Polydopamine Nanocapsule: A Theranostic Agent for Photoacoustic

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Polydopamine Nanocapsule: A Theranostic Agent for Photoacoustic Imaging and Chemo-Photothermal Synergistic Therapy Hanqiong Zhuang, Huiling Su, Xuexin Bi, Yuting Bai, Lu Chen, Dongtao Ge, Wei Shi, and Yanan Sun ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00260 • Publication Date (Web): 30 Jun 2017 Downloaded from http://pubs.acs.org on July 3, 2017

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ACS Biomaterials Science & Engineering

Polydopamine Nanocapsule: A Theranostic Agent for Photoacoustic Imaging and Chemo-Photothermal Synergistic Therapy Hanqiong Zhuang, Huilin Su, Xuexin Bi, Yuting Bai, Lu Chen, Dongtao Ge, Wei Shi and Yanan Sun*

Key Laboratory of Biomedical Engineering of Fujian Province University/Research Center of Biomedical Engineering of Xiamen, Fujian Key Laboratory of Materials Genome, Department of Biomaterials, College of Materials, Xiamen University, No. 422, Siming South Road, Xiamen 361005, P. R. China

* E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Polydopamine capsule has aroused widely attention since its emerging, because of its biocompatibility and the great potential as drug delivery carrier. However, preparing the nanometer PDA capsule (PDAC) is still remained a challenge, especially with the size below 300 nm. Moreover, there is little research about its photoacoustic imaging (PAI) and photothermal therapy (PTT) effect. In this paper, we reported an improved DMDES emulsion template method to obtain 200 nm PDAC, and act as highly efficient theranostic agent for photoacoustic imaging (PAI) and chemo-photothermal synergistic therapy. Due to its hollow structure and the higher photothermal conversion efficiency (η), the PDAC showed excellent PAI ability as its PA intensity was far outweigh the PBS and over two folders than the same size polydopamine particles (PDAP) at the same concentration in vitro. The animal experiment also verified this conclusion. Then the anticancer drug-doxorubicin (DOX) was loaded on PDAC via electrostatic interaction and π-π stacking. Moreover, the drug release was pH responsive and NIR laser responsive to minimize the side effect, and this system was proved to efficiently ablate the tumor in vitro and in vivo experiments. This research highlights the great potential of PDA capsule as a new theranostic agent.

KEYWORDS: polydopamine nanocapsule, hollow structure, photoacoustic imaging, photothermal therapy, chemo-photothermal synergistic therapy


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INTRODUCTION In the last decade, theranostics, as a combination of diagnostics and therapeutics has attracted widespread interest in research field since it can achieve a more specific and individualized treatment.1-3 Traditionally, diagnosis and therapy were two separate processes, which patients need take contrast agents or drugs in different times. The concept of theranostics offers the potential to relieve the patients’ pain and simultaneously to treat the disease more accurately.4-6 Photothermal therapy (PTT), famous for its non-invasiveness, utilized photo-absorbant agents to turn the near infrared (NIR) light into heat in specific site and thus to kill the cancer cells. Currently, photothermal agents mainly include gold-based nanomaterials,7-11 carbon-based nanomaterials12-13 and organic polymers.14 Some of these agents also exhibit excellent photoacoustic (PA) contrast abilities, since the PA imaging is photo-triggered and the transferred heat will cause acoustic emission, which may figure out different tissues.15-17 Owing to this, the PTT agents offer great potential to act as theranostics of the combination of PTT and PA imaging. Polydopamine (PDA), formed by the simple oxidative self-polymerization of dopamine, a hormone and neurotransmitter occurred in human body, was widely used in many fields,18-19 especially in biomedical field for its unique good biocompatibility. Recently, it has also been exploited to apply in PPT,20-21 due to its strong NIR absorbance. PDA capsules (PDAC) were recognized as drug delivery22-23 as their low toxicity and high drug loading. They were usually prepared by template approach, including soft or hard template, which was then sacrificed. Caruso et al.24 successfully



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synthesised the PDAC with tuned size between 400 nm to 2.4 µm. However, the diameter of PDAC was too large in vivo research. Much efforts was devoted to reduce the size of PDAC. Zhu et al.25 employed the 250 nm polystyrene sphere as hard template to fabricate 330 nm PDAC. Ni et al.26 utilized a miscible tetrahydrofuran–tris buﬀer mixture to obtain a 200 nm hollow PDAC. Yeroslavsky G. et al.27 obtained the 227±25 nm PDAC through a sonochemical method. Their method all need toxic organic solvent (THF etc.) or time-costing (10 days), although the sonochemical method was rather “green”. In general, so preparing a hollow PDAC with nanometer by a simple method now was still a challenge. Furthermore, given that the PA imaging was a combination of thermal imaging and ultrasound imaging,28 and much ultrasound imaging contrasts are hollow space,29-31 the PA imaging ability of hollow PDAC was believed to be excellent. However, up to now, there is little literature about the PA imaging of the PDAC. Here, we innovatively prepared a 200 nm hollow PDAC by adding sodium dodecyl sulfate (SDS) to the dimethyldiethoxysilane (DMDES) soft templates. SDS, a common stabilizer, here was to reduce the size of emulsion droplets and thus minimize the diameter of final PDAC. Owing to the strong NIR absorbance of PDA and the hollow interface, we then investigate its PTT and PAI application. As is expected, PDA capsule showed excellent photothermal conversion efficiency and PA intensity. We then loaded an anticancer drug on this capsule and explore the chemo/photothermal therapy effect in vitro and in vivo. Our results all prove that this PDA capsule offers great potential of being a theranostic agent, and works well.


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EXPERIMENTAL SECTION Materials. Dopamine hydrochloride was purchased from Sigma–Aldrich (USA), Doxorubicin hydrochloride (DOX) was supplied by Shanghai Hao Yun Chemical Technology Co., Ltd. Dimethyldiethoxysilane (DMDES) was supplied by J&K Scientific Co., Ltd. Sodium dodecyl sulfate (SDS) was bought from Shantou Dahao Fine Chemical Co., Ltd. Ammonium hydroxide (NH3 content 28%–30%), ethanol were all obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Synthesis

of

polydopamine

nanocapsule.

The

nanometer

scale

of

polydopamine capsule was obtained by a modified DMDES emulsion templates method according to a published article.24 Briefly, 200 µL DMDES and 200 µL ammonia solution (final concentration, 1% v/v) was added into SDS solutions (final concentration, 10% w/v), followed by vigorous manual shaking for 1min. The emulsions were left to stand for 6 h at room temperature. Then, it was added into 24 mL TRIS buffer contained 35 mg dopamine. The mixture was shaking for 24 h. After reaction, 44 mL of ethanol was poured into the solution and stayed overnight. To obtain polydopamine nanocapsules, centrifugation was performed. Characterization of polydopamine nanocapsule. The morphology of resulting PDA nanocapsules were analyzed by scanning electron microscope (SEM, SU-70) and transmission electron microscopy (TEM, JEOL TEM-1400). TEM samples were prepared by dipping sample suspensions on a silicon slices or 400 mesh carbon-coated copper grid, and then air-dried. Dynamic light scattering (DLS, Mastersizer 2000) was performed to measure the average size, polydispersity index and zeta potential of



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nanocapsules. All the measurements were tested in triplicate (n = 3) and at room temperature. UV1750 spectrophotometer (SHIMADZU, Japan) was conducted to acquire the UV/VIS absorption spectra of PDA nanocapsules in the range 200-1000 nm. Measurement of photothermal performance. 1mL different concentrations of PDA capsules (25 µg/mL-200 µg/mL) and nanaoparticles were placed in a glass tube and exposed to an 808 nm laser with an irradiation intensity of 2 W/cm2 for 8 min. Then, the temperature of various samples was recorded every 1 minute. In vitro PA imaging. To compare the PAI difference of polydopamine capsule (PDAC) and nanoparticles (PDAP), a real-time multispectral optoacoustic tomographic imaging system (MSOT, inVision 128, iThera Medical GmbH) was performed. Samples of PDAC and the PDAP between the concentrations of 200 µg/mL – 800 µg/mL were placed in phantom, which was to mimic the real mouse and the laser wavelength was designed to be 680-900 nm. The image was acquired and the PAI was analyzed by the software. Drug loading and release of the PDAC. DOX, as a model drug, was loaded into the PDAC through a vigorous stirring process. In specific, different volume of DOX solution (1 mg/mL) was added into the PDAC suspension and stirred vigorously for 24 h in the dark. To acquire PDAC-DOX sample, adequate centrifugation (1300 rpm, 10min) was carried out to remove the unloaded DOX. The amount of DOX was determined by the absorbance of 480 nm and the drug loading efficiency (LE) and encapsulation efficacy (EE) was calculated by the following formula:


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‫ ܧܧ‬ሺ%ሻ = ‫ܧܮ‬ሺ%ሻ =

୅ି୆ ୅ ୅ି୆ େ

× 100

(1)

× 100

(2)

In which A was the added amount of DOX, B was the amount of DOX in supernatant, and C was the amount of PDAC. To evaluate the drug released behavior, a typical dialysis bag method was taken. 1 mL PDAC-DOX solution was sealed in the dialysis bag, and then soaked in 5 mL phosphate buffered saline (PBS) media at different pH (pH 7.4 or pH 5.0). At predetermined time intervals, 5 mL media was withdrawn and fresh PBS was added. The released amount of DOX was measured by the UV–Vis spectrometer. Cytotoxicity assay. To access the cytotoxicity of the PDA capsule, MCF-7 cells were used. They were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, at 37°C in a 5% CO2 humidified atmosphere. Here, a typical method-MTT was carried out. In specific, cells (1×104 per well) were seeded onto 96-well plates and incubated overnight. Then, the medium was removed and changed to fresh medium containing different concentrations of PDA capsules solution. After another 24 h incubation, the medium was removed and PBS washed twice. Then, culture medium without FBS and with 10% MTT was added onto the cell. Afterwards, 4 h incubation was allowed to form formazan dye, which was then dissolved in DMSO. Finally, enzyme-linked immunosorbent assay reader was performed to measure the absorption of formazan at 570 nm. In Vitro Hyperthermia and Chemotherapy. The MCF-7 cells seeded on the



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96-well plate were incubated with different concentrations of the PDA capsules and PDAC-DOX samples, for photothermal therapy groups, it was exposed to 808 nm laser for 5 minutes with an irradiation intensity of 2 W/cm2. Then, it was proceed incubating for 24 h, and the cell viability was measured by the MTT method. To observe the hyperthermia more visually, the cell was stained with Calcein-AM and PI after irradiation and examined with the fluorescence microscope. Cellular Uptake. To more visually investigated the cellular uptake of PDAC, MCF-7 cells were seeded onto a plate (Ø 6cm) and grown. After 24 h incubation, culture medium was exchanged to fresh medium containing 100 µg/mL PDAC and incubated for 6 h to make the PDAC enter into the cell enough. Next, cells were collected by centrifugation and immersed in glutaraldehyde fixed solution for 2 h. Then, it was fixed with OsO4 and dehydrated in an ethanol, acetone and resin series. Afterwards, it was observed by a Tecnai-G2 Spirit Twin instrument (FEI Co., The Netherlands). To observe the cellular uptake of DOX-loaded PDA capsules, 2 × 105 of MCF-7 cells were incubated per glass tissue plate and grown. Afterwards, they were treated with PBS, DOX, or PDAC-DOX for 0.5 or 2 h. Next, the cells were washed by PBS adequately and fixed by 4% formaldehyde. Furthermore, the cells were stained with DAPI for 10 min and imaged with a confocal laser scanning microscopy (CLSM, Leica TCS SP5, Germany). In Vivo imaging and chemo-photothermal therapy. The BALB/c nude mice (6-week old) were obtained from Animal Center of Xiamen University. All animal


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experiments were carried out in accordance with the guidelines of the Regional Ethics Committee for Animal Experiments. To prepare MCF-7 model tumors, 1 × 107 cells dispersed in 50 µL PBS were subcutaneous injected into the back of the female mice. About 1.5 weeks later, the tumor size reached approximately 100mm3 and then was used. Several tumor-bearing mice were picked to investigate the PA imaging thermal imaging. For PA imaging, tumor-bearing mice were anesthetized with 1-2% isoflurane and the PA imaging was acquired with the laser wavelength 700 nm before injected the sample. Then they were intratumorally injected with 100 µL PDA capsules (2 mg/mL) and PDA nanoparticles (2 mg/mL), respectively and imaged again after 30 min. For the PDAC-DOX group, 200 µL PDAC-DOX solution (2 mg/mL) was intravenously injected, and the PA image was acquired at pre-injection, post 0.5 h, 2 h, 6 h and 24 h. As for the thermal imaging mice, they were intratumorally injected with 50 µL PBS (control), PDAC (2 mg/mL), PDAP (2 mg/mL), PDAC-DOX (2 mg/mL) respectively. Then the mouse was fixed and the tumor was exposed to 808 nm laser with a power of 2 W/cm2 for 5 minutes, the thermal image was captured by an infrared camera every minute. 30 tumor-bearing mice were randomly divided into 6 groups (n=5) and were intratumorally injected with 50 µL PBS (control), PDA capsules (2 mg/mL), PDAC-DOX (2 mg/mL, 10 µg DOX was loaded on100 µg PDA), DOX (0.2 mg/mL), separately. For the laser group, tumors were exposed to 808 nm laser with a power of 2 W/cm2 for 5 minutes every day. During the treatment period, body weight and



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tumor width and length were measured, the tumor volume was calculated as the formula: V = (tumor length) × (tumor width) 2 /2, and the relative tumor volume was calculated as V/V0 (V0 was defined as the initial tumor volume). After treated for 7 days, several tumor-bearing mice were sacrificed, the tumors and the major organs of PBS group and PDAC-DOX+Laser group harvested and fixed with paraformaldehyde, dehydrated and sliced for H&E staining and then was observed with a microscope (Leica). Results and Discussion Polydopamine nanocapsule (PDAC) was prepared by surfactant-DMDES emulsion templates method with SDS as a surfactant. Here, SDS can decrease the DMDES emulsion droplets and thus decrease the average size of the PDA capsule.32 Typical scanning electron microscopy (SEM) and transmission electron microscopy (TEM) image showed that the as-prepared PDAC was hollow sphere (Fig.1A, B), with the average diameter of around 200 nm, shell thickness about 30 nm. And it was normal to slightly smaller than the hydrodynamic size 216.8 ± 5.6 nm (Fig.1C). The zeta potential of PDAC was measured as -19.8 mV (FigS1) , which may be attributed from the catechol moiety of PDA and indicates the capsule was quite stable.33 This 200 nm PDAC, to the best of our knowledge, was first synthesized by the simple, green method, as only mild organic solvent - ethanol was used to form the hollow structure.


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Figure 1. Characterization of the as-prepared PDA capsules (PDAC). A) TEM image and B) SEM image of PDAC. C) Size distribution of PDAC. D) UV–vis absorption of different concentrations of PDAC. E) Photothermal elevation curves of different concentrations of PDAC.

Like other PDA materials,34 the PDAC showed broad absorbance from ultraviolet (UV) to NIR region (Fig. 1D). Owing to this, the PDAC has a certain potential on the PPT and thus the photothermal effect was investigated. Various



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concentrations of PDAC were irradiated under the NIR laser with the power of 2 W/cm2 for 8 minutes. The temperature showed significant dependence of the irradiation time and the concentrations of the sample (Fig. 1E). With the rise of irradiation time and the concentration of sample, the temperature was greatly increased. After irradiation for 8 minutes, the temperature of pure water was only up by 4.1°C, while the PDAC (200 µg/mL) was up by 43°C and reach 66.3°C, which was far more than 50°C, a temperature believed to efficiently kill the cancer cell.35 Then the photothermal conversion efficiency (η) of PDAC was measured according to a published method.34 To compare the η between the hollow PDAC and the common PDAP, the same 200 nm diameter PDAP was obtained according to a published method.34 Both PDAC and PDAP have a strong absorption in the NIR region (Fig. 2A, S3A), which indicates they have the potential to be used as PTT agents. The η was calculated to be 40.43%, which was slightly more than the same 200 nm diameter PDA nanoparticles (PDAP) (η was 37.14%, Fig.S3C, D). Moreover, it should be noted that the η was significantly higher than Au nanorods (η was 22%),34 a material is widely used for photothermal therapy. This feature of PDAC makes itself a highly promising PTT agent. In general, the photothermal agents can also be used as photoacoustic constrast agents due to the strong absorbance in NIR region. In fact, polydopamine has been used for PA imaging recently, an advanced technology to diagnose tumors.36-38 To verify whether the hollow PDAC can have a higher PA signal than the common PDAP, the PA spectra of PDAC and PDAP were detected between the wavelength of


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680-1000 nm at different concentration (Fig. 2B). Both of them have the maximum PA intensity at 700 nm. This wavelength makes them suitable for in vivo PA imaging due to the low absorption of oxygenated hemoglobin (HbO2) in this wavelength. In addition, the PA intensity was increased with the concentration of PDAC and PDAP. The representative PA image of PDAC and PDAP (200-800 µg/mL) at 700 nm were acquired (Fig. 2C), and the PA intensity was linear relevant to the concentration of PDAC (R2=0.992) and PDAP (R2=0.982) (Fig. 2D). However, the PDAC exhibited a rather excellent PA effect, for the PA intensity was over two folders than the PDAP at the same concentration. A possible reason for the higher PA intensity of PDAC may be due to the hollow structure of PDAC, which can produce harmonic imaging in a certain ultrasound output power39-40 and thus to enforce the ultrasonic signal, a base of PAI signal.41 Moreover, the higher photothermal conversion efficiency (η) of PDAC is also responsible for the good PAI effect to some degree.



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Figure 2. PAI ability of PDAC.

A) UV–vis absorption and B) PA spectra of PDAP

and PDAC in 1× PBS at pH = 7.4. C) Representative PA images of PDAP and PDAC solutions in the range of concentrations 200–800 µg mL−1, each excited by pulsed laser at 700 nm. D) PA intensities as a function of PDAP and PDAC concentrations in PBS.

In order to kill the cancer cells more thoroughly, we then loaded anticancer drug on this theranostic agents - PDAC. Doxorubicin hydrochloride (DOX) was chosen as a model drug and was loaded on the carrier by simply mixing with PDAC solutions for 24 h. The unbound DOX was removed by centrifugation repeated and the sample


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was then investigated by UV–vis spectroscopy. Clearly, a broad absorption peak at around 490 nm, which was characteristic absorption peak of DOX appeared over the PDAC background (Fig.3A), indicating the successful formation of PDAC-DOX. The encapsulation efficiency (EE) and loading efficiency (LE) was calculated by measuring the absorbance of the supernatant at 480nm. They could be regulated by adding the different amount of DOX into the PDAC solutions (Fig.S4). Notably, the EE was decreased with the increase of the amount of added DOX, while the LE was increased. We next study the in vitro drug released profile at different conditions by dialysis bag method (Fig.3B). At pH7.4, a simulated physiological condition, DOX was released from PDAC-DOX system at a very slow rate, only 16% was released within 24 h and with the irradiation of NIR laser, it can reach to 18%, little additional release was observed. However, at pH5.0, a simulated tumor cells condition, approximately 35.1% DOX was released within 24 h, which was over 2-folder faster than it was at physiological pH. Moreover, the NIR laser can also significantly promote the release behavior, and it was finally up to 45%. This was presumably attributed to that DOX was bound to the PDAC through electrostatic interaction and

π-π stacking.42 The lower pH or higher temperature could decrease the interaction between PDAC and DOX and thus facilitate the release of DOX.25 Furthermore, the heat induced from laser can accelerate the rate of release behavior on the other hand. All of these results showed that the PDAC-DOX platform was pH and NIR laser responsive, which would maximize its anticancer effect. To evaluate the cytotoxicity of PDAC, we investigated the biocompatibility by a



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traditional MTT assay. A certain concentration of PDAC was co-cultured with MCF-7 cells at suitable 5% CO2 humidified atmosphere for 24 h, the relative viability was then determined. As is expected, the PDAC (0-200 µg/mL) showed no obvious toxicity, giving that the cell viability all remained over 85% (Fig. 3C). This result was quite consistent with the other PDA materials.43 On the other hand, the cell viability decreased drastically under the irradiation of 808 nm laser with the power 2 W/cm2 for 5 minutes (Fig. 3C). Moreover, the cell viability was constantly decreased as the concentration of PDAC increased. When the PDAC was 100 µg/mL, about 50% cells retained alive. However, only 16.5% live cells was detected at the concentration up to 200 µg/mL, demonstrating that PDAC can efficiently kill cancer cells utilizing the photothermal behavior. To make the result more vividly, calcein AM and propidium iodide (PI) were used to stain the MCF-7 cells (Fig. 3E). We can easily observe that almost all the cells were green (alive) after incubated with PBS or PDAC (200 µg/mL) for 24 h without the irradiation of NIR laser. In contrast, most of the cells in control group still remained green (alive) under the irradiation, while the PDAC group were red (dead) especially at the irradiation spot. This result was further evidenced by the cell image taken at bright field. The morphology of cells round up and was detached at the laser spot of PDAC group, while the other cells were attached and with clear edges 2D monolayer cell morphology. This good biocompatibility and excellent photothermal performance makes PDAC a great potential PTT agents. We then investigate the chemotherapy and the synergistic photothermal-chemo therapy effect of this delivery system (PDAC-DOX) by standard MTT assay (Fig. 3D).


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Free DOX, a widely used anticancer drug, exhibited significant cytotoxicity at low concentration. However, the relative viability almost kept 25% when the dose of DOX reached 5 µg/mL and more. PDAC-DOX showed certain degree cytotoxicity as the cell viability decreased constantly along the increase of concentration although the efficiency was always lower than the free DOX. It was presumably due to that DOX loaded on the PDAC cannot release completely within 24 h. As is described previously, even in the cancer cell condition, almost 65% DOX still retained in this system. However, the phenomena changed with the external irradiation of 808 nm laser, since the cell viability substantially decreased. Especially at 5 µg/mL, less than 10% cancer cells stay alive, this cytotoxicity far outweigh the free DOX. The heat induced by irradiation can not only accelerate the release of drug, but also kill the cells directly.35



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Figure 3. The drug loading and

releasing ability of PDAC. A) UV-vis-NIR

absorbance spectra of PDAC, free DOX and PDAC-DOX in water. B) DOX release from PDAC-DOX overtime in buffers at the different pH values (5.0 and 7.4) and the NIR irradiation (2 W/cm2, 5 min at different time points) triggered release of DOX from PDAC- DOX at pH values of 5.0 and 7.4. C) Relative viability of MCF-7 cells with/without irradiation (808 nm, 2 W/cm2, 5 min) after incubation at different concentration of PDAC. D) Relative viability of MCF-7 cells after treated with


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different concentrations of free DOX or the PDAC-DOX (808 nm, 2W/cm2, and 5 min). E) Photothermal destruction of MCF-7 cells with or without PDAC and NIR laser (808 nm, 2 W/cm2, 5 min) treatments, the scale bar is 500 µm. P values were calculated by ANOVA with Tukey’s post-test: NS (non-significant difference) P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

The uptake of PDAC and PDAC-DOX by cancer cells was further studied with TEM observation (Fig.4A) and confocal laser scan microscopy (CLSM) observation (Fig.4B). For PDAC sample, TEM was carried out after PDAC or PBS was incubated with the cell for 6 h. We can observe directly that much PDAC was inside of the cell compared to the control, while some of them are deformed, which strongly evidenced the internalization of the PDAC by MCF-7 cells. As for the PDAC-DOX system, MCF-7 cells were treated with PBS, free DOX or PDAC-DOX at the equivalent dose for 0.5 or 2 h, and then DAPI was used to stain the cell nuclei. The red fluorescence of free DOX was overlapped to the blue fluorescence of cell nuclei. The red fluorescence became strong and more focused with prolongation of time, which indicated that free DOX was fully localized within cell. The red fluorescence was also observed and more focused to blue fluorescence over time in PDAC-DOX, although it was little weaker than free DOX for only about 15% DOX was released from the platform within 2 hours in the cancer cell surrounding. These results verified that PDAC-DOX can efficiently deliver the drug to cancer cells.



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Figure 4. Celluar uptake of PDAC and PDAC-DOX. A) TEM characterization of celluar uptake PBS or PDAC by MCF-7 cells, the scale bar is 2 µm. B) CLSM of MCF-7 cells after incubation with PBS, free DOX and PDAC at equivalent DOX concentration of 5 µgmL−1 for 0.5 or 2 h, the scale bar is 25 µm.


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Finally, the in vivo imaging and the combined effect of chemotherapy and photothermal effect was explored. MCF-7 xenograft mice tumor model was established first. To compare the in vivo PA intensity between PDAC and PDAP, model mice were injected 100 µL (2 mg/mL) the capsule or nanoparticle in tumor (Fig. 5A). The PA image of pre-injection and post 30min were acquired at the 700 nm laser wavelength and the PA intensity was analyzed by the software (Fig. 5B). It is observed that both the PDAP and PDAC can be used as PAI contrast agents, because their PA intensity all enhanced significantly in comparison to the pre injection. In specific, their PA signal intensity after injection was increased 4.16 ± 0.14 a.u. and 6.56 ± 0.27 a.u., respectively. Moreover, the PA signal in PDAC group was higher greatly than the PDAP group, which was in accordance with the in vitro result and firmly verified the outstanding PAI ability of PDAC. Furthermore, we investigated the PA intensity of the PDAC loaded with anticancer drug (PDAC-DOX) by injecting 200 µL (400 µg) into the tail vein of the tumor-bearing mice. The PA image and intensity of tumor site at different time was assessed. As we can see from the Fig. 5C, D, the PA intensity was enhanced about 8 a.u. after post injection 30min, and proceed increased to the maximum 11.6 ± 0.2 a.u. at 2 h, even at 24 h , the PA intensity was about 2 a.u., higher than the pre-injection. On these grounds we have come to the conclusion that the PDAC-DOX can reach the tumor site quickly by the enhanced permeability and retention (EPR) effect44 and the loading of DOX did not render the PA intensity.



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Figure 5. In vivo PAI. A) Representative optoacoustic images and B) average PA signal changes in transverse section of tumor before and 30 min after intratumor injection of 0.1 mL of PDAP and PDAC with concentration of 2 mg/mL in breast tumor bearing mice (n = 4) at 700 nm. C) Representative photoacoustic images and D)


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average PA signal changes upon intravenous administration of 0.2 mL of PDAC-DOX with concentration of 2 mg/mL in breast tumor bearing mice (n = 4) at 700 nm. All data are expressed as mean ± SD. P values were calculated by ANOVA with Tukey’s post-test: NS (non-significant difference) P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

In order to evaluate the possible of photothermal therapy of the PDAC in vivo, we use the thermal image to investigate photothermal conversion effect of the PDAC (Fig. 6A, B), tumor-bearing nude mice were administrated by PBS, PDAC, PDAP and PDAC-DOX with 50 µL (2 mg/mL), respectively. Then they were tied and exposed to an 808 nm laser with a power of 2 W/cm2 for 5 minute. The tumors’ thermal image was taken every minute by a thermal camera. Obviously, the temperature in PBS group elevated slightly, only 6°C higher than the normal body temperature after irradiation, while the other group elevated significantly, even over 35°C was elevated at the irradiation spot of tumor site. Moreover, the temperature elevated in PDAC group was higher than PDAP group, though they can both used as an photothermal imaging agents. As for the PDAC-DOX, the DOX loading did not affect the photothermal behavior as the temperature of PDAC-DOX (90 µg PDAC) was only little lower than the pure PDAC group (100 µg). To determine if this chemo-photothermal synergistic therapy system would impact the tumors growth in vivo, the tumor-bearing mice were divided into 6 groups. They were named PBS, PBS+Laser, DOX, PDAC-DOX, PDAC+Laser and



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PDAC-DOX+Laser, and then they were intratumorally injected with 50 µL (2 mg/mL) samples. For the laser group, tumor was exposed to an 808nm laser for 5 minutes every day during the first 7 days. The relative tumor volume was calculated every day (Fig. 6C). The tumors of PBS and the laser alone group grew rapidly and steadily, and their relative tumor volumes were approximately 15.5 folders and 13.0 folders. While the DOX and PDAC-DOX group restricted the tumor to some degree, and the anticancer effect of PDAC-DOX was little superior to free DOX, for their relative tumor volume were about 8.4 folders and 6.8 folders, respectively. It is worth noting that only PDAC-DOX+Laser group (chemo-photothermal synergistic therapy) can eradicate the tumor completely, while the tumor in PDAC group was inhibited and tended to grow again at the 11th day. This result was consistent with the photograph taken before and after treatment (Fig. 6E). As we can see that tumor was scabbed in all the laser group, even the DOX group exhibited dark skin for hypoxia. However, the other group can receive a tumor, while nothing can get in PDAC-DOX+Laser group after sacrificed these treating mice (Fig. 6F). Body weight was recorded as it was an indicator for toxicity (Fig. 6D). All the mice was heavier than pre-treating, though DOX group showed a roughly undulate, which was presumably for its’ toxicity. Furthermore, tumors in these group and main organs of PBS and PDAC-DOX+Laser group were dissected and stained with hematoxylin and eosin (H&E) (Fig. 6G). Cells in PBS and laser alone group almost kept intact, while the others were apoptosis to some degree. Especially the PDAC-DOX+Laser group, cells were significantly damaged. Furthermore, there was no distinct difference in the main organs between


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PDAC-DOX+Laser group and control group. This indicates the PDAC-DOX was biocompitable and can be used as a good chemo-photothermal therapy agents.

Figure 6. The chemo-phototherapy synergistic therapeutic effect in vivo. A) IR thermal images of MCF-7 tumor-bearing mice recorded by an IR camera. B)



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Temperature changes of tumors monitored by the IR thermal camera in different groups during laser irradiation. C) The growth of tumors in different groups of mice after various treatments indicated. The relative tumor volumes were normalized to their initial sizes. D) Body weight of mice after various treatments indicated. E) Photographs of tumor-bearing mice before and after treatment. F) The tumors collected from mice at the end of treatments (day 14). G) The H&E-stained tumor slices collected from mice post various treatments. P values were calculated by ANOVA with Tukey’s post-test: NS (non-significant difference) P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

CONCLUSIONS Overall, we first reported a modified way to prepare PDA hollow capsules with the diameter ca. 200 nm, which is smaller than much of the other ways. The capsule was stable and has strong absorbance in NIR region, which makes it have good photothermal performance. Moreover, for the first time, we compare the PA intensity between PDA capsule (PDAC) and nanoparticle (PDAP). The results confirmed that capsules showed better PA imaging ability than the nanoparticles, no matter in vitro or in vivo. In addition, due to the reactivity of the PDA surface, the capsule can load anticancer drug and localize into the cell, and thus destroy the cancer completely through chemotherapy and photothermal therapy. In a word, this research highlights the remarkable potential of PDAC-DOX as a theranostic agent with PAI and chemo-photothermal synergistic therapy.


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ASSOCIATED CONTENT Supporting Information. Calculation of the photothermal conversion efficiency of PDAC; Synthesis and characterization of PDA nanoparticle (PDAP); Zeta potential of PDAC, The photothermal response of the PDAC; SEM image and UV-Vis absorbance of PDAP; The photothermal response of the PDAP; DOX Encapsulation and loading efficiency of PDAC; The H&E-stained slices of major organs in PBS and PDAC-DOX group.

AUTHOR INFORMATION

Corresponding Author * E-mail: [email protected] ORCID Hanqiong Zhuang: 0000-0003-1834-5288

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (31271009, 81271689 and 30900305), the Fundamental Research Funds for the Central Universities (no.20720150087), the Natural Science Foundation of Fujian Province (2012J05066), and the Program for New Century Excellent Talents in



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University, and the Program for New Century Excellent Talents in Fujian Province University.

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Table of Contents Graphic

Polydopamine Nanocapsule: A Theranostic Agent for Photoacoustic Imaging and Chemo-Photothermal Synergistic Therapy Hanqiong Zhuang, Huilin Su, Xuexin Bi, Yuting Bai, Lu Chen, Dongtao Ge, Wei Shi and Yanan Sun*


Polydopamine Nanocapsule: A Theranostic Agent for Photoacoustic

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