Hydrophobic IR-780 Dye Encapsulated in cRGD-Conjugated Solid

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Hydrophobic IR-780 dye Encapsulated in cRGD-conjugated Solid Lipid Nanoparticles for NIR Imaging-guided Photothermal Therapy Ye Kuang, Kunchi Zhang, Yi Cao, Xing Chen, Kewei Wang, Min Liu, and Renjun Pei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16705 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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ACS Applied Materials & Interfaces

Hydrophobic IR-780 dye Encapsulated in cRGD-conjugated Solid

Lipid

Nanoparticles

for

NIR

Imaging-guided

Photothermal Therapy

Ye Kuang1, Kunchi Zhang2*, Yi Cao1, Xing Chen3, Kewei Wang1, Min Liu1* and Renjun Pei1*

1

CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, Suzhou

Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China. 2

Shanghai University of Medicine & Health Sciences, Shanghai 200120, China

3

Public Health of Guangxi Medical University, Nanning 530021, China

Keywords: IR-780 dye, Solid Lipid Nanoparticles, c(RGDyK), Near-infrared, Imaging-guided Therapy, Photothermal Therapy

1

ACS Paragon Plus Environment


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ABSTRACT: It is extremely demanded to enhance the accumulation of near-infrared theranostic agents in the tumor region, which is favorable to the effective phototherapy. Compared with indocyanine green (a clinically applied dye), IR-780 iodide possesses higher and more stable fluorescence intensity and can be utilized as an imaging-guided PTT agent with laser irradiation. However, lipophilicity and short circulation time limit its applications in cancer imaging and therapy. Moreover, solid lipid nanoparticles (SLNs) conjugated with c(RGDyK) was designed as efficient carriers to improve the targeted delivery of IR-780 to the tumors. The multifunctional cRGD-IR-780 SLNs exhibited a desirable monodispersity, preferable stability and significant targeting to cell lines over-expressing αvβ3 integrin. Additionally, the in vitro assays such as cell viability and in vivo PTT treatment denoted that U87MG cells or U87MG transplantation tumors could be eradicated by applying cRGD-IR-780 SLNs under the laser irradiation. Therefore, the resulted cRGD-IR-780 SLNs may serve as a promising NIR imaging-guided targeting PTT agent for cancer therapy.

2


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1. INTRUDUCTION Imaging-guided therapy means integrating imaging diagnosis into therapy. However, how to meet the precise treatment requirements, 1,2 improve the therapeutic effect and reduce the undesirable side-effects, have become the focus of attention. 3-5 In order to endow theranostic platforms with an efficient imaging-guided therapy, some vital factors

are

usually

contained

in

design.

Typically,

the

biocompatibility,

physiologically stability, and highly specificity to tumor tissues are the foremost focus when fabricating the theranostic systems.

4,6-8

Recently, molecular imaging

technology has become the main tool in medical diagnosis, including near-infrared (NIR) fluorescence imaging, positron emission tomography (PET), X-ray computed tomography (CT), magnetic resonance imaging (MRI), and ultrasonic imaging. NIR fluorescence imaging, a technique with sensitive, non-invasive, non-radiant, is usually applied for real-time observing of biological information in vivo. 9,10 In addition, NIR fluorescence imaging also possesses relatively low photon tissue attenuation and auto-fluorescence.

11,12

Yuan et al. fabricated a self-assembled PEG-IR-780-C13

micelle, which can be utilized for NIR imaging that can verify the effective organ accumulation through fluorescence signal. imaging materials,

14,15

13

Compared with other NIR fluorescence

NIR dyes (ICG, IR-783, IR-820, and IR-780 iodide) are

excellent theranostic materials which are comprised of NIR fluorescence imaging and photothermal therapy.

5,16,17

Moreover, NIR dyes are capable of fluorescent imaging

in vivo and provide better guided theranostic effect with high sensitivity because of evidently less background from tissue auto-fluorescence. 3, 18 3



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Photothermal therapy (PTT), which uses light absorbing materials that are transported to tumors before the treatment and causes thermal ablation of cancer cells with photo irradiation, has become an important treatment modality. 19,20 Compared to other treatment methods, PTT possesses the superior selectivity and minor invasiveness. 3 Several tumor models have been considered using various PTT agents, such as NIR dyes,

9, 21,22

gold nanomaterials,

23

graphene oxide,

20

and carbon

nanotubes. 24 Specially, NIR dyes are able to absorb radiation in a transparent window for organisms (wavelength range 700-900 nm).

18,25,26

However, the poor aqueous

stability, quick exclusion from the body, and inadequate target specificity confines its further biological applications.

9,27

Thus, various nanomaterials have been

investigated to address this deficiency by encapsulating IR-780. For example, polymeric micelles, 3,8,25,28 human serum albumin nanoparticles, 19 silica nanoparticles 29

and heparin-folic acid NPs 9 have been utilized to deliver IR-780 to the tumor area.

In the last decade, due to the low toxicity, the good stability and biocompatibility, low cost and ease of scale-up preparation and excellent loading efficiency of hydrophobic drugs, solid lipid nanoparticles (SLNs) have been considerably studied in drug-delivery systems. 30,31 Although the preparation method of SLNs is similar to oil-in-water emulsion, a solid lipid is used to replace the liquid lipid of the emulsion during the synthesizing process and thus offers many advantages such as sufﬁcient protection of entrapped agents.

27

Banerjee et al. encapsulated paclitaxel (PTX) into

Tyr-3-octreotide-modified SLNs to enable dual-targeting chemotherapy of tumor cells and tumor neovasculature, which enhanced the anticancer efficacy of PTX. 32 4


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Therapy effectiveness of PTT is confined by the possible nonspecific heating of healthy tissues. It was reported the stability and tumor accumulation of NIR dye could be enhanced by utilizing of various nanocarriers. However, it is still difficult for these nanocarriers to accumulate into the specific tumor area since the interstitial fluid pressure is altered in most of solid tumors. 33 Recently, due to the excellent specificity for αvβ3 integrin positive cells and tumor angiogenic vessels, cycle RGD peptide was modified on many nanomaterials to improve their targeting specificity. conjugated c(RGDyC)-peptide onto the surface of Gd- and

99

34

Yang et al.

mTc-labeled AuNPs

(RGD@AuNPs-Gd-99mTc), the as-prepared nanoprobes obviously improved the cellular uptake ratio and thus enhanced radio sensitization.

35

Luo et al. fabricated

ultrasmall Fe3O4 nanoparticles with RGD peptide and confirmed its targeting towards U87MG cells overexpressing αvβ3 integrin.

33

It was reported non-targeted SLNs,

RGD-SLNs and blocked RGD-SLNs were prepared to encapsulate near-infrared quantum dots for live animal imaging in the paper of Wu’s group.

36

They concluded

that the targeted SLNs showed a massive specific retention in tumor vascular. Recently, the same group has found the highest tumor accumulation with prolonged tumor retention by conjugating 1% cRGD on the surface of SLNs.

37

In addition,

Dong et al. developed RGD/PEG-PUE-SLN by loading puerarin in RGD modiﬁed and PEGylated SLNs to enhance bioavailability of PUE. 38 These hybrid nanoparticles displayed evident targeting specificity to αvβ3-overexpressing cells in vitro or biological systems. 39,40 Different from the above RGD-SLNs used for delivering chemotherapeutic drugs 5



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or quantum dots, we reported a convenient method to encapsulate IR-780 in cRGD-conjugated SLNs (cRGD-IR-780 SLNs), which was applied as the targeted NIR imaging-guided photothermal therapy system for U87MG xenograft tumors.33 cRGD-IR-780 SLNs was characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and ultraviolet-visible (UV-vis) spectrophotometry. Further, the PTT performance of cRGD-IR-780 SLNs was evaluated in vitro and in vivo. Finally, the PTT effect of cRGD-IR-780 SLNs was also evaluated by injection of cRGD-IR-780 SLNs into U87MG tumor-bearing mice.

2. EXPERIMENTAL SECTION 2.1.

Materials.

Palmitic

acid

(PA), IR-780

iodide,

1-(3-(dimethylamino)

propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and phosphotungstic acid was purchased obtained from Sigma-Aldrich. c(RGDyK) peptide (cyclo (Arg-Gly-Asp-d-Tyr-Lys)) was obtained from GL biochem (Shanghai) Ltd.. GIBCO Life Technologies provided the fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM). 1, 2-distearoyl-snglycero-3phosphoethanolamine-N-[carboxy (polyethylene glycol)-2000] (DSPE-PEG2000 -carboxylic acid) and all the other reagents were acquired from domestic suppliers.

2.2. Synthesis of cRGD-IR-780 SLNs. The cRGD-IR-780 SLNs were fabricated using a slightly modified solvent-diffusion method as described earlier. 30,41 Concisely, 400 mg PA and IR-780 (500 µL, 10 mg/mL in trichloromethane) were dissolved in 6


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methanol (10 mL) to form lipid phase at 400 rpm and 75 oC. In order to conjugate targeting ligands onto the SLNs surface, DSPE-PEG2000-carboxylic acid (125 mg) was added into the above lipid phase. Afterwards, 40 mL aqueous solution comprising of P407 (200 mg) and Tween 80 (200 mg) in ultrapure water was prepared. Finally, 10 mL of the lipid phase containing DSPE-PEG2000-carboxylic acid was gradually added into 40 mL of P407-Tween 80 mixture at 400 rpm and 75 oC for 5 min. Then, the mixed fluid was combined into 500 mL ultrapure water at 1000 rpm and 4 oC for at least 15 min. After removing any large particles via filtrating, the filtrate with solidified HOOC-IR-780 SLNs was concentrated to 10 mL through centrifugal filter devices (10K MWCO, Millipore Corp.) at 5000 rpm and 4 oC. Finally, the filtrate was lyophilized with D-mannitol to obtain the final product (HOOC-IR-780 SLNs). The IR-780 SLNs without DSPE-PEG2000-carboxylic acid was prepared according to the same procedure above. EDC (100 µL, 1 mM) and NHS (100 µL, 1.5 mM) were added into 10 mL HOOC-IR-780 SLNs at 4 oC for 1 h. Furthermore, the obtained solution was added dropwise into 5 mL cRGD solution (1 mg) under vigorous stirring at 4 oC for 6 h. Afterwards, the unreacted cRGD was eradicates by numerous washes using centrifugal filter devices (10K MWCO, Millipore Corp.) at 4 oC. The obtained product (cRGD-IR-780 SLNs) was then lyophilized with D-mannitol and stored at 4 o

C.

2.3. Characterization of cRGD-IR-780 SLNs. The mean diameter, size distribution 7



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and zeta potential of IR-780 SLNs, HOOC-IR-780 SLNs and cRGD-IR-780 SLNs were investigated using DLS (Zetasizer Nano, Malvern instrument, UK). 0.2% (w/v) cRGD-IR-780 SLNs was dripped on a 230-mesh carbon-coated copper grid and then

10 µL phosphotungstic acid solution (10%, w/v) was dropped onto the copper grid to stain cRGD-IR-780 SLNs. The particle geometry of cRGD-IR-780 SLNs was characterized using TEM (HT7700, Hitachi, Japan). The absorptions spectra of free IR-780 and cRGD-IR-780 SLNs were recorded using an UV/vis spectrophotometer (Lambda 25, PerkinElmer, USA). The content of the IR-780 in IR-780 SLNs, HOOC-IR-780 SLNs and cRGD-IR-780 SLNs was calculated according to the standard curve. The loading content and encapsulation efficiency of IR-780 were amounted by: 9,19 IR-780 loading content (%) =(weight of IR-780 in SLNs/weight of SLNs)×100% IR-780 encapsulation efficiency (%) = (weight of IR-780 in SLNs /weight of total added IR-780)×100%

2.4 Release of IR-780 from cRGD-IR-780 SLNs at different pH 1 mL HOOC-IR-780 SLNs containing 0.05% (w/v) sodium azide was added into a dialysis bag, which was immersed in a flask of 100 mL containing 20 mL of PBS at pH 5.5 or pH 7.4. Hydrochloric acid and sodium hydroxide were used to control the pH value. A bath reciprocal shaker at 80 rpm and 37oC was performed over 48 h. At a certain time point, 100 µL of the sample containing the released IR-780 was measured by UV/vis absorption spectra according to the standard curve. 100 µL of fresh PBS 8


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was immediately added into the flask to recover the solution volume. The cumulative percentage of the released IR-780 was calculated by： PIR-780 (%) =(Cumulative weight of released IR-780 in dissolution medium/Total weight of IR-780 in fresh HOOC-IR-780 SLNs)×100%

2.5. Photothermal properties of cRGD-IR-780 SLNs aqueous solution. The photothermal properties of the free IR-780 and cRGD-IR-780 SLNs aqueous solution under laser irradiation in a 24-well plate were determined with a thermocouple needle. 1 mL IR-780 or cRGD-IR-780 SLNs at different concentrations (15 mg/L and 50 mg/L IR-780) was put into the wells. Then, the NIR 808 nm laser was used to irradiate every well from the top at 1 W/cm2. The temperature change in every well was recorded for 5 min.

2.6. In vitro cellular uptake. U87MG cells were seeded into 24-well plates with glass slices at a density of 5×104 cells per well. When the cells grew to 60% confluency, the cells were washed with PBS. Furthermore, 1 mL fresh medium containing (a) Free IR-780, (b) HOOC-IR-780 SLNs, (c) cRGD-IR-780 SLNs or (d) cRGD-IR-780 SLNs, but the cells were pretreated with free cRGD for 1h was added, respectively. The concentration of IR-780 in each sample was maintained at 4 mg/L. After 0.5 h, the cells were washed thrice with PBS and dual-stained with Hoechest 33258 and lyso-tracker for 15 min. Finally, the cells were washed thrice with PBS and then observed by confocal laser scanning microscope (CLSM, Leica TCS SP5, Germany). 9



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The excitation wavelength of IR-780 is located at 633 nm.

2.7. Cytotoxicity of cRGD-IR-780 SLNs. The cytotoxicity of free IR-780, HOOC-IR-780 SLNs and cRGD-IR-780 SLNs with laser irradiation were performed against U87MG cells through MTT assay. U87MG cells were cultured with free IR-780, HOOC-IR-780 SLNs or cRGD-IR-780 SLNs at two different concentrations (2.5 or 5.0 mg/mL IR-780) for 2 h, followed by 808 nm laser irradiation (0.5 W/cm2) for 5 min each well. The cells are treated with free IR-780, HOOC-IR-780 SLNs or cRGD-IR-780 SLNs except laser irradiation and the cells without any treatment were both taken as controls. The wells were washed twice with PBS after removing the cultured medium and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5 mg/mL, 10 µL) was subsequently added. After incubation at 37 °C for 4 h, the remaining media were removed and 150 µL DMSO was added to dissolve the intracellular blue-violet formazan crystals. The optical density (OD) value was calculated with a cell imaging microplate reader (Cytation 3, BioTek) at 570 nm wavelength. The cell viability (%) was counted as a percentage of the control culture value by: (ODsample-ODblank/ODcontrol-ODblank) × 100%. When the cells reached 60% confluency, the medium was replaced with fresh medium containing cRGD-IR-780 SLNs (4 mg/L of IR-780) and HOOC-IR-780 SLNs (equivalent to material of cRGD-IR-780 SLNs). After 1 h incubation, the plate was placed on the Labnet Accublock digital dry bath incubator so that the wells remained at 37 oC before the utilization of the laser. The cells were irradiated with a 10


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0.5 W/cm2 808 nm laser for 5 min and then washed thrice with PBS after 2 h. Furthermore, then stained with a mixed solution of calcein AM and PI for 15 min. After washing with PBS for thrice, these stained cells were observed using the fluorescence microscope. The excitation wavelength was set at 490 nm for calcein AM and 545 nm for PI.

2.8. In vivo imaging and biodistribution analysis. Female athymic nude mice (5 weeks, 20 g) were obtained from Nanjing Sikerui Biological Technology Co. LTD and acclimated for at least 1 week. The suitable water and standard pellet diet were used. All animal experiments were performed in compliance with the relevant laws and institutional guidelines. U87MG cells were subcutaneously inoculated into the nude mice. When the volume of tumor grew to about 100-200 mm3, free IR-780, HOOC-IR-780 SLNs or cRGD-IR-780 SLNs (1 mg/kg for IR-780, n=5) was intravenously injected into these mice. The corresponding NIR images were captured at 0 h, 0.5 h, 1 h, 2 h, 24 h, and 48 h using a vivo imaging system (DXS4000pro, Kodak, USA). During 24 h post injection, the organs including heart, liver, spleen, lung, kidney and tumor were collected from the nude mice and analyzed by the Kodak vivo imaging system. The excitation wavelength of IR-780 was set at 704 nm and 740-950 nm for the emission spectrum.

2.9. Temperature measurements and photothermal therapeutic effect in vivo. To directly evaluate the photothermal effect of free IR-780, HOOC-IR-780 SLNs, and 11



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cRGD-IR-780 SLNs in vivo, a visual IR thermometer (Fluke Corporation) was applied to detect the temperature change of mice under laser irradiation. The tumor-bearing mice were randomly divided into six groups (n=5) and intravenously injected with free IR-780, HOOC-IR-780 SLNs or cRGD-IR-780 SLNs. The dose was maintained at 1 mg/kg for IR-780. Meanwhile, mice were performed as a control by treating with physiological saline. Afterwards, tumors on the mice treated with free IR-780, HOOC-IR-780 SLNs or cRGD-IR-780 SLNs were irradiated with 808 nm wavelength laser (0.5 W/cm2) at 2 h post injection, while the rest were then treated the same as above after 24 h injection. Temperature changes and the IR images were acquired at 0, 0.5, 1, 2, 3, 4, and 5 min. Furthermore, the changes of tumor size and body weight were then recorded to evaluate the PTT efficacy. Briefly, when the volume of tumor grew to 100-200 mm3, the mice were intravenously injected with free IR-780, IR-780 SLNs, or cRGD-IR-780 SLNs (1 mg/kg for IR-780, n=5). The same volume of physiological saline (n=5) was injected as the control groups. 24 h post-injection, tumors on the mice were irradiated with 808 nm wavelength laser (0.5 W/cm2, 5 min). Tumor diameters were measured by a vernier caliper, and the weights of mice were recorded every 3 days. The tumor volume (V) was estimated by: V=D×d2/2 (where D is the longest diameter of tumor and d is the shortest diameter of tumor).

2.10. Statistical analysis. Mean±SD values were utilized for the expression of data. Student’s t test was used for statistical analyses of data. Differences of P

Hydrophobic IR-780 Dye Encapsulated in cRGD-Conjugated Solid

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