Targeting Delivery of Lidocaine and Cisplatin by Nanogel Enhances

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Biological and Medical Applications of Materials and Interfaces

Targeting Delivery of Lidocaine and Cisplatin by Nanogel Enhances Chemotherapy and Alleviates Metastasis Xiurong Gao, Hui Yang, Min Wu, Kun Shi, Cheng Zhou, Qian Yang, and JinRong Peng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09376 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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ACS Applied Materials & Interfaces

Targeting Delivery of Lidocaine and Cisplatin by Nanogel Enhances Chemotherapy and Alleviates Metastasis Xiurong Gao a, #, Hui Yang a, #, Min Wu a, Kun Shi b, Cheng Zhou c, Qian Yang

a,

*,

Jinrong Peng b, *,

a

School of Pharmacy, Collaborative Innovation Center of Sichuan for Elderly Care and

Health, Sichuan Province College Key Laboratory of Structure-Specific Small Molecule Drugs, Chengdu Medical College, No. 783, Xindu Avenue, Xindu District, Chengdu, Sichuan, 610500, P. R. China. b

State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and

Collaborative Innovation Center, No. 17, Section 3, Southern Renmin Road, Chengdu 610041, Sichuan, P. R. China c

Laboratory of Anesthesia & Critical Care Medicine, Translational Neuroscience

Center, West China Hospital of Sichuan University, Chengdu 610041, Sichuan, P.R. China.

KEYWORDS: nanogels; combinational therapy; metastasis; tumor-targeting; adverse effects

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ABSTRACT Tumor growth inhibition and adverse effect reduction together with metastasis alleviation are still the challenges need to be overwhelmed in cancer chemotherapy. Combinational therapy provides an alternative solution for these challenges. And nanoparticles are the ideal carriers for combinational therapy due to their versatile drugs loading capacity and versatile tumor-targeting strategies. In this study, a cRGDfk modified nanogel system has utilized to co-load lidocaine, a voltage-gated Na+ channels inhibitor, and cisplatin, a common anticancer drug to obtain a tumor-targeted dual drugs-loaded nanogel system. The introduction of lidocaine not only promotes the cisplatin-induced apoptosis in vitro and in vivo, but also alleviates the metastasis of MDA-MB-231 breast cancer cells in mouse model. Besides, the body weight loss caused by cisplatin has also been relieved, and higher dose with less body weight loss can be achieved, which indicated the adverse effect caused by cisplatin-mediated chemotherapy has been alleviated. Furthermore, the introduction of peptide segment-cRGDfk, which presents high affinity to αvβ3 integrin, further increases the enrichment of drugs-loaded nanogel in tumor site. It favors the primary tumor growth inhibition. The results demonstrate the co-loading of lidocaine and cisplatin by ligand-modified nanogels is a promising strategy for αvβ3 integrin-overexpressing breast cancer combinational therapy.


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INTRODUCTION

Although the therapeutic outcome of some novel cancer treatments in recent years has been proved to be the promising choices for cancer therapy, chemotherapy is still the widest-used manner for cancer therapy.1 However, the unsatisfactory tumor growth inhibition companying with severe adverse effects are impeding the applications of chemotherapy and lowering the living quality of cancer-bearing patients.2 Besides, the main reason for the death of cancer-bearing patient is metastasis, but chemotherapy alone cannot inhibit the tumor metastasis efficiently. It is another major drawback of chemotherapy.3 How to improve the therapeutic outcome, particularly strengthen the tumor metastasis inhibition during chemotherapy is the major challenge for cancer chemotherapy.4 Combinational therapeutic strategies have provided infinite possibilities for the improvement of chemotherapy.5 The progress and metastasis of cancer depend on numerous pathways and mechanisms, so as to get enough nutrient from the host and avoid the surveillance and elimination of immune system6-9. On the opposite, it also provides numerous targets for tumor growth inhibition. Many chemodrugs have been invented and used in clinic. However, by administrating with single chemodrug cannot obtain expected therapeutic outcome, and the high drug-dosage causes unexpected adverse effect, which may also be another lethal threat to the patients. The administration of the combinations of different chemodrugs may enhance the tumor growth inhibition, but the adverse effects may also be aggravated. In recent years, the introduction of some other drugs which is used to treat non-cancer diseases has provided an alternative choice for chemotherapy improvement.10 For example, the introduction of metformin not only is favor of prolonging tumor growth remission by decreasing the dose of chemodrugs, but also exhibits synergistic effects with paclitaxel and doxorubicin in cancer therapy.11-12 Although the non-chemodrugs still exist some adverse effects, they are more gentle and can be alleviated, which make the non-chemodrug a promising candidate for cancer combinational therapy. In general, metastasis involves three steps: first, cancer cells detach from the primary site, then they invade into the surrounding tissues, and finally establish metastasis in other tissues by disseminating to distant sites.13-14 And the movement of the cancer cell



membrane involves in every step of the metastasis of cancer cells, and acts as one of the controllers. Several signal pathways and mechanisms which contribute to the movement of cancer cell membrane have been identified.15-16 Ion channels, particular the voltage-gated Na+ channels (VGSCs) are attracting enormous attentions due to the development of VGSCs inhibitors which have been widely used in many non-cancer diseases, such as epileptic attack, arrhythmia, and acute myocardial infarction, etc. It has also been revealed in pre-clinical studies that VGSCs can promote metastasis in metastasis cancer cells by regulating the cell behaviors which include migration and invasion.17 VGSCs are the potential targets for reducing local invasion and metastasis. Kinds of VGSCs inhibitor have been developed and applied in clinic, including lidocaine, mexiletine, phenytoin and quinidine etc.18-19 Among them, it has been revealed that the lidocaine can inhibit the morphological change of cancer cells and inhibit microtentacle attachment, microfilament organization and cell adhesion, etc. in vitro.20-21 Furthermore, the lidocaine can also enhance the cisplatin-induced apoptosis.22-23 However, because of the rapid clearance of lidocaine and cisplatin, the combinational therapeutic outcome of lidocaine and cisplatin in vivo remains unclear. The development of nanomaterials and nanotechnology may provide an option for evaluating this combinational therapeutic strategy. As drug delivery system, nanocarriers have proved their potentials.24-25 They not only can be served as ideal carriers for combinational therapy due to their drugs-encapsulated capacity, but also be the carriers for multi-functionalizations, such as photothermal conversion, magnetic resonance imaging, and active targeting, etc.26 The passive targeting, which is the result of enhanced permeability and retention (EPR) effect, plus with active targeting, which can be accomplished by ligand modification on the surface of nanoparticle, provide versatile strategies for tumor targeting of drug-loaded nanocarriers.27 In our previous reports, we have developed a pH-thermal nanogel system for cisplatin delivery. By the introduction of this nanogel system, the tumor growth inhibition of cisplatin has been enhanced in vitro and in vivo, and the adverse effect of cisplatin (kidney damage) has also been alleviated.28 But the tumor growth inhibition of cisplatin-loaded nanogel still needs to be improved due to the absence of tumor targeting ligand modification. Then, we used the similar nanogel system which was modified with peptide segments (KLWVLPKGGGC) targeting to type IV to load hydrophobic drug-rapamycin. The obtained type IV-targeted


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rapamycin-loaded nanogels exhibited enhanced alleviation of vascular restenosis in vivo.29 The results demonstrate that the nanogel systems can be served as nanocarriers for hydrophilic and hydrophobic drugs as well as active targeting. It further indicates that the nanogel can be used to co-load lidocaine (hydrophobic form) and cisplatin. Herein, based on our previous studies, we have designed and synthesized a nanogels system conjugated with cyclic (Arg-Gly-Asp-D-Phe-Lys) (cRGDfk) peptide which showed a high affinity for αvβ3 integrin, an overexpressed protein on most of tumor cells, such as mammary carcinoma cells,30 and the targeted nanogels system was utilized for cisplatin (CDDP) and lidocaine (Lido) co-delivery (scheme 1A). Meanwhile, the controlled drug release efficiency which accelerated the release of both chemotherapeutic moieties in an acidic tumor environment, helped to improve drug distribution, then enhanced therapeutic outcome of chemotherapy in vitro and in vivo (scheme 1B). Thus, the ligand-modified and dual drugs-co-loaded nanogel (NG) system enables the enhancement of targeting delivery of chemodrugs as well as alleviation of metastasis by combinational therapy.

MATERIALS AND METHODS Materials N-isopropylacrylamide

(NIPAm),

methacrylic

acid

(MAA),

poly(ethylene

glycol)methacrylate (PEGMA, Mn 360), N,N-methylene bis(acrylamide) (BIS), 2,20-azoisibutyronitrile (AIBN), N-hydroxysuccinimide (NHS), 1-(3-(dimethylamino) propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), methyl thiazolyl tetrazolium (MTT), were purchased from Sigma-Aldrich (St. Louis, Missouri) and used as received. Ferric chloride hexahydrate and ferrous chloride tetrahydrate were purchased from Aladdin (china). Cisplatin (CDDP, MW: 300.3) was obtained from Dalian Meilun Biotechnology, CO., LTD. And the lidocaine hydrochloride was purchased from Fortune Zhaohui Pharmaceutical Co. Ltd. (Shanghai, China). The culture medium such as Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 Medium were purchased from Gibco (Thermo Fisher Scientific, USA) as well as antibiotic/antimycotic solution and fetal bovine serum. All other solvents and reagents used in this study were analytical reagent grade. Cell lines: Kinds of cell lines, including MCF-7, MDA-MB-231 and HUVEC cell lines, were all purchased from American Type Culture Collection (ATCC, Rockville,



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MD) and passaged or preserved for further used. Cell culturing procedure is similar with our previous report.31 The culture medium is of DMEM medium supplemented with 10% of FBS, 1% of penicillin/streptomycin, respectively. Animals: The animals (balb/c-nu mice, 4-6 weeks old) were purchased from Beijing Weitong Lihua Bioscience Co. Ltd, China. All animal care and experimental protocols were performed in compliance with Guide for the Care and Use of Laboratory Animals in Chengdu Medical College. Construction of nanogels Synthesis of the cRGD conjugated nanogel (cRGD-NG): The p(NIPAM-co-MAA) nanogel (NG) was synthesized by emulsion polymerization according to the previous report.32 After 5 days’ purification by dialysis, the obtained NG (10 mg/mL) was incubated in 100 mM MES for 2 h at room temperature in the presence of EDC (115 mM) and NHS (30 mM). The activated NG were then incubated with cRGDfk peptide (3.5 mg/mL) overnight at room temperature. The as-prepared cRGD-NG were purified by dialysis (MWCO: 8,000-14,000 Da) for 24 hours at room temperature. Preparation of CDDP-coordinating nanogel (cRGD-NG-Pt and NG-Pt), and Lido-loaded cRGD-NG-Pt (Lido/cRGD-NG-Pt): The obtained cRGD-NG desperation was pre-iced and stirred for 10 min, and then a predetermined amount of CDDP was added into the above solution with adjusting its pH to 8.0 with NaOH (50 mM) subsequently. The mixture was maintained vigorously stirring for another 24 h in the dark. To remove free CDDP, the mixture was dialyzed against water with a dialysis (MWCO: 7,000 Da) for 4 h and followed by lyophilization in the dark, yielding cRGD-NG-Pt. The NG-Pt without cRGD modification was also prepared as a control from bared NG. The Lido-loading procedure was followed the method mentioned above by adding an aqueous solution of lidocaine hydrochloride dropwise into the pre-iced basic solution, which

means

the

Lido-loading

and

CDDP-incorporating

were

conducted

simultaneously. The obtained Lido/cRGD-NG-Pt desperation was also dialyzed and followed by lyophilization in the dark. For an intracellular uptake study, we chose coumarin-6 (C6) as the fluorescence tracker and C6-loaded NG (C6/NG, C6/cRGD-NG and C6/cRGD-NG-Pt) were prepared by the drug-loading method for hydrophobic drug described in our previous


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work. While for in vivo imaging studies, the DID as near-infrared (NIR) fluorescent probe was loaded into NG system with the same method as C6-loaded NG prepared. The content of CDDP in different formulation was calculated by platinum concentration which measured by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES, SPECTROARCOS, Spectro, Germany). The content of Lido in Lido/cRGD-NG-Pt was determined by reversed-phase high performance liquid chromatography (HPLC) system (HPLC 1260, Agilent, US, with a C18 column, 4.6 mm x 150 mm, 5 mm). The reported results of each sample were the average of three replicates. The drug loading capacity and entrapment efficiency were calculated according to the following formula: Drug loading capacity (DL) = (Mass of drugs loaded in nanogels / Total mass of drugs and nanogels) * 100% Entrapment efficiency (EE) = (Experimental drug loading / Theoretical drug loading) * 100% Morphology characterizations: Particle size distribution and zeta-potential were determined by dynamic light scattering (DLS) analysis using a Malvern Nano-ZS 90 analyzer. The temperature was kept at 25oC during measurement. All results are presented as the mean of three test runs and expressed as the mean ± SD. The

surface

morphology

of

cRGD-NG,

NG-Pt,

cRGD-NGs-Pt

and

Lido/cRGD-NGs-Pt were observed by transmission electron microscope (TEM, H-6009IV, Hitachi, Japan). The accelerating voltage is of 180 kV. Fourier Transform Infrared Spectroscopy (FT-IR): The surface chemistry of the lyophilized NG formulations was evaluated using FT-IR analyses on a Nicolet 6700 FTIR spectrometer (Thermo Scientific). Molecular Binding Energy: The existence of Pt(II) moiety valence states in cRGD-NGs-Pt structure was confirmed by the binding energy of platinum measured by X-ray photoelectron spectroscopy (XPS, XSAM 800, Kratos, UK). In vitro Drug release profile of the Lido/cRGD-NG-Pt



In

vitro

release

studies

were

carried

out

at

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physiological

temperature.

Lido/cRGD-NG-Pt dispersion and CDDP (or lidocaine hydrochloride) solution were placed separately in a dialysis bag (MWCO: 3500 Da) and sealed. At each determined time point, the releasing medium was collected and replaced by fresh medium. The collected samples were determined concentration of CDDP by ICP-AES and that of Lido by HPLC, respectively. Both pH 7.4 and 5.5 PBS buffer (0.01 M, contained 150 mM NaCl and 0.5% of Tween-80 at 37 oC) were chosen to investigate the influence of environment to drug release profile. In Vitro Cytotoxicity Cells Uptake Assay: For the cells uptake assay, MDA-MB-231 and MCF-7 cells were seeded onto glass slips in 6-well plate and cultured for 24 h. After incubated with predicted doses of C6/NG, C6/cRGD-NG, C6/cRGD-NG-Pt for 4 h, cells were further labeled with DAPI (5 ug/mL) after being washed with PBS, then fixed with 4% paraformaldehyde consequently. The cellular uptake imaging were visualized by fluorescence microscope (Zeiss OBSERVER D1/AX10 cam HRC). Quantificational evaluation of cellular uptake was conducted by flow cytometry. After incubated with different formulations for various durations, cells were washed by PBS and then collected for flow cytometric analysis (NovoCyteTM Flow Cytometer, ACEA Bioscience. Inc., USA). Cytotoxicity: The cytotoxicity of blank cRGD-NG or different formulation of CDDP was evaluated by MTT assay. The MDA-MB-231and MCF-7 cells were seeded in 96-well plates (5 × 103 cells/well) in 100 µL of culture medium and pre-incubated for 24 h. The culture medium was replaced with 200 µL of fresh medium containing with different

concentration

of

cRGD-NG,

free

CDDP,

cRGD-NG-Pt,

and

Lido/cRGD-NG-Pt. The survivals of the cells were measured by MTT assay after being incubated for another 24 h, 48 h and 72 h. The IC50 of each sample was calculated based on the mean percentage of cell survival, and all data were presented as means ± SD (n = 6). Cell Apoptosis Analysis: The drug-induced cell apoptosis was detected by flow cytometry

with

commercial

Annexin

V-FITC/PI

apoptosis

detection

kit.

5

MDA-MB-231cells were seeded in 6-well plates (5 × 10 cells/well) and pre-incubated for 24 h. Sequentially, cells were treated with free Lido, Lido/cRGD-NG,


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cRGD-NG-Pt and Lido/cRGD-NG-Pt, respectively, and the untreated cell was used as negative control. The final concentration of CDDP and Lido in various NG formulations settled as 10 µM (3 µg/mL) and 50 µM (11.7µg/mL), respectively. Before flow cytometry analyzing, the cells were further cultured for 24 h, then were trypsinized, collected and stained with fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC) and propidium iodide (PI), and incubated in the dark for another 15 min (RT). The fluorescence intensity was analyzed by NovoCyteTM Flow Cytometer (ACEA Bioscience. Inc., USA) and 10,000 events collected. In vivo MDA-MD-231 tumor targeting efficiency study The enrichment of cRGD-NG system in MDA-MD-231 tumors established on Balb/c-nu mice was visually and semi- quantitatively evaluated by in vivo NIR-fluorescence imaging. NIR fluorescent dye, DID, was used for better distinguishing. When the tumor volume reached 150 mm3, mice were administered intravenously with 5% glucose, free DID, DID/NG, and DID/cRGD-NG (approximately at 100 µg/kg of DID). Fluorescence living-image was carried out after 0, 1, 2, 8 and 24 h with the IVIS Lumina XR system (excitation = 645 nm, emission = 715 nm long pass). After 24 h, the major organs of the mice were eviscerated for ex-vivo fluorescence imaging. In vitro and in vivo MR imaging We used Fe3O4 nanoparticles as a substitute for drug loading to visualize the cRGD-NG MRI imaging. 20 mg of the purified NG or cRGD-NG were pre-iced for 10 min, and 500 µL prepared Fe3O4 nanoparticles chloroform solution was added dropwise. Then, the solution was vortexed vigorously for 10 min followed by magnetically stirring and slowly warmed to room temperature overnight. The MRI signals of Fe3O4/cRGD-NG in aqueous solution with different concentrations were measured by Animal MRI instrument

(BioSpec70/20USR,

Bruke).

To

test

the

contrast

ability,

the

Fe3O4/cRGD-NG dispersions at various concentrations of Fe (quantitatively determined by TGA) were evaluated, and water was used as control. Magnetic field strength is 7T, and turbo RARE sequence for T2. Same animal MRI instrument was used to evaluate the enrichment of Fe3O4/cRGD-NG in tumor site in vivo. The mice for tumor imaging were all anesthetized by general inhalation anesthetic (1.5% isoflurane in a mixture of O2/N2). The T2-weighted MR



images of the mice at pre-injection time-point were collected. Then, 200 µl dispersion of Fe3O4/NG or Fe3O4/cRGD-NG (as the Fe content of 2 mg/kg in each sample determined by TGA) was injected intravascularly, respectively, and the T2-weight MR images were also collected at 8 h and 24 h post-injection, respectively. After 24h, the major organs of the mice were harvested for quantitative analysis of the bio-distribution of the nanomaterials by ICP-AES. Anticancer performance of Lido/cRGD-NG-Pt Antitumor activity of Lido/cRGD-NG-Pt was evaluated in MDA-MB-231 subcutaneous tumors bearing-Balb/c nu mice. The animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals of Chengdu Medical College, China. After the tumors growing until reaching the average volume of 70 mm3, mice were divided randomly into six groups (n=5), and subsequently, injected with 5% glucose, free CDDP (3 mg/kg), free CDDP (3 mg/kg) plus Lido (12 mg/kg), cRGD-NG-Pt (3 mg/kg on a CDDP base), Lido/cRGD-NG-Pt (with 3 mg/kg of CDDP and 12 mg/kg of Lido, respectively) and Lido/cRGD-NG-Pt (with 6 mg/kg of CDDP and 24 mg/kg of Lido, respectively). Drug administrations were carried out on days 0, 2, 6 and 12 by intravenous injection. The dose of CDDP solution (3 mg/kg) was decided by the tolerable body weight loss for treatment. Tumor volumes and body weight for each group were measured in one-day interval. After 24 days’ record, mice were sacrificed and the main organs include tumors were excised for further study. Histological and immunohistochemical analyses All the tumors issues were fixed in 4% paraformaldehyde for 24 h, and then exposing to 70% ethanol overnight and embedding in paraffin. Apoptotic cells within tumors were detected by a commercial terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling kit (TUNEL, Promega, Madison, WI, USA). The TUNEL assay was performed following the manufacturer's protocols, and the apoptosis cells were identified by observing the bright green fluorescence via fluorescence microscope (400 x magnification). The lung metastasis of each group was evaluated by counting the naked-eye visible metastasis node in lung tissue, and immersed in formalin (4%, pH=7.4) for


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hematoxylin eosin (H&E) staining and assessed histological alterations by microscope (400 x magnification). Statistics analysis SPSS software was used for statistical analysis. All results were present as mean ± SD. ANOVA method was employed for significant difference identification (while p < 0.05 were considered statistically significant). RESULTS AND DISCUSSIONS Construction and Characterization of cRGD-NG and CDDP&Lido-Based Drug loading. The PNIPAM-co-MAA nanogels with 25% ratio of MAA is prepared from previous synthesis method.32 cRGDfk peptide sequence was conjugated with carboxylates groups (–COOH) of MAA under the sequent activation of EDC and NHS, yielding cRGD-NG. The molar ratio for cRGD was set at 20% of the total number of carboxylates groups in the NG molecular, leading to conjugation of the cRGDfk onto the surface of NG system. cRGD-NG-Pt was constructed by ligand exchange of Clwithin CDDP and –COO- from resident –COOH of MAA and Asp, under a basic condition. Finally, Lido was loaded mainly by deprotonating and entrapping in the NG network at a basic condition (Scheme 1 A).33 The NG-Pt and cRGD-NG-Pt both had narrow size distribution with hydrodynamic diameters of 136.5 ± 4.8 nm and 148.1 ± 3.6 nm, respectively, which were all slight smaller than that of the cRGD-NG (162.2 ± 4.5 nm), suggesting the shrinkage of nanogel caused by the interaction between Pt moiety and carboxylic group (Figure 1A). And the surface charge of Pt-incorporated NGs were about -8.8 mV and -7.3 mV, respectively, which present mild increase to that of the cRGD-NG (-13.5 mV) since the incorporation with Pt moieties (Figure 1B). The dual-drug loaded NG (Lido/cRGD-NG-Pt) with average size of 157.6 ± 7.4 nm and Zeta potential of -9.2 mV was prepared for further investigation (Figure 1A and B, Table S1). From the TEM imaging, the obtained NG-Pt, cRGD-NG-Pt and Lido/cRGD-NG-Pt still maintained typical spheres architecture and homogeneously distributed, as the bared NG did. While being compared with bared NG, the dark grey morphology of CDDP-loaded particles which is the results of ultrasmall Pt clusters formed inside the nanogel structure indicated that the Pt moiety was successfully coordinated with the



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carboxylates groups on NG system, which was also consistent with the appearance of the NG dispersion changed from transparence opalescence to transparence gray, when compared with the NG (Figure 1 C). 34 All the structural compositions of NG-based materials were further evaluated by FTIR and XPS measurement. The disappearance of -COOH adsorption bands in FTIR spectrum indicates the success of cRGD and CDDP modification (Figure S-1A). Meanwhile, the peaks (72.6 eV and 74.4 eV) corresponding to the binding energy of the divalent Pt in XPS analysis of cRGD-NG-Pt further illustrates the incorporation of –COOH and Pt (Figure S-1B). The content of Pt was measured by ICP-AES, and the final calculated CDDP-loading capacity of NG-Pt and cRGD-NG-Pt were 16.2 % and 15.7 %, respectively. For further in vitro and in vivo investigation, the loading content of CDDP and Lido were settled at 6.3% and 23.5% respectively, by varying the drugs feeding ratio (Table S1).

In vitro drug release In addition to targeting effect of cRGD peptide, the pH-dependent drug release from the drug carriers was monitored over 72 h at 37 °C with pH of 7.38 and 5.5, which mimicked the pH values of the circulation and tumor endosomes, respectively.35 The CDDP release in the buffer solutions at the pH of 5.5 and 7.38 were calculated by the concentration

of

Pt

monitored

by

ICP-AES.

The

CDDP

release

from

Lido/cRGD-NG-Pt were determined to be 54.9% and 29.2% at the pH of 5.5 and 7.38, respectively, in the first 8 h. After 72 h investigation, the Lido/cRGD-NG-Pt present more cumulative release of CDDP at pH 5.5 (64.4%) than that of pH 7.4 (38.5%) (Figure 1D, left). That is, the carboxylate ligand coordination was exposed to the Clattacking directly, which made parts of CDDP release from the Lido/cRGD-NG-Pt, even in the pH 7.38 buffer.36-37 The mean release rate of CDDP at the pH of endosome (5.5) was accelerated by a factor of two, which compared with that at the pH of the cell culture medium (7.38), indicating the acid-responsive CDDP release, which can be attributed to a further decrease in the stability of the ligand−platinum coordination. Thus, a small amount of CDDP released in the blood circulation (pH7.38) and enhanced drug release occurs when the NG systems are internalized into endosomal compartments, which would result in significantly reduced systemic toxicity.


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Meanwhile, the residual amount of Lido in NG-based formulation at physiological condition (pH 7.38) was larger than that of tumor endosome (pH 5.5), after 72 h. On the other hand, Lido/cRGD-NG-Pt at pH 5.5 also exhibited the pH controlled drug release profile with total Lido released up to 60% within the initial 8 h, and followed by continuous slow release (Figure 1D, right). The rather fast diffusion of Lido from the NG

network

demonstrated

acidic environment.

31, 38

that

Lido was almost entirely ionized in the

Furthermore, at pH 7.4, the release of CDDP is much faster

than that of lidocaine. The reason may be that the release of CDDP at pH 7.4 mainly depended on the concentration of Cl- in body circulation, resulted in the nearly 30% of drug release in the first 8 h (Figure 1D, left). While most of the lidocaine was deionized and hydrophoblic at physiological condition, so the release of lidocaine from the nanogel system present much slower than that of CDDP at pH7.4. Even through, the cRGD-NG still can be used as a pH-responsive drug carrier.

In vitro cytotoxicity The NG system with C6-loading was used for cell labeling and intracellular tracking. The cellular uptake was performed by MDA-MB-231 cells and MCF-7 cells for cubulation for 4 h, and the majority fluorescent signal were localized in cytoplasm of the cells form the merge images. As exhibited in Fig. 2A, cRGD-NG showed significant cellular uptake enhancement than NG in MDA-MB-231 cells, while the nearly equal amount of fluorescence signal was observed in MCF-7cells. Quantitative analysis (Figure 2B and S-2A) also demonstrated the similar result. So it was indicated that cRGD-NG exhibited a greater binding efficacy to αvβ3 integrin which highly expressed on MDA-MB-231cells, compared to MCF-7 cells with negative αvβ3 expression (confirmed by western blotting assay, figure 2C). In addition, compared to MDA-MB-231cells treated with cRGD-NG-Pt at pH 7.4, more effective internalization was observed when that at pH 6.5, which indicated that the cRGD peptide partly shielded by CDDP can be regenerated in acidic extracellular microenvironment (Figure 2D and E).39 The cytotoxicity of Lido/cRGD-NG-Pt was evaluated by MTT assay. Firstly, the cell survival of MDA-MB-231, MCF-7 and HUVEC cells co-cultured with blank cRGD-NG were usually higher than 85%, even with a high concentration of 1000



ug/mL, indicating the biosafety of the nanocarriers (Figure 3A). Moreover, we also investigated the chemotherapeutic effect of different CDDP formulation on the survival of MDA-MB-231 and MCF-7 cells (Figure 3B). The IC50 of the cRGD-NG-Pt was higher than that of free CDDP in both breast cancer cells, and the cytotoxicity of cRGD-NG-Pt to these cells increased significantly when the incubating time increased, which were well correspondent with the cellular uptake behavior of cRGD-NG-Pt and the in vitro drug release behavers mentioned above. However, the same dose of CDDP in Lido/cRGD-NG-Pt treatment was demonstrated significantly increased the inhibition effect for MDA-MB-231 cells proliferation, but not as so for MCF-7 cells. Furthermore, the MDA-MB-231 cells growth was significantly inhibited by the dual drug-loaded formulation, and the combination index were 1.25, 1.24 and 1.22 (the combination index q was calculated by the formula, q = E(CDDP+Lido) / (ECDDP + ELido – ECDDP * ELido); E was the efficacy of different NG formulation),40 while the correspondent CDDP concentrations were 20, 40 and 80 µM, respectively (Figure S-2B). The above results suggest that Lido enhanced the cytotoxicity of CDDP and the NG-based CDDP formulation transported within cells by acidic responded endocytosis process. The apoptosis data analysis of MDA-MB-231 cells showed that treatment with Lido/cRGD-NG-Pt significantly increased cell apoptosis compared with treatment with Lido/cRGD-NG or cRGD-NG-Pt alone (Figure 3C and D). These data suggest that Lido enhanced CDDP-induced apoptosis of MDA-MB-231 cells and exhibited a synergistic effect when combined with CDDP. In vivo fluorescence Tumor targeting To investigate the targeting efficiency of NG system, in vivo imaging system (IVIS) was utilized to monitor the fluorescence of DID/cRGD-NG and its non-targeting variant DID/NG on MDA-MB-231 tumor bearing nude mice. The Fig. 4A showed that, after administering the same dose of DID, NG formulation treated groups displayed enrichment of fluorescence signal in the tumor site at 2 h postinjection. However, the highest accumulation was found in liver tissue. As the time was prolonged, much higher tumor accumulation of DID/cRGD-NG treated group was strengthened in comparison with DID/NG treated group, while the fluorescence intensity of free DID group became weaken in tumor region indicating the fast elimination in circulation. After 24 h, the strong signal at tumor site of DID/cRGD-NG group demonstrated the


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specific internalization of cRGD peptide. Then all the main organs and tumors of the mice were collected for biodistribution evaluation (Figure 4B and C). The results were further proved that after cRGDfk peptide conjugation, the enrichment of NG system in MDA-MB-231 tumor was further enhanced. It indicates the introduction of cRGDfk favor the targeting delivery of drugs-loaded nanogel.

In vivo MR Imaging The encapsulation of Fe3O4 NPs enabled the NG systems as MR contrast agents for in vivo tumor detection.

41

After emulsification procedure, Fe3O4 NPs encapsulated into

the nanogels network to form the and Fe3O4/cRGD-NG and its non-targeting variant Fe3O4/NG, both of which were collected and purified by magnetic adsorption.42 As increased concentration of Fe in Fe3O4/cRGD-NG dispersions, an obvious darkening of T2-weighted MRI was observed. By evaluated via linear least-square fitting of 1/T2 versus Fe concentration, the r2 relaxivity was calculated about 173.4 mM−1 S−1 (Figure 5 A). The integrity of the Fe3O4/cRGD-NG further demonstrated the potential capacity for T2-weighted MR imaging applications.43 Furthermore, T2-weighted MR imaging of on MDA-MB-231 tumor-bearing mice was utilized to evaluate the enhancement effect of NG. As shown in Fig. 5B, visible signal enhancement at the MDA-MB-231 tumor region were present in both groups since

8 h after administration, and the significant signal increase from

Fe3O4/cRGD-NG treated mice was observed compared with that of Fe3O4/NG treated group, demonstrated the enhanced accumulation. Moreover, the increase T2-weight signal shown in T2-weighted MR images of the tumors treated with Fe3O4/cRGD-NG as time prolonged indicated the further tumor targeting and accumulation of Fe3O4/cRGD-NG. The results of the biodistribution of Fe in major organs and tumors which measured by ICP-AES, as shown in Fig. 6C, showed that the content of Fe in the tumors treated by Fe3O4/cRGD-NG was approximate 1.66 times of that of the Fe3O4/NG. It indicates more Fe3O4 was delivered by Fe3O4/cRGD-NG to the tumor tissues and cRGDfk modification helped to improve the targeting delivery efficiency of nanogel. Therefore, the cRGD modified NG would become a promising drug carrier with beneficial for tumor-targeted therapy. Some studies have found that the targeting capacity of tumor-targeting ligands can be attenuated by the influence of protein corona.44-46 And some recent studies have also



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revealed that the targeting capacity of ligands can be partially retained after in vivo corona formation.47 From the results in vitro and in vivo we obtained, it indicates the modification can improve the enrichment of drug-loading nanogel in tumor site. The improvement may be ascribed to the cRGDfk-mediated tumor targeting and penetration ability of nanomedicines. The protein corona may impede the direct interaction between ligand and receptor, but the composition of protein corona may also be changed after ligand modification. The changed protein corona may favor tumor-targeting.48 How the protein corona formed on the ligand-modified nanoparticle influence the targeting capacity still remains unclear.

In vivo antitumor studies of Lido/cRGD-NG-Pt Herein, the in vivo antitumor efficacy of the Lido/cRGD-NG-Pt was evaluated in xenograft models of MDA-MB-231 tumor. The tumor-bearing mice with the tumor volume of ~70 mm3 were divided to five groups and treated with 5% glucose, free CDDP, Free CDDP plus Lido, cRGD-NG-Pt, Lido/cRGD-NG-Pt (low dose) and Lido/cRGD-NG-Pt (high dose), respectively (Figure 6A). Since free CDDP which was used to be 6 mg/kg in the preliminary investigation caused all mice death after the third administration, and on the other hand, we purposed to notably highlight the effectiveness of dual-drug chemotherapy, the dose of CDDP in all the formulations (except the high-dose Lido/cRGD-NG-Pt), was lowered to 3 mg/kg in our research. From the results (Figure 6), we found that in either low-dose of CDDP or CDDP plus Lido treated group, the tumor grew slower in the initial stage, however, the inhibition of tumor growth couldn’t maintain after the 4th administration (Figure 6B). What’s worse, the serious side effects of free CDDP caused severe emaciation of each mice indicating systemic toxicity of CDDP (Figure 6C). Different NG-based CDDP formulations present significant inhibition performance on tumor growth compared with the free CDDP treated group, which indicated the targeting NG system could deliver CDDP to the tumor site effectively.49 Furthermore, no significant change was observed from the body weight of mice treated with the NG-based CDDP formulations. Tumors

volume

of

the

mice

treated

with

dual-drugs

treatment

group

(Lido/cRGD-NG-Pt) demonstrated better tumor growth inhibition than that of cRGD-NG-Pt chemotherapy group, however, recurrence happened in some treated mice due to the low dosage of CDDP, as shown in Fig. 6B. Obvious regression of the tumors treated with high dosage of Lido/cRGD-NG-Pt, and one of the tumor was


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diminished without recurrence. Consistent with the tumor growth curves, the mean weight of the tumors in the high dosage of Lido/cRGD-NG-Pt treated group was the lowest among all treatments (Figure 6D). The enhanced anticancer performance of Lido/cRGD-NG-Pt was further proved by the immunofluorescence analysis of the tumor tissues. As showed in Fig. 7A and B, apoptosis of tumor cells was clearly observed after dual-drugs treatments, especially in the Lido/cRGD-NG-Pt treated groups. More importantly, by H&E staining the lung tissues and counting the naked-eye visible metastasis nodes in lung tissues, we further demonstrated that the targeted delivery of CDDP and Lido not only inhibited the primary tumor growth, but also alleviated the metastasis (Figure 8A and B). In conclusion, the high dose of Lido/cRGD-NG-Pt treatment suppressed 89.9% of tumor growth and inhibited 93.3% of lung metastasis.

CONCLUSION In summary, a tumor-targeted nanogel-based drug delivery system was designed and prepared for tumor chemotherapy. The dual drugs-loaded nanogel system displayed good stability with suitable size distribution and pH-responsive drug release profiles. By in vitro evaluation, it demonstrated that the as-prepared Lido/cRGD-NG-Pt exhibited higher selective cellular uptake after incubating with MDA-MB-231 cell line and then triggered by the intracellular acidic environment to accelerate the release of cisplatin to eliminate cancer cells effectively. Beside delivering anticancer drugs, the local anesthetics--lidocaine was explored for enhancing cisplatin-induced apoptosis for metastatic breast cancer cells and exhibited a synergistic effect for combined therapy. Meanwhile, in vivo NIR fluorescence imaging or MR imaging by DID or Fe3O4 nanoparticle-loaded NG systems further confirmed the desired tumor targeting effect on MDA-MB-231 tumor-bared mice. Moreover, this nanosystem was served as dual-drug delivery system achieved synergistic therapeutic effect of cisplatin and lidocaine, not only the growth of primary tumor can be inhibited, but also the metastasis can be alleviated. This combinational therapy might have comprehensive potential for metastatic cancer therapy.



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FIGURES Scheme

Scheme 1. Schematic illustration of tumor targeting therapy and metastasis prevention by the dual drugs-loaded nanogels system. (A) Construction of Lido/cRGD-NG-Pt.

(B)

The

drugs-loaded

nanogels(Lido/cRGD-NG-Pt)

by

introduction of peptide segment-cRGDfk, which presents high affinity to αvβ3 integrin, demonstrate the increased enrichment in tumor site, which also alleviated the adverse effect caused by cisplatin-mediated chemotherapy. And it favors the primary tumor growth inhibition and lung metastasis prevention.


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Figure 1. Characterization of NGs. (A) Size distribution of the cRGD-NG, NG-Pt, cRGD-NG-Pt and Lido/cRGD-NG-Pt. (B) Zeta-potential of the cRGD-NG, NG-Pt, cRGD-NG-Pt and Lido/cRGD-NG-Pt. (C) Morphology of different NGs captured by TEM, the scale bar represents 100 nm. (D) In vitro drug release profiles.



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Figure 2. (A) The confocal images and (B) Semi-quantitative analysis of C6/NG and C6/cRGD-NG by MCF-7 cells or MDA-MB-231 cells for 4 h incubations. The blue fluorescence represents the nucleus, the green fluorescence represents C6-loaded NG; Scale bar represent 20 µm for MCF-7 cells and 50 µm for MDA-MB-231 cells, respectively. Error bar indicates SD (n = 3, “**” P < 0.01 was considered as significant

difference).

(C)

The

western

blot

identification

of

the

αvβ3

integrin-overexpressing on different breast cancer cell lines. (D) The confocal images and (E) Semi-quantitative analysis of C6/cRGD-NG and C6/cRGD-NG-Pt uptake by MDA-MB-231 cells at different pH environment. The blue fluorescence represents the nucleus, the green fluorescence represents C6-loaded NG; scale bar represents 50 µm (n = 3, “*” P < 0.05 was considered as significant difference).


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Figure 3. Cytotoxicity of formulations in vitro. (A) MTT assays for blank cRGD-NG, (B) IC50 of the different CDDP formulations on MCF-7 cells or MDA-MB-231 cells. (C) Apoptosis analysis of MDA-MB-231 cells after 24 h incubation with different formulations. (D) Percentage of early apoptotic, apoptotic, necrotic and survivance MDA-MB-231 cells summarized from flow cytometry analysis.



Figure 4. Biodistribution in vivo. (A) In vivo NIR fluorescence imaging of the distribution of DID/NG or DID/cRGD-NG in MDA-MB-231 tumor-bearing mice after intravenous injection. (B) Ex vivo fluorescence imaging of tumor and major organs harvested from the MDA-MB-231 tumor-bearing mice 24 h after intravenous administration of NGs. (C) Semi-quantification of fluorescent intensity of the main organs and tumors measured by Living Image® Software (“*” p

Targeting Delivery of Lidocaine and Cisplatin by Nanogel Enhances

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