iRGD-modified co-loaded curcumin piperine liposomes inhibit angiogenesis in non-small cell lung cancer through the VEGFR2/P38/MK2 signaling axis.

Introduction
Lung cancer is the second most common cancer in the world, with high incidence and mortality rates [1]. The World Health Organization divides lung cancer into two major categories; non-small cell lung cancer (NSCLC) accounts for 80%–85% of all lung cancer cases [2–4]. Chemotherapy is effective in NSCLC but has unavoidable toxicities and side effects [5, 6]. Numerous studies have shown that combination therapy has enormous potential value in treating lung cancer. The combination of targeted or chemotherapy drugs with natural compounds may improve the efficacy of drug therapy and reduce adverse reactions [7]. The anti-cancer properties of curcumin (CUR) are currently a research hotspot [8, 9], but its chemical properties are unstable [10, 11]. The combination of piperine (PIP) and CUR can improve its bioavailability [12]. Therefore, our research group successfully prepared liposomes containing iRGD-LP-CUR-PIP in the early stage, and we evaluated and validated their anti-tumor efficacy via in vitro and in vivo experiments [13]. However, the mechanism of their action is unclear, and further exploration is needed to clarify their roles.
The growth of tumors relies on abundant blood vessels to provide nutrients, and abnormal angiogenesis is a key factor in tumor progression.As one of the most effective vascular growth factors, vascular endothelial growth factor (VEGF) [14]. These VEGFs bind to VEGF receptors (VEGFRs, including VEGFR-1-3) and neurokinins (neurokinin-1 and − 2), leading to self-phosphorylation of tyrosine residues in these receptors, which can subsequently activate cellular substances such as members of the mitogen-activated protein kinase (MAPK) superfamily, thereby regulating vascular endothelial cell proliferation, differentiation, and survival [15, 16]. Natural compounds play an important role in regulating tumor angiogenesis through VEGF [17], such as CUR, resveratrol, green tea polyphenols, and PIP can synergistically act with VEGF/VEGFR2 inhibitors to enhance their anti-angiogenic effects [18]. Thus, blocking-up the VEGF/VEGFR2 signaling pathway can effectively curb tumor angiogenes.
The tumor-targeting lipid system, utilizing iRGD peptides to modify natural compounds, offers a highly promising drug delivery system [19–21]. Therefore, iRGD coupling and combined anti-angiogenic natural products can enhance the active targeting effect on tumor angiogenesis and reduce toxic side effects. In our previous study, we successfully constructed iRGD-LP-CUR-PIP and verified in vitro and in vivo that it had anti-NSCLC activity. However, the mechanism of action of CUR-PIP on NSCLC is unknown. In this study, we found that iRGD peptide surface-modified liposomes effectively enhanced uptake and anti-angiogenic activity of A549 tumor cells. Mechanism studies have shown that iRGD-LP-CUR-PIP inhibits tumor endothelial cell proliferation and differentiation by inhibiting the VEGFR2/P38/MK2 signaling axis, thereby inhibiting neovascularization. In addition, iRGD-LP-CUR-PIP inhibits the growth and angiogenesis of A549 tumors in nude mice by downregulating VEGFR2/P38/MK2.

Materials and methods

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.

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).

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].

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.

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.

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.

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:

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).

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].

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).

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.

Results

iRGD-LP-CUR-PIP May inhibit tumor angiogenesis via the VEGFR2/P38/MK2 pathway
In this study, the mechanism of action of iRGD-LP-CUR-PIP on the tumors of A549 tumor-bearing nude mice was investigated (Fig. 1A). We measured the anti-angiogenesis effects by using immunofluorescence of CD31, which is an endothelial marker that allows us to quantify vessels. Our results showed that iRGD-LP-CUR-PIP had a significantly decreasing effect on vascular density as measured by CD31 in tumor mice (Fig. 1B and C). The average fluorescence intensity of VEGF and VEGFR2 in the tumor tissues was detected using immunofluorescence. We observed that iRGD-LP-CUR-PIP could effectively reduce the fluorescence expression intensity of VEGF and VEGFR2 in the tumor tissues of A549 hormonal nude mice (Fig. 1D and F). WB confirmed that iRGD-LP-CUR-PIP reduced the protein expression of VEGF and VEGFR2 in tumor tissue (Fig. 1E and G). The VEGF-VEGFR2 signaling axis could regulate phosphorylation activation of p38 MAPK. Mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2 or MK2), the downstream substrate of p38 MAPK, governs the activation and deactivation of heat shock protein 27 (HSP27). A previous study reported that the activation of the p38MAPK/MK2/HSP27 axis promotes angiogenesis [25]. In this study, we found that iRGD-LP-CUR-PIP inhibited the expression of p-P38, p-MK2, and HSP27 in tumor tissues (Fig. 1H and J). Interestingly, iRGD-LP-CUR-PIP and iRGD-LP-CUR decreased VEGF and VEGFR2 protein levels equally. However, for the phosphorylation of downstream proteins (p-P38 and p-MK2), iRGD-LP-CUR-PIP decreased protein levels more than iRGD-LP-CUR. In summary, iRGD-LP-CUR-PIP inhibited NSCLC angiogenesis, and its anti-tumor effects may be related to downregulating the VEGFR2/P38/MK2 signaling pathway.

iRGD-LP-CUR-PIP targets tumor cells
To determine the uptake of LP by cells, we incubated A549 with different DiR-labeled liposome formulations for 2 h. We used DiR (red fluorescence) to track the cell distribution of LP and DAPI (blue fluorescence) to stain the nucleus. As shown in Fig. 2A and B, tumor cells treated with non-targeted LP showed weak red fluorescence. To support RGD integrin αvβ3, we co-treated free iRGD (fiRGD) and iRGD-LP into A549 cells to investigate their interactions during iRGD-LP uptake. Figure 2C and D show the competitive combination of fiRGD and iRGD-LP αvβ3. At elevated concentrations of fiRGD, the uptake of iRGD-LP decreased accordingly. Overall, iRGD-LP could be effectively absorbed by A549 cells, which is of great significance for delivering natural products of anti-lung cancer effects into cells.

iRGD-LP-CUR-PIP reduced VEGF in Td-ECs
The above results indicated that iRGD-LP-CUR-PIP inhibited angiogenesis and had a targeted effect on tumor cells. When we used HUVECs for administration, we found that iRGD-LP-CUR-PIP had no effect on the generation of VEGF in HUVECs (Fig. 3A). Therefore, Td-ECs were prepared using Transwell cells to construct a co-culture system of A549 and HUVECs. Compared with HUVECs, Td-ECs underwent a series of ultrastructural changes. SEM results showed that the elongation of Td-ECs was reduced while the filamentous pseudopods on the cell ends became few and short, which indicated that the cells were in an active state and suitable for migration (Fig. 3B). TEM results showed that Td-ECs exhibited more vesicular structures and obvious mitochondrial vacuolization than the other cells (Fig. 3C). WB results showed that Td-ECs highly expressed tumor endothelial cell-specific antigens TEM1 and TEM8 (Fig. 3D and G). We observed that iRGD-LP-CUR-PIP effectively reduced the expression of VEGF in Td-ECs by immunofluorescence and WB (Fig. 3H and J). Therefore, iRGD-LP-CUR-PIP was effective on tumor endothelial cells without affecting normal endothelial cells. Blood vessels present in a specific tumor are actually embedded blood vessels composed of endothelial cells and tumor cells, which are interdependent. Therefore, targeting tumor blood vessels is closely aligned to the internal environment of the organism, cutting off the nutritional supply of the tumor by inhibiting tumor angiogenesis, rendering it unable to continue growing and ultimately achieving tumor inhibition.

iRGD-LP-CUR-PIP downregulated VEGFR2 and inhibited phosphorylation of P38 and MK2 in vitro
To further specify the anti-angiogenesis effects of iRGD-LP-CUR-PIP in vitro, we conducted cell migration experiments on Td-ECs co-cultured with A549/HUVECs. In the cell migration experiments, compared with the control group, different liposomes containing CUR all had larger wound areas, with iRGD-LP-CUR-PIP being more significant, indicating a greater anti-migration effect. The migration rates of co-cultured cells treated with iRGD-LP-CUR and iRGD-LP-CUR were 12.4% and 13.4%, respectively, which were much lower than those of LP-CUR-PIP (18.6%) and control group (25.4%) (Fig. 4A and B). Signaling pathway proteins of Td-ECs were detected by WB (Fig. 4C and F). iRGD-LP-CUR-PIP reduced the protein expression of VEGFR2, inhibited the phosphorylation of P38 and MK2, and downregulated HSP27. In addition, treatment with iRGD modification had better pharmacological effects than other groups. Similarly, in vivo, for VEGF and VEGFR2, iRGD-LP-CUR-PIP and iRGD-LP-CUR decreased protein levels equally. However, for the phosphorylation of downstream proteins (p-P38 and p-MK2), iRGD-LP-CUR-PIP decreased protein levels more than iRGD-LP-CUR.

PIP inhibited CTR1 to affect the downstream signaling pathway of VEGFR2
We found that the iRGD-LP-CUR-PIP group and iRGD-LP-CUR group could significantly downregulate the expression of VEGF and VEGFR2 proteins equally. However, for the expression of downstream phosphorylation, the difference between the two groups was obvious. Copper transport 1 (CTR1) participated in VEGFR2 signaling transduction. Upon VEGF stimulation, CTR1 is rapidly sulfonylated at the cytoplasmic C-terminus of Cys189, which induces the formation of the CTR1-VEGFR2 disulfide bond and co-internalization with early nuclear endosomes, driving sustained VEGFR2 signaling and stimulating downstream phosphorylation secretion, enhancing tumor angiogenesis [26] (Fig. 5D). To further investigate the potential binding interactions of PIP and CTR1, we performed molecular docking analysis to assess the binding affinity of PIP to CTR1. The molecular docking score (kcal/mol− 1) was used to show the binding strength. In our results, all of the binding energies were below − 4.0 kJ mol− 1, and these results indicated that CUR had very strong binding activity to VEGF targets and PIP had moderate to strong binding activity to key CTR1 targets. The estimated docking binding energy was 5.7297 kcal/mol. In this conformation, the ligand mainly contacted the protein through hydrogen bonds. The ligand could form hydrogen bonds with the N atom of the Asn 104 side chain of the protein, which was conjugated with the side chain hydrocarbon atom XingchengCH benzene ring of Ile101 (Fig. 5A). The results of ex vivo WB showed that the addition of PIP significantly increased the inhibitory effect of liposome drugs on CTR1 in vivo and vitro (Fig. 5B and C). Thus, PIP may affect the downstream signaling pathway of VEGFR2 by inhibiting CTR1.

Effect of iRGD-LP-CUR-PIP treatment and VEGFR2 silencing alone and in combination on VEGFR2/P38/MK2 signaling in Td-ECs.
Knockdown of VEGFR2 expression by ShRNA to block the downstream signaling pathway of VEGFR2 further validated that iRGD-LP-CUR-PIP inhibited tumor angiogenesis through the VEGFR2/P38/MK2 signaling pathway. The results were as follows: the shRNA-VEGFR2 silencing plasmid was constructed (Fig. 6A), and the results verified by WB showed that the silencing plasmid significantly inhibited the expression of VEGFR2 in Td-ECs compared with the control group at 24 h post-transfection. We chose this plasmid for the cell experiments (Fig. 6B). Given that iRGD-LP-CUR-PIP treatment of Td-ECs inhibited VEGF signaling, we asked whether such effects of iRGD-LP-CUR-PIP would be abrogated with silencing of VEGFR2. We observed that VEGFR2 silencing almost completely inhibited the phosphorylation of P38 and MAPK2, and the combination of iRGD-LP-CUR-PIP with VEGFR2 silencing did not augment the inhibition of the phosphorylation of P38 and MK2 (Fig. 6C and F). These observations proved the regulation of the iRGD-LP-CUR-PIP on the VEGFR2/P38/MK2 signaling pathway.

Discussion
iRGD is a tumor-targeting and tumor-penetrating peptide that binds with αν integrins. Our research results showed that A549 cells co-cultured with iRGD-LP exhibited strong red fluorescence, and they were clustered around the cell membrane. This result was attributed to the interaction between integrin αvβ3 on the cell surface and RGD on iRGD-LP, which reportedly occurs during the internalization of RGD ligands into cells through receptor-mediated endocytosis [27, 28]. In addition, when iRGD was co-administered with nanoformulations, the bystander effect of iRGD activated a large amount of drugs to be transported, which could improve the bioavailability and efficacy of drug delivery [29, 30]. In our previous study, we designed and prepared an iRGD-LP-CUR-PIP with iRGD as the functional targeting substrate, CUR and PIP as the model drugs, and LP as the delivery system. We verified its anti-NSCLC activity in vitro and in vivo, and we found that drugs with iRGD had good anti-tumor efficacy, and the combination of CUR and PIP was superior to CUR alone. However, its mechanism remains unclear.
To further investigate the inhibitory effect of iRGD-LP-CUR-PIP on tumor angiogenesis through the VEGFR2/P38/MK2 pathway, we used WB and immunofluorescence methods to validate the results in vivo. The results showed that iRGD-LP-CUR-PIP inhibited the expression of p-P38, p-MK2, and HSP27 in tumor tissue. Td-ECs were constructed in vitro, and their microstructure and specific antigens were detected using SEM, TEM, and WB. The results showed that tumor-induced phenotypic changes occurred in vascular endothelial cells, which was consistent with the current understanding of the interaction between tumor cells and vascular endothelial cells. Thus, we successfully constructed a co-culture model for preparing tumor endothelial cells. Unlike normal endothelial cells, tumor vascular endothelial cells are located in the tumor microenvironment, enhancing their ability to proliferate, migrate, and invade. Tumors and blood vessels are interdependent. Tumors provide vascular growth factors to endothelial cells, accelerating the occurrence and development of angiogenesis, whereas endothelial cells provide necessary nutrients for tumor proliferation and migration [31].Therefore, treating tumor blood vessels as targets must occur close to the growth environment within the body. By inhibiting the regeneration of tumor blood vessels, the nutritional supply to the tumor can be cut off, preventing its growth and achieving anti-tumor effects. The WB results further confirmed that iRGD-LP-CUR-PIP was effective on tumor endothelial cells without affecting normal endothelial cells.
An interesting finding in the validation of in vitro and in vivo mechanisms was that iRGD-LP-CUR-PIP and iRGD-LP-CUR both reduced VEGF and VEGFR2 protein levels. However, for downstream proteins (p-P38MAPK, p-MK2, and HSP27), iRGD-LP-CUR-PIP was more effective in reducing protein levels than iRGD-LP-CUR, and its anti-tumor effect may be related to the downregulation of the VEGFR2/P38/MK2 signaling pathway. According to reports, CTR1 is involved in VEGFR2 signaling transduction. Under VEGF stimulation, CTR1 rapidly undergoes sulfonylation at the cytoplasmic C-terminus of Cys189, inducing the formation of disulfide bonds between CTR1-VEGFR2 and co-internalizing with early endosomes, driving the sustained VEGFR2 signaling pathway and stimulating downstream phosphorylation secretion to enhance tumor angiogenesis [26]. Molecular docking results also confirmed that PIP may affect the downstream signaling pathway of VEGFR2 by inhibiting CTR1. In subsequent plasmid transfection experiments, we observed that VEGFR2 silencing almost completely inhibited the phosphorylation of P38 and MAPK2, whereas the binding of iRGD-LP-CUR-PIP to VEGFR2 silencing did not enhance the inhibition of P38 and MK2 phosphorylation. These observations further confirmed the regulatory effect of iRGD-LP-CUR-PIP on the VEGFR2/P38/MK2 signaling pathway.

Conclusion
The results of this study indicated that surface modification of iRGD peptides loaded with CUR-PIP could effectively enhance the cellular uptake and anti-angiogenic ability of Td-ECs. Research on its mechanism suggested that iRGD-LP-CUR-PIP inhibited angiogenesis by inhibiting the VEGF-VEGFR2 signaling axis and inducing cell migration in Td-ECs. In addition, iRGD-LP-CUR-PIP inhibited the growth and angiogenesis of A549 tumors in nude mice by downregulating VEGF-VEGFR2. Our research results indicated that using iRGD peptide-functionalized CUR-PIP as a carrier for anticancer drugs may be an efficient way to achieve tumor-targeted anti-angiogenic synergistic effects by eliminating pro-angiogenic signaling pathways.

Supplementary Information
Below is the link to the electronic supplementary material.