Doxorubicin and Indocyanine Green Loaded Hybrid Bicelles for

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Doxorubicin and Indocyanine Green Loaded Hybrid Bicelles for Fluorescence Imaging Guided Synergetic Chemo\Photothermal Therapy Li Lin, Xiaolong Liang, Yunxue Xu, Yongbo Yang, Xiaoda Li, and Zhifei Dai Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00407 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Bioconjugate Chemistry

Doxorubicin and Indocyanine Green Loaded Hybrid Bicelles

for

Fluorescence

Imaging

Guided

Synergetic Chemo\Photothermal Therapy Li Lin, Xiaolong Liang, Yunxue Xu, Yongbo Yang, Xiaoda Li, Zhifei Dai * Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China *

Corresponding author

Tel.: +86-10-62767059; fax: +86-10-62767059. Email: [email protected]

ABSTRACT: Hybrid bicelles have been demonstrated to have great potential for hydrophobic drug delivery. Herein, we report a near-infrared light-driven, temperature-sensitive hybrid bicelles co-encapsulating hydrophobic doxorubicin (DOX) and indocyanine green (ICG) (DOX/ICG@HBs). Encapsulation of ICG into the lipid bilayer membrane of DOX/ICG@HBs results in higher photostability than free ICG. DOX/ICG@HBs exhibited temperature-regulated drug release behavior and significant photothermal cytotoxicity. After tail vein injection, such discotic nanoparticles of DOX/ICG@HBs were found to accumulate selectively at the tumor site and act as an efficient probe to enhance fluorescence imaging greatly. The in vivo experiments

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showed that the DOX/ICG@HBs mediated chemo- and photothermal combination therapy was more cytotoxic to tumor cells than the photothermal treatment or the chemotherapy alone due to the synergistic effect, reducing the occurrence of tumor metastasis. Therefore, DOX/ICG@HBs can act as a powerful nanotheranostic agent for chemo-photothermal therapy of cancer under the guidance of near-infrared fluorescence imaging.

Introduction Nowadays, chemotherapy remains the main cancer treatment option1. However, most commonly used chemotherapeutic drugs are not speciﬁcally toxic to cancer cells and are also toxic to normal tissues they contact so they produce undesirable side effects, such as doxorubicin2. One strategy for reducing the side effect of DOX-chemotherapy and improving therapeutic efficiency is to minimize the drug dose by combining it with other treatment modalities such as photothermal therapy (PTT), photodynamic therapy (PDT), gene therapy and immunotherapy3-13. Furthermore, it has demonstrated that hyperthermia aided the functionality of chemotherapeutic agents in vitro and in vivo7,

14-16

and may reverse drug resistance

17

. In

addition, nanoparticle (NP) anticancer drug delivery promises a disruptive technology to accommodate the multiple drugs or therapeutic modalities to achieve synergistic therapeutic effect, to assess vivo drug biodistribution, to non-invasively visualize the drug release from a given nanoparticle, to predict and monitor therapeutic outcome in real-time18, 19. Therefore, the development of image-guided nanoparticle drug delivery systems has attracted intensive interests from both public and private research institutes and entrepreneurs. As a FDA-approved drug, indocyanine green (ICG) has been widely used for human medical imaging and diagnosis20, 21. It has a maximum absorption peak at around 780 nm in aqueous


2

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solution and 800 nm in the lipid environment22, 23. The fluorescence emission peak of ICG is about 810-830 nm, where fluorescence interference from blood and tissue is minimal24, 25. In addition, ICG is attractive for localized hyperthermia because of its ability to convert excitation energy into heat even in deep tissue7, 21. However, due to the oxidation of ICG double bonds, it degrades rapidly in aqueous solutions, and it can be quickly cleared by the liver (t1/2=2-4 min) because of the combination with plasma proteins26, 27. So application of free ICG is limited because it is prone to aggregation and fluorescence quenching due to protein binding, thermal degradation and photobleaching22,

28, 29

. In order to improve its stability, ICG is often

encapsulated into nanoparticles29-31. It was found that packing ICG with phospholipids may reduce the self-quenching of the dye molecules, hence increase the fluorescence yield

32-34

.

Furthermore, co-encapsulating ICG and anticancer drug in nanoparticles with an optimal dose can integrate real-time tracking, near infrared (NIR) laser-driven drug release, synergistic chemo-photothermal therapy, affording optimized efficacy to inhibit tumor growth through enhanced passive targeting and combinatorial therapeutic mechanisms in tumor intracellular circumstance and extracellular matrix1, 34-36. Organic-inorganic hybrid bicelles (HBs) were comprised of a mixture of short-chain amphiphiles, such as 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, segregated to edge regions of high curvature), and long-chain cerasome-forming lipid (CFL)37,

38

. Such hybrid

bicelle has attracted attention since its higher stability than conventional phospholipid bicelles3943

. Besides, evidences indicate that disc-like nanoparticles display higher circulation time

higher cellular uptake ability42,

46-48

and higher microvascular adhesion49,

50

44, 45

,

than spherical

nanoparticles. Also the experimental data have shown that the disc-like bicelle was very suitable for encapsulation of hydrophobic anti-tumor drugs42,

43, 48

. In order to further minimize the


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chemotherapeutic drug dose, improve the therapeutic efficacy and achieve fluorescence imaging tracking, the present article reported the fabrication of hydrophobic doxorubicin (DOX) and ICG loaded HBs (DOX/ICG@HBs) from CFL and DHPC and 1, 2-distearoyl-sn-glycero-3phosphoethanolamine-N-PEG2000 (DSPE-PEG2000) at the designated molar ratio in combination with sol-gel reaction and self-assembly process (Fig. 1). The ratio of DOX and ICG was optimized to achieve effective NIR fluorescence imaging guided chemotherapy combined with photothermal therapy.

Fig. 1 Schematic representation of DOX/ICG@HBs for fluorescence imaging guided chemophotothermal therapy of cancer. RESULTS AND DISCUSSION


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Characterization of DOX/ICG@HBs. The different ratio of DOX and ICG were loaded into the lipid bilayer of hybrid bicelles which were formed with CFL, DSPE-PEG2000 and DHPC. As shown in Fig. S1, the DOX/ICG@HBs solutions exhibited different color at the different ratio of DOX to ICG. TEM images showed that the ratio of DOX to ICG had no apparent effect on the shape of DOX/ICG@HBs and all of them had the nanodisc shape showing rod like (edgeon, red arrows) and ellipsoidal shape(face-on) (Fig. 2). Then, the hydrodynamic diameter and zeta potential of DOX/ICG@HBs were summarized in Table 1. The diameter of bicelles was about 65 nm and the potential was about -15 mV. No obvious change was observed by varying the ratio of DOX to ICG.

Fig. 2 TEM images of DOX/ICG@HBs with different molar ratio of DOX to ICG, (a) 4:1; (b) 2:1; (c)1:1; (d) 1:2; (e)1:4 The drug loading content (DLC) of ICG and DOX were detected by the traditional spectrophotometric method, respectively. As shown in Table 1, the DLC of DOX increased while the DLC of ICG decreased with increasing the ratio of DOX. The actual ratio of DOX to ICG loaded into the bicelles was calculated to be 4.21:1, 1.96:1, 0.91:1, 0.21:1 and 0.08:1 when


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the feeding ratio was 4:1, 2:1, 1:1, 1:2 and 1:4. The DLC of DOX and ICG was 1.62 and 1.18 at the feeding ratio of 2:1, respectively. The actual ratio of DOX/ICG was 1.96:1, quite similar to the feeding ratio. Nevertheless, the actual ratio of DOX/ICG showed an obvious reduction when the feeding ratio was 1:2 and 1:4. Probably, the ICG molecules produced a certain competition to DOX loading with increasing the ICG loading content. When the feeding ratio of ICG increased from 20% to 80%, the percentage of ICG in the actual package varied from 19.2% to 92.6%. The results provided a good evidence for the design of a bicellar vector with a suitable drug ratio. Table1 Characterization of DOX/ICG@HBs with different molar ratio of DOX to ICG Feeding ratio of DOX/ICG

1:0

4:1

2:1

1:1

1:2

1:4

0:1

Diameter (nm)

63.95 ± 7.90

59.24 ± 9.26

61.22 ± 12.76

65.37 ± 11.12

65.93 ± 8.55

65.85 ± 10.97

63.48 ± 7.52

Zeta potential (mV)

-16.81 ±1.55

-17.05 ± 3.86

-16.99 ± 1.51

-12.40 ± 1.99

-13.28 ± 1.48

-17.17 ± 0.96

-19.30 ± 1.20

DLC of ICG (%)

0

0.67 ± 0.14

1.18 ± 0.69

1.29 ± 0.35

1.64 ± 0.31

2.02 ± 0.72

2.84 ± 0.67

DLC of DOX (%)

2.54 ± 0.58

1.98 ± 0.51

1.52 ± 0.19

0.82 ± 0.12

0.27 ± 0.09

0.11 ± 0.03

0

4.21:1

1.96:1

0.91:1

0.21:1

0.08:1

Actual ratio of DOX/ICG

The UV-vis-NIR spectra of DOX/ICG@HBs (Fig. 3a) displayed a broad absorption band around 490 nm and 784 nm, attributed the characteristic absorption peak of DOX and ICG, respectively. Upon excitation wavelength of 480 nm and 745 nm, the emission peak of DOX/ICG@HBs appeared around 590 nm and 810 nm, consistent with free DOX and free ICG, respectively (Fig. 3b and c). The data indicated that both the DOX and ICG kept their optical properties after encapsulation into the bicelles.


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Fig. 3 DOX/ICG@HBs with different ratio of DOX to ICG: (a) UV-vis-NIR absorption spectra, (b) fluorescence spectra upon excitation at 480nm wavelength; (c) fluorescence spectra upon excitation at 745 nm wavelength. Temperature elevation and photostability of DOX/ICG@HBs. ICG is an FDA approved dual-functional agent for optically mediated diagnostic and photothermal therapy. To evaluate the photothermal efficiency of DOX/ICG@HBs, the temperature change was monitored upon 808 nm NIR laser irradiation at 2 W/cm2 for 10 min by keeping the concentration of DOX/ICG@HBs at 0.13 µg/mL and varying the feeding ratio of DOX/ICG (Fig. 4a). No obvious temperature change was observed for water and DOX/ICG@HBs with the feeding ratio of DOX/ICG (1:0 and 4:1). In contrast, aqueous dispersion of DOX/ICG@HBs with the feeding ratio of DOX/ICG (2:1, 1:1, 1:2, 1:4) achieved temperature elevation of 13.6℃, 17.9℃, 38.8℃ and 53.2℃, respectively. The influence of concentration of DOX/ICG@HBs on photothermal


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properties was also evaluated. As shown in Fig. 4b, the temperature elevation of DOX/ICG@HBs (2:1 of DOX/ICG) was 11.4℃, 13.6℃, 17℃, 27.3℃ and 28.7℃ at the ICG concentration of 0.1 µM, 0.2 µM, 0.4 µM, 0.8 µM and 1 µM, respectively. This verified the excellent photothermal efficiency of DOX/ICG@HBs. At an ICG concentration of 0.2 µM or more, the aqueous dispersions of DOX/ICG@HBs can be easily heated up to above 42℃, which is sufficient to kill tumor cells. It indicated that DOX/ICG@HBs could act as NIR-light absorbers for photothermal tumor therapy.

Fig. 4 The temperature change of aqueous dispersion of DOX/ICG@HBs with different feeding molar ratios of DOX/ICG (a), different ICG concentrations (b), and three cycles of laser on/off upon 808 nm NIR laser irradiation at 2 W/cm2 for 10 min. To compare the photostability of DOX/ICG@HBs with the free ICG, the dispersion of DOX/ICG@HBs and free ICG were irradiated with NIR laser for 10 min (laser on), followed by


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naturally cooling to room temperature with no laser irradiation for 30 min (laser off). Then this cycle was repeated for three times to investigate the photostability of ICG molecules before and after loading into DOX/ICG@HBs. As shown in Fig. 4c, the temperature elevation of 25.5℃, 17.7℃ and 13℃ for DOX/ICG@HBs and 25.3℃, 13.3℃ and 7.3℃ for free ICG was achieved after sequential laser on/off cycles, respectively. The higher elevated temperature indicated that ICG molecules loaded into the lipid bilayer of DOX/ICG@HBs show enhanced photostability than free ICG. From the results obtained above, the DOX/ICG@HBs with the feeding ratio of DOX/ICG (2:1) was selected as the follow-up experimental study since it is almost similar to the actual ratio of DOX/ICG, and the photothermal effect would be sufficient to kill tumor cells. Drug release behavior of DOX/ICG@HBs. The drug release behavior of DOX/ICG@HBs was investigated in PBS solutions at 37℃, 42℃, 50℃ (Fig. 5). It was found that the drug release rate increased with increasing the temperature. Only 49.94% of DOX was released from DOX/ICG@HBs at 37℃ after 48 h. When the temperature increased to 42℃ and 50℃, the percentage of the released drug from DOX/ICG@HBs increased to 70.85% and 85.85%, respectively. It indicated that DOX/ICG@HBs was heat-sensitive and a higher temperature caused a higher drug release rate. As we know, DHPC was a kind of heat-sensitive phospholipid. So, it is easily understood that the DHPC included DOX/ICG@HBs so temperature can be used to control the release behavior of the drug encapsulated within lipid vesicles. The heat generated in the process of photothermal therapy would accelerate the release of drug encapsulated into DOX/ICG@HBs to realize


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effective chemotherapy combined with photothermal therapy. DOX/ICG@HBs can achieve a more accurate drug delivery by applying the NIR laser to regulate drug release behavior.

Fig. 5 In vitro release behavior of DOX from DOX/ICG@HBs at different temperature. The cell experiment in vitro. Cellular uptake is an important characterization of drug carrier in biological application. In this study, the cellular uptake of DOX/ICG@HBs was observed by confocal laser scanning microscopy (CLSM) after incubating MDA-MB-231 cells with DOX/ICG@HBs for 4 h at 37℃. As shown in Fig. 6a, a large amount of drug gathered around the nucleus (blue represents DAPI labeled nuclei, red represents doxorubicin fluorescence). This confirmed the effective internalization of DOX/ICG@HBs into the cells and the free DOX was released into the nuclei to kill tumor cells. To further investigate the combined effects of photothermal cytotoxicity and chemotherapy of DOX/ICG@HBs, the MDA-MB-231 cells were incubated with different concentration of DOX/ICG HBs with or without 3 min laser irradiation at 808 nm. Then, the cell viability was quantitatively evaluated by MTT assay after further incubation for 24 h, 48 h and 72 h. As shown in Fig. 6 (b, c, d), the cytotoxicity increased with increasing the drug concentration and


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incubation time in spite of no laser irradiation, suggesting the effect of DOX/ICG@HBs acting as chemotherapeutic drug. When exposing the MDA-MB-231 cells to DOX/ICG@HBs in combination with NIR laser irradiation, obvious decrease of cell viability was observed at the same concentration compared to that with no laser irradiation. It indicated that the combined chemo- and photothermal therapy was more cytotoxic to cancer cells than the photothermal therapy or the chemotherapy alone.

Fig. 6 (a) CLSM images of cellular uptake of DOX/ICG@HBs at 37℃. The photothermaltoxicity (3min irradiation at 808 nm) and chemo-toxicity of DOX/ICG@HBs to MDA-MB-231 cells after incubation for 24h (b), 48 h (c), 72h (d). (*p≤0.05, **p≤0.01)


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In vivo fluorescence imaging and biodistribution study. The biodistribution and tumor accumulation of DOX/ICG@HBs were evaluated by whole body NIR fluorescence imaging approach. DOX/ICG@HBs was intravenously injected into MDA-MB-231 tumor-bearing BALB/c nude mice. The variations of fluorescence intensity at tumor were monitored within 24 h. Fig. 7 showed the fluorescence signal and intensity distributions as a function of time for DOX/ICG@HBs and free ICG delivered systemically via intravenous (i.v.) injections. Fig. 7a showed that no significant accumulation at the tumor region after 0.5 h injection of free ICG and DOX/ICG@HBs. The fluorescence intensity of the tumor region gradually increased with time, indicating accumulation of free ICG and DOX/ICG@HBs at tumor site. Nevertheless, DOX/ICG@HBs showed much higher fluorescence intensity at the tumor site than free ICG group after injection for 4 h, 12 h and 24 h, respectively. The quantitative analysis showed that the averaged tumor fluorescence intensity of DOX/ICG@HBs was 0.88, 2.61, 2.85 and 3.24 times higher than that of free ICG, respectively (Fig. 7b). The data further confirmed that the loading ICG into DOX/ICG@HBs could significantly reduce clearance from blood circulation and DOX/ICG@HBs could accumulate at tumor site through enhanced permeation retention (EPR) effect. In addition to this, the tumor fluorescence signals of DOX/ICG@HBs increased with increasing post-injection time from 0 to 12 h, then began to fall away. Therefore, 12 h postinjection was chosen as the optimal time point for photothermal therapy in order to obtain the maximum efficacy. To further evaluate the biodistribution of the injected nanoparticles, the major organs were collected at 24 h post-injection. As shown in Fig. 7c and 7d, the fluorescence signal of the tumor in the DOX/ICG@HBs group was much stronger than that of free ICG. In addition, the fluorescence imaging of major organs suggested that DOX/ICG@HBs obviously promoted the


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accumulation of ICG in tumor. The averaged intensity of DOX/ICG@HBs in tumor was 1.9 times higher than that of free ICG. Compared with DOX/ICG@HBs, free ICG exhibited weaker fluorescence intensity in major organs likely due to its faster clearance rate or rapid fluorescence quenching in vivo.

Fig. 7 In vivo fluorescence imaging and biodistribution of MDA-MB-231 tumor-bearing BALB/c nude mice after intravenously injection of free ICG and DOX/ICG@HBs: (a) timelapse NIR fluorescence images; (b) quantification analysis of tumor fluorescence intensities after injection for different time; (c) NIR fluorescence images of major organs after 24 h injection; (d) quantification analysis of organ fluorescence intensities after 24 h injection. Data shown as mean standard deviation (SD), n=6.


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In vivo chemo-photothermal therapy and biocompatibility. The in vivo chemo-photothermal therapy was carried out on 4T1 tumor-bearing BALB/c mice with different treatments (Fig. 8). When the tumor volume reached approximately 200 mm3, the mice were randomly divided into six groups: saline only, saline + laser, free DOX, free ICG + laser, DOX/ICG@HBs only, DOX/ICG@HBs + laser (n=6 for each group). According to the results of NIR fluorescence imaging, NIR laser irradiation (808 nm, 2W/cm2, 10 min) was carried out at 12 h after i. v. injection of saline, free ICG and DOX/ICG@HBs (150 µL; ICG dose: 100 µg/mL; DOX dose: 140 µg/mL). The in vivo therapy efficacy was evaluated by measuring the tumor volume every 3 days after different treatments. It was found that tumor volume increased rapidly from original 200 mm3 to approximately 1962 mm3, 1787 mm3, 1580 mm3 and 1395 mm3 within 24 days in the mice treated with saline only, saline + laser, free DOX and DOX/ICG@HBs only, respectively. These results indicated that laser irradiation or free DOX or DOX/ICG@HBs alone at the experimental condition did not cause potential destructive effects. For free ICG + laser group, the size of tumor decreased with time in the first 12 days after treatment; after reaching to a tumor size of 53 ± 9 mm3, the tumor restore growth and increased to about 433 ± 38 mm3 in the next 12 days, suggesting an insufficient hyperpyrexia for ablating tumor. In marked contrast, DOX/ICG@HBs plus laser irradiation caused a complete tumor ablation, leaving a black scar at the original tumor site after 6 days and gradually disappeared (Fig. 8c). Scars fell off after 42 days of treatment (Fig. S2). After 24 days of different treatments, the tumors was removed and weighed. As shown in Fig. 8b, the mean tumor weight was 2.27 ± 0.78 g, 1.71 ± 0.50 g, 1.29 ± 0.04 g, 0.76 ± 0.29 g and 1.09 ± 0.09 g after treatment with saline, saline + laser, free DOX, free ICG + laser and DOX/ICG@HBs, respectively. Excitingly, the combined chemo-photothermal therapy with DOX/ICG@HBs plus laser irradiation was more cytotoxic to tumor cells than the


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photothermal treatment or the chemotherapy alone due to the synergistic effect. The hyperpyrexia can enhance significantly the sensitivity of the cancer cells toward DOX, resulting in the improved drug efficacy. Moreover, the photothermal effect may also induce a rapid drug release to reach a high effective DOX concentration in the tumor. We further evaluated the in vivo biocompatibility of DOX/ICG@HBs by body weight change and histopathologic examination. The body weights of mice were recorded every 3 days after i. v. injection of different solutions. As shown in Fig. 8d, body weight had no significant change among all of the groups within 24 days of observation. To assess the toxicity of DOX/ICG@HBs to major tissues in vivo, the organs including heart, liver, spleen, lung and kidney of the treated mice were collected at 24 days after treatment. The hematoxylin and eosin (H&E) staining showed no noticeable organ damage or inflammation (Fig. 8e). It indicated that DOX/ICG@HBs did not cause evident side effects to the vital organs which is a critical factor for drug vector. It is reported that 4T1 tumor-bearing BALB/c mice was prone to lung cancer metastasis 54. The H&E staining results showed that the lung cancer metastasis (red arrow) were observed for saline group, free DOX group and group of free ICG with laser irradiation, but no obvious tumor metastasis was found the group treated with DOX/ICG@HBs combined NIR laser irradiation. The results demonstrated that DOX/ICG@HBs can effectively prevent the occurrence of tumor metastasis. It is reported that 4T1 tumor-bearing mice tend to produce tumor-related splenomegaly 55 so the relative spleen size was observed after different treatments. As shown in Fig. S3, different degrees of splenic enlargement can be found in all the groups except for the group treated with DOX/ICG@HBs combined laser irradiation. It indicated that the DOX/ICG@HBs mediated chemo-photothermal therapy could effectively reduce the likely


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cancer-related splenomegaly. Therefore, DOX/ICG@HBs can act as a powerful chemophotothermal therapeutic agent for in vivo therapy of cancer.

Fig.8 In vivo chemo-photothermal therapy and biocompatibility of DOX/ICG@HBs on 4T1 tumor-bearing BALB/c mice upon the 808 nm laser irradiation (2W/cm2, 10 min). (a) Tumor volume change with time. Data shown as mean SD, n=6. (b) The tumor weight after different treatments for 24 days. (c) The representative photographs of 4T1 tumor-bearing BALB/c mice after treatment for 0 day, 6 days, 15 days and 24 days. (d) Body weight of mice in groups after


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different treatments; (e) H&E stained images of the heart, liver, spleen, lung and kidney from treated mice after 24 day post-injection. Data shown as mean SD, n=6. CONCLUSION In order to minimize the chemotherapeutic drug dose, improve the therapeutic efficacy and achieve fluorescence imaging tracking, the present article reported the fabrication of hydrophobic DOX and ICG loaded discotic nanoparticles of DOX/ICG@HBs from cerasomeforming lipid, DHPC and DSPE-PEG2000 at the designated molar ratio in combination with solgel reaction and self-assembly process. DOX/ICG@HBs exhibited temperature-regulated drug release behavior and significant photothermal cytotoxicity. It was found that the DOX/ICG@HBs could release more than 70.85% and 85.85% drug at 42 ℃ and 50 ℃ , respectively. After systemic administration of DOX/ICG@HBs, such discotic nanoparticles of DOX/ICG@HBs were found to accumulate selectively at the tumor site and act as an efficient probe to enhance fluorescence imaging greatly. As a result, DOX/ICG@HBs in combination with NIR laser irradiation not only enable the delivery of higher concentrations of chemotherapy drugs directly to the tumor, achieve maximum therapeutic efficacy and minimal side effects, reducing the occurrence of tumor metastasis and splenomegaly, but also avoid the damage to the healthy tissues causing by systemic administration of drugs under the fluorescence imaging guidance. Therefore, DOX/ICG@HBs in combination with NIR laser irradiation hold great potential for efficient local chemo-photothermal therapy of cancer. The DOX/ICG@HBs platform also has the capability to be employed to a variety of drug products. MATERIALS AND METHODS


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Materials. Indocyanine green was obtained from Shenyang Bomei New Medical Technology Development Co. Ltd.. DHPC was purchased from Avanti Polar Lipids Inc. Doxorubicin hydrochloride (DOXHCl) was obtained from Beijing Huafeng United Technology. DSPEPEG2000 was got from Shanghai Advanced Vehicle Technology Pharmaceutical L. T. D.. Methylthiazolyl tetrazolium (MTT) was from Sigma-Aldrich. All chemicals and reagents were commercially obtained and used without further purification. Preparation of DOX/ICG@HBs. CFL was synthesized according to the literature51. Hydrophobic DOX was prepared by treating DOXHCl with triethylamine according to the report methods.52 The DOX/ICG@HBs were prepared from the lipid mixture of CFL, DSPEPEG2000 and DHPC at the molar ratio of 6.65:0.35:2. The lipids were dissolved in chloroform, followed by addition of hydrophobic DOX. ICG was dissolved in methanol and added into the lipid solutions. Then, the solvent was removed by using a vacuum rotary evaporator at 45℃ and the thin lipid film formed on the wall of the flask. Then, ultrapure water was added into the flask and hydrated for 30 min~1 h at 55~65℃. After ultrasonic water bath for 10 min, the ultrasonic waves of probe-type sonicator were applied to the resultant dispersion for 5 min at the output amplitude of 30. The obtained hybrid bicelles were incubated overnight at room temperature. Characterization of DOX/ICG@HBs. The morphology of hybrid bicelles was examined by transmission electron microscopy (TEM, Hitachi, H-7650). The hydrodynamic diameter and zeta potential of DOX/ICG@HBs were analyzed with a 90Plus/BI-MAS DLS analyzer (Brookhaven Zeta PALS instruments). UV-vis-NIR absorption spectra and fluorescence spectra of DOX/ICG@HBs

were

tested

by

UV-vis

spectrophotometer

(Varian

4000)

and

fluorospectrophotometer (Cary Eclipse, Varian).


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Evaluation of drug loading content. The amount of DOX loaded in the DOX/ICG@HBs was measured spectrophotometrically53. Firstly, the freshly prepared nanodiscs of DOX/ICG@HBs (0.1 mL) were suspended in 0.9 mL HCl solution (1 M). After vigorous stirring overnight, the fluorescence intensity of the DOX solutions was measured at an excitation wavelength of 480 nm and an emission wavelength of 590 nm. The amount of DOX was determined using fluorescence spectrophotometer and the corresponding standard calibration curve. The drug loading content (DLC %) was evaluated by following formulas:

DLC % =

Amount of drug in bicelles ×100% Total amount of bicelles

The amount of ICG loaded in the DOX/ICG@HBs was measured with the same method. Temperature elevation and photostability of DOX/ICG@HBs. The DOX/ICG@HBs of different concentrations were put into cuvettes (volume of 3.0 mL). Then, the 808 nm NIR laser (2 W/cm2) light was delivered through the cuvette to irradiate for 10 min. The temperature of solutions was measured by a digital thermometer probe every 10 seconds. The water was used as control. For detecting the photostability of DOX/ICG@HBs, free ICG solutions and DOX/ICG@HBs solutions were irradiated with 808 nm NIR laser light for 10 min, followed by naturally falling to room temperature without laser irradiation for 30 min. The temperature of solutions was recorded every 10 seconds and the cycle repeated for three times. In vitro release behavior of DOX from DOX/ICG@HBs. In order to investigate the effect of temperature on drug release behavior, 1 mL DOX/ICG@HBs solution was sealed in a dialysis bag (molecular weight cut off 8000~14000 Da, Viacase, American) and immersed in 25 mL of


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PBS (pH 7.4) which was preheated to 37℃, 42℃ and 50℃, respectively. At predetermined time intervals, 2 mL of sample was taken and replaced with the same amount of PBS. The amount of DOX released was determined by a fluorescence spectrophotometer. All experiments were carried out in triplicate. Cellular uptake and chemo-photothermal toxicity of DOX/ICG@HBs in vitro. MDA-MB231 cells were cultured in 6-well plate for 24 h. Then, the cells were incubated with 2.0 mL DOX/ICG@HBs at 37℃ for 4 h. After staining with DAPI, the cells were imaged using a confocal laser scanning microscope (Leica TCS SP5 CLSM, Heidelberg, Germany). The chemo-photothermal toxicity of DOX/ICG@HBs was evaluated on breast cancer cell, MDA-MB-231. The cells were seeded into 96-well plate (1×104 cells/well) in 200 µL complete DMEM medium. After incubation with different concentrations of DOX/ICG@HBs for 4 h, the cells were irradiated with 808 nm NIR laser for 3 min and cultured for different time. The viability of cells was detected by MTT method. Tumor fluorescence imaging in vivo and biodistribution analysis. DOX/ICG@HBs and free ICG were injected into tumor bearing mice through tail vein and fluorescence images were taken by IVISTM-200 (Xenogen Corp.) after injection at 0.5, 4, 12, and 24 h. A filter set (Ex=745 nm, Em=840 nm) was used for the measurement of ICG. The mice were kept on the imaging stage under anesthetized condition with 2.5% isoflurane gas in oxygen flow during the imaging process. After injection at 24 h, the mice were sacrificed and main organs (heart, liver, spleen, lung, kidney, brain and tumor) were collected for imaging and semi-quantitative biodistribution analysis of nanoparticles in vivo.


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In vivo chemothermal, photothermal and chemo-photothermal therapy. The mice were divided into eight groups (six per group) that were iv injected with 150 µL saline with or without laser irradiation for 5min, free DOX (67 µg/mL) with or without laser irradiation, free ICG (55 µg/mL) with or without laser irradiation, and DOX/ICG@HBs (containing 67 µg/mL DOX and 55 µg/mL ICG) with or without laser irradiation. The tumor volumes and changes in body weight of mice were recorded every three days. To further detect the in vivo chemo-photothermal effect, mice were sacrificed after 30 days and representative organs including heart, liver, lung, spleen, kidney and tumor were collected, fixed in formaldehyde solution, embedded in paraffin, followed by section, stained with hematoxylin and eosin (H&E) and observed by optical microscope. All the animal experiments were approved by institutional animal use committee and carried out ethically and humanely. ACKNOWLEDGMENTS This work was financially supported by National Key Research and Development Program of China (No. 2016YFA0201400), State Key Program of National Natural Science of China (No. 81230036), National Natural Science Foundation of China (No. 81371580) and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 81421004). SUPPORTING INFORMATION The Supporting Information is avaliable free of charge on the ACS Publications website at DOI:


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Figures for photographs of DOX/ICG@HBs solutions, representative photographs of 4T1 tumor-bearing mice after treatment with free ICG +laser and DOX/ICG@HBs +laser for 42 days, and representative spleen photographs of mice after 24 days of treatment. AUTHOR INFORMATION Author Contributions All authors have given the approval to the final version of the manuscript.

Corresponding Author *

Zhifei Dai

Tel.: +86-10-62767059; fax: +86-10-62767059. Email: [email protected] ORCID Li Lin: 0000-0003-3840-4265 Notes The authors declare no competing finanicial interest.

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TOC graphic:

Hybrid bicelles co-encapsulating hydrophobic doxorubicin (DOX) and indocyanine green (ICG) (DOX/ICG@HBs) act as a powerful nanotheranostic agent for chemo-photothermal therapy of cancer under the guidance of near-infrared fluorescence imaging.


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Doxorubicin and Indocyanine Green Loaded Hybrid Bicelles for

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