pH-Responsive NIR-Absorbing Fluorescent Polydopamine with

May 8, 2017 - pH-Responsive NIR-Absorbing Fluorescent Polydopamine with Hyaluronic Acid for Dual Targeting and Synergistic Effects of Photothermal and...
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pH-Responsive NIR-Absorbing Fluorescent Polydopamine with Hyaluronic Acid for Dual Targeting and Synergistic Effects of Photothermal and Chemotherapy Sung Han Kim,† Insik In,*,†,‡ and Sung Young Park*,†,§ †

Department of IT Convergence, ‡Department of Polymer Science and Engineering, and §Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju 380-702, Republic of Korea S Supporting Information *

ABSTRACT: In cancer therapy, optimizing tumor-specific delivery, tumor distribution, and cellular uptake of a drug is important for ensuring minimal toxicity and maximum therapeutic efficacy. This study characterized the therapeutic efficacy of a stimulus responsive and dual targeting nanocarrier for a bioimaging-guided photothermal and chemotherapeutic platform. Hyaluronic acid (HA) conjugated with triphenylphosphonium (TPP) and boronic acid (BA) diol-linked βcyclodextrin (β-CD) forms an inclusion complex with paclitaxel (PTX), creating a shell-like composite on a core of carbonized fluorescent polydopamine nanoparticles (FNPs-pDA) applicable for photothermal therapy as well as bioimaging. The successful diol cross-linking between core@shells generates nanocarriers [FNPs-pDA@HA-TPP-CD-PTX] that can be used as an extracellular HA- and intracellular TPP-mediated dual targeting system. The carbonized FNPs-pDA was cross-linked with the boronic acid groups of HA-TPP-CD-PTX to promote the formation of boronate esters for pH-mediated photothermal activity, which have shown time dependent complete PTX release along with a photothermal mediated response. The in vitro dual bioimaging and photothermal-chemotherapeutic activities were compared between cancer and normal cells. Lysosomal escape and live/dead cells staining confocal images highlight the promise of this system, which might open up a new approach, a simple and versatile method for site-specific synergetic drug delivery.

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INTRODUCTION Improving cellular uptake and controlling subcellular fate is critical for intracellularly acting drugs including conventional chemotherapeutics, siRNA, and gene therapies.1,2 Active targeting through inclusion of ligands that bind to overexpressed tumor receptors has had inconsistent effects in nanoparticlebased drug carriers, but is capable of improving the efficacy of drug delivery by enhancing passive accumulation in tumor tissue.3 Moreover, in tumor target delivery, it is critical to quantify not only the total accumulation of the drug inside solid tumors, but also the amount reaching the site of action, as many.1,4 chemotherapeutic agents exert their effect in the nucleolus, mitochondria, or other organelles. Therefore, both active and passive targeting can increase accumulation of therapeutic cargo; otherwise, intracellular drug activity cannot be exploited to ensure that the drug reaches the appropriate organelle. The appropriate delivery of chemotherapeutics to the intracellular compartment should not only accelerate the onset of action but also minimize unwanted side effects, through manipulating cellular metabolism in a specific way. The nucleus and mitochondria of tumor cells are emerging as promising intracellular targets for chemotherapeutic drug delivery systems (DDS), as these organelles have important roles in apoptosis.1 Typically, a high-density positive charge produced from © 2017 American Chemical Society

triphenylphosphonium cations (TPP) when attached to drug carrier surfaces promoted mitochondrial accumulation through accumulation in negatively charged intracellular components.5,6 Although mitochondrial targeting has yet to yield promising results, undesirable consequences may arise from carriers interacting with normal tissue.5,7 Hence, using extracellularly active target ligands can be a cooperative tool for restricting the display of positive charges following cellular uptake. Hyaluronic acid (HA), which is the active binding ligand for the CD44 receptor, has been widely used in chemotherapeutic delivery owing to overexpression of CD44 by many growing tumors.8,9 HA adheres to the tumor cells, and convenient functionalization has made it the most preferable choice in tumor targeted diagnosis and DDS. Following delivery, the release of the drug from endosomal vehicles and lysosomal compartments into the intracellular space requires a pH responsive carrier sensitive to acidic environments.10−12 A simple means of exploiting the pH responsive carrier is conjugation of the drug through a pH liable linker, such as a boronic acid (BA) diolcomplex or a hydrazone bond.1 Of these, BA has produced remarkable results in the sensing and detection of carbohydrate Received: February 22, 2017 Revised: April 10, 2017 Published: May 8, 2017 1825

DOI: 10.1021/acs.biomac.7b00267 Biomacromolecules 2017, 18, 1825−1835

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conjugated HA, first we conjugated ethylene diamine into HA backbone as source of the amino group for bound to TPP trough amine coupling mechanism (EDC/NHS).18 Ethylene diamine (8 mg) dissolved in 120 mL of 2× PBS (pH 3−4) was activated using EDC (25 mg) and NHS (15 mg) for 15 min and then reacted with 1 g of HA for 24 h at room temperature. The amine group-conjugated HA was purified by dialysis (MW: 3500) against water and finally freeze-dried. Subsequently, ethylene diamine-conjugated HA (0.5 g) with excess amounts of EDC and NHS were reacted with BA (6.7 mg) in 30 mL of 2× PBS (pH 3−4) at room temperature for 24 h. The product was purified by dialysis (MW: 3500) against water and finally freeze-dried. Afterward, to the ethylene diamine and BA-conjugated HA, 29.2 mg of TPP first dissolved in 30 mL of 2× PBS (pH 3−4) was added. Then, the EDC (13 mg) to activate the carboxyl group of TPP and NHS (7.9 mg) for activation of amino group in ethylene diamine was added to the mixing solution for 15 min and then reacted with for 24 h at room temperature.18 The final product was purified by dialysis (MW: 3500) against water and finally freeze-dried. Conjugation of β-CD on HA-TPP-BA [HA-TPP- CD]. HA-TPPBA (0.8 g) was dissolved with β-CD (37.3 mg) in a PBS buffer at pH 11.0. The solution was allowed to react overnight at room temperature to obtain HA-TPP-CD. Following the reaction, the solution was dialyzed (MWCO: 3500) for 24 h and then freeze-dried. The HA repeating unit and β-CD ratio was maintained at 10:1, and the final product yield was 92.6%. Synthesis of FNPs-pDA Decorated with HA-TPP-CD shells [FNPs-pDA@HA-TPP-CD]. The HA-TPP-CD (0.1 g) was dissolved with FNPs-pDA (0.2 g) in PBS buffer with pH 11.0. The solution was allowed to react overnight at room temperature to obtain FNPs-pDA@ HA-TPP-CD. After the reaction, the solution was purified by centrifugation and freeze-drying. The final yield of products was 81.3%. Inclusion Complexation of Paclitaxel (PTX) with β-CD via FNPs-pDA@HA-TPP-CD [FNPs-pDA@HA-TPP-CD-PTX]. PTX was loaded into FNPs-pDA@HA-TPP-CD following a widely used inclusion complex method. In detail, 100 mg of FNPs-pDA@HATPP-CD and 1 mg of PTX were dissolved in 20% (v/v) ethanol solution to obtain FNPs-pDA@HA-TPP-CD-PTX. This solution was stirred for 12 h at room temperature (25 °C), before centrifugation and freezedrying. The resulting products were stored at −20 °C before use. The loading content = [(FNPs-pDA@HA-TPP-CD-PTX)weight − FNPspDA@HA-TPP-CD weight] × 100/[(FNPs-pDA@HA-TPP-CDPTX)weight], and was found to be 5.03%. The loading efficiency = [PTX(total) − PTX(noncomplex)] × 100/[PTX(total)], and was found to be 83.9%. Release Kinetics of PTX from [FNPs-pDA@HA-TPP-CD-PTX] Nanocarriers. The release behavior of PTX from FNPs-pDA@HATPP-CD-PTX was evaluated at pH 7.4, 6.0, and 5.0 in PBS (10 mmol) inside dialysis chambers. The chambers were immersed in 30 mL of release medium at pH 7.4, 6.0, and 5.0, respectively. Finally, the samples were placed in a shake incubator at 37 °C for the entire experimental period. To determine the amount of PTX released, a 3 mL solution from the surrounding release medium was taken, and the amount of drug released was calculated using absorbance measurements taken using UV spectroscopy. To evaluate drug release following irradiation with NIR light, FNPspDA@HA-TPP-CD-PTX was dissolved in the respective pH buffer and treated for 5 min with NIR irradiation before input into the dialysis chambers. The percentage of accumulated PTX release was then measured using the method described above. MTT Assay. Cytotoxicity was measured using an MTT assay. Here, 200 μL of MDA-MB-231 cells (human breast cancer cells) and MDCK (Madin-Darby canine kidney epithelial) cells, at a density of 2 × 105 cells/mL, were placed in each well of a 96-well plate. Afterward, the cells were incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere. To determine the cellular viability, stock solutions of FNPs-pDA@HATPP-CD-PTX and solution of PTX were used. The PTX has very good solubility in DMSO up to 400 mg/mL and in ethanol up to 40 mg/mL; it is very poorly soluble in water. In buffer, serum, and additive water, solubility is estimated to be about 10−20 μM.10 To dissolve the PTX in media, first we dissolved it in DMSO at 5 mg/mL and further diluted up

derivatives through diol-complexation. Recently, carbohydrate derivatives, such as β-cyclodextrin (β-CD) have demonstrated promising outcomes in DDS by improving bioavailability of poorly water-soluble drugs, and stabilizing active drugs through protection from light-induced, thermal, and oxidative degradation.13 This system has gained considerable acceptance, especially when β-CD is reversibly included in complexes with paclitaxel (PTX), which thermodynamically allows the complete release of free PTX into cytosolic compartments.13,14 In our recent report, we synthesized fluorescent carbon nanoparticles (FNPs) using polydopamine (pDA) via a carbonization method using an acidic catalyst. We demonstrated that the remaining catechol moieties generated photothermal heat in response to near-infrared (NIR) light.15 Recently, Lee and co-workers reported that marine mussels dopamine (DA) derivatives conjugates hyaluronic acid can exhibit both adhesiveness for easy functionalization of the surface of materials and cohesiveness to build 3D hydrogel by the external pH conditions.16,17 Based on this study, our present work explored the use of FNPs-pDA core nanoparticles decorated with TPP and BA-conjugated HA diol-linked β-CD, inclusion complexed with PTX. The core FNPs-pDA allows fluorescent bioimaging and photothermal cytotoxicity activities, whereas the shell of the integrated structure regulates biodirection for delivery of PTX to the tumor site. Extracellular CD44-HA responsive target delivery and TPP-mediated intracellular localization reasonably maximized cytosolic PTX activity. Moreover, chemotherapeutic and photothermal cytotoxic effects are combined when using these fluorescent nanocarriers to enhance drug efficacy against devastating malignant tumors.

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EXPERIMENTAL SECTION

Materials. Paclitaxel (Taxol), [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (MTT), N,N′-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), 4-(dimethylamino)pyridine (DMAP), dichloromethane (MC), triphenylphosphonium (TPP), ethylene diamine, 3-amino phenyl boronic acid (BA), β-cyclodextrin (CD or β-CD), and hyaluronic acid (HA) MW 230 kDa were purchased from SigmaAldrich, Korea. Penicillin−streptomycin, fetal bovine serum (FBS), 0.25% (w/v) trypsin, 0.03% (w/v) EDTA (ethylene diamine tetra acetic acid) solution, and RPMI-1640 medium were purchased from Gibco BRL (Carlsbad, CA). FNPs-pDA were obtained from our previous study, synthesized using dehydration of polydopamine (pDA) in the presence of concentrated sulfuric acid.15 1 H NMR spectra were recorded using a Bruker Advance 400 MHz spectrometer with deuterium oxide (D2O) and deuterium dimethyl sulfoxide (d6-DMSO) as the solvent. The UV−vis spectra were recorded using an Optizen 2020UV; Mecasys Co. Infrared camera (NEC Avio, Thermo Tracer TH9100), and particle size was measured using dynamic light scattering (DLS) (Zetasizer Nano, Malvern, Germany). Photoluminescence (PL) spectra were obtained using a L550B luminescence spectrometer (PerkinElmer). The NIR laser was 808 nm (PSU-III-LRD, CNI Optoelectronics Tech. Co. LTD, China). A multimode microplate reader, Filter MaxF3 (Molecular Devices, LLC.), was used for the MTT assay. Transmission electron microscopy (TEM) (JEM-2100F, JEOL) was carried out with an 80−200 kV electron gun. Propidium iodide (PI), calcein acetoxymethyl ester (calcein AM), LysoTracker Green DND-26, and MitoTracker Red CMXRos were purchased from Molecular Probe, Life Technologies (Invitrogen). Confocal laser scanning microscope images of the samples were recorded using an LSM510 confocal microscope (Carl Zeiss, Germany). Conjugation of 3-Amino Phenyl Boronic Acid (BA) and Triphenylphosphonium (TPP) on HA [HA-TPP-BA]. To perform 3amino phenyl boronic acid (BA) and triphenylphosphonium (TPP) 1826

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Biomacromolecules to 5 μg/mL using RPMI medium. The media was removed and the cells were treated with the different concentrations of FNPs-pDA@HA-TPPCD-PTX and PTX. The cells were then incubated for another 24 h. The media containing FNPs-pDA@HA-TPP-CD-PTX and PTX was then replaced with 180 μL fresh medium and 20 μL of a stock solution containing 15 mg of MTT in 3 mL PBS and incubated for another 4 h. Finally, the medium was removed, 200 μL MTT solubulizing agent was added to the cells, and accurate shaking was performed for 15 min. Absorbance was measured at a wavelength of 570 nm using a microplate reader. The relative cell viability was measured by comparing with the absorbance of the cell-only control. To evaluate the in vitro effects of NIR light irradiation, FNPs-pDA@ HA-TPP-CD-PTX was dissolved in the respective pH buffer and irradiated for 5 min before input into the cell culture system. The percentage cell viability was measured following the method mentioned above. Quantitative Evaluation of Cellular Uptake. To quantify the cellular uptake of FNPs-pDA@HA-TPP-CD-PTX, cancerous MDAMB-231 and normal MDCK cells were selected and seeded in a 96-well plate at a concentration of 0.5 × 105 cells/mL. After 24 h of incubation, the media was removed, and the cells were treated with 0.2 mg/mL of FNPs-pDA@HA-TPP-CD-PTX in media. Cells were then incubated for another 4 h. At the end of the period, the media was removed, and the cells were washed several times with PBS (1 × , pH 7.4). Triton X-100 (1%) was then used to lyse the cells. The relative amount of accumulated FNPs-pDA@HA-TPP-CD-PTX within the cell interior was determined by measuring the fluorescence intensity at an excitation wavelength of 365 nm and an emission wavelength of 465 nm, in a multimode microplate reader (Filter MaxF3, Molecular Devices LLC.). The extent of in vitro cellular uptake was calculated by comparison with the intensity of the initial total content, used as control (100%). Confocal Imaging. The cellular uptake of composite photothermal materials was analyzed using confocal imaging. The MDA-MB-231 and MDCK cells were plated over a cover slide on an eight-well plate at a density of 2 × 105 cells/mL per well and were incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere. Afterward, the cells were treated with the composite material at 0.01 mg/mL for 30 min, in fresh culture media. The cells were then washed with PBS several times to remove any unbound FNPs-pDA@HA-TPP-CD-PTX nanoparticles and costained with LysoTracker Green and MitoTracker Red. Finally, the cells were examined at 20× magnification using an LSM510 confocal laser scanning microscope (Carl Zeiss, Germany). Calcein AM and Propidium Iodide Cells Staining Assay. Cell viability imaging of photothermal-chemotherapy was evaluated through the calcein AM and PI staining method as described in the literature.19 To assess the photothermal effect, the MDA-MB-231 and MDCK cells were incubated in 8-well plates at 37 °C containing FNPs-pDA@HATPP-CD-PTX at a concentration of 0.2 mg/mL for 30 min. Following incubation, the cells were irradiated with an 808 nm laser at a power density of 2 W/cm2 for 5 min. The cells were then stained with both calcein AM and PI. Finally, an LSM510 confocal laser scanning microscope (Carl Zeiss, Germany) was used at 10× magnification to image the stained (live/dead) cells.

potential with photothermal heat generation under acidic pH conditions, with HA-TPP-CD-PTX decorating FNPs-pDA nanoparticles as a shell, utilizing a diol cross-linked core@shell structure (FNPs-pDA@HA-TPP-CD-PTX). This system therefore a dual targeting, bioimaging guided DDS with a photothermal-mediated hyperthermally cytotoxic theragnostic platform. In addition, the most important is how to facilitate the early release of our system from endosomes into the cytosol through endosomal escape. The protonation some domains of amino acids at low pH can trigger interaction between the peptides and the lipid bilayer of endosomes to form pores by interaction with phospholipid, leading the membrane fusion and lysis resulting in endosome escape. In the cases of water-soluble pH sensitive polymer, upon entering the acidic endosomes, they become too polar to escape rapidly from the endosome through the membrane.22 The details involved in design and development are illustrated in Figure 1.

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RESULTS AND DISCUSSION The desired goal of designing the nanoparticles for DDS is to improve the therapeutic index by controlling biodistribution while limiting the effects on healthy organs. In cancer treatments, strategies that simultaneously incorporate chemical as well as physical methods have revealed the unique potential of nanomedicine.1,3,20 Furthermore, real time bioimaging of the treatment area allows physicians to adjust the dose and type of therapy to individual patients.10,21 To achieve this novel goal, the extracellular targeting ligand, HA, was conjugated to the intracellular binding ligand of TPP for complete control of biodirection, while BA diol-linked β-CD preferentially released PTX from the inclusion complex HA-TPP-CD-PTX. The nanocarrier core, FNPs-pDA had demonstrated bioimaging

Figure 1. Schematic illustration of the synthesis of boronic acid (BA) diol-linked β-cyclodextrin (CD) and triphenylphosphonium(TPP) conjugated hyaluronic acid (HA) [HA-TPP-CD]. The conjugated composites were assembled on the core surface of carbonized fluorescent polydopamine nanoparticles (FNPs-pDA) [FNPs-pDA@ HA-TPP-CD]. Finally, paclitaxel (PTX) was integrated with these core@shells nanoparticles using a CD-PTX bond created via a host− guest inclusion complex mechanism [FNPs-pDA@HA-TPP-CD-PTX]. The lower scheme illustrates selective target delivery of core@shells nanoparticles and intracellular site specific release of PTX. 1827

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Figure 2. (a) 1H NMR spectra (400 MHz, D2O) of HA-TPP-CD, FNPs-pDA@HA-TPP-CD, FNPs-pDA@HA-TPP-CD-PTX, and FNPs-pDA@HATPP-CD-PTX at pH 5.0 (Inset pictures show BA and TPP peaks in d6-DMSO). (b) 1H NMR spectra (400 MHz, D2O) of FNPs-pDA@HA-TPP-CD, and FNPs-pDA@HA-TPP-CD-PTX. (c) UV−vis-NIR absorption spectra of HA-TPP-CD, FNPs-pDA@HA-TPP-CD, and FNPs-pDA@HA-TPPCD-PTX (concentration is 0.5 mg/mL and ratio of PTX loading is 1:100).

Figure 3. (a) X-ray diffraction patterns of pure PTX, FNPs-pDA@HA-TPP-CD, and FNPs-pDA@HA-TPP-CD-PTX. Red dots represent specific peak of β-CD. (b) Wide-scan XPS spectra and (c) narrow-scan of C 1s spectra of FNPs-pDA@HA-TPP-CD-PTX.

peaks. Subsequently, PTX was inclusion complex with the β-CD of HA-TPP-CD, resulting in shifts in the NMR signals of the H-3 and H-5 protons located in the cavity of β-CD.10,23 As the boronic diol linkage is highly pH sensitive, this stimulusresponsive behavior has been evaluated using 1H NMR integration peaks prepared by dialysis under acidic pH shock followed by freeze-drying of the remaining materials, showing the absence of β-CD and PTX peak. In the UV−vis-NIR absorption spectra, the HA-TPP-CD exhibited a sharp absorption band at around 240 nm and a weak peak in the range 275−390 nm, which refers to amphiphilic boronic acid compound and aromatic group in TPP. After bounding with

HA conjugated with BA and TPP were synthesized from 3amino phenyl boronic acid and carboxyl group activated HA were confirmed in the 1H NMR spectrum (Figure 2). The peaks at 1.9 ppm [3H, −NH−CO−CH3], 3.6−3.7 ppm [2H, −CH2− OH], and 4.2−4.3 ppm [1H, −CH−] are indicative of HA.9 The integral peaks at around 7.1−8.0 ppm of aromatic groups confirmed 18 unit of BA and 20 units of TPP in the HA polymer backbone. Following the conjugation of HA-TPP-BA, the β-CD was integrated via interaction between the boronic acid and the diol groups of β-CD, indicated by 1H NMR peaks around 3.00− 4.00 ppm (H-4, 3.45; H-2, 3.50; H-5, 3.75 and H-3, 3.85 ppm), wherein peak area depicts 10 unit of β-CD compared to HA 1828

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Figure 4. (a) DLS measurements taken from FNPs-pDA@HA-TPP-CD-PTX nanocarriers to measure the change in hydrodynamic radius distribution in response to pH change (5.0 to 7.4). (b) Selected magnified high resolution TEM image (HR-TEM). (c) Fluorescence emission intensity of FNPspDA@HA-TPP-CD-PTX at different excitation wavelengths (320 to 500 nm) at pH 7.4. (d) Fluorescence lifetime curve of FNPs-pDA@HA-TPP-CD and FNPs-pDA@HA-TPP-CD-PTX at 375 nm wavelength. The τ value indicates respective fluorescence lifetime.

FNPs-pDA, the absorption properties of HA-TPP-CD show obvious increasing in the NIR region, referring to π−π* electron transitions in phenolic compounds after carbonization. The specific addition of PTX into HA-FCN-CD leads broader NIR absorption properties at long wavelengths (400−900 nm) due to the light harvest contribution of the assembled aromatic group.10,24 Due to the solubility behavior, we choose ratio 1:100 of PTX loading for further use as shown in Figure S1, which shows great solubility in water. In addition, the surface charge (±mV) of FNPs-pDA@HA-TPP-CD-PTX in aqueous environment was observed using a zeta potential analyzer as shown in Figure S2. The FNPs-pDA@HA-TPP-CD-PTX shows positive (+mV), owing to TPP that contains highly electropositive charges, which conjugation to negative (−mV) potential of carboxylic groups on surface of the FNP-pDA in physiologic pH. This means that we have successfully functionalized the TPP to the FNP-pDA core. The X-ray diffraction (XRD) pattern in Figure 3a of FNPspDA@HA-TPP-CD-PTX was studied in the 1−60° (2θ) area, where the peaks associated with the crystalline structure of β-CD almost disappeared. Moreover, the characteristic XRD reflections (2θ) between the 10−20° (2θ) region, in terms of peak shape and relative intensities, indicate possible conformation changes arising from the β-CD-PTX inclusion complex.25 The FNP-pDA@HA-TPP-CD-PTX inclusion complex DDS was characterized by X-ray photoelectron spectroscopy (XPS) to evaluate structural composition. The XPS wide-scan and narrowscan C 1s spectra of FNPs-pDA@HA-TPP-CD-PTX show

different carbon groups including CC, C−C, C−O, C−O−C, CO, and O−CO at around 283.8, 284.2, 286, 287, 288.2, and 289.5 eV, respectively.26 The structural formula of carbonized FNPs-pDA and other composites may give rise to the diverse C 1s binding energies (eV) (Figure 3b, c). The size and arrangement of constituent composites in nanocarriers determine delivery mechanisms, from efficient endocytosis to drug release.27,28 In an aqueous medium (pH 7.4), the average size (diameter, d) of nanocarriers was 172.8 nm with PTX loading. The constructed boronate ester in FNPs-pDA@ HA-TPP-CD-PTX nanocarriers is highly sensitive under acidic conditions; therefore, diol linkages between FNPs-pDA, HATPP-BA, and β-CD were weakened and finally dissociated, resulting in decreased particle diameters of 138.4 nm at pH 6.0 and 114.5 nm at pH 5.0, respectively (Figure 4a). As a control, Figure S3 showed slightly change in size of FNPs-pDA, indicating the pH responsive appeared from diol linkages between FNPs-pDA and HA-TPP-CD not from core of FNPspDA. These results clearly demonstrate that the FNPs-pDA@ HA-TPP-CD-PTX delivery system is pH responsive, which might be a prerequisite for escape from lysosomal compartments. Additionally, the TEM image clearly shows small spherical particles of 115 ± 6 nm in size, supporting the above DLS measurements (Figure 4b). The red box in the Figure 4b shows the lattice distance of a small area near the particle center. The lattice size about ∼0.31 to 0.329 nm refers to graphene like particles, suggesting internalized carbon after carbonization.29 1829

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Figure 5. (a) Quantitative in vitro cellular uptake of FNPs-pDA@HA-TPP-CD-PTX in MDCK and MDA-MB-231 cells. (b) Confocal laser scanning microscope (CLSM) images of MDA-MB-231 (MDA-MB-231) and MDCK cells treated with FNPs-pDA@HA-TPP-CD-PTX. Scale bars represent a distance of 50 μm.

In response to tumor-specific stimuli, the nanoparticle DDS achieved intratumoral drug release in the presence of overexpressed matrix, and enhanced the permeability and retention (EPR) effect.31,32 The cellular internalization of FNPs-pDA@ HA-TPP-CD-PTX therapeutic carriers was studied in MDAMB-231 and MDCK cells (Figure 5). As HA works as an extracellular binding ligand to the CD44 receptor, which is extensively found on the surface of cancer cells, the internalized DDS was found in malignant MDA-MB-231 cells to a greater extent compared with normal growing MDCK cells (36% and 22%, respectively). Additionally, the confocal laser scanning microscopy (CLSM) images showed the mitochondria targeting of FNPs-pDA@HA-TPP-CD-PTX, demonstrating the bright fluorescence of polymer inside the mitochondria MDA-MB-231 cells at merge images. This was compared with CLSM images of polymer without TPP compartments, which only localized within the cell not specific at mitochondria area.33 pH has been widely used as a stimulus to enhance drug release from early endosomes into the cytoplasmic compartment through the lysosomal escape process.34,35 For instance, PTX released from nanocarriers was observed using LysoTracker (green) and MitoTracker (red) costaining methods at different time intervals. As shown in Figure 6, the amount of colocalized

This means that we successfully developed multi responsive drug matrix with fluorescent properties based dehydration process. In nanomedicine, the combination of chemo and phototherapy with real time bioimaging in a DDS has been explored in order to take full advantage of the potential of pH-responsive nanocarriers. The core FNPs-pDA and the HA-TPP-CD-PTX shell were strategically functionalized to optimize target delivery while allowing bioimaging. To demonstrate the capability for bioimaging, the carbonized nanocarriers were excited at different wavelengths and the spectral fluorescence intensities were recorded, showing different intensities under different buffer conditions (pH 5.0, 6.0, and 7.4). Under acidic conditions (pH 5.0 and pH 6.0) (Figure S4), the intensity of the emission wavelengths was quite similar. However, fluorescence wavelengths have been found to widen with lower intensity in neutral buffers (Figure 4c). As the surface volume controlled energy trapping, the pH-stimulus responsive core@shell FNPs-pDA and HA structure contributes to increased/decreased surface volume resulting in fluctuation in fluorescence emission peaks.30 Additionally, as shown in Figure 4d, the fluorescence lifetimes measured at 375 nm without and with PTX inclusion in the complex were 4.34 and 4.62 ns, indicating the surface volume difference phenomenon. 1830

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Figure 6. Confocal laser scanning microscope (CLSM) images of MDA-MB-231 cells incubated with FNPs-pDA@HA-TPP-CD-PTX for 30 min, 1, 2, 4, and 6 h (FNPs-pDA@HA-TPP-CD-PTX concentration: 1 mg/mL). Blue, FNPs-pDA@HA-TPP-CD-PTX; green, LysoTracker; red, MitoTracker. Scale bars represent a distance of 50 μm.

whereas the shell composite, HA-TPP-CD-PTX, appears relatively less responsive to NIR light (ΔT = 15 °C) at pH 7.4. The increased temperature generated from NIR irradiation at lower pH might be due to the absence of the HA-TPP-CD-PTX shell, arising from the consolidated π−π conjugation/sticking structure of the FNP-pDA core.10 In the core structure of FNPs-pDA, a specific amount of PTX might be loaded based on hydrophobic interaction of π−π interaction, but because the cavity of β-CD was located in the shell, the PTX was more easily bounded to the β-CD than FNPspDA. To prove this study, we provide the UV−vis of FNPspDA@HA-TPP-CD before and after 60 h release of PTX in Figure S5a. The PTX absorbance is loss after 60 h, demonstrating efficient drug release because an amount of PTX release from the CD cavity in acid condition as a function of pH linkages responses. Further, we observed the UV−vis spectra of PTX in loaded FNPs-pDA in Figure S5b, showing the slight change of UV−vis spectra, which indicates the existence of a small amount of PTX. Furthermore, we have tested PTX release profile from FNPs-pDA in different pH conditions (Figure S5c). In the release profile, we did not find any PTX release from FNPs-pDA

signal incrementally increases over time, indicating particle accumulation. However, the rate of accumulation slows down by the 4 h time point. This is due to DDS escape from the liposomal vehicle into the cytoplasm, resulting in minimal fluorescent signal from cells between 4 and 6 h. Combating this most complicated disease, cancer, relies on a simultaneous treatment strategy so that the tumor is completely eradicated, preventing recurrence. The design and development of photothermal and chemotherapy combined treatments are a promising synergistic therapeutic approach, where hyperthermal heat and chemicals combine to completely destroy the target cancer cells.28,29 The pH-responsive boronate ester linkage between FNPs-pDA and HA-TPP-CD-PTX, together with the beneficial effects of photothermal heat, has provided a pHcontrolled anticancer drug delivery and remote NIR anticancer therapy system. As the carbonized FNPs-pDA had been optimized to release a considerable amount of thermal heat, the developed nanocarrier DDS were engineered to generate high temperatures in response to NIR light.15 The NIR irradiated core@shell composite, FNPs-pDA@HA-TPP-CD-PTX, generates sufficient heat (ΔT = 25 °C) irrespective of buffer acidity, 1831

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Figure 7. Temperature elevation curve of an aqueous solution of (a) FNPs-pDA@HA-TPP-CD and (b) FNPs-pDA@HA-TPP-CD-PTX as a function of NIR irradiation time (808 nm laser, 2 W cm−2), at pH values between 5.0 and 7.4. In vitro release profiles of FNPs-pDA@HA-TPP-CD-PTX in PBS at pH 7.4, 6.0, and 5.0, (c) without NIR irradiation, and (d) with NIR irradiation (5 min, 808 nm, 2 W cm−2).

cell phenotype such as both migration and invasion.38 Furthermore, free PTX in cell medium exhibited moderate cytotoxicity in both cell lines owing to inappropriate delivery and aqueous instability while different pH did not causes any changes to cytotoxic profile which was stably over range of pH values from pH 5−7.4 (Figure S6). Interestingly, the NIR irradiated treatments elicited noticeable moderate to mild cytotoxicity in both normal MDCK (90−70%) and cancerous MDA-MB-231 (90−55%) cells.39 However, PTX-loaded delivery matrices have demonstrated concentration and pH dependent ability to damage living MDA-MB-231 and MDCK cell lines. Moreover, the HA-based control PTX activity was less pronounced in MDCK cells (80 to 50%) and profound in malignant MDA-MB231 (58 to 20%) cells, in a pH and concentration dependent manner. The localized photothermal mild cytotoxicity and PTX mediated chemo-toxicity combined to produce synergetic effects, augmenting toxicity for application in nanobiomedicine.40,41 Finally, trigger-targeted and stimulus responsive chemophotothermal activity was further evaluated using CLSM images in Figure 9 of live and dead costained cells. The cooperative photothermal and chemotherapeutic DDS selectively increased the number of dead cells (red color) in MDA-MB-231 cells, whereas decreased numbers of dead cells were observed for the treated normal MDCK cells.10,42 The extracellular HA-based target delivery was restricted to in vitro biological activity in noncancer groups and was more prominent at higher concentrations against cancerous cells. This could be a useful treatment strategy to prevent chemotherapeutic resistance. In general, tumor cells located deep inside the tumor possess heterogeneous morphology, and are frequently resistant to chemotherapy owing to poor drug delivery efficiency and the fact that some drugs need oxygen for their cytotoxicity. Temperatureinduced DDS would lead to several physiological changes, including oxygenation, pH variation, and increased blood flow. Additionally, increasing blood flow to the target area enhances

core, indicating inefficient drug release behavior due to the absence of pH responsive moieties and a small amount of PTX in the FNPs-pDA core. At the same time, the in vitro drug release of PTX from prepared matrix in PBS buffers with different pH values and one 5 min NIR irradiation period was studied.10,36 The amount of PTX released depended on pH and NIR intensity, and varied with time. The early release period was completed during the first 10 h interval, with ∼10% more of the drug released in acidic conditions than at normal physiological pH. These differences in release behavior are due to the pHresponsive boronate ester bond between FNPs-pDA and BA in the HA polymer backbone, which hinders PTX release from βCD. However, the dissociation of the boronate ester under acidic conditions results in the freeing of PTX from the β-CD cavity, increasing the percentage release of the drug. After NIR irradiation, PTX release dramatically increased by 10−15%, owing to the synergetic effects of temperature and pH stimuli (Figure 7). These results are consistent with the increased solubility of β-CD in response to rising thermal energy, which in turn creates repulsive forces against hydrophobic PTX in the host−guest complexing system. In addition, the cleavage of boronate ester linkages induce the HA with CD cavity away from core, results in lessening hydrophobicity of complex matrix. This renders the freeing of PTX from the β-CD cavity because of the increased solubility of β-CD in free cell medium. The newly generated unfavorable hydrophilic environments decomplexed the PTX and caused early release from the nanocarrier DDS.13 In nanoscopic therapeutic platforms, efficient delivery of therapeutic ingredients directly to cancer cells and biocompatibility are two vital features.3,37 To evaluate biocompatibility, an MTT assay shown in Figure 8 was performed, revealing concentration (1−0.001 mg/mL) and pH (5.0, 6.0, and 7.4) dependent viability in both MDA-MB-231 and MDCK cells. Even though the viability of cells in different pH (5.0, 6.0 and 7.4) is only slightly changed for each pH condition as control (see magnification of Figure 8), mild acidic pH can cause effects on 1832

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Figure 8. MTT assay for the concentration-dependent in vitro biocompatibility study of MDCK (a, c, e) and MDA-MB-231 (b, d, f) cells treated with FNPs-pDA@HA-TPP-CD (a, b); FNPs-pDA@HA-TPP-CD with NIR irradiation (c, d); and FNPs-pDA@HA-TPP-CD-PTX with NIR irradiation (e, f).

boronate ester bond from boronic acid, together with β-CDlinked FNPs-pDA@HA-TPP for PTX controllable release and FNPs-pDA@HA-TPP-CD-PTX for a remotely controlled photothermal effect, we have successfully developed an extracellular and intracellular dual targeting DDS. Combining both HA and TPP creates a dual targeting DDS that selectively regulates release of PTX only at the target tumor site and protects healthy surrounding tissues from side effects. Its pH-responsive behavior was confirmed by fluorescence emission wavelengths and intensity, which could have potential in application as

the migration of immune cells that would subsequently mitigate tumor burden.43 As the treatment of malignant tumors is considered very complex, photothermal-chemotherapy using a bioimaging-guided DDS can pave the way for the design and development of next generation drug carriers.

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CONCLUSIONS This system can synergistically trigger pH-responsive photothermal and chemotherapeutic platforms in the extracellular tumor environment. Therefore, using a combination of a 1833

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Figure 9. Calcein AM and PI stained live/dead cells for fluorescence imaging of FNPs-pDA@HA-TPP-CD-PTX-treated MDA-MB-231 and MDCK cells. All scale bars represent 100 μm. NIR irradiation was carried out for 5 min with an 808 nm laser at a power density of 2 W cm−2. All studies were performed at normal physiological pH.

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ACKNOWLEDGMENTS This study was supported by Grant Nos. 10062079, R0005303, and R0005237 from the Ministry of Trade, Industry & Energy (MOTIE), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1A2B2002365).

bioimaging probes. The in vitro release profile from the pH triggered and NIR irradiated system demonstrated preferentially controlled therapeutic activity, with the targeting ligand establishing a clear safety barrier between malignant MDAMB-231 and normal growing MDCK cell lines. In this therapeutic system, the photothermal heat originating from carbonized FNPs-pDA and the chemotoxicity arising from optimal release of PTX could open new opportunities for the development of other potential therapeutic system in the near future.

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ABBREVIATIONS HA, hyaluronic acid; TPP, triphenylphosphonium; BA, boronic acid; CD, β-cyclodextrin; PTX, paclitaxel; FNPs-pDA, carbonized fluorescent polydopamine nanoparticles; FNPs-pDA@HATPP-CD-PTX, hyaluronic acid conjugated with triphenylphosphonium and boronic acid diol-linked β-cyclodextrin forms an inclusion complex with paclitaxel, creating a shell-like composite on a core of carbonized fluorescent polydopamine nanoparticles (FNPs-pDA)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00267. UV−vis-NIR absorption spectra of FNPs-pDA@HATPP-CD-PTX; zeta-potential values of FNPs-pDA, HATPP-CD, and FNPs-PDA@HA-TPP-CDDPTX; DLS measurements; fluorescence emission spectra of FNPspDA@HA-TPP-CD-PTX; UV−vis-NIR absorption spectra of FNPs-pDA@HA-TPP-CD and FNPs-pDA@HATPP-CD-PTX before and after 60 h release of PTX; MTT assay for the concentration-dependent in vitro biocompatibility study of MDCK and MDA-MB-231 cells treated with PTX (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Sung Young Park: 0000-0002-0358-6946 Notes

The authors declare no competing financial interest. 1834

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