Metastatic status of sentinel lymph nodes in breast cancer determined with photoacoustic microscopy via dual-targeting nanoparticles

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000, ammonium salt) were obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). HA with different molecular weights of 5 and 15 kDa were obtained from Lifecore Biomedical LLC (MN, USA) (cat#: HA5K-1, HA15K-1). 1-Ethyl-3-[3-dimethyl)aminopropyl]carbodiimide (EDAC) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The near-infra-red dye DiR-BOA (1,1′-dioctadecyl-3,3,3′,3′-tetra-methylindotricarbocyanine iodide bis-oleate) was synthesised as previously described51. R4F (an ApoA1-mimetic peptide, Ac-FAEKFKEAVKDYFAKFWD) was synthesised by Shanghai Apeptide Co., Ltd. (Shanghai, China).

Mice and cells

Albino-C57BL/6 transgenic mice were obtained from the Jackson Laboratory (Bar Harbor, ME). BALB/c female mice were purchased from the Hubei Research Center of Laboratory Animals (Hubei, China). All animal studies were conducted in compliance with protocols that had been approved by the Hubei Provincial Animal Care and Use Committee and in compliance with the experimental guidelines of the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology (IACUC number: 2160). The mouse mammary adenocarcinoma 4T1 cell line was kindly provided by Professor Li Su (Huazhong University of Science and Technology). 4T1-tfRFP cells were obtained by transfecting 4T1 cells with a plasmid containing the tfRFP gene52. The B16F10 cell line was purchased from BOSTER Company (Wuhan, China), and the CT26 cell line was obtained from the American Type Culture Collection (Manassas, USA). These cells were cultured in complete RPMI-1640 medium (Gibco, Thermo Fisher Scientific, USA) containing 10% foetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco) in a cell incubator (Thermo, USA) with 5% CO2 and 95% air at 37 °C.

Synthesis of HA-DMPE

HA-DMPE was synthesised as described previously using a modified reaction53. Briefly, 14 mg of HA was dissolved in 5 ml of distilled water, and 6 mg of EDAC was added at pH 4 to preactivate the HA (5 and 15 kDa) for 2 h at 37 °C. Subsequently, a suspension of DMPE (0.5 mg) was added to the preactivated HA solution, and the pH was adjusted to 8.6 with 0.1 M borate buffer (pH 9.4). This reaction proceeded for 24 h at 37 °C. The mixture was purified by dialysis (MWCO 3 kDa) four times for 12 h each time. Then, the purified solution was lyophilised, and 1 ml of chloroform–methanol (4:1; v/v) was added to the lyophilised sample. Next, the sample was centrifuged at 12,000 rpm for 10 min at 4 °C; this step was repeated three times. The free HA precipitate was removed, and the supernatant of the HA-DMPE solution was collected and stored at 4 °C. The structures of HA, DMPE and the HA-DMPE copolymers were determined using 1H NMR (AV400, Bruker, Switzerland) in D2O and DMSO-d6.

Synthesis and characterization of the HA-HPPS core loaded with DiR-BOA

HA-HPPS complexes were synthesised as follows. DMPC (3 μmol), cholesterol oleate (CO, 0.1 μmol), DiR-BOA (0.2 μmol), DSPE-PEG2000 (0.0114 μmol) and HA-DMPE (5 or 15 kDa, 0.04 μmol) in chloroform (400 μl) were dried under a nitrogen stream to form a uniform film. Then, 2 ml of PBS solution was added to the dried film and vortexed for 5 min. Subsequently, the mixture was sonicated for ~1 h at 48 °C. R4F (0.78 μmol) was dissolved in 1 ml of PBS, added dropwise to the lipid emulsion and stored overnight at 4 °C. After concentration using centrifugal filter units (30 kDa, Millipore, USA), the nanoparticles were purified using the Akta FPLC system with a HiLoad 16/70 Superose 6 column (General Electric Healthcare, NY, USA). The peptide concentration was measured using a CBQCA protein quantitation kit (Invitrogen Corporation, CA, USA). The morphologies of the nanoparticles were analysed by TEM (TECNAI G2, FEI Company, OR, USA). The size distributions of the nanoparticles were measured by DLS on a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK). The stability of the nanoparticles was evaluated using seminative SDS–PAGE.

Cytotoxicity test

The cytotoxicity of the samples to 4T1 cells was determined by the MTS assay. In brief, 1 × 104 4T1 and RAW264.7 cells (in 100 μl of medium) were seeded in each well of a 96-well culture plate and incubated at 37 °C with 5% CO2 for 24 h. The medium in each well was replaced with culture medium (100 μl) containing different concentrations (1–40 μM) of HA-HPPS. After incubation for 24 h, 20 μl of MTS was added to each well, and incubation was continued for 1 h at 37 °C. Then, absorption at 490 nm was measured using a Bio-Tek Epoch microplate spectrophotometer (Winooski, Vermont, USA).

Confocal imaging

To illustrate the 4T1 cell dual-targeting ability of the nanoparticles in vitro, 4T1 cells, RAW264.7 cells, BMDMs and BMDCs were seeded into 8-well chambers covering the glass bottoms (Nunc Lab-Tek, Thermo Scientific) (2 × 104 cells/well). Then, the cells were incubated with 5K-HA-HPPS, HPPS or 5K-HA-e at a DiR-BOA concentration of 10 μM for 3 h, and Hoechst 33258 (0.5 μg ml−1) was added 15 min before washing. For imaging of the tissue sections, the LNs were fixed in 4% paraformaldehyde for 10 h at 4 °C and then dehydrated in a 30% sucrose solution. The LNs were then frozen in OCT compound (Sakura, Torrance, CA, USA) and sectioned into 10-μm-thick slices using a freezing microtome (Leica, Germany). For staining, slides were washed once with PBS, and the sections were immunostained with Lyve-1 (eBioscience, clone ALY7, 1:200), F4/80 (Clone: BM8, 1:200), CD11c (Clone: N418, 1:100), CD3 (Clone: 17A2, 1:200) and B220 (Clone: RA3-6B2, 1:200). Fluorescence images were acquired using an LSM 710 laser confocal scanning microscope (Zeiss, Germany) with an excitation wavelength of 405 nm for Hoechst 33258 and DAPI, 488 nm for FITC and Lyve-1 and 633 nm for DiR-BOA.

FCM analysis

For 4T1 cell-targeting ability testing, 4T1 cells, B16 cells, CT26 cells and E0771 cells were plated in 96-well flat-bottom culture plates (5 × 104 cells/well), and 5K-HA-HPPS, HPPS or 5K-HA-e were incubated with the cells at various DiR-BOA concentrations for 0.5 or 3 h. Regarding the competition assay, 4T1 cells were precultured with excessive amounts of HDL protein and free HA. The mass ratio of HDL to R4F in 5K-HA-HPPS and HPPS was 10:1 and that of free HA to HA in 5K-HA-HPPS and 5K-HA-e was 250:1. Then, the cells were incubated with 5K-HA-HPPS, HPPS or 5K-HA-e at a DiR-BOA concentration of 10 μM for 3 h. The total volume of the solution in the wells was 200 μl for FCM. Fluorescence signals were quantified by a Guava easyCyte 8HT flow cytometer (Millipore Corporation, Billerica, MA, USA). The data were analysed using FlowJo.

LN metastasis and inflammation models

To establish the LN metastasis model of 4T1 murine breast cancer, 5 × 105 4T1 cells or 4T1-tfRFP cells in 20 μl of PBS were injected into the left hock area of BALB/c mice. Albino-C57BL/6 mice were used to develop the pLN inflammation model by intra-lymph node injection of LPS (5 ml kg−1, Sigma) 2 days before imaging experiments11. In vivo imaging experiments were performed using a custom-made whole-body optical imaging system.

In vivo fluorescence imaging

For in vivo fluorescence imaging of the LNs, 25 nmol of 5K-HA-HPPS, HPPS or 5K/15K-HA-e were injected into one side of the footpad of normal Albino-C57BL/6 mice, and nanoparticle accumulation in pLNs and sLNs was detected and analysed by wide-field fluorescence imaging at 10 min, 1, 3, 6 and 12 h after injection of the probe. For fluorescence imaging of Inf-LNs and T-MLNs, 5K-HA-HPPS, HPPS or 5K/15K-HA-e were injected into both sides of the footpad of inflammatory Albino-C57BL/6 mice and the hock area of BALB/c mice. The fluorescence signals of N-LNs, T-MLNs and Inf-LNs were imaged at 0.5, 3, 6, 12 and 24 h after injection of the probes. Fluorescence images of DiR-BOA were acquired with an NIR filter set (excitation: 716/40 nm; emission: 800/40 nm; exposure time: 10 or 30 s). The mice were anaesthetised with 3% isoflurane/O2 (v/v) and maintained on isoflurane/O2 at 1.5% (v/v) throughout the experiments. For LN imaging, 3 weeks after hock inoculation of 4T1-tfRFP tumour cells, resected T-MLNs were imaged 6 h after intratumoural injection of FITC–5K-HA-HPPS and FITC–HPPS. Fluorescence images of tfRFP and FITC were acquired with a filter set (excitation: 562/40 nm, emission: 640/40 nm; and excitation: 496/40 nm, emission: 562/40 nm, respectively). Regarding the orthotropic breast cancer model, tumour-bearing mice were subjected to fluorescence imaging on day 30 after tumour inoculation (n = 3). A single dose of 5K-HA-HPPS or HPPS (DiR-BOA: 25 nmol) in 50 μl of sterile PBS was injected intratumourally. Imaging of brachial and axillary LNs was performed 6 h after injection of the probes.

In vitro and in vivo PAM

For in vitro PAM of 4T1 cells and BMDMs, 4T1 cells and BMDMs were seeded into 15 mm dishes and incubated with 5K-HA-HPPS, HPPS or 5K-HA-e for 3 h in vitro.

For in vivo PAM analysis of different statuses of LNs, the skin was excised and pLNs were exposed 6 h after injection of 5K-HA-HPPS, HPPS or 5K-HA-e (DiR-BOA: 25 nmol). Photoacoustic (PA) maximum amplitude projection images (image field of view: 5 mm × 5 mm) were taken in vivo with the custom-made acoustic resolution PAM (AR-PAM) system with 744 nm pulsed lasers at 600 nJ. The images were acquired by performing a B-scan, with each B-scan including 500 steps with a step size of 10 μm. The maximum image depth of AR-PAM was 1.6 mm. The spatial resolution of AR-PAM was ~45 μm. To quantitatively analyse the distribution of PA signals in LNs with different statuses, we extracted PA signals along the transverse (dotted yellow line) and longitudinal (dotted white line) diameters of each LN and constructed cross-sectional intensity profiles to reduce error from single section measurement. The LN centre (C) was defined as 40% of the diameter (20% on each side of the centre point). The LN periphery (P) was defined as 60% of the diameter (30% on each side of the edge). The average ratio of PA intensity in the LN centre (C) to that of the LN periphery (P) was calculated by the formula R = (RT + RL)/2. RT and RL represent the ratio of transverse (T) and longitudinal (L) PA intensity, respectively, and were calculated as follows:

$$R_T = C_1/left( {frac{{P_1 + P_2}}{2}} right),$$

$$R_L = C_2/left( {frac{{P_3 + P_4}}{2}} right),$$

where C1 and C2 represent the PA intensities of the LN centre in the transverse and longitudinal sections, respectively; P1 and P2 represent the PA intensities of the LN periphery in the transverse section on the left and right sides, respectively; and P3 and P4 represent the PA intensities of the LN periphery in the longitudinal section on the top and bottom, respectively (Supplementary Fig. S9).

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, CA). Student’s t test (two tailed) was used for the in vitro and in vivo studies. Data are presented as the mean ± SD. Significant differences between or among the groups are indicated as follows: ns for no significant difference, * for P < 0.05, ** for P < 0.01, and *** for P < 0.001.

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