Polydopamine based multifunctional platform for combined

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Polydopamine based multifunctional platform for combined photothermal therapy, chemotherapy and immunotherapy in malignant tumor treatment Rui Chen, Chenqi Zhu, Yaojie Fan, Wenna Feng, Jingjing Wang, Erning Shang, Qin Zhou, and Zhipeng Chen ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00718 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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ACS Applied Bio Materials

Polydopamine based multifunctional platform for combined photothermal therapy, chemotherapy and immunotherapy in malignant tumor treatment Rui Chen1#, Chenqi Zhu1,2#, Yaojie Fan1, Wenna Feng1, Jingjing Wang1, Erning Shang2, Qin Zhou2, Zhipeng Chen1* 1 College

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of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China

Department of Pharmacy, Suzhou Hospital Affiliated to Nanjing Medical University,

Suzhou 215002, China #These authors contributed equally.

*Corresponding Author: Zhipeng Chen, Email:[email protected] KEYWORDS: polydopamine; folate; photothermal therapy; immunotherapy; combined therapy.

ABSTRACT: We designed a multifunctional platform by co-loading DOX, an antitumor drug, and imiquimod (R837), an immune adjuvant against Toll-like-receptor-7 (TLR-7), onto polydopamine nanoparticles (PDA NPs), a photothermal therapy (PTT) agent, to

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develop PDA/DOX&R837 NPs used as combined photothermal therapy, chemotherapy and immunotherapy in order to enhance the cancer therapeutic effects. For high delivery to malignant tumors, a folate ligand–receptor recognition molecule was grafted to the nanoparticle surface for higher cellular uptake efficiency. The particle size, zeta potential, morphology, drug loading content and drug release profiles of FA-PDA/DOX&R837 NPs were investigated. The antitumor effects under near infrared (NIR) laser radiation were evaluated, and our results showed that a three-mode strategy combined therapy was significantly superior to single mode therapy for tumor suppression. The synergetic toxicity of hyperthermia and DOX almost completely eliminated tumors, and together with R837, they further promoted the maturation of dendritic cells to induce a strong antitumor immune response, making tumor recurrence substantially lower than that without R837. This platform can be used as a potential targeted drug delivery system for combined cancer therapy.

Introduction With the difficult cure and easy recurrence of malignant tumors, the combination of multiple therapies is a potential strategy for cancer treatment.

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PTT is a technique involving the

transformation of light energy into heat energy that is cytotoxic to cancer cells and has attracted considerable interest recently due to local tumor destruction and few side effects. 3 Various PTT agents, such as gold nanoparticles, 4 graphene, 5 and carbon nanotubes, 6 have been reported to treat malignant tumors with moderate success. However, PTT is a local treatment strategy, and the tumor cells around the radiation target likely remain hidden, posing a potential recurrence danger. Therefore, the combination of PTT with chemotherapy is highly desirable because it was reported that some chemotherapeutic agents have enhanced cytotoxicity at elevated temperatures.

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Although the mechanism of heat-induced enhancement in cytotoxicity is not completely understood, chemotherapeutic drugs applied with photothermal reagents have been widely studied in many reports. 8-12 However, the toxicity of high-dose chemotherapy continues to pose the risk of side effects, while low-dose chemotherapy is ineffective on diffused stem cells. Therefore, a new treatment needs to be developed as a complementary strategy to PTT or chemotherapy. Cancer immunotherapy, the use of the patient’s own immune system to recognize and destroy cancer cells, is the safest and most effective way to treat cancer. 13 After PTT treatment, tumorassociated antigens are released along with cancer cell apoptosis, and immune responses could be triggered in the body. Such antitumor immune signals are likely to be effectively amplified with the assistance of immune adjuvants, and the application of immunotherapy after PTT can potentiate host antitumor immunity to protect the body from tumor rechallenge. 14, 15 However, the simple mixture of multiple drugs for combined therapy may cause poor solubility, poor bioavailability and even negative effects. Therefore, how to improve the cooperation of the co-delivery systems is significant for tumor-combined treatment. First, the selection of carrier materials must be biocompatible with easy surface modification, so that various drugs can be heavily loaded to the carrier with a strong interaction. Second, these drugs need to accurately target and have high delivery to tumor cells dependent on the active target, which is primarily based on specific binding of receptors to ligands on the cell membrane (e.g., folic acid, 16

hyaluronic acid, 17 peptide, 18 and aptamer 19). In this study, we prepared polydopamine (PDA) nanoparticles grafted to the folic acid (FA)

molecule. FA is a high affinity ligand of folate receptors, which are frequently over-expressed in cancer cells but expressed at a low level in normal tissues. Because the structure of polydopamine is similar to the natural substance melanin in the body, PDA has excellent biocompatibility and low cytotoxicity, making it a promising candidate for a biomaterial. 20 In addition, it also has been reported that the surface of PDA is chemically reactive and can be fabricated with various biomolecules with amine or thiol groups via Michael addition or Schiff base formation. 21 Hence, the modification of PDA NPs is easy and convenient, 22-24 and PDA-based materials used as a drug delivery system, 25-27 also have unique properties, including ease of preparation, excellent drug loading capacity, controlled release, 28 and biodegradability. 29 It is reported the aromatic drugs or organic dyes were easily facilitated by the abundant aromatic ring backbones of PDA though π−π stacking interactions, while metal ions interact through coordination bonding. Moreover, PDA is


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zwitterionic due to the protonated amine groups and dissociated phenolic hydroxyl groups. 30 The charged drug molecule can be pH-dependent, loaded or released from the PDA carriers under different pH conditions. 31 Because of these benefits, PDA can be selected as a bioinspired drug delivery system for high loading and stimuli-responsive release. 32 Based on these concepts, our strategy for a combined treatment platform is summed up in Figure 1. Briefly, dopamine was polymerized into PDA nanoparticles as a biocompatible PTT agent according to the reported method. 33 Then, FA molecules, as a targeting moiety, were grafted to the PDA surface though Michael addition at pH 8.5. Finally, a large amount of DOX and R837 was adsorbed to the PDA surface though π−π stacking and electrostatic attraction interactions in PBS at pH 7.4. When internalized by the cell though receptor-ligand action, PDA can produce heat triggered by the NIR laser and DOX is subsequently released under acidic conditions. The hyperthermic and chemotherapeutic toxicity are both destructive to the tumor cell, with an R837induced antitumor immunity response.

Through combining DOX, R837 and PDA into a single

platform, a common delivery of various therapeutic reagents can be achieved to play synergistic effect in the same site, making an outstanding advantage which separate delivery of reagents cannot achieve.

Results Preparation and Characterization of FA-PDA/DOX&R837 NPs The PDA NPs was prepared by self-polymerization under alkaline conditions according to the reported method. 33 The size of the PDA NPs can be adjusted by the ammonia concentration or the ratio of the ethanol/water solution.

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FA molecules were grafted to PDA via the Michael

addition reaction in an active targeting strategy. The obtained PDA NPs can load R837 and DOX after 24h incubation. The morphology of all nanoparticles including PDA, FA-PDA, FAPDA/R837, FA-PDA/DOX&R837 were spherical (Figure 2A) with the size of 209.8 nm, 220.5 nm, 225.4 nm and 287.3 nm (Figure 2B), while the zeta potential was -24.7 mV, -42.5 mV, -35.8 mV and -17.0 mV, respectively (Figure 2C). The stability of nanoparticles in PBS 7.4 was good during a week observation. FT-IR (Figure S1) proved the successful preparation of PDA and FAPDA. The new characteristic peak of the PDA curve at 3344 cm-1 (stretching vibration of phenolic


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O-H and N-H), 1613 cm-1 (aromatic C-C bonds), 1508 cm-1 (C-N bending)，and 1583 cm-1 and 1436 cm-1 (C=C in benzene rings) compared to the DA curve proved that the polymerization occurred. The broad absorbance between 3400 and 3100 cm-1 of the FA-PDA curve and the new peak at 1442 and 1325 cm-1 indicate successful incorporation of the FA molecules on the surface of PDA NPs. The excellent photothermal conversion property of FA-PDA was investigated by NIR 808 nm radiation at 2 W/cm2. The thermal images over ten minutes are displayed in Figure S2A and the temperature data are plotted in Figure S2B. Compared to water, the temperature of FA-PDA can reach 54.2°C, which is high enough to kill cells. Moreover, they exhibited excellent photostability during the three repeated heatings (Figure S2C).

Drug Loading and Release To evaluate the drug loading capacity of nanoparticles, R837 or DOX were mixed with 1 mg FAPDA in PBS (pH = 7.4) for 24 h under different drug feeds, and the drug loading content was determined by high-performance liquid chromatography (HPLC). The drug can be highly loaded onto the nanoparticle due to the adhesiveness of PDA, and the highest drug loading reached 51.2% for DOX and 40.6% for R837 in our test (Table S1). Since R837 was an immune adjuvant, the dosage did not need to be too large. At the optimized conditions, the loading capacities of DOX and R837 on FA-PDA NPs were 16.5% and 1.0% for the following test. The high drug loading was due to the strong π−π stacking interactions and electrostatic interaction, as revealed by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS).

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In the FT-IR spectra (Figure 3A), the new peaks of FA-

PDA/R837 at 1645 and 759 cm-1 were attributed to the presence of R837. After loading DOX, the additional peaks at 813 and 1106 cm-1 were seen on the curve of FA-PDA/DOX&R837. As a result, the high efficiency adsorption of DOX made the FA-PDA NPs a high drug loading option. The XPS spectrum was also analyzed to determine the interaction at the interface of the materials. In Figure 3B, the XPS spectra shows the most informational regions (C 1s, N 1s and O 1s) of the FA-PDA NPs before and after drug adsorption. For FA-PDA, the effective peak at 284.9 eV, 285.9 eV and 287.85 eV were due to the aromatic C-H, C-N and C=O, respectively (Figure 3C). The low intensity peak at 289.15 eV of FA-PDA assigned to the π−π transition in the aromatic ring, which was weaker in the FA-PDA/DOX&R837 (Figure 3D), gave an indication of the π−π interactions between PDA and the drug. Meanwhile, the binding energies of the N 1s spectra of -NH2 (400.0


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eV) and -NH (399.625 eV) found at FA-PDA peaks (Figure 3E) were lower after drug adsorption (Figure 3F). In contrast, the corresponding O 1 s spectrum of C=O (532.4 eV) and C-OH (532.78 eV) of FA-PDA were higher after drug adsorption (Figure 3G and H), which may be because the O atom donated electron pairs while the N atom accepted the electron. This finding further indicated that the adsorption process occurred mainly due to the electrostatic interactions between FA-PDA and the drug. The elemental analysis of XPS (Table S3) and the composition changes also demonstrated the successful conjugation of the drug onto FA-PDA NPs. On the other hand, because of the zwitterionic properties of polydopamine, the electrostatic interaction between the drug and polydopamine is also influenced by pH. When drugs loaded NPs came to acidic conditions, the protonation of amino group on PDA surface made NPs positive charge and rejected the drugs molecules, displaying an accelerated drug release. However, the strong mutual adsorption based on π-π stacking may result in an incomplete release of drugs finally. 36 To evaluate the drug release behavior, the in vitro cumulative release from FA-PDA NPs was investigated at pH 4 or 7.4. The pH-dependent release kinetics of DOX and R837 are displayed in Figure S3. In general, drug-loaded FA-PDA exhibited sustained release properties with a relatively fast release at the initial stage. However, the release rate in the neutral solution (pH 7.4) is much lower than that in the acidic solution (pH 4) within 72 h. For DOX, a significantly greater total release from the FA-PDA at pH 4 was observed than at pH 7.4, which was probably the result of a decrease in the interaction between the PDA and DOX upon protonation of the amino and catechol groups of the PDA at low pH. 37 From this point of view, the pH-responsive drug release behavior could reduce premature drug release during circulation but specifically enhance intracellular drug release, which is definitely beneficial for effective cancer treatment.38

Cell uptake A cell uptake assay was used to investigate cell targeting function in HeLa cells. From Figure 4A, all groups of DOX, PDA/DOX&R837 and FA-PDA/DOX&R837 displayed a concentrationdependent increases in cellular uptake. The difference amount between PDA and FA-PDA indicated that the cellular uptake pathway of these two types of nanoparticles may be different. In Figure 4B, the uptake of PDA was present within 4 h, while FA-PDA prolonged to 8 h. The temperature effect from 37°C to 4°C was investigated in Figure 4C due to the inhibition of cell transport proteins at 4°C. In the DOX group, no significant change was seen at 4 °C or 37 °C,


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which indicated passive diffusion uptake. However, in the FA-PDA group, a two times higher uptake occurred at 37 °C compared to that at 4 °C, which indicated an energy-dependent process involved in protein transport. Finally, the cell uptake mechanism was investigated using different inhibitors (Figure 4D). The cellular internalization mechanism can be investigated by their corresponding inhibitors such as chlorpromazine (clathrin-mediated endocytosis), nystatin (caveolae-mediated endocytosis), sodium azide (energy-dependent endocytosis) and amiloride (micropinocytosis). All inhibitors had no obvious impact on DOX uptake, and a decrease caused by sodium azide and amiloride in group of PDA or FA-PDA, which indicated possible energy metabolism. At the same time, nystatin inhibited the uptake of only PDA and mannitol inhibited the uptake of only FA-PDA, which indicated the different entry pathways of PDA and FA-PDA. All results proved that the enhanced cellular uptake of FA-PDA was relevant to FA receptormediated endocytosis.

Tumor target Because of the enhanced cellular uptake of FA-PDA NPs by cancerous cells with high expression of FA receptor on surface, we believed that there was more drug at the tumor site that in other regions. The fate of FA-PDA/DOX&R837 NPs in vivo was investigated using a fluorescence imaging system labeled with NIR 797 dye. From the results (Figure 5A), we could see that the NPs quickly targeted the tumor and reached the highest intensity at 8 hours. After 72 h, only the tumor area was fluorescent. Then, the mouse was euthanized and the isolated tissues were harvested and imaged (Figure 5B). The tumor and the liver had a strong signal, indicating that the NPs can target the tumor tissues due to FA decoration. To obtain quantitative drug concentration distributions, the amount of DOX was determined by fluorescence spectrophotometry after extraction from the organs. In Figure 5C, we can see that most DOX was located in the liver, spleen and kidney due to the capture of nanoparticles by the reticuloendothelial system (RES). 39 The amount of DOX in the tumor was as high as ~6%, which proved that DOX was selectively delivered to the tumor site by the FA-PDA NPs.

Immune response The generation of an immune response could be attributed to T cell activation, which is


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responsible for directly killing the target cells. The immune response effect of FA-PDA/R837 NPs was assessed using immature dendritic cells (DCs). DCs are a type of antigen-presenting cells (APCs), which can be stimulated to mature by capturing antigens to activate T cells. The upregulation of CD80 and CD86 on the DCs surface could affect the level of DC maturation and is evaluated by flow cytometry analysis. Figure 6A shows the different abilities of the NPs to stimulate DC maturation. The percentage of mature DCs (CD80+ and CD86+) treated with R837 nanoparticles was higher than free R837, whereas PDA nanoparticles without R837 loading triggered no appreciable immune response to DCs. In addition, interleukin 12 (IL-12p40) and tumor necrosis factor α (TNF-α) were considered as the immune-related indicator for DC activation. These cytokines levels secreted from DCs response to different formulations were evaluated by enzyme-linked immune sorbent assay (ELISA). After R837 treatment, the IL-12p40 and TNF-α levels were higher than those with free R837 (Figure 6B), and without inducing potentially toxic systemic cytokine release. All triggered significant DC maturation, found to be stronger than the other two. Thus, FA-PDA/R837 NPs had a strong effect on triggering DC maturation and antitumor immune activation.

In vivo tumor inhibition effect To assess the treatment effect on solid tumors, an H22 tumor-bearing mouse model was established and six formulations, including saline, FA-PDA, FA-PDA/DOX and FAPDA/DOX&R837 with or without NIR radiation, were used in the antitumor study. The tumor growth and mouse survival were measured for the following 37 days (Figure 7A and B). An uninhibited tumor growth was seen in the saline group, with a large size of approximately 5.8×103 mm3 at day 15, and the mice were all dead at day 16. The tumor size of the FA-PDA (+) group was 1200 mm3 at Day 15, much smaller than that of the saline group. Interestingly, it was also slightly smaller than that of the FA-PDA/DOX group (1500 mm3 at Day 15), which demonstrated that the inhibition effect of PTT alone is better


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than the chemotherapeutic agent alone. As the survival rate depends on the tumor growth, the survival time of the former was longer than that of the latter. On the other hand, the PTT combined chemotherapy FA-PDA/DOX(+) group revealed a more significant tumor suppressive effect and the longest survival time. Most significantly, when combined with immunotherapy, FA-PDA/DOX&R837(+) was more effective in eradicating tumors, and half of the mice were alive on Day 37. However, the behavior of the FAPDA/DOX&R837 group without PTT was worse than PTT alone. These results demonstrate that R837 could amplify PTT-induced antigen signals but not produce them to call T cells; in the absence of PTT, only R837 seems useless. The combined treatment of PTT and immunotherapy has a significant ability to suppress the tumor and prolong the lifetime of mice. H&E staining (Figure 7C) and TUNEL immunofluorescence staining (Figure 7D) were used to examine the apoptotic and necrotic extent in the tumor regions after different treatments. From the H&E stained images, we can see most of the tumor tissue was necrotic in the irradiation- or DOX-treatment groups compared to the saline group, suggesting that both heat and chemical toxicity have a general antitumor effect. In particular, vascular expansion and congestion were observed in the two irradiation groups. Similarly, TUNEL staining images reflected significant tumor cell apoptosis in all treatment groups compared to the saline group. In addition, a more severe cell apoptotic


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and necrotic effect was detected in the FA-PDA/DOX&R837(+) group, which had the best antitumor effect compared with the other groups.

Conclusion In summary, our work reports an active target platform with a high co-loading of a photothermal conversion agent, chemotherapy drug and immune adjuvant for integrating PTT, chemotherapy and immunotherapy to achieve synergistic effect in combating malignant tumors. The chemotherapeutic drug DOX and the immune adjuvant R837 were absorbed by FA-PDA NPs through π-π stacking and electrostatic interaction with high efficiency, and the FA molecule offer selective targeting to cancer cell for a high delivery of all drugs. Our FA-PDA/DOX&R837 nanoparticles could be used for NIR-induced thermochemotherapy to destroy local tumors and a systemic immune response to protection from tumor recurrence. Therefore, this three-mode combination strategy provides a comprehensive treatment plan for malignant tumors, not only directly eliminating primary tumors by the 808-nm laser irradiation but also efficiently protecting the body from tumor recurrence through the strong antitumor immune responses. Importantly, this platform has a number of unique advantages, including easy fabrication, full biocompatibility, and the high efficiency co-delivery capability for therapeutic drugs, which has great potential application in clinical biomedicine.

Materials and methods Materials Dopamine hydrochloride (DA), folic acid (FA), and ammonia solution （ 25% ） were purchased from Sigma-Aldrich (St. Louis, USA). Doxorubicin (DOX) was purchased from Nanjing Keygen Biotechnology Co. Ltd. (Nanjing, China). Imiquimod (R837) was purchased from MedChemExpress. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sunshine Biotech. Co. Ltd. (Nanjing, China). Tris(hydroxymethyl)aminomethane (BioFroxx, Germany) and phosphate buffered saline (PBS, 0.01 M) contained 136.7 mM NaCl, 2.7 mM KCl, 8.7 mM Na2HPO4 and 1.4 mM KH2PO4. All other reagents were of analytical grade.


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All aqueous solutions were prepared using ultrapure water from a Milli-Q system (Millipore, USA).

Preparation of FA-PDA NPs PDA was prepared using an oxidation reaction under alkaline conditions. Dopamine hydrochloride (50 mg) was dissolved in deionized water (1 mL) and then injected into the solution of ethanol (4 mL) and deionized water (9 mL). Under mild stirring, an aqueous ammonia solution (0.25 mL, 28-30%) was added. The colorless solution immediately turned to pale yellow and gradually changed to dark brown. The reaction was allowed to proceed for 24 h; then, the PDA nanoparticles were collected by centrifugation and washed with water thrice. The PDA NPs (1 mg/mL) were resuspended in Tris buffer (10 mM, pH 8.5) containing folate ligands. After 30 min incubation at room temperature while rotating, folate-grafted polydopamine nanoparticles were collected by centrifugation and washed with water thrice.

Characterization of FA-PDA NPs The morphology of the PDA NPs and FA-PDA NPs was observed by transmission electron microscopy (JEM-100S, JEOL, Japan). The size distribution and zeta potentials were determined using a Malvern Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., UK). To measure the photothermal conversion performance of the FA-PDA NPs, a 1 mL aqueous dispersion of FAPDA NPs at different concentrations (0-200 μg mL-1) was introduced in a quartz cuvette and irradiated with an 808-nm NIR laser at a power density of 2 W cm-2 for 10 min. The temperature was recorded every 10 s by a digital thermometer (Fluke 561, USA) and observed using a thermal image device (FLIR Plus One 2, USA).

Drug Loading and Release To evaluate the drug loading, the solution of FA-PDA nanoparticles and R837 (FAPDA:R837, 100:1,10:1,1:1,1:2) or DOX (FA-PDA:DOX, 10:1, 5:1, 2:1, 1:1) was shaken for 24 h in the dark with different feeding ratios at weight. Then, the drug-loaded FA-PDA NPs were separated by centrifugation (12000 rpm, 5 min) and the remaining drug in the supernatant was measured by HPLC (LC-2010A, Shimadzu, Japan). The drug loading content (LC) and encapsulation efﬁciency (EE) were calculated as follows:


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Loading content (%)=

M drugs on NPs M NPs +M drugs on NPs

Encapsulation efficiency (%) =

100%

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(1)

M total drugs  M drugs in supernatant M total drugs

100%

(2)

The freeze-dried samples were analyzed using FTIR (Nicolet, USA) and XPS (PHI 5000 VersaProbe, UlVAC-PHI, Japan) to demonstrate that DOX was loaded onto FA-PDA. The drug

release test was performed by suspending 200 μg of FA-PDA/DOX NPs in 1.0 mL of buffer solution with different pH values (7.4, 4) at 37 °C. To determine the release amount at any given time, 1.0 mL of the solution was withdrawn after centrifugation, and the same volume of buffer was added to keep the volume constant. The drug concentration in the withdrawn solution was analyzed via HPLC. The drug release profiling was repeated at least two times, and the cumulative drug release percentage as a function of time was recorded.

Cell uptake First, 1×105 HeLa cells per well were seeded into 24-well plates with Dulbecco’s Modified Eagle Medium (DMEM) at 37 °C under 5% CO2 atmosphere, supplemented with L-glutamine (2 mM), penicillin (100 units/mL), streptomycin (100 mg/mL), and 10% fetal bovine serum (FBS). Then, different formulations containing DOX, PDA/DOX and FA-PDA/DOX&R837 were added to the cells for a 4 h co-incubation. After washing with PBS thrice, the cells were collected and centrifuged to determine the DOX concentration by fluorescence spectroscopy. The protein concentration was determined using an enhanced BCA protein assay kit (Beyotime). The cellular uptake of DOX was calculated as the weight of DOX / protein. For a systematic study of cellular uptake, different concentrations (1, 10, 25, 50 μg/mL), co-incubation times (2, 4, 8 h), temperatures (4 and 37 °C) and different kinds of endocytosis inhibitors, such as nystatin (15 μg/mL), chlorpromazine (20 μg/mL), amiloride (50 μM), and sodium azide (1 mg/mL), were investigated. The inhibitors were added 1 h before drugs followed by another 4 h incubation, and the inhibitor caused decline on uptake compared to that without inhibitor represented the corresponding endocytosis pathways.


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Tumor target To establish the tumor-bearing mouse model, 0.1 mL saline with 5×105 murine hepatic H22 cells were subcutaneously injected into the right armpit of each mouse. The tumor was allowed to develop until the volume reached approximately 100 mm3 at 7 days. Then, 100 μL NIR797-labeled FA-PDA/DOX&R837 NPs solution (1 mg/mL) was intravenously injected into the mouse, and the fluorescence signals of the isoflurane anesthetized mouse at 1 h, 2 h, 4 h, 8 h, 24 h, 48 h and 72 h were monitored using the Maestro in vivo fluorescence imaging system (CRi Inc. Woburn, MA). After 72 h, the mouse was euthanized, and the heart, liver, spleen, lung, kidney, tumor, stomach, intestine and brain were harvested for isolated imaging. To obtain a quantitative DOX concentration distribution, the mice were euthanized, and the tissues were homogenized. The DOX amount after extraction from organs was determined by fluorescence spectrophotometry.

In vitro DC activation and cytokine analysis The bone-marrow-derived dendritic cells (DCs) were harvested from C57bl/6 mice and the experimental method was based on the reported paper using antibody against CD80-APC/CD86PE (Miltenyi Biotec GmbH) and ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.). 40

In vivo assessment of antitumor effect Sixty tumor-bearing mice were randomly divided into six groups for in vivo antitumor experiments. When the tumor volume was 100 mm3 on average, two groups were intravenously injected with saline and FA-PDA NPs, two groups with FA-PDA/DOX NPs, and two groups with FA-PDA/DOX&R837 NPs. The groups needing NIR laser irradiation were anesthetized with chloral hydrate (5%, 0.1 mL per mice) and then irradiated under an 808-nm laser at 2 W/cm2 for 5 min. The irradiation was implemented 8 h post-injection, and the time was set as Day 1. During the 37 days observation, the tumor volume and survival of each mouse was recorded every other day. The tumor volume was calculated as V =a2×b/2, where a is the shortest diameter and b is the longest diameter of the tumor. Another six mice were used for H&E staining and terminal deoxynucleotidyl transferase

(TdT)-mediated dUTP nick end labeling

(TUNEL)

analysis. Each mouse was euthanized at Day 7, and the tumor tissue was removed for sectioning.


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Statistical analysis All the experiments were repeated at least three times and the results were expressed as mean ± SD. The statistical significance of all the results was determined by the Student’s t-test. P < 0.01 and P < 0.001 was considered to be statistically significant. Supporting Information Drug loading of FA-PDA NPs at different feeding, elemental Ratio in XPS, FTIR spectra of Dopamine (DA), PDA, Folate (FA) and FA-PDA, the thermal images of FA-PDA NPs solution under 808nm laser radiation during ten minutes, the center point temperature of the thermal images and the thermal circulation of FA-PDA NPs solution, release profiles of DOX or R837 from FAPDA/DOX&R837 NPs at different pH values.

(PDF)

Acknowledgments This work was supported by the National Natural Science Foundation (No. 81601598, 81773662, 81473147). Competing Interests The authors have declared that no competing interest exists

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Figure 1: Scheme of FA-PDA/DOX&R837 NPs preparation and the in vivo PTT treatment process.


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Figure 2: (A) TEM images, (B) size and (C) zeta potential for the nanoparticles of PDA, FA-PDA, FA-PDA/R837 and FA-PDA/DOX&R837.


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Figure 3: (A) FTIR and (B) XPS spectra of FA-PDA before and after drug loading. Peakfitting spectra for C 1 s of (C) FA-PDA and (D) FA-PDA/DOX&R837; N 1 s of (E) FA-PDA


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and (F) FA-PDA/DOX&R837; O 1 s of (G) FA-PDA and (H) FA-PDA/DOX&R837.

Figure 4: Cellular uptake in HeLa cells after treatment with DOX, PDA/DOX&R837 or FAPDA/DOX&R837 at (A) different time, (B) different drug concentration, (C) different temperature (**p

Polydopamine based multifunctional platform for combined

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