Materials
Poly (lactic-co-glycolic acid)-carboxylic acid (PLGA-COOH) with a molecular weight of 20,000 Da (LA:GA = 50/50) was obtained from Jinan Daigang Biotechnology Co., Ltd. (Jinan, China). The MMP-2 substrate peptide (EGPLGVRGK) and PEG3000-COOH were custom synthesized by Qiyue Biotechnology Co., Ltd. (Xi’an, China). FITC-labeled MMP-2-PEG was also custom synthesized. DSF was obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Recombinant mouse MMP-2 was purchased from Abcam Inc. (Abcam, Cambridge, Britain), and p-aminophenyl mercuric acid (APMA) was obtained from Genmed Scientifics, Inc., USA.
Synthesis of PFP@PDM-PEG and PFP@PD
To synthesize the PEGylated MMP-2 peptide (EGPLGVRGK-PEG), we first used Fmoc solid-phase peptide synthesis (SPPS) following a known method to create the MMP-2 peptide (EGPLGVRGK). Next, PEG3000-COOH was introduced, and the reaction vessel was sequentially washed with dimethylformamide (DMF), dichloromethane (DCM), and methanol before drying. The mixture was then cleaved using a combination of trifluoroacetic acid (TFA), water, 1,2-ethyldimercaptan (EDT), and triisopropyl silane (TIS), followed by sequential evaporation to eliminate the organic solvent DCM. Finally, the crude product was subjected to dialysis against distilled water (MWCO 3,500 Da) for purification. Then, the PEGylated MMP-2 peptide was affixed to the surface of the PLGA-COOH shell using the standard carbodiimide method, resulting in the formation of PLGA-MMP-2-PEG. This material was subsequently subjected to freeze-drying for further use.
For the next stage, a mixture comprising PLGA-MMP-2-PEG, DSF, and PFP was dissolved in dimethyl sulfoxide (DMSO) and subjected to ultrasonication in an ice bath for 3 min (100 W, on:off = 1:1) using a sonicator (Sonics & Materials Inc., Newtown, CT, USA). Next, 8 ml of a polyvinyl alcohol solution (4% w/v) was introduced, and the ultrasonication process was repeated under the same conditions. Subsequently, a solution of isopropyl alcohol (10 ml, 2% v/v) was added, and the mixture was magnetically stirred in an ice bath for 3 h to remove the organic solvent. After this process, the PFP@PDM-PEG nanodroplets were obtained by centrifugation (4 °C, 12,000 rpm, 8 min), followed by washing and resuspension.
To optimize the synthesis of the nanodroplets, we tested different feeding ratios of DSF and PLGA-MMP-2-PEG, including DSF/PLGA-MMP-2-PEG (w/w) ratios of 1:2.5, 1:7.5, 1:12.5, 1:17.5, and 1:22.5. Notably, the feeding ratio of 1:12.5 (w/w) yielded the highest encapsulation efficiency (EE) (Additional file 1: Fig. S1). Consequently, we utilized this specific feeding ratio in subsequent experiments.
The release curve of DSF was assessed using the dialysis membranes diffusion technique. First, PFP@PDM-PEG solution was equivalently poured into two dialysis membranes (MW cut-off = 2000 Da, n = 3), one of which was irradiated with LIFU (2 W cm−2 for 3 min), and the other one was without any treatment. Then, the dialysis membrane was placed into an 80 ml buffer solution containing Tween-80 (v/v = 0.1%) and ethanol (v/v = 30%). Next shake slowly on a shaker (120 rpm, 37 °C). At specific times (0.5, 1, 2, 4, 8, 16, 24, 48 h), 500 μL buffer solution was collected for measurement by the HPLC system and replaced by 500 μL of a fresh buffer solution. Finally, the cumulative release ratio of DSF in the two groups was calculated.
For the synthesis of the PFP@PD nanodroplets, a mixture containing PLGA-COOH (50 mg), DSF (5 mg), and PFP (200 µl) was dissolved in DMSO and subjected to ultrasonication. The subsequent steps mirrored the procedure described above. Additionally, fluorescent dyes, such as DiI, DiR or FITC, were introduced during the synthesis process to confer the nanodroplets with fluorescence imaging capability.
Characterization of the PFP@PDM-PEG and PFP@PD nanodroplets
The morphology of the PFP@PDM-PEG and PFP@PD nanodroplets was examined using transmission electron microscopy (TEM, Hitachi H-7500, Tokyo, Japan) and scanning electron microscopy (SEM, Hitachi SU8010, Tokyo, Japan). Particle size and zeta potential were determined using a Malvern Zetasizer Nano ZS90 (UK). Nuclear magnetic resonance (1H NMR) spectra were recorded on an NMR spectrometer (Bruker AVANCE NEO 400, Bruker BioSpin GmbH, Rheinstetten, Germany) to confirm the successful formation of covalent bonds between PLGA-COOH, the MMP-2 substrate peptide, and PEG.
To quantitatively evaluate the binding rate of FITC-labeled MMP-2-PEG to PLGA-COOH, flow cytometry (FCM) was employed (Sonic SH800, Japan), using PFP@PD nanodroplets as a control. The excitation wavelength was set to 488 nm.
Cell culture and animal model
The 4T1 cell line (a mouse TNBC cell line) was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, v/v) and 1% penicillin‒streptomycin (v/v) under standard conditions (5% CO2, 37 °C). All experiments involving mice were conducted following the approval of the Animal Care and Use Committee of Chongqing Medical University. Female BALB/c mice (6–8 weeks old) were procured from Chengdu Dossy Experimental Animals Co., Ltd. The 4T1 tumor-bearing model was established by subcutaneously injecting 1 × 107 4T1 cells into the right lower abdomen of each mouse.
In vitro cellular uptake and in vivo biodistribution
In vitro cellular uptake
In the in vitro cellular uptake study, 4T1 cells were seeded in 3.5 cm confocal dishes at a density of 2 × 104 cells per dish. DiI-labeled PFP@PDM-PEG (with an equal PLGA concentration of 1.0 mg mL−1) was added for incubation at 37 °C for 1 h in the absence or presence of APMA-activated MMP-2 (1 µg mL−1). Subsequently, the pretreated nanodroplets were added to each well for 1, 2, or 4 h of coincubation. After a 20-min incubation with DAPI, the cells were examined using confocal laser scanning microscopy (CLSM, OLYMPUS confocal microscope CSU-W1, Japan). Additionally, flow cytometry (FCM, Sonic SH800, Japan) was employed to assess the impact of MMP-2-triggered PEG deshielding on 4T1 cell uptake.
In vivo biodistribution
For the in vivo biodistribution experiment, tumor-bearing mice were randomly divided into two groups (n = 3): the PFP@PDM-PEG group and the PFP@PD group. Each group of mice received an intravenous injection of DIR-labeled nanodroplets at a dose of 10 mg kg−1 DSF. Small animal fluorescence imaging equipment (AniView 600 pro, Guangzhou, China) was used to monitor the accumulation of nanodroplets in the tumor region at various time points (0, 3, 6, 12, 24, and 36 h after injection). Additionally, three mice from each group were euthanized after 24 h, and their major organs (heart, liver, spleen, lungs, kidneys) and tumor tissues were collected for fluorescence imaging. Quantitative analysis was performed on the obtained fluorescence images.
Drug penetration
To evaluate the ability of the drug delivering nanodroplets to penetrate from the surface to the core, 3D spheroid models of 4T1 cells were constructed. Using 6-well ultralow adherent plates, 1 × 106 4T1 cells were cultured for 10 days. The cells were then incubated with FITC-labeled PFP@PDM-PEG (with an equal PLGA concentration of 1.0 mg mL−1) at 37 °C for 1 h in the absence or presence of APMA-activated MMP-2 (1 µg mL−1). Subsequently, pretreated nanodroplets were added to 3D cell spheroids with or without LIFU irradiation. After 4 h of coincubation, CLSM was used for observation. Scanning various layers from the surface to the interior of the tumor spheroids was conducted to quantify the penetration depth. For the assessment of intratumoral drug penetration, mice bearing 4T1 tumors were randomly divided into three groups: the PFP@PD group, the PFP@PDM-PEG group, and the PFP@PDM-PEG + LIFU group. In the PFP@PDM-PEG + LIFU group, a LIFU device (LMSC051 ACA; Institute of Ultrasound Imaging, Chongqing Medical University, Chongqing, China) was employed. The nanodroplets were labeled with the fluorescent dye DiI. Ultrasound irradiation was administered 24 h after injection. Confocal laser scanning microscopy (CLSM) was performed on frozen tumor slices to directly observe drug penetration after staining with DAPI.
In vitro and in vivo ultrasound imaging (USI) performance
In vitro phase transition confirmation
To confirm the acoustically induced phase transition capability of the PFP@PDM-PEG, the nanodroplets were excited by LIFU, and their phase transition was observed under an optical microscope. In Vitro Ultrasound Imaging (USI) Performance: The in vitro ultrasound imaging (USI) performance of PFP@PDM-PEG was assessed by diluting a sample of PFP@PDM-PEG and placing it in a perforated 3% agarose gel phantom. The sample was then exposed to pulsed-wave LIFU irradiation for durations ranging from one to four minutes at power levels of 1–4 W cm−2. Ultrasound images were captured before and after each LIFU treatment to determine the most suitable LIFU parameters for in vivo evaluation.
In vivo study
4T1 tumor-bearing mice were randomly divided into two groups. Each group received an intravenous injection of PFP@PD or PFP@PDM-PEG (200 μL, with an equal PLGA concentration of 1.0 mg mL−1). After 24 h, US mode, contrast-enhanced ultrasound (CEUS) mode, and peak intensity (PI) mode images were acquired at the tumor site both before and after LIFU treatment (2 W cm−2, 3 min). The analysis of ultrasonic intensity was conducted using DFY ultrasound imaging analysis software developed by the Institution of Ultrasound Imaging at Chongqing Medical University, China.
Antitumor efficacy of PFP@PDM-PEG in vitro.
In vitro cytotoxicity evaluation
4T1 cells were seeded in 96-well plates at a density of 4 × 103 cells per well and incubated for 24 h. Subsequently, the cells were cultured for an additional 24 h in fresh medium (2% FBS) containing varying concentrations of DSF or PFP@PDM-PEG (equivalent DSF concentrations of 250 nM, 500 nM, 750 nM, 1000 nM, 1500 nM, and 2000 nM). The PFP@PDM-PEG group was further divided into two subgroups, with or without APMA-activated MMP-2 pretreatment (1 µg mL−1). After a 2-h incubation with 10 μL of CCK-8 reagent, the viabilities of the cells in each well were determined using a microplate reader (Cytation 3, BioTek, Vermont, USA) at a wavelength of 450 nm. The cell viability at the half maximal inhibitory concentration (IC50) was calculated using the provided equation considering the background absorbance of the medium.
Cell apoptosis assay
4T1 cells were seeded into a six-well plate and cultivated for 24 h. Subsequently, 4T1 cells were randomly assigned to different groups (n = 3): (i) Control group, (ii) LIFU group (exposed only to LIFU), (iii) DSF group, (iv) PFP@PDM-PEG group, (v) PFP@PDM-PEG with APMA-activated MMP-2 (1 µg mL−1), and (vi) PFP@PDM-PEG with APMA-activated MMP-2 (1 µg mL−1) + LIFU group (LIFU irradiation was applied after a 4-h incubation. After 24 h, all cells were collected, stained with Annexin V-FITC/PI apoptosis detection kit, and analyzed for cellular apoptosis using FCM.
Assessment of cell migration and invasion
Cell Migration: 4T1 cells (2.5 × 105 per well) were initially seeded in 6-well plates. When the cell density reached 80–90%, a precise denuded area was created in the center of each well using a 10 μl pipette tip. Subsequently, the detached cells were washed away with PBS. The cells were then subjected to various treatments, including serum-free medium, DSF, and PFP@PDM-PEG (with an equivalent DSF concentration of 1000 nM). The PFP@PDM-PEG group was further subdivided into two groups for pretreatment with APMA-activated MMP-2 (1 µg mL−1) or not. To exclude any potential effects of LIFU on cell migration, all groups received LIFU irradiation (2 W cm−2, 3 min) after a 4-h incubation. After 24 h, microscopic observations were made, image collection was performed, and the scratch width was quantitatively analyzed using ImageJ software (version 1.52 g, Java 1.80_172, NIH).
Cell Invasion: For the cell invasion study, 1 × 105 cells were plated on Matrigel-coated Transwell chambers (Costar, Corning, NY, USA). Following the same treatment conditions as described above, 4T1 cells were treated with serum-free medium, DSF, or PFP@PDM-PEG for 24 h. Cells that had migrated to the lower surface of the membrane were fixed with 75% ethanol at 4 °C for 20 min. Then, the cells were stained for 15 min with hematoxylin. Subsequently, the cells in random objective fields were observed at × 10 magnification after being washed three times.
VM formation assay
For the VM formation assay, an 8-well culture slide (Falcon®, Corning, USA) was coated with 30 μL of Matrigel (Corning Cat. No. 354234) using a thin gel coating method at 37 °C for 15 min. The cells were suspended in serum-free Opti-MEM (Life Technologies, USA) supplemented with 1% GlutaMAXTM (Life Technologies, USA) and placed on the Matrigel-coated surface. Each well received 2.5 × 105 cells and was subjected to various treatments based on the experimental group (such as those in the cell migration study). The cells were then incubated for 24 h. After incubation, the slides were examined and photographed using an inverted microscope (OLYMPUS, Tokyo, Japan). Quantitative analysis of VM formation was performed using ImageJ software.
In vivo VM targeted therapy
To evaluate the antitumor efficacy of PFP@PDM-PEG due to its inhibition of VM formation in vivo, 4T1 tumor-bearing mice were randomly assigned to different groups (n = 5):
(i) Control group, (ii) LIFU group (exposed only to LIFU), (iii) DSF group, (iv) PFP@PD group, (v) PFP@PDM-PEG group, and (vi) PFP@PDM-PEG + LIFU group. Mice were administered 200 μL of normal saline, PFP@PD, PFP@PDM-PEG, or DSF suspension via the tail vein (equivalent DSF concentration: 10 mg kg−1). LIFU irradiation (2 W cm−2, 3 min) was applied to the tumor area 24 h postinjection. Tumor dimensions were measured every two days, and tumor volume was calculated using the formula (length × width^2)/2 mm3. After two weeks, blood samples were collected from the orbit to assess routine blood indices and biochemical indices. Subsequently, the mice in each group were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) were stained with H&E to evaluate histopathological toxicity. The metastatic burden in both the lungs and liver was evaluated by counting the number of metastatic nodules in the histopathological whole-slide images (WSIs). After staining with CD34/PAS (Abcam, ab8536, Cambridge, MA), COL1 (Affinity Biosciences, AF7001, Jiangsu, China), or activated MMP-2 (Abcam, ab92536, Cambridge, MA), the tumor tissues were examined to determine the mechanism by which PFP@PDM-PEG inhibits VM development. Furthermore, the protein levels of COL1 and active MMP-2 in tumor tissues were quantified through Western blot analysis.
Statistical analysis
The results are presented as the mean ± standard deviation of the mean for each sample. Statistical analysis was performed using one-way analysis of variance (ANOVA) and t tests in GraphPad Prism 9.0 software (GraphPad Prism software, San Diego, CA, USA) to compare data between different groups.