Polydopamine Nanoparticles Enhance Drug Release for Combined

Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 21125−21136

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Polydopamine Nanoparticles Enhance Drug Release for Combined Photodynamic and Photothermal Therapy Barbara Poinard,† Samuel Zhan Yuan Neo,‡ Eugenia Li Ling Yeo,§ Howard Peng Sin Heng,§ Koon Gee Neoh,†,∥ and James Chen Yong Kah*,†,§ †

NUS Graduate School of Integrative Sciences and Engineering, National University of Singapore 117456, Singapore School of Life Sciences & Chemical Technology, Ngee Ann Polytechnic 599489, Singapore § Department of Biomedical Engineering, National University of Singapore 117583, Singapore ∥ Department of Chemical and Biomolecular Engineering, National University of Singapore 117585, Singapore

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‡

S Supporting Information *

ABSTRACT: Our study shows a facile two-step method which does not require the use of core templates to load a hydrophobic photosensitizer drug chlorin e6 (Ce6) within polydopamine (PDA) nanoparticles (NPs) while maintaining the intrinsic surface properties of PDA NPs. This structure is significantly different from hollow nanocapsules which are less stiff as they do not possess a core. To our knowledge, there exist no similar studies in the literature on drug loading within the polymer matrix of PDA NPs. We characterized the drug loading and release behavior of the photosensitizer Ce6 and demonstrated the therapeutic efficacy of the combined photodynamic (PDT) and photothermal therapy (PTT) from Ce6 and PDA, respectively, under a single wavelength of 665 nm irradiation on bladder cancer cells. We obtained a saturated loading amount of 14.2 ± 0.85 μM Ce6 in 1 nM PDA NPs by incubating 1 mg/mL dopamine solution with 140 μM of Ce6 for 20 h. The PDA NPs maintained colloidal stability in biological media, whereas the pi−pi (π−π) interaction between PDA and Ce6 enabled a release profile of the photosensitizer until day 5. Interestingly, loading of Ce6 in the polymer matrix of PDA NPs significantly enhanced the cell uptake because of endocytosis. An increased cell kill was observed with the combined PDT + PTT from 1 nM PDA−Ce6 compared to that with PTT alone with 1 nM PDA and PDT alone with 15 μM equivalent concentration of free Ce6. PDA−Ce6 NPs could be a promising PDT/PTT therapeutic agent for cancer therapy. KEYWORDS: polydopamine, photosensitizer, drug delivery, photodynamic therapy, photothermal therapy

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chemical and thermal stability11 on practically any surface.12 Surface coating with PDA confers hydrophilicity and zwitterionic property with its isoelectric pH of 4.511,13 that could potentially facilitate mucopenetration of PDA-coated surfaces at physiological pH for the mucosal delivery of hydrophobic payloads, which would otherwise interact with the hydrophobic and negatively charged moieties in mucus to limit their delivery. Furthermore, it is well-established that the surface hydrophobicity of NPs dictates immune recognition by macrophages, hence the importance of designing hydrophilic PDA coating to reduce undesirable immunostimulation.14 It was only recently in 2009 that PDA was developed in a nanoparticulate form,15,16 with the chemical and structural properties similar to those of the naturally occurring black melanin particles17 found in human skin. PDA NPs thus

INTRODUCTION Multitudes of nanomaterials have been developed as nanocarriers for drug delivery, such as liposomes, polymers, organic, and inorganic nanoparticles (NPs). Although many of these nanocarriers have demonstrated the loading of photosensitizer drugs,1−3 polydopamine (PDA) maintained its uniqueness over the other types of nanocarriers in its ability not just to encapsulate and load drugs, but also in possessing photothermal properties, while being a nature-inspired coating. PDA is the oxidative self-polymerized form of the biomolecule dopamine in alkaline conditions,4 and dopamine was derived from the early observations of the adhesiveness of invertebrate mussels to surfaces in wet marine conditions5,6 because of their catechol-containing compound 3,4-dihydroxyphenyl-L-alanine and lysine located in their foot threads.7,8 PDA was therefore originally developed as a surface modification that relied on similar catecholamine functional groups5 to provide the adhesiveness to form organic nanometer-scale thin films with low toxicity9,10 and high © 2018 American Chemical Society

Received: March 24, 2018 Accepted: June 5, 2018 Published: June 6, 2018 21125

DOI: 10.1021/acsami.8b04799 ACS Appl. Mater. Interfaces 2018, 10, 21125−21136

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ACS Applied Materials & Interfaces

Figure 1. Schematic of the preparation of PDA NPs and loading of hydrophobic photosensitizer Ce6 into the polymer matrix of PDA NPs.

possess excellent biocompatibility,18,19 aqueous solubility, and strong optical absorption to enable photothermal property,11 particularly in the near-infrared (NIR) region for photothermal therapy (PTT) in vivo.20,21 The combination of photothermal properties and versatile surface chemistry have therefore motivated the conjugation of photosensitizers on the surface of PDA NPs to enable combined PTT and photodynamic therapy (PDT) under dual laser irradiation,22 which has been demonstrated to be more efficacious than individual therapy.23−25 Others have made use of the core−shell structures with gold26 or carbon core27 and a PDA shell modified to bind the photosensitizers via electrostatic and π−π stacking interactions on its surface. The advantages of such surface loading approaches lie in the facile control of the exact drug amount loaded on the NP surface. However, not only were the capacities for surface loading limited, but the surface properties of PDA were also lost with the bound drugs. To retain the unique surface properties of PDA without surface blocking by drugs, others have developed PDA nanocapsules for loading small-molecule drugs inside the capsular structure.28,29 Such a complex preparation involved hazardous etching reagents to create the hollow interior, and the resulting PDA capsules were fragile and prone to folding, distortion, or collapse after the removal of the templates or during subsequent treatment processes because of their reduced mechanical rigidity.30 Furthermore, such a weak stiffness impacts cellular uptake by hindering endocytosis.31 Here, we developed a facile two-step method to prepare PDA NPs loaded with a hydrophobic photosensitizer chlorin e6 (Ce6) in its polymer matrix instead of its surface or in capsular form, while maintaining the hydrophilicity and negative surface charge of PDA. Our protocol generated PDA NPs with a PDA core stiffer than the other reported nanocapsules. We characterized the drug loading and release behavior of Ce6 and subsequently demonstrated improved therapeutic efficacy of PDT and PTT from Ce6 and PDA, respectively, under a single wavelength of 665 nm irradiation in vitro. There are presently no studies that have loaded a hydrophobic photosensitizer in the polymer matrix of PDA NPs to our knowledge.

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Briefly, 1 mg/mL of dopamine hydrochloride (Sigma-Aldrich, Missouri, USA) was dissolved in 10 mM Tris (Sigma-Aldrich, Missouri, USA) at pH 10.5 and allowed to polymerize at room temperature (25 °C) over time. The synthesized PDA NPs were then washed with Tris buffer (pH 10.5) by repeated centrifugation at 16 100g for 4 min and stored for use. The concentration of PDA NPs was determined from their absorbance at 808 nm (εPDA = 7.3 × 108 M−1 cm−1).21 Ce6 was dissolved in Tris buffer (pH 10.5) at 0.5 mg/mL to form a 837 μM Ce6 stock solution before being added to the as-synthesized PDA colloid formed after 5 h of polymerization at a final Ce6 concentration of 140 μM (Figure 1). The mixture was allowed to incubate for 20 h at 25 °C for Ce6 to be loaded into the PDA NPs. The Ce6-loaded PDA NPs (PDA−Ce6) were washed thrice after incubation by repeated centrifugation at 16 100g for 4 min to remove the residual unloaded Ce6 molecules. The morphology of PDA and PDA−Ce6 NPs was examined under transmission electron microscopy (TEM) (JEM-1220, JEOL Ltd, Japan), and their hydrodynamic diameter (DH) and zeta potential (ζ) were measured using a Zetasizer (Nano ZS, Malvern, UK) at 25 °C in different media including Milli-Q water, RPMI, RPMI + 10% fetal bovine serum (FBS), human serum, and urine to examine their colloidal stability in different commonly used biological media. The size of the particles was quantified with ImageJ and its macro plugin NS-size analyzer. The scale of the images’ pixel to nanometer ratio was entered, and the different particles were delimited and the diameter was measured. The hydrophilicity of PDA and PDA−Ce6 NPs was determined from both rose bengal (RB) assay for surface hydrophobicity33,34 and partition coefficient measurements of their log P value based on their partition into water and 1-octanol. The concentration of PDA NPs in both water and octanol phase was determined from their absorbance at 808 nm (εPDA = 7.3 × 108 M−1 cm−1)21 and the log P value was log[PDA] calculated from the equation: log P = log[PDA]octanol . water

Quantification of Ce6 Loading and Passive Release. We determined the amount of Ce6 loaded in the PDA NPs by subtracting the amount of unloaded Ce6 remaining in the supernatant from the initial 140 μM Ce6 added to the synthesized PDA NPs, as measured from the fluorescence of Ce6 against its concentration calibration curve. We also confirmed the amount of loaded Ce6 by thermally releasing Ce6 from PDA−Ce6 NPs in a water bath at 100 °C over 5− 60 min.35 A saturation in the released Ce6 was observed after 10 min. The heated sample was centrifuged at 16 100g for 4 min, and the fluorescence of Ce6 (λEx/λEm = 400/665 nm) in the supernatant was measured using a microplate reader (Tecan, Switzerland) to determine its concentration being released. The passive release of Ce6 from PDA−Ce6 at different pH values of 5, 7, and 10.5 and different commonly used biological media (RPMI with 10% FBS, human serum, and urine) was also examined

MATERIALS AND METHODS

Synthesis and Characterization of PDA and PDA−Ce6 NPs. PDA NPs were synthesized using a previously established protocol.32 21126


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Figure 2. Characterization of PDA and PDA−Ce6 NPs with TEM images showing (A) PDA NPs without Ce6 and (B) PDA−Ce6 NPs. (C) Hydrodynamic diameter (DH) histogram distribution profile of PDA NPs (black) and PDA−Ce6 (red). (D) Zeta potential of PDA NPs with (red) and without (black) Ce6 loaded under different pH conditions. (E) Characterization of hydrophilicity and hydrophobicity using RB. PDA NPs with (red) and without (black) Ce6 were resuspended in RB solution for 2 h. The colloidal suspension was then centrifuged, and the absorbance of the supernatant was measured. The absorbance of the supernatant without any PDA NPs is shown as a reference (blue). (F) The partition coefficient of the PDA NPs incubated with varying concentrations of Ce6 to form PDA−Ce6 NPs. (G) Fluorescence emission spectra after excitation at 404 nm of 1 nM PDA NPs (black); 1 nM PDA NPs loaded with Ce6 (red); and 15 μM free Ce6 (green) at the same concentration as the amount of Ce6 loaded onto PDA−Ce6. (H) Loading capacity of 1 nM of PDA in the presence of varying initial Ce6 concentrations after 20 h of incubation. to 200 μL of 1 nM PDA−Ce6 before irradiation, and the supernatant of PDA−Ce6 after irradiation was then measured for its APF fluorescence (Safire2, Tecan, Switzerland). To compare if the ROS generation from Ce6 was affected by their loading in PDA NPs, the PDA−Ce6 NPs were heated at 100 °C for 10 min to thermally release the loaded Ce6, which was subsequently irradiated to determine its ROS generation. The amount of ROS generation from free Ce6 at the same concentration as that released from the thermal treatment was used as our control. Similarly, the effect of pH in ROS generation was examined by irradiating 1 nM of PDA−Ce6 in three different pH conditions (pH 5, 7, and 10.5) and measuring the amount of ROS generation as described above. Cellular Uptake of PDA−Ce6. Human urothelial carcinoma T24 cells (ATCC, Manassas, USA) were cultured in RPMI-1640, supplemented with 10% FBS, 1 mM L-glutamine, and 1% penicillin. The culture was maintained at 37 °C in a humidified 5% CO2 atmosphere. The T24 cells were seeded at 5 × 105 cells per well in sixwell culture plates for 24 h before 1 mL of PDA−Ce6 in culture media was then added to each well at varying final concentrations of 0.10, 0.25, 0.50, 0.75, or 1 nM with 1 mL of 15 μM free Ce6 concentration equivalent to that loaded in 1 nM of PDA as a control

over time from 3 h to day 17 after preparation. At each time point, the sample was centrifuged at 16 100g for 4 min, and the fluorescence of Ce6 in the supernatant was measured to determine the concentration of the passively released Ce6 over time against a calibration curve specific to the pH or media. Photothermal Heating and Reactive Oxygen Species Production by PDA−Ce6 in Buffer. We irradiated 200 μL of 1 nM PDA and PDA−Ce6 in 10 mM of Tris buffer in a 96-well plate with a 665 nm continuous wave (CW) laser (Photonitech, Singapore) for 15 min at 250 mW/cm2. The light dose was delivered directly above the plate via an optical fiber, with a laser spot size of 12.57 mm2, giving a total fluence of 28.3 J. The temperature of the colloid was monitored by a thermocouple microprobe (Fluke, USA). The Tris buffer alone served as a control. The same PDA−Ce6 was prepared by adding varying initial Ce6 concentration (0, 28, 140, and 280 μM) to the synthesized PDA NPs and irradiated under the same conditions as described above. The reactive oxygen species (ROS) generated from the loaded Ce6 in PDA−Ce6 NPs was measured based on the fluorescence of a ROS indicator dye 30-(p-aminophenyl)fluorescein (APF) (λex/λem = 490/ 515 nm) (Invitrogen, USA). Briefly, 0.8 μL of 5 mM APF was added 21127


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ACS Applied Materials & Interfaces and incubated over time from 2 to 24 h at 37 °C. The cellular uptake of PDA−Ce6 in T24 was examined by flow cytometry and absorbance measurements of the remaining PDA NPs recovered from the cell culture supernatant after incubation. The supernatant in each well was removed after incubation, and the concentrations of PDA−Ce6 and free Ce6 were quantified by absorbance measurements at 808 nm (εPDA = 7.3 × 108 M−1 cm−1) and fluorescence measurements (λex/ λem = 404/665 nm), before being subtracted from the initial concentration of PDA−Ce6 or Ce6 added. The concentration of PDA−Ce6 was determined from its absorbance measurements (calculated with εPDA = 7.3 × 108 M−1 cm−1) measured against its concentration calibration curve. The concentration of free Ce6 uptaken in cells was determined from its fluorescence measured against its concentration calibration curve. For flow cytometry, the cells were rinsed three times in phosphatebuffered saline (PBS) (pH 7.4) after incubation to remove the excess PDA−Ce6, trypsinized and fixed with 4% formaldehyde for 15 min. The Ce6 fluorescence in cells was acquired with a FACSCanto flow cytometer using FACSDiva software (Becton, Dickinson, USA). The data were then analyzed based on at least 1.5 × 104 cells using FlowJo version 7.2.2 (FlowJo, USA). The qualitative analysis of cell uptake was also performed with fluorescence imaging by seeding 105 cells on a glass cover slip in 24well plates for 24 h. The T24 cells were dosed with 1 nM of PDA− Ce6 in culture media or 15 μM of free Ce6 and incubated over time from 2 to 24 h at 37 °C. The cells were then fixed with 4% formaldehyde for 15 min after incubation, washed thrice in PBS, and stained with DAPI (4′,6-diamidino-2-phenylindole) for 5 min before being imaged by a camera (SCMOS PcoEdge, Einst Technology Pte. Ltd., Singapore) mounted on a fluorescence microscope (Nikon, Eclipse, Ci) equipped with a 60× oil-immersion objective (N.A., 1.25) and appropriate filters. The Ce6 fluorescence intensities of the fluorescence images showing Ce6 uptake in cells were quantified with ImageJ by converting the images to gray scale and calculating their mean gray intensity level. Therapeutic Efficacy of PDA−Ce6 on Bladder Cancer Cells. The cultured T24 cells were seeded at 3 × 104 cells per well in 96-well culture plates for 24 h before 200 μL of PDA−Ce6 and PDA NPs in culture media were added to each well at varying final concentrations of 0.1, 0.5, and 1 nM, and incubated for 24 h at 37 °C. Equivalent concentrations of free Ce6 as that loaded (15 μM) and released (1.5 μM) were also dosed to cells as controls. These two concentrations were chosen for their relevance to 1 nM of PDA−Ce6, with 15 μM of free Ce6 being the amount loaded and 1.5 μM being the free Ce6 that could be released in RPMI with 10% FBS. The excess PDA−Ce6 and free Ce6 were removed, and the cells were rinsed three times with PBS (pH 7.4) after incubation before being irradiated by a 665 nm CW laser for 15 min at 250 mW/cm2 to initiate PDT with free Ce6, PTT with PDA NPs, and combined PDT + PTT with PDA−Ce6 NPs. The cell viability was measured 24 h post treatment with PrestoBlue based on its absorbance at 570 nm normalized to 600 nm and compared with the dark toxicities of free Ce6, PDA, and PDA− Ce6 NPs without laser irradiation.

residues were covalently linked to the quinone moieties to result in steric hindrance or increased nucleophilicity. A linear increase in PDA NP concentration was observed over time as they stopped growing (Figure S2, Supporting Information) because of the continuous synthesis of new PDA NPs from quinones, unhindered by the Tris residues. The successful synthesis of PDA NPs was determined by Fourier transform infrared spectroscopy, which showed the presence of the characteristic primary and secondary amine peaks at 1650 and 1500 cm −1 , respectively (Figure S3, Supporting Information). PDA NPs were synthesized from 1 mg/mL dopamine in alkaline Tris buffer (pH 10.5) and used for loading Ce6 after 5 h of polymerization. We observed a maximum of 14.2 ± 0.85 μM of Ce6 loaded on 1 nM PDA NPs after 20 h of incubation when Ce6 was added at an initial concentration of 140 μM (∼10% loading of Ce6) (Figure 2H). The loaded amount reached saturation and did not increase as the initial Ce6 concentration added to 1 nM PDA NPs was increased further beyond 140 μM. Here, Ce6 possesses aromatic rings similar to PDA, which allowed them to interact and be stably bound in the PDA polymer matrix via π−π stacking interactions. The loading saturation occurred when the limited number of π−π interacting sites in the PDA NPs was reached. The prepared PDA and PDA−Ce6 NPs were monodispersed with an average size of 141.3 ± 4.3 and 142.4 ± 4.1 nm, respectively. No significant change in the size of NPs was observed with Ce6 loading (Figure 2A,B). However, the hydrodynamic diameter (DH) of unloaded PDA NPs (244.2 ± 10.1 nm) was slightly larger than that of PDA−Ce6 NPs (200.1 ± 8.5 nm) (Figure 2C), whereas the polydispersity index of both PDA NPs and PDA−Ce6 NPs was in the range of 0.2 which is generally accepted as a proof of the polymers’ homogeneity and monodispersity. The larger value obtained from the dynamic light scattering (DLS) measurements over TEM was likely attributed to the swelling of PDA NPs as they hydrated in an aqueous solution because of their hydrophilicity, whereas the hydrophobic Ce6 in PDA−Ce6 NPs minimized the hydration to result in less swelling and smaller increase in DH. Nonetheless, the DH values of PDA and PDA− Ce6 generally agreed well with the size from TEM images, with the differences attributed to the swelling of hydrophilic PDA in solution during the DLS measurements and shrinkage when the same samples were dried for TEM. Such an observation was common among polymers.37,38 Both PDA and PDA−Ce6 NPs were negatively charged in Tris buffer at pH 10.5 with a zeta potential of −48.7 ± 1.3 and −45.45 ± 1.4 mV, respectively, because of the deprotonation of phenolic and amino groups on PDA (Figure 2D). The similarity in their surface potential also supported the loading of Ce6 within the polymer matrix via π−π stacking instead of surface loading. In both cases, the zeta potential became less negative as the pH decreased toward the zwitterionic isoelectric pH of 4.5 for PDA, before flipping to a positive surface charge of 35.3 ± 2.4 and 31.2 ± 2.0 mV for PDA and PDA−Ce6 NPs, respectively, as the pH crossed the isoelectric pH to pH 3. This confirmed the zwitterionic surface property of PDA NPs that was not lost with Ce6 loading in the polymer matrix. Apart from surface charge, the hydrophilicity of PDA was also critical to their biological response. We determined this using the RB assay for surface hydrophobicity where the negatively charged hydrophobic RB dye molecule would

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RESULTS AND DISCUSSION Synthesis of PDA NPs and Loading of Ce6. PDA NPs were synthesized from polymerizing its monomer dopamine at high alkaline pH with Tris buffer. We synthesized a range of PDA NP sizes by varying both the dopamine concentration and the pH of Tris buffer, where the increase in dopamine concentration and lowering of basic pH led to larger sizes (Figure S1, Supporting Information). In all cases, the growth of PDA NPs slowed after 20 h. Della Vecchia et al. attributed the mechanism of polymerization and growth to the sequential chain elongation and aggregation steps of dopamine monomers oxidized to quinone monomers which acted as electrophilic acceptors for the existing oligomers.36 The growth slowed as this process was subsequently hindered as the Tris 21128


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Figure 3. (A) Colloidal stability of PDA NPs in different media over time as measured by their hydrodynamic diameter, DH, in different media. (B) Table summarizing the average DH and surface charge of PDA NPs in different media after 12 h.

Figure 4. Passive release of Ce6 from 1 nM of PDA−Ce6. (A) Release of Ce6 from PDA−Ce6 over 10 days in Tris buffer of different pH. (B) Release of Ce6 from PDA−Ce6 NPs in RPMI with 10% FBS, human serum, and urine over a period of 2 weeks. In all cases, PDA−Ce6 was formed by incubating PDA NPs with 140 μM of Ce6 for 20 h. (C) Burst release profile in urine over a span of 1 day of PDA NPs incubated with 140 μM of Ce6. (D) Amount of Ce6 thermally released after heating 1 nM of PDA−Ce6 at 37 and 45 °C for different durations.

lower compared to free Ce6 at the same loaded concentration (Figure 2G). The PDA NPs showed negligible fluorescence on their own because of their very low quantum yield (0.2%). They also possessed strong fluorescence quenching from the π−π stacking interaction between the monomeric units.4,11 As Ce6 was loaded in PDA NPs, the π−π stacking of their aromatic groups with those of PDA could have resulted in the fluorescence quenching of 5.5 times over free Ce6 as measured by the change in fluorescence intensity in Figure 2G. Colloidal Stability of PDA NPs and Passive Release Characteristics of Ce6. The colloidal stability of NPs in biological media is crucial to their biological utility. As the PDA NPs were reconstituted in RPMI alone, they aggregated after 3 h with their DH increased from 259.2 ± 1.8 to 885.82 ± 32.1 nm (Figure 3A). Their zeta potential was undetermined because of the severe aggregation. The aggregation of PDA NPs in RPMI alone was not unexpected because the RPMI media typically presented a high ionic environment that screened the electrostatic repulsive charges between the NPs. However, the PDA NPs became colloidally stable even up to 12 h with a DH of 292.0 ± 6.0 nm in cell culture media when serum proteins were incorporated into RPMI. Such a colloidal stability in RMPI with 10% FBS was more significant as the presence of serum proteins in biological media was more

interact with a hydrophobic NP and be pelleted to result in a reduced absorbance of the dye in the supernatant. Here, electrostatic interaction between the negatively charged RB dye and PDA NPs was ruled out. The RB assay showed a similar peak absorbance at 542 nm between the supernatant of PDA, PDA−Ce6, and equivalent concentration of RB solution (Figure 2E), suggesting a minimal hydrophobic interaction and hence hydrophilicity of both PDA and PDA−Ce6 NPs. The hydrophilicity of PDA−Ce6 NPs was further confirmed from their negative log P value, which became slightly less negative from −1.40 ± 0.16 for PDA NPs to −1.12 ± 0.04 when an increasing Ce6 concentration was added to PDA NPs (Figure 2F). The hydrophilicity of PDA−Ce6 also confirmed that Ce6 was loaded within the polymer matrix instead of the NP surface as Ce6 was hydrophobic with a log P of 0.940 ± 0.005. The hydrophilic PDA−Ce6 NPs would decrease the immune recognition by macrophages as the hydrophobic NPs are preferentially uptaken by macrophages.14 The negative surface charge and hydrophilicity of PDA NPs could also potentially confer them good mucopenetration properties with minimal interactions with the hydrophobic and negatively charged regions of mucins.39−42 The PDA NPs became fluorescent after Ce6 loading, although the fluorescence of PDA−Ce6 was significantly 21129


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Figure 5. Photothermal and photodynamic properties of PDA and PDA−Ce6 NPs. (A) Absolute temperature obtained after irradiating 1 nM PDA NPs in different pH conditions and 10 mM Tris buffer without any NPs as a control with a 665 nm CW laser at 250 mW/cm2 for 15 min. (B) Absorbance spectra of PDA, PDA−Ce6, 15 μM free Ce6 and Tris buffer without any NPs. ROS generation with (C) increasing PDA−Ce6 concentrations at physiological pH and (D) increasing pH at fixed concentration of 1 nM PDA−Ce6. The PDA−Ce6 NPs were either subjected to heat (100 °C for 10 min) (red, dotted) to release the maximal amount of Ce6 for irradiation or unheated to leave the Ce6 loaded within the PDA NPs during irradiation (blue, dotted). Free Ce6 at an equivalent concentration as that released from the heating was included as a comparison (green, dotted). The samples were either kept in the dark (solid lines) or irradiated with a 665 nm CW laser of 250 mW/cm2 for 15 min (dotted lines).

zwitterionic property of PDA, which became less negative at lower pH, thus reducing the electrostatic repulsion with the negatively charged Ce6 molecule because of its deprotonation of the pendant carboxyl groups. This suggested that pH could be a handle to modulate the release behavior of loaded therapeutic compounds. Although a good drug release was typically preferred at lower pH for cancer treatment as the tumor microenvironment was known to possess a slightly acidic pH of ∼6, the same low release at a slightly acidic pH of ∼6 could be beneficial in developing PDA NPs as a drug delivery vector in intravesical instillation for the treatment of bladder malignancies. In this context, the release of Ce6 needs to be minimized not just in the slightly acidic urine during instillation, but also during the mucopenetration through the acidic bladder mucus layer, before reaching the acidic tumor microenvironment, and entering the cancer cells through the acidic endosomes. Until here, a minimum release of Ce6 would be preferred until PDA−Ce6 escapes the endosomes and enters the cytoplasm with physiological pH which triggers a greater release of Ce6 for PDT in the cytoplasm. Previous studies have demonstrated that the PDA-coated NPs could enable the escape from the endosomes to the cytosol.21 Regardless of the pH, the amount of Ce6 that could be released from 1 nM PDA NPs passively over time still represented a low percentage (2.67−20%) of the 14.2 ± 0.85 μM Ce6 loaded (Figure 2H). It appeared that PDA NPs did not release all their Ce6 payload passively over time with a significant Ce6 concentration which remained trapped in the PDA polymer matrix possibly because of the strong π−π stacking loading mechanism as discussed earlier. A similar first-order burst release profile of Ce6 was also observed in various biological media for the first 5 days. Here, RPMI with 10% FBS at pH 7.4 resulted in the highest release

physiologically relevant in the biological systems. These serum proteins from 10% FBS formed a protein corona25,43,44 on the PDA NPs to confer steric stabilization. Although PDA NPs could potentially be used as a drug delivery vector in a wide range of contexts, urine is one biological medium of interest, especially in the context of PDA NPs as a drug delivery vector in intravesical instillation, which is the preferred delivery mode for the treatment of bladder malignancies. In bladder cancer where the tumor is usually localized on the luminal surface of the bladder urothelium, intravesical delivery is preferred over systemic delivery to minimize the systemic side effects. Here, drugs could be instilled directly into the bladder via the ureter, and the bladder urothelium with a mucus layer serves as a physical barrier to limit the systemic absorption of drugs, thereby reducing the host toxicities. As the PDA NPs would be in contact with urine before being uptaken by the bladder cancer cells, we examined their colloidal stability in urine to ensure minimum aggregation. The PDA NPs were also colloidally stable in urine (DH = 296.8 ± 6.7 nm), demonstrating their biological applicability in the biological media. The DH of PDA NPs in different media after 12 h is summarized in Figure 3B. With 14.2 μM of Ce6 loaded on 1 nM PDA NPs, we examined their passive release over time in different pH and biological media. At a high basic pH of 10.5 in Tris buffer, Ce6 showed a first-order burst release profile in the first 5 days, before the release rate slowed and stopped after 7 days (Figure 4A). The burst release became less pronounced at physiological pH 7 and eventually switched toward a zeroth-order linear release profile as the pH was further reduced to a mild acidic pH 5. The final amount of Ce6 passively released after 10 days also reduced with pH from 3.0 ± 0.13 μM (pH 10.5) to 1.46 ± 0.09 μM (pH 7) and 0.51 ± 0.09 μM (pH 5). The pH-dependent release profile of Ce6 could be explained by the 21130


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ACS Applied Materials & Interfaces of Ce6 (1.59 ± 0.12 μM) after 17 days, followed by human serum (1.03 ± 0.06 μM) and urine (0.96 ± 0.04 μM) (Figure 4B). It seemed that the formation of a protein corona around PDA−Ce6 NPs in RPMI with 10% FBS and human serum did not affect the Ce6 release profile or the release amount as the amount of Ce6 released was comparable or even higher than that in Tris buffer at pH 7 (Figure 4A). Urine induced the lowest amount of Ce6 released, possibly because of its slightly acidic pH of ∼6 and a shorter burst release which took place in the first 10 h (Figure 4C). The photothermal properties of PDA could induce an increase in the temperature of the microenvironment surrounding the PDA NPs to >37 °C. Here, we investigated if this heating could be exploited to trigger the release of Ce6 from PDA NPs. We observed a significant increase in the concentration of Ce6 released over time at higher temperatures of 37 °C (body temperature) and 45 °C (after photothermal heating by laser irradiation, as discussed in the next section) compared to 25 °C (room temperature) (Figure 4D). A further increase in temperature to 100 °C by heating the solution led to an even more significant concentration of Ce6 being released from PDA−Ce6 (Figure S4, Supporting Information). It appeared that the release of Ce6 from PDA−Ce6 was temperature-dependent, in which higher temperature enabled more disruption of the π−π stacking interactions between Ce6 and PDA, leading to a more significant increase in Ce6 release. This demonstrated that the photothermal heating of PDA NPs upon laser irradiation could modulate the release of Ce6 by disrupting the π−π stacking bonds. In general, the burst release behavior of Ce6 that we observed in PDA NPs was similar to the other drug delivery systems such as CaCO3 and lactoferrin which when loaded with Ce6 exhibited a release profile reaching a plateau at 545 and 6 h,46 respectively. This faster saturation compared to that of PDA NPs (5 days) presents limitations as the nanocarriers would have released most of their payload before reaching the target cells. Photothermal Behavior of PDA and PDA−Ce6 NPs. Being chemically similar to the naturally occurring black melanin particles found in human skin, Zhu et al. showed strong optical absorption and photothermal conversion in PDA NPs when irradiated with an 808 nm laser.20,47 We observed that a different wavelength source of 665 nm used to excite Ce6 at 250 mW/cm2 could also induce a significant increase in the temperature of 1 nM PDA NPs colloid over a buffer solution in a pH-independent manner after irradiation for 15 min. Here, a significant increase in temperature from 23.9 ± 1.12 to 42.1 ± 1.5 °C was observed (Figure 5A). We further demonstrated that the photothermal property of PDA NPs was concentration-dependent with an observed saturation at >3 nM (Figure S5A, Supporting Information). There was also no significant difference in the temperature rise between 1 nM of PDA−Ce6 (43.33 ± 0.83 °C) and PDA NPs (42.10 ± 0.47 °C) when irradiated with the same 665 nm laser for 15 min (Figure S5B, Supporting Information). This significant temperature rise was largely attributed to the strong optical absorption in the NIR region, particularly at the 665 nm wavelength of irradiation (Figure 5B), leading to rapid absorption of photons and heat conversion by PDA and PDA−Ce6.48,49 This enabled a rapid temperature rise that was otherwise not possible with the buffer alone.

We also determined the photothermal efficiency of 1 nM PDA and PDA−Ce6 under this irradiation condition to be ∼60.4% (input energy of 28.3 J and output energy of 17.1 J, see the Supporting Information for detailed calculations). Comparatively, gold NPs present a photothermal efficiency which is inversely proportional to their size,50 with smaller sizes of 15 nm range having ∼100% of photothermal efficiency, which decreased gradually to 55% when the size increased to 100 nm.51 The use of 665 nm laser irradiation to effect the temperature rise not only matched the excitation of Ce6 and potentially allowed simultaneous PDT and PTT, but was also biologically advantageous as the wavelength also provided for a deeper optical penetration in the tissue to reach the subepithelial tumor tissue.52 The use of a single 665 nm laser demonstrated significant therapeutic efficacy with a near-complete tumor eradication in in vivo experimental models in our previous study by Yeo et al.53 Although the typical absorbance peak of Ce6 at ∼665 nm was not clearly observed, this was not because of its low loading capacity of Ce6 as we have earlier determined that 15 μM of Ce6 was loaded on 1 nM PDA, giving a ratio of ∼15 000 Ce6 molecules loaded on each PDA NP. The absence of a strong Ce6 absorbance peak at 665 nm in the absorption spectrum of PDA−Ce6 was likely a combination of Ce6 molecules loaded inside the PDA polymer matrix, coupled with the PDA NPs by themselves having a strong absorbance spectrum across the entire visible wavelengths (Figure 5B), which masked the absorbance of Ce6 in PDA. This strong absorbance of PDA in itself was not surprising, hence its use as an effective photothermal agent. The results also suggested that the photothermal heating of PDA−Ce6 was not affected by the loaded Ce6 as there was no significant difference in the temperature measured between PDA and PDA−Ce6. Hence, PDA−Ce6 remained effective as a PTT agent even after Ce6 loading. The temperature reached (42.4 ± 0.5 °C) was within the range of 41−50 °C known to cause hyperthermia and cell kill by thermolysis in cancer cells, which were known to be more susceptible to heat than the more heat-tolerant healthy cells.54,55 ROS Production by PDT with PDA−Ce6. Most photosensitizers such as Ce6 were hydrophobic, which posed not just aqueous solubility issues, but also poor delivery in biological barriers such as the mucus. Here, PDA NPs provided a mean to load and deliver hydrophobic Ce6 in a highly aqueous and hydrophilic environment to target tissues for PDT. When irradiated by laser, Ce6 generated ROS which were capable of eliciting cancer cell kill by oxidative stress. We treated PDA−Ce6 thermally by heating them in a water bath at 100 °C to release a maximum of 2.8 ± 0.06 μM Ce6 from 1 nM PDA−Ce6 after heating for 10 min (Figure S4, Supporting Information) as the π−π stacking between the aromatic cycles of Ce6 and PDA molecules was noncovalent and could be disrupted by heating.35 This amount of thermally released Ce6 from 1 nM PDA−Ce6 NPs was comparable to the maximum amount that was passively released over time (Figure 4A). The thermally released Ce6 was irradiated and the level of ROS generated was comparable to that from the same concentration of free Ce6 (Figure 5C, green and red dotted lines), suggesting that the Ce6 molecules that were originally loaded did not lose their photodynamic properties with loading. In the absence of thermal treatment to leave the Ce6 loaded within the PDA NPs, we also observed an increased level of ROS generation from 1 nM PDA−Ce6 after irradiation by a 21131


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Figure 6. Cellular uptake of PDA−Ce6 NPs over a range of PDA−Ce6 concentration dosed to T24 cells and incubation time as quantified by (A) subtracting the absorbance measurement of nonuptaken PDA−Ce6 NPs recovered from cell culture supernatant from the initial absorbance of PDA−Ce6 added to cells and (B) by measuring the fluorescence intensity of Ce6 within cells using flow cytometry. (C) Comparison of cellular uptake of 15 μM of free Ce6 and 15 μM of Ce6 loaded in 1 nM of PDA−Ce6 in terms of Ce6 concentration uptaken and the uptake percentage in T24 cells over the concentration dosed for varying incubation times as quantified via the supernatant loss absorbance measurement technique in (A).

Figure 7. Fluorescence imaging of the cellular uptake and localization of (A) 15 μM free Ce6 and (B) 1 nM PDA−Ce6 loaded with the same 15 μM Ce6 over a range of incubation time. Ce6 fluorescence was shown in the red channel, whereas the cell nucleus (DAPI-stained) was shown in the blue channel. Scale bar = 20 μm. (C) Quantification of Ce6 fluorescence intensity of the different fluorescent images using ImageJ.

665 nm CW laser at 250 mW/cm2 for 15 min compared to the nonirradiated PDA−Ce6 in the dark (Figure 5C, blue lines). This was due to the excitation of Ce6 as the level of ROS generated increased with a higher concentration of Ce6 loaded on PDA NPs that could be effectively released from the PDA NPs. However, the amount of ROS generated was much lower when Ce6 was loaded in the PDA NPs (fluorescence intensity = 2684.3 ± 136.9, blue dotted) than when the same equivalent concentration of Ce6 was thermally released (fluorescence intensity = 39 236 ± 954.9, red dotted). The Ce6 loaded in the PDA polymer matrix generated a much lower level of ROS compared to the amount of Ce6 that

was thermally released, even though the amount of Ce6 loaded (14.2 ± 0.85 μM) was ∼5 times higher than that released thermally (2.8 ± 0.06 μM). This was likely due to a combination of the strong optical absorption of irradiated light by PDA that reduced the excitation of Ce6 loaded in the polymer matrix and the reduced oxygenation in the polymer matrix needed for ROS generation. Nonetheless, our observations not only further proved that Ce6 was loaded within PDA and not on its surface, but also suggested that the passive release of Ce6 over time could support fractionated PDT at chosen time points with a single PDA−Ce6 dosing. 21132


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amount of free Ce6 was already observed despite the quenching of Ce6 by PDA. Hence, the enhancement in Ce6 uptake with PDA−Ce6 would be even more significant if the quenching was corrected for. We determined the quenching effect of PDA on Ce6 fluorescence to be ∼5.5 times (Figure 2G). Hence, at 24 h, the corrected fluorescence gray intensity level for PDA−Ce6 would be ∼220 compared to 15.7 for free Ce6 alone. The 8.3-fold higher Ce6 uptake with PDA NPs as determined from the supernatant loss absorbance measurement technique (Figure 6C) could be explained by the endocytosis internalization pathway. It was demonstrated that stiffer NPs are predominantly uptaken by endocytosis,31 whereas small molecules have limited receptor-mediated endocytosis.56,57 T24 bladder cancer cells maintained a high cell viability of >80% when dosed with up to 1 nM PDA and PDA−Ce6 NPs for 24 h (Figure 8, solid lines). Upon irradiation with a 665 nm

We also characterized the ROS generation from PDA and PDA−Ce6 NPs at different pH, with either prior thermal heating or no thermal heating to release Ce6. PDA did not generate any ROS, regardless of irradiation (Figure 5D, black lines). As observed earlier, the absence of Ce6 released from PDA NPs and nonirradiation of the samples resulted in a low level of ROS generation (Figure 5D, solid lines), whereas the irradiated samples with Ce6 thermally released from PDA− Ce6 (Figure 5D, red dotted) resulted in a much higher ROS generation compared to PDA−Ce6 with no Ce6 released (Figure 5D, blue dotted). More importantly, the ROS generation from PDA−Ce6 increased with the pH increased. This increase in ROS generation with pH correlated well with the increase in Ce6 released with pH, as previously shown in Figure 4A. Cell Uptake and Therapeutic Efficacy. The uptake of Ce6 in T24 bladder cancer cells with 1 nM PDA−Ce6 carrying 15 μM of Ce6 was significantly higher than the uptake of an equivalent 15 μM free Ce6 (Figure 6C). By using two different quantitative techniques of flow cytometry and subtraction of the absorbance measurements of the nonuptaken PDA−Ce6 NPs recovered from the cell culture supernatant from the initial concentration of PDA−Ce6 added, we showed that the uptake of PDA−Ce6 into the T24 cells was not just dosedependent, but also increased over time up to a maximum after 24 h of dosing (Figure 6A,B). Here, the fluorescence of Ce6 was used to determine its concentration uptake by the cells instead of its absorbance as the Ce6 absorbance would be masked by the absorbance of PDA. This was observed in Figure 5B with the disappearance of the Ce6 characteristic peak at 665 nm on loading in PDA−Ce6. Such dose- and time-dependent uptake was not observed with free Ce6 at the same equivalent concentration, thus demonstrating the merit of exploiting PDA NPs for Ce6 delivery into cells (Figure 6C). For the same Ce6 concentration of 15 μM, 35.7% was uptaken by the T24 bladder cancer cells when loaded in PDA NPs compared to the 4.3% dosed in its free form. We confirmed the improved Ce6 uptake in cells with PDANPs from fluorescence microscopy images of the Ce6 uptake over time. With an increasing incubation time of 15 μM free Ce6 (equivalent amount loaded in 1 nM PDA−Ce6), we observed a peak in the uptake of Ce6 after 6 h with the localization mainly confined to the cytoplasm (Figure 7A,C). The peak uptake agreed well with the supernatant loss absorbance measurement technique (Figure 6C). In a similar manner, the Ce6 fluorescence in cells dosed with 1 nM of PDA−Ce6 agreed with our absorbance measurement and flow cytometry results, in which a peak uptake was observed after 24 h with the localization mainly confined to the cytoplasm (Figure 7B,C). In comparing the uptake between Ce6 loaded in PDA-NPs and free Ce6 at the same concentration, a much higher Ce6 fluorescence in cells were observed when delivered with 1 nM PDA−Ce6 NPs as compared to the delivery of the same Ce6 concentration in its free form (Figure 7) under the same excitation and acquisition parameters. After 24 h of incubation, the measured intensity from the quantitative image analysis showed free Ce6 uptake with a mean gray level intensity of 15.7 ± 1.2 compared to the PDA−Ce6 uptake with a mean gray level intensity of 39.9 ± 2.0. Here, a significantly higher uptake of Ce6 in cells (as indicated by higher fluorescence intensity in cells) using PDA−Ce6 compared to the equivalent

Figure 8. Cell viability after dosing T24 bladder cancer cells with varying concentrations of PDA NPs, PDA−Ce6 NPs, and the equivalent free Ce6 in a medium supplemented with serum for 24 h and irradiating them with a 665 nm CW laser at 250 mW/cm2 for 15 min. Using PrestoBlue cell viability assay, the dark toxicity of PDA NPs, PDA−Ce6 NPs, and free Ce6 was minimal (high cell viability >80%). The viability decreased drastically with the combined PDT + PTT with 1 nM PDA−Ce6 (dotted red) compared to that with PTT alone with 1 nM PDA (dotted black) and PDT alone with 15 μM free Ce6 (dotted green).

CW laser at 250 mW/cm2 for 15 min after dosing for 24 h, we observed dose-dependent cell kill with PDA NPs, PDA−Ce6 NPs, and free Ce6 at the equivalent concentration loaded (Figure 8, dotted lines). Among the three therapeutic agents, the highest cell kill was observed with the combined PTT + PDT from 1 nM PDA−Ce6 (76.0 ± 3.4% cell kill), followed by PDT with 15 μM Ce6 (57.8 ± 7.1%) and PTT with 1 nM PDA NPs (47.1 ± 7.4%). This was true not just for the highest concentration of these therapeutic agents, but also across all the concentrations studied (Figure 8, dotted lines). However, at physiological pH, the amount of Ce6 which was effectively released from the 1 nM PDA−Ce6 particles (i.e. ∼1.5 μM) resulted in ∼32.6% of cell death. We have shown earlier that ROS generation correlated to the amount of Ce6 released from PDA−Ce6 (Figure 5C); thus, Ce6 that remained unreleased in the PDA polymer matrix were unable to generate ROS for PDT. This level of cell death from PDT of the released Ce6 (i.e. 32.6%), if added to the cell death from PTT by PDA NPs alone (i.e. 47.1%), would result in an additive total of 79.7% cell kill. This similarity in cell kill level observed in the combined PTT + PDT with PDA−Ce6 (i.e. 76.0%) suggested an additive effect between PTT alone and PDT 21133


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endocytosis and therapeutic efficacy of the combined PDT and PTT from PDA−Ce6 under a single laser irradiation. By maintaining the surface charge and hydrophilicity of PDA NPs, these NPs could avoid uptake by the macrophages and potentially be mucopenetrative to enable more effective cancer phototherapy in the organs requiring mucosal drug delivery such as bladder, intestinal, and nasopharyngeal cavity, which is currently limited by the poor mucopenetration of hydrophobic photosensitizers. Although recent advances have suggested PDA as a viable nanomaterial in nanomedicine applications, many aspects of PDA NPs remained as scientific gaps, including their mucopenetrative properties, immunogenic response, and biodegradation behavior, which needed to be addressed before eventual clinical utility. This formed our motivation for subsequent further studies.

alone, which when combined together resulted in an enhanced therapeutic effect over a single treatment. As discussed above, the enhanced cell kill by PDA−Ce6 was likely attributed to the combination of PTT by PDA, coupled with the ROS generation from loaded Ce6 for PDT and enhanced uptake of Ce6 by PDA−Ce6, which collectively resulted in the enhanced killing of T24 bladder cells. PDT by Ce6 is oxygen-dependent for generating ROS. As the oxygen level in cells and tissues was gradually depleted from PDT over time, its efficacy decreased and the T24 cells became more sensitive to heat.58 Under such circumstances, the oxygenindependent PTT by PDA played a complementary role in maintaining a high therapeutic efficacy.59,60 Therefore, the combination of PTT by PDA and PDT by Ce6 played significant roles in creating a strong enhancement in therapeutic efficacy by PDA−Ce6 over a single treatment. Such cell kill levels were not observed in the absence of irradiation where T24 cells maintained a high viability of >80% when dosed with up to 1 nM of PDA and PDA−Ce6 containing an equivalent Ce6 concentration of 15 μM (Figure 8, solid lines). This showed that PDA NPs and PDA−Ce6 NPs possessed low levels of dark toxicity, similar to free Ce6, and was expected since PDA was known to have good biocompatibility with an LD50 of 400−585 mg/kg in vivo in a murine model as determined by Liu et al.21,61 Although effective cell kill was also demonstrated by Zhang et al., the studies were performed on a different cell line (human liver cancer HepG2) and a dual laser irradiation by 665 and 808 nm lasers operating at a much higher laser power intensity (50 mW/cm2 for 665 nm laser and 2 W/cm2 for 808 nm laser) was used in their therapy22 compared to our single laser operating at a lower power intensity (250 mW/cm2). Furthermore, Ce6 was covalently conjugated on the surface of PDA NPs, thus altering the desirable hydrophilic and zwitterionic properties of PDA NPs, making them more hydrophobic. The release characteristics of these covalently bound Ce6 were also not characterized. Here, the loading of Ce6 in the PDA polymer matrix preserved the hydrophilicity and zwitterionic properties of PDA NPs and allowed for a significantly higher payload and gradual release of Ce6 over a few days. Such a drug loading and release behavior afforded by PDA NPs would enable fractionated PDT at different time points, which was not possible with surface loading.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b04799. Synthesis and characterization of PDA and PDA−Ce6 and active release of Ce6 triggered by heating PDA−Ce6 NPs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Koon Gee Neoh: 0000-0002-2700-1914 James Chen Yong Kah: 0000-0002-2247-6929 Funding

The funding used to support the research of the manuscript was from the Ministry of Education (MOE) AcRF Tier 2 Grant (MOE2014-T2-2-147). B.P. would like to acknowledge the scholarship support from the NGS Graduate scholarship. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The funding used to support the research of the manuscript was from the Ministry of Education (MOE) AcRF Tier 2. B.P. acknowledges scholarship support from NUS Graduate School of Integrative Sciences and Engineering. The authors also acknowledge Laurent Bekale, Lanry Yung, and Giorgia Pastorin for helpful discussions.

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CONCLUSION We have developed a facile two-step method to load a hydrophobic photosensitizer Ce6 within the polymer matrix of a biopolymer PDA NPs which has not been demonstrated to date. Such a drug loading approach removed the need for complex preparation involving hazardous etching reagents to create the hollow interior of nanocapsules and maintained the mechanical and structural integrity of PDA NPs as well as their intrinsic surface hydrophilicity and zwitterionic properties, which was not possible with the surface conjugation of hydrophobic drugs. We also extensively characterized the colloidal stability and the drug loading and release behavior of Ce6 over a prolonged period of a few days and in different biological media. Such data provided new insights on the characteristics of PDA NPs as a nanocarrier which have been lacking in the literature to date. It appears that the π−π stacking interaction between PDA and Ce6 could be exploited for prolonged drug release. With our approach to loading Ce6 in PDA NPs, we also demonstrated enhanced cell uptake by

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ABBREVIATIONS APF, 3′-(p-aminophenyl)fluorescein; Ce6, chlorin e6; CW, continuous wave; DH, hydrodynamic diameter; DLS, dynamic light scattering; EPR, enhanced permeability and retention; FBS, fetal bovine serum; FTIR, Fourier transform infrared spectroscopy; HS, human serum; NIR, near infrared; NP, nanoparticle; PDA, polydopamine; PDT, photodynamic therapy; PBS, phosphate-buffered saline; PTT, photothermal therapy; ROS, reactive oxygen species; TEM, transmission electron microscopy

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Polydopamine Nanoparticles Enhance Drug Release for Combined

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