RGD peptide-functionalized micelles loaded with crocetin ameliorate doxorubicin-induced cardiotoxicity

Abstract

Doxorubicin (Dox)-induced cardiotoxicity presents a significant challenge to fully harnessing its chemotherapeutic potential. Crocetin (Cro), a dicarboxylic acid found in the crocus flower and gardenia fruit, has shown remarkable antioxidant and anti-inflammatory activities. However, its poor aqueous solubility and limited cellular uptake severely constrain its further application in treating diseases. In this study, we developed Arg-Gly-Asp (RGD) peptide-decorated nanomicelles delivering Cro to alleviate Dox-induced cardiac injury. The RGD@M(Cro) nanomicelles exhibited excellent aqueous solubility and a drug-loading efficiency of 93.3 %. RGD-decorated micelles could enhance the cellular uptake of Cro in cardiomyocytes and inhibit approximately 60 % of HL-1 cell apoptosis through efficient reactive oxygen species (ROS) scavenging. In a cardiomyopathy mouse model, RGD@M(Cro) substantially reduced cardiac damage and improved cardiac indicators. This study highlights the great potential of RGD-decorated micelles in treating cardiac injury and other diseases.

Keywords: Antioxidant effect; Cardiotoxicity; Crocetin; Drug delivery; RGD peptide.

Graphical abstract

Scheme 1

Schematic illustration of RGD@M(Cro) nanoparticles to alleviate Dox-induced cardiotoxicity.

Fig. 1

Preparation and characterization of RGD@M(Cro). A: Schematic illustration of RGD@M(Cro) preparation. B: Morphology analysis of RGD@M(Cro) nanomicelles detected by TEM. Scale bar: 100 nm. C: Size distribution of RGD@M(Cro) determined by dynamic light scattering (DLS). D: Zeta potential analysis of RGD@M(Cro) micelles.

Fig. 2

Cellular uptake of RGD@M(Cro) nanoparticles in HL-1 cells. A: The internalization of RGD@M(Cro) nanoparticles in HL-1 cells by CLSM imaging. Cy3-labeled M(Cro) or RGD@M(Cro) nanoparticles containing equal Cro (20 μM) were incubated with HL-1 cells for 2 h. Scale bar: 10 μm. B: Flow cytometric analysis of HL-1 cells treated with M(Cro) or RGD@M(Cro) nanomicelles containing 20 μM Cro for 2 h at 37 °C.

Fig. 3

RGD@M(Cro) nanoparticles alleviated cardiotoxicity in HL-1 cells. A: The protective analysis of RGD@M(Cro) nanoparticles containing different Cro centrations (0, 2.5, 5, 10, 20, 40 μM) in HL-1 cells via CCK8 assay. B: Quantitive analysis of apoptotic cells in TUNEL analysis. C: TUNEL analysis of cell apoptosis treated with Cro, M(Cro), or RGD@ M(Cro) containing 40 μM Cro. Scale bar: 100 μm. D: ROS scavenging analysis of Cro, M(Cro), or RGD@M(Cro) micelles containing 40 μM Cro. Scale bar: 20 μm. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01 compared to the Dox group; ^##P< 0.01 compared to the Normal Group.^▲P < 0.05, ^▲▲P < 0.01 compared to the M(cro) group.

Fig. 4

RGD@M(Cro) nanoparticles alleviated Dox-induced cardiac damage in vivo. The mice were administered Cro, M(Cro), or RGD@M(Cro) (20 mg/kg) for one week, followed by a single intraperitoneal injection of Dox (15 mg/kg) on the seventh day. A: Representative echocardiographic images in mice treated with G1(Control), G2(Dox), G3(Dox + Cro), G4(Dox + M(Cro)), G5(Dox + RGD@M(Cro)). B: Ejection fraction (EF%) analysis in different groups; C: Fractional shortening (FS%) analysis in different groups; D: Left ventricular end-diastolic diameter (LVEDd) analysis; E: Left ventricular end-systolic diameter (LVESd) analysis. All data are reported as mean ± standard deviation (SD), with n = 6 mice per group. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01 compared to the Dox group; ^##P < 0.01 compared to the Normal Group.

Fig. 5

RGD@M(Cro) nanoparticles on cardiac tissue staining and detection of cardiac injury indicators. The mice were administered Cro, M(Cro), or RGD@M(Cro) (20 mg/kg) for one week, followed by a single intraperitoneal injection of Dox (15 mg/kg) on the seventh day. A&B: H&E staining; C: Masson staining; D: TUNEL staining of cardiac tissue in mice. Serum BNP(E), CK (F), and LDH (G) of Dox-induced cardiotoxicity in mice. Data are presented as mean ± SD; n = 6, ^⁎P < 0.05, ^**P < 0.01 compared with the Dox group, ^##P < 0.01 compared with Normal Group. The red arrow represents inflammatory cell infiltration, and the green arrow represents myocardial cell arrangement. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)