Smart pH-Sensitive Nanogels for Enhancing Synergistic

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Biological and Medical Applications of Materials and Interfaces

Smart pH-Sensitive Nanogels for Enhancing Synergistic Anticancer Effects of Integrin ## Specific Apoptotic Peptide and Therapeutic Nitric Oxide v

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Yurui Xu, Lei Sun, Shujun Feng, Jianmei Chen, Ya Gao, Leilei Guo, Xueying An, Yuanyuan Nie, Yu Zhang, xiaoxuan Liu, and Xinghai Ning ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10830 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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ACS Applied Materials & Interfaces

Smart pH-Sensitive Nanogels for Enhancing Synergistic Anticancer Effects of Integrin αvβ3 Specific Apoptotic Peptide and Therapeutic Nitric Oxide Yurui Xu,a Lei Sun,a Shujun Feng,a Jianmei Chen,a Ya Gao,a Leilei Guo,b Xueying An,c Yuanyuan Nie,a Yu Zhang,*a Xiaoxuan Liu,*b Xinghai Ning*a

aNational

Laboratory of Solid State Microstructures, College of Engineering and Applied

Sciences, Nanjing University, Nanjing 210093, China.

bState

Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug

Discovery for Metabolic Diseases, Center of Drug Discovery, Center of Advanced Pharmaceutics and Biomaterials, China Pharmaceutical University, Nanjing 210009, China

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cState

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Key Laboratory of Pharmaceutical Biotechnology, Department of Sports Medicine

and Adult Reconstructive Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210093, China

KEYWORDS: nanogels, acid-responsive, integrin αvβ3 agonist, apoptotic peptide, synergistic anticancer

ABSTRACT: Apoptotic peptide (kla), which can trigger the mitochondria-mediated apoptotic program cell death, has been widely recognized as a potential anticancer agent. However, its therapeutic potential has been significantly impaired by its poor biostability, lack of tumor specificity, and particularly low cellular uptake. Herein, a linear peptide Arg-Trp-D-Arg-Asn-Arg (RWrNR) was identified as an integrin αvβ3 specific ligand with a nanomolar dissociation constant (Kd=0.95nM), which can greatly improve kla antitumor activity (IC50=8.81μM) by improving its cellular uptake, compared to the


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classic integrin-recognition motif c-RGDyK (IC50=37.96μM). Particularly, the RWrNR-kla conjugate can be entrapped in acidic sensitive nanogels (RK/Parg/CMCS-NGs), composed of poly-L-arginine (Parg) and carboxymethyl chitosan (CMCS, pI=6.8), which can not only carry out controlled release of RWrNR-kla in response to tumor acidic microenvironment, and consequently enhance its tumor specificity, cell internalization, but trigger tumor associated macrophages to generate nitric oxide, leading to enhanced synergistic anticancer efficacy. Importantly, RK/Parg/CMCS-NGs have been proved to effectively activate apoptosis signaling pathway in vivo, and significantly inhibit tumor growth with minimal adverse effects. To be summarized, RK/Parg/CMCS-NGs are a promising apoptotic peptide based therapeutics with enhanced tumor accumulation, cytosolic delivery and synergistic anticancer effects, thereby holding a great potential for the treatment of malignant tumors.


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Introduction

Peptides are endogenous signaling molecules that are a critical factor of cell biological process, and have been considered as excellent drug candidates, due to their attractive pharmacological profiles and intrinsic properties.1-3 Anticancer peptides (ACPs) that induce intrinsic cellular apoptosis pathway have been proved to be an ingenious strategy for anticancer treatment, and are expected to provide higher selectivity for cancer cells without disturbing native biological functions.4-6 Particularly, apoptotic peptide (kla) with a sequence of (D-Lys-D-Leu-D-Ala-D-Lys-D-Leu-D-Ala-DLys)2, a positively charged amphiphilic α helix polypeptide, shows effective anticancer capability by depolarizing mitochondrial membrane and activating apoptotic caspase pathway.7 Nevertheless, kla has some intrinsic limitations such as poor cellular uptake, unsatisfied cytotoxicity, low biostability and tumor specificity, thereby impairing its therapeutic efficacy.8 Various biotechnologies have been utilized to improve its therapeutic potential, including cell-penetrating peptide conjugates,7,

9-11

combined

therapeutic strategies and delivery scaffolds.12-15 Therefore, the development of a


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multifunctional kla based therapeutics for facilitating its tumor targeting, cellular uptake and anticancer activity, may significantly improve anticancer treatment.

Growing evidences show that the conjugation of kla with tumor specific ligands has the potential of improving its cellular uptake. Particularly, the ligands for integrin αvβ3, which is highly expressed on neovascularization endothelial cells and cancer cells, and exhibits important roles in tumor progression, metastasis, angiogenesis and associated inflammation, can greatly improve kla anticancer efficacy by enhancing its intracellular transport.16-17 Integrin αvβ3 interacts with ECM proteins through binding the RGD tripeptide motif, and many efforts have been made in the development of RGD-mimetic peptides for selectively binding to integrin αvβ3 receptor.17-18 Particularly, cilengitide (c(RGDfv)), as a potent integrin αvβ3 ligand, has been reported as a promising anticancer chemotherapeutic agent for glioblastoma patients.19-20 However, it has failed in the Phase III trial evaluating because of its low affinity and specificity for integrin αvβ3.21 In addition, RGD-containing peptides also recognize integrin αvβ5 and α5β1, which are expressed on normal cells, leading to the impedance of their tumor specific


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potential.22 Therefore, it would be of great benefit to develop a novel integrin αvβ3 ligand with promising specificity and affinity.

In addition to low cellular uptake, kla is a positively charged polypeptide and easily absorbed by plasma components, leading to rapid clearance from the bloodstream and poor tumor accumulation. To prolong systemic circulation and improve tumor specificity of kla, many peptide delivery systems have been developed to improve kla anticancer efficacy. Nanogels are a type of nanosized hydrogel with crosslinked hydrophilic polymer networks, which can be controlled to modulate their properties, such as swelling,

degradation,

and

chemical

functionality.23 Particularly,

biodegradable

hydrogels show many advanced properties as attractive drug delivery nano system, such as good biocompatibility, responsible degradation, and effective drug loading.24 For example, pH-sensitive nanogels are designed to target tumor microenvironment, and exhibit the acid-dependent drug release profiles due to special dynamic swelling behavior of polyelectrolyte in components.25-27 Therefore, pH-sensitive nanogels provide


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a functional platform for delivery of peptide drugs in tumor tissues with good stability and decreased potential immunogenicity.

Here, we have developed a linear peptide ligand RWrNR for integrin αvβ3 with high affinity and specificity, which can promote the intracellular transportation of RWrNR-kla conjugate into cancer cells by targeting cancer integrin αvβ3, leading to cancer cell apoptosis. In addition, therapeutic potential of RWrNR-kla can be further improved by incorporating in acid-sensitive nanogels (RK/Parg/CMCS-NGs), composed of poly-Larginine

(Parg)

and

carboxymethyl

chitosan

(CMCS,

pI=6.8)

(Figure

1).

RK/Parg/CMCS-NGs can carry out controlled release of RWrNR-kla in response to tumor acidic microenvironment, and consequently enhance its tumor specificity, cell internalization. Importantly, Parg is not only a component of nanogels, but provides Larginine substrate of nitric oxide synthase (iNOS) for stimulating tumor associated macrophages (TAMs) to generates the cytotoxic nitric oxide (NO),28-29 leading to enhanced synergistic anticancer effects of apoptotic kla and therapeutic NO. To be summarized,

RK/Parg/CMCS-NGs

are

a

promising

apoptotic

peptide

based


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therapeutics with enhanced tumor targeting, cellular uptake and synergistic anticancer effects, thereby holding a great potential for the treatment of malignant tumors.

Experimental Section

Materials and Instrument. All chemicals were obtained from Aladdin and Sigma Company. B16F10 cell lines, NCTC 1469 cell lines and RAW 264.7 cell lines were obtained from Cell Bank of Chinese Academy of Science. B16F10 cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and penicillin/streptomycin (1%, w/v) respectively. RAW 264.7 and NCTC 1469 cell lines were

cultured

in

DMEM/HG

medium

supplemented

with

10%

FBS

and

penicillin/streptomycin (1%, w/v) respectively. Balb/c nude mice (6–8 weeks, 18–22 g) were purchased from Nanjing Qinglongshan Experimental Animal Center. All animal experiments were performed according to the National Institute of Health Guidelines under the protocols, approved by the ethics committee at the Affiliated Drum Tower Hospital of Nanjing University Medical School.


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Terminal-free peptides (KLAKLAK)2 (L-KLA), (klaklak)2 (kla), cRGDyK-GG-(klaklak)2 (RGD-kla) and RWrNR-GG-(klaklak)2 (RWrNR-kla) and their RhB or DiR labeled peptides (>95%) were purchased from KareBay Biochem, Inc. (Ningbo, China). Carboxymethyl chitosan (CMCS, molecular weight of 50,000 Da; 85% deacetylation; 60% carboxymethyl substitution) was purchased from Jinan Haidebei Biological Engineering Co. (Jinan, China). Poly-L-arginine (Parg, average MW = 7000-15000 Da) was purchased from Yuanye Biotechnology Co. (Shanghai, China). Hoechst, MitoGreen, Nitric oxide Detection Kit and Mitochondrial Detection Kit were purchased from Beyotime Institute of Biotechnology Co. LLC. (Nantong, China). Carboxy-H2DCFDA was purchased from MedChem Express (USA). 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Merck Life Science, Shanghai, China). MTT assays were carried out on a microplate reader (Tecan Group). Transmission electron microscope (TEM) images were performed on an H-800 transmission electron microscope (Hitachi). Dynamic light scattering (DLS) was detected on a Zetasizer Nano ZS (Malvern). Fluorescent images were obtained using an inverted fluorescence microscope (Nikon Instruments), and confocal laser scanning


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microscopy (CLSM) (Zeiss, L710, Germany). The pH values were measured by a digital pH-meter (pH 301, HANNA Instruments). In vivo images were conducted on PerkinElmer IVIS Spectrum (PerkinElmer).

Microscale thermophoresis assay. Binding affinity of RWrNR or c-RGDyK to recombinant integrin αvβ3 (R&D Systems) were measured using the microscale thermophoresis binding assay (NanoTemper Technologies). NT-647 fluorescencelabeled (Monolith NTT Protein Labeling Kit, NanoTemper Technologies) integrin αvβ3 was incubated with RWrNR or c-RGDyK. Thermophoresis measurements were performed with NanoTemper Software (NanoTemper Technologies) to calculate the Kd values.

Characterization of RWrNR-kla. The concentration of kla, RGD-kla and RWrNR-kla were analyzed by HPLC (SHIMADZU, JAPAN). HPLC column: VP-ODS C18 column, 150 × 4.6 mm, 5 µm. Flow rate and solvent: 1mL min-1, solvent: solvent A: 0.1% trifluoroacetic acid in water, solvent B: 0.1% trifluoroacetic acid in 80% acetonitrile and 20% water; gradient 28%→48% B for kla, 35%→55% for RGD-kla, 30%→50% for


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RWrNR-kla mobile phase B from 0-20 min. The detection was conducted at wavelength of 220 nm. CD spectrums of kla, RGD-kla and RWrNR-kla in PBS buffer (pH 7.4, 10 mM) containing 25 mM of sodium dodecylsulfate were measured using a Jasco J-810 spectropolarimeter (Jasco, Japan). The serum stability of kla, RGD-kla and RWrNR-kla was investigated by incubating in PBS buffer (pH 7.4) containing FBS (50% v/v) at 37 ℃ for 24 h, and concentrations were measured by HPLC.

Cell viability assay. In vitro cytotoxicities of kla, RGD-kla and RWrNR-kla peptides or peptides loaded nanogels were evaluated in B16F10 and NCTC 1469 cells by MTT assay. Briefly, 5000 cells per well were seeded into 96-well plates and cultured at 37°C for 24 h. Then the medium was replaced with new medium containing peptides or peptides loaded nanogels at different pH condition (regulated by lactic acid). MTT solution (20 μL) was added to the culture medium and incubated for 4h, followed by removing the medium and adding dimethyl sulfoxide (150 μL). The UV absorbance intensity was measured at 570 nm using a microplate reader.


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Cellular uptake. B16F10 and NCTC 1469 cells, 2.5 × 105 cells per well were seeded into 24-well plates and cultured at 37°C for 24 h, followed by culturing with RhB labeled peptide or nanogels for additional 24 h at different pH condition (regulated by lactic acid). The cells were washed by PBS, and the intracellular fluorescence intensity was analyzed and imaged by flow cytometry (BD FACS Calibur) and fluorescence microscope (Ex/Em=555/580 nm), respectively.

The measurement of mitochondrial membrane potential. The mitochondrial membrane potential changes were measured using a mitochondrial detection kit (Beyotime C2006). 5 × 105 cells per well were seeded into confocal dishes (Costar, Washington, DC, USA) and cultured at 37°C for 24 h, followed by replacing with 10 μM different peptides for 24 h. Depolarization of the mitochondrial membrane is characterized by a shift from red fluorescence to green fluorescence. The simultaneous measurement of fluorescence was performed by CLSM (Leica Microsystems, Wetzlar, Germany). Green fluorescent emission of J-monomer (Ex/Em = 514/529 nm) and red fluorescent emission (Ex/Em = 585/590 nm) of J-aggregate was measured and analyzed using ImageJ programs.


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Preparation and characterization of RK/Parg/CMCS-NGs. CMCS (2 mg/mL) and Parg (2mg/ml) were individually dissolved in deionized water. Aqueous solution of CMCS was dropped into Parg solution at different volume ratio under stirring for 10 min at room temperature to obtain Parg/CMCS-NGs. To prepare RK/Parg/CMCS-NGs, different volumes of RWrNR-kla solution (0.5 mg/mL) were added to the CMCS solution to obtain RK-CMCS solution, followed by adding Parg solution under stirring for 10 min at room temperature. Finally, the suspension was filtered through a 0.45 μm cellulose nitrate membrane to obtain peptides loaded nanogels.30-31

The physical properties of nanogels including the particle size distribution and zeta potential were measured using Zetasizer Nano ZS (Malvern). RK/Parg/CMCS-NGs morphology was measured by TEM. The ultrafiltration method was used to determine EE% and DL%. In brief, 1 mL of peptide-loaded nanogels was centrifuged for 30 min at 10,000 rpm using an ultrafiltration centrifuge tube (MW cutoff [MWCO] of 10 kDa), followed by analyzing peptides with HPLC.

The DL% and EE% were calculated as follows:


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DL (%) = Wp1/Wt × 100%

EE (%) = Wp1/Wp2 × 100%

Wp1 represents the total amount of peptides in nanogels; Wp2 represents the initial amount of peptides used to prepare nanogels; Wt represents the total amount of peptides loaded nanogels.

The pH-sensitive release of RK/Parg/CMCS-NGs. The in vitro release behavior of RK/Parg/CMCS-NGs was carried out in PBS (pH 7.4 or pH 6.5) using a dialysis method. Briefly, 1 mL of RK/Parg/CMCS-NGs was transferred into dialysis bags (MWCO of 10 kDa), followed by immersing into 50 mL of PBS solution under stirring at 37°C. Dialysis medium (0.1 mL) was collected at different time points (0.25, 0.5, 1, 2, 4, 8, 12, 24h), which were measured with HPLC system to calculate the peptide concentrations.

Plasma stability of RK/Parg/CMCS-NGs. Different nanogels were mixed with equal volume of FBS (filtered by a 0.22 μm millipore filter membrane) and incubated for 24h at


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37◦C. the particle sizes were measured different time points to determine the stability of RK/Parg/CMCS-NGs.

Intracellular trafficking of RK/Parg/CMCS-NGs. 5×105 B16F10 cells per well were cultured in confocal microscope dishes at 37°C for 24 h, followed by adding RhBlabeled RWrNR-kla loaded nanogels (diluted by RPMI 1640 cell culture medium at pH 6.5) for additional 2 h or 12 h. The cells were washed with PBS, and stained with Hoechst 33342 and MitoGreen for labeling nucleus and mitochondria, respectively.

Cellular uptake of RK/Parg/CMCS-NGs by macrophages. RAW 264.7 cells, 2.5 × 105 cells per well were cultured into 24-well plates at 37°C for 24 h, followed by culturing with FITC labeled Parg (100 nM) of nanogels at different pH condition (regulated by lactic acid). The cells were washed by PBS, and the intracellular fluorescence intensity was detected and imaged by flow cytometry and fluorescence microscope (Ex/Em=494/518 nm).

NO production induced by RK/Parg/CMCS-NGs in macrophages. Nitric oxide Detection Kit was used to quantify the NO generated in RAW 264.7 macrophage. RAW 264.7


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cells, 2.5 × 105 cells per well were cultured in 24-well plates at 37°C for 24 h, followed by adding LPS (100 ng/mL) for inducing expression of iNOS in RAW 264.7 cells. Then the cells were cultured with medium containing different nanogels at different pH condition 6.5 or 7.4 (regulated by lactic acid) for 12 h. The supernatant was collected for measuring the nitrite concentration.

Synergistic anticancer effects of RK/Parg/CMCS-NGs in vitro. The RAW 264.7 cells were co-cultured with B16F10 cells for analyzing the synergistic anticancer effects of RK/Parg/CMCS-NGs in vitro. In brief, 2.5 × 105 B16F10 cells per well were cultured in 24-well plates at 37°C for 24 h. 5000 RAW 264.7 cells per well were seeded in transwell, followed by adding LPS (100 ng/mL) at 37°C for 8 h. RAW 264.7 cells were transferred to the B16F10 seeded 24-well plates. 400 μL of culture medium (pH 6.5) containing different nanogels (equivalent to10 μg of RWrNR-kla) were transferred into donor chamber and cultured at 37◦C for 24 h, followed by measuring with MTT assay.

Measurement of intracellular levels of ROS. The RAW 264.7-B16F10 cells co-culture model was established as above described. 400 μL of culture medium (pH 6.5)


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contained different nanogels (equivalent to10 μg of RWrNR-kla) were transferred into filters (donor chamber) and incubated at 37◦C for 24 h. After incubation, B16F10 cells were washed with PBS, followed by incubating with Carboxy-H2DCFDA (1 μL, 10 mM) for 10 min, and imaged by CLSM (Ex/Em = 495/529 nm).

In vivo biodistribution. Male Balb/c nude mice were subcutaneously injected with 1×107 B16F10 cells. Mice were divided into 3 different groups (n=6) for investigating the biodistribution of RK/Parg/CMCS-NGs when tumor volume reached ~100 mm3. Mice were intravenously administrated with DiR-labeled peptides loaded nanogels, and imaged at 24 h. The major organs were harvested for evaluating biodistribution of nanogels.

Pharmacokinetic (PK) studies in mice. Healthy male Balb/c nude mice (n=6) were used to evaluate PK profiles of RWrNR-kla after i.v. injection of kla, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs (5 mg/kg of kla). Blood samples were collected at predetermined intervals, followed by centrifuging at 10000 rpm for 10 min. The supernatant was mixed


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with acetonitrile to precipitate proteins, and measured by HPLC to determine the concentration of RWrNR-kla. PK data was evaluated using Kinetica 4.4.

In vivo antitumor effects of RK/Parg/CMCS-NGs. Male Balb/c nude mice bearing B16F10 tumors were randomly divided into 4 groups (n = 5), which were injected with saline (as control), Parg/CMCS-NGs, RK/Plys/CMCS-NGs or RK/Parg/CMCS-NGs (5mg/kg of kla) every 3 days for 13 days. Tumor sizes and body weights were monitored every 3 days. At the end of the experiments, tumors and major organs were collected after mice were sacrificed. To evaluate the apoptotic profiles in tumor tissues, caspase-3 and TUNEL stains were applied. The major organs were analyzed by H&E stains for biosafety evaluation. In addition, the survival rates of mice (n=10) treated with saline, Parg/CMCS-NGs, RK/Plys/CMCS-NGs or RK/Parg/CMCS-NGs (5mg/kg of kla) were monitored.

Statistics. All experiments were redone 3 times with 6-12 biological samples. Data are represented as mean ± SD. Error bars show standard error of the mean from independent samples. GraphPad Prism 6 software was used for statistical analysis.


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Unpaired Student’s t-test or one-way ANOVA was used to calculate statistical significance. * represents p < 0.05, ** represents p < 0.01 and ***represents p < 0.001.

Results and Discussion

The design and binding affinity of RWrNR for integrin αvβ3. Integrin αvβ3 has been identified as a key factor for tumor initiation, progression and metastasis, making it an attractive target for cancer therapy.32 In order to develop a specific ligand for integrin αvβ3, crystal structure (PDB ID: 1L5G) was selected to design targeting peptides using structure-based pharmacophore method integrated with molecular docking. Six candidates were identified by ranking docking score and free binding energy. As shown in Figure 2A, pentapeptide RWrNR exhibited lowest docking score (-22.067), calculated by Triangle Matcher method, suggesting it is a potential integrin αvβ3 ligand. In addition, the binding free energy of RWrNR and c-RGDyK with integrin αvβ3 was computed using the MM/GBVI method (Figure 2B). RWrNR (-266.029 kcal/mol) had a lower binding free energy than c-RGDyK (-147.913 kcal/mol), suggesting its high affinity for integrin αvβ3. Furthermore, Figure 2C and Figure S1 show that RWrNR formed more hydrogen


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bonding and hydrophobic interaction with the integrin αvβ3 than c-RGDyK. MicroScale Thermophoresis (MST) was used to evaluate the dissociation constant (Kd) between RWrNR and integrin αvβ3, which reflects the binding affinity at molecular level (Figure 2D). The sigmoidal binding curves showed that the Kd values of RWrNR and c-RGDyK of 0.95±0.02 nM and 7.56±0.06 nM, respectively, indicating that RWrNR possesses higher specific binding affinity for integrin αvβ3 than c-RGDyK.

Synthesis and characterization of RWrNR-kla. Peptide drugs exhibit several advantages in the management of malignant tumors, including optimized pharmacokinetics and pharmacodynamics.33 Apoptotic peptide kla has been identified as a potential anticancer agent that induces destruction of mitochondrial membranes.7 However, its anticancer effects are hindered because of its poor tumor targeting and cell membrane permeability.34 In addition, the highly positive charge of kla is inconvenient for systemic administration due to strong adsorption of plasma protein, leading to poor bioavailability and fast clearance.35 Therefore, the development of a targeting ligand for improving its cellular uptake and systemic bioavailability is necessary for increasing its anticancer


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capacity. Here, we coupled RWrNR with kla via a GG bridge to obtain integrin-targeted apoptotic peptide RWrNR-kla, the structure was accredited by mass spectrum (Figure S2), and the purities were tested by HPLC (more than 95%, Figure S3). Since the specific α-helical structure of kla determine its apoptotic activity, we therefore tested the secondary structure of RWrNR-kla, RGD-kla and kla using circular dichroism (CD) spectrum. Figure 2E shows that RWrNR-kla, RGD-kla and kla, with around 222 nm and 208 nm peak, exist typical α-helical conformation in a PBS buffer (pH 7.4, 10 mM) containing 0.7% sodium dodecylsulfate, indicating that integrin-targeted peptide do not affect the structure of kla. Furthermore, Figure 2F shows that no changes in the concentration of RWrNR-kla, RGD-kla and kla were observed after 24h-incubation in FBS at 37 °C, indicating that these peptides have good biostability, and suitable for in

vitro and in vivo applications.

Cytotoxicity of RWrNR-kla. MTT assay was carried out to evaluate the cytotoxicity of RWrNR-kla. Melanoma B16F10 cells with high integrin αvβ3 expressed on cell surface and normal hepatocyte NCTC 1469 cells without expression of integrin αvβ3 were


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incubated with different concentrations of RWrNR-kla, RGD-kla and kla, and the cell viability was measured at different time points.36 Figure 3A exhibits that 24h treatment of RWrNR-kla induced higher cell death in B16F10 cells, compared to RGD-kla and kla. For example, RWrNR-kla had IC50 of 8.81 µM, which is 5-fold lower than RGD-kla (IC50 of 37.96 µM), indicating that the high binding affinity of RWrNR for integrin αvβ3 can effectively improve anticancer activity of kla. However, RWrNR-kla displayed minimal cytotoxic effects on NCTC 1469 cell (Figures 3B and 3D), indicating that RWrNR-kla has low cell membrane penetration, and is transported through integrin αvβ3 mediated cellular uptake. In addition, RWrNR-kla displayed time-dependent cytotoxicity to B16F10 cells (Figure 3C). These data prove that RWrNR-kla has a favorable cytotoxicity for integrin αvβ3 expressed cancer cells, and produces minimal side effects on normal cells.

Cellular uptake of RWrNR-kla. The low cellular uptake is one of key problem for applications of kla.7 We therefore performed flow cytometry to investigate uptake profiles of RWrNR-kla, RGD-kla and kla in B16F10 and NCTC 1469 cells for 24h


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treatment (Figures 3E-F). In comparison of NCTC 1469 cells, which had minimal intracellular concentration of RWrNR-kla, B16F10 cells displayed an obvious concentration-dependent internalization of RGD-kla and RWrNR-kla, which is consistent with cytotoxic results in NCTC 1469 and B16F10 cells. In addition, RWrNR-kla was rapidly transported into B16F10 cells, and generated 3-fold higher intracellular concentration than RGD-kla after 24h incubation, indicating that RWrNR endows kla with high specificity and affinity for cancer cells by targeting integrin αvβ3, thereby exhibiting higher anticancer effects.

The mitochondrial membrane potential assay. Many studies report that the apoptotic activity of kla is related to the disruption of mitochondrial membranes via strong electropositivity.37 We then utilized the JC-1 assay, which is widely used to indicate changes of mitochondrial membrane potentials, to investigate the cell apoptosis process. JC-1 dye can generate J-aggregates and exhibits red fluorescence accumulated in normal mitochondria, whereas it shifts to green fluorescence after mitochondrial disruption.38 Figures 3G-H show that RWrNR-kla significantly disrupted


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mitochondria in B16F10 cells, and the red fluorescence intensity was 71.56% of RGDkla. In contrast, low green fluorescence of JC-1 dye was observed in NCTC 1469 cells after incubation with either RWrNR-kla or RGD-kla, indicating that RWrNR-kla is a potent anticancer agent with minimal effects on healthy cells.

Design, preparation and characteristics of pH-sensitive RK/Parg/CMCS-NGs. In comparison of chemical drugs, peptide drugs show many promising pharmaceutical prospects, such as minimal side-effects, weak drug tolerance and high specificity, thereby attracting a lot of research attention on the development of anticancer drugs.4 However, they are susceptible to proteolytic enzymes, and can elicit an adverse immune response within a host. Particularly, it is difficult to effectively deliver into tumors due to fast systemic clearance and lack of tumor targeting. Here, we have developed a new type of pH-sensitive nanogels (RK/Parg/CMCS-NGs), composed of Parg, CMCS (pI=6.8) and RWrNR-kla, for enhancing tumor specificity and circulation half-life

of

RWrNR-kla.

RK/Parg/CMCS-NGs

are

designed

target

tumor

microenvironment to selectively release RWrNR-kla and Parg due to charge reverse of


24

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CMCS. RWrNR-kla can be rapidly transported into cancer cells by targeting integrin αvβ3, and Parg can trigger NO mediated apoptotic pathway by activating iNOS in macrophage,28 leading to enhancing synergistic anticancer effects.

The particle sizes and zeta potential of Parg/CMCS-NGs are the key factors for their therapeutic effects, and we therefore optimized the formulation of nanogels by mixing different mass ratios of Parg and CMCS. Figure 4A shows that Parg/CMCS-NGs formed compact particles with the particle sizes of 140 nm when the mass ratio of Parg to CMCS was ranging from 2:1 to 1:5. In contrast, the particle sizes of Parg/CMCS-NGs with mass ratio of 1:7 reached to more than 400 nm, indicating that large excess of CMCS affect compactness of nanogels. Meanwhile, the zeta potential of Parg/CMCSNGs varied from positive to negative when the mass ratio of CMCS increased, and reached to -15mV at 1:5 ratio, indicating the emplacement of CMCS on the Parg/CMCS-NGs surface. Therefore, we identified that Parg/CMCS-NGs with 1:5 mass ratio is the optimized formulation, which have the particle size of 137.81±1.2 nm (PDI=0.16±0.036) and zeta potential of -18.44±2.5 mV. In addition, we confirmed that


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the maximal encapsulation efficiency (EE) and drug loading rate (DL) of RWrNR-kla in RK/Parg/CMCS-NGs were 92.10±0.21 and 7.06±0.19, respectively, suggesting that nanogels have high peptide loading efficacy through electrostatic adsorption (Figure 4B). Furthermore, Figure 4C shows that RK/Parg/CMCS-NGs had negatively charged surface with zeta potential of –15 mV, indicating successful encapsulation of RWrNR-kla in nanogels. Importantly, encapsulation of RWrNR-kla or polylysine (Plys, instead of Parg) nanogels (RK/Plys/CMCS-NGs) did not change size and zeta potential of nanogels (Figure 4D). The TEM image further suggested that RK/Parg/CMCS-NGs were almost spherical without any aggregation (Figure 4E). Therefore, we successfully prepared RK/Parg/CMCS-NGs with optimal pharmaceutical profiles, which allow for systemic administration.

pH-response of RK/Parg/CMCS-NGs. Tumor acidic microenvironment is one of the key characteristics of solid tumors, which mainly results from the high glycolytic rate of tumor cells, along with a disorganized vasculature and inefficient perfusion and clearance, generating substantial acid-outside pH gradients (pH 6.5-6.8).39 Tumor acidic


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microenvironment has a number of important sequellaes that are germane to cancer, including abnormal tumor angiogenesis, peripheral immune tolerance, cellular metabolism, and tumor metastasis, indicating that it is a promising anticancer target.40 RK/Parg/CMCS-NGs were composed of CMCS with pI=6.8, which can be disaggregated in tumor acidic microenvironment to release RWrNR-kla.41

We therefore investigated the pH-responsive profile of RK/Parg/CMCS-NGs. Figure 4E shows that the appearance of colloid solution of RK/Parg/CMCS-NGs was translucent and opalescent at pH 7.4, whereas the colloid solution became clear and transparent after incubating in pH 6.5 PBS buffer. In addition, DLS studies displayed that the average particle sizes of RK/Parg/CMCS-NGs changed from 140 nm to 15 nm within 30 min in pH 6.5 buffer (Figure 4F), suggesting rapid decomposition of RK/Parg/CMCS-NGs in tumor acidic microenvironment. Furthermore, we also investigated the drug-release profiles RK/Parg/CMCS-NGs, RK/Parg/CMCS-NGs and RK/Plys/CMCS-NGs at pH 6.5 in PBS using a dialysis method (Figure 4G and Figure S4). RK/Parg/CMCS-NGs showed a sustained release at pH 7.4, and no more than


27


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20% RWrNR-kla was released within 24h. On the contrary, RWrNR-kla was released quickly at pH 6.5, and the release reached plateau (more than 60%) after 12h, as well as RK/Plys/CMCS-NGs. The structure of RK/Parg/CMCS-NGs could be disintegrated in acidic environment owing to the low isoelectric point of CMCS, leading to expulsion of RWrNR-kla.

Therefore,

RK/Parg/CMCS-NGs

could

be

stable

in

physiological

environment but exhibited quick release under tumor acidic microenvironment, making them a potent tumor-targeting acid-responsive drug delivery system.

Plasma stability of RK/Parg/CMCS-NGs. The plasma dispersion ability of nanosized delivery system greatly effects in vivo behavior of nanoparticles due to plasma protein adsorption induced self-aggregation.42 We therefore performed experiments to investigate the stability of RK/Parg/CMCS-NGs in fetal bovine serum (FBS). Figure S5 shows that no changes in particle sizes of nanogels were observed after 24h incubation, indicating that strong negative charge on the surface of nanogels can prevent adsorption of plasma proteins. Therefore, RK/Parg/CMCS-NGs with CMCS coating are stable in the plasma and have a long circulation time.


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Cytotoxicity of RK/Parg/CMCS-NGs. Nanogels are a type of nanosized hydrogel with crosslinked hydrophilic polymer networks, which can be controlled to modulate their properties, such as swelling, degradation, and chemical functionality.23 Nanogels are an effective platform for delivery of peptide drugs, allowing for transporting active peptides without affecting

bioactivity, biostability and immunogenicity.43-44 We therefore

investigated the cytotoxicity of RK/Parg/CMCS-NGs in B16F10 and NCTC 1469 cells using MTT assay. Figure 5A shows that RWrNR-kla displayed significant cytotoxicity to B16F10 cells at pH 7.4, and whereas RK/Parg/CMCS-NGs had minimal anticancer effects at pH 7.4. On the contrary, RK/Parg/CMCS-NGs exhibited similar cytotoxity to RWrNR-kla at pH 6.5 due to integrin αvβ3 mediated intracellular transport after releasing from nanogels, indicating that RK/Parg/CMCS-NGs have pH-dependent anticancer profiles. More importantly, RK/Parg/CMCS-NGs showed no cytotoxicity to NCTC 1469 cells and macrophages at physiological condition, indicating their good tumor specificity and biocompatibility (Figure S6 and Figure S7).


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Cellular uptake of RK/Parg/CMCS-NGs. Acidic responsive RK/Parg/CMCS-NGs have a potential of enhancing tumor targeting by disaggregating and releasing RWrNR-kla in acidic condition (pH 6.5), consequently improving cellular uptake through targeting integrin αvβ3. Therefore, we investigated and compared cellular uptake of RhB labeled RK/Parg/CMCS-NGs in B16F10 and NCTC 1469 cells at pH 6.5 and 7.4, followed by measuring with flow cytometry and fluorescence microscope. As shown in Figure 5C and Figure S8, B16F10 displayed obvious red fluorescence at pH 6.5 after 24h incubation of RK/Parg/CMCS-NGs, and generated 3.2-fold higher intracellular fluorescent intensity than NCTC 1469, demonstrating that RWrNR-kla can be released from RK/Parg/CMCS-NGs in acidic condition and efficiently internalized by targeting integrin αvβ3. In contrast, minimal accumulation of RK/Parg/CMCS-NGs in cancer cells was observed at pH 7.4, indicating that RK/Parg/CMCS-NGs can selectively target tumor acidic microenvironment, and prevent nonspecific uptake of RWrNR-kla in healthy tissues expressed integrin αvβ3.


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Intracellular trafficking of RK/Parg/CMCS-NGs. kla can disrupt the mitochondrial membrane mainly due to its α-helical strong positive electricity, leading to cell apoptosis.7 Therefore, selective delivery of kla to cancer cell mitochondria is a potent mitochondria-dependent cancer therapy. We further studied the intracellular location of RWrNR-kla peptide. B16F10 cells were incubated with RhB-labeled RK/Parg/CMCSNGs at pH 6.5, followed by visualizing with confocal laser scanning microscopy (CLSM) at 2h or 12h. As depicted in Figure 5D and Figure S9, RWrNR-kla peptide was selectively anchored on the cell membrane after 2h incubation, indicating that RWrNRkla peptide could be dissociated from nanogels, and can be embedded to cell membrane integrin αvβ3 receptors. However, RWrNR-kla selectively accumulated in mitochondria after intracellular uptake, due to positive charge of RWrNR-kla peptide, which can bind to the negative charged mitochondrial lipid membrane. This targeting ability of RWrNR-kla to integrin αvβ3 and mitochondria ensure it with highly specific and effective anticancer effects.


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Cellular uptake of RK/Parg/CMCS-NGs by macrophages. L-arginine that is the substrate of iNOS can stimulate the NO generation in macrophages.45 In recent years, various polyarginine peptides and peptidomimetics have been identified as highly efficient NO stimulators.28,

46

Particular, Parg and Parg conjugates can facilitate the

transduction of peptide, protein, and nucleic acid conjugates into a wide range of mammalian cell types, and especially macrophages. We therefore used FITC labeled Parg to prepare RK/Parg/CMCS-NGs, and investigated cellular uptake of Parg in RAW 264.7 cells at pH 6.5 and 7.4, followed by measuring with flow cytometry and fluorescence microscope. Meanwhile, we also mearsured cellular uptake of FITC labeled Plys of RK/Plys/CMCS-NGs, which was utilized as a control. As shown in Figure 5F and Figures S10-11, only minimal fluorescent intensity of Parg was detected in RAW 264.7 at pH 7.4 after 24h incubation. Whereas, RAW 264.7 displayed time-dependent cellular uptake of Parg at pH 6.5, and the obvious green fluorescence was observed after 24h incubation, generating 2.5-fold higher intracellular fluorescent intensity than cell incubated at pH 7.4. In addition, RK/Plys/CMCS-NGs displayed similar cellular uptake to RK/Parg/CMCS-NGs, demonstrating that both RK/Parg/CMCS-NGs and


32

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RK/Plys/CMCS-NGs can selectively release cationic polypeptide in response to tumor acidic condition, allowing Parg for targeting TAMs.

NO production induced by RK/Parg/CMCS-NGs in macrophages. NO is a ubiquitous free radical, and exhibits a critical function in various physiological and pathological processes, and especially in tumor angiogenesis, apoptosis, invasion and progression, making it a potential anticancer agent.47-50 Strategies for regulating in vivo production and exogenous delivery of NO attract more attention for improving therapeutic effects. Herein, we tested the ability of RK/Parg/CMCS-NGs to stimulate the production of NO by activated macrophages, and RK/Plys/CMCS-NGs were used as control. RAW 264.7 cells, pretreated with LPS for inducing iNOS expression in the cells, were incubated with RK/Parg/CMCS-NGs for 24h at pH 6.5 or 7.4, followed by measuring the levels of NO using NO kits. As shown in Figure 5G, both Parg/CMCS-NGs and RK/Parg/CMCS-NGs could promote NO production in macrophages at pH 6.5, and generated about 4-fold higher levels of NO, compared to the cells at pH 7.4. The accelerated production of NO from the Parg/CMCS-NGs and RK/Parg/CMCS-NGs were caused by acid-mediated


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liberation of Parg from nanogels, indicating that RK/Parg/CMCS-NGs can induce tumor specific NO therapeutics. However, RK/Plys/CMCS-NGs did not stimulate the production of NO, demonstrating that Parg is the essential substrate for iNOS in macrophages.

Synergistic anticancer effects of RK/Parg/CMCS-NGs in vitro. The combination therapy has been recognized as one of the most efficient anticancer treatment due to its potential of significantly improving therapeutic effects compared with single modality treatment. NO is an important intrinsic gas medium, and can affect many biological pathways, including cell apoptosis and proliferation. Therefore, NO-mediated anticancer method is a promising ideal strategy for chemotherapeutics. There is increasing evidence for the potential function of NO to increase toxic chemosensitizing.49 However, its application potential is hindered due to the low bioavailability, poor NO capacity and fast systemic clearance of NO-donors. Therefore, the stimuli responsive vehicles that can trigger the dose-controllable NO release have outstanding advantages in precise NO therapy. We therefore investigated the synergistic anticancer effects of either


34

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RK/Parg/CMCS-NGs or RK/Plys/CMCS-NGs (control) on B16F10 cells co-cultured with RAW 264.7 cells using transwell method (Figure 5E).

RAW 264.7 macrophages were seeded in transwell plate and pretreated with LPS for 12 h, followed by co-incubating with B16F10 cells co-cultured with RK/Parg/CMCS-NGs or RK/Plys/CMCS-NGs at pH 6.5. As shown in Figure 5H, no cytotoxicity was observed in B16F10 cells treated with Parg/CMCS-NGs in the absence of RAW 264.7 cells. In contrast, Parg/CMCS-NGs induced apparent cytotoxicity in the presence of RAW 264.7 cells, indicating that Parg can trigger the macrophage to initiate NO-mediated apoptosis. In addition, RK/Parg/CMCS-NGs displayed enhanced anticancer effects in B16F10 cells co-incubated with RAW 264.7 cells, compared with B16F10 cells alone. However, RK/Plys/CMCS-NGs displayed similar cytotoxicity to B16F10 cells with or without RAW 264.7 cells, indicating that Parg stimulated NO production provides a remarkable klaNO cooperative anticancer effects. Furthermore, we also identified that B16F10 cells generated higher intracellular ROS levels in the presence of NO, produced by macrophages with stimulation of RK/Parg/CMCS-NGs, in comparison of B16F10


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without NO activation (Figure 5I). Since RWrNR-kla elevated ROS levels, which consequently oxidase NO to form highly biocidal peroxynitrite molecules (ONOO−), NO cannot only cooperatively enhance the efficacy of chemotherapy, but directly kill cancer cells through the nitrosation of mitochondria and DNA.51

Inhibitory effects of RK/Parg/CMCS-NGs on tumor growth in vivo. kla has been identified as an effective agent for depolarizing mitochondrial membrane potential, but its anticancer effects are limited by low cellular uptake, unfavorable biostability and poor tumor specificity. Nanogels as an applicable peptide-loaded delivery system exhibits good biocompatibility, minimal systemic decomposition and enhanced tumor targeting, endowing peptides with in vivo practical potential.23 Importantly, pH-sensitive nanogels can effectively release apoptotic peptides at tumor acidic microenvironment, thereby improving antitumor effects. To investigate tumor accumulation ability, DiR-labeled RWrNR-kla, RK/Parg/CMCS-NGs and RK/Plys/CMCS-NGs were intravenously injected in B16F10 tumor-bearing mice, and Maestro EX fluorescence imaging system was used to image mice after 24h injection. Figure 6A shows that both RK/Parg/CMCS-NGs and


36

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RK/Plys/CMCS-NGs generated higher fluorescent signals in tumor regions than RWrNR-kla peptide, indicating that nanogels can improve the tumor accumulation of kla, and consequently enhance its therapeutic efficacy. In addition, biodistribution study shows that, in comparison of direct injection of RWrNR-kla, nanogels can effectively accumulate in tumor tissues. Importantly, nanogels were removed from major organs such as liver and spleen, suggesting that nanogels have the great potential in protecting RWrNR-kla from system clearance and accumulating RWrNR-kla in tumor tissues (Figure 6B and Figure S12).

In addition, the nanogels can enhance peptide biostability by protecting positive kla from hydrolase. We therefore measured the plasma concentration-time curves of RWrNR-kla after i.v. injection of RWrNR-kla solution, RK/Parg/CMCS-NGs and RK/Plys/CMCS-NGs in mice at a dose of 5 mg/kg of kla (Figure 6C). The blood concentration of RWrNR-kla quickly decreased from 409.33 ng/mL (5min) to 48.72 ng/mL after 2h administration of RWrNR-kla, indicating that RWrNR-kla has fast systemic clearance, which is consistent with pharmacokinetic profiles of peptide drugs.52


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However, RK/Parg/CMCS-NGs could improve the circulation of RWrNR-kla in the blood, and 1546.05 ng/mL of RWrNR-kla was detected after 5min administration, which is 4fold higher than direct administration of RWrNR-kla. In addition, RK/Plys/CMCS-NGs exhibited similar pharmacokinetics as RK/Parg/CMCS-NGs, further confirming that nanogels are a potent peptide delivery system. We also calculated the area under the curve (AUC0−inf) and clearance rate by Kinetica 4.4 (Table S1). Compared with RWrNRkla solution, nanogels dramatically increased AUC0−inf (around 8 folds) and significantly extended mean residence time (MRT, around 4 folds). These data demonstrate that nanogels can prevent rapid removal of peptide drugs from the body, and enhance their anticancer effects.

Furthermore, subcutaneous B16F10 xenograft model were used to investigate antitumor effects of nanogels. Mice were administrated intravenously with saline (control), RWrNR-kla, RK/Plys/CMCS-NGs, RK/Parg/CMCS-NGs, every 3 days for 13 days. Figure 6D shows that tumor growth could be effectively inhibited by RK/Parg/CMCS-NGs, and displayed 4.3-fold higher inhibition activity than RWrNR-kla,


38

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indicating that Parg based nanogels can effectively improve antitumor effects of RWrNR-kla. Importantly, RK/Parg/CMCS-NGs exhibited higher antitumor effects, compared to RK/Plys/CMCS-NGs, confirming enhanced synergistic antitumor effects of NO. In addition, at the end of study, mice were sacrificed and the tumors were collected. Figures 6E and 6G show that the tumor sizes in the RK/Parg/CMCS-NGs group were smaller than other treatment groups, suggesting that RK/Parg/CMCS-NGs exhibit strong inhibitory effects on tumor growth. Furthermore, RK/Parg/CMCS-NGs provided survival benefits compared to all the other groups (Figure 6F). For example, mice with untreatment had increased tumor growth and a median survival of about 15 days. In contrast, mice treated with RK/Plys/CMCS-NGs had an apparent decrease in tumor sizes with a median survival of about 20 days. Particularly, RK/Parg/CMCS-NGs significantly prolong mouse survival, and mice lived an average of 30 days, indicating that combination therapy of kla and NO is a potent antitumor strategy.

Moreover, RK/Parg/CMCS-NGs showed good biosafety, and no apparent decrease in body weights, tissue damages and other factors associated with toxic effects were


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observed during the treatment (Figures S13 and S14). It was reported that kla possesses effective anticancer capability by depolarizing mitochondrial membrane and activating apoptotic caspase pathway, and we therefore measured necrosis and apoptosis in tumors by staining caspase-3 and TUNEL. Figures 6H-I show that RK/Parg/CMCS-NGs significantly induced cell apoptosis and death in tumors through the caspase-3 pathway. Therefore, RK/Parg/CMCS-NGs are a desirable functional delivery system for kla, and hold a great potential for enhancing synergistic antitumor effects.

Conclusion

In summary, we have successfully developed a novel class of pH-sensitive nanogels (RK/Parg/CMCS-NGs), for enhancing synergistic anticancer effects of integrin αvβ3 specific apoptotic peptide and therapeutic nitric oxide. RK/Parg/CMCS-NGs are composed of Parg, CMCS (pI = 6.8) and a novel integrin αvβ3 peptide antagonist conjugated apoptotic peptide (RWrNR-kla), endowing great anticancer application potential. RK/Parg/CMCS-NGs cannot only prolong systemic circulation of RWrNR-kla,


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but allowing for selectively targeting tumor by responding to tumor acidic microenvironment and recognizing integrin αvβ3. Particularly, RK/Parg/CMCS-NGs can stimulate TAMs to generate NO, which consequently promotes anticancer effects of kla

in vitro and in vivo. Therefore, RK/Parg/CMCS-NGs are a desirable functional delivery system for kla, and hold a great potential for the treatment of malignant tumors.


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FIGURES

Figure 1. Smart acidic responsive nanogels (RK/Parg/CMCS-NGs) for enhancing synergistic anticancer effects of integrin αvβ3 specific apoptotic peptide (kla) and therapeutic nitric oxide (NO). pH-Sensitive nanogels, composed of RWrNR-kla, poly-Larginine (Parg) and carboxymethyl chitosan (CMCS, pI = 6.8), can not only effectively


42

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deliver kla into cancer cells by sequentially targeting tumor acidic microenvironment and cancer integrin αvβ3, but stimulate the generation of macrophage mediated NO, leading to highly synergistic anticancer effects.


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Figure 2. Development of an integrin αvβ3 antagonist (RWrNR) and its apoptotic peptide conjugate (RWrNR-kla). (A) The docking scores of peptide candidates. (B) The binding free energy of RWrNR and c-RGDyK. (C) The binding model between RWrNR and integrin αvβ3. (D) The binding affinity of RWrNR and c-RGDyK with integrin αvβ3. (E) CD spectrum of kla, RGDkla, RWrNR-kla and L-KLA in a PBS buffer (pH 7.4, 10 mM) containing 25 mM of sodium dodecylsulfate (SDS). (F) Serum stability of kla, RGD-kla and RWrNR-kla in 50% FBS. The data are presented as means ± s. d. (n=6).


44

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Figure 3. Cytotoxity of RWrNR-kla. (A, B) Dose-dependent cytotoxity of B16F10 cells and NCTC 1469 cells incubated with kla, RGD-kla and RWrNR-kla for 24h. (C, D) Timedependent cytotoxity of B16F10 cells and NCTC 1469 cells incubated with 10μM kla, RGD-kla and RWrNR-kla. (E, F) Cellular uptake profiles of kla, RGD-kla and RWrNR-kla in B16F10 cells and NCTC 1469 cells for 24h.

(G, H) Mitochondrial membrane

potentials (JC-1 assay) in B16F10 cells and NCTC 1469 cells determined by CLSM and flow cytometry after 24h incubation of 10μM kla, RGD-kla and RWrNR-kla. The data are


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presented as means ± s. d. (n=6). ***p < 0.001 (Student’s t test, two tails)


46

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Figure 4. Preparation and characterization of RK/Parg/CMCS-NGs. (A) Particle sizes and zeta potential of Parg/CMCS-NGs with different mass ratios of Parg to CMCS. (B) Particle sizes and zeta potential of RK/Parg/CMCS-NGs with different mass ratios of RWrNR-kla to CMCS, the ratio of Parg to CMCS fixed at 1:5. (C) Zeta potential of RWrNR-kla and RK/Parg/CMCS-NGs. (D) Particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency (EE) and drug loading rates (DL) of Parg/CMCS-NGs, RK/Parg/CMCS-NGs and RK/Plys/CMCS-NGs. (E) The TEM image and photograph of


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RK/Parg/CMCS-NGs. (F) Size distributions of RK/Parg/CMCS-NGs in PBS at pH 7.4 or 6.5. (G) In vitro drug release of RK/Parg/CMCS-NGs in PBS at pH 7.4 or 6.5 at 37 ℃. The data are presented as means ± s. d. (n=6).


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Figure 5. In vitro anticancer activity of RK/Parg/CMCS-NGs. (A) Cell viability of B16F10 cells treated with Parg/CMCS-NGs, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs in medium of pH 7.4. (B) Cell viability of B16F10 cells treated with Parg/CMCS-NGs, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs in medium of pH 6.5. (C) The cellular


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uptake of RhB labeled RWrNR-kla (3μM) of RK/Parg/CMCS-NGs in B16F10 and NCTC 1469 cells at pH 6.5 or 7.4. (D) Confocal laser scanning microscopy images of B16F10 cells 2h or 12h after incubation with RK/Parg/CMCS-NGs (3μM) at pH 6.5. (E) Synergistic effects of RWrNR-kla and NO therapeutics on B16F10 cells, co-cultured with RAW 264.7 cells using the transwell method. (F) The cellular uptake of FITC labeled Parg (100 nM), released from RK/Parg/CMCS-NGs, in RAW 264.7 cells at pH 6.5 or 7.4. (G) The production of nitric oxide in RAW 264.7 incubated with Parg/CMCSNGs, RK/Plys/CMCS-NGs, RK/Parg/CMCS-NGs (equivalent to10 μM of kla) in medium of pH 6.5 or 7.4. (H) Cell viability of B16F10 cells, co-cultured with RAW 264.7 cells, and

treated

with

Parg/CMCS-NGs,

RK/Plys/CMCS-NGs,

RK/Parg/CMCS-NGs

(equivalent to10 μM of kla) in medium of pH 6.5. (I) The fluorescence images of intracellular ROS in B16F10 cells stained by ROS dye Carboxy-H2DCFDA, co-cultured with

RAW

264.7

cells,

treated

with

Parg/CMCS-NGs,

RK/Plys/CMCS-NGs,

RK/Parg/CMCS-NGs in medium of pH 6.5. Values are the mean ± SD, n = 6. ***p < 0.001 (Student’s t test, two tails).


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Figure 6. In vivo anticancer effects of RK/Parg/CMCS-NGs in mice bearing B16F10 xenograft tumor. (A) In vivo fluorescent images of mice 24h after intravenous injection of DiR-labled RWrNR-kla, RK/Plys/CMCS-NGs, RK/Parg/CMCS-NGs (1mg/kg DiR). (B)

Ex vivo fluorescent image of fluorescence intensity of major organs and tumors. (C)


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Time-dependent plasma concentration of RWrNR-kla after intravenous injection of RWrNR-kla, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs at a dose of 5 mg/kg of kla. (D) Inhibition of tumor growth in mice treated with saline (Control), RWrNR-kla, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs. (E) Average weights of tumors after 13day treatment. (F) Survival curves of mice treated with saline (Control), RWrNR-kla, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs (n = 10). (G) Representative images of B16F10 xenograft tumors after 13-day treatments. (H) Histological analysis of tumor tissues, stained with Caspase-3 for apoptosis. (I) Histological analysis of tumor tissues, stained with TUNEL for apoptosis. Values are the mean ± SD, n = 5. ***p < 0.001 (oneway ANOVA).


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ASSOCIATED CONTENT

Supporting Information. The interaction of c-RGDyK with integrin αvβ3, mass spectrum of RWrNR-kla, HPLC analysis of kla, RGD-kla and RWrNR-kla, drug release of RK/Plys/CMCS-NGs, stability of Parg/CMCS-NGs, RK/Parg/CMCS-NGs and RK/Plys/CMCS-NGs, cell viability of NCTC 1469 cells treated with Parg/CMCS-NGs, RWrNR-kla and RK/Parg/CMCS-NGs, fluorescent images of RK/Parg/CMCS-NGs in B16F10 and NCTC 1469 cells, colocation of RhB-labeled RWrNR-kla and MitoGreen of B16F10 cells, cellular uptake and fluorescent images of FITC labeled Parg, in vivo biodistribution, body weights, histopathological analyses of major organs and pharmacokinetic parameters. (PDF)

AUTHOR INFORMATION

Corresponding Author * E-mail: [email protected] (Y. Zhang)

* E-mail: [email protected] (X. Liu)


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* E-mail: [email protected] (X. Ning)

Author Contributions X.N. and X.L. designed and developed the research. Y.X., L.S., S.F. and J.C. performed the cell experiments. Y.G., L.G., X.A. and Y.N. contributed to animal experiments. Y.X., Y.Z., and X.N. wrote the manuscript. All authors contributed to general discussions and edited and reviewed the manuscript.

Funding Sources The works were supported by the National Key Research and Development Program of China (Grant No. 2018YFB1105400), the National Natural Science Foundation of China (Grant No. 21708019), the Thousand Talents Program for Young Researchers, the Natural Science Foundation of Jiangsu Province (Grant No. BK20180334), the Shuangchuang Program of Jiangsu Province, the Fundamental Research Funds for Central Universities Nanjing University and the Scientific Research Foundation of Graduate School of Nanjing University (Grant No. 2017ZDL04).

Notes


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The authors declare no competing financial interest.

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Figure 1. Smart acidic responsive nanogels (RK/Parg/CMCS-NGs) for enhancing synergistic anticancer effects of integrin αvβ3 specific apoptotic peptide (kla) and therapeutic nitric oxide (NO). pH-Sensitive nanogels, composed of RWrNR-kla, poly-L-arginine (Parg) and carboxymethyl chitosan (CMCS, pI = 6.8), can not only effectively deliver kla into cancer cells by sequentially targeting tumor acidic microenvironment and cancer integrin αvβ3, but stimulate the generation of macrophage mediated NO, leading to highly synergistic anticancer effects.



Figure 2. Development of an integrin αvβ3 antagonist (RWrNR) and its apoptotic peptide conjugate (RWrNRkla). (A) The docking scores of peptide candidates. (B) The binding free energy of RWrNR and c-RGDyK. (C) The binding model between RWrNR and integrin αvβ3. (D) The binding affinity of RWrNR and c-RGDyK with integrin αvβ3. (E) CD spectrum of kla, RGD-kla, RWrNR-kla and L-KLA in a PBS buffer (pH 7.4, 10 mM) containing 25 mM of sodium dodecylsulfate (SDS). (F) Serum stability of kla, RGD-kla and RWrNR-kla in 50% FBS. The data are presented as means ± s. d. (n=6).


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Figure 3. Cytotoxity of RWrNR-kla. (A, B) Dose-dependent cytotoxity of B16F10 cells and NCTC 1469 cells incubated with kla, RGD-kla and RWrNR-kla for 24h. (C, D) Time-dependent cytotoxity of B16F10 cells and NCTC 1469 cells incubated with 10μM kla, RGD-kla and RWrNR-kla. (E, F) Cellular uptake profiles of kla, RGD-kla and RWrNR-kla in B16F10 cells and NCTC 1469 cells for 24h. (G, H) Mitochondrial membrane potentials (JC-1 assay) in B16F10 cells and NCTC 1469 cells determined by CLSM and flow cytometry after 24h incubation of 10μM kla, RGD-kla and RWrNR-kla. The data are presented as means ± s. d. (n=6). ***p < 0.001 (Student’s t test, two tails)



Figure 4. Preparation and characterization of RK/Parg/CMCS-NGs. (A) Particle sizes and zeta potential of Parg/CMCS-NGs with different mass ratios of Parg to CMCS. (B) Particle sizes and zeta potential of RK/Parg/CMCS-NGs with different mass ratios of RWrNR-kla to CMCS, the ratio of Parg to CMCS fixed at 1:5. (C) Zeta potential of RWrNR-kla and RK/Parg/CMCS-NGs. (D) Particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency (EE) and drug loading rates (DL) of Parg/CMCS-NGs, RK/Parg/CMCS-NGs and RK/Plys/CMCS-NGs. (E) The TEM image and photograph of RK/Parg/CMCS-NGs. (F) Size distributions of RK/Parg/CMCS-NGs in PBS at pH 7.4 or 6.5. (G) In vitro drug release of RK/Parg/CMCS-NGs in PBS at pH 7.4 or 6.5 at 37 ℃. The data are presented as means ± s. d. (n=6).


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Figure 5. In vitro anticancer activity of RK/Parg/CMCS-NGs. (A) Cell viability of B16F10 cells treated with Parg/CMCS-NGs, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs in medium of pH 7.4. (B) Cell viability of B16F10 cells treated with Parg/CMCS-NGs, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs in medium of pH 6.5. (C) The cellular uptake of RhB labeled RWrNR-kla (3μM) of RK/Parg/CMCS-NGs in B16F10 and NCTC 1469 cells at pH 6.5 or 7.4. (D) Confocal laser scanning microscopy images of B16F10 cells 2h or 12h after incubation with RK/Parg/CMCS-NGs (3μM) at pH 6.5. (E) Synergistic effects of RWrNR-kla and NO therapeutics on B16F10 cells, co-cultured with RAW 264.7 cells using the transwell method. (F) The cellular uptake of FITC labeled Parg (100 nM), released from RK/Parg/CMCS-NGs, in RAW 264.7 cells at pH 6.5 or 7.4. (G) The production of nitric oxide in RAW 264.7 incubated with Parg/CMCS-NGs, RK/Plys/CMCS-NGs, RK/Parg/CMCS-NGs (equivalent to10 μM of kla) in medium of pH 6.5 or 7.4. (H) Cell viability of B16F10 cells, co-cultured with RAW 264.7 cells, and treated with Parg/CMCS-NGs, RK/Plys/CMCS-NGs, RK/Parg/CMCS-NGs (equivalent to10 μM of kla) in medium of pH 6.5. (I) The fluorescence images of intracellular ROS in B16F10 cells stained by ROS dye Carboxy-H2DCFDA, co-cultured with RAW 264.7 cells, treated with Parg/CMCS-NGs, RK/Plys/CMCS-NGs, RK/Parg/CMCS-NGs in medium of pH 6.5. Values are the mean ± SD, n = 6. ***p < 0.001 (Student’s t test, two tails).



Figure 6. In vivo anticancer effects of RK/Parg/CMCS-NGs in mice bearing B16F10 xenograft tumor. (A) In vivo fluorescent images of mice 24h after intravenous injection of DiR-labled RWrNR-kla, RK/Plys/CMCSNGs, RK/Parg/CMCS-NGs (1mg/kg DiR). (B) Ex vivo fluorescent image of fluorescence intensity of major organs and tumors. (C) Time-dependent plasma concentration of RWrNR-kla after intravenous injection of RWrNR-kla, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs at a dose of 5 mg/kg of kla. (D) Inhibition of tumor growth in mice treated with saline (Control), RWrNR-kla, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs. (E) Average weights of tumors after 13-day treatment. (F) Survival curves of mice treated with saline (Control), RWrNR-kla, RK/Plys/CMCS-NGs and RK/Parg/CMCS-NGs (n = 10). (G) Representative images of B16F10 xenograft tumors after 13-day treatments. (H) Histological analysis of tumor tissues, stained with Caspase-3 for apoptosis. (I) Histological analysis of tumor tissues, stained with TUNEL for apoptosis. Values are the mean ± SD, n = 5. ***p < 0.001 (one-way ANOVA).


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