Materials. Z-His-Obzl (ZHO) was purchased from Bachem (Bubendorf, Switzerland). Indocyanine green (ICG), IR 806, IR 140, Rhodamine B (RhB), ZnCl2, methanol were purchased from Sigma-Aldrich Inc.. Bovine serum albumin (BSA) was purchased from Solarbio Biotechnology Co. Ltd.. Dulbecco's Modified Eagle's Medium (DMEM), heat-inactivated fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (PBS), trypsin-EDTA, and penicillin-streptomycin were purchased from BioLegend Co.. Other materials were purchased from Beijing Chemical Co. Ltd. unless otherwise noted.
Preparation of two-photon absorption nanoprobes (TPA NPs). ICG NPs were typically prepared as follows: 10 µL dimethyl sulfoxide(DMSO) solution of Z-His-Obzl (ZHO, 264 mM) was mixed with 985 µL aqueous solution of indocyanine green (ICG, 1.41 mM), followed by the addition of 5 µL ZnCl2 solution (100 mM) solution into the above mixed solution. The final concentration of ZHO, ICG and Zn2+ was 2640 µM, 139 µM and 500 µM, respectively. Based on the opalescence of the samples, nanostructures formed immediately and the pH value was 6.5, approximately. The obtained ICG NPs were stored at 4°C in the dark for 24 h. The aged NPs were centrifuged with a centrifugal force (RCF) of 9391 g for 10 min and were dispersed in pure water.
RhB NPs and IR 806 NPs were prepared with the same method. The final concentration after preparation of ICG, RhB and IR 806 was kept the same.
IR 140 NPs were prepared as follows: 10 µL DMSO solution of ZHO (264 mM) was mixed with 10 µL DMSO solution of IR 140 (14.1 mM), then 975 µL pure water and 5 µL Zn2+ (100 mM) solution were added to the stirred solution to obtain IR 140 NPs with a pH value of 6.5. The aging and washing procedures of IR 140 NPs were the same as for ICG NPs.
Morphological and spectral characterization. An aliquot of a suspension of two photon absorption (TPA) NPs was spread on a silica plate and dried in vacuum at room temperature. S-4800 (HITACHI, Japan) with 10 kV accelerating voltage was used for scanning electron microscopy (SEM) measurements. Transmission electron microscopy (TEM) was performed by a JEOL JEM-1011 at 100 kV with a drop of sample carefully applied to a carbon-coated copper grid and dried in vacuum. The size distribution and zeta potential were determined using a Malvern dynamic laser scattering (DLS) instrument. CLSM images were acquired by an Olympus FV500 microscope equipped with a Ti: Sapphire oscillator laser (Mai Tai, USA). TPA NPs were excited by a femtosecond (fs) pulse laser (λ = 808 nm) and the signal channels used were 495–540 nm and 575–630 nm. UV-Vis absorption spectra were recorded using a Shimadzu UV-2600 spectrophotometer with a quartz cuvette of 1 mm path length. A fluorescence spectrometer (Hitachi F-4500) equipped with Xenon lamp as excitation source was used to measure the fluorescence (808 nm) of the samples with a quartz cuvette of 1.0 cm. The fluorescence spectra based on TPA were measured on a home-made optical platform. For excitation a fs Ti: Sapphire oscillator laser (λ = 808 nm, 100 fs, SP-5W, Spectra physics, USA) equipped with a short-pass filter (730 nm) was applied onto the sample in a 1.0 cm quartz cuvette, and spectra were recorded by an Omni-λ300 monochromator/spectrograph (Zolix instruments Co., Ltd., China) equipped with a PMT (PMTH-S1C1-CR131, Zolix instruments). Electron spin resonance (ESR) measurements were conducted on a spectrometer (ESP-300, Bruker) equipped with 808 nm laser at room temperature. 2,2,6,6-Tetramethylpiperidinooxy (TEMPO) agent was added into the samples to capture the singlet oxygen signal. Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a TENSOR 27 FTIR spectrometer (BRUKER), with the samples prepared using the KBr pellet method. The molecular weight mass charge ratio (m/z) of ICG NPs were determined by ESI mass spectrometry.
Entrapment efficiency (EE) of ICG NPs. The suspension solution of ICG NPs was centrifuged at a centrifugal force (RCF) of 18,400 for 20 min, and the precipitate was suspended in methanol. The absorption intensity of ICG was measured by UV-Vis absorption spectroscopy and the corresponded concentration of ICG molecules was determined by calibration absorption curves with a range of known standard concentrations. ZHO concentration were analyzed by high-pressure liquid chromatography (HPLC) using calibration absorption curves with a range of known standard ZHO concentrations. The drug loading efficiency (LE) and encapsulation efficiency (EE) were calculated according to the following formula:
$$LE=\frac{weigℎt of ICG in tℎe precipitate}{weigℎt of tℎe precipitate}\times 100\%$$
$$EE=\frac{weigℎt of ICG in tℎe precipitate}{weigℎt of ICG added}\times 100\%$$
Two-photon absorption (TPA) cross section measurement. TPA cross sections were measured with rhodamine B (RhB) in methanol as a reference. The fs Ti: Sapphire oscillator laser was used. The fluorescence spectra based on TPA were recorded in a 1.0 cm quartz cuvette, where the ICG NPs concentration was kept as 20 µM in methanol (sample) and RhB NPs concentration was kept as 2 µM in methanol (reference)[1]. The experimental fluorescence excitation and detection wavelengths (400 nm-700 nm) of samples and references were kept constant. The TPA cross section of the probes was calculated at each wavelength according to the following formula:
$${\delta }_{sample}={\delta }_{reference}\frac{{\varnothing }_{\left(reference\right)}{I}_{\left(sample\right)}{C}_{\left(reference\right)}{\eta }_{\left(sample\right)}^{2}{P}_{\left(reference\right)}^{2}}{{\varnothing }_{\left(sample\right)}{I}_{\left(reference\right)}{C}_{\left(sample\right)}{\eta }_{\left(reference\right)}^{2}{P}_{\left(sample\right)}^{2}}$$
Where I is the integrated fluorescence intensity, C is the concentration, η is the refractive index, ∅ is the quantum yield, and P is the incident power on the sample, subscript “reference” indicates reference and “sample” indicates sample, respectively.
Mechanism demonstration of photo-oxidation enhanced emission. A suspension of ICG NPs with a concentration of 20 µM was added to a 1.0 cm quartz cuvette with a sealed cap. The solution was saturated with high-purified argon (Ar) or oxygen (O2) gas. A portable dissolved oxygen measurement apparatus (JPBJ-608) was used to detect the dissolved oxygen concentration. ICG NPs solution in air atmosphere was used as control. Alternatively, glutathione (GSH, 5mM), H2O2 and H2O were added to the ICG NPs solution to mimick the oxygen-depleted, oxygen-saturated, and control groups, respectively.
Computational simulation. The molecular dynamics (MD) simulations were performed using Gromacs package (Version 5.1.2). The general AMBER force field (GAFF) was used to model the ICG and ZHO molecules. To obtain the GAFF force field parameters, the geometry optimization and molecular electrostatic potential of both the molecules were achieved through density functional theory (DFT) calculations with B3LYP functional[2] and 6-31G(d) basis set using Gaussian 09 (Revision D.01) package[3]. The Antechamber package was used to compute atomic charges according to the restrained electrostatic potential (RESP) formalism. Water molecules were modeled using the tip3p potential. We performed MD simulations on a selected model system consisting of 2 ICG and 32 ZHO molecules in a water box sized 8.2 ⋅ 8.2 ⋅ 8.2 nm3. The systems were firstly minimized utilizing the conjugate-gradient algorithm, and then equilibrated through running for 500 ps NVT simulations followed by 500 ps NPT simulations. Production runs in the NPT ensemble were then run for 150 ns at 298 K and 1.0 bar, employing the leapfrog algorithm with a time step of 2 fs to integrate the equations of motion. The electrostatic forces were treated with the particle-mesh Ewald approach. Both the cutoff values of van der Waals forces and electrostatic forces were set to be 1.2 nm. The LINCS algorithm was utilized to preserve bonds.
Physiological stability and biocompatibility of ICG NPs. The physiological stability test was conducted by incubating ICG NPs at 37°C for 24 h in 10-fold (v/v) dilutions of ICG NPs suspensions with FBS. Morphological changes were characterized by TEM and DLS.
Biocompatibility of NPs was verified by a standard MTT assay. Human breast cancer cells (MCF-7) were seeded in 96-well plates (1×104 cells well− 1) and incubated for 24 h. Then, the media were replaced with 200 µL of DMEM containing different concentrations of the RhB NPs and ICG NPs. After incubation for another 24 h, the cells were washed three times with PBS, infused with fresh media and the cell viability was examined.
In vitro TPA fluorescence imaging. In vitro TPA fluorescence imaging was conducted as follows. MCF-7 cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) containing 10% FBS. They were seeded onto Petri dishes (5×103 cells well− 1) and incubated for 24 h at 37°C in humidified ambiance of 5% CO2. Then, the medium was replaced with 2 mL of DMEM medium containing ICG NPs and RhB NPs (equivalent concentration of 25.8 µM) and 5% glucose solution (control group). Then cells were incubated for 24 h at 37 oC. The intracellular localization of ICG was determined using an Olympus FV500 microscope equipped with a Ti: Sapphire oscillator laser. The fluorescence of ICG NPs was excited by a fs Ti: Sapphire oscillator laser (λ = 808 nm) and the corresponding signal channel was located in 495–540 nm. The fluorescence of RhB NPs was excited by a fs Ti: Sapphire oscillator laser (λ = 808 nm) and the corresponding signal channel was located in 575–630 nm.
In vivo TPA fluorescence imaging. Before in vivo TPA fluorescence imaging, the tumor models were established. All animal experiments were conducted in accordance to the protocols approved by the Ethical Committee in compliance with the Chinese law on experimental animals. Female BALB/c-nude mice (5 weeks old, Beijing HFK Bioscience Co. Ltd.) were housed in an environmentally controlled animal facility with regular 12/12 cycle. MCF-7 cells were collected and suspended in PBS with a concentration of 6 × 107 cells mL− 1. Each mouse was injected with 100 µL cellular suspension in the right sub-dermal dorsal area. The tumor dimensions were measured using a caliper every day. The tumor volume was determined using the following formula:
$$tumor volume=\frac{lengtℎ\times widtℎ\times widtℎ}{2}$$
Approximately 1 week after inoculation, the tumors approximately grew to the volume of 150 ± 30 mm3. 200 µL 5% glucose solution of ICG NPs and RhB NPs (equivalent concentration of 258 µM) and pure 5% glucose solution (control group) were intravenously injected into the tumor bearing mice via the tail vein. After injection, the mice were anesthetized with 4% (w/w) chloral hydrate (10 mL kg− 1 body) and the skin was peeled off. The flow and accumulation of ICG NPs in tumor tissue was detected on a water mirror (APO 25×/0.95) by a Leica multiphoton (MP) laser scanning confocal microscope (TCS-SP8, Germany). The NPs were excited by a Ti: Sapphire oscillator laser (Mai Tai, USA) at 808 nm. An area of 300 × 300 µm was randomly selected as site of interest to perform the Z axis analysis as to observe the spatial distribution of the NPs within the tumor tissue. The Z axis crosscutting was continued until the signal disappeared. The obtained images were analyzed by Leica application suite X (LAS X) software.
Histological analysis. Tumor tissues were excised from the mice after imaging of ICG NPs accumulation after 24 h. The frozen tissue was sliced into 20 µm slices which were analyzed by TCS-SP8 microscopy.
References
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