In vivo inhibition of angiogenesis by htsFLT01/MiRGD nano complex

Highlights

• •htsFLT01 is a novel fusion protein that targets and neutralizes both VEGF and PlGF, showing potential for advanced anti-angiogenic cancer therapies.
• •MiRGD nano carrier is an innovative nanoparticle-based system that has shown significant potential in delivering nucleic acids for gene therapy.
• •the htsFLT01/MiRGD nano complex was effectively delivered to MCF7 breast cancer cells via iRGD targeting.
• •Protein analysis confirmed both intracellular production and extracellular secretion of htsFLT01.
• •Gene expression studies indicated the complex neutralizes VEGF and positively regulates genes related to apoptosis, inflammation, and metabolism.
• •In animal models, tumor size decreased, angiogenesis and cell division were reduced, and cancer cell necrosis increased after treatment.
• •Immunofluorescence confirmed decreased VEGF, VEGFR, and CD31 protein levels.

Abstract

The inhibition of angiogenesis is a crucial therapeutic strategy in cancer treatment, as it limits tumor growth and metastasis. In this study, we investigate the anti-angiogenic potential of a novel htsFLT01/MiRGD nanocomplex, designed to target key angiogenesis markers in cancer. This nanocomplex integrates the anti-angiogenic fusion protein htsFLT01 with the MiRGD peptide to enhance its efficacy. Our findings demonstrate that htsFLT01/MiRGD effectively suppresses angiogenesis both in vitro and in vivo, particularly in breast cancer models. Histological and molecular analyses reveal a significant reduction in blood vessel formation, accompanied by structural changes in tumor tissue. Furthermore, the expression levels of key angiogenesis-related genes, including VEGF, VEGFR, and CD31, are markedly downregulated, highlighting the therapeutic potential of this nanocomplex. Beyond its anti-angiogenic effects, the treatment also induces apoptosis and inhibits tumor cell proliferation, reinforcing its role as a promising targeted therapy for angiogenesis-dependent malignancies. These results underscore the potential of htsFLT01/MiRGD in cancer treatment and pave the way for future clinical applications in anti-angiogenic therapies.

Introduction

Cancer and related diseases remain among the most pressing health challenges of the 21st century [1]. Under normal physiological conditions, angiogenesis plays a crucial role in wound healing, embryonic development, and tissue regeneration [2,3]. However, in pathological conditions such as cancer, dysregulated and excessive angiogenesis promotes tumor growth, metastasis, and resistance to therapy [4]. Tumor cells exploit angiogenesis to secure nutrients and oxygen, facilitating their proliferation and invasion into distant tissues [5]. Consequently, targeting angiogenesis has emerged as a promising strategy in cancer therapy.

Vascular endothelial growth factor (VEGF) is a key regulator of tumor-associated angiogenesis and serves as a critical target for therapeutic interventions [6]. Several anti-angiogenic agents, including monoclonal antibodies (e.g., Bevacizumab) and tyrosine kinase inhibitors, have been developed to inhibit VEGF signaling [7]. However, conventional approaches face significant limitations, such as drug resistance, limited efficacy, and systemic side effects, necessitating the development of alternative therapeutic strategies [4,8].

The fusion protein sFLT01, which targets VEGF and placental growth factor (PlGF), has demonstrated potential in inhibiting angiogenesis [[9], [10], [11]]. However, concerns related to inflammation and immunogenicity remain due to its structural composition [12,13]. To address these challenges, a modified version, htsFLT01, has been developed with enhanced efficacy in suppressing angiogenesis [12]. The htsFLT01 sequence was designed by Fahimeh Zakeri et al. (2023) and has been validated for its therapeutic potential in vitro and in vivo, particularly in ocular neovascularization [12]. This engineered protein was derived from sFLT01 through truncation (sFLT01 short hinge), incorporating a strategically designed flexible linker composed of nine glycines and a short hinge between the FLT1 domain 2 and IgG1 Fc to optimize its function [12]. This study aims to evaluate the therapeutic efficacy of htsFLT01 as a novel anti-angiogenic agent in breast cancer.

In parallel, drug nanocomplexes have emerged as powerful tools for targeted gene/drug delivery and precision therapy. Among these, the htsFLT01/MiRGD gene delivery nanocomplex has been developed as a tumor-targeting nanoparticle system with enhanced therapeutic potential. The MiRGD peptide incorporates functional motifs such as Histone H1, nuclear localization signal (NLS), Gp41, and iRGD, which play essential roles in overcoming gene delivery barriers [14]. These motifs facilitate various biological functions, including endosomal escape, DNA binding, cellular uptake, and tumor tissue infiltration [[15], [16], [17]]. Leveraging these properties, htsFLT01/MiRGD enhances the feasibility and effectiveness of gene therapy for cancer treatment.

Previous studies have demonstrated that this nanocomplex effectively inhibits angiogenic processes in vitro. Therefore, the present study aims to evaluate both in vitro and in vivo effects of this nanocomplex on angiogenesis suppression and assess its therapeutic impact on tumor progression. The findings of this research may contribute to the development of novel therapeutic strategies and ultimately enhance treatment outcomes and the quality of life for cancer patients.

Materials and methods

MiRGD expression and purification

The DNA coding sequence of the MiRGD peptide was previously synthesized and cloned into the pET28a plasmid. The recombinant plasmids containing the genes were transformed into E. coli BL21 (DE3) pLysS (Novagen) [14,16].

To create a preculture, E. coli BL21 bacteria were incubated in 10 mL of LB culture containing kanamycin at 37°C for 12 hours while shaking at 220 × g. One liter of 2xYT liquid medium containing kanamycin was inoculated with 10 ml of fresh culture medium and shaken at 220 × g. After reaching an OD₆₀₀ of 0.6-0.8, expression was induced with 1 ml of 0.6 mM IPTG and incubated at 37°C. Six hours later, MiRGD-containing bacteria were harvested by centrifugation [15]. The cell pellets were placed in a solution of 1000 μl lysis buffer, as outlined in Supplementary Table S1. The resulting solution was sonicated for 10 minutes (10 pulses followed by a 20-second rest) while the sample was kept on ice. The lysate baths were spun at a speed of 12,000 × g for 20 minutes. The supernatants were spun in the centrifuge for an additional 20 minutes. The chromatography columns (Bio-Rad) were pre-equilibrated with lysis buffer and incubated for 1 hour at room temperature. The columns were run six times with different washing buffers according to the protocol (Supplementary Table S1) [18]. The purity and size of the collected washes were evaluated using sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The peptides were separated from the solution using cold PBS with 10 % glycerol in a 3.5 kDa dialysis tube. Then, the concentration of the purified peptide was checked using a spectrophotometer, and the isolated peptides were stored at -20°C for further tests.

Preparation of htsFLT01/MiRGD nano complex

A nano complex was created by combining htsFLT01 with MiRGD peptide-based carriers at varying N/P ratios. The N/P ratio refers to the ratio of positive charges on the peptide to the negative charges on the DNA. The N/P ratio was determined using the given equation:

$$N/P=\frac{\frac{\frac{weightofpeptide}{molecularweightofpeptide}}{numberofpositivecharge}}{\frac{weightofhtsFLT01}{molecularweightofhtsFLT01}}$$

As an illustration, a nano complex with an N/P ratio of 4 was created by combining 2 μg of htsFLT01 and 4.8 μg of peptide-based carrier and allowing the mixture to incubate at room temperature for 25 minutes. The peptide's capacity to attach to htsFLT01 was tested through agarose gel retardation experiments.

Transfection of htsFLT01/MiRGD into MCF7 cancer cells and investigation of cytotoxicity

To determine the best N/P ratio, 2 × 10⁵ MCF7 cells were grown in DMEM medium supplemented with 10 % fetal bovine serum and penicillin-streptomycin in a humidified 5 % CO₂ incubator for 48 hours before the experiment started. Then, the medium was replaced with DMEM medium containing htsFLT01 alone or as a complex with MiRGD peptide in different N/P ratios. Additionally, to compare the transfection efficiency of the constructed vectors, the polyethyleneimine (PEI) branch (25 kDa) was used as a positive control. To evaluate the toxicity of the htsFLT01/MiRGD nano complex, the MTT test was used as follows: MCF-7 cells were cultured in a 96-well plate with 10,000 cells per well. Nano complexes fabricated from peptide and plasmid were added to the cells according to the N/P ratio used in previous experiments. After incubation, ten μl of MTT solution (5 mg/ml) was added to the cultures. Dark blue formazan crystals were revealed by adding dimethyl sulfoxide (50 μl). A microplate reader (Bio-Tech) measured absorbance at 540 nm.

Western blotting

Conditioned media (CM) and cell lysates from the MCF7 cell line transfected with the htsFLT01/MiRGD (N/P = 14) Nano complex were analyzed by western blot to investigate the secretion of the htsFLT01 protein [12]. Cell lysates were prepared according to laboratory protocols by placing the cell culture dish on ice and rinsing the cells with cold PBS. The cells were then treated with trypsin to break them down, then washed with PBS. After centrifugation to remove any remaining trypsin, the cell pellet was mixed with 1 mL of PBS, observed under a microscope, and lysed with 300 microliters of RIPA + protease inhibitor buffer. The lysate was centrifuged and stored at -70°C for long-term storage. For western blotting, SDS gel electrophoresis was performed at 90 volts to separate proteins based on size on SDS-PAGE gel. The loading of lysate and conditioned medium was determined based on Lowry concentration. The samples were then transferred to a PVDF membrane after electrophoresis, and the membrane was blocked in a solution containing PBS, BSA, and Tween 20 for 1 hour at room temperature. Following washes with TBST, the membrane was exposed to the primary antibody (R&D systems, cat# AF321-S) overnight in the refrigerator, followed by three washes with TBST. The membrane was then incubated with the secondary antibody solution (sc-2768) for 2 hours at room temperature, washed three times with TBST, and bands were visualized using the electrochemiluminescence (ECL) method, resulting in bands on Ray-X film. All conditioned media and cell lysates were prepared without serum.

Gene expression analysis

Gene expression analysis was conducted to investigate the effect of htsFLT01/MiRGD nano complex on the expression of essential genes (VEGF, HIF-1, IL-1, COX-2, NF-KB, TNF-α, IL-6, IL-17, IL-8, Bcl-2, BAX, MMP-2, MMP-14, MMP-9, SLC16A8, and APOE) using quantitative reverse transcription polymerase chain reaction (RT-qPCR). MCF7 cells were transfected with htsFLT01/MiRGD nanocomplex, and after 72 hours, RNA was extracted using TRIzol. Total cDNA was then synthesized using H-minus MMLV, and the relative expression of the studied genes and GAPDH in MCF7 cells was analyzed with specific primers (Supplementary Table S2) in the presence of SYBR Green using the Step One Real-Time PCR system (Applied Biosystems).

Animal induction of breast cancer model

Healthy female BALB/c mice (15–20 g) were obtained from the Pasteur Institute Laboratory Animal Center of Iran and were housed under standard laboratory conditions with access to food and water ad libitum. The ethics control committee of the National Institute of Genetic Engineering and Biotechnology of Iran approved all experimental protocols (IR.NIGEB.EC.1402.11.29.C). 4T1 cells (1 × 10⁶) were injected into the mice via heterotopic injection to induce tumor growth. Tumors became visible on the 10th day post-injection. When the average tumor diameter reached 6 mm, the animals were divided into four groups: tumor control (PBS injection), tumor control (MiRGD injection), tumor treatment (plasmid htsFLT01 injection), and tumor treatment (htsFLT01/MiRGD injection). The complex amount was determined based on cell studies (N/P = 14) and instructions, then administered to the tumor-bearing mice four times at 5-day intervals. Upon reaching an average tumor diameter of 20 mm in the control group, mice were euthanized, and the tumors were excised for pathological and histological analysis.

Histological analysis of mouse models of breast cancer

Tumor tissues were excised from the mouse models and fixed in 10 % paraformaldehyde, followed by decolorization, paraffin embedding, and sectioning at a thickness of 3–5 µm. The sections were stained with hematoxylin and eosin (H&E) and examined under an optical microscope (LABOMED). For immunofluorescence (IF) analysis, the paraffin sections underwent dehydration, dewaxing, and rehydration. Antigen retrieval was performed by incubating the sections in citrate buffer and subjecting them to microwave heating for 2 minutes. Samples were subsequently washed three times with PBS (P4417-Sigma) at 5-minute intervals. Primary antibodies (VEGF: orb191500, VEGFR: orb11556, and CD31: orb676197) were diluted 1:100 in PBS and applied to the samples, followed by incubation at 2–8°C for 24 hours. Following primary antibody incubation, the sections were treated with the secondary antibody (orb688925), diluted 1:150, and incubated at 37°C for 90 minutes in the dark. After three additional PBS washes, DAPI (D9542, Sigma) was applied for 20 minutes, followed by a final PBS wash. A glycerol/PBS mounting medium was used, and coverslips were placed on the sections. Fluorescent imaging was performed using an Olympus microscope, and quantitative image analysis was conducted using ImageJ software (National Institutes of Health, USA). The analysis incorporated specialized plugins such as Angiogenesis Analyzer, NeuronJ, TUNEL Assay Analysis, Cell Counter, CellProfiler, and Dead Cell Count. The final interpretation of histological findings was reviewed by a pathologist to ensure accuracy and reliability.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics (version 22, IBM, USA) and REST 2009 software. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for comparisons between experimental groups. Statistical significance was defined as p < 0.05 (*) and p < 0.01 (**), while non-significant results were reported as ns. Data were expressed as mean ± standard deviation (SD) from three independent experiments. No randomization was applied in this study.

Results

Production and characterization of the htsFLT01/MiRGD nanocomplex

In previous studies, the accuracy of gene synthesis and cloning was verified using restriction enzyme digestion and sequencing analyses [14,19]. Following the addition of IPTG (0.5 mg/L), bacterial expression of the MiRGD peptide was induced, and the peptide was purified using nickel-NTA affinity chromatography. SDS-PAGE analysis confirmed high yield and purity, consistent with prior investigations on peptide-based carrier production in bacterial systems (Supplementary Fig. S1). A critical factor in gene therapy is the ability of a carrier to efficiently bind and deliver therapeutic genes. To assess this, we experimentally evaluated the binding capacity of the MiRGD peptide to htsFLT01.

Cytotoxicity, cellular uptake, and intracellular release of the htsFLT01/MiRGD nanocomplex

Efficient intracellular delivery of large biomolecules such as htsFLT01 requires overcoming multiple biological barriers, including the negatively charged cell membrane, nuclear membrane, and endosomal compartments. As illustrated in Fig. 1A, MCF7 cells treated with the htsFLT01/MiRGD nanocomplex (N/P = 14) exhibited a strong fluorescent signal, indicating effective cellular uptake. A positive control (htsFLT01/PEI at N/P = 14) was included for comparison (Fig. 1B). Our findings revealed that htsFLT01 alone exhibited limited cellular uptake (Fig. 1F). However, when complexed with MiRGD, cellular internalization was significantly enhanced (Fig. 1A). Transfection efficiency analysis demonstrated that Group B (PEI + htsFLT01) achieved a significantly higher transfection rate of 85 %, compared to Group A (MiRGD + htsFLT01), which exhibited an efficiency of 75 % (p < 0.0001) (Fig. 1E). Despite its superior transfection efficiency, PEI-based transfection was associated with considerably higher cytotoxicity, as evidenced by the cytotoxicity assay (Fig. 1C). In contrast, the MiRGD/htsFLT01 nanocomplex, while exhibiting a slightly lower transfection rate (75 %), demonstrated markedly reduced toxicity, making it a more viable candidate for gene therapy applications. These findings highlight the potential of MiRGD-based gene delivery systems in advancing safe and effective anti-angiogenic cancer therapies.

Fig. 1.

Compatibility, cytotoxicity, cellular uptake, and intracellular release of the htsFLT01/MiRGD nanocomplex at the optimal N/P ratio. (A) The N/P ratio of 14 was selected for further analysis based on the nanocomplex's size and absorption properties. (B) Plasmid/polyethylene imine (PEI) was used as a control for comparison. (C) Cytotoxicity was evaluated across various N/P ratios in comparison to the control, with assessments conducted in triplicate at 24, 48, and 72 hours. (D) Western blot analysis confirmed the expression and presence of htsFLT01 in MCF7 cell lysates and conditioned medium. MCF7 cells were transfected with either the htsFLT01/MiRGD nanocomplex (N/P = 14) or MiRGD alone, and Western blotting was performed to detect htsFLT01 expression. A protein molecular weight marker was used to estimate the size of the detected protein. The arrows indicate a specific band at approximately 49 kDa, corresponding to htsFLT01. The "+" symbol denotes presence, while the "-" symbol indicates absence. Sample conditions: 1. Protein molecular weight marker. 2. Conditioned medium from MCF7 cells transfected with htsFLT01/MiRGD (N/P = 14). 3. Conditioned medium from MCF7 cells transfected with MiRGD (N/P = 14). 4. MCF7 cell lysate from cells transfected with htsFLT01/MiRGD (N/P = 14). 5. MCF7 cell lysate from cells transfected with MiRGD (N/P = 14). 6. Untransfected MCF7 cell lysate (negative control). (E) As demonstrated in the comparison, Group B (PEI + htsFLT01) exhibited a higher transfection efficiency, averaging approximately 85 %, compared to Group A (MiRGD + htsFLT01), which achieved an average efficiency of approximately 75 %. The images illustrate the negative control for transfection, where the htsFLT01 plasmid was used as a negative control in the absence of a carrier. (F) The images depict the negative control for transfection in cells. In this experiment, the htsFLT01 plasmid was used as a negative control, transfected without a carrier.

Additionally, an increase in the N/P ratio of the nano complex led to a higher fluorescence signal intensity and more cells emitting fluorescence. This observation supported the idea that the MiRGD nano complex enhanced the cellular uptake of htsFLT01 in an N/P ratio-dependent manner. The N/P ratio-dependent cellular uptake of the nano complex can be explained by the increased number of iRGD targeting motifs on the surface of the nano complex, leading to enhanced cell uptake and plasmid transfection efficiency. A N/P ratio of 14 was chosen for future analysis based on the size and absorption rate of the nano complex.

Furthermore, as depicted in Fig. 1A and 1B, the nano complex released its htsFLT01 content within MCF7 cells, with maximum release observed 72 hours post-treatment. This demonstrated the stability of the nano complex in the extracellular environment and its reversible binding property for the appropriate intracellular release of htsFLT01.

Optimal expression and release of the htsFLT01 protein

To verify the production and release of the secretory protein htsFLT01, a western blot was conducted under reduced conditions using human VEGFR1/FLT1 polyclonal antibodies (see Fig. 1D). The data revealed that htsFLT01 was secreted at its highest level in the medium of MCF7-transfected cells without the additional concentration or purification. A lysate obtained from MCF7 cells treated with MiRGD was used as a control in the experiment. The results indicated increased secretion of the htsFLT01 protein outside the cell. To prevent unintended consequences in the serum and potential cross-reactions, a conditioned medium was created in a serum-free environment.

Effects of htsFLT01 on the expression of critical genes related to angiogenesis, migration, apoptosis, metabolism, and inflammation

In cancer cells, the balance of gene expression is disrupted. Investigating the effects of new drugs on the expression changes of these genes can provide valuable insights into cancer treatment. Results from RT-qPCR comparing gene expression revealed that after transfection with the anti-angiogenic gene htsFLT01, there was a decrease in the expression of hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) genes, which are essential for stimulating angiogenesis. Additionally, the expression of MMP2, MMP9, and MMP14, crucial genes in the migration pathway, decreased in response to the htsFLT01/MiRGD complex, although the change in MMP14 expression was insignificant. The BAX/BCL2 ratio indicated an increased susceptibility to apoptosis post-transfection. Molecular studies also demonstrated that the htsFLT01/MiRGD complex inhibited interleukin-1 (IL-1) and decreased the expression of interleukin-6 (IL-6) and interleukin-17 (IL-17). Furthermore, there was a significant decrease in interleukin-8 (IL-8) gene expression in breast cells treated with the htsFLT01/MiRGD complex. The expression of the COX-2 gene was also significantly reduced. The study indicated decreases in NF-kB and TNF-α gene expression, though results for NF-kB were insignificant. Lastly, the expression of active genes in the metabolic pathway (SLC16A8 and APOE) decreased following gene delivery of the htsFLT01/MiRGD complex. The modulation of migration, angiogenesis, apoptosis, inflammation, and metabolic pathways demonstrate the anti-angiogenic and anti-metastatic effects of the htsFLT01/MiRGD complex (see Fig. 2A, B, C, D).

Fig. 2.

Evaluation of the impact of htsFLT01/MiRGD nanocomplex on essential gene expression in MCF7 cells treated with htsFLT01/MiRGD compared to the control and housekeeping gene GAPDH using RT-qPCR. (A) Relative expression of HIF-1α, VEGF, MMP2, MM9, MMP14. (B) Relative expression of BAX, BCL2. (C) IL-1β, COX-2, NF-κB, TNF-α, IL-6, IL-17, IL-8. (D) Relative expression of SLC16A8, APOE.

Delivery of the htsFLT01/MiRGD nano complex and its impact on breast cancer growth and invasiveness in a mouse model

The htsFLT01/MiRGD nanocomplex effectively inhibited tumor growth and invasion in a mouse model. Following the initial injection, tumor progression was monitored across all experimental groups. As expected, the PBS and MiRGD groups exhibited continuous tumor enlargement, reaching approximately 20 mm by the end of the experiment. In contrast, the htsFLT01 group demonstrated moderate tumor growth suppression, while the MiRGD/htsFLT01 combination treatment (N/P = 14) resulted in a significant reduction in tumor size and inflammation, particularly after the second injection. This reduction became statistically significant after the third injection. Notably, three mice in the htsFLT01 group did not survive beyond the second injection, suggesting potential toxicity or sensitivity to the treatment. These findings indicate that, although htsFLT01 alone exerts an anti-tumor effect, its combination with MiRGD significantly enhances therapeutic efficacy, potentially improving treatment outcomes.

The htsFLT01/MiRGD complex and its impact on angiogenesis in mouse tumor tissue

The data obtained from hematoxylin and eosin (H&E) staining demonstrate the significant effects of the htsFLT01/MiRGD combination on various cellular processes (Fig. 4A). The percentage of mitotic cells remained unchanged among the PBS, MiRGD, and htsFLT01 groups; however, treatment with htsFLT01/MiRGD resulted in a significant reduction in cell proliferation (Fig. 4E). Additionally, the htsFLT01/MiRGD combination markedly increased the percentage of apoptotic cells compared to the other groups, indicating its pro-apoptotic effects (Fig. 4D). Furthermore, angiogenesis was significantly inhibited in the htsFLT01/MiRGD group, while remaining unchanged in the other conditions, highlighting its strong anti-angiogenic potential (Fig. 4C). The percentage of necrotic cells was also significantly elevated in the htsFLT01/MiRGD group, whereas no significant differences were observed in the other treatment groups. This suggests a restoration of normal stromal architecture within the tumor tissue (Fig. 4B). Overall, quantitative analysis using ImageJ underscores the potent antitumor properties of the htsFLT01/MiRGD combination, characterized by reduced proliferation, enhanced apoptosis, suppressed angiogenesis, and increased necrosis, reinforcing its therapeutic potential.

Fig. 4.

Comparative Analysis of the Effects of htsFLT01/MiRGD, PBS, MiRGD, and htsFLT01 on Angiogenesis, Necrosis, and Apoptosis. (A) Representative H&E staining images. Image 1 is captured at 50 × magnification, while Images 1 and 2 are at 10 × magnification. (B) Quantitative analysis of necrosis levels in treated cells. The htsFLT01/MiRGD nanocomplex (N/P = 14) induced a significant increase in necrosis compared to PBS, MiRGD, and htsFLT01. No significant difference was observed among the PBS, MiRGD, and htsFLT01 groups. (C) Comparison of Angiogenesis Across Experimental Groups. The htsFLT01/MiRGD group exhibited a significant reduction in angiogenesis compared to the other groups. (D) Apoptosis Rate Analysis Across Experimental Groups. The htsFLT01/MiRGD group demonstrated a significantly higher rate of apoptosis compared to the other groups. (E) Mitotic Cell Percentage Across Experimental Groups. The PBS, MiRGD, and htsFLT01 groups showed similar levels of cell division, whereas the htsFLT01/MiRGD group exhibited a significant decrease in mitotic activity.

Impact of the htsFLT01/MiRGD nanocomplex on the expression of VEGF, VEGFR, and CD31 in mouse tumor tissue

Immunofluorescence (IF) analysis was performed to assess the expression levels of VEGF, VEGFR, and CD31 in mouse tumor tissues. The results revealed a significant reduction in the expression of these proteins following administration of the htsFLT01/MiRGD nanocomplex, compared to the PBS, MiRGD, and htsFLT01 groups (Figs. 5B, 6B, 7B). Furthermore, treatment with the MiRGD nanocomplex alone did not produce any notable changes in the expression of these proteins (Figs. 5B, 6B, 7B). The IF-based protein analysis was consistent with findings from in vitro studies and H&E staining of tumor tissues in vivo (Fig. 4A–E). This highlights the distinct roles of these factors in vascular modulation, with potential implications for the development of targeted angiogenesis therapies.

Fig. 5.

Comparative Analysis of htsFLT01/MiRGD, PBS, MiRGD, and htsFLT01 on VEGF Expression in Breast Cancer Tumors. (A) Comparative analysis of the effects of htsFLT01/MiRGD in comparison to PBS, MiRGD, and htsFLT01 on VEGF protein expression in breast cancer tumors. Data are presented as mean ± SD, with n = 3 tumors per group. (B) Immunofluorescence (IF) analysis showing a significant reduction in VEGF protein expression following injection of the htsFLT01/MiRGD nanocomplex (N/P = 14) compared to PBS, MiRGD, and htsFLT01. The results indicate that PBS and MiRGD treatments do not significantly alter VEGF expression, with no notable difference between these two groups. However, both are significantly different from the htsFLT01/MiRGD and htsFLT01 groups.

Fig. 6.

Comparative Analysis of htsFLT01/MiRGD, PBS, MiRGD, and htsFLT01 on VEGFR Expression in Breast Cancer Tumors. (A) Comparative analysis of the effects of htsFLT01/MiRGD versus PBS, MiRGD, and htsFLT01 on VEGFR protein expression in breast cancer tumors. Data are presented as mean ± SD, with n = 3 tumors per group. (B) Immunofluorescence (IF) analysis showing a significant reduction in VEGFR protein expression following injection of the htsFLT01/MiRGD nanocomplex (N/P = 14) compared to PBS, MiRGD, and htsFLT01. The results indicate that PBS, MiRGD, and htsFLT01 do not significantly alter VEGFR expression and do not differ from each other. However, all three groups exhibit statistically significant differences compared to the htsFLT01/MiRGD group.

Fig. 7.

Comparative Analysis of htsFLT01/MiRGD, PBS, MiRGD, and htsFLT01 on CD31 Expression in Breast Cancer Tumors. (A) Comparative analysis of CD31 protein expression in breast cancer tumors treated with htsFLT01/MiRGD, PBS, MiRGD, and htsFLT01. Data are presented as mean ± SD, with n = 3 tumors per group. (B) Immunofluorescence (IF) analysis demonstrates a significant reduction in CD31 protein expression following injection of the htsFLT01/MiRGD nanocomplex (N/P = 14) compared to PBS, MiRGD, and htsFLT01. The results indicate that PBS, MiRGD, and htsFLT01 treatments do not significantly alter CD31 expression and do not differ from each other, while all three groups exhibit a statistically significant difference compared to the htsFLT01/MiRGD group.

Discussion

In this study, we utilized the MiRGD peptide nanocomplex, which incorporates key functional motifs: Histone H1 for DNA compression and charge neutralization. Gp41 motif (from HIV) for endosomal escape [20], Nuclear localization signal (NLS) (from SV40 virus) for nuclear membrane translocation [21]. iRGD motif for targeting vascular endothelial cells expressing the receptor at high levels. The results confirmed successful GFP reporter gene transfer using the nanocomplex (Fig. 1A). Additionally, Western blot analysis validated the expression, translation, and secretion of htsFLT01 in cancer cells (Fig. 1D). These findings are consistent with previous studies demonstrating the effectiveness of similar motifs in gene delivery [14,16,18,19]. Cytotoxicity assessments indicated that the nanocomplex exhibited minimal or no toxicity, making it a safer alternative to lipid and polymer-based nanocarriers (Fig. 1C) [[18], [19], [20], [21], [22], [23]]. Although PEI/htsFLT01 demonstrated higher transfection efficiency compared to MiRGD/htsFLT01 (Fig. 1E), it also exhibited greater cytotoxicity, which is consistent with prior research on PEI's high transfection efficiency but inherent toxicity (Fig. 1C). In contrast, MiRGD/htsFLT01, despite a slightly lower transfection efficiency (75 % vs. 85 %, Fig. 1E), represents a more suitable candidate for gene therapy due to its superior biocompatibility and reduced toxicity (Fig. 1C). These findings suggest that incorporating targeting ligands such as MiRGD can optimize the balance between transfection efficiency and biocompatibility, making it a clinically viable nanocarrier for gene therapy. Further examination of the nanocomplex’s tumor penetration ability in a mouse model confirmed successful MiRGD-mediated tumor targeting compared to non-targeted complexes (Fig. 3A). The anti-angiogenic potential of htsFLT01 holds significant therapeutic promise in oncology. Tumor growth analysis (Fig. 3B) demonstrated that while tumors in the PBS group continued uninterrupted growth, the htsFLT01 and htsFLT01/MiRGD groups exhibited significant tumor suppression, with MiRGD/htsFLT01 demonstrating the greatest inhibition. These findings indicate a synergistic effect between htsFLT01 and MiRGD, enhancing angiogenesis inhibition and tumor suppression. Histological H&E staining and IF analyses corroborated these findings, showing reduced blood vessel density and size in htsFLT01/MiRGD-treated tumors, confirming its strong anti-angiogenic effects. Previous research has optimized htsFLT01 for molecular stability while minimizing immune responses associated with prolonged drug exposure. Given that VEGF and PlGF are overexpressed in age-related macular degeneration, diabetic retinopathy, and inflammatory disorders, htsFLT01 has potential therapeutic applications beyond oncology [12]. In conditions where PlGF contributes to pathogenesis, htsFLT01 may offer additional benefits, expanding its clinical utility. Angiogenesis inhibition strategies such as Sorafenib and Sunitinib non-selectively target PlGF and VEGFR1, while also affecting PDGF, VEGFR2, Flt3, and c-Kit pathways [24]. Aflibercept, a structurally similar agent to htsFLT01, is undergoing clinical trials for multiple cancers [25,26]. Additionally, the successful intracellular and extracellular transport of htsFLT01 via MiRGD was confirmed (Fig. 1A, 1D). Western blot analysis further demonstrated significant extracellular secretion of htsFLT01 in MCF7 cells (Fig. 1D).

Fig. 3.

A) Suppression of tumor growth and angiogenesis by administering htsFLT01/MiRGD nanocomplex: images displaying tumor volume. B) Tumor growth dynamics across different experimental groups over time. Tumor size (mm) was measured at various time points following treatment initiation (Day 0). The PBS and MiRGD groups exhibited continuous tumor progression, whereas the htsFLT01 group demonstrated a moderate reduction in tumor size. Notably, the combination therapy (MiRGD/htsFLT01) resulted in a significant tumor size reduction, particularly following the second injection.

Solid tumors frequently experience hypoxia, leading to HIF-1 activation and upregulation of VEGF and MMPs [27,28]. The observed downregulation of VEGF, HIF-1, MMP2, and MMP9 suggests that htsFLT01/MiRGD exerts a modulatory effect on hypoxia-driven angiogenesis (Fig. 2A). Moreover, alterations in Bax/Bcl-2 expression indicate an increase in apoptosis following htsFLT01/MiRGD transfection, as evidenced by the elevated BAX/BCL2 ratio (Fig. 2B). These results were further confirmed by H&E staining, which showed increased apoptosis and necrosis in treated tumors (Fig. 4B).

Additionally, IL-8, a key driver of cell migration and tumor progression, was significantly downregulated following htsFLT01/MiRGD treatment (Fig. 2C), while COX-2—a marker linked to cancer progression—was also significantly reduced [[29], [30], [31]]. (Fig. 2C). The reduction of TNF-α, an essential regulator of epithelial-mesenchymal transition (EMT) and metastasis, further supports the therapeutic potential of htsFLT01/MiRGD (Fig. 2C). Our gene expression analysis also demonstrated a downregulation of SLC16A8 and APOE, which have been linked to cancer metabolism and progression (Fig. 2D) [[32], [33], [34], [35], [36]]. Additionally, prior studies have established that VEGF and VEGFR-1 expression are significantly elevated in breast cancer [[37], [38], [39]]. and VEGFR-1 knockdown has been shown to reduce tumor survival via AKT inhibition [40].

Our IF experiments confirmed a decrease in VEGF and VEGFR protein expression following htsFLT01/MiRGD treatment (Figs. 5B, 6B). The significant reduction in CD31 expression in htsFLT01/MiRGD-treated tumors indicates suppressed angiogenesis, consistent with prior research demonstrating CD31’s role in tumor invasion [41] (Fig. 7B). Additionally, H&E staining confirmed a reduction in angiogenesis and mitotic activity, alongside increased apoptosis and necrosis, in htsFLT01/MiRGD-treated tumors (Fig. 4B, C, D, E).

Conclusions

htsFLT01/MiRGD represents a novel, next-generation anti-angiogenic agent, capable of binding to VEGF and PlGF while mitigating toxicity and modulating key oncogenic pathways. Our findings reinforce its therapeutic potential for treating breast cancer and angiogenesis-dependent malignancies.

Data availability

Data will be made available upon reasonable request.

CRediT authorship contribution statement

Mohadeseh Khoshandam: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Zahra-Soheila Soheili: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Funding acquisition, Data curation. Saman Hosseinkhani: Writing – review & editing, Validation, Supervision, Project administration, Methodology. Shahram Samiee: Validation, Software, Resources. Hamid Latifi-Navid: Writing – review & editing, Validation, Methodology. Hamid Ahmadieh: Writing – review & editing, Conceptualization. Hossein Soltaninejad: Writing – review & editing, Writing – original draft, Visualization, Conceptualization. Babak Jahangiri: Methodology.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.