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A Biopolymer-Drug Conjugate Nanotheranostics for Multimodal Imaging-Guided Synergistic Cancer Photothermal-Chemotherapy Chang Du, Jiwen Qian, Linzhu Zhou, Yue Su, Rong Zhang, and Chang-Ming Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10163 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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ACS Applied Materials & Interfaces

A

Biopolymer-Drug

Conjugate

Nanotheranostics

for

Multimodal

Imaging-Guided Synergistic Cancer Photothermal-Chemotherapy |‖

|‖

|‖

|‖

Chang Du , , Jiwen Qian , , Linzhu Zhou , , Yue Su , , Rong Zhang

‖,†,

*, Chang-Ming

| ‖,

Dong , * |

School of Chemistry and Chemical Engineering, Shanghai Key Laboratory of Electrical

Insulation and Thermal Aging, Shanghai Jiao Tong University, Shanghai 200240, P. R. China ‖

Joint Research Center for Precision Medicine, Shanghai Jiao Tong University & Affiliated

Sixth People's Hospital South Campus, Shanghai 200240, P. R. China †

Joint Research Center for Precision Medicine, Shanghai Fengxian Hospital, Southern

Medical University, Shanghai 201400, P. R. China

Submitted as an Article to ACS Appl. Mater. Interfaces

Address correspondence to: Professor Chang-Ming Dong, Joint Research Center for Precision Medicine, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China; E-mail: [email protected] Prof Rong Zhang, Joint Research Center for Precision Medicine, Shanghai Fengxian Hospital, Southern Medical University, Shanghai 201400, P. R. China; E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT Some of biomedical polymer-drug conjugates are being translated into clinical trials; however, they intrinsically lack photothermal and multi-imaging capabilities, hindering them from imaging-guided precision cancer therapy and complete tumor regression. We introduce a new concept of all-in-one biopolymer-drug conjugate nanotheranostics and prepare a kind of intracellular pH-sensitive polydopamine-doxorubicin conjugate nanoparticles (PDCNs) under mild conditions. Significantly, this strategy integrates polymeric prodrug-induced chemotherapy, near infrared light (NIR)-mediated photothermal therapy, and triple modalities including doxorubicin (DOX) self-fluorescence, photothermal and photoacoustic imaging, into one conjugate nanoparticle. The PDCNs present excellent photothermal property, dual stimuli-triggered drug release behavior, and about 12.4-fold blood circulation time compared to free DOX. Small animal fluorescent imaging technique confirms PDCNs have preferential tumor-accumulation effect in vivo, giving a 12.8-fold DOX higher than the control at 12 h post injection. Upon NIR laser irradiation (5 min, 808 nm, 2 W/cm2), the PDCNs mediated photothermal effect can quickly elevate the tumor over 50 oC, exhibiting good photothermal and photoacoustic imaging functions, of which the photoacoustic amplitude is 3.6-fold greater than the control. In vitro and in vivo assays persuasively verify that intravenous photothermal-chemotherapy of PDCNs produces synergistic antitumor activity compared to single photothermal therapy or chemotherapy, achieving complete tumor ablation during the evaluation period. Keywords: polymer-drug conjugate, combination treatment, photothermal therapy, polydopamine, theranostics. 2


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INTRODUCTION In contrast to small molecular anticancer drugs, the physically drug-loaded polymeric nanoparticles (i.e., the nanomedicine formulations) have made stride in safe and effective cancer treatment because of the enhanced drug solubility and maximal tolerated drug dose, the prolonged circulation half-life, and the preferential tumor accumulation via the enhanced permeation

and

retention

(EPR)

effect.1-12

However,

new

generation

polymeric

nanotherapeutics including NK-105 and BIND-014 very recently present disappointing clinical results in efficacy and safety, which are greatly correlated with the multi-drug resistance, the tumor complexity and heterogeneity, and the increased tumor recurrence and metastasis.1-4 Compared to the physically loaded nanomedicine, the polymer-drug conjugates demonstrate distinct characteristics including high drug-loading capacity, no free drug crystals within nanoparticles, and no leaky and premature drug release.13-25 Recently, scientists have made great progress on the polymer-drug conjugates, including backbone-degradable poly(N-2-hydroxypropyl methacrylamide)-drug conjugate (e.g., DOX and paclitaxel),15 biodegradable poly(lactic acid)-drug conjugate (e.g., camptothecin, paclitaxel,

docetaxel,

and

DOX),17,18

and

the

reduction-sensitive

poly(ethylene

glycol)-polycarbonate-drug conjugate (e.g., camptothecin and chlorambucil).19 However, the multiple synthesis steps and limited yields, the potential toxicity and immunity, and the polydispersities of the polymer-drug conjugates become major drawbacks, which hinder their scale-up formulation and clinical translation.13-17 To avoid potential toxicity and immunity, the biomolecular prodrugs (e.g., peptide, antibody, and DNA) and drug-drug conjugate provide novel strategies to construct self-delivery nanoparticles for cancer therapy.26-30 3



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On the other hand, the combination treatments (e.g., surgery or radiotherapy with chemotherapy) are widely applied for cancer therapy in the clinic.31,32 Especially, the combination strategies including the emerging photothermal therapy (PT) with chemotherapy (CT) can utilize individual advantages to produce synergistic antitumor effect, holding great potentials for next generation cancer therapy.33-51 Compared to conventional surgery, the PT implemented by near-infrared light (NIR, 650−950 nm) and nanoparticle can pinpoint ablate tumor and even induce antitumor immunity.33-40 Moreover, the photothermal effect can simultaneously enlarge the porous blood vessels of tumor and enhance the permeability of cell membrane, thus facilitating the extravasation, accumulation and penetration of nanoparticle in tumor and subsequent cellular internalization. Although some of biomedical polymer-drug conjugates are being translated into clinical trials, they intrinsically lack PT and multi-imaging functions, hindering them from imaging-guided precision cancer therapy and complete tumor ablation.5-7, 13-17 Consequently, to make the polymer-drug conjugate with intrinsic PT attribute will provide an innovative strategy for the combination PT-CT treatment of cancer, which might outperform current combination CT with surgery or radiotherapy.33-40, 48-51 As a biomimetic polymer to natural melanin in human body, polydopamine (PDA) recently attracts increasing attention for biomaterials, cancer therapy and imaging applications, providing an opportunity to construct NIR-absorbing polymer-drug conjugate.52-71 Very recently, we and others fabricated various kinds of physically DOX-loaded PDA systems for targeted cancer therapy and theranostics;66-71 however, the dopamine-derived prodrug and the related PDA-drug conjugate nanoparticles have not been investigated until now. 4


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Figure 1. Synthesis of the prodrug DA-DOX (A) and the polydopamine-doxorubicin conjugate nanoparticles of PDCNs (B); Illustration of one conjugate nanoparticle of PDCN25 for synergistic PT-CT treatment with fluorescent, photothermal and PA imaging in vivo (C). To overcome the shortcomings of current polymer-drug conjugates (i.e., lacking photothermal attribute and multi-imaging capabilities) and address the challenges in physically drug-loaded polymeric cancer nanomedicine (i.e., the compromised anticancer efficacy and severe side effect), herein, we introduce a new concept of all-in-one biopolymer-drug conjugate nanotheranostics. To this purpose, we for the first time design a biomolecular dopamine-doxorubicin (DA-DOX) prodrug and in one-pot synthesize a novel kind of intracellular pH-sensitive polydopamine-doxorubicin conjugate nanoparticles 5



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(PDCNs) under mild conditions (Figure 1A-B). The PDCNs exhibit the following advantages: (1) without the necessity of additional assembly procedure used for amphiphilic polymer-drug conjugates,5-7, 13-17 the PDCNs can be directly prepared by the precipitation copolymerization of the designed DA-DOX prodrug with DA,66 (2) besides playing a drug-carrier role, the PDA backbone possesses intrinsic NIR-absorbing ability for photothermal conversion, and especially (3) the polymeric prodrug-induced CT, the NIR-mediated PT, and triple modalities including DOX self-fluorescence, photothermal and photoacoustic (PA) imaging, are integrated into one biopolymer-drug conjugate nanoparticle that can achieve multimodal imaging-guided synergistic PT-CT of cancer, as illustrated in Figure 1C. The PDCNs are fully characterized and exhibit excellent NIR-mediated photothermal property and both endo-lysosomal pH-sensitive and photothermally triggered drug release profiles. In vitro and in vivo biological assays including multimodal imaging and comparative antitumor treatments are thoroughly investigated. Compared to single CT or PT, the combination PT-CT treatment of PDCN25 (the subscript denotes DOX weight percentage) presents synergistic antitumor activity with complete tumor ablation during the therapeutic period. EXPERIMENTAL SECTION Materials. Dichloromethane (99.5%), dimethylformamide (DMF, 99.5%), ethyl acetate (99.5%),

and

methanol

(99.5%)

were

distilled

before

use,

respectively.

Dicyclohexylcarbodiimide (DCC, 99.2%, Aldrich), 3, 4-dihydroxy-benzenepropanoic acid (98%, Aldrich), dopamine hydrochloride (98%, Aldrich), 1-hydroxybenzotriazole (HOBT, 99%, GLS), tert-butylcarbazate (Boc-NHNH2, 99%, Acros), and trometamol (Tris, ≥99.9%, 6


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Sigma) were used as received. Dulbecco’s modified eagle medium (DMEM, PAA laboratory), fetal bovine serum (FBS, PAA laboratory), methylthiazolyldiphenyl-tetrazolium bromide (MTT, ultrapure, Aldrich), hoechst33342 (ultrapure, Aldrich) were used as received. HeLa (a human uterine cervix carcinoma cell line) and L929 (a mouse fibroblastic cell line) were received from Shanghai Institute of Biochemistry and Cell Biology. Methods. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer at room temperature. 1H NMR (400 MHz) and

13

C NMR (100

MHz) spectra were recorded on a Varian Mercury-400 spectrometer at room temperature using tetramethylsilane as an internal standard. Time-of-flight mass spectrometry (TOF-MS) was performed on a Waters Acquity UPLC/Premier QTOF MS Premier. The elemental analysis (EA) was determined by using a Vario EL Cube instrument. X-ray photoelectron spectroscopy (XPS) was conducted on a VG ESCALAB MKII spectrometer and obtained by using XPS PEAK software (Version 4.1) to deconvolute the spectra. The mean hydrodynamic diameter and polydispersity index (PDI) of nanoparticles were determined by dynamic light scattering (DLS), during which the samples were measured for five times on a Malvern ZS90 instrument at 25 °C. Transmission electron microscopy (TEM) was performed on a JEM-2010 instrument at 200 kV accelerating voltage, during which Samples were dropped on the 300 mesh Formvar-carbon film-coated copper grids. The UV-Vis-NIR spectroscopy was recorded on a Perkin-Elmer Lambda 750S spectrometer at room temperature. The fluorescent spectroscopy was recorded on a Perkin-Elmer LS-50B spectrometer at room temperature. Electron spin resonance (ESR) spectroscopy was performed on a Bruker BioSpin EMX-8 spectrometer at room temperature. A continuous wave diode laser (Shanghai 7



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SFOLT Corp., FC-960-6000-MM; wavelength: 808 nm, power: 0−1650 mW) with a fiber optic patch cable (FC/PC/200UM/1M) and a fiber collimator (to control the spot size) was applied for the NIR irradiation. Synthesis of DBA-CONHNH2. 3, 4-Dihydroxy-benzenepropanoic acid (DBA, 2.5 mmol, 455.5 mg), HOBt (3 mmol, 405.4 mg), and DCC (3 mmol, 619.2 mg) was dissolved in DMF (20 mL) and stirred for 4 h at room temperature. Boc-NHNH2 (3 mmol, 396.5 mg) in DMF (1 mL) was then added dropwise into the above solution and the reaction mixture was vigorously stirred for another 20 h. DMF was removed by rotary evaporation and the residues were purified by silica column chromatography (ethyl acetate: petroleum ether, 3:1). Finally, the purified intermediate was dried in vacuum (50% yield). 1H NMR (400 MHz, DMSO-d6, δ), 1.39 (s, 9H, C(CH3)3), 2.26 (dd, J=8.4, 7.6 Hz, 2H, CH2CO), 2.63 (dd, J=7.2, 8.4Hz, 2H, ArCH2), 6.57-6.22 (m, 3H, Ar), 8.66-8.75 (t, 3H, CONH and OH), 9.52 (s, 1H, NHCOO). 13C NMR (100 MHz, CD3OD, δ), 171.28 (C=O), 155.37 (C=O), 145.06 (C-OH), 143.44 (C-OH), 131.87 (Ar), 118.77 (Ar), 115.66 (Ar), 115.51 (Ar), 79.08 (C(CH3)), 35.45 (CH2CO), 30.28 (CH2CH2), 28.14 (CH3). FT-IR (KBr, cm-1): 3298 (νOH), 2981 (νC-H), 1716 (νCONH), 1675 (νCONH), 1606 (νN-H), 1522 (νphenyl), 1176 (νC–O–C). The above-obtained intermediate (0.3 mmol, 88.8 mg) was dissolved in dichloromethane (5 mL) and then TFA (2 mL) was added dropwise and stirred for 2 h at room temperature. The DBA-CONHNH2 product was quantitatively obtained. 1H NMR (400 MHz, DMSO-d6, δ), 2.38 (dd, J=7.6, 7.6 Hz, 2H, CH2CO), 2.64 (dd, J=7.2, 8.0 Hz, 2H, ArCH2), 3.55 (s, 2H, NH2), 6.61-6.48 (m, 3H, Ar), 8.75 (m, 3H, CONH and OH). FT-IR (KBr, cm-1): 3409 (νOH), 2981 (νC-H), 1676 (νCONH), 1607 (νN-H), 1527 (νphenyl), 1145 (νC–O–C). 8


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Synthesis of the dopamine-doxorubicin (DA-DOX) prodrug. DOX hydrochloride (0.2 mmol, 116.0 mg) and DBA-CONHNH2 (0.3 mmol, 58.8 mg) were dissolved in 30 mL of anhydrous methanol. A drop of TFA was added into the above solution and then the reaction was stirred in dark at room temperature for 96 h. The solution was concentrated and acetonitrile (10 mL) was added until slight turbidity appearance, and then the resulting suspension was kept at −20oC for 24 h. The red solid was collected by centrifugation and washed with methanol-acetonitrile (1:10, v:v) to give a yield of 67.5%. The purified DA-DOX was fully characterized by means of 1H NMR, 13C NMR, FT-IR, and TOF-MS. 1H NMR(400 MHz, CD3OD, δ): 1.29 (m, 3H, CHCH3), 1.85-3.25 (m, 10H, CCH2COH, OHCCH2CHO, CH2CHOCH and CH2CH2CO), 3.52-3.75 (m, 2H, NH2CHCHOH), 4.02 (s, 3H, OCH3), 4.34 (m, 1H, CHCH3), 4.71 (s, 2H, CH2OH), 5.07 (dd, J=17.6, 2.8 Hz, 1H, CHOCHO), 5.44 (dd, J=6.0, 6.0 Hz 1H, OCHO), 6.18-6.72 (m, 3H, Ph), 7.25-7.86 (m, 3H, Ph).

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C NMR (100 MHz, CD3OD, δ) 186.55 (C1), 185.50 (C2), 175.99 (C3), 161.00 (C4),

156.25 (C5), 154.54 (C6), 153.75 (C7), 144.64 (C8), 143.03 (C9), 135.74 (C33), 135.23 (C34), 134.85 (C35), 134.59 (C36), 132.50 (C32), 119.75 (C28), 119.22 (C29), 119.01 (C30), 118.69 (C31), 115.34 (C26), 115.23 (C27), 110.75 (C24), 110.52 (C25), 99.45 (C23), 81.22 (C22), 74.36 (C22), 72.41 (C21), 69.39 (C20), 66.41 (C19), 57.43 (C18), 55.73 (C17), 38.51 (C16), 33.78 (C15), 33.28 (C14), 30.31 (C13), 29.61 (C12), 28.12 (C11), 15.64 (C10). FT-IR (KBr): 3427 (νOH), 2981 (νC-H), 1703 (νC=N), 1673 (νCONH), 1616 (νN-H), 1521 (νphenyl), 1115 (νC–O–C). TOF-MS m/z: [M + H]+ calcd for C36H39N3O13, 722.2483; found, 722.2549. Synthesis of pH-sensitive PDA-DOX conjugate nanoparticles (PDCNs). The PDCNs and PDA nanoparticles were synthesized according to our previous publication.66 As a typical 9



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example, both DA-DOX (5 mg) and DA hydrochloride (12.5 mg) that were pre-dissolved in deionized water (1 mL) were added into aqueous solution (20 mL) of Tris (410 mg) at 30 °C, and then stirred under air for 24 h. The resultant suspension was dialyzed (molecular weight cutoff: 3500) against deionized water or PBS (10 mM, pH 7.4) for 2 days to give the nanoparticles solution and then stored at −4oC for further use. NIR-mediated photothermal properties of nanoparticles. Briefly, the PDA or PDCNs solution (200 µL) in a 96-well plate was vertically irradiated by the NIR laser (5 min, 808 nm, power intensity = 1, 2 or 4 W·cm-2), during which the solution temperature was recorded by a digital thermometer via a thermocouple probe. After repeated NIR laser irradiation (heating for 5 min) and non-irradiation (naturally cooling for 10 min) for three times, the temperature change curve was recorded for determining the photothermal conversion efficiency (η).66 The Vis-NIR spectra of the irradiated samples were also measured for studying the photostability. In vitro DOX release. As for NIR-triggered release, the PDCNs in PBS (1 mL, 10 mM, pH 7.4) was put into a dialysis bag (MWCO = 3500 Da). The sample was vertically irradiated by the NIR laser (5 min, 808 nm, 2 W·cm-2) each one hour, immersed in 10 mL of PBS with different pH values (i.e., pH 5.0, 6.0 and 7.4) at 37 °C, and then kept in a horizontal shaker at 150 rpm. Then 10 mL of dialysis solution was removed at predetermined time intervals and same volume of fresh PBS solution was replenished. The DOX release without irradiation was similarly tested. The released DOX amount was measured by fluorescence spectroscopy (DOX: excitation at 495 nm and emission at 590 nm). All release experiments were carried out in duplicate and each sample was measured at least for 3 times. For monitoring the size change of the PDCNS nanoparticles upon the combination stimuli 10


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of pH and NIR irradiation, 5 mL of PDCNS at different pH values (i.e., pH 5.0 and 7.4) and at 37 °C was irradiated by the NIR laser each 30 min (5 min, 808 nm, 2 W·cm-2) and then monitored by DLS. In vitro photothermal therapy (PT), chemotherapy (CT), and PT-CT. HeLa was chosen as a model cancer cell line. HeLa or L929 was cultured at 37 °C under a humidified atmosphere of 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin. Briefly, 200 µL of HeLa cells in DMEM was seeded in 96-well plate (104 cells per well) and incubated overnight at 37 °C. 200 µL of PDA or PDCNs with gradient concentrations was added and then incubated for 48 h. After incubation and twice washing with PBS, 200 µL of fresh culture medium containing MTT (20 µL) was added and further incubated for 4 h to allow full formation of formazan. Finally 100 µL of DMSO was added into the well for 20 min and the absorbance at 490 nm was recorded by a Microplate Reader (Elx800, BioTek Company). The cell viability was calculated by comparing with negative control. To evaluate PT, CT, and PT-CT, the nanoparticle solutions with gradient concentrations were added separately into a 96-well plate and incubated for 4 h at 37 °C before the NIR laser irradiation. After that, the cells were further incubated 12 h for PT and 48 h for CT and/or PT-CT, respectively. Half maximal inhibitory concentration (IC50) was calculated by GraphPad Prism 6 software using eight samples. As for CT and the combination CT, IC50 was calculated on the basis of DOX concentration; and the IC50 values for PT and the combination PT were calculated on the basis of the concentration of pure PDA or the PDA in PDCNs. The combination index (CI) was calculated by the following equation: CI = [IC50 11



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(combination CT)/IC50 (CT)] + [IC50 (combination PT)/IC50 (PT)].44, 72 Both flow cytometry and inverted fluorescence microscopy were used to monitor the cell internalization process of DOX (excitation at 495 nm and emission at 590 nm) or PDCNs. Briefly, HeLa cells (5.0×105cells per well) were treated with same drug concentration of free DOX or PDCNs (10 µg/mL DOX equiv.) for predetermined time at 37 °C or further irradiated by the NIR laser. After removal of the incubation media, the cells were rinsed with PBS for three times and then treated with trypsin. Finally the collected data for 1.0 ×104 gated events were analyzed by FlowJo software. For inverted fluorescence microscopy, HeLa cells were fixed with 4 % formaldehyde for 30 min and then stained by Hoechst 33342 for 5 min after similar treatment. Animals. The animal experiments were performed according to the guidelines for the care and use of laboratory animals and approved by the Animal Ethics Committee of Shanghai Jiao Tong University. 5-week-old male Balb/c nude mice (~20 g) and Sprague−Dawley (SD) rats (~200 g) were purchased from Chinese Academy of Sciences (Shanghai, China). HeLa cells (~106 cells) in PBS were implanted subcutaneously into each Balb/c nude mouse and the HeLa tumor-bearing nude mice with tumor volumes (100 −150 mm3) were used for cancer treatment study. Pharmacokinetics. 5-week-old SD rats (~200 g) were randomly divided into two groups (n=4) and each mouse was injected through tail vein with 2.5 mg/kg free DOX or PDCN25 equivalent. The blood samples (0.3 mL) were obtained at prescribed time intervals (i.e., 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h). Plasma samples were harvested by immediate centrifugation (10 min, 1000 rpm) and frozen at −20°C for analysis. The pharmacokinetic parameters were 12


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calculated by using OriginPro 8. In vivo multimodal imaging and biodistribution. The HeLa tumor-bearing nude mice were intravenously injected via tail vein with 200 µL of PDCN25 or free DOX (2.5 mg/kg). In vivo fluorescent imaging was monitored at 1, 2, 4, 6, 8, 10, 12 and 24 h post-injection using a Kodak multimode imaging system (DOX excitation wavelength: 530 nm, DOX emission wavelength: 600 nm). After 12 h post-injection, the tumor site was further irradiated by the NIR laser (5 min, 808 nm, 2 W·cm-2), in vivo fluorescence imaging was recorded at 30 min, 60 min and 90 min. For ex vivo distribution, tumor and major organs were carefully collected at 2, 6 and 12 h post-injection, washed with saline, and then imaged by a Kodak multimode imaging system. After imaging, weighing, and being homogenized in 0.5 mL of 1% Triton X-100, 1.2 mL of the extraction solution (HCl-IPA) was added, and then the samples were incubated at −20 °C overnight. After vortexing and centrifugation at 15, 000 ×g for 15 min, the DOX concentration was determined by fluorescence spectroscopy. Data are presented as the percentage injected dose per gram tissue (% ID/g). As for in vivo photothermal effect and imaging, the mice (n = 4) were intravenously injected with 200 µL (1 mg/mL) of PDCN25 or PDA or the control PBS, and then irradiated by the NIR laser (5 min, 808 nm, 2 W/cm2) at 12 h post-injection, respectively. The thermographic image and the temperature in tumor site were recorded by an infrared thermal camera (AXT100, Ann Arbor Sensor Systems). After PDCN25 (200 µL, 1mg/mL) or the control PDA was intravenously injected into the mice, the PA imaging was acquired at 1, 2, 4, 6, 8, 10, 12 and 24 h post-injection using a commercial Endra Nexus 128 PA tomography system (Endra Inc., Ann Arbor, Michigan) 13



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equipped with a tunable nanosecond pulsed laser (808 nm wavelength, 7 ns pulse, 7 mJ/pulse, 2 Hz pulse repetition frequency) and a 128 unfocused ultrasound transducer with a 5 MHz center frequency. The PBS injection alone was used to eliminate the background signal for obtaining the PA signals of both PDA and PDCNs groups, and the PDA control group was used to compare with the PDCNs group. In vivo antitumor activity. The HeLa tumor-bearing nude mice were randomly divided into six groups (n = 4). All the mice were intravenously injected twice at a time point of 0 and 4 days with 2.5 mg/kg DOX or nanoparticles (PDA or PDCN25, 200 µL, 1 mg/mL) or PBS. As for the PDA + NIR and PDCN25 + NIR groups, the tumor sites were irradiated by the NIR laser (5 min, 808 nm, 2 W/cm2) at 12 h and 24 h post-injection, respectively. The tumor size and body weight of each mouse were measured by using a caliper or a balance every two days. Tumor volume (V) was calculated according to the equation: V (mm3) = 1/2 × length (mm) × width (mm) × width (mm). The tumor inhibitory rates (TIR) of various treatments are calculated by the equation: TIR (%) = 100 × (mean tumor weight of the PBS group − mean tumor weight of others)/(mean tumor weight of the PBS group). Data are represented as average ± standard error. When the tumor volume of the PBS group reached ~1400 mm3 after 16-day treatment, all the mice were sacrificed. Finally the tumors as well as major organs (heart, liver, spleens, lung and kidneys) were dissected, washed with saline, weighted, photographed, and fixed in 4% formaldehyde for histology, TUNEL, and PCNA assays.

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Table 1. Characterization of the PDCNs and PDA nanoparticles. Samplea Diameterb (nm) PDA PDCN16 PDCN25

75 ± 3 89 ± 3 87 ± 2

TEM Morphology

Zeta IC50 (PT)c b potential (µg/mL) (mV)

sphere sphere sphere

IC50 (CT)c (µg/mL)

IC50 (PT-CT)d (µg/mL)

CIe

− 4.65 5.85

− 9.22 + 1.76 5.63 + 1.88

− 0.55 0.43

53.76 53.70 53.84

−33.1 −27.0 −28.5

a

the subscript denotes the weight percentage of DOX within PDCNS ; bthe mean diameter and the zeta potential of nanoparticle in PBS are determined by DLS; cIC50 is calculated from Figure 5 A and Figure S8 by using a GraphPad Prism 6 software; das for the combination PT-CT, the former value is the IC50 of the combination PT and the latter one as that of the combination CT; eCI denotes the combination index of PT-CT.

RESULTS AND DISCUSSION Synthesis of pH-sensitive polydopamine-doxorubicin conjugate nanoparticles (PDCNs). The biomolecular prodrug of dopamine-doxorubicin (DA-DOX) dyad with an acid-labile hydrazone bond was newly designed and synthesized in three steps,12, 14 and its molecular structure was confirmed by means of FT-IR, 1H NMR, 13C NMR, and TOF-MS spectroscopy (see experimental and supporting information, Figure S1-2). To achieve efficient NIR-mediated PT by utilizing nanoparticle, two basic parameters including the size and the temperature-elevating ability (∆T) are the most important for nanoparticle.33-40 As the nanotherapeutics with a sub-100 nm diameter have good extravasation and accumulation ability in solid tumor via the EPR effect,9, 10 we have optimized experimental conditions for the

fabrication

of

PDCNs

with

suitable

size

of

sub-100

nm

and

excellent

temperature-elevating ability. Thereafter, the precipitation copolymerizations of DA-DOX with DA in different ratios were performed to produce the PDCNs in aqueous solution of tris at 30 oC for 24 h and the detailed data are summarized in Table S1. Taking the size and ∆T into consideration, both PDCN16 and PDCN25 (the subscript denotes DOX weight percentage) are used for the next detailed investigation. 15



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The microstructure of PDCNs was fully characterized by various techniques. FT-IR spectra of PDCNs show a typical vibration peak at 1703 cm-1 for the hydrazone bond linker, besides other characteristic peaks of PDA including the indole structure at 1625−1640 cm-1, the amide (CONH) at 1660 cm-1, and the ether (C-O-C) at 1110 cm-1 (Figure S3). Owing to the

π-electron radicals delocalized in the quinone residue, the ESR spectra of PDCNs give single peak with g-factor of 2.0037−2.0064, which is reminiscent of melanin and PDA.54-56 Both XPS and elemental analysis further confirm that the DA-DOX dyad was successfully copolymerized with DA to form PDCNs, and the DOX weight percentage was basically consistent with the feed ratio (Table 1 and Table S2). The XPS of PDCNs shows the binding energy peaks of C=N-N at 402 eV (C=N) and 398 eV (N-N), verifying the acid-labile hydrazone bond still retained in PDCNs. As shown in Figure 2, both DLS and TEM analyses show that PDCNs have nearly spherical morphology with hydrodynamic diameters ranging from 70 nm to 100 nm, and the size determined by TEM is in agreement with that measured by DLS. Taken together, the biopolymer-drug conjugate nanoparticles of PDCNs can be facilely prepared in one-pot by the precipitation copolymerization of the DA-DOX prodrug with DA under mild conditions, to the best of our knowledge, which have not been designed.52-71

16


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Figure 2. DLS (A, B) and TEM (C, PDCN16; D, PDCN25) results for PDCNS. To assess the physiological stability, the PDCNs nanoparticles were dispersed well in PBS (10 mM, pH 7.4) or with fetal bovine serum (FBS, 1%, 5%, and 10%) at 37 °C and then monitored by DLS. The PDCNs in PBS hardly changed for two months while the hydrodynamic size in FBS still kept < 350 nm after 48 h incubation (Figure S4). This is due to the PDCNs having negative zeta potential of about −(27.0−28.5) mV, which is slightly higher than −33.1 mV of the PDA counterpart. Note that the size increase of nanoparticle is because the albumins in FBS would be absorbed onto the surface of nanoparticle to form the so-called “protein corona”.73 The PDCNs stability in FBS suggests they might be intravenously administered for in vivo antitumor study.

17



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Figure 3. (A) The NIR spectra of the nanoparticles (300 µg/mL) and those upon the laser irradiation; (B) The temperature-elevating magnitude of the nanoparticle solution (300 µg/mL) upon NIR laser irradiation over time; (C, D) The temperature change curves of the nanoparticle solution during repeated laser on/off cycles. Photothermal effect and dual stimuli-triggered drug release of PDCNs. Owing to strong π-π stacking of indole units, the melanin and PDA nanomaterials often exhibit NIR absorption at 650−900 nm.52-56 As expected, the PDCNs present broad and strong NIR-absorbing spectroscopy similar to the PDA counterpart (Figure 3A). This indicates that they have an intrinsic photothermal conversion capability and the covalent-linked DOX has no apparent effect on the NIR absorbance. To study the NIR-mediated photothermal effect, the PDCNs solution was irradiated by a continuous-wave diode laser for 5 min at room temperature and/or at 37 °C (to mimic the human body temperature), respectively. For instance, upon the NIR laser irradiation (5 min, 808 nm, 2 W/cm2), the samples PDA, PDCN16 and PDCN25 heated their solutions by 25.7 °C, 23.5 °C and 24.6 °C respectively 18


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while the control PBS increased by only 4.0 °C (Figure 3B). The ∆T magnitude can be easily tuned by the laser power density and the nanoparticle concentration (Figure S5). These data indicate that ∆T is mainly induced by the NIR-absorbing PDCNs or PDA. Moreover, the heating-cooling cycle experiments show excellent photo-stability of the PDCNs with no change in photothermal curves (Figure 3C-D), which is also verified by the NIR-absorbing spectra before and after irradiation. Notably, both PDCN16 and PDCN25 have a good photothermal conversion efficiency (η) of 34.7 % and 38.5 %, respectively, which is slightly lower than 40.3 % of PDA.66 In contrast, the well-known gold nanorod and nanoshell have a lower η of 22% and 13%; and the gold nanorod would be melted to some extent and lose partial photothermal ability after repeated irradiation.34 Collectively, these experiments evidence that the PDCNs possess excellent NIR-absorbing capability, high photothermal conversion efficiency and better photostability, enabling them as superior polymeric photothermal nanoagents.33-40 The physically drug-loaded polymeric nanomedicine often has a burst and premature drug release behavior, which compromises the anticancer efficacy and induces safety concern.2-5 Do the PDCNs have a minimized drug release at pH 7.4 and at 37 oC but exhibit an intracellular pH-sensitive drug release behavior? The PDCNs were incubated in PBS at 37 oC under different pH conditions; to mimic the bloodstream and intracellular endo-lysosome milieu, the pH value was set at 7.4, 6.0 and 5.0, respectively. Taking PDCN25 as example (Figure 4A), only 15.3 % of DOX released at pH 7.4 for 130 h while it significantly increased to 63.7 % at pH 6.0 and to 89.3 % at pH 5.0. This is because the hydrazone bond that linked DOX with PDA was cleaved quickly at acidic pH 6.0 or 5.0, resulting in a rapid 19



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DOX release.14 Similar trend was observed for PDCN16 (Figure 4B). Compared to physically drug-loaded nanoparticles with burst drug release at pH 7.4, the PDCNs possess an endo-lysosomal pH-sensitive drug release profile, endowing intracellular enhanced efficacy and minimized side effect for chemotherapeutics.20, 50, 51 In addition, the PDCNs at pH 7.4 maintained initial size even upon the NIR irradiation during the drug release process (Figure S6). When pH was switched to 5.0, the size of the PDCNs significantly increased at 3.5 h and then kept stable at 10 h. This pH-dependent size change of PDCNs also suggests that the hydrophobic DOX was quickly detached from nanoparticles due to the acidic cleavage of hydrazone linker. This results in the swelled nanoparticle with a loose structure and a bigger size because of the decreased π-π stacking and hydrophobic interactions. Note that the drug release of PDCNs was controlled by pH-sensitive hydrazone bond cleavage and drug diffusion from nanoparticle; however, the size change was controlled mainly by pH-activated swelling of nanoparticle.2 Thus the drug release might be delayed compared to the size change of nanoparticle. Whether does the intrinsic photothermal attribute of PDCNs affect on the drug release behavior? This is a key point for attaining the combination PT-CT efficacy.45-48 As shown in Figure 4C, without NIR irradiation the 12 h cumulative DOX release of PDCN25 was 7.9 %, 25.4 %, and 47.2 % at pH 7.4, 6.0, and 5.0, respectively; however, upon irradiation it increased to 16.5 %, 43.7 %, and 82.5 %, which is almost 2-fold greater than that without the stimulus. Similar photo-heating-enhanced drug release profile was found for PDCN16 because the photothermal effect can accelerate DOX diffusion from nanoparticles, inducing a fast drug release (Figure 4D).66-71 Collectively, the PDCNs present both endo-lysosomal 20


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pH-sensitive and NIR-triggered DOX release profiles, which will simultaneously enhance the CT efficacy.

Figure 4. The pH-sensitive (A, B) and the NIR-triggered (C, D) drug release profiles of PDCNS in PBS at 37 oC (n=6). In vitro PT-CT and in vivo pharmacokinetics. To evaluate the polymeric prodrug release induced CT, the intrinsic PT, and the combination PT-CT of PDCNs in vitro, the cell viability was measured by a standard MTT method. Upon the NIR laser irradiation (5 min, 808 nm, 2 W/cm2) and/or the incubation with PDA without irradiation, both HeLa and L929 cells kept alive above 95 % (Figure S7). The control tests indicate that both the NIR laser irradiation and PDA itself induced less cytotoxicity to these healthy and carcinoma cell lines. As for PT, CT, and PT-CT, the cytotoxicity of PDCNs or PDA is dose-dependent and the cell viability gradually decreases over the drug or nanoparticle concentration (Figure 5A). As for single PT, both PDCN16 and PDCN25 gave a half maximal inhibitory concentration (IC50) of 53.70 µg/mL and 53.84 µg/mL on the basis of PDA concentration (Figure S8), which is comparable to 53.76 µg/mL of the control PDA. Based on the DOX concentration, both 21



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PDCN16 and PDCN25 gave an IC50 of 4.65 µg/mL and 5.85 µg/mL for single CT, respectively (Table 1). As for the combination PT-CT, PDCN16 gave an IC50 of 9.22 µg/mL for the combination PT and that of 1.76 µg/mL for the combination CT; however, PDCN25 showed that of 5.63 µg/mL and 1.88 µg/mL for the combination PT and the combination CT, respectively. The combination effect between different drugs or therapies is generally evaluated by the combination index (CI).32, 44, 72 The CI

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