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. Author manuscript; available in PMC: 2019 Dec 12.
Published in final edited form as: Nano Res. 2018 Sep 29;12:273–279. doi: 10.1007/s12274-018-2210-x

A theranostic agent for cancer therapy and imaging in the second near-infrared window

Zhuoran Ma 1,§, Hao Wan 1,§, Weizhi Wang 2,§, Xiaodong Zhang 3,§, Takaaki Uno 4, Qianglai Yang 5, Jingying Yue 1, Hongpeng Gao 1, Yeteng Zhong 1, Ye Tian 1, Qinchao Sun 1, Yongye Liang 5, Hongjie Dai 1
PMCID: PMC6907162  NIHMSID: NIHMS1019732  PMID: 31832124

Abstract

Theranostic nanoparticles are integrated systems useful for simultaneous diagnosis and imaging guided delivery of therapeutic drugs, with wide ranging potential applications in the clinic. Here we developed a theranostic nanoparticle (~ 24 nm size by dynamic light scattering) p-FE-PTX-FA based on polymeric micelle encapsulating an organic dye (FE) fluorescing in the 1,000–1,700 nm second near-infrared (NIR-II) window and an anti-cancer drug paclitaxel. Folic acid (FA) was conjugated to the nanoparticles to afford specific binding to molecular folate receptors on murine breast cancer 4T1 tumor cells. In vivo, the nanoparticles accumulated in 4T1 tumor through both passive and active targeting effect. Under an 808 nm laser excitation, fluorescence detection above 1,300 nm afforded a large Stokes shift, allowing targeted molecular imaging tumor with high signal to background ratios, reaching a high tumor to normal tissue signal ratio (T/NT) of (20.0 ± 2.3). Further, 4T1 tumors on mice were completed eradicated by paclitaxel released from p-FE-PTA-FA within 20 days of the first injection. Pharmacokinetics and histology studies indicated p-FE-PTX-FA had no obvious toxic side effects to major organs. This represented the first NIR-II theranostic agent developed.

Keywords: theranostic nanoparticles, second near-infrared window, fluorescence imaging, cancer therapy

1. Introduction

Theranostic nanoparticles that combine both diagnostic and therapeutic agents on a single platform have been considered as a promising strategy for cancer detection and treatment [1, 2]. Generally, cancer theranostic nanoparticles accumulate in tumors through passive uptake which utilizes enhanced permeability and retention (EPR) effect [3] as well as molecular targeting that employs ligand binding to specific receptors [4]. The imaging agents help to pinpoint tumor locations by increasing the signal ratio between the tumor and its surrounding normal tissues. Meanwhile, the therapeutic agents are released from the nanocarrier to kill the tumor cells.

Different imaging modalities have been explored with theranostic nanoparticles, including magnetic resonance imaging (MRI) [5, 6], positron emission tomography (PET) [7, 8], and optical imaging [9–11]. Compared to other imaging modalities, fluorescence imaging offers high spatial and temporal resolution [12]. While fluorescence imaging in the visible (400–700 nm) suffers from poor tissue penetration [13], imaging in the conventional near infrared windows (700–900 nm) benefits from low tissue absorption and background fluorescence, allowing for higher penetration depth [14–16]. Recently, fluorescence imaging in the second near-infrared window (NIR-II, 1,000–1,700 nm) has demonstrated significant improvements in spatial resolution and imaging depth, due to further reduced autofluorescence, tissue scattering, and absorption [17–21].

On the other hand, appropriate nanocarriers need to be used to deliver and release aqueous insoluble drug molecules. Although different formulations of chemotherapeutic agents have been developed, most of these formulations could cause significant adverse effects at a high dose [22, 23]. Thus, it is highly desired to explore novel drug delivery vehicles that can afford high efficacy in suppressing tumor growth while exhibiting minimum side effects.

We recently developed a NIR-II nanofluorophore by encapsulating an organic dye (FE) [24] in micelles of an amphiphilic polymer poly(styrene-co-chloromethyl styrene)-graft-poly(ethylene glycol) (PS-g-PEG) [25]. The nanoparticle, named p-FE exhibited good water solubility and biocompatibility while retained the high quantum yield (QY) of the FE dye. Due to its amphiphilic nature, the same polymer could also be employed as a vehicle to trap and deliver hydrophobic anti-cancer drug molecules.

Here, we described the synthesis of a novel theranostic system based on carboxylated poly(styrene-co-chloromethyl styrene)-graft-poly(ethylene glycol) (PS-g-PEG-COOH), the NIR-II dye FE and paclitaxel. We encapsulated FE and the hydrophobic anti-cancer drug paclitaxel (PTX) into the polymer to produce a water-soluble nanoparticle p-FE-PTX. Folic acid (FA) was conjugated to p-FE-PTX to allow targeted molecular imaging and cancer therapy. In vivo administration of the complex to murine 4T1 tumor bearing mice was performed. A high tumor to normal tissue signal ratio (T/NT) was achieved, and 4T1 tumors were completely eliminated. No obvious damage to main organs was observed. The high T/NT in fluorescence imaging, tumor suppression effect at low injection dose, and low toxic side effect made p-FE-PTX-FA a promising system for cancer theranostics.

2. Results

2.1. Synthesis and characterization of p-FE-PTX and p-FE-PTX-FA

p-FE-PTX nanoparticles were prepared using a modified method reported recently [25] (Fig. 1(a) and see Method). In brief, hydrophobic NIR-II dye FE, PTX and amphiphilic polymer PS-g-PEG were dissolved in chloroform. All the substances were reconstituted after the solvent was evaporated. There were absorption peaks at ~ 770 and ~ 230 nm, which corresponded to the absorption of FE [24, 25] and PTX [26, 27], respectively (Figs. 1(b) and 1(c)).

Figure 1.

Figure 1

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Synthesis and characterization of p-FE-PTX and p-FE-PTX-FA. (a) Scheme of synthesis of p-FE-PTX and p-FE-PTX-FA. (b) Absorption spectra of p-FE-PTX, p-FE-PTX-FA and PTX (200–500 nm). p-FE-PTX and p-FE-PTX-FA were dissolved in PBS buffer, and PTX was dissolved in methanol. The peak of the absorption spectrum was normalized to 1. (c) The absorption (600–900 nm) and emission (900–1,500 nm) spectra of p-FE-PTX and p-FE-PTX-FA. The peak of the absorption spectrum was normalized to 1. The excitation was provided by an 808 nm laser diode, and a 900 nm long-pass filter was used as the emission filter. The emission spectra of p-FE-PTX and p-FE-PTX-FA were normalized by the absorption at 808 nm. (d) Dynamic light scattering analysis of p-FE-PTX and p-FE-PTX-FE in PBS buffer.

About 10% of the PEG chains in PS-g-PEG were carboxylated, which allowed facile conjugation to targeting ligands. Ethylene diamine was conjugated to the nanoparticles to convert the carboxyl groups to amino groups (see Method), and then FA was conjugated to the nanoparticles through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry to impart targeted molecular imaging and therapy (Fig. 1(a)). There was a peak at ~ 280 nm (Fig. 1(b)) in the absorption spectrum of p-FE-PTX-FA, indicating that the FA was successfully conjugated to the nanoparticles [28, 29]. p-FE-PTX-FA showed an emission peak at ~ 1,010 nm under the excitation of an 808-nm laser (see Fig. 1(c) for absorption and emission spectra of p-FE-PTX-FA), and the relative QY of p-FE-PTX-FA in aqueous solution was ~ 0.8%–8% (based on QY of IR-26 reference was 0.05%–0.5% [30–33]), which was ~ 20 times higher than that of single-walled carbon nanotubes [34, 35] (Fig. 1(c)).

Dynamic light scattering (DLS) analysis revealed that p-FE-PTX and p-FE-PTX-FA nanoparticles had a hydrodynamic size of ~ 15 and ~ 24 nm, respectively (Fig. 1(d) and Fig. S1 in the Electronic Supplementary Material (ESM)). The small size of p-FE-PTX and p-FE-PTX-FA nanoparticles made them suitable for in vivo bioimaging and cancer therapy.

2.2. In vitro cell imaging and toxicity study

We next investigated p-FE-PTX-FA as a NIR-II probe capable of targeting specific receptors on the cell surface for performing molecular imaging of cancer cells. As-prepared p-FE-PTX and p-FE-PTX-FA were incubated at 4 °C with murine 4T1 breast cancer cells, which expressed folate receptor (FR) [36–39]. The FR-negative HEK-293T cells were used as a negative control. Cells were imaged by a home-built confocal system [25, 40, 41] with a 785-nm excitation laser, and the NIR-II fluorescence signal above 1,050 nm was detected selectively on 4T1 cells treated with p-FE-PTX-FA, but not on the negative HEK-293T cells, or 4T1 cells incubated with p-FE-PTX (Fig. 2(a)). Cell imaging using p-FE-PTX-FA showed a positive/negative ratio of ~ 5.6.

Figure 2.

Figure 2

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In vitro cell imaging and toxicity study. (a) Molecular imaging of 4T1 cells which overexpress folate receptor, and HEK-293T cells (folate receptor negative) with p-FE-PTX or p-FE-PTX-FA. The cells were imaged by a home-built confocal microscope system. FE was excited by a 785-nm laser, and detected with an 1,050-nm long-pass filter (red channel on the images). QRAQ5 was excited by a 658-nm laser, and detected with a 699-nm long-pass filter and an 850-nm short-pass filter (blue channel on the images). (b) Relative viabilities of 4T1 cells after being treated with p-FE-PTX or p-FE-PTX-FA at different concentrations for 24 h (n = 6 for each condition).

We next tested the in vitro efficacy for killing cancer cells of different formulations of PTX. 4T1 cells were incubated with p-FE-PTX or p-FE-PTX-FA at different concentrations at 37 °C for 24 h, and a standard cell viability assay using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) [42, 43] was performed. Compared to p-FE-PTX with no FA conjugation, the p-FE-PTX-FA nanoparticles exhibited a lower 50% growth inhibition concentration (IC50) of ~ 15 μM (Fig. 2(b)), indicating increased cell binding and internalization after ligand conjugation. The IC50 of p-FE-PTX-FA was similar to that of free PTX reported previously [44], suggesting that PTX in this formulation be as an effective chemotherapeutic drug.

2.3. In vivo molecular imaging using p-FE-PTX-FA

We performed in vivo molecular imaging using p-FE-PTX-FA. C57BL/6 mice (n = 5) inoculated with 4T1 tumors were intravenously injected with p-FE-PTX-FA at a dose of 5 mg/kg PTX. Under an excitation of 808 nm, fluorescence signal above 1,300 nm was collected to minimize the influence of tissue scattering and autofluorescence [18, 25]. The fluorescence of tumor could be clearly visualized 2 h post injection (p.i.), and T/NT reached a maximum of (20.0 ± 2.3) at 24 h p.i. (Figs. 3(a) and 3(b)). In comparison, the non-targeted p-FE-PTX showed a lower maximum T/NT of (12.9 ± 1.4) at 24 h p.i. (Figs. S2(a) and S2(b) in the ESM). While p-FE-PTX with no FA conjugation showed a high T/NT due to EPR effect, the uptake of PTX in tumor could be increased by utilizing specific targeting ligands.

Figure 3.

Figure 3

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In vivo fluorescence imaging and treatment of 4T1 tumor bearing mice with p-FE-PTX-FA. (a) Wide-field NIR-II fluorescence imaging of mice inoculated with 4T1 tumors after different time points post intravenous injection. C57BL/6 mice (n = 5) were injected with p-FE-PTX-FA at a dose of 5 mg/kg PTX. The mice were excited by an 808 nm laser at a power density of 70 mW/cm2, and the fluorescence signal above 1,300 nm was collected. (b) Time course variation of T/NT of the mice (n = 5) injected with p-FE-PTX-FA. (c) Representative photographs of 4T1-bearing C57BL/6 mice treated with p-FE-PTX-FA. (d) Tumor growth curves of 4T1-bearing mice (n = 5) treated with p-FE-PTX-FA or p-FE-FA (n = 5 each group). The same dose of PTX (5 mg/kg) was injected on days 0, 7 and 14 (marked by arrow) for the p-FE-PTX-FA group.

2.4. In vivo therapy of 4T1 tumor-bearing mice with p-FE-PTX-FA

We next investigated the in vivo therapeutic effect of the nanoparticles. The same dose of p-FE-PTX-FA (5 mg/kg PTX) was injected intravenously in 4T1-tumor bearing C57BL/6 mice (n = 5) on days 0, 7 and 14 post treatment. The sizes of the subcutaneous tumors were measured over a course of 4 weeks. The size of the tumors started to decrease 3 days after the initialization of the treatment, and the tumors were completely eradicated after 20 days (Figs. 3(c) and 3(d)). Tumors treated with p-FE-PTX were eliminated within 25 days of the first injection (Fig. S2(c) in the ESM). In contrast, p-FE-FA showed no inhibition effect on the growth of 4T1 tumors (Fig. 3(d) and Fig. S3 in the ESM), and the sizes of the tumors increase by (5.9 ± 0.3) fold within 20 days, indicating PS-g-PEG or FE dye alone did not have therapeutic effect. The p-FE-PTX-FA treated group showed a greater inhibition of tumor growth than the p-FE-PTX treated group at an equivalence dose of PTX. This could be explained by an increased uptake of PTX as suggested by the in vivo imaging experiments.

2.5. Pharmacokinetics and biodistribution of p-FE-PTX-FA

To investigate the pharmacokinetics of p-FE-PTX-FA, C57BL/6 mice (n = 5 each group) were injected with p-FE-PTX or p-FE-PTX-FA at the same dose as used for in vivo imaging. The fluorescence signal in blood vessels was plotted as a function of time (Fig. 4(a)), and blood half-time was determined based on a previous method [41, 45]. The p-FE-PTX and p-FE-PTX-FA nanoparticles demonstrated a blood half-time of 16.6 ± 2.6 and 13.1 ± 1.8 h, respectively, which was similar to that of previously reported p-FE nanoparticles [25]. The long circulation time of the particles facilitate uptake into tumors. Biodistribution at 24 h p.i. indicated that other than the tumor, liver was the main organ for accumulation of both p-FE-PTX and p-FE-PTX-FA. Compared with the nontargeted particles, p-FE-PTX-FA showed a significantly increased accumulation from 1.5 %ID/g to 9.0 %ID/g in tumors at 24 h p.i. (Fig. 4(b)). The fluorescence in liver decreased gradually in long-term imaging experiments (Figs. S4 and S5 in the ESM), suggesting p-FE-PTX-FA could be cleared from the body through the hepatobiliary pathway [25].

Figure 4.

Figure 4

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Pharmacokinetics, biodistribution and toxicity of p-FE-PTX and p-FE-PTX-FA. (a) Normalized fluorescence intensity of p-FE-PTX and p-FE-PTX-FA in blood over the course of 72 h. (b) Biodistribution of p-FE-PTX and p-FE-PTX-FA in 4T1 tumor bearing mice at 24 h post intravenous injection. (c) Change of body weight of mice treated with p-FE-PTX and p-FE-PTX-FA. No obvious loss of body weight was observed in both groups. (d) H&E stained histological sections of the liver and spleen from healthy mice and the mice treated with p-FE-PTX-FA.

To evaluate the toxicity of p-FE-PTX and p-FE-PTX-FA formulations, we measured body weight of the mice (n = 5 each group) in the process of cancer treatment. Mice in both groups showed a steady body weight during treatment (Fig. 4(c)), suggesting low toxicity of the formulations. Furthermore, histological studies of hematoxylin and eosin (H&E) stained sections of major organs from the mice (n = 5) treated with p-FE-PTX-FA were examined, and no obvious damage in major organs was observed (Fig. 4(d) and Fig. S6 in the ESM). Especially, organs with high uptake of p-FE-PTX-FA, such as liver and spleen, showed no detectable injury during the procedure (Fig. 4(d)).

3. Discussion

Here we developed the first NIR-II theranostic system for simultaneous tumor imaging in the 1,000–1,700 nm range and drug delivery. Fluorescence imaging benefits from micro-scale spatial resolution and high frame rate (> 25 frames per second), which would be difficult to achieve by other medical imaging modalities [5–8]. U.S. Food and Drug Administration (FDA)-approved fluorescent molecules in the conventional near infrared window (NIR-I window, 700–900 nm), such as indocyanine green (ICG) and methylene blue, have been used for tumor imaging and therapy [15, 16]. However, the NIR-I fluorescence-based theranostic nanoparticles usually suffer from high background signal and low T/NT ratio ~ 5 [14, 46], much lower than T/NT ~ 20 achieved here with p-FE-PTX-FA in NIR-II. Compared to fluorescence imaging in the NIR-I window, in vivo imaging in the NIR-II window exhibits improved imaging clarity and spatial resolution, owing to reduced photon scattering in biological tissues, which scales with λ−α (λ is the wavelength, α = 0.2–4) [47]. The FE dye showed an absorption peak at ~ 760 nm, while the main peak of the emission spectrum was ~ 1,010 nm. The large Stokes shift of the NIR-II fluorescent dyes helped to reduce the background signal caused by autofluorescence. Furthermore, molecular targeting by folic acid facilitated the specific accumulation of the nanoparticles in the folate expressing 4T1 murine breast tumor. Hence, an impressive T/NT ratio ~ 20 was achieved by p-FE-PTX-FA.

It should be noted that p-FE-PTX without FA conjugation also showed high uptake in 4T1 tumors and good therapeutic efficacy, although the cell experiments suggested it did not bind to 4T1 cells specifically ex vivo at 4 °C and the IC50 was much higher than that of p-FE-PTX-FA due to the lack of strong nanoparticle-cell interaction without the specific ligands. The discrepancy between the in vitro cell experiment and the in vivo animal experiment could be attributed to the long blood circulation of the nanoparticles in vivo and the EPR effect. In vivo, PEGylation could reduce the uptake of the nanoparticles by the reticuloendothelial system (RES), and increase the blood circulation half time [48]. The long blood circulation of p-FE-PTX facilitated its accumulation in tumors through the EPR effect, and thus a high T/NT could be achieved without targeting ligands. What’s more, the long retention time in tumors allowed cellular uptake of the nanoparticles through endocytosis pathways [49]. While p-FE-PTX-FA could be internalized by receptor-mediated endocytosis pathway, p-FE-PTX without FA conjugation could utilize other non-specific pathways. Without a targeting ligand, long circulating p-FE-PTX could still work as a theranostic agent in vivo, and its tumor homing effect can be further enhanced by conjugation to targeting ligands.

Although our current study focused on imaging and treating 4T1 breast tumors with FA conjugated theranostic nanoparticles, similar ideas can be applied to other tumor models by using different targeting ligands/antibodies and therapeutic molecules that are poorly water soluble. Visible and NIR-I fluorescent probes have been conjugated to targeting ligands, such as antibodies [50, 51], peptides [52–54], and nucleic acid aptamers [55, 56] for in vivo molecular imaging of tumors. It is possible to employ these ligands to p-FE-PTX through bioconjugation chemistry, such as EDC chemistry demonstrated in this work, and higher T/NT ratios achievable than in NIR-I. Besides PTX, other hydrophobic anti-cancer drugs can also be delivered using the system. The poor water solubility of many anti-cancer molecules interferes the bioactivity and therefore hinders their clinical applications [22, 57]. The amphiphilic polymer PS-g-PEG helps to deliver the hydrophobic molecules in aqueous environment, and the therapeutic efficacy could be retained.

4. Methods

4.1. Synthesis of PS-g-PEG-COOH

The poly(styrene-co-chloromethyl styrene) (PS) backbone was synthesized using a reversible addition-fragmentation chain transfer (RAFT) reaction described previously [25]. To prepare carboxylated polyethylene glycol (PEG-COOH), Zn(OAc)2 (0.045 g, 0.25 mmol) and polyethylene glycol (Mn ~ 1,000, 2.5 g, 2.5 mmol) were dissolved in 10 mL dichloromethane (DCM) and di-tert-butyl decarbonate (0.55 g, 2.5 mmol) was added, and the mixture was stirred for 17 h at 40 °C under nitrogen. The crude product was purified by silica-gel column chromatography. To graft both poly(ethylene glycol) methyl ether (mPEG) and PEG-COOH onto the PS backbone, the copolymer (0.05 g), mPEG (Mn ~ 1,000, 0.31 mmol), PEG-COOH (0.04 g, 0.04 mmol) and NaOH (0.02 g) were added to dry THF (3 mL), and the mixture was stirred for 18 h at room temperature under nitrogen. The reaction mixture was filtered and the filtrate was concentrated under vacuum. Three droplets of trifluoroacetic acid were added and the solution was stirred at room temperature for 2 h to remove the tert-butyl protection group. The crude product was dialyzed against water using a 10k membrane for 2 days and lyophilized to remove the solvent.

4.2. Synthesis of p-FE-PTX-FA

First, p-FE-PTX was prepared. 28 μg FE dye (synthesized according to our previous protocol [24, 25]), 100 μg paclitaxel and 10 mg PS-g-PEG-COOH were dissolved in 100 μL chloroform. The solvent was evaporated at room temperature overnight. All the substances were reconstituted with water under sonication for 10 min. Precipitates were removed by centrifugation at 4,400 rpm for 30 min.

To synthesize p-FE-PTX-FA, as-prepared p-FE-PTX was dissolved in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) solution at a concentration of 10 mg/mL PS-g-PEG-COOH. In a typical reaction, 50 μL ethylene diamine was added to 200 μL p-FE-PTX solution. 15 mg EDC was added to the solution, and the solution was stirred at room temperature overnight. Excess ethylene diamine was removed by washing the solution with 10 mM MES solution using an 100k centrifugal filter (4,400 rpm, 20 min) for 6 times. The sample was diluted with 10 mM MES to 2.85 mL, and mixed with 150 μL FA solution (11.76 mg/mL in dimethyl sulfoxide (DMSO)). 15 mg EDC was added to the solution, and the solution was stirred at room temperature overnight. To remove excess folic acid, 200 μL NaOH (1 M) was added to the solution, and the solution was washed with phosphate buffered saline (PBS) buffer using an 100k centrifugal filter (4,400 rpm for 20 min) for 6 times.

4.3. Cell staining and confocal imaging

4T1 cells and HEK-293T cells were cultured in RPMI-1640 and Dulbecco’s modified Eagle’s medium (DMEM) high glucose medium, respectively. All the cells were seeded in chamber slides, and cultured at 37 °C with 5% CO2 for 24 h before staining. Cells were then incubated with p-FE-PTX or p-FE-PTX-FA (contained 20 μM FE) at 4 °C for 30 min. Cells were washed with PBS buffer for 3 times to remove unbounded fluorophores, and fixed by 4% paraformaldehyde (PFA) at room temperature for 20 min. To label the nuclei, the fixed cells were incubated with DRAQ5 (5 μM) at room temperature for 30 min.

A home-built galvo mirror scanning confocal microscope system was used for cell imaging in both NIR-I and NIR-II windows. An 100× objective (Olympus, oil immersion, NA 0.8) was used to focus the excitation light onto the sample, and the emission light went through a dichroic and emission filters before being detected by PMTs (Hamamatsu H7422–50 for NIR-I imaging and Hamamatsu H12397–75 for NIR-II imaging). The scanning rate of the galvo mirror was 2.5 ms/pixel. An 150-μm pinhole was used to block the out-of-focus signals. For DRAQ5 channel, a 658-nm laser diode (Thorlabs) was used as the excitation source, and a 699-nm long- pass and an 850-nm short-pass filter were used as the emission filters. For FE channel, a 785-nm laser (RPMC lasers) was used as the excitation source, and an 1,050-nm long-pass filter was used as the emission filter. The images were analyzed by ImageJ.

4.4. Cytotoxicity of p-FE-PTX and p-FE-PTX-FA

Cytotoxicity of p-FE-PTX and p-FE-PTX-FA was determined with a MTS assay using a CellTiter96 kit (Promega). About 5000 4T1 cells were cultured in each well with 100 μL RPMI-1640 medium, and incubated with serially diluted p-FE-PTX or p-FE-PTX-FA solution (n = 6 for each concentration). The cells were kept at 37 °C with 5% CO2 for 24 h. The cell culture medium was removed, and replaced with fresh medium before addition of 15 μL CellTiter96 in each well. The cells were incubated for 1 h, and the absorbance at 490 nm was recorded using a 96-well plate reader. The absorbance of wells without adding p-FE-PTX or p-FE-PTX-FA was used as control. Cell viability was calculated as a fraction of the absorbance of the control wells.

4.5. Mouse handling

All animal experiments were approved by Stanford Institutional Animal Care and Use Committee (IACUC). C57BL/6 mice were obtained from Charles River. 4T1 tumors were generated by subcutaneous injection of 2 × 106 cells in 50 μL PBS into the right hindlimb. The mice were used for imaging and treatment when the volume of the tumor reached 50 mm3 (about 5 days post inoculation). For imaging and treatment, p-FE-PTX or p-FE-PTX-FA was dispersed in PBS buffer, and injected intravenously into the mice (n = 5 each group). The injection dose of PTX was 5 mg/kg for each mice. The same dose of p-FE-PTX or p-FE-PTX-FA was injected on days 0, 7 and 14 after the treatment was initialized. The size of the tumor was measured every other day using a caliper, and the volume of the tumor was calculated using the following formula: Volume = (tumor length) × (tumor width)2/2. Relative tumor size was calculated as the V/V0 (V0 is the volume of the tumor at the beginning of the treatment). Body weight of the mice was measured every other day, and normalized to the initial weight.

4.6. In vivo fluorescence imaging in the NIR-II window

NIR-II fluorescence images were recorded by a liquid-nitrogen-cooled two-dimensional InGaAs array (Princeton Instrument). The excitation light was provided by an 808 nm fiber-coupled diode laser (RPMC lasers) at a power density of 70 mW/cm2. The excitation light was filtered through an 850-nm short-pass and an 1,000-nm short-pass filter (Thorlabs). Mice were placed on a stage beneath the laser. The emission light was filtered using a 900-nm long-pass and an 1,300-nm long-pass filter (Thorlabs), and focused by a lens set onto the camera. All images were analyzed by MATLAB.

4.7. Pharmacokinetics and biodistribution studies

4T1 tumor-bearing C57BL/6 mice (n = 5 each group) were used for pharmacokinetics and biodistribution studies. All mice were intravenously injected with p-FE-PTX or p-FE-PTX-FA at the same dose for in vivo imaging. The mice were imaged using an 808-nm diode laser (RPMC lasers), and the concentration of the particles in blood was estimated based on the NIR-II fluorescence intensity (filtered by an 1,300 nm long-pass filter) of the vessels. Biodistribution was performed 24 h post injection. Major organs including heart, liver, spleen, lung, kidney, testis and tumor were collected. Fluorescence signal in tissue homogenate was used to quantify the amount of nanoparticles.

For pathology experiments, 4T1 tumor-bearing C57BL/6 mice (n = 5 each group) were sacrificed 28 days after the initialization of the cancer treatment. Main organs including liver, spleen, heart lung, kidney, brain, stomach, and intestine were collected. The organs were fixed in 10% formalin for 48 h, and made into paraffin sections. The sections were treated according to a standard H&E protocol, and was examined by an optical microscope (Olympus).

Supplementary Material

Supplemental

Acknowledgements

This study was supported by National Institutes of Health NIH DP1-NS-105737, the Deng family gift, and the Shenzhen Peacock Program Grant KQTD20140630160825828.

Footnotes

Electronic Supplementary Material: Supplementary material (hydrodynamic size of the nanoparticles as a function of time, fluorescence imaging using p-FE-PTX and p-FE-FA, and histology images) is available in the online version of this article at https://doi.org/10.1007/s12274-018-2210-x.

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