancing tumor penetration in an orthotopic breast cancer.


Page 12 of 34

Page 13 of 34

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


Figure 1. (A) The synthetic routes to Hydrazine-FPD and Hydrazine-HPD. (B) The reaction schemes of the amphiphilic FPD-HZN-Dex, HPD-HZN-Dex and FPD-Dex polymers.

The MeO-FPD or MeO-HPD was obtained through amidation reaction between MeO-PD and heptafluorobutyric anhydride or butyric anhydride. The resulting MeO-FPD or MeO-HPD was further coupled with an excess hydrazine hydrate to gain Hydrazine-FPD or HydrazineHPD, as evidenced using mass spectra (MS) (Figure 2A and Figure S2). The most abundant peaks at m/z = 1223.12 and 695.28 were corresponding to the [M + Na]+ signal from Hydrazine-FPD and [M - H]- signal from Hydrazine-HPD, respectively. The MS data also confirmed that the desired four fluorocarbon or hydrocarbon chains have been successfully attached to the dendron. The pH-sensitive amphiphilic FPD-HZN-Dex or HPD-HZN-Dex polymers were prepared through conjugation of Hydrazine-FPD or Hydrazine-HPD to Dex13 ACS Paragon Plus Environment


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

CHO using hydrazone bonds. Meanwhile, the insensitive FPD-Dex was obtained via the reduction of hydrazone bonds in FPD-HZN-Dex with sodium borohydride. The degree of substitution (DS) of dendrons at dextran was optimized to the similar level, as revealed by the elemental analysis in Table 1.

Table 1. Elemental analysis the degree of substitution (DS) of amphiphilic polymers. DS (%) FPD-HZN-Dex

34.07 ± 3.23

HPD-HZN-Dex

39.24 ± 1.82

FPD-Dex

33.24 ± 3.45

Comparing the DSC of all polymers in Figure 2B, no obvious thermomechanical behavior of the HPD-HZN-Dex, Dex-CHO and dextran could be found in the temperature range from 25 to 200℃. However, there were two peaks at 133℃ and 137℃ in both FPD-HZN-Dex and FPD-Dex curves, respectively, which may be due to the unique properties of the fluorinated dendrons. The structures of FPD-HZN-Dex, FPD-Dex and HPD-HZN-Dex were further confirmed using FTIR analysis in Figure 2C. Dex-CHO was successfully prepared as the evidence of 1733 cm-1 for the aldehyde groups. For FPD-HZN-Dex and FPD-Dex curves, the strong signals at 1702 and 1666 cm-1 were from carbonyl groups, while 1353 and 1231 cm-1 came from the CF3 and CF2 groups owing to the fluorinated dendrons. As compared with FPD-HZN-Dex, there was no peak at 1624 cm-1 in the spectrum of FPD-Dex, because of the reduction of hydrazone bonds. As to the IR spectrum of HPD-HZN-Dex, the peak at 2961 cm1

was the stretching vibration of -CH3 in the alkylated dendron, and just one strong signal

appeared at 1649 cm-1 corresponding to the carbonyl groups (C=O) in the alkylated dendrons. Therefore, the FPD-HZN-Dex, FPD-Dex and HPD-HZN-Dex were successfully synthesized, as demonstrated in the FTIR spectra.


Page 14 of 34

Page 15 of 34

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


Figure 2. (A) Mass spectra of Hydrazine-FPD and Hydrazine-HPD. (B) DSC analysis and (C) FTIR spectra of (a) Dextran, (b) Dex-CHO, (c) FPD-HZN-Dex, (d) HPD-HZN-Dex and (e) FPD-Dex.

3.2. Stability and stimulus responsive properties of SFNs/DOX SFNs self-assembled from the amphiphilic FPD-HZN-Dex polymer in aqueous medium. The CAC value is one of the major thermodynamic constants determining the stability of the selfassemblies.38-40 Thus, the stability of the so-formed SFNs was assessed by the determination of CAC using pyrene as a fluorescent probe. As presented in Figure 3A, the CAC value of the SFNs at room temperature was 4.73 µg/mL, which was lower than that of SHNs (52.02 µg/mL). This result implies that the SFNs with fluorocarbon chains could be formed at a lower polymer concentration than the SHNs consisting of hydrocarbon analogues. The ultralow CAC of SFNs could be reasonably attributable to the inherent super-hydrophobicity or “fluorous effect” of fluorocarbons and amplification effects of dendrons. 15 ACS Paragon Plus Environment


When DOX-loaded SFNs and SHNs are injected, the concentration of nanocarriers will be diluted by the blood, causing the dissociation of self-assemblies. Thus, we investigated the stability of DOX-loaded SFNs and SHNs against dilution (Figure 3B). The dilution of SHNs/DOX resulted in significantly increased particle size and disassembly in the solution. No obvious change in particle size was detected for SFNs/DOX, even after 1000-fold dilution. The reason was that the SHNs/DOX concentration was 10 µg/mL when diluted to 1000 folds, which is below the CAC value (52.02 µg/mL) of the amphiphilic HPD-HZN-Dex polymer. It is important to mention that the water- and oil-repellency of hydrophobic inner cores and the ultra-low CAC of the SFNs may be beneficial to the extracellular stability and endure the extensive dilution of the blood after intravenous injection, thus leading to good thermodynamic stability in circulation.

0.95

(a) SFNs

0.90

I338/I334

I338/I334

A 0.95

CAC: 4.73 µg/mL

-2

0.90 CAC: 52.02 µg/mL

-1 0 1 Log C (µg/mL)

2

-2

3

C 30

B 700

SFNs/DOX SHNs/DOX

25 Number (%)

600 500 400 300 200

-1 0 1 Log C (µg/mL)

SFNs/DOX SHNs/DOX

2

3

SFNs/DOX post-storage SHNs/DOX post-storage

20 15 10 5

100 0

(b) SHNs

0.85

0.85

Particle Size (nm)

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 34

1

10 100 Dilution Folds

1000

0 101

102 103 Size (d.nm)

104

Figure 3. (A) The critical association concentration of (a) SFNs and (b) SHNs measured using the fluorescent dye pyrene. (B) Particle size of SFNs/DOX and SHNs/DOX diluted with PBS (pH 7.4). (C) Size distribution of SFNs/DOX and SHNs/DOX after storage for 1 week. In addition, the highly-branched architecture of peptide dendrons end-capped with four fluorocarbon tails enable SFNs to provide a safe and spacious harbor for the hydrophobic drugs. These amphiphiles and the hydrophobic anticancer drug DOX self-assembled into the core-shell nanocarriers in aqueous medium. Also of note is that, SFNs/DOX still maintained a 16 ACS Paragon Plus Environment

Page 17 of 34

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


narrow size distribution and dispersed well in PBS buffer (pH 7.4) after 1 week storage (Figure 3C), whereas SHNs/DOX cannot keep its initial state. These phenomena demonstrated the superiority of SFNs/DOX in stability. Meanwhile, the drug loading content and the encapsulation efficiency of SFNs/DOX, IFNs/DOX, and SHNs/DOX were further investigated and shown in Table 2. The drug loading content and encapsulation efficiency of SFNs were 20.42 ± 3.11% and 52.73 ± 1.32%, respectively. The drug loading content of all nanocarriers was adjusted to the same level of 10% for the following studies.

Table 2. Drug loading content (LC) and encapsulation efficiency (EE) of nanocarriers. LC (%)

EE (%)

SFNs/DOX

20.42 ± 3.11

52.73 ± 1.32

IFNs/DOX

20.09 ± 2.83

50.10 ± 2.49

SHNs/DOX

10.60 ± 3.37

30.30 ± 1.98

SFNs/DOX was spherical in shape with 110 ± 11 nm diameter (polydispersity index: 0.18). The insensitive IFNs/DOX was prepared in identical conditions with the similar size and morphology to the SFNs/DOX in PBS buffer (pH 7.4), as demonstrated using DLS and TEM (Figure 4a and b). The stability dilemma of drug delivery systems involves the extracellular stability in the blood circulation and intracellular dissociation for drug release. To verify the on-demand intracellular dissociation ability of the SFNs/DOX, both SFNs/DOX and IFNs/DOX were incubated in PBS buffer at pH 5.0 and monitored using DLS and TEM. As expected, the size distribution of SFNs/DOX exhibited an obvious change from narrow to wide, and the SFNs/DOX turned into irregular aggregates, revealing the endo/lysosome pH triggered dissociation ability of SFNs/DOX (Figure 4c). By contrast, there were no significant changes in the size and morphology of IFNs/DOX formulation in the acid condition (Figure 4d). Considering the circulation stability and acid responsive behavior of



the SFNs/DOX, one can conclude that the stimuli responsive designed SFNs/DOX could

a 30

b 30

Number (%)

Number (%)

definitely beneficially balance the extracellular stability and intracellular dissociation.

20 10 2

10

3

10

Size (d.nm)

10

10 0 1 10

4

c 30

d 30

Number (%)

0 1 10

pH 7.4

20

Number (%)

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 34

20 10 0 1 10

pH 5.0 10

2

10

3

Size (d.nm)

4

10

pH 7.4 2

10

3

10

Size (d.nm)

4

10

20 10 0 1 10

pH 5.0 2

10

3

10

Size (d.nm)

4

10

Figure 4. Size distribution and TEM images of SFNs/DOX (a and c) and IFNs/DOX (b and d) at pH 7.4 and pH 5.0, respectively. 3.3. In vitro drug release SFNs/DOX generated from the amphiphilic FPD-HZN-Dex polymer containing hydrazone linkages, it should show the endo/lysosomal pH-sensitive DOX release. We thus investigated the in vitro drug release profiles of pH-sensitive SFNs/DOX and the iRGD optimized formulation SFNs/DOX/iRGD in the neutral physiological pH 7.4, the tumor extracellular pH 6.8 and the endo/lysosome pH 5.0. As displayed in Figure S3 and Figure 5A, the SFNs/DOX/iRGD in PBS buffer (pH 7.4) have brought about approximately 35% of DOX released in 1 day. In contrast, after 1 day incubation at endo/lysosomal pH 5.0, about 80% of DOX were released from the SFNs/DOX/iRGD. The cleavage of hydrazone linkages of SFNs and the protonation of DOX at pH 5.0 may facilitate the drug release. The endo/lysosomal pH triggering drug release behavior will endow the SFNs/DOX/iRGD with a preferential drug release profile inside cancer cells , whereas keeping stable during extracellular transport.


Page 19 of 34

A Cumulative release (%)

80 70

**

60 50 40 30 20

SFNs/DOX/iRGD pH 5.0 SFNs/DOX/iRGD pH 6.8 SFNs/DOX/iRGD pH 7.4

10 0 0

2

4

6

8

10 12 14 16 18 20 22 24

Time (h)

B 120

SHNs SFNs

IFNs SFNs/iRGD

Cell Viability (%)

100 80 60 40 20 0 0.01

0.1

1

10

5

50

100

200

Concentration (µg/mL) C 100

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


80

60

40

DOX SHNs/DOX IFNs/DOX SFNs/DOX SFNs/DOX/iRGD DOX.HCl

20

0 -4

10

-3

10

10

-2

-1

10

10

0

10

1

2

10

Dox Concentration (µg/mL)

Figure 5. (A) In vitro drug-release profile of SFNs/DOX/iRGD in different pH conditions (pH 7.4, 6.8 and 5.0) at 37°C. (B) The cell viability of blank nanocarriers on 4T1 cells. (C) In vitro anticancer efficacy on 4T1 cells.

3.4. In vitro anticancer efficacy of SFNs/DOX/iRGD Encouraged by the on-demand intracellular drug release behavior of SFNs/DOX/iRGD and the tumor homing and penetrating abilities of iRGD, we went on to the assessment of the in

vitro cytotoxicity of blank nanocarriers and the anticancer efficacy of iRGD involved 19 ACS Paragon Plus Environment


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

SFNs/DOX nanocarriers in 4T1 mammary breast cancer cells. As presented in Figure 5B, the blank nanocarriers including SFNs, SHNs, and IFNs, as well as SFNs/iRGD showed the high viability (>80%) against 4T1 cells, demonstrating the negligible cytotoxicity of these formulations. SFNs/DOX/iRGD with 0.5 mM iRGD, SFNs/DOX, SHNs/DOX and IFNs/DOX formulations showed the similar tendency as DOX·HCl group at the DOX concentrations from 0.0001 to 100 µg/mL. Specifically, the IC50 value of SFNs/DOX/iRGD against 4T1 cells was 1.45 µg/mL (Figure 5C), which was 1.52 folds and 4.54 folds lower than that of SFNs/DOX and free DOX, respectively. These findings obviously confirmed that co-administration of iRGD to SFNs/DOX could improve the anticancer efficacy.

3.5. Cellular uptake of SFNs/DOX/iRGD To explore the mechanism underlying the improved anticancer efficacy of SFNs/DOX/iRGD, we investigated the intracellular uptake of the SFNs/DOX/iRGD in 4T1 cells using confocal laser scanning microscopy (CLSM) and flow cytometry. Observably, the strong red fluorescence from the SFNs/DOX group was observed in cells after incubation 0.5 h. The red signal from the SFNs/DOX/iRGD group was much stronger than that from SFNs/DOX group (Figure 6A). Flow cytometry also showed that SFNs/DOX/iRGD drastically enhanced the internalization of DOX into 4T1 cells within 0.5 h (Figure 6B). The internalization of SFNs/DOX/iRGD, SFNs/DOX and IFNs/DOX was much faster and more effective than that of SHNs/DOX. These results can be attributable to the synergistic effect of the fluorocarbon chains having good membrane permeability and iRGD having tumor-homing ability, which could facilitate the efficiency of drug delivery system. The fast and effective internalization profile is also a key factor for in vivo application, because the contact between target cells and nanocarriers is usually transient.


Page 20 of 34

Page 21 of 34

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


Figure 6. Cellular uptake of DOX-loaded nanocarriers after incubation with 4T1 cells in the medium containing 10% FBS for 0.5 h (DOX: 10 µg/mL). (A) Confocal laser scanning microscopy images, overlay of DOX fluorescence channel over bright field (Row 1), fluorescence images (Row 2). Scale bars are 10 µm. (B) Flow cytometry analysis.

3.6. Penetration in multicellular spheroids The traditional nanocarriers with poor tumor penetration can give rise to a portion of tumor cells with insufficient drug doses, increasing the risk of not only drug resistance but also tumor recurrence. Recent studies have revealed that co-administrating a cyclic tumor penetrating peptide, iRGD, to nanocarriers can improve the tumor penetration capacity. The good cell membrane permeability of fluorocarbon modified peptide dendrons and the superiority of iRGD motivated us to evaluate the potential of SFNs/DOX/iRGD for enhancing penetration. In this study, 4T1 tumor spheroids were used as a 3D tumor model, which could in part simulate the intracellular drug delivery for tumor in vivo. Thanks to the red fluorescence of DOX, we could distinguish the penetration efficiency from the intensity of the red signal. Tumor spheroids were incubated with SFNs/DOX/iRGD, SFNs/DOX, and SHNs/DOX, as well as DOX for 3 h, respectively. Then, the penetration ability was assessed using CLSM with Z-stacking scanning (Figure 7). The red signal from SFNs/DOX group was much stronger than both SHNs/DOX and DOX groups. The slight red fluorescence of SHNs/DOX and DOX groups had mostly located on the periphery of tumor spheroids through serial sections. It is important to mention that the fluorocarbon functionalized nanocarriers are particularly beneficial for penetration in tissues because the fluorocarbon functionalized peptide dendrons can effectively facilitate the permeability of cell membrane. The fluorescent 21 ACS Paragon Plus Environment


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

intensity of SFNs/DOX/iRGD inside the tumor spheroids was much stronger than that of SFNs/DOX group at each scanning depth, effectively affecting the cells far from the periphery, demonstrating the deep penetration and uniform distribution of SFNs/DOX/iRGD — evidence that could indeed be ascribed to the joint contribution of a tumor penetrating peptide iRGD and the good membrane permeability of fluorocarbon.


Page 22 of 34

Page 23 of 34

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


Figure 7. Drug Penetration of DOX-loaded nanocarriers into 3D-cultured 4T1 tumor spheroids was observed using confocal laser scanning microscopy after incubating 3 h (DOX: 10 µg/mL, scale bar = 50 µm). The images of the spheroids were taken from the bottom to the middle per 10 µm. Overlay of DOX fluorescence channel over bright field (Row 1), fluorescence images (Row 2).



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

3.7. In vivo biodistribution There is a close relationship between the stability of self-assemblies and their biodistribution performance in vivo. To further verify the stability and tumor accumulation properties of SFNs, we monitored the biodistribution of DiR encapsulated nanocarriersusing a BABL/c nude mice model bearing the orthotopic breast cancer, which tends to spread from the primary site in mammary gland to lungs.41 Lung metastases were detected and showed in Figure S4. Mice were then administrated intravenously with saline, free DiR, SHNs/DiR, SFNs/DiR and SFNs/DiR/iRGD formulations. In vivo biodistribution exhibited that the fluorescent signals from SFNs/DiR and SFNs/DiR/iRGD groups were concentrated at tumor sites, whereas the negligible fluorescent signals were detected in free DiR and SHNs/DiR groups, indicating the poor stability and fast clearance of SHNs/DiR. In addition, the fluorescent intensity increased at 2 h post-administration (Figure 8A). The ex vivo fluorescence images further confirmed that the fluorescent pixels can be detected not only in the primary tumor site but also in lungs with metastases lesions (Figure 8B). Specifically, when combined with iRGD, SFNs/DiR/iRGD group showed the increased fluorescent intensity at tumor and lungs compared to SFNs/DiR group. The further quantitative analysis in Figure 8C showed that the fluorescence at tumor from SFNs/DiR/iRGD group was 2.2-fold and 3.0-fold stronger than that of SFNs/DiR group and SHNs/DiR group at 2 h post-injection. Moreover, the fluorescence in lungs from SFNs/DiR/iRGD group was also superior to that of SFNs/DiR and SHNs/DiR. Indeed, these phenomena remarkably demonstrated that the SFNs/DiR/iRGD group is able to effectively accumulate at tumor site and lungs with metastases lesions, which can be ascribed to the synergism of the excellent stability of SFNs and the enhanced permeability and retention (EPR) effect, as well as the tumor homing and penetrating peptide iRGD. The EPR effect is considered to be a crucial factor for tumor-targeting.42 It was expected that the increased stability of SFNs in the blood circulation allows nanocarriers to accumulate at 24 ACS Paragon Plus Environment

Page 24 of 34

Page 25 of 34

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


the primary and metastasized tumor sites in orthotopic breast cancer model after an intravenous injection through the EPR effect. Lungs contain linear blood vessels with abundant blood flow. Moreover, the leakage and high permeability of the blood vessels in solid tumors could be more beneficial for the accumulation in lung metastases. Importantly, iRGD could activate a transport channel that allows SFNs/DiR extravasated from vessels to penetrate into the tumor sites in a αvβ3 integrin and neuropilin-1 dependent manner.34 Therefore, a combination of SFNs and iRGD could remarkably improve the accumulation not only in the primary tumor but also in lung metastases. These results also confirmed that SFNs/DiR/iRGD could be used for the orthotopic breast cancer with metastasis formed at other organs.

Figure 8. In vivo biodistribution of DiR encapsulated nanocarriers (a) saline, b) DiR, c) SHNs/DiR, d) SFNs/DiR and e) SFNs/DiR/iRGD) through intravenous injection in the lung metastasis orthotopic breast cancer model. (A) Fluorescence images of tumor accumulation at 0.5 h and 2 h post-administration, (B) Ex vivo fluorescence images of tissues at 2 h postadministration, (C) Quantitative analysis of the fluorescence intensity of tissues at 2 h postadministration (**P < 0.01). White circles indicate the position of tumors.



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

3.8. In vivo anticancer efficacy and anti-metastasis activity Encouraged by the excellent in vitro performance of SFNs/DOX/iRGD, including the improvement of cellular uptake and penetration, in vitro antitumor efficacy, on-demand the intracellular dissociation and stable in physiologic condition, as well as the good biocompatibility, we performed in vivo tumor therapy through the evaluation of the tumor growth and metastasis suppression in BABL/C nude mice bearing orthotopic breast cancers. Because the tumor growth, metastasis, and sensitivity to DOX are tightly linked to the environments of orthotopic and ectopic organ.43 Therefore, the orthotopic breast cancer models were employed to study the tumor growth and metastasis in this study. Saline, SFNs/DOX, SFNs/DOX/iRGD with 4 µmol/kg free iRGD, and DOX·HCl, as well as blank SFNs/iRGD were intravenously injected into mice bearing orthotopic breast cancers, respectively.


Page 26 of 34

C 100

B 25 20 **

15

Saline SFNs/DOX/iRGD SFNs/DOX DOX.HCl SFNs/iRGD

10 5

Survival Rate (%)

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


Body Weight (g)

Page 27 of 34

80 60 Saline SFNs/DOX/iRGD SFNs/DOX DOX.HCl SFNs/iRGD

40 20 0

0

2 4 6 8 10 Days Post Treatment

12

0

2 4 6 8 10 Days Post Treatment

12

Figure 9. In vivo anticancer efficacy and anti-metastasis activity against orthotopic breast cancer. (A) Tumor volume, (B) Body weight, (C) Mice survival rate, (D) Immunohistochemical analysis for tumor tissues were investigated using H&E, CD31, Ki-67, and TUNEL assays, and (E) Histological analysis of heart, liver and lungs after treatment. 27 ACS Paragon Plus Environment


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 28 of 34

To evaluate the in vivo antitumor efficacy and toxicity, we measured the tumor volume and the body weight every 2 days during the treatment, as well as analyzed histological and immunohistochemical patterns after the treatment (Figure 9). After the entire treatment period, the tumor volume of the saline group and the blank SFNs/iRGD group increased to 5fold than the initial volume at the first treatment (Figure 9A), indicating that the blank SFNs/iRGD alone did not have any therapeutic effect. Whereas all the mice administrated with DOX-related groups exhibited slower tumor growth than the saline administrated mice. The moderate inhibition on tumor growth was observed at mice treated with DOX·HCl. Notably, the treatment with SFNs/DOX/iRGD formulations showed remarkably better anticancer activity than other formulations in terms of suppressing about 80% of the aggressive tumors growth. It should be noted that the anticancer efficacy of co-administrating iRGD to SFNs/DOX formulations was much better than that of SFNs/DOX used alone, and SFNs/DOX group showed certain anticancer efficacy about 50% inhibition in the tumor growth compared with the saline treated mice. Additionally, all mice treated with saline, blank SFNs/iRGD, SFNs/DOX, and SFNs/DOX/iRGD did not show any abnormal behavior during therapy, and the body weight of these mice showed slight increase (Figure 9B), suggesting low systemic toxicity of these formulations. However, considerable weight loss was observed at mice with DOX·HCl. Moreover, some mice treated with DOX·HCl cannot survive (Figure 9C), indicating serious side effects of free DOX·HCl.

Because the orthotopic 4T1 breast cancers grow much faster and are more likely to metastasize than the same type of subcutaneously grown cancer.44,

45

Histological and

immunohistochemical analyses were performed to further study the anticancer ability of SFNs/DOX/iRGD. As shown in Figure 9D, the tumor slices of both blank SFNs/iRGD and saline groups showed the intact nuclei morphology, suggesting almost no apoptosis or necrosis. Whereas SFNs/DOX groups treated mice displayed a certain degree of necrosis or 28 ACS Paragon Plus Environment

Page 29 of 34

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


apoptosis in tumor, this efficacy can be dramatically improved by co-administrating iRGD to SFNs/DOX formulations. Therefore, much necrosis area, the maximum degree of nuclei deficient in tumor of SFNs/DOX/iRGD treatment groups was observed, as confirmed using H&E staining. Meanwhile, SFNs/DOX/iRGD induced a significantly greater number of apoptotic cells (TUNEL-positive cells) in the tumor tissues after therapy. The in vivo anticancer ability was also demonstrated using CD-31 and ki-67 assay, SFNs/DOX/iRGD treatment groups more significantly reduced the CD31-positive blood vessels formation and suppressed the cell proliferation of Ki-67 positive tumor cells compared with other treatment groups. These results are quite consistent with the inhibition of tumor volume.

The DOX associated with toxicity and anti-metastasis activity were further studied using H&E staining. Severe cardiac toxicity such as necrosis and myocardial fiber breakage can be observed in DOX·HCl group. In contrast, for the mice administrated with SFNs/DOX/iRGD, SFNs/DOX, and blank SFNs/iRGD, as well as saline, did not induce any obvious damage to heart, spleen and kidneys (Figure S5). The dramatically reduced toxicity in normal organs can be reasonably explained by the tumor accumulation ability of SFNs/DOX/iRGD through the EPR effect and iRGD tumor-homing property. However, severe breast cancer metastasis to the lungs and liver were detected in the control group (Figure 9E). A certain degree of lungs and liver metastases can also be monitored in SFNs/DOX and DOX·HCl groups, whereas no visible metastasis was found in SFNs/DOX/iRGD group, demonstrating that SFNs/DOX/iRGD could effectively suppress the metastasis of the aggressive 4T1 orthotopic breast cancer. Collectively, the above in vivo findings confirm that SFNs/DOX/iRGD systems drastically enhance the anticancer effect and suppress the tumor metastasis, remarkably reducing the systemic toxicity. The superiority of SFNs/DOX/iRGD nanosystems can be ascribed to overcoming the stability dilemma, and iRGD-enhanced penetration, as well as the excellent membrane permeability of fluorocarbon. 29 ACS Paragon Plus Environment


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

4. CONCLUSIONS In summary, we have developed the original pH-sensitive fluorocarbon functionalized nanocarriers (SFNs) with a tumor homing and penetrating peptide iRGD for overcoming the stability dilemma and enhancing tumor penetration in orthotopic breast cancer. SFNs is based on the super-hydrophobic fluorocarbon functionalized peptide dendrons and intracellularly cleavable linkages. The resulting highly stable SFNs can not only provide a safe and spacious harbor for the hydrophobic drugs, but also balance the on-demand intracellular dissociation for drug release and the extracellular stability in the blood circulation. Additionally, the cellular uptake efficiency and the tumor penetration capacity have been dramatically enhanced in 3D multicellular spheroids, because of the synergistic effect between coadministration of iRGD and good cell membrane permeability of fluorocarbon. In vivo findings also confirm that SFNs/DOX/iRGD systems can effectively accumulate at tumor site and lungs with metastases lesions, drastically enhance anticancer efficacy and anti-metastasis activity, remarkably reducing the systemic toxicity. This study opens perspectives in the design of highly stable fluorinated drug delivery systems for combating cancer.

ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of China (NSFC, No. 51133004, 81361140343 and 31500810), the Joint Sino-German Center for Research Promotion (GZ905), the Young Scholar Program of Sichuan University (2015SCU11038), and the Foundation for Talent Introduction from Sichuan University (YJ201464).

ASSOCIATED CONTENT Supporting Information


Page 30 of 34

Page 31 of 34

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 and HPLC Data, photos of lung metastasis breast cancer model, and histological analysis of organs after treatment. This information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION *Corresponding Author E-mail: [email protected] (Dr. Yiyan He) [email protected] & [email protected] (Prof. Zhongwei Gu)

REFERENCES (1) Chen, W.; Zheng, R.; Baade, P. D.; Zhang, S.; Zeng, H.; Bray, F.; Jemal, A.; Yu, X. Q.; He, J. Cancer Statistics in China, 2015. CA: Cancer J. Clin. 2016, 66, 115-132. (2) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2016. CA: Cancer J. Clin. 2016, 66, 7-30. (3) Butcher, N. J.; Mortimer, G. M.; Minchin, R. F. Drug Delivery: Unravelling the Stealth Effect. Nat. Nanotechnol. 2016, 11, 310-311. (4) Deng, C.; Jiang, Y.; Cheng, R.; Meng, F.; Zhong, Z. Biodegradable Polymeric Micelles for Targeted and Controlled Anticancer Drug Delivery: Promises, Progress and Prospects. Nano Today 2012, 7, 467-480. (5) 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, 503-535. (6) Zhuang, J.; Gordon, M. R.; Ventura, J.; Li, L.; Thayumanavan, S. Multi-Stimuli Responsive Macromolecules and Their Assemblies. Chem. Soc. Rev. 2013, 42, 7421-7435. (7) Nowag, S.; Haag, R. Ph‐Responsive Micro‐and Nanocarrier Systems. Angew. Chem. Int. Ed. 2014, 53, 49-51. (8) Torchilin, V. P. Multifunctional, Stimuli-Sensitive Nanoparticulate Systems for Drug Delivery. Nat. Rev. Drug Discovery 2014, 13, 813-827. (9) Minchinton, A. I.; Tannock, I. F. Drug Penetration in Solid Tumours. Nat. Rev. Cancer 2006, 6, 583-592. (10) Jain, R. K.; Stylianopoulos, T. Delivering Nanomedicine to Solid Tumors. Nat. Rev. Clin. Oncol. 2010, 7, 653-664. (11) Zhang, Z.; Zhang, X.; Xu, X.; Li, Y.; Li, Y.; Zhong, D.; He, Y.; Gu, Z. Virus‐Inspired Mimics Based on Dendritic Lipopeptides for Efficient Tumor‐Specific Infection and Systemic Drug Delivery. Adv. Funct. Mater. 2015, 25, 5250-5260. (12) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater: 2013, 12, 991-1003. (13) Li, Y.; Wang, S.; Zhu, D.; Shen, Y.; Du, B.; Liu, X.; Zheng, Y. Reversibly Cross-Linked Poly(Ethylene Glycol)–Poly(Amino Acid)S Copolymer Micelles: A Promising Approach to Overcome the Extracellular Stability Versus Intracellular Drug Release Challenge. RSC Adv. 2015, 5, 20025-20034. (14) Chen, W.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z. Facile Construction of Dual-Bioresponsive Biodegradable Micelles with Superior Extracellular Stability and 31 ACS Paragon Plus Environment


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

Activated Intracellular Drug Release. J. Controlled Release 2015, 210, 125-133. (15) Wu, Y.; Chen, W.; Meng, F.; Wang, Z.; Cheng, R.; Deng, C.; Liu, H.; Zhong, Z. CoreCrosslinked Ph-Sensitive Degradable Micelles: A Promising Approach to Resolve the Extracellular Stability Versus Intracellular Drug Release Dilemma. J. Controlled Release 2012, 164, 338-345. (16) Liu, K. C.; Yeo, Y. Extracellular Stability of Nanoparticulate Drug Carriers. Arch. Pharm. Res. 2014, 37, 16-23. (17) Dai, L.; Yu, Y.; Luo, Z.; Li, M.; Chen, W.; Shen, X.; Chen, F.; Sun, Q.; Zhang, Q.; Gu, H. Photosensitizer Enhanced Disassembly of Amphiphilic Micelle for Ros-Response Targeted Tumor Therapy in Vivo. Biomaterials 2016, 104, 1-17. (18) Chandler, D. Interfaces and the Driving Force of Hydrophobic Assembly. Nature 2005, 437, 640-647. (19) Xiong, S. D.; Li, L.; Jiang, J.; Tong, L. P.; Wu, S. L.; Xu, Z. S.; Chu, P. K. Cationic Fluorine-Containing Amphiphilic Graft Copolymers as DNA Carriers. Biomaterials 2010, 31, 2673-2685. (20) Chauhan, V. P.; Stylianopoulos, T.; Martin, J. D.; Popović, Z.; Chen, O.; Kamoun, W. S.; Bawendi, M. G.; Fukumura, D.; Jain, R. K. Normalization of Tumour Blood Vessels Improves the Delivery of Nanomedicines in a Size-Dependent Manner. Nat. Nanotechnol. 2012, 7, 383388. (21) Chauhan, V. P.; Martin, J. D.; Liu, H.; Lacorre, D. A.; Jain, S. R.; Kozin, S. V.; Stylianopoulos, T.; Mousa, A. S.; Han, X.; Adstamongkonkul, P.; Popovic, Z.; Huang, P.; Bawendi, M. G.; Boucher, Y.; Jain, R. K. Angiotensin Inhibition Enhances Drug Delivery and Potentiates Chemotherapy by Decompressing Tumour Blood Vessels. Nat. Commun. 2013, 4, 2516. (22) Zhou, H.; Fan, Z.; Deng, J.; Lemons, P. K.; Arhontoulis, D. C.; Bowne, W. B.; Cheng, H. Hyaluronidase Embedded in Nanocarrier Peg Shell for Enhanced Tumor Penetration and Highly Efficient Antitumor Efficacy. Nano Lett. 2016, 16, 3268-3277. (23) Wang, X.; Zhen, X.; Wang, J.; Zhang, J.; Wu, W.; Jiang, X. Doxorubicin Delivery to 3d Multicellular Spheroids and Tumors Based on Boronic Acid-Rich Chitosan Nanoparticles. Biomaterials 2013, 34, 4667-4679. (24) Zhen, X.; Wang, X.; Xie, C.; Wu, W.; Jiang, X. Cellular Uptake, Antitumor Response and Tumor Penetration of Cisplatin-Loaded Milk Protein Nanoparticles. Biomaterials 2013, 34, 1372-1382. (25) Li, Y.; Lai, Y.; Xu, X.; Zhang, X.; Wu, Y.; Hu, C.; Gu Ms, Z. Capsid-Like Supramolecular Dendritic Systems as Ph-Responsive Nanocarriers for Drug Penetration and Site-Specific Delivery. Nanomedicine 2016, 12, 355-364. (26) Li, H. J.; Du, J. Z.; Liu, J.; Du, X. J.; Shen, S.; Zhu, Y. H.; Wang, X.; Ye, X.; Nie, S.; Wang, J. Smart Superstructures with Ultrahigh Ph-Sensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration. ACS Nano 2016, DOI, 10.1021/acsnano.1026b02326. (27) Wong, C.; Stylianopoulos, T.; Cui, J.; Martin, J.; Chauhan, V. P.; Jiang, W.; Popovic, Z.; Jain, R. K.; Bawendi, M. G.; Fukumura, D. Multistage Nanoparticle Delivery System for Deep Penetration into Tumor Tissue. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2426-2431. (28) Li, H.-J.; Du, J.-Z.; Du, X.-J.; Xu, C.-F.; Sun, C.-Y.; Wang, H.-X.; Cao, Z.-T.; Yang, X.Z.; Zhu, Y.-H.; Nie, S. Stimuli-Responsive Clustered Nanoparticles for Improved Tumor Penetration and Therapeutic Efficacy. Proc. Natl. Acad. Sci. U. S. A.2016, 113, 4164-4169. (29) Sun, Q.; Sun, X.; Ma, X.; Zhou, Z.; Jin, E.; Zhang, B.; Shen, Y.; Van Kirk, E. A.; Murdoch, W. J.; Lott, J. R.; Lodge, T. P.; Radosz, M.; Zhao, Y. Integration of Nanoassembly Functions for an Effective Delivery Cascade for Cancer Drugs. Adv. Mater. 2014, 26, 76157621. (30) Ju, C.; Mo, R.; Xue, J.; Zhang, L.; Zhao, Z.; Xue, L.; Ping, Q.; Zhang, C. Sequential 32 ACS Paragon Plus Environment

Page 32 of 34

Page 33 of 34

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


Intra‐Intercellular Nanoparticle Delivery System for Deep Tumor Penetration. Angew. Chem. Int. Ed. 2014, 53, 6253-6258. (31) Lee, J.; Kim, J.; Jeong, M.; Lee, H.; Goh, U.; Kim, H.; Kim, B.; Park, J.-H. LiposomeBased Engineering of Cells to Package Hydrophobic Compounds in Membrane Vesicles for Tumor Penetration. Nano Lett. 2015, 15, 2938-2944. (32) Neil, E.; Marsh, G. Towards the Nonstick Egg: Designing Fluorous Proteins. Chem. Biol. 2000, 7, R153-R157. (33) Banerjee, S.; Vidya, V. M.; Savyasachi, A. J.; Maitra, U. Perfluoroalkyl Bile Esters: A New Class of Efficient Gelators of Organic and Aqueous–Organic Media. J. Mater. Chem. 2011, 21, 14693. (34) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science 2010, 328, 1031-1035. (35) Skinner, M. Therapy: Drugs Hitch a Ride. Nat. Rev. Cancer 2010, 10, 384-385. (36) Ma, S.; Zhou, J.; Wali, A. R.; He, Y.; Xu, X.; Tang, J. Z.; Gu, Z. Self-Assembly of PhSensitive Fluorinated Peptide Dendron Functionalized Dextran Nanoparticles for on-Demand Intracellular Drug Delivery. J. Mater. Sci. Mater. Med. 2015, 26, 5550. (37) Zhao, P.; Xia, G.; Dong, S.; Jiang, Z. X.; Chen, M. An Itep-Salinomycin Nanoparticle That Specifically and Effectively Inhibits Metastases of 4t1 Orthotopic Breast Tumors. Biomaterials 2016, 93, 1-9. (38) Zhang, Y.; Ding, J.; Li, M.; Chen, X.; Xiao, C.; Zhuang, X.; Huang, Y.; Chen, X. OneStep "Click Chemistry"-Synthesized Cross-Linked Prodrug Nanogel for Highly Selective Intracellular Drug Delivery and Upregulated Antitumor Efficacy. ACS Appl. Mater. Interfaces 2016, 8, 10673-10682. (39) Owen, S. C.; Chan, D. P.; Shoichet, M. S. Polymeric Micelle Stability. Nano Today 2012, 7, 53-65. (40) Lu, J.; Owen, S. C.; Shoichet, M. S. Stability of Self-Assembled Polymeric Micelles in Serum. Macromolecules 2011, 44, 6002-6008. (41) Lelekakis, M.; Moseley, J. M.; Martin, T. J.; Hards, D.; Williams, E.; Ho, P.; Lowen, D.; Javni, J.; Miller, F. R.; Slavin, J. A Novel Orthotopic Model of Breast Cancer Metastasis to Bone. Clin. Exp. Metastasis 1999, 17, 163-170. (42) Fang, J.; Nakamura, H.; Maeda, H. The Epr Effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Delivery Rev. 2011, 63, 136-151. (43) Wilmanns, C.; Fan, D.; O'Brian, C. A.; Bucana, C. D.; Fidler, I. J. Orthotopic and Ectopic Organ Environments Differentially Influence the Sensitivity of Murine Colon Carcinoma Cells to Doxorubicin and 5‐Fluorouracil. Int. J. Cancer 1992, 52, 98-104. (44) Shan, S.; Sorg, B.; Dewhirst, M. W. A Novel Rodent Mammary Window of Orthotopic Breast Cancer for Intravital Microscopy. Microvasc. Res. 2003, 65, 109-117. (45) Su, J.; Sun, H.; Meng, Q.; Yin, Q.; Tang, S.; Zhang, P.; Chen, Y.; Zhang, Z.; Yu, H.; Li, Y. Long Circulation Red-Blood-Cell-Mimetic Nanoparticles with Peptide-Enhanced Tumor Penetration for Simultaneously Inhibiting Growth and Lung Metastasis of Breast Cancer. Adv. Funct. Mater. 2016, 26, 1243-1252.



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


Page 34 of 34

Highly Stable Fluorinated Nanocarriers with iRGD for Overcoming the

Recommend Documents