Coadministration of iRGD with Multistage Responsive Nanoparticles

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Functional Nanostructured Materials (including low-D carbon)

Coadministration of iRGD with Multistage Responsive Nanoparticles Enhanced Tumor Targeting and Penetration Abilities for Breast Cancer Therapy Chuan Hu, Xiaotong Yang, Rui Liu, Shaobo Ruan, Yang Zhou, Wei Xiao, Wenqi Yu, Chuanyao Yang, and Huile Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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ACS Applied Materials & Interfaces

Coadministration of iRGD with Multistage Responsive Nanoparticles Enhanced Tumor Targeting and Penetration Abilities for Breast Cancer Therapy Chuan Hu, Xiaotong Yang, Rui Liu, Shaobo Ruan, Yang Zhou, Wei Xiao, Wenqi Yu, Chuanyao Yang and Huile Gao*

Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, 610041, China

* Corresponding author: [email protected]; [email protected]

KEYWORDS: iRGD, drug delivery, size-shrinkable, NO donor, deep penetration, antitumor

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ABSTRACT

Limited tumor targeting and poor penetration of nanoparticles are two major obstacles to improving the outcome of tumor therapy. Herein, coadministration of tumor-homing peptide iRGD and multistage-responsive penetrating nanoparticles for the treatment of breast cancer are reported. This multistage-responsive nanoparticle, IDDHN, was comprised of NO donor-modified hyaluronic acid shell (HN) and small-sized dendrimer, namely Dendri-Graft-L-Lysine conjugated with doxorubicin and indocyanine (IDD). The results showed that IDDHN could be degraded rapidly from about 330 nm to a smaller size that was in a size range of 35 nm to 150 nm (most at 35~60 nm) after hyaluronidase (HAase) incubation for 4 hours, in vitro cellular uptake demonstrated that iRGD could mediate more endocytosis of IDDHN into 4T1 cells, which was attributed to the overexpression of αvβ3 integrin receptor. Multicellular spheroids penetration results showed synergistically enhanced deeper distribution of IDDHN into tumors, with the presence of iRGD, HAase incubation and NO release upon laser irradiation. In vivo imaging indicated that coadministration with iRGD markedly enhanced the tumor targeting and penetration abilities of IDDHN. Surprisingly, coadministration of IDDHN with iRGD plus 808 nm laser irradiation nearly suppressed all tumor growth. These results systematically revealed the excellent potential of coadministration of iRGD with multistage-responsive nanoparticles for enhancing drug delivery efficiency and overcoming the 4T1 breast cancer.


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1. INTRODUCTION

Cancer treatment built on nanomedicines remains restricted due to the poor drug delivery efficiency and limited tumor penetration. In addition to cancer cell physiology, the tumor microenvironment (TME) also plays an essential role in drug delivery.1, 2 Nanomedicines can deliver anticancer agents to solid tumor via the enhanced permeability and retention (EPR) effect, which is based on the leaky tumor vasculature and poor lymphatic drainage.3-5 However, there is a long way to go before nanomedicines arrive at the tumor vasculature. iRGD is a widely used tumor-homing penetration peptide, which can increase vascular and tissue permeability in a tumor-specific and neuropilin-1-dependent manner.6, 7 Many studies have verified that coadministration of iRGD enhanced the tumor targeting and penetration ability of nanomedicines, and consequently increased the treatment efficacy.8-12 Thus, it is a simple and efficient way to enhance the efficiency of tumor targeted delivery just by coadministration of iRGD. However, the heterogeneous nature of tumor microenvironment hinders the deep penetration of nanomedicine into tumor parenchyma. What’s more, because of the abnormal vasculature, high interstitial fluid pressure (IFP) and high solid tissue pressure (STP) in solid tumor become formidable barriers to nanoparticles delivery and distribution.13 To this end, many researchers have focused on overcoming the heterogeneous tumor microenvironment to improve the therapy efficiency.14-20 Nitric oxide (NO) is a multifunctional gaseous mediator, which involves in many physiological and pathological processes of tumor,21 especially in vascular functions 3 ACS Paragon Plus Environment


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such as enhancing vascular permeability and consequently enhancing the therapeutic efficacy of chemotherapy.22-24 A number of NO donors with the property of stimuli-responsive NO release have been developed for cancer therapy.25-28 Nonetheless, the accumulation and distribution of nanoparticles in tumors are a complex and multifaceted problem that needs to be addressed, significantly affected by physicochemical properties of nanomedicines, such as size, surface features, elasticity and so on.29-32 Size plays an important role in the retention and penetration of nanoparticle within the tumor microenvironment. Generally, large sized nanoparticles hold higher retention ability to accumulate in the vicinity of blood vasculature but have poor penetration and fail to distribute into the dense tumor matrix,9, 33 while small sized nanoparticles possess greater tumor interstitial transport ability but may suffer short blood half-life time and poor tumor retention.31, 34 The dilemma has promoted the construction of size-changeable nanoparticles, which can respond to various tumor-specific stimuli (for example, enzyme, acidic pH, and UV light).35-37 We recently developed an enzyme sensitive size-changeable and laser-enhanced NO release nanoparticles (IDDHN) based on hyaluronidase (HAase), triggering the degradation of Hyaluronic acid (HA) shell.38 The particle size of IDDHN could shrink from over 300 nm to less than 40 nm. Foremost, upon 808 nm laser irradiation, the high photothermal conversion efficiency of indocyanine green (ICG) could induce strong hyperthermia (HT) effect, which could not only perform antitumor effect but also enhance NO release. In return, released NO could enhance the vascular permeability and further improve the tumor targeting drug delivery. However, the accumulation of 4 ACS Paragon Plus Environment

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programmed IDDHN at the tumor site primarily relied on the EPR effect, thus, to fulfill the purpose of our nanoparticles in the tumor. The high accumulation of IDDHN within the tumor was more important than the following size shrinking procedure and deep penetration. To test our hypothesis, we demonstrated that co-administrated iRGD could endow IDDHN with both active and passive targeting abilities, which could home them into tumor and guarantee higher accumulation of nanoparticles at tumor site, and then followed by precise and effective size shrinkage. 2. EXPERIMENTAL SECTION 2.1. Materials. Dendri-Graft-L-Lysine (DGL-G3) dendrimer was obtained from Colcom (Montpellier Cedex, France) and sodium hyaluronate (MW = 65 kDa) was purchased from Freda Biopharm Co., Ltd (Jinan, China). Hyaluronidase (C3867-1VL) was obtained from Sigma-Aldrich (Shanghai, China). Doxorubicin hydrochloride was purchased from Beijing Huafeng United Technology Co., Ltd. (Beijing, China). Indocyanine green (ICG, >94.3%) was obtained from Dalian Meilun Biotech Co., Ltd (Dalian, China). Rabbit anti-integrin beta-3 was obtained from Abcam Ltd. (HongKong, China). Rabbit neuropilin-1 polyclonal antibody (NRP-1) was purchased from 4A Biotech Co., Ltd. (Beijing, China). Mouse mammary breast tumor cell line (4T1) and A2780 cell line were purchased from Shanghai Institute of Cell Biology (Shanghai, China). Other chemical reagents were obtained from J&K Scientific Ltd (Shanghai, China), all of them were analytical grade and without further purification. BALB/C mice (Female, 18 ± 2 g) were purchased from Dashuo Biotechnology Co.,



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Ltd, (Chengdu, China). All animal experiments were performed under the guidelines approved by the experiment animal administrative committee of Sichuan University. 2.2. Synthesis of Dendrimeric Prodrugs of Doxorubicin and Indocyanine Green (IDD): DOX·HCl (3.8 mg, 6.6 µmol) and DGL-G3 (11 mg, 0.5 µmol) were dissolved in dimethyl sulfoxide and triethylamine was added to neutralize the hydrochloride. The mixture reacted for 48 h and was purified by dialysis (cutoff molecular weight = 7000 Da). The fluorescence intensity of connected DOX was measured by a RF-5301PC spectrofluorophotometer (Shimadzu, Japan). Next, ICG (1 mg/mL in 5% glucose solution) loaded on the DD via electrostatic absorption. The hydrated diameter and zeta-potential of DD and IDD were characterized by a Malvern Zetasizer (Malvern, NanoZS, UK). 2.3. Synthesis of Nitrooxyacetic Acid (NO Donor)-Modified Hyaluronic Acid (HN): First, 2-(Nitrooxy)acetic acid was synthesized as the NO donor. Silver nitrate (3.70 g, 21.8 mmol) and Bromoacetic acid (2.00 g, 14.3 mmol) were dissolved in anhydrous acetonitrile (80 mL). 18 h later, the precipitates and acetonitrile were removed by filtration and evaporation in vacuo, respectively. Then dichloromethane (100 mL) was added to dissolve product. 2 h later, filtered again, and the dichloromethane was removed in vacuo to afford nitrooxyacetic acid as yellow oil (1.15 g, 64%). 2-(Nitrooxy)acetic acid (20 µL, 0.25 mmol), EDC (60 mg, 0.3 mmol), NHS (36 mg, 0.3 mmol), and DMAP (7 mg, 0.06 mmol) were added in 2 mL phosphate buffer (pH 6.0). Then, hyaluronic acid (29 mg, 0.45 µmol) was added and the pH value was 6 ACS Paragon Plus Environment

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elevated to 8.0. After stirring for 18 h, the mixtures were purified with centrifugal filters (10 K MWCO) (Millipore) and fixed to 1.0 mL. The degree of substitution (DS) was determined to be about 21% by elemental analysis (Euro EA 3000, Lehman-Liebers, US) and calculated by Equation. (1). DS % = [(%age of N element in HN)-(%age of N element in HA)] / [(theor %age of N element in HN)-(%age of N element in HA)] × 100

(1)

2.4. Preparation and Characterization of IDDHN. Briefly, 100 µL of prepared HN was added dropwise in the 140 µL of prepared IDD solution and mixed 60 seconds with a vortex. The unencapsulated DOX and ICG were purified with centrifugal filters (10 K MWCO) (Millipore), the concentration of DOX and ICG in ultrafiltrate were detected to determine the drug loading capacity and encapsulation efficiency. The morphology was captured by transmission electronic microscopy (TEM) (Tecnai G2 F20 S-TWIN, FEI, USA). The hydrodynamic diameter and zeta-potential were determined by a Malvern Zetasizer (NanoZS, Malvern, UK). 2.5. Cellular Uptake. 4T1 cells and A2780 cells were inoculated in 12-well plates at a density of 5 × 104 cells per well. After incubation for 24 h, IDDHN and IDDHN/iRGD were added to the wells respectively, with the same DOX concentration of 10 µg mL-1. The concentration of iRGD was 43.3 µg mL-1 in IDDHN/iRGD group. After incubation for 1 h and 4 h, the cells were harvested. The fluorescence intensity of DOX between different groups was measured by a flow cytometer (Beckman Conlter, USA). Meanwhile, 4T1 cells and A2780 cells were inoculated onto glass coverslips in 6-well plates at a density of 5×105 cells per well. After incubation for 24 h, IDDHN and 7 ACS Paragon Plus Environment


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IDDHN/iRGD were added as described in previous steps. After incubation for 1 h and 4 h, the cells were washed and fixed, and then DAPI (0.5 µg mL-1) was added to stain the nuclei. The coverslips were placed on the slides and confocal images were observed by confocal microscope (A1+, Nikon, Japan). 2.6. The Enhanced Thermal Effect of IDDHN/iRGD in Vitro. 4T1 cells were incubated with IDD, IDDHN and IDDHN/iRGD (equivalent 20 µg mL-1 ICG) for 4 h, and were rinsed with PBS, then exposed to 808 nm laser irradiation at 1.5 W/cm2 for 5 min. After that, the cells were further cultured with fresh medium for 4 h. Then, cells were stained by Calcein AM and PI solution and observed under the fluorescence microscope. In addition, differences in 4T1 metabolic activity treated with laser irradiation were assessed by MTT assay. 2.7. Multicellular Spheroids. 4T1 multicellular spheroids (MCSs) were prepared as our previous studies.39 Briefly, 4T1 cells were seeded in 96-well pre-coated with 80 µL of 2% low-melting-temperature. Then they were incubated with IDDHN, IDDHN/iRGD, HAase + IDDHN (pretreated with 150 IU/mL HAase for 4 h) and HAase + IDDHN/iRGD (pretreated with 150 IU/mL HAase for 4 h) at an equivalent DOX concentration (10 µg/mL) for 4 h. Laser groups were carried out with laser irradiation (1.5 W/cm2, 5 min). Afterwards, the MCSs were rinsed and fixed, fluorescent intensity was observed by a confocal microscope (FV1000, Olympus, USA).


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2.8. Animals. Female BALB/C mice (6 weeks old) were bought from Dashuo Biotechnology Co., Ltd, (Chengdu, China). All animal experiments were approved by the ethics committee of Sichuan University. 2.9. Tumor Models. Female BALB/c mice were anesthetized and 4T1 cells (2 × 106 mL-1，100 µL) were slowly injected into the left upper thigh subcutaneous region. The mice were used for in vivo fluorescence imaging and antitumor efficacy until the tumor volumes achieved ~200 mm3 and ~70 mm3, respectively. 2.10. In vivo Fluorescence Imaging. The xenograft 4T1 breast-bearing mice were randomly divided into IDDHN and IDDHN/iRGD groups. Mice were intravenously injected with the corresponding drugs at the dose of ICG 2 mg kg-1, the mice of IDDHN/iRGD group were coadministration with iRGD at a final dose of 4 µM kg-1. The ICG fluorescence signal (Ex 780 nm, Em 845 nm) was photographed by IVIS Spectrum system (Caliper, USA) at 24 h and 36 h. The tumor-bearing mice were sacrificed and perfused with PBS followed with 4% (w/v) paraformaldehyde at 36 h post-injection, and then the tumors and major organs were excised for ex vivo imaging.

2.11. In Vivo Tumor Distribution. The tumors of mice obtained in section 2.10 were fixed with 4% (w/v) paraformaldehyde for immunofluorescence study. The tumors were sectioned at a thickness of 10 µm using a freezing microtome (Leica, Germany). DAPI (5 µg/mL) was used to stain the nuclei, Cy3-labeled anti-CD34 antibody (Diluted 100 times) was used to stain tumor vasculature. Finally, the slices were observed by a confocal microscope (A1+, Nikon, Japan).



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2.12. Photothermal Imaging. The xenograft 4T1 breast-bearing mice were randomly divided into IDDHN and iRGD/IDDHN groups. Mice were intravenously injected with 5% glucose, IDDHN and IDDHN/iRGD, the corresponding drugs at the dose of ICG 2 mg kg-1, the mice of IDDHN/iRGD group were coinjected with iRGD at a final dose of 4 µM/kg. 24 h-post injection, the tumors were exposed to the 808 nm laser (1.5 W/cm2, 5min). Thermal imaging of maximum temperatures was photographed by an IR camera (Fotric 220, ZXF, USA). 2.13. Pharmacokinetics study. The female Balb/c mice (n=6 for each group) were intravenously injected with IDDHN and IDDHN/iRGD at a dosage of 3 mg/kg of ICG, respectively, followed by collection of 20 µL blood sample from retro-orbital at 0.083, 0.167, 0.5, 1, 2, 4, 6, 10 h post injection. The blood sample were diluted in PBS (pH 7.4, 80 µL), the ICG fluorescence signal of each sample was analyzed by a microplate reader (SPARK 10M, Tecan, Switzerland). 2.14. In vivo Antitumor Efficacy. When the tumor size reached ~70 mm3, the tumor-bearing mic were randomly divided into 5% glucose, IDDHN, IDDHN/iRGD, IDDHN with laser, and IDDHN/iRGD with laser groups (n = 5). 5% glucose group was treated with 5% glucose appropriately. Other groups were treated with corresponding drugs at the dose of ICG 2.0 mg kg-1 and DOX 2.8 mg kg-1. The mice of IDDHN/iRGD with or without laser groups were coinjected with iRGD at a final dose of 4 µM kg-1 every 3 days. 24 h post-injection, the mice of laser groups were irradiated by 808 nm laser (1.5 W/cm2 for 5 min). The weight and tumor size of mice were measured every 2 days. The tumor volume (V) was calculated by the formula V = L × W2 / 2, where L and 10 ACS Paragon Plus Environment

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W are the length and width of the tumor, respectively. Mice were sacrificed one day after the last irradiation. Tumors and major tissues were collected and fixed with 4% (w/v) paraformaldehyde for hematoxylin and eosin (HE) staining. 2.15. Statistical Analysis. The results were expressed as means ± standard deviations (s.d.) of the mean. N-value in vitro of our studies represented for triplicate experiments. Difference between two groups was analyzed by two-tailed Student's t-test under SPSS statistical program version 20 (SPSS Inc., Chicago, IL). 3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of IDDHN. DD determined by dynamic light scattering (DLS) was around 28.2 nm, with a zeta potential of + 47.9 mV. After coated with ICG, IDD had a slight size increase to 34 nm, while zeta-potential decreased to + 37.3 mV. HN was then attached onto the surface of the IDD, mainly owing to electrostatic adsorption. The drug loading capacity of DOX and ICG were 2.3 ± 0.2% and 1.6 ± 0.7%, respectively. The encapsulation efficiency of DOX and IGG were 84.3 ± 3.8% and 100.0 ± 0.0%, respectively (Table S1). The release of DOX from IDDHN was very slow, the cumulative release of DOX from IDDHN at pH 7.4 was only 8.9% after 168 h incubation. Normally, pH in normal tissue and blood was 7.4. The low release of DOX at pH 7.4 mimicked the DOX release profiles during blood circulation and in normal tissues. The low release of DOX in pH 7.4 could reduce the side effect on normal tissues. The charge of IDDHN was - 23.5 mV and the hydration diameter was 311.2 nm (Figure 1ABC). The hydrate particle size of IDD and IDDHN were consistent with the TEM images (Figure 1DE). Notable changes in zeta-potential 11 ACS Paragon Plus Environment


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and size between IDD and IDDHN indicated that HN was successfully coated on the surface of IDD and the nanoparticles were established. In addition, size changes in IDDHN were observed by TEM after incubation with HAase at pH 6.8 for 4 hours (Figure 1F). These results showed IDDHN reduced sharply from about 330 nm to smaller size which vary from 35 nm to 150nm) (most at 35~60 nm), which was close to the original size of IDD, indicating the fully degradation of HN shell under HAase incubation, and the result was consistent with our previous study.8, 38, 39

Figure 1. Characterization of nanoparticles. Hydrodynamic size distribution of (A) IDD and (B) IDDHN. (C) Zeta-potential of the DD, IDD and IDDHN. Representative TEM images of (D) IDD, (E) IDDHN, and (F) IDDHN after 4 h HAase incubation.


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3.2. Cellular Uptake and Apoptosis in vitro. To study tumor-homing and cell penetration capacities of iRGD peptide cotreated with IDDHN, high αvβ3-expressing 4T1 cells and low αvβ3-expressing A2780 cells were used to investigate cellular uptake of IDDHN in vitro (Figure 2B). Both flow cytometry and confocal images indicated that coincubated with iRGD contributed to a higher cellular internalization of IDDHN in 4T1 cells (Figure 2AC). In contrast, coincubation of iRGD had no effect on the uptake of A2780 cells (Figure 2AD). Those results verified that iRGD could home to 4T1 cells through the αvβ3 receptor，thus facilitating the internalization of nanoparticles and tumor active targeting ability.

Figure 2. Cellular uptake and apoptosis. (A) Confocal images of cellular uptake on 4T1 cells and A2780 cells after being treated with IDDHN and IDDHN/iRGD for 1 h and 4 h, bars represent 50 µm. (B) Western blotting analysis of the integrin beta 3 receptor. The quantitative analysis of the fluorescence intensity after being treated with IDDHN and IDDHN/iRGD on 4T1 cells (C) and A2780 cells (D) for 1 h and 4 h. Data were 13 ACS Paragon Plus Environment


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presented as mean ± SD ( n = 3), * P < 0.05. (E) Temperature changes of photothermal effects for 5% glucose, free ICG, IDD and IDDHN (equivalent 20 µg/mL ICG) after 808nm laser irradiation (5 min, 1.5 W/cm2). (F) Fluorescence images of Calcein AM/PI stained 4T1 cells incubated with PBS, IDD, IDDHN and IDDHN/iRGD respectively for 4 h after the irradiation of 808 nm lasers (5 min, 1.5 W/cm2), bar represents 50 µm. (G) Relative viability of 4T1 cells incubated with IDD, IDDHN, IDDHN/iRGD after the irradiation of 808 nm laser irradiation (5min, 1.5 W/cm2), which was determined by MTT assay. Data were presented as mean ± SD (n = 3), * P < 0.05.

Enhanced cellular uptake triggered by coincubating of iRGD paved the way for the further use of IDDHN-based PTT/chemotherapy for killing 4T1 cells. First, we investigated photothermal effects of IDDHN in aqueous solutions. An aqueous solution of PBS, ICG, IDD and IDDHN (equivalent 20 µg/mL ICG) were irradiated with an 808 nm laser. The temperature between ICG and IDD had no significant difference during the process of irradiation. Interestingly, the temperature of IDDHN group was much higher than other groups, which might be ascribed to improved photostability of ICG when it was encapsulated in the nanoparticles (Figure 2E).40 Then, 4T1 cells were treated with IDD, IDDHN or IDDHN/iRGD; besides, all groups were irradiated with 808 nm laser (1.5 W/cm2, 5 min) and examined by dead-live staining assay. As expected, coincubation with iRGD, IDDHN exhibited a distinct therapeutic effect as shown in Figure 2F. With laser irradiation, we could observe almost all cells died in the IDDHN/iRGD group. In line with the results of dead-live staining assay, MTT 14 ACS Paragon Plus Environment

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assay results showed that IDDHN/iRGD with laser group produced a higher cytotoxicity to 4T1 cells (Figure 2G). Those results indicated that overexpressed αvβ3 receptors in 4T1 cells mediated more nanoparticles endocytosis. Subsequently, IDDHN induced prominent apoptosis ability on 4T1 cells via the combined PTT/chemotherapy upon NIR irradiation. 3.3. In vivo biodistribution. Ideally, coadministration of iRGD with IDDHN was expected to improve the tumor targeting capability and produce higher therapeutic effects. Before the PTT/chemotherapy, the pharmacokinetic profiles of both IDDHN and IDDHN/iRGD were investigated firstly after intravenous injection into Balb/c mice (Table S2). The mean residence time (MRT0-t) for IDDHN/iRGD was 1.37 ± 0.34 h, which was 4.57-fold higher than that of IDDHN group (p < 0.05). The result suggested that coadministration iRGD with IDDHN contributed longer blood retention. Then, we estimated enhanced tumor targeting and accumulation abilities mediated by coadministration of iRGD with IDDHN in vivo using IVIS imaging system (Caliper, USA). As shown in Figure 3ABC, HAase-mediated degradation of HN shell and emancipation of NO ameliorated the EPR effect in 4T1 breast tumor, suggesting IDDHN had good tumor distribution ability, as demonstrated in our previous study.34 However, 36 h post-injection, the ICG signal in the tumor region was gradually reduced. As expected, coadministration of iRGD could significant enhance the tumor targeting and accumulation abilities of nanoparticles, evidenced by higher ICG fluorescent signals of IDDHN/iRGD in the tumor site relative to IDDNH at 24 h and 36 h. From typical images of major organs, it could be concluded that the fluorescence signals were 15 ACS Paragon Plus Environment


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mainly observed in liver, kidney, lung and tumor. On the one hand, the liver and kidneys are major metabolic organs, on the other hand, some liver and lung cells also express integrin receptors, nanoparticles could also home to there, which may produce side effects. The ICG fluorescence signal intensity of IDDHN/iRGD group in the tumor was 2.1-fold than IDDHN at 36 h post-injection, suggesting that iRGD could efficiently mediate homing to 4T1 tumor delivery of nanoparticles. Moreover, the result was further confirmed by an infrared thermal imager. An 808 nm laser irradiation was performed 24 h post tail vein injection of 5% glucose, IDDHN and IDDHN/iRGD. As shown in Figure 3D, the tumor temperatures of IDDHN and IDDHN/iRGD were 55.1 oC and 60.7 oC respectively after 5 min irradiation. While a moderate temperature increase in the tumor was observed in the 5% glucose group (39.9 oC), which was inadequate to cause any damages. Those results indicated that coadministration of iRGD with IDDHN enhanced the tumor targeting and homing more IDDHN to the tumor region.


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Figure 3. The increased accumulation of IDDHN in 4T1 tumor bearing mice. (A) The in vivo biodistribution in tumor bearing mice under in vivo imaging system at different time points after injection. (B) Ex vivo imaging of the major organs. (C) Radiant efficiency of the organs measured by ex vivo imaging. (D) IR thermal images of post-injection of 5% glucose, IDDHN and IDDHN/iRGD in the 808 nm laser irradiation process (5min, 1.5 W/cm2).

3.4. iRGD-mediated deep Penetration in vitro. To investigate whether cotreated with iRGD further enhanced the nanoparticles penetration in tumors, we first used MCSs as 3D tumor models to evaluate the drug distribution in vitro. MCSs could simulate the tumor microenviroment and have been widely used to study the the tumor biology and therapeutic efficacy of nanoparticles in vitro.41 The disribution of IDDHN in the MCSs was measured post incubation 8 h. Figure 4A showed that, without laser irradiation, IDDHN/iRGD penetrated deeper than IDDHN group into 60 µm from top of the MCSs, while IDDHN were mainly located in edge regions rather than in the central region at 80 µm from the top of MCSs with or without iRGD. Pre-incubation with HAase triggered the size shrink of IDDHN and significantly improved the penetration of IDDHN. As expected, enhanced penetration of cotreated with iRGD could be found at 80 µm from the top of MCSs. The DOX fluorescence intensity of HAase + IDDHN/iRGD (pre-4 h incubation with HAase) group was 1.4-, 2.6-, and 4.4-fold higher than that of HAase + IDDHN (pre-4 h incubation with HAase), IDDHN/iRGD and IDDHN groups (p < 0.001) (Figure 4B), respectively. With laser irradition, deep penetration triggered by laser-enhanced NO release could be fully 17 ACS Paragon Plus Environment


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demonstrated compared with the same depth without laser irradiation groups. As espected, the DOX fluorescence intensity of HAase + IDDHN/iRGD (pre-incubation with HAase) was the highest among all groups (p < 0.001). All of these results clearly elucidated that overexpressed αvβ3 integrin in 4T1 cells contributed to enhancing the effect of iRGD on the targeting and penetration of IDDHN.

Figure 4. (A) Confocal images of IDDHN and IDDHN/iRGD (with/without HAase) distribution in MCSs of 4T1 cells, bars represent 100 µm. (B) Fluorescence intensity of the MCSs central region versus Z-axis distance (means ± SD, n=3, ***p < 0.001).


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3.5. iRGD-mediated deep Penetration in vivo. To further investigate the deep penetration of IDDHN/iRGD in the deep tumor, we adopted immunofluorescence technique to observe the intratumoral distribution of nanoparticles. 36 h-post injection, the tumors were cryo-sectioned and the cryo-sectioned were stained with DAPI (blue) and tumor neovessels marker CD34 (red). In the captured images, IDDHN were expressed as green fluorescence signals for the DOX. Surprisingly in IDDHN/iRGD group, iRGD significantly enhanced the tumor distribution of IDDHN and coadministration with iRGD showed the highest DOX intensity and diffused far away from the red signal (CD34 stained tumor neovessels) under the laser irradiation (Figure 5A). The green fluorescence intensity of IDDHN/iRGD + Laser group was 1.56-, 1.83-, and 2.23-fold higher than IDDHN + Laser, IDDHN/iRGD, and IDDHN group, respectively (Figure 5B). This could be contributed to the home ability of iRGD onto overexpressed αvβ3 integrin and neuropilin-1 in 4T1 cells and vascular endothelial cells, and thus enhancing the targeting and penetration abilities of IDDHN in 4T1 tumor (Figure 5C).



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Figure 5. iRGD-mediated deep penetration in vivo. (A) Fluorescence distribution of different formulations in 4T1 tumor section after staining with anti-CD34 antibodies, bars indicate 50 µm. (B) Semi-quantitative intensity of DOX fluorescence signals from the tumors (means ± SD, n=3, ***p < 0.001). (C) The expression of αvβ3 integrin and neuropilin-1 in 4T1 tumors, and bars represent 50 µm.

3.6. Antitumor Efficacy. Motivated by the superior tumor accumulation and penetration of IDDHN/iRGD, in vivo PTT/chemotherapy were performed using 4T1 tumor-bearing mice. The mice were randomized into five groups (n = 5), and respectively treated with different formulations at a single dose of 2.8 mg kg-1 of DOX and 2 mg kg-1 of ICG. iRGD groups were coinjected with iRGD at a final dose of 4 µM kg-1. As shown in Figure 6A, control group (administered with 5% glucose) showed a rapid tumor growth. IDDHN and IDDHN/iRGD group exhibited a moderate


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antitumor efficiency due to the monotherapy and slow NO release without laser irradiation. Upon 808 nm laser irradiation, IDDHN could inhibit tumor growth to a certain extent while with less recurrence of the tumor, validating the synergistic effects for laser-enhanced NO release and HAase-triggered size shrink for deep tumor penetration. Excitingly, coadministration with iRGD of IDDHN completely inhibit tumor growth (Figure 6ABD and Figure S2), which could be put down to more nanoparticles homed to the tumor site mediated by iRGD and synergistic antitumor efficiency by a combination of DOX-mediated chemotherapy and ICG-mediated PTT. Moreover, no significant body weight loss was monitored in all groups (Figure 6C), illustrating the satisfactory biocompatibility of IDDHN and iRGD.



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Figure 6. In vivo antitumor effect in 4T1 tumor bearing mice. (A) Tumor growth curves after injected with different formulations. (B) The tumor weight at the endpoint. (C) Change in body weight over the treatment. (D) Representative photographic image of tumors from the 4T1 tumors-bearing mice sacrificed after being treated with different formulations for 16 days. Data were presented as mean ± SD ( n = 5), * P < 0.05, ** P < 0.01, *** P < 0.005.

3.7. Immunohistochemistry analysis. To further reveal the better antitumor effect of IDDHN/iRGD, hematoxylin and eosin (H&E) staining was carried out. As showed in Figure S3, after treated with IDDHN/iRGD under 808 nm laser irradiation, most of tumor cells were apoptotic or necrotic. In addition, compared to the 5% glucose group, neither significant damage nor inflammation of major organs was observed from treatment groups. These results indicated that coadministration of iRGD with IDDHN could greatly suppress tumor progression without noticeable side effects. 4. CONCLUSIONS

In summary, we demonstrated the strategy of co-administrating tumor penetration peptide iRGD with size-shrinkable and NO donor-modified multistage responsive nanoparticles to enhance the tumor targeting and penetration abilities and further enhanced the antitumor efficiency. In vitro cellular study and in vivo optical imaging indicated that coadministration of iRGD could home more IDDHN to the 4T1 cells. MCSs penetration experiment and in vivo slice distribution demonstrated that coadministration with iRGD showed a synergistic effect of significantly enhanced


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accumulation and penetration in the depth of tumor, resulting in the most effective antitumor effect. iRGD, which mediated more IDDHN to the tumor site, became the foundation of our multistage responsive nanoparticles. The combination with iRGD could be an effective strategy for enhancing drug delivery efficiency and improving the therapy outcome. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected]

Present Addresses Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy, Sichuan University, 610041, China

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

The work was granted by National Natural Science Foundation of China (31571016) and Young Elite Scientists Sponsorship Program by CAST (2017QNRC001), also supported by Research Center for Public Health and Preventive Medicine, West China School of Public Health, Sichuan University.

AUTHOR CONTRIBUTIONS



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H.G. supervised the study. C.H. and H.G. designed the experiments. C.H. performed all experiments with assistance from X.Y., R.L., S.R., Y.Z., W.X., W.Y., and C.Y., C.H. and H.G. analyzed the data and wrote the paper.

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Abstract Graphic 35x15mm (300 x 300 DPI)

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Figure 1. Characterization of nanoparticles. Hydrodynamic size distribution of (A) IDD and (B) IDDHN. (C) Zeta-potential of the DD, IDD and IDDHN. Representative TEM images of (D) IDD, (E) IDDHN, and (F) IDDHN after 4 h HAase incubation. 110x73mm (300 x 300 DPI)


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Figure 2. Cellular uptake and apoptosis. (A) Confocal images of cellular uptake on 4T1 cells and A2780 cells after being treated with IDDHN and IDDHN/iRGD for 1 h and 4 h, bars represent 50 µm. (B) Western blotting analysis of the integrin beta 3 receptor. The quantitative analysis of the fluorescence intensity after being treated with IDDHN and IDDHN/iRGD on 4T1 cells (C) and A2780 cells (D) for 1 h and 4 h. Data were presented as mean ± SD ( n = 3), * P < 0.05. (E) Temperature changes of photothermal effects for 5% glucose, free ICG, IDD and IDDHN (equivalent 20 µg/mL ICG) after 808nm laser irradiation (5 min, 1.5 W/cm2). (F) Fluorescence images of Calcein AM/PI stained 4T1 cells incubated with PBS, IDD, IDDHN and IDDHN/iRGD respectively for 4 h after the irradiation of 808 nm lasers (5 min, 1.5 W/cm2), bar represents 50 µm. (G) Relative viability of 4T1 cells incubated with IDD, IDDHN, IDDHN/iRGD after the irradiation of 808 nm laser irradiation (5min, 1.5 W/cm2), which was determined by MTT assay. Data were presented as mean ± SD (n = 3), * P < 0.05. 100x57mm (300 x 300 DPI)



Figure 3. The increased accumulation of IDDHN in 4T1 tumor bearing mice. (A) The in vivo biodistribution in tumor bearing mice under in vivo imaging system at different time points after injection. (B) Ex vivo imaging of the major organs. (C) Radiant efficiency of the organs measured by ex vivo imaging. (D) IR thermal images of post-injection of 5% glucose, IDDHN and IDDHN/iRGD in the 808 nm laser irradiation process (5min, 1.5 W/cm2). 86x46mm (300 x 300 DPI)


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Figure 4. (A) Confocal images of IDDHN and IDDHN/iRGD (with/without HAase) distribution in MCSs of 4T1 cells, bars represent 100 µm. (B) Fluorescence intensity of the MCSs central region versus Z-axis distance (means ± SD, n=3, ***p < 0.001). 127x115mm (300 x 300 DPI)



Figure 5. iRGD-mediated deep penetration in vivo. (A) Fluorescence distribution of different formulations in 4T1 tumor section after staining with anti-CD34 antibodies, bars indicate 50 µm. (B) Semi-quantitative intensity of DOX fluorescence signals from the tumors (means ± SD, n=3, ***p < 0.001). (C) The expression of αvβ3 integrin and neuropilin-1 in 4T1 tumors, and bars represent 50 µm. 109x72mm (300 x 300 DPI)


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Figure 6. In vivo antitumor effect in 4T1 tumor bearing mice. (A) Tumor growth curves after injected with different formulations. (B) The tumor weight at the endpoint. (C) Change in body weight over the treatment. (D) Representative photographic image of tumors from the 4T1 tumors-bearing mice sacrificed after being treated with different formulations for 16 days. Data were presented as mean ± SD ( n = 5), * P < 0.05, ** P < 0.01, *** P < 0.005. 119x90mm (300 x 300 DPI)


Coadministration of iRGD with Multistage Responsive Nanoparticles

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