Hyaluronic AcidâMethotrexate Conjugates Coated Magnetic

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Hyaluronic Acid-Methotrexate Conjugates Coated Magnetic Polydopamine Nanoparticles for Multimodal ImagingGuided Multistage Targeted Chemo-Photothermal Therapy Qi Li, Jiayong Yang, Yilin Chen, Xinyi Zhou, Dengyue Chen, Yang Li, and Xuan Zhu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00473 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Molecular Pharmaceutics

Hyaluronic Acid-Methotrexate Conjugates Coated Magnetic Polydopamine Nanoparticles for Multimodal Imaging-Guided Multistage Targeted Chemo-Photothermal Therapy Qi Lib,1, Jiayong Yanga,1, Yilin Chenb, Xinyi Zhoub, Dengyue Chenb, Yang Lic,* Xuan Zhub,* a

Xiang'an Branch, the First Affiliated Hospital of Xiamen University， Xiamen

361001， China; b

Fujian Provincial Key Laboratory of Innovative Drug Target Research, School of

Pharmaceutical Science, Xiamen University, Xiamen 361002, China c

Department of Biomaterials, College of Materials, Xiamen University, Xiamen

361005, China Corresponding Authors *Yang Li, E-mail: [email protected], telephone number: (+86) 05922189650 *Xuan Zhu, E-mail: [email protected], telephone number: (+86) 05922881181 ABSTRACT

Combination cancer therapy with various kinds of therapeutic approaches could improve the effectiveness of treatment while reducing side effects. Herein, we elaborately developed a theranostics nano-platform based on magnetic

polydopamine

acid-methotrexate

(MPDA)

conjugates

coated

with

hyaluronic

(MPDA@HA-MTX) 1

ACS Paragon Plus Environment

for

Molecular Pharmaceutics 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

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chemo-photothermal treatment (PTT). In this nano-platform, Fe3O4 served as the core was applied as contrast agent for T2-weighted magnetic resonance imaging (MRI) and early-phase magnet targeting. Meanwhile PDA was used as a versatile shell for effective loading of chemotherapeutic doxorubicin (DOX) to achieve controlled release and PTT simultaneously. Moreover, HA-MTX conjugates could offer later-phase specific cellular dual-targeting ability during the therapy. Both in

vitro

and

in

vivo

studies

demonstrated

that

DOX-loaded

MPDA@HA-MTX (MPDA/DOX@HA-MTX) exhibited the preferential tumor accumulation, enhanced specificity to target tumor cells, pH-/laser-responsive release, and high tumor cell-killing efficiency. By combined chemo-PTT under the guidance of fluorescence/MR imaging, the tumors in mice were completely eliminated after treatment, indicating that MPDA@HA-MTX nanoparticles have great potentials as a novel drug-loading

platform

for

imaging-guided

multistage

targeted

chemo-photothermal combination therapy.

KEYWORDS:

magnetic

polydopamine;

multistage

targeted;

chemo-photothermal therapy; fluorescence/magnetic resonance imaging

2


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1. INTRODUCTION

Chemotherapy is widely used as a common strategy for cancer therapy nowadays, while severe side effects such as poor bioavailability 1 and multidrug resistance

2,

3

are inevitable during the therapy.

Nanoparticles acting as drug carriers are able to target tumor region or cells, control drug release, and even be used for imaging, hence the great advantages of nanoparticles are of great interest to investigators. Compared

with

the

conventional

chemotherapeutic

4-8

drug,

nanoparticles-based delivery system could provide various synergistic effects as well as allowing the multimodality of cancer therapy. 9-11 Photothermal therapy (PTT) has been introduced as a promising non-invasive methodology in clinical multimodality theranostics when applied with chemotherapy or other therapy modalities.

12, 13

So far,

numerous of nanomaterials have been explored as potential light absorbing agents with excellent photothermal conversion property which utilized the heat generated from near infrared (NIR) laser irradiation to ablate cancer cells.

14-16

A mild photothermal heating (nearly 45°C) can not only

enhance the cellular uptake of nanoparticles but also help the drug release from nanoparticles to further improve the chemotherapeutic efficiency. 17, 18

Recently, polydopamine (PDA) nanoparticles are drawing increasing

attention due to their great biocompatibility and excellent NIR absorption 3



ability.

19

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The PDA films are formed by dopamine (DA) as thin and

surface-adhesiveness material via self-polymerization process, which can happen to the surface of a wide range of inorganic and organic materials with the presence of oxygen. 20 A large number of functional groups make PDA shells load aromatic therapeutic drugs by means of π-π stacking and hydrophobic interactions. 21, 22 In addition, Fe3O4 nanoparticles are approved by Food and Drug Administration (FDA) for in vivo application and introduced as a magnetic resonance (MR) imaging agent due to low toxicity and great biocompatibility.

23-25

Moreover, Fe3O4 nanoparticles can be used for

magnetically targeted drug delivery. 26 Therefore, magnetic polydopamine (MPDA) nanoparticles prepared by in situ self-polymerization of polydopamine shells on the surface of Fe3O4 nanoparticles could be served as a potential nanoplatform for tumor physical targeting and MR imaging-guided PTT in combination cancer therapy. Furthermore, hyaluronic acid (HA) is an anionic and hydrophilic natural material, containing repeating units with functional groups such as hydroxyl and carboxyl that can be functionalized with specific moieties covalently. 27, 28 HA acts as extracellular matrix component in vivo, which shows excellent biocompatibility and specific binding affinity to the CD44 receptor overexpressed on the surface of the tumor cells. great

advantages

such

as

extensive

sources,

4


29

Due to the

biocompatibility,

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biodegradability, and non-toxicity, HA has been extensively introduced to drug delivery system as an active targeting moiety in cancer treatment. The cellular uptake and internalization of nanoparticles would be thus enhanced with the aid of tumor targeting ability, increasing the intracellular drug concentration. 30, 31 It was well known that folate receptor as the specific receptor was overexpressed on the membrane of tumor cells such as ovarian, kidney, breast, lung, brain, endometrial, and colon cancer cells. 32, 33So it is a good marker for targeted drug delivery to tumor site. 34, 35 Methotrexate (MTX), an analogue of folic acid, not only could act as anticancer drug to kill the cancer cells, but also an active targeting moiety that specifically bounds to folate receptor on the cancer cells surface and are internalized through receptor-mediated endocytosis.

36-38

Moreover, high binding affinity

towards folate receptor could still remain when jointed to a foreign molecule.

39

Thus, a drug carrier with dual-targeting effects is designed

based on the conjunction of MTX and HA through chemical reaction, in which the tumor targeting efficiency and tumor accumulation could enhanced to a certain degree by the unique targeting effect of therapeutic MTX via folate receptor and the targeting capacity of HA materials via CD44 receptor. Hence, we developed a multi-functional carrier based on dual-active targeting strategy to carry both anticancer drugs and imaging agent. When 5



Page 6 of 48

supported with magnetic field and NIR laser, the drugs could be delivered and released in a controlled manner at targeted location and specific time, which help precisely treat the tumor under the guidance of multimodal imaging. The schematics of the preparation and delivery mechanism of MPDA@HA-MTX

were

described

conceptually

in

Figure

1.

MPDA@HA-MTX targeting or accumulating to tumor sites would accelerate via magnetic targeting effect and enhanced penetration and retention (EPR) effect following intravenous injection in mice. Meanwhile, folate receptor and CD44 receptor dual-targeting would enhance the uptake of MPDA@HA-MTX in tumor cells, and drug release at tumor site would be accelerated under the simulation of NIR laser and tumor acidic pH condition. Simultaneously, fluorescence/MR imaging could be used to guide PTT, and cancer cells could be killed by the multimodal treatment. These features ensure that the as-fabricated MPDA/DOX@HA-MTX could be used as a theranostic agent to realize a great

synergistic

therapeutic

effect

by

the

fluorescence/MR

imaging-guided chemo-photothermal combination treatment with reduced side effects both in vitro and in vivo. Additionally, the novel design of MPDA/DOX@HA-MTX consists of biodegradable PDA and HA, which serve as endogenous biomaterials and could be highly beneficial to in vivo cancer theranostics.

6


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Figure 1. Schematic illustration of (A) the synthesis process of MPDA/DOX@HA-MTX nanoparticles. (B) NIR fluorescence/MR imaging-guided chemo-thermal combination therapy through intravenous injection. 7



Page 8 of 48

2. EXPERIMENTAL METHODS 2.1. Materials FeCl2•4H2O, FeCl3•6H2O, dimethyl sulfoxide (DMSO, >99.8%), ammonia

solution

(NH4OH,

25-28%),

N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, 99.0%), 4-dimethylaminopyeidine (DMAP), indocyanine green (ICG, 75%), formamide, and dialysis bag (3500 Da) were purchased from Aladdin (Shanghai, China). Doxorubicin hydrochloride (DOX) was obtained from Huafeng United Technology Co., Ltd. (Beijing, China), Hyaluronic acid was bought from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China), Tris was attained from Energy Chemical (Shanghai, China). FluoroshieldTM with DAPI and dopamine hydrochloride (DA) were obtained from Sigma (USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium penicillin-streptomycin

bromide solution

were

(MTT), purchased

trypsin, from

and Beyotime

Biotechnology (Shanghai, China). Fetal bovine serum (FBS) was purchased from Biological Industries (Israel). RPMI medium and DMEM medium was acquired from Thermo Scientific (USA). HeLa cells and 4T1 cells were provided by American Type Culture Collection (ATCC). Methotrexate (MTX) was purchased from Bio Basic Inc. (Markham, Ontario, Canada). All other chemicals were analytical grade and used without further purification. 8


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2.2. Synthesis of Fe3O4 Nanoparticles and Drug-Loaded Magnetic Polydopamine Nanoparticles (MPDA/DOX) Fe3O4 nanoparticles were prepared by chemical co-precipitation method as reported40,

41

with mild adjustments under N2 atmosphere.

FeCl2•4H2O and FeCl3•6H2O at the ratio of 1.5: 1 were mixed in 20 mL of deionized water with mild stirring, and then heated to 50°C. 5 mL of ammonia solution was added dropwisely, and the temperature was adjusted to 70°C. The product was magnetically separated, washed with deionized water twice, and then lyophilized for further use. Magnetic polydopamine nanoparticles (MPDA) were prepared as reported with mild adjustments.

42, 43

Firstly, 300 mg of dopamine was

added to 100 mL of 10 mM Tris (pH 8.5) containing 100 mg of Fe3O4 nanoparticles and the solution was then mixed by sonication. After stirring at room temperature for 12 h, MPDA were obtained by magnetic separation and washed with deionized water for three times. The final obtained MPDA nanoparticles were re-dispersed in water for further use. The as-obtained MPDA nanoparticles were re-dispersed in water for further use. MPDA (1 mg/mL) was firstly dissolved in PBS (10 mM) at pH 8.0. Then DOX at different weight ratios with MPDA (DOX: MPDA = 0.25, 0.5, 1, and 2) was added. After the stirring process for 12 h in the dark condition, the above solutions were centrifuged and washed with PBS until the supernatant was colorless. 9



Page 10 of 48

2.3. Preparation and Characterization of HA-MTX Conjugates HA-MTX was synthesized by previously described method

44

with

minor modification. Briefly, MTX (42 mg, 0.92 mmoL), EDC (76 mg, 0.40 mmoL), and DMAP (45 mg, 0.37 mmoL) were dissolved in 3 mL of DMSO, and then mildly stirred for over 30 min to activate the carboxyl group of MTX in the dark condition. Then HA (50 mg, 0.0125 mmoL) was dissolved in 15 mL of formamide and mixed with MTX solution under vigorous stirring for the esterification reaction. The reaction was kept at room temperature for more than 24 h in the dark condition. Thereafter, the production was purified by dialysis in deionized water for 5 days and then lyophilized for 24 h. The final product was analyzed by 1H NMR (400 Hz, Bruker, Switzerland), HA was dissolved in D2O, and MTX and HA-MTX were dissolved in DMSO-d6. The fourier transform infrared (FT-IR) spectroscopy spectra of HA, MTX, the physical mixture of HA and MTX, and the MTX-HA conjugate were analyzed using an infrared spectrometer (Bruker, Switzerland). The X-ray diffraction (XRD) spectra of the samples were also obtained using an X-ray diffractometer (Rigaku Ultima IV). And after HA, MTX, the physical mixture of HA and MTX, and the MTX-HA conjugate were dispersed in water, the UV-vis-NIR spectra were determined

by

Perkin Elmer Lambda 750 UV-vis-near-infrared

spectrophotometer (UV-vis-NIR, Perkin-Elmer, Norwalk CT).

10


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2.4.

Preparation

of

HA-MTX

Coated

Drug-Loaded

MPDA

Nanoparticles (MPDA/DOX@HA-MTX) MPDA/DOX was mixed with HA solution or HA-MTX solution (5 mg/mL) for 4 h, and the solution was then centrifuged for three times to fully remove the excess unbound HA or HA-MTX. And the FT-IR spectroscopy spectra of MPDA and MPDA@HA were analyzed using an infrared spectrometer.

2.5. Physicochemical Characterization The crystallinity and phase composition of Fe3O4 nanoparticles were determined by X-ray diffraction (XRD, Rigaku Ultima IV). A vibrating sample magnetometer (VSM, Lake Shore, USA) was used to measure the magnetic properties of Fe3O4 nanoparticles and MPDA@HA-MTX. The morphology of Fe3O4 nanoparticles and MPDA@HA-MTX was characterized by a transmission electron microscope (TEM, Tecnai G, Spirit, FEI, Hong Kong). The particle size and zeta potential of the as-synthesized nanoparticles were measured by dynamic light scattering

（DLS, Nano-ZS, Malvern, Instruments, Ltd., U. K.). The weight ratio of Fe3O4 nanoparticles in MPDA/DOX@HA-MTX was measured by inductive coupling plasma mass spectrometry (ICP-MS, 7700x, Aglient, Instruments, Ltd., USA). MPDA/DOX@HA-MTX was dissolved in aqua regia to determine the Fe concentration by ICP-MS and then the weight ratio of Fe3O4 nanoparticles in MPDA/DOX@HA-MTX was calculated. 11



2.6. Measurement of Photothermal Efficacy and Stability To confirm the photothermal effect, 200 µL of solutions with different concentrations were exposed to 808 nm laser (STL 808CFS-10W, China) under the power density of 1 W/cm2 for 300 s. The temperature of each sample was measured by a thermocouple thermometer (TES-1310, Taiwan) to identify the photothermal stability of nanoparticles by recording the temperature changes of the solutions under irradiation of the 808 nm laser under the power density of 1 W/cm2 for 5 cycles (5 min per cycle).

2.7. Drug Loading Capacityand Releasing The DOX loading capacity was detected by fluorescence at 475/565 nm intensity using high throughput drug screening system (PerkinElmer OperettaTM, USA) and UV-vis-NIR spectra. Drug release at different conditions was carried out. The experiments were divided into three groups at pH 5.0, pH 7.4, and pH 5.0 with NIR laser (808 nm, 1 W/cm2, 5 min) respectively, and the solutions were collected at different time points. The concentration of DOX released from MPDA/DOX@HA-MTX was also measured by fluorescence at 475/565 nm intensity using high throughput drug screening system.

2.8. Hemolysis Test Hemolysis test was performed on MPDA and MPDA@HA-MTX which were dispersed in PBS and mixed with identical volume of 2% (v/v) 12


Page 12 of 48

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rabbit red blood cells (RBCs). The final concentrations of nanoparticles were prepared at 5, 12.5, 25, 50, 125, 250, and 500 mg/mL. The mixtures were then vortexed and incubated at 37°C for 1 h followed by the centrifugation at 8, 000 rpm for 5 min. The absorbance values of supernatant were measured at 560 nm by a Multiskan FC microplate reader. An equal volume of PBS was set as a negative control and deionized water as a positive control. The hemolysis percentage was calculated

by the

following

formula:

hemolysis

percentage

=

(experimental sample absorbance - negative control sample absorbance)/ (positive control sample absorbance - negative control sample absorbance) × 100%.

2.9. Blood Circulation 200 µL of MPDA/DOX@HA-MTX was intravenously injected into tumor-free healthy SD mice. At each time point, 0.5 mL of blood was collected from eyes and then centrifuged at 10, 000 rpm for 10 min at 4°C. 0.1 mL of supernatant was added with acid ethanol (concentrated hydrochloric acid mixed with dehydrate alcohol at 1: 20 in volume) to extract DOX. Subsequently, the mixture was incubated in dark overnight. After the centrifugation to obtain the DOX in supernatant, the concentration of remaining DOX was determined by fluorescence intensity.

2.10. Cellular Experiments 13



To investigate the in vitro cancer targeting efficiency of nanoparticles, HeLa cells were seeded in the 12 wells plate and incubated in DMEM medium. After the incubation for 24 h, the medium was replaced with fresh DMEM containing MPDA/DOX@HA-MTX or MPDA/DOX@HA (DOX dose: 10 µg/mL) and incubated for another 1 h and 4 h, respectively. HeLa cells were then washed with PBS for three times and fixed with 4% paraformaldehyde while DAPI was used for nuclei staining. The stained HeLa cells were observed by confocal laser scanning microscopy (CLSM, Leica Microsystems, Germany). In addition, to test the NIR laser-triggered cell uptake of nanoparticles, HeLa cells were incubated with free DOX or MPDA/DOX@HA-MTX with the same amount of DOX (10 µg/mL), and then exposed with or without NIR laser (808 nm, 1 W/cm2) for 20 min. After the exposure, the cells were immediately washed with PBS to remove excess DOX and nanoparticles. The uptake efficiency was also determined by CLSM. To measure the internalization of MPDA/DOX@HA-MTX or MPDA/DOX@HA, HeLa cells were incubated in 6 wells plate for 24 h and cultured with MPDA/DOX@HA-MTX or MPDA/DOX@HA (DOX dose: 10 µg/mL) for 1 h or 4 h. Then the cells were washed with PBS. The cellular DOX fluorescence was analyzed by a flow cytometry (Beckman Coulter, CA, USA). In addition, to test the NIR laser-triggered cell uptake of nanoparticles, HeLa cells were incubated with the free DOX or 14


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MPDA/DOX@HA-MTX with same amount of DOX (10 µg/mL), and then exposed with or without NIR laser (808 nm, 1 W/cm2) for 20 min. Excess DOX and nanoparticles were removed by washing cells with PBS. The uptake efficiency was determined by flow cytometry. MTT assay was performed to test the cytotoxicity of free MTX, HA-MTX

conjugate,

DOX-free

MPDA@HA-MTX,

DOX-free

MPDA@HA, MPDA/DOX@HA-MTX, and MPDA/DOX@HA against HeLa cells with or without NIR laser (808 nm, 1 W/cm2, 5 min). HeLa cells were seeded in 96 wells plate, in which different concentrations of nanoparticles were added and irradiated by NIR laser (808 nm, 1 W/cm2, 5 min). After incubation under 37°C and 5% CO2 condition for another 24 h, the cell viability was measured by MTT assay and untreated cells were used as control.

2.11. Mouse Tumor Model Female BALB/c nude mice and BALB/c mice were bought from Xiamen University Laboratory Animal Center. Animal experiments were performed under protocols approved by Xiamen University Laboratory Animal Center. To generate tumor model, 1×106 4T1 cells were subcutaneously injected to the back of mice, and the tumor size reached nearly 20 mm3 after one week.

2.12. Fluorescence/MR Imaging

15



To

measure

the

combined

tumor

Page 16 of 48

targeting

effect

of

HA/MTX/constant magnetic field (CMF) and obtain the in vivo distribution of the prepared nanoparticles, 4T1 tumor-bearing female BALB/c nude mice were divided into three groups (three mice for each group). Owing to the amino groups existing on the surface of PDA nanoparticles, they showed positive charges in the acidic solution (isoelectric point ≈4.6, Supplementary Figure S1). Thus at the acid solution (pH 2~3), ICG with negative charges could be easily adsorbed on the surface of PDA, likely which may be drive by both electrostatic and hydrophobic interactions.11 What’ more, aromatic ICG could be loaded onto PDA nanoparticles by means of π-π stacking. Indocyanine green (ICG, trace amounts of ICG)-labeled MPDA/DOX@HA and ICG-labeled MPDA/DOX@HA-MTX were prepared by mixing ICG solution (1 mL, 0.5 mg/mL) with DOX-loaded MPDA under ultrasonic condition. After stirring for 2 h and washing by deionized water for several times, the excess ICG was removed and the ICG-labeled MPDA/DOX nanoparticles were obtained. 200 µL of ICG-labeled MPDA/DOX@HA-MTX was intravenously injected into mice at some ICG concentrations (0.2 mg/kg) for in vivo fluorescence images to investigate targeting efficiency. Two groups of mice were injected with MPDA/DOX@HA-MTX and about 5 mm diameter magnets were stuck to the tumors in one group. Mice in another group were injected with ICG (trace amounts of ICG)-labeled 16


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MPDA/DOX@HA. The ICG concentration in all groups was equivalent. The fluorescence signals from the tumor site were obtained by Maestro in

vivo imaging system (Cambridge Research & Instrumentation, Woburn, MA, USA) to track fluorescence of ICG at 1 h, 4 h, 8 h, and 24 h. Different concentrations of MPDA/DOX@HA-MTX were dissolved in deionized water and scanned by a 7T MRI scanner (Varian, Palo Alto, CA) with MSME sequences. Animal MR imaging were conducted under the same MR scanner. To investigate the CMF targeting efficiency, tumor-bearing mice were divided into two groups with one group fixed with 5 mm diameter magnets before MRI.

2.13. Photothermal Therapy Efficiency In vivo cancer treatment with MPDA/DOX@HA-MTX was performed to test the PTT efficiency. Tumor-bearing female BALB/c mice were divided into two groups: (1) MPDA/DOX@HA-MTX, (2) MPDA/DOX@HA-MTX + CMF. The mice in each group were intravenously injected with nanoparticles through the tail vein (DOX dose =4 mg/kg)45. After 12 h, magnets were removed and the mice were irradiated by the NIR laser (808 nm, 1 W/cm2 for 5 min). The temperature changes of the tumors during irradiation were monitored by an IR thermal camera.

2.14. In Vivo Combination Therapy

17



For evaluating in vivo combination therapy, 18 mice were divided into six groups equally and randomly. Mice in each group were treated with PBS, free DOX, MPDA/DOX@HA-MTX, MPDA/DOX@HA-MTX + CMF, MPDA/DOX@HA-MTX + light, MPDA/DOX@HA-MTX + CMF + light (DOX dose = 4 mg/kg) in every two days. CMF was given immediately and after 12 h, the tumors were supported with NIR laser (808 nm, 1 W/cm2 for 5 min). The body weight and tumor volume of every mice were monitored for 2 weeks. Tumor volumes were calculated by the equation: tumor volume = length × width2/2.

2.15. Histopathological Examination All mice were sacrificed after in vivo combination therapy, and their tissues including kidneys, heart, liver, spleen, and lung were harvested and fixed in 4% paraformaldehyde at 4°C for over 24 h. Then the tissues were embedded in paraffin blocks and sectioned into slices. The obtained tissue slices were stained with hematoxylin and eosin, respectively. The stained slices were examined and observed by a digital microscopy (D-35578 Wetzlar, Leica Microsystems CMS GmbH, Germany).

2.16. Statistic Analysis Data were expressed as means ± standard deviation (SD). To determine the significance level among the tested groups, the analysis of variance was performed and P values less than 0.05 were considered to be statistically significant. 18


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3. RESULTS AND DISCUSSION 3.1. Preparation of MPDA Figure 1A schematically illustrated the synthesis process of MPDA/DOX@HA-MTX. Fe3O4 nanoparticles as the core particles were prepared by chemical co-precipitation method, and coated with PDA shells by mild stirring under alkaline condition. The Fe concentration was measured by ICP-MS, and the weight ratio of Fe3O4: PDA in MPDA nanoparticles was measured to be approximately 0.35: 1. PDA possessed several functional groups such as amino and catechol groups,

46

which

could facilitate the further functionalization for prolonged blood circulation and tumor cell targeting. Additionally, MPDA nanoparticles were negatively charged in the alkaline solution as the isoelectric point of MPDA was about 4.5 (Supplementary Figure S1). Due to the delocalized π-electron structures, MPDA could be used to load aromatic molecules such as commonly used chemotherapy drug DOX by hydrophobic interaction and π-π stacking interaction 19 when the MPDA was dispersed in PBS (pH = 8.5). The drug-loading content on MPDA increased along with the increase of the weight ratios of DOX: PDA. In addition, HA-MTX conjugates with carboxyl and amide groups were coated onto MPDA via hydrogen bond interaction and electrostatic adsorption due to

19



the amino and catechol groups existing on the surface of MPDA nanoparticles. 47

3.2. Preparation and Characterization of HA-MTX Conjugates HA-MTX conjugates were prepared for dual-targeting to CD44 and folate receptor. The carboxyl group of MTX was conjugated to the hydroxyl group of HA via an esterification reaction (Figure 2A). As shown in Figure 2B, 2C and 2D, the 1H NMR spectrum of HA and HA-MTX exhibited the peak at 1.8 ppm, which was ascribed to the N-acetyl group from HA. In addition, the characteristic proton peaks of benzene ring and pteridine ring from MTX were shown at 6.8, 7.5, and 8.6 ppm in the 1H NMR spectrum of MTX and HA-MTX. These results demonstrated the successful conjugation of MTX to HA. In FT-IR results (Figure 2E), compared with pristine MTX, HA, and physical mixture of MTX and HA, HA-MTX conjugates showed new absorption bands at 1696.92 cm−1, which was ascribed to the ester carbonyl (C=O) stretching vibrations. This result indicated that MTX was successfully conjugated to HA via ester linkage. XRD pattern in Figure 2F exhibited that several sharp crystalline peaks existed in MTX result (yellow) and no obvious peaks were found in HA result (black), indicating the crystalline form of MTX and the amorphous form of HA. Certain crystalline peaks were still detectable in the XRD pattern of the physical mixture (blue), indicating that MTX 20


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remained in their physical mixture in crystalline form. However, in XRD pattern of HA-MTX conjugates (red), all of the crystalline peaks disappeared, implying a less ordered structure and crystal defects. This result further indicated the successful conjugation of MTX to HA. For UV-vis-NIR analysis (Figure 2G), the characteristic absorption peak of free MTX and physical mixture of MTX and HA at 308 nm were observed in UV-vis-NIR spectra, while a characteristic absorption peak of HA-MTX conjugate was detected around 350 nm. The results also demonstrated the successful synthesis of HA-MTX conjugate. These results shown above confirmed the successful synthesis of HA-MTX

conjugates

that

could

be

potentially

used

surface-functionalize the nanoparticles for dual-targeting delivery.

21


to


22


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Figure 2. Characterization of HA-MTX conjugate. (A) Synthetic routes of HA-MTX through esterification. 1H NMR spectra of (B) HA, (C) Free MTX, (D) HA-MTX conjugate. (E) FT-IR spectra of HA, MTX, HA+MTX mixture and HA-MTX conjugate. (F) XRD pattern of HA, MTX, HA+MTX mixture and HA-MTX conjugate. (G) UV-vis-NIR spectra of HA, MTX, HA+MTX mixture, and HA-MTX conjugate.

3.3. Synthesis and Characterization of MPDA@HA-MTX As observed in TEM (Figure 3A), Fe3O4 nanoparticles were uniformly dispersed and their diameters were about 10 nm. As shown in the Supplementary Table S1, the Fe3O4 nanoparticles and MPDA had a hydrodynamic diameter of 81.5 nm and 167.10 nm respectively, due to the existence of a hydration layer in aqueous solutions. After loading DOX into MPDA, the hydrodynamic diameter of nanoparticles changed dramatically. Additionally the zeta potential increased from -25.3±0.67 mV to 24.9±3.12 mV, indicating that the driving force for DOX loading was mainly by electrostatic interaction.

The TEM

image of

MPDA@HA-MTX (Figure 3B) showed that the self-polymerization of DA occurred on the surface of Fe3O4 nanoparticles. In addition, the synthesized nanoparticles displayed a preferable monodispersed nature. And

the

inserted

image

in

Figure

3B

23


revealed

that

every


MPDA@HA-MTX encapsulated more than one Fe3O4 nanoparticle. It was found that MPDA@HA-MTX was monodispersed with a hydrodynamic particle size of 236.5 ± 21.5 nm (Figure 3C) and obvious tyndall effect. Additionally, the zeta potential of MPDA@HA-MTX was determined to be -22.5 ± 0.4 mV (Figure 3D) by DLS. The particle size of MPDA/DOX@HA and MPDA/DOX@HA-MTX are 267.1 and 236.50 nm. The FT-IR spectra (Supplementary Figure S2) of MPDA@HA had a broad peak around 3400 cm−1 ascribed to C−NH, C=NH, and −OH stretching vibrations. The coatings also presented the peaks at 1050 cm−1 (C−O−C stretching vibrations), suggesting that the HA were successfully prepared on to MPDA and HA coatings may be due to the strong electrostatic interaction between PDA and HA. Because the amino group of PDA and the carboxyl group of HA have electrostatic interaction or hydrogen bonding or a combination of both effect. As shown in XRD results (Figure 3E) of Fe3O4 nanoparticles, the diffraction peak at 30.2°, 35.5°, 43.2°, 54.9°, 57.3°, and 62.8°, which was coincided well with the (220), (311), (400), (422), (511), and (440) facets of Fe3O4 respectively, indicating that the prepared Fe3O4 nanoparticles were of cubic spinal structure. The VSM studies (Figure 3F) conducted on Fe3O4 nanoparticles and MPDA@HA-MTX showed that they both displayed good magnetic properties. The decrease in the magnetization saturation values after the coating of PDA and HA-MTX conjugates could be explained by the 24


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functionalization of Fe3O4 nanoparticles, as the same weight of samples contained less Fe3O4 nanoparticles, reducing the magnetic properties to a certain extent. As shown in photothermal heating curve (Figure 3G), the temperature of

MPDA@HA-MTX

dispersion

increased

in

an

irradiation

time-dependent manner. The final temperature which increased by 38°C was detected at the concentration of 100 µg/mL, while the temperature increase in water was only 7°C. The result indicated that the temperature increase was mainly due to the strong NIR absorption ability of PDA 45 and photothermal conversion ability of the novel nanoparticles. And in photothermal stability experiment (Figure 3H), no obvious temperature decrease was detected for each NIR irradiation during the measuring period, which suggested that these prepared materials had excellent photothermal stability. Therefore, those unique properties made the nanoparticles effective photothermal agents that could be applied for PTT to cancer.

3.4. Drug Loading Capacity and Release Studies As shown in the schematic (Figure 4A), the test of stimuli-responsive drug release from MPDA/DOX@HA-MTX was carried out under the stimuli condition including acidic pH and NIR laser. In this case, DOX was loaded by simple incubation with MPDA in alkaline aqueous solution overnight.

45

The unloaded drug was removed and the concentration of 25



DOX in supernatant was confirmed by high throughput drug screening system (Figure 4B). A largest drug-loading capacity was measured to achieve nearly 150% (DOX: MPDA, w/w). The drug-loading capacity of nanoparticles increased due to the increase of weight ratios of feeding DOX and PDA. After the loading of the drug into MPDA, a typical absorption peak at 490 nm for DOX was detected in the UV-vis-NIR spectra (Figure 4C), and the peak intensity gradually increased due to the increase of drug-loading amount. The result demonstrated that DOX was successfully loaded within MPDA. MPDA/DOX@HA-MTX samples in PBS at pH 5.0 and 7.4 were irradiated by the NIR laser (808 nm, 1 W/cm2 for 5 min) at different time points to test the drug release behavior. After six cycles of NIR laser irradiation, ~34% of DOX was released from nanoparticles under pH 5.0 for 4 h (Figure 4D), which was nearly 7.5-fold of that under pH 7.4 without NIR laser irradiation (~4.5%). And ~20% of DOX was released under pH 5.0 without NIR laser irradiation, which was almost 4-fold of that in neutral buffer. Such acid-triggered drug release was possibly due to the protonation of amine groups on the MPDA and/or on the DOX molecules, which partially destroyed the π-π and hydrophobic interactions between MPDA materials and DOX. The results showed that the nanoparticles would be stable during the neutral condition and the pH-responsive release

26


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behavior would occur once the nanoparticles were internalized in the acidic endo/lysosomes inside tumor cells. 48

27



28


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Figure 3. Characterization of Fe3O4 nanoparticles and MPDA@HA-MTX. (A) TEM image of Fe3O4 nanoparticles. (B) TEM image of MPDA@HA-MTX.

(C)

Size

distribution

of

MPDA@HA-MTX

determined by DLS (inset: tyndall effect). (D) Zeta potential distribution of MPDA@HA-MTX determined by DLS. (E) XRD pattern of Fe3O4 nanoparticles. (F) Magnetization loops of Fe3O4 nanoparticles and MPDA@HA-MTX.

(G)

Photothermal

heating

curves

of

MPDA@HA-MTX with varied concentrations irradiated by NIR laser at a power density of 1 W/cm2. (H) Photothermal stability curves of MPDA@HA-MTX at 50 µg/mL irradiated by NIR laser at a power density of 1 W/cm2.

29



Page 30 of 48

Figure 4. Drug loading and stimuli-responsive drug release profiles. (A) Schematic illustration of the multi-responsive controlled release from MPDA/DOX@HA-MTX triggered by NIR laser and pH. (B, C) Measurement of DOX loading at different ratios of DOX and MPDA. (D) Accumulative

drug

release

at

different

pH

conditions

from

MPDA/DOX@HA-MTX triggered by NIR laser irradiation at a power density of 1 W/cm2. *p

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