Biodegradable Natural Product-Based Nanoparticles for Near-Infrared

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Biological and Medical Applications of Materials and Interfaces

Biodegradable Natural Product-Based Nanoparticles for NearInfrared Fluorescence Imaging-Guided Sonodynamic Therapy Xiuli Zheng, Weimin Liu, Jiechao Ge, Qingyan Jia, Fuchun Nan, Ying Ding, Jiasheng Wu, WenJun Zhang, Chun-Sing Lee, and Pengfei Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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ACS Applied Materials & Interfaces

Biodegradable

Natural

Product-Based

Nanoparticles

for

Near-Infrared Fluorescence Imaging-Guided Sonodynamic Therapy Xiuli Zheng,a,b Weimin Liu,a,b Jiechao Ge,a,b* Qingyan Jia,a,b Fuchun Nan,a,b Ying Ding,a,b Jiasheng Wu,a Wenjun Zhang,c Chun-Sing Lee,c and Pengfei Wanga,b* a Key Laboratory of Photochemical Conversion and Optoelectronic Materials and CityU-CAS Joint Laboratory of Functional Materials and Devices, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, People's Republic of China b School of Future Technology, University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China c Center Of Super-Diamond and Advanced Films (COSDAF) & Department of Materials Science and Engineering, City University of Hong Kong, Hong Kong SAR, People's Republic of China Keywords: natural product, sonosensitizer, biodegradability, fluorescence imaging, sonodynamic therapy

Abstract Natural products show the high potential clinical translation due to their specific biological activities and molecular structure diversities. Sonosensitizers that originate from natural products play a crucial role as anti-inflammatory and anti-cancer agents. Herein, hypocrellin derivative nanoparticles (APHB NPs) were constructed for synchronous near-infrared fluorescence (NIR FL) imaging and sonodynamic therapy (SDT) for deep-seated tumor in vivo. The prepared APHB NPs exhibit excellent water solubility, FL in the NIR region, and effective reactive oxygen species generation under ultrasound stimulation. Furthermore, the APHB NPs show excellent biocompatibility, suitable biodegradation rate, and enhanced tumor accumulation. Therefore, the APHB NPs exhibit promising clinical potential as novel safe and precise NIR FL imaging and SDT agent for deep-seated tumor therapy.

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1. INTRODUCTION Reactive oxygen species (ROS)-mediated tumor therapy (e.g., sonodynamic therapy (SDT) and photodynamic therapy (PDT)) is attracting considerable attention because of its high selectivity, non-invasiveness, and negligible drug resistance.1-7 PDT, which takes advantage of ROS through singlet-to-triplet transition from a photosensitizer that induces death of cancer cells, is an emerging alternative cancer therapy method to traditional cancer radiotherapy and chemotherapy.8-13 Recently, the near-infrared (NIR) light-responsive photosensitizer approach to solve penetration depth issues have been proposed, and a great progress has been achieved, however, the acquired PDT efficiency is still need to be improved for deep-seated tumor.14 SDT is a non-invasive therapeutic form based on PDT and can be applied to therapy of deep tissue sites. In SDT, sonosensitizers generate ROS that causes cell apoptosis and necrosis by low-energy ultrasound activation instead of light energy activation.15,16 Compared with light irradiation applied to PDT, ultrasound has an excellent tissue-penetrating capability in soft tissues, which allows it to reach deep-seated tumor and locally activate the sonosensitizers.17-20 In the past few decades, various organic sonosensitizers, including porphyrins,21,22 methylene blue,23,24 and rose bengal,25 have been proposed and achieved great progresses. Considering the water dispersibility and potential toxicity of the traditional sonosensitizer, it is also need to develop new efficient/biocompatible sonosensitizers for clinical translation. The in-depth application of SDT is limited by not only deficient visualization of the distribution, delivery, and metabolism but also deficient pinpoint and compositive


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assessment of SDT effect toward tumor tissue. Thus, in the past decade, different modal imaging-guided SDT nanotheranostics that contain imaging and therapy on a single nanoparticles have been widely proposed to achieve the precision nanomedicine.17,22 NIR Fluorescence (FL) imaging has great application prospects due to its minimal tissue scattering and autofluorescence.26-28 FL imaging can monitor in vivo sonosensitizers distribution and tumor detection in real time by means of spectrum-resolved and contrast enhancement technology.29-31 Despite the great progress in this area, explore new multifunctional nanotheranostics with important potentials in NIR FL imaging-guided SDT are still needed. Compared with artificial dyes, natural products, such as porphyrins, hypocrellins, and curcumin, show high potential clinical translation due to the diversity and complexity of their molecular structure.32,33 Natural products often exhibit specific biological activities and high selectivity because of their ability to regulate multiple signaling pathways.34,35 To date, natural products and their derivatives are increasingly applied not only in PDT but also in SDT. Sonosensitizers that originate from natural products play a crucial role as anti-inflammatory and anti-cancer agents. Among them, hypocrellins, metabolites present in a traditional Chinese medicinal fungus Hypocrella bambusae, are obtaining considerable attention due to their high singlet oxygen quantum yields, excellent biocompatibility, and rapid metabolism from the normal tissues.36,37 Nevertheless, the water insolubility and ineffective absorption in NIR region of HB greatly limit their its clinical application for anti-cancer treatments.38 For decades, substantial efforts have been conducted for improving their



intrinsic issues by chemical modification or construct novel nanostructure.39-42 Unfortunately, the as-obtained hypocrellin derivatives and hypocrellin nanostructures scarify the biological activity or increase pharmacokinetic complexity. Therefore, preparing a multifunctional and new hypocrellin derivative nanoplatform with NIR FL imaging and SDT for deep-seated cancer therapy is still urgently required. In the last few years, we have synthesized several hypocrellin derivatives, applied them to phototherapy, and achieved excellent results.43 We found that such hypocrellin derivatives can be used as sonosensitizers for SDT. Thus, in the present work, we modified hypocrellin B with 1,2-diaminopropane to form hypocrellin derivatives (APHB). Then, hypocrellin derivative nanoparticles (APHB NPs) were fabricated using PEG-PLGA and APHB through self-assembly. As expected, the prepared APHB NPs exhibit excellent water solubility, FL in the NIR region, and effective ROS generation under ultrasound stimulation. Moreover, the APHB NPs show excellent biocompatibility, suitable biodegradation rate, and enhanced tumor accumulation. The APHB NP could be served as a NIR FL imaging agent and show effective SDT for deep cancer treatment in vivo. Therefore, the APHB NPs exhibit promising clinical potential as novel safe and precise NIR FL imaging and SDT agent for deep-seated tumor therapy. 2. EXPERIMENTAL SECTION 2.1. Synthesis of APHB. 1,2-diaminopropane (10 mL) and hypocrellin B (500 mg) were added in two-necked flask, and stirred at 55 ℃ for 10 hours under dark condition. The solvent was then


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eliminated with reduced pressure. The black powder products were dealt with the column chromatography with dichloromethane to obtain APHB. 1H NMR (400 MHz, CDCl3): δ 16.89 (s, 1H), 11.88 (s, 1H), 6.41 (s, 1H), 6.29 (s, 1H), 6.18 (s, 1H), 5.19 (s, 1H), 4.15 (s, 3H), 4.01 (s, 3H), 3.95 (s, 3H), 3.74-3.19 (m, 3H), 2.30 (s, 3H), 1.61 (s, 3H), 1.45 (d, 3H).

13C

NMR (100 MHz, CDCl3) δ 204.57, 186.49, 184.59, 168.98,

168.50, 165.82, 144.98, 144.94, 142.99, 141.84, 136.63, 135.14, 124.40, 123.70, 123.04, 122.15, 119.37, 118.89, 116.23, 107.43, 106.68, 103.64, 101.54, 61.28, 56.33, 56.00, 46.18, 45.43, 32.76, 27.87, 24.53, 23.15. TOF-EI calcd for [C32H28N2O7] m/z 552.1897 (M), found 552.1893. 2.2. Preparation of APHB NPs. Under sonication in the ice bath for 3 min, 4 mL of PVA aqueous solution (0.1%) was added into 2 mL of CHCl3 solution, which contains 2 mg of APHB and 14 mg of PEG-PLGA (Mw: 1000-1000). 40 mL PVA aqueous solution (0.5%) was added into solution mentioned above and stirred for 3 min, and then pass the vacuum distillation eliminate the CHCl3 to acquire APHB NPs. 2.3. Measurement of sonodynamic performance. HeLa cells were cultured in cell culture dishes and APHB NPs (100 μg mL−1) were exchanged to the cells and incubated another 12 h. Next, the APHB NPs were removed and washed three times with PBS. Subsequently, cells were stained with 1 mg mL−1 of 2,7-dichlorofluorescein diacetate (DCFH-DA, 1 μL) for 5 min and then under ultrasound stimulation (Frequency: 1 MHz, Duty cycle: 20%, Intensity: 0.6 W cm−2) for 0.5 min or 1 min. The control group is the cells were incubated with free



DCFH-DA. FL images were gained by a Nikon C1si laser scanning confocal microscope (LSCM). 2.4. SDT in vitro. To determine the SDT efficacy, the 4T1 cells or HeLa cells were cultured in the 96-well plates with 104 per well and cultured overnight. Subsequently, the cells medium was exchanged for different concentrations of APHB NPs (0, 5, 20, 50, and 100 μg mL−1) for 12 h and were treated using ultrasound (0.6 W cm−2, 1 MHz, 1 min). Then, incubated for another 12 h, the cell viability was determined by MTT assays. The samples were analyzed using similar approaches except for the ultrasound to determine the cell toxicity assays in dark. 2.5. Animal models. The animal studies in this work are all comply with the experimental animal law and with approval from the China Committee for Research and Animal Ethics. To establish tumors in aged 4 weeks nude female mice (15–20 g), the anesthetized mice was subcutaneously injected with 50 μL of 4T1 cells (2 × 106, PBS) into the buttock of mice. Tumors were allowed to reached approximately 50 mm3 before in vivo experiments. 2.6. FL imaging in vivo. The mice were injected with APHB NP solution (100 μL, 2.0 mg mL−1) though tail vein and the FL imaging was acquired with In Vivo Imaging System (FX Pro, Kodak, Japan). Scans were carried out at 0, 3, 7, 12, 24, and 48 h post-injection. After 48 h intravenously (i.v.) injection, the mice were sacrificed and the FL intensities of major


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organs and tumor were measured. 2.7. SDT in vivo. For in vivo SDT, we randomly divided the mice into three groups (n=5 in per group). Among them, a group of mice were i.v. injected with APHB NP solution (100 μL, 2.0 mg mL−1). At 7 h post-injection, the tumors were blocked with a 1 cm-thick pork tissue and then ultrasound stimulated (0.6 W cm−2, 1 MHz, 5 min, SDT). The mice were i.v. injected with APHB NP solution only (APHB NPs only) or PBS under ultrasound stimulation (0.6 W cm−2 + PBS of ultrasound) as control groups. Tumor volume was calculated using the following formula: volume = (tumor length) × (tumor width)2/2. 2.8. H&E Staining. Tumors at 4 h after ultrasound stimulation were extracted and fixed with 4% formaldehyde, treated conventionally implanted in a paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histological analysis. 3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of APHB NPs. Figure 1a shows the synthetic route of the APHB. After purification, the structures of APHB was identified by mass spectrometry, 1H NMR spectrum and 13C NMR spectrum. The fluorescence quantum yield and maximum molar extinction coefficient of APHB in DMF were measured to be 1.3% and 32000 M−1 cm−1 (Figure S1), respectively. To obtain water soluble APHB for biological systems, PEG-PLGA was added through self-assembly via the hydrophobic-hydrophobic interactions



between the APHB and PEG-PLGA. Transmission electron microscope (TEM) image indicated that APHB NPs has an average diameter of 82.1 ± 23.2 nm (Figure 1b). The scanning electron microscopy (SEM) image in Figure 1c confirms that the APHB NPs is sphericity and has a diameter of 75.5 ± 24.5 nm. In addition, the APHB NPs exhibit an average hydration diameter of around 98.5 nm, as shown in dynamic light scattering results (Figure 1d). This finding strongly agrees with the TEM and SEM results. The zeta potential of the APHB NPs is −26.4 mV in water, −23.1 mV in DMEM, and −27.5 mV in PBS (Figure 1e). The suitable hydrated diameter and the negatively charged surface of APHB NPs could passive targeting of solid tumors by enhanced permeability and retention effects.44,45 As shown in Figure 1f, the APHB NPs performed a main absorption at around 630 nm and a NIR emission peak at 690 nm under 580-nm excitation, which provide the APHB NPs with capability of NIR FL imaging. Electron spin resonance (ESR) technique was applied to test ROS production of the APHB NPs. In ESR measurements, 2,2,6,6-tetramethyl-4-piperidone (TEMP) was used as ROS trappers. The intensity of this trapper increases with the growth of the ultrasound stimulation time (0.6 W cm−2) (Figure 1g). In addition, the free TEMP inconsiderably changes (Figure S2), which confirms the APHB NPs could effective generate ROS under ultrasonic stimulation. A standard fluorescent indicator, DCFH-DA, was applied to further evaluate the ROS production of the APHB NPs in vitro. Figure 1h shows that the DCFH-DA FL intensifies with the increase in stimulation time in DCFH-DA + APHB NP groups, whereas free DCFH-DA as a control exhibits no FL. This result


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indicates that APHB NPs can generate ROS efficiently under ultrasound stimulation. All the results show that the water-dispersible APHB NPs can be served as NIR FL imaging and SDT agents because of their NIR emission and effective ROS generation.

Figure 1. (a) The synthetic route of APHB. (b) TEM image and (c) SEM image of APHB NPs. (d) The DLS analysis of APHB NPs in aqueous solution. (e) Zeta potential of APHB NPs in different solwents. (f) Absorption spectrum and FL spectrum of APHB NPs in DMEM. (g) Time-dependent ESR signals of APHB NPs under ultrasound stimulation (0.6 W cm−2). (h) FL images of ROS generation tested by DCFH-DA in HeLa cells at different ultrasonic stimulation time. Scale bar: 20 µm.

3.2. Biodegradation behavior of the APHB NPs The biodegradable behavior of the APHB NPs in the PBS was assessed. Previous reports have indicated that the ester linkage of the PLGA in PBS degrades by hydrolysis to segments with less molecular weights, oligomers and monomers and then to carbon dioxide and water.38, 39 The SEM images show that the morphology of



the nanoparticles has only slight morphological changes at 6 days. Further incubation results in collapse of most nanoparticles at 12 days. After 20 days, disrupted nanoparticles can be observed due to the complete biodegradability of PLGA (Figure 2a). As shown in Figure 2b, the color of the APHB NPs solution lightens with the increase in incubation time. This phenomenon indicates that the hydrophobic APHB is aggregated and precipitated in a physiological environment. Accordingly, the absorbance of APHB NPs decreases as incubation time prolongs (Figure 2c). Hence, the distinct biodegradability of the APHB NPs could be metabolism from the normal tissue without damage after cancer therapy.

Figure 2. (a) SEM images, (b) the corresponding photographs and (c) Absorbance of the APHB NPs at different time degradation in PBS.

3.3. In vitro FL imaging and SDT. Figure S3a,b show the confocal FL images at excitation wavelengths of 638 nm. The red FL of the APHB NPs internalizes into the HeLa cells, and stained in the cytoplasm, and this phenomenon indicates cellular internalization of APHB NPs and presumably by endocytosis. The cells were stained by APHB NPs and Hoechst 33342 (nucleus dye) or Dio(DioC18(3)) (cell membrane dye), from which we can see clearly


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that the APHB NPs are primary located in the cytoplasm (Figure S4). This result indicates that APHB NPs can serve as a NIR FL imaging agent for guided SDT. To investigate the SDT effect of APHB NPs in vitro, the ability of cellular uptake was investigated. HeLa cells were incubated with APHB NPs for different time intervals (0.5, 1, 3, 7, and 12 h), and imaged with LSCM. As shown in Figure 3a,b, the intracellular FL increases with the increase in incubation time. This condition indicates the prefect cellular uptake of APHB NPs. Next, the cytotoxicity induced by APHB NPs was determined by MTT assay. No evident cytotoxicity is observed in HeLa cells and 4T1 cells even at 100 μg mL−1 (Figure 3c and Figure S5). The result indicates that APHB NPs alone unable cause cancer cell apoptosis and further confirms their excellent biocompatibility and low cytotoxicity. However, approximately 90% cell death is found at the concentration of 100 μg mL−1 under ultrasound stimulation (0.6 W cm−2, 60 s). By contrast, no cell death is observed under ultrasound (0.6 W cm−2) treatment without APHB NPs (Figure 3d). This phenomenon suggests the excellent SDT efficacy on cancer cells. Calcein AM (green, live cells) and PI (red, dead cells) co-staining was used to testify their excellent SDT efficacy. In the ultrasound only (0.6 W cm−2) and APHB NP only groups, no cell death is found on account of all the cells show green FL. However, in the SDT group, many cells are destroyed when incubated with APHB NPs (100 μg mL−1) and stimulated with ultrasound (0.6 W cm−2). When time is prolonged to 60 s, nearly all cells are destroyed, as demonstrated by the red FL, these results agree well with the MTT analysis of SDT (Figure 3e). All the results prove that the APHB NPs



can be used as a sonosensitizer for SDT cancer treatment.

Figure 3. (a) FL imaging of the cell uptake of APHB NPs in living HeLa cells at different incubation time. Scale bar: 20 μm. (b) FL intensities of the cell uptake of APHB NPs at different incubation time. Cell viability of HeLa cells (c) incubated with APHB NPs in the dark or under ultrasound stimulation (0.6 W cm−2) and (d) under ultrasound treatment alone at different power densities. (e) FL images of HeLa cells incubated with APHB NPs (100 μg mL−1) after ultrasound stimulation (0.6 W cm−2) using calcein AM/PI staining. Scale bar: 100 μm.

3.4. In vivo FL imaging. The excellent in vitro results suggest the feasibility of APHB NPs for in vivo FL imaging. As the tumors increased to around 50 mm3, the 4T1 tumor-bearing nude mice were i.v. injected with APHB NPs. Then, the FL images were obtained at various time intervals. The FL intensities of the APHB NPs at the tumor site rapidly increase and attain a plateau after 7 h i.v. injection, and the FL intensity evidently


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decreases at 48 h post-injection (Figure 4a,b). The ex vivo major organs and tumor were obtained and imaged to more precisely assess the FL intensity within tissues after 48 h i.v. injection (Figure 4c). The APHB NPs mainly accumulate in tumor, and the tumor shows stronger FL intensity than other organs, as shown in Figure 4d. This phenomenon indicates that i.v. injected APHB NPs accumulate at the tumor through passive targeting. All the FL imaging results indicate that the APHB NP can be served as a superior NIR FL imaging agent for guiding SDT tumor treatment in vivo. Meanwhile, the optimal time point for SDT is 7 h post-injection of APHB NPs.

Figure 4. (a) FL images of the mice and (b) FL intensities of the tumor at different time points post-injection of APHB NPs. Ex vivo (c) FL images and (d) FL intensities of major organs and tumor at 48 h post-injection of APHB NPs.

3.5. In vivo SDT. We further investigated whether the APHB NPs can be used as a sonosensitizer for deep-tissue tumor treatment in vivo. We blocked tumor on mice with a 1 cm-thick pork tissue to imitate internal tumor located inside the body during different treatments to demonstrate the advantage of SDT in vivo for deep tumor tissue



treatment (Figure 5a). Three groups of 4T1 tumor-bearing mice were applied in experiments, as follows: APHB NPs only, PBS + ultrasound, and APHB NPs with ultrasound (0.6 W cm−2, 1.0 MHz, SDT group). Among them, in the treatment group, the mice were i.v. injected with APHB NPs (100 μL, 2.0 mg mL−1) and radiated by ultrasound (0.6 W cm−2) for 5 min after 7 h i.v. injection. On the contrary, the control group was administered with APHB NPs without stimulation (APHB NPs only) or treated with PBS and exposed to the ultrasound stimulation (PBS + ultrasound). The variation of the tumor volume was sostenuto monitored for 14 days. No evident inhibited effect of the tumor is observed in control groups (Figure 5b,c). This result suggests that either the APHB NPs or ultrasound does not affect the tumor growth. However, the SDT group shows a considerably decreased tumor growth rate, which demonstrates the excellent capability of SDT for deep-tissue tumor treatment. To further evaluate the SDT efficacy of APHB NPs, tumor slices were stained and investigated by H&E at 4 h after SDT. Figure 5d shows the tumor slices in the SDT group with remarkable nucleus and cytoplasm separation, which indicates that the cancer cells are dramatically damaged in the SDT group. The tumor slices have no noticeable cell damage in the control groups. Thermographic analysis of tumor sites temperature using IR thermal mapping apparatus was measured during ultrasound stimulation. No significant temperature changes in tumor, showing no thermal damage toward the tumor (Figure S6). All the results indicate that the APHB NPs have effective SDT for deep cancer treatment in vivo. The side toxicity of APHB NPs was assessed by analyzing the results of tissue H&E-stained slices from healthy mice


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post-injection with APHB NPs. There are little inflammation and cell apoptosis in major organs, indicating that the APHB NPs had no side toxicity to mice, as shown in Figure S7.

Figure 5. (a) Schematic of in vivo experimental design. Tumors were blocked pork slice (1.0 cm) during the different treatment. (b) Tumor growth curves of mice after different treatments (n = 5; P < 0.05 for each group). (c) The photographs of mice after different treatments (n = 5). (d) H&E-stained slices of the tumor. Scale bar: 50 μm.

4. CONCLUSION In this study, we designed and constructed APHB NPs as novel versatile theranostic agent for NIR FL imaging-guided SDT for deep-seated tumor in vivo. The prepared APHB NPs exhibit excellent water solubility, FL in the NIR region, and effective ROS generation under ultrasound stimulation. The APHB NPs also show excellent biocompatibility, suitable biodegradation rate, and enhanced tumor



accumulation. Therefore, this study not only provides novel APHB NPs for highly efficient NIR FL imaging and SDT, but also considerably broadens the biomedical applications of hypocrellins for efficient deep-seated tumor therapy. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/((please add manuscript number)) UV absorption value of calibration curve of APHB; ESR signals of TEMP; FL images, Z-stack image and colocalization images of APHB NPs; relative viability of 4T1 cells incubated with APHB NPs in the dark or under ultrasound stimulation; IR camera recorded the tumor temperature during ultrasound stimulation; and H&E-stained images of major organs. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected] ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 61720106014), the International Partnership Program of Chinese Academy of Sciences (No. GJHZ1723), and the Instrument Developing Project of the Chinese Academy of Sciences (No. YJKYYQ20170015). REFERENCES (1) Li, X.; Peng, X.-H.; Zheng, B.-D.; Tang, J.; Zhao, Y.; Zheng, B.-Y.; Ke, M.-R.; Huang, J.-D. New Application of Phthalocyanine Molecules: from Photodynamic Therapy to Photothermal Therapy by Means of Structural Regulation Rather than Formation of Aggregates. Chem. Sci. 2018, 9, 2098-2104.


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Biodegradable Natural Product-Based Nanoparticles for Near-Infrared

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