2.1 Materials

The reagents used for preparing liposomes have been indicated in the published articles of this project [13]. VEGF rabbit mAb, VEGFR2 rabbit mAb, TEM1 mouse mAb, TEM8 mouse mAb, P38 MAPK polyclonal antibody rabbit mAb, phospho-P38 MAPK rabbit mAb, MAPKAPK2 polyclonal antibody, phospho-MAPKAPK2 rabbit mAb, HSP27 rabbit mAb, and CTR1 rabbit mAb were used in this study.

2.2 Cells and animals

The A549 and human umbilical vein endothelial cell (HUVEC) lines were obtained from the Chinese Academy of Sciences Stem Cell Bank (Shanghai, China). Male BALB (Bagg Albino)/c nude mice, were provided by the Experimental Animal Center of Fujian University of Traditional Chinese Medicine (Fuzhou, China), grant number: FJTCM IACUC 2022063).

2.3 Preparation of iRGD-LP-CUR-PIP

iRGD-LP-CUR-PIP was prepared by thin-film hydration. In our previous study, We optimized the formulation parameters by single factor test and Box-Behnken design combined with response surface methodology, and morphology was observed by transmission electron microscopy. The release characteristics of iRGD-LP-CUR-PIP in vitro were investigated by membrane dialysis [13].

2.4 Western blotting

The protein concentrations were measured by a BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and denatured at 100 °C for 10 min. Approximately 50 µg of protein was separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). After incubation with the specified primary antibodies for 12 h at 4 °C and secondary antibodies for 60 min at room temperature, the membranes were washed three times with TBST. The blots were visualized using a two-color infrared imaging system (Odyssey, USA). Expression levels of proteins included VEGF (1 : 2000, 19003-1-AP, Proteintech), VEGFR2 (1:5000, 26415-1-AP, Proteintech), Integrin Alpha V + Beta3 (1 : 1000, bs-1310R, Bioss), TEM8 (1:1000, 15091-1-AP, Proteintech), TEM1 (1:2000, 60170-1-lg, Proteintech), MK2 (1:1000, 13949-1-AP, Proteintech), p-MK2 (1:1000, BS4902, Bioword), HSP27 (1:1000, 50353, Cell Signaling), P38 (1:1000, 14064-1-AP, Cell Proteintech), p-P38 (1:1000, AF4001, Proteintech), GAPDH (1:1000, 2118, CST), and CTR1 (1:1000, 16023-H02H, Sino Biological). These proteins were normalized to GAPDH expression. In the experiment, after successful transfer, we trimmed the desired band of interest based on the molecular weight of the antibody. We confirm that no image processing operations (such as stitching, erasing, or selective enhancement) have been performed other than uniform cropping and brightness/contrast adjustments for the entire image using Image Lab software.

2.5 Cellular uptake

We inoculated A549 cells into a cell culture dish (5 × 104 cells/culture dish). The cells were co-incubated with fluorescent iodide (DiR)-labeled solution, DiR-labeled LP, or DiR-labeled iRGD-LP for 2 h and washed twice with PBS [22]. Fixed with 4% paraformaldehyde for 10 min, washed with PBS, and stained with anti fluorescence quencher containing DAPI for 10 min, the cell uptake of different treatment groups was observed by using a confocal laser scanning microscope. All measurements were repeated three times.

2.6 Co-culturing system

In short, the cell co-culture model involved co-culturing HUVEC and A549 in a Transwell chamber co-culture system containing matrix gel, providing a tumor microenvironment for the growth of HCVECs and inducing their transformation into tumor-derived endothelial cells (Td-ECs). The specific steps were as follows: A549 cell suspension with a density of 1 × 104/mL was prepared and inoculated in the upper chamber to prepare 1 × 105/mL HUVECs, which were inoculated into the lower lumen. The culture was continued every 24 h to form monolayer cells in a subfusion state (80%–90%) [23]. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed to identify changes in cell characteristics under co-culture conditions to determine whether the co-culture model was successfully constructed. At the same time, HUVECs cultured separately were used as control cells, and their inoculation density, culture conditions, and time were the same as those of co-cultured cells.

2.7 Cell migration assays

We inoculated Td-ECs at 6 × 105cell/well in a 6-well plate, which was incubated at 37 °C for 24 h. We then scraped three lines with a 200 µL pipette tip for each well and washed them with PBS three times. Subsequently, different drugs were added to various wells for 24 h, each with a volume of 10–60 µL. The control group was added with 60 µL of culture medium. At 0 and 24 h, the width was captured using a Nikon eclipse Ti-S Microscope. The anti-migration effect of treatment groups was evaluated by the percentage of gap closure, which was calculated according to the following formula:

$$ \:Migration\:rate = 1 - \frac} - Width_} }}} }} \times \:100\% $$

2.8 Immunofluorescence analysis

Fix tumor tissue with 4% paraformaldehyde, then slice and detect protein fluorescence intensity using CD31, VEGF, and VEGFR2 antibodies. Inoculate Td-ECs into a cell culture dish containing 5 × 104 cells/dish, incubate with the drug containing formula for 24 h, and incubate at room temperature for 1.5 h using fixation, permeabilization, blocking, VEGF primary antibody binding, and secondary antibody. Add 10–20 µL of DAPI dropwise and stain in the dark for 10 min. All samples are observed under a fluorescence microscope (LEICA, USA).

2.9 Molecular docking verification between the core substances and key targets

Download and screen the three-dimensional structure of PIP active compounds through pubchem, find the corresponding protein ID in uniport, search for the pdb format of the corresponding structure in PDB, and use PyMol software to remove water molecules and existing ligands from the receptor. Finally, molecular docking was performed using MOE 2019 software, and < − 5.0 kcal/mol suggested strong binding activity [24].

2.10 Plasmid construction and transfection

To silence VEGFR2 in Td-ECs, we cloned VEGFR2-shRNA (NM_002253.2) into the VB191 vector (APExBIO, Shanghai, China). For transient knockdown studies, VEGFR2-shRNA and control shRNA (sh-NC) plasmids were transfected for 24 h using Lipo 3000™ reagent (ThermoFisher, L3000015, USA).

2.11 Statistical analysis

Statistical analysis was performed using Prism 8.0 (GraphPad). All experimental data were shown by mean ± standard deviation (SD). For comparisons between two groups, a two-tailed Student’s t test was applied. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used and statistical significance was set at P < 0.05.