Materials
NH2-PLGA-NH2 (lactide:glycolide, 50/50, MW: 8 kDa) was obtained from Xi’an Rui-Xi biological technology Co. Ltd. (Xi’an, Shaan Xi, China). 1-butyl-3-methylimidazolium-L-lactate (BML, MW: 228.29 Da) was provided by Cheng Jie Chemical Co. Ltd. (Shanghai, China). Hyaluronic acid (HA, MW: 3.8 kDa) was purchased from Dalian Meilun Biotechnology Co. Ltd. (Dalian, Liao Ning, China). Paclitaxel (Ptx, MW: 853.93 Da) was purchased from Shanghai Aladdin Biotechnology Co. Ltd. (Shanghai, China). 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC, MW: 734.1 Da), polyvinyl alcohol (PVA, MW: 30-70 kDa), 2-(N-morpholino) ethane sulfonic acid (MES), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). All reagents used in this work were of analytical grade without further purification.
Preparation of HA-BNPs@Ptx
The double emulsification method was used to synthesize Ptx & BML loaded DPPC-PLGA hybrid nanoparticles (BNPs@Ptx) following the previous report [29]. Briefly, 10 mg DPPC, 20 mg NH2-PLGA-NH2 and 2 mg Ptx were thoroughly dissolved in 2 mL dichloromethane (DCM). 400 µL BML (0.5 mg/mL, in deionized water) was mixed in the solution. The Ptx and BML solution was emulsified on ice bath (2 min, with a 5 s on-off duty cycle) using an ultrasonic oscillation instrument (SCIENTZ-IID, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, Zhejiang, China) at 12% power (24 kHz, 600 W). 8 mL PVA (2% w/w, in water) solution was further added to the emulsion and emulsified as before. DCM was then evaporated in a fume hood for 3 h under magnetic stirring. Finally, BNPs@Ptx was collected by centrifugation at 10000 g for 10 min and washed twice with double distilled water. Nanoparticles targeting CD44 were prepared by covalently coupling hyaluronic acid to BNPs@Ptx via amino groups on PLGA [30]. Briefly, 2 mg HA was dissolved in 195 mg MES buffer (pH = 5.5). 27 mg EDC and 8.6 mg NHS were then added to HA solution. Subsequently, the mixture was incubated with 1 mL BNPs@Ptx suspension under continuous stirring for 24 h on ice bath. HA-BNPs@Ptx was collected and purified by centrifugation and washing with double distilled water 2 times.
Characterization of HA-BNPs@Ptx
Morphology and physicochemical properties of HA-BNPs@Ptx
Transmission electron microscopy (TEM, Tecnai G2 F30, FEI Co., Ltd., Hillsboro, Oregon State, USA) and scanning electron microscope (SEM, SU8020, Hitachi, Ltd., Tokyo, Japan) were used to observe the morphology of nanoparticles. The size distribution, polydispersity index (PDI) and zeta potential were obtained using dynamic light scattering (DLS, Brookhaven Omni, Brookhaven Instruments Inc., Holtsville, NY, USA). The Fourier transform infrared spectrometer (FTIR, Nicolet iS10, Thermo Fisher Scientific Co., Ltd, Massachusetts, USA) was used to record the spectra of HA, BNPs@Ptx and HA-BNPs@Ptx in the wavelength range of 500–4000 cm−1. The drug loading (DL) and encapsulation efficiency (EE) of Ptx were quantified using a standard calibration curve measured at 229 nm on a visible ultraviolet spectrophotometer (L5S, INESA Analytical Instrument Co., Ltd., Shanghai, China, see Additional file 1, Methods 1.1 for details).
MW induced thermal effect and Ptx release of HA-BNPs@Ptx
To evaluated thermal effect of MW irradiation with sensitizer BML, 1 mL HA-BNPs@Ptx or HA-NPs@Ptx suspensions were added to 24-well plate and exposed to MW energy instrument (WZY-1, Beijing Muheyu Electronics co., ltd, Beijing, China). The suspensions were irradiated for 4 min at different MW power (0.4, 0.8, 1.2 and 1.6 W cm-2, 450 MHz). The temperature change of HA-BNPs@Ptx suspension was recorded every minute using an infrared thermal mapping instrument (FLUKE 572-2, Hawk-IR International, Inc., Everett, WA, USA). Ptx release kinetics upon MW heating was evaluated by using visible ultraviolet spectrophotometer.
In-vitro biocompatibility and targeting ability of HA-BNPs@Ptx
Cytotoxicity assay
Mouse breast cancer cell line 4T1, mouse macrophage cell line J774 and human umbilical vein endothelial cells (HUVEC) were purchased from Cellcook (Guangzhou Cellcook Cell Biotechnology, LTD., Guangzhou, Guangdong, China) and cultured according to the supplier's instructions. 4T1 cells and HUVEC were seeded into a 96-well plate (1×104 cells per well) and incubated overnight. 5 duplicate holes were set in each group. Subsequently, 100 µL HA-BNPs@Ptx at Ptx concentrations (2, 5, 10, 20 and 50 µg/mL) was introduced to each group. The cytotoxicity of HA-BNPs@Ptx was examined by CCK-8 viability assay (Boster Biological Technology Co., Ltd., California, USA). The optical density (OD) was measured at 450 nm by Varioskan Flash microplate reader (Synergy Mx, BioTek Instruments, Inc., Winooski, VT, USA). The cytotoxicity treated with HA-NPs (without drug loaded NPs) was evaluated using the same method.
Hemolytic activity
2% suspension of chicken red blood cells were used to determine hemolytic activity of HA-BNPs@Ptx. 100 µL of phosphate buffer saline (PBS), double distilled water or HA-BNPs@Ptx suspensions at Ptx concentrations of 2, 5, 10, 20 and 50 µg/mL were added to erythrocyte suspensions (100 µL). PBS was used as negative control group, and double distilled water was used as positive control group. All suspensions were incubated at 37 °C for 1 h and centrifuged at 800 g for 10 min. The OD was measured at 550 nm using the Varioskan Flash microplate reader.
CD44 receptor mediated nanoparticles targeting
4T1 cells were used to verify CD44 targeting ability of nanoparticles. The CD44 expression level of 4T1 cells was evaluated by an inverted fluorescence microscope (see Additional file 1, Methods 1.2 for details). The targeting test was performed using BNPs@Ptx, HA-BNPs@Ptx and HA-BNPs@Ptx with the cells in which CD44 receptor was presaturated by an excess amount of free HA [31]. To obtain stained nanoparticles, 1 mL BNPs@Ptx or HA-BNPs@Ptx suspensions were stained with 10 µL DiO (1:100). After overnight culture, 4T1 cells were co-incubated with 100 µL BNPs@Ptx or HA-BNPs@Ptx for 6 h. The targeting ability was visualized by the inverted fluorescence microscope (AX10 imager A2/AX10 cam HRC, Carl Zeiss, Co., Ltd, Jena, Germany). Flow cytometry (FCM, Cyto Flex, Beckman Coulter, Inc., CA, USA) was used to further evaluate the ability of HA-BNPs@Ptx to target 4T1 cells at different time intervals (30 min, 1, 3, 6, and 24 h).
In-vitro cellular uptake and intracellular tracking
To examine cellular uptake and intracellular trafficking, BNPs@Ptx and HA-BNPs@Ptx were labelled with a red fluorescence probe Dil. Endocytosis/phagocytosis experiment were performed in 4T1 cells and J774 cells (murine macrophages), respectively. 1×104 4T1 cells per well were seeded in confocal dishes. After 24 h, 100 µL DiI-BNPs@Ptx and DiI-HA-BNPs@Ptx were added to co-incubate with 4T1 cells for 1, 3 and 6 h, respectively. At each time point, 20 µL LysoTracker Green was added to each confocal dish to stain endo/lysosomes. 1.5 h after incubated with LysoTracker Green, all cells were fixed with 4% paraformaldehyde for 10 min. 10 µL 4,6-diamidino-2-phenylindole (DAPI, blue) was finally added in fixed cells to stain the nuclei. Confocal fluorescence images of the fixed cells were obtained by a laser scanning confocal microscope (LSCM, A1R+MP, Nikon Co., Tokyo, Japan).
J774 cells were treated in the same way as 4T1 cells, except that 5×104 cells and 10 µL of PMA (100 ng/ml) were added to each dish and incubated for 48 h.
In-vitro synergistic anti-tumor ability
Synergistic anti-cancer activity of thermal-chemotherapy was evaluated with 4T1 cells in-vitro. The cells were seeded into 6-well plate (7.5 × 105 cells per well) for 24 h and then incubated with (i) PBS, (ii) HA-BNPs@Ptx, (iii) MW, (iv) BNPs@Ptx + MW, (v) HA-BNPs@Ptx + MW for 6 h. In order to optimize the thermal effects of nanoparticles, HA-BNPs@Ptx/4T1 cells were further exposed to MW irradiation at different power (0.4, 0.8, 1.2 and 1.6 W cm-2) for 1, 2, 3 and 4 min, respectively (see Additional file 1, Methods 1.3 for details). After incubation, the cells of the last 3 groups were exposed to MW irradiation (0.8 W cm-2, 4 min). The anti-cancer activity of each group was qualitatively examined by using Calcein-AM/propidium iodide (PI) double stain kit (Beyotime Biotechnology® Inc., Suzhou, Jiangsu, China). After the staining of Live/Dead cells, all cell samples were imaged under the inverted fluorescence microscope.
To quantitatively assess the synergistic chemo-thermal therapy, 4T1 cells were stained by Annexin V-(FITC)/PI apoptosis detection kit (4A Biotech Co., Ltd, Peking, China) and analyzed by flow cytometer.
In vivo TNBC models, biodistribution and targeting ability
BALB/c mice (female, 18-20 g) were provided by Dashuo Biological Technology (Chengdu, Sichuan, China). All processes were in accordance with the Chinese Society of Laboratory Animals on animal welfare and approved by the Animal Use and Care Management Advisory Committee of West China Hospital of Sichuan University (Approval No. 2017014A). 4T1 cells (1 × 106 /wells) were implanted into the second axillary mammary fat pad on the right side. When the volume of tumor reached about 100 mm3, 4T1 breast tumor-bearing mice were randomly assigned into two groups (6 mice per group) to compare CD44-targeted (DiI-HA-BNPs@Ptx) and non-targeted (DiI-BNPs@Ptx) nanoparticles. Nanoparticles (0.5 μL/g) were injected into 4T1 breast tumor-bearing mice via tail vein. In vivo fluorescence images were collected before and after injection of nanoparticles at 1, 3, 6 and 24 h using IVIS Spectrum system (Lumina XR, Caliper Life Sciences, Boston, Massachusetts, USA). To examine the biodistribution of nanoparticles, the main organs (heart, liver, spleen, lungs, and kidneys) and tumors were isolated from mice to perform ex-vivo imaging using the same IVIS Spectrum.
Blood chemistry analysis of BALB/c mice after 7 days intravenously injected with HA-BNPs@Ptx (see Additional file 1, Methods 1.4 for details).
In vivo mild hyperthermia - the 1st MW irradiation
The 1st MW irradiation aimed to generate mild hyperthermia to alter the TME to increase the uptake of nanoparticles. 6 mice in each group received mild hyperthermia at the tumor site with a MW power of 0.8 W cm-2. The temperature of tumor was monitored in real time using the infrared thermal mapping instrument. After mild hyperthermia, DiI-HA-BNPs@Ptx (0.5 μL/g) were immediately injected into 4T1 breast tumor-bearing mice via tail vein. The mice were euthanized and the tumor tissue was removed after 24 h. The retention of nanoparticles (red fluorescence) in tumor tissues were revealed using a pathological section scanner Pannoramic DESK (P-MIDI-P250, 3D HISTECH, Budapest, Hungary).
To further explore changes in TME, intratumoral perfusion and micro-vessel density of tumor were evaluated using contrast-enhanced ultrasound imaging (CEUS) and CD31 immunohistochemical analysis. Microbubbles (0.2 mL/kg, SonoVueTM, Bracco, Italy) were injected via tail vein 24 h after the 1st MW irradiation. CEUS was performed using an ultrasound scanner (iU22, Koninklijke Philips N.V., Eindhoven, Netherlands) with a 12-5 MHz transducer. Subsequently, mice were euthanized and tumors were sectioned and stained with CD31 (dilution 1:50, Wuhan Service bio Co., Ltd, Wuhan, Hubei, China) to evaluate the micro-vessel density. The pathological sections were imaged using a fluorescence microscope.
In vivo anti-tumor efficiency – the 2nd MW irradiation
The 2nd MW irradiation was dedicated to activate the sensitizer BML and to release the chemotherapy agent Ptx in the nanoparticles. Thirty 4T1 breast tumor-bearing mice were divided into 5 groups: (1) PBS (control group, G1), (2) 1st MW + HA-BNPs@Ptx (G2), (3) 1st MW + HA-BNPs + 2nd MW (G3), (4) HA-BNPs@Ptx + 2nd MW (conventional SDDS, G4), and (5) 1st MW + HA-BNPs@Ptx + 2nd MW (G5). After treated with mild hyperthermia (4 min MW exposure at 0.8 W cm-2, G2 G3 and G5), PBS, HA-BNPs or HA-BNPs@Ptx (0.5 mL/kg) were immediately injected into 4T1 breast tumor-bearing mice intravenously. 24 h after injection, the mice received a 2nd MW irradiation (0.8 W cm-2) for 4 min. The tumor temperature was monitored in real time by infrared thermal mapping instrument. The body weight and tumor volumes were recorded every three days. The tumor suppression rate (TSR) was calculated using the following formula:
TSR (%) = (Vc-Vx)/Vc×100%
Vc: volume of PBS group; Vx: volume of treatment group.
On day 18 after treatment, tumors and main organs were excised and fixed overnight in 10% buffered formalin. The tumors were sectioned and stained with H&E, Ki-67 antibody and TdT-mediated dUTP nick-end labeling (TUNEL) staining. And the main organs were sectioned and stained with H&E. The expression level of CD3+, CD4+, CD8+ of immune cells in tumor tissues were evaluated by immunofluorescence. The slides of tumor tissue were incubated with Anti-CD3 Rabbit pAb (P22646,1:700), Anti-CD4 Rabbit pAb (P06332 1:800), and Anti-CD8 Rabbit mAb (P10966, 1:500), following the standard procedure of Wuhan Service bio Co., Ltd, (Wuhan, Hubei, China). Nucleus was labeled with DAPI. The images were obtained by using the pathological section scanner Pannoramic DESK.
In addition, the peripheral blood was collected from treated mice before euthanasia to analyze the percentages of tumor antigen-specific CD3+, CD4+ and CD8+ T cells. Serum cells were incubated with 3 µL FITC anti-mouse CD3 antibody, 5 µL PE anti-mouse CD4 and 10 µL APC anti-mouse CD8 (4A Biotech Co., Ltd, Peking, China) for 1 h at room temperature, respectively. Finally, the cells were resuspended in PBS and analyzed by flow cytometry and Flow Jo software.
Statistical analysis
All data were expressed as mean ± standard deviation (SD). The statistical analysis was carried out with GraphPad Prism Version 8.0 software (GraphPad, USA). Comparisons among multiple groups were performed by one-way analysis of variance (ANOVA). And two-group comparisons were performed by Student’s t-test. Statistical significance was indicated by * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.