In vitro assessment of varying peptide surface density on the suppression of angiogenesis by micelles displaying αvβ3 blocking peptides

Abstract

Ligand targeted therapy (LTT) is a precision medicine strategy that can selectively target diseased cells while minimizing off-target effects on healthy cells. Integrin-targeted LTT has been developed recently for angiogenesis-related diseases. However, the clinical success is based on the optimal design of the nanoparticles for inducing receptor clustering within the cell membrane. The current study focused on determining the surface density of Ser-Asp-Val containing anti-integrin heptapeptide on poly (ethylene glycol)-b-poly(propylene sulfide) micelles (MC) required for anti-angiogenic effects on HUVECs. Varying peptide density on PEG-b-PPS/Pep-PA MCs (Pep-PA-Peptide-palmitoleic acid) was used in comparison to a random peptide (SGV) and cRGD (cyclic-Arginine-Glycine-Aspartic acid) construct at 5%-density on MCs. Immunocytochemistry using CD51/CD31 antibody was performed to study the integrin blocking by MCs. In addition, the expression of VWF and PECAM-1, cell migration and tube formation was evaluated in the presence of PEG-b-PPS/Pep-PA MCs. The results show PEG-b-PPS/SDV-PA MCs with 5%-peptide density to achieve significantly higher αvβ3 blocking compared to random peptide as well as cRGD. In addition, αvβ3 blocking via MCs further reduced the expression of vWF and PECAM-1 angiogenesis protein expression in HUVECs. Although a significant level of integrin blocking was observed for 1%-peptide density on MCs, the cell migration and tube formation were not significantly affected. In conclusion, the results of this study demonstrate that the peptide surface density on PEG-b-PPS/Pep-PA MCs has a significant impact in integrin blocking as well as inhibiting angiogenesis during LTT. The outcomes of this study provides insight into the design of ligand targeted nanocarriers for various disease conditions.

Keywords: LTT; PEG-b-PPS; anti-angiogenic peptide; micelles; αvβ3.

FIGURE 1

Peg‐b‐PPS/ Pep‐PA MCs synthesis and characterization. (A) The LCMS spectrum of SDV heptapeptide construct, SGV heptapeptide construct, and cRGD peptide construct indicates the 99% purity of the modified peptide constructs. (B) Schematic representation of synthesis of PEG‐b‐PPS/Pep‐PA MCs with varying peptide surface density. (C) DLS spectrum for (i) blank‐MC (ii) PEG‐b‐PPS/SDV‐PA MCs, (iii) PEG‐b‐PPS/SGV‐PA MCs, (iv) PEG‐b‐PPS/cRGD‐PA MCs. (D) Morphology of the (i) blank‐MC and (ii) PEG‐b‐PPS/ Pep‐PA MCs MCs by CryoTEM analysis. (E) Graph showing the zeta potential values of the PEG‐b‐PPS/Pep‐PA MCs with different densities on the MCs (SDV‐1 SDV‐3, SDV‐5 SGV‐5, and cRGD‐5 represents a different percentage [1%, 3% and 5%] of respective peptides on the MCs)

FIGURE 2

Inhibition of CD51/CD61 antibodies to αvβ3 receptor after short‐term incubation with PEG‐b‐PPS/Pep‐PA MCs. (A) confocal images of CD51/CD61 antibody staining and (B) quantitative expression of Integrin αvβ3 by ImageJ, where the color intensity recorded from individual cell and values were expressed as mean ± SD, n = 15 (SDV‐1 SDV‐3, SDV‐5 SGV‐5, and cRGD‐5 represent a different percentage [1%, 3% and 5%] of respective peptides on the MCs. The term “Pep” in figure B and C represents corresponding peptide sequence). p < .05 was considered significant. Statistical significance compared to control, and between the samples was determined by one‐way ANOVA, Tukey's model (*0.05, **0.01, ****0.0001). The scale bar is 75 μm

FIGURE 3

Expression of Platelet Endothelial Cell Adhesion molecule (PECAM‐1) and von Willebrand factor (vWF‐red) bu HUVEC cells. (A) Confocal microscopic image of HUVEC cells expressing PECAM‐1 (green) and vWF (red) after PEG‐b‐PPS/Pep‐PA MCs and Blank MCs. Quantitative expression of vWF and PECAM‐1 by ImageJ, where the color intensity recorded from individual cell and values were expressed as mean ± SD, n = 20 (B) PECAM‐1 and (C) vWF. (SDV‐1 SDV‐3, SDV‐5 SGV‐5, and cRGD‐5 represent different percentages [1%, 3% and 5%] of respective peptides on the MCs. The term “Pep” in figure B and C represents corresponding peptide sequence). p < .005 was considered significant. Statistical significance compared to control (ψ), and between the samples (*) was determined by one‐way ANOVA, Tukey's model. The scale bar is 75 μm

FIGURE 4

Inhibition of HUVEC cell migration after PEG‐b‐PPS/Pep‐PA MCs treatment. (A) Microscopic image of HUVECs treated with Blank MCs and PEG‐b‐PPS/Pep‐PA MCs at 0 and 24 h. (B) The percentage of wound healing was analyzed using ImageJ software, where the area of the scratched space was recorded and values were expressed as mean ± SD, n = 3). (SDV‐1 SDV‐3, SDV‐5 SGV‐5, and cRGD‐5 represent different percentages [1%, 3% and 5%] of respective peptides on the MCs. The term “Pep” in figure B and C represents corresponding peptide sequence). The scale bar is 100 μm. Statistical significance compared to untreated control (ψ), between the samples (*) and compared to blank‐MCs (#), was determined by one‐way ANOVA, Tukey's model. p < .05 is considered significant

FIGURE 5

Suppression in endothelial tube formation in vitro by PEG‐b‐PPS/Pep‐PA MCs. (A) Representative image of the effect of density of peptide on PEG‐b‐PPS MCs on the tube formation after 4 h of incubation. Where SGV‐5, cRGD‐5, SDV‐1, SDV‐3, and SDV‐5 are representing corresponding PEG‐b‐PPS/Pep‐PA MCs. (SDV‐1 SDV‐3, SDV‐5 SGV‐5, and cRGD‐5 represent different percentages [1%, 3% and 5%] of respective peptides on the MCs. The term “Pep” in figure B and C represents corresponding peptide sequence)). Scale bar 100 μM. (b) The bar graph represents the quantification of the tubes formed using ImageJ an angiogenesis analyzer plugin where values were expressed as mean ± SD, n = 3, p < .05 was considered significant. Statistical significance between the samples (*) and compared to control (ψ) was determined by one‐way ANOVA, Tukey's model