Precise atorvastatin delivery by cardiac homing peptide functionalized nanoliposomes for myocardial damage repair after myocardial infarction

ABSTRACT

Objective

To develop a novel lipid nanoparticle (Ato@DSPE-PEG-CHP) for targeted delivery to ischemic myocardium to treat myocardial infarction.

Methods

Ato@DSPE-PEG-CHP was prepared using the thin-film dispersion method. Physicochemical properties were characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS). In vitro targeting, uptake, and cytotoxicity were evaluated in OGD/R-treated HL-1 cells using confocal microscopy and CCK8. In vivo, a mouse MI model was established by ligating the left anterior descending coronary artery. The targeted distribution was observed using small animal in vivo imaging. Efficacy was evaluated using TTC, HE, Masson and TUNEL staining. Biosafety was evaluated through hemolysis assays and histopathological analysis.

Results

Ato@DSPE-PEG-CHP with uniform morphology, excellent dispersibility, and high stability were successfully prepared. This material can target myocardial ischemia sites both in vitro and in vivo. Ato@DSPE-PEG-CHP reduced infarct size, cell necrosis, inflammation, fibrosis, and apoptosis; decreased levels of tumor necrosis factor-α, interleukin-6, and malondialdehyde; and increased the enzyme activity of superoxide dismutase. Additionally, it demonstrated good biocompatibility and exhibited no significant toxicity to major organs.

Conclusion

Ato@DSPE-PEG-CHP specifically targeted ischemic myocardium, alleviated damage through its anti-inflammatory and antioxidant effects, and demonstrated superior efficacy and safety, presenting a promising treatment strategy for MI.

1. Introduction

Myocardial infarction (MI) referred to the necrosis of myocardial tissue resulting from prolonged ischemia and hypoxia of the coronary arteries. It was one of the leading causes of mortality and disability globally. In China alone, nearly 700,000 individuals died from acute myocardial infarction (AMI) each year [1]. Although modern medicine can restore blood supply to myocardial cells through revascularization techniques, such as interventional therapy and thrombolysis, the short half-life of these drugs, their lack of targeting, and the inevitable ischemia-reperfusion injury (IRI) that occured as a consequence of revascularization further exacerbate inflammatory responses, oxidative stress, and apoptosis in myocardial cells. This cascade of events ultimately led to adverse ventricular remodeling [2,3]. Therefore, it was essential to develop targeted treatment strategies that effectively mitigated IRI and suppressed inflammatory responses.

Nanoliposomes were a type of nanolipid drug delivery system characterized by particle sizes ranging from 10 to 1000 nm. They utilized natural or synthetic solid lipids, such as phosphatidylcholine and triacylglycerol, as carriers to encapsulate or sandwich drugs within the lipid core [4]. On the one hand, nanoliposomes were spherical vesicles made up of phospholipid bilayers that can simultaneously encapsulate both hydrophilic and hydrophobic drugs. This dual capability enhanced drug efficacy by prolonging circulation time in the body and minimizing toxic side effects [5]. On the other hand, modifying the surface of nanoliposomes enabled active targeted drug delivery, allowing for precise “navigation” of the drug to the site of the lesion. This approach increased local drug concentration while minimizing damage to healthy tissue [6]. Currently, nanoliposome drug delivery systems are extensively utilized in the treatment of cancer [7], cardiovascular diseases [8], and ophthalmic conditions [9], rendering them an optimal choice for drug delivery.

Cardiac homing peptide (CHP) was a short peptide with the sequence CSTSMLKAC that specifically bound to ischemic myocardial tissue [10]. Studies have demonstrated that CHP was a safe and effective technology capable of delivering therapeutic drugs to ischemic myocardial cells without causing significant alterations in blood counts, blood chemistry, or cardiac function [11]. CHP-functionalized exosomes or nanoparticles have been extensively utilized in the treatment of conditions such as myocardial infarction and myocardial hypertrophy [12,13]. They were anticipated to facilitate precise, targeted therapies for ischemic myocardium.

Atorvastatin (Ato) was a broad-spectrum inhibitor of 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. In addition to its well-known lipid-lowering effects, atorvastatin also possessed anti-inflammatory, antioxidant, endothelial function-enhancing, and atherosclerotic plaque-stabilizing properties [14]. However, traditional oral atorvastatin had a low absolute bioavailability (12%) and a high systemic clearance rate (80%) [15], which made it challenging to achieve effective therapeutic concentrations locally in the heart. Recent studies have shown that the intravenous administration of atorvastatin during AMI can limit infarct size and adverse cardiac remodeling, thereby reducing cardiac damage in subsequent myocardial infarctions [16]. Therefore, this study aimed to develop a novel CHP-functionalized, atorvastatin-loaded PEGylated liposome nanomedicine system (Ato@DSPE-PEG-CHP) to facilitate the targeted accumulation of atorvastatin in ischemic myocardial tissue via intravenous administration. This approach sought to maximize the myocardial protective effects of atorvastatin and offered an efficient, low-toxicity treatment option for MI.

2. Methods

2.1. Preparation and characterization of Ato@DSPE-PEG-CHP

2.1.1. DSPE-PEG2000-CHP synthesis

Dissolve 1 mg CHP (HY-P4094, MedChemExpress, USA) and 0.1 g NHS (MM3703, Maokang Biotechnology, CHN) in 5 mL dimethyl sulfoxide (DMSO, 67-68-5, Wokai Pharmaceutical, CHN). Stir the mixture at room temperature for 30 min. Then, add 50 mg DSPE-PEG2000-COOH (HY-170706, MedChemExpress, USA) and 0.2 g EDC (25952-53-8, Sangon Biotech, CHN), and continue stirring at room temperature for 6 h. After the reaction, dialysis was conducted using a 2000 Da dialysis bag for a duration of 48 h. The resulting solution was freeze-dried to yield a white, powdery solid known as DSPE-PEG2000-CHP. The preparation process for DSPE-PEG2000 followed the same steps as outlined above, with the exception of the addition of CHP.

2.1.2. Liposome preparation and drug loading

Nanoliposomes were prepared using the thin-film dispersion technique [17]. In brief, 15 mg DSPC (HY-W040193, MedChemExpress, USA) and 5 mg cholesterol (14606, Sigma, USA) were dissolved in 5 mL chloroform (PHR1552, Merck, USA), followed by the addition of 5 mL of an ethanol solution containing either DSPE-PEG2000 or DSPE-PEG2000-CHP. The mixture was evaporated using a rotary evaporator (RE-2000B, Jinan Hepu Instrument Equipment Co., Ltd., CHN) at 30°C and 100 rpm to remove excess solvent, and then it was vacuum-dried overnight to obtain a uniform lipid film. Add 5 mg/mL of atorvastatin (HY-B0589, MedChemExpress, USA) to the lipid film, sonicate in a water bath for 5 min, and then transfer the mixture to a liposome extruder (610000, Avanti Research™, USA) to obtain Ato@DSPE-PEG-CHP or Ato@DSPE-PEG nanoliposomes. The preparation process for blank liposomes followed the same steps as described above, but without the incorporation of atorvastatin. The resulting product was verified using infrared spectroscopy.

2.1.3. Physical and chemical characterization of Ato@DSPE-PEG-CHP

The prepared nanoliposomes were diluted 100-fold with distilled water, and 10 μL was utilized for sample preparation. Double staining with uranium acetate (SPI-02624, HEAD Biotechnology, CHN) and lead citrate (L885990, Macklin, CHN) was performed, and the morphological structure of the nanoliposomes was examined using a transmission electron microscope (TEM, JEM-1400Flash, JPN). The particle size, zeta potential, and degree of dispersion of nanoliposomes were measured using a dynamic light scattering (DLS, NANOTRAC FLEX, Microtrac, USA) instrument. The drug encapsulation efficiency and drug loading capacity of atorvastatin (246 nm) in nanoliposomes were assessed using a UV spectrophotometer (UV-2365, Uniqo, CHN). The formula for calculation was as follows:

$$\mathit{Drug}\mathit{encapsulation}\mathit{efficiency}=\frac{{\mathit{m}}_{\mathit{encapsulated}\mathit{drugs}}}{m_{\mathit{administered}\mathit{drugs}}}\times 100\mathrm{\%}$$

$$\mathit{Drug}\mathit{loading}\mathit{capacity}=\frac{{\mathit{m}}_{\mathit{encapsulated}\mathit{drugs}}}{m_{\mathit{blank}\mathit{liposomes}}}\times 100\mathrm{\%}$$

2.1.4. Release characteristics and stability assessment in vitro

Place the Ato@DSPE-PEG liposome solution and the Ato@DSPE-PEG-CHP liposome solution into separate dialysis bags, and seal the openings securely. Then, place the bags in a phosphate-buffered saline (PBS) solution containing 30% methanol and stir uniformly at a speed of 100 rpm. At various time points (1, 2, 3, 4, 5, 6, and 7 h), remove 0.5 mL PBS and filter it through a 0.22 μm microporous membrane (F513134-0001, Sangon Biotech, CHN). After filtration, utilize a UV spectrophotometer (UV-2365, Uniqo, CHN) to determine the concentration of atorvastatin in the PBS solution. After sampling, replenish the PBS solution with 30% methanol until the solution returns to its initial volume.

In addition, Ato@DSPE-PEG liposomes and Ato@DSPE-PEG-CHP liposomes were placed in PBS solution. Samples were taken daily for 6 days to measure particle size and dispersion, which were used to evaluate the stability of the liposomes.

2.2. Cell experiments

2.2.1. Cell culture

Mouse cardiomyocytes HL-1 were purchased from STEM RECELL (STM-CL-6052). HL-1 cells were cultured in HL-1 cell-specific culture medium, supplemented with claycomb medium (51800C, Sigma, USA), 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (P/S), 0.1 mM Norepinephrine, and 2 mM L-Glutamine. All cells were cultured in an incubator maintained at 5% CO₂ and a temperature of 37°C.

2.2.2. Oxygen-glucose deprivation/reperfusion (OGD/R) treatment

HL-1 cells were cultured until they reached approximately 90% confluence, after which they were subjected to OGD/R treatment. Discard the original medium and wash the cells three times with PBS. Next, add FBS-free, sugar-free DMEM medium and incubate in a hypoxic cell culture chamber (94% N₂, 5% CO₂, 1% O₂, 37 °C) for 6 h [18,19]. After this period, return the cells to normal culture conditions for continued growth.

2.2.3. OGD/R model validation

To verify the successful construction of the OGD/R cell model, the following experiments were conducted:

Cell viability was assessed using the CCK8 assay. Briefly, HL-1 and OGD/R cells were seeded at a density of 5 × 10³ cells per well in a 96-well plate, with 100 μL of medium added to each well, followed by incubation for 24 h. Subsequently, 10 μL of CCK8 solution (40203ES60, Yeasen, CHN) was added, and the plates were incubated for an additional hour at 37°C. Absorbance at 450 nm was measured using a microplate reader (Multiskan SkyHigh, ThermoFisher, USA).

Cell reactive oxygen species (ROS) levels were measured using flow cytometry (CytoFLEX, Beckman, USA). Briefly, HL-1 and OGD/R cells were uniformly seeded into six-well plates to achieve a cell density of 80%–95%. Cells were then harvested, suspended in a diluted DCFH-DA solution (S0033S, Beyotime, CHN), and incubated at 37°C in a cell culture incubator for 20 min. Centrifuge the sample to collect the cells, resuspend them in pre-chilled PBS, and transfer to flow cytometry tubes for analysis.

Collect HL-1 and OGD/R cell supernatants, and measure MDA and SOD levels using a colorimetric assay. A microplate reader (Multiskan SkyHigh, ThermoFisher, USA) was used to measure absorbance values at 532 nm and 560 nm, respectively.

2.2.4. Evaluation of cell uptake and targeting ability of liposomes in vitro

Replace the fluorescent dye Rhodamine B (RhB, HY-Y0016, MedChemExpress, USA) with atorvastatin encapsulated in DSPE-PEG or DSPE-PEG-CHP liposomes to investigate the cellular uptake and targeting ability of liposomes in vitro.

To investigate the cellular uptake capacity of liposomes in vitro, logarithmic phase cells were seeded into 96-well plates (100 μL per well). Subsequently, a medium containing RhB@DSPE-PEG and RhB@DSPE-PEG-CHP liposomes was added. Cells were collected at 0.5 h, 4 h, and 24 h post-incubation. To investigate the targeting ability of CHP-functionalized liposomes for myocardial ischemia, RhB@DSPE-PEG-CHP was added to the culture medium of HL-1 and OGD/R cells, and the cells were incubated for 24 h. After collecting cells from the two experiments mentioned above, they were sequentially fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (P0096, Beyotime, CHN), and washed with PBS. FITC Phalloidin (40735E875, Yeasen, CHN) was utilized to stain the cytoskeleton, while DAPI was employed to label the cell nuclei. Fluorescence signals were observed and analyzed at a wavelength of 495 and 513 nm using a fluorescence confocal microscope (STELLARIS 5&8, Leica, GER).

2.2.5. Cytotoxicity assay

CCK8 assay was employed to evaluate the toxicity of both blank liposomes and drug-loaded liposomes. In brief, HL-1 and OGD/R cells were seeded into 96-well plates at a density of 5 × 10³ cells per well, with 100 μL of medium added to each well, and incubated for 24 h. Subsequently, CHP, DSPE-PEG, DSPE-PEG-CHP, Ato, Ato@DSPE-PEG, and Ato@DSPE-PEG-CHP were added to each well. After incubating for 24 h, add 10 μL of CCK8 solution (40203ES60, Yeasen, CHN) and then incubate at 37°C for an additional hour. Measure the absorbance at 450 nm using a microplate reader (Multiskan SkyHigh, ThermoFisher, USA).

2.3. Animal experiments

2.3.1. MI mouse model

Seventy-three male ICR mice (7–8 weeks old, 25–30 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (China) (License No. SCXK (Lu) 20230045). All mice were acclimatized for 7 days under conditions of 22°C to 25°C with a 12 h light/dark cycle. Mice were anesthetized with sodium pentobarbital (60 mg/kg) via intraperitoneal injection. Mice from the model group were placed on an experimental platform preheated to 37°C. Following shaving and skin preparation, tracheal intubation was performed. Mechanical ventilation was then initiated using a MiniVent ventilator (845, Harvard Apparatus, USA), with real-time monitoring of respiratory status. The thoracic cavity was accessed by incising the pleura along the third or fourth intercostal space, following the path of the intercostal muscles. The left lung was isolated using a sterile pad, and the pericardium was exposed. The pericardium was incised, and the left anterior descending artery was ligated with a 5–0 silk suture to establish a mouse model of myocardial infarction. To validate the successful establishment of the MI model, 12 ICR mice were randomly divided into two groups: a sham group and a model group, with six mice in each. Twenty-four hours after surgery, fresh cardiac tissue was collected. Three specimens were subjected to TTC staining, while the remaining three were embedded, sectioned, and processed for H&E staining and flow cytometry.

2.3.2. Animal grouping and treatment

To compare the efficacy of three different formulations – Ato@DSPE-PEG-CHP, Ato, and Ato@DSPE-PEG – in treating myocardial infarction, 45 ICR mice were randomly divided into five groups: Sham group, Model group, Ato group, Ato@DSPE-PEG group, and Ato@DSPE-PEG-CHP group. After successful modeling, mice in the Ato group were administered atorvastatin (28 mg/kg/day) via oral gavage for a duration of 4 weeks [20]. Mice in the Ato@DSPE-PEG group and the Ato@DSPE-PEG-CHP group were administered 14 mg/kg of nanoparticles via intravenous injection once a week for four consecutive weeks [21]. At the end of the experiment, all mice were euthanized using a carbon dioxide system. Blood and heart tissue were subsequently collected. All animal procedures were approved by the Ethics Committee of Binzhou Hospital (Ethics Approval Number: 2024-L068).

To evaluate the safety of Ato@DSPE-PEG-CHP, 10 normal ICR mice were randomly assigned to control group and Ato@DSPE-PEG-CHP group. The treatment group received a single intravenous injection of Ato@DSPE-PEG-CHP (14 mg/kg), while the control group was administered an equivalent volume of physiological saline. General observations were conducted on mice in each group. On the seventh day after treatment, all mice were euthanized using a carbon dioxide system, and liver and kidney tissues were collected.

All tissues were fixed with paraformaldehyde, dehydrated, cleared, and subsequently embedded in paraffin before being sectioned into 5 μm slices. The sections were then dewaxed in a xylene solution, dehydrated using a gradient of ethanol, and subjected to subsequent histological staining.

2.3.3. Hemolysis test

In vitro hemolysis experiments were conducted according to the protocol previously reported in the literature [22,23]. In brief, collect fresh anticoagulated mice blood and centrifuge at 4°C for 10 min at 1000 rpm for 15 min to collect red blood cells. Add approximately ten times the volume of physiological saline to wash the red blood cells until the supernatant was no longer red. Use physiological saline to prepare a 2% suspension of red blood cells. Mix the Ato@DSPE-PEG-CHP solution with the 2% red blood cell suspension in a 1:1 volume ratio, and transfer the mixture to a 1.5 mL centrifuge tube. Incubate in an oscillator and use physiological saline (0% hemolysis) as a negative control and 2% Triton X-100 (100% hemolysis) as a positive control. After incubating for 3 h, centrifuge the sample, observe the results, and photograph them to determine whether hemolysis has occurred. Following the methodology described in previous studies, small volumes of blood samples incubated with different reagents from each group were placed in a microplate reader (Multiskan SkyHigh, ThermoFisher, USA), and the absorbance values were quantitatively measured at 540 nm.

2.3.4. In vivo targeted validation

Intravenous injection of RhB@DSPE-PEG and RhB@DSPE-PEG-CHP were performed to validate the targeted delivery of CHP-functionalized liposomes to myocardial ischemia sites. Fluorescent signals of RhB were monitored using a small animal in vivo imaging system (NightOwl II LB 983, Berthold, GER). Twenty-four hours after the last medication administration, major organs (heart, liver, spleen, lung, and kidney) were collected from euthanized mice for imaging to assess the active targeting ability and biodistribution of RhB@DSPE-PEG-CHP liposomes.

Additionally, we employed fluorescence confocal microscopy to observe the distribution of RhB fluorescence in cardiac, hepatic, and renal tissues according to the protocol previously reported in the literature [24,25]. A microplate reader was used to quantitatively analyze the OD values of isolated hearts, livers, and kidneys from mice in different treatment groups, thereby further confirming that RhB@DSPE-PEG-CHP specifically targeted myocardial infarction regions.

2.3.5. TTC staining

Twenty-four hours after molding and 24 h following the final drug administration, mice were anesthetized with sodium pentobarbital (60 mg/kg), and their thoracic cavities were opened. Perform cardiac perfusion using physiological saline, then excise the heart. Quickly cut the heart into 1 mm-thick slices below the ligature and immerse them in a 1% TTC staining solution (C0652, Beyotime, CHN). Incubate at 37°C for 30 min, wash twice with PBS, and then scan and photograph the heart slices.

2.3.6. Histopathological examination

The following staining kits were used sequentially: hematoxylin and eosin (H&E) staining kit (C0105S, Beyotime, CHN), Masson’s trichrome staining kit (G1340, Solarbio, CHN), and TUNEL apoptosis detection kit (C1091, Beyotime, CHN) to assess morphological changes, collagen fiber deposition, and apoptosis in mice myocardial tissue. In brief, heart tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm slices. HE staining was performed sequentially, with hematoxylin used for nuclear staining and eosin for cytoplasmic staining. Masson’s trichrome staining employed lichun red acid fuchsin staining solution to stain muscle fibers, Aniline Blue to stain collagen fibers, and Weigert’s iron hematoxylin solution to stain cell nuclei. TUNEL staining was conducted by sequentially applying proteinase K (ST532, Beyotime, CHN), 3% hydrogen peroxide solution, terminal deoxynucleotidyl transferase (TdT), and biotin-dUTP reaction solution to label apoptotic nuclei. After DAB staining, the sections were counterstained with hematoxylin. Finally, all sections were dehydrated and analyzed under an optical microscope (DM2700M, Leica, GER).

2.3.7. Inflammation and oxidative damage detection

Collect serum samples from each group of mice and conduct the experiment following the instructions provided with the IL-6 (PI326, Beyotime, CHN) and TNF-α ELISA kits (PT512, Beyotime, CHN). Use a microplate reader (Multiskan SkyHigh, ThermoFisher, USA) to measure the absorbance values of the standards and samples from each group. Then, establish a standard curve and calculate the levels of the inflammatory factors IL-6 and TNF-α in each group of samples. Collect mouse heart tissue and cells, and measure MDA and SOD levels using a colorimetric assay. A microplate reader (Multiskan SkyHigh, ThermoFisher, USA) was used to measure absorbance values at 532 nm and 560 nm, respectively.

2.3.8. Flow cytometry

Twenty-four hours after modeling, fresh mouse heart tissue was harvested, thoroughly washed in pre-chilled PBS, minced, and digested with trypsin. Following centrifugation and filtration, a single-cell suspension was prepared, with the cell concentration adjusted to 1 × 10⁷ cells/mL. Next, an appropriate volume of the cell suspension was incubated with fluorescently labeled anti-F4/80 antibody (ab60343, Abcam, UK) at 4°C in the dark for 30 min, including both negative and positive controls. After incubation, the mixture was washed three times with PBS to remove any unbound antibodies. The labeled cell suspension was subsequently analyzed using flow cytometry (CytoFLEX, Beckman, USA). FITC fluorescence signals from F4/80⁺ cells were detected using the FITC channel, and the proportion of macrophages in acute myocardial infarction heart tissue was subsequently calculated.

2.4. Statistical analysis

Data were statistically analyzed using GraphPad Prism version 9. Quantitative data are expressed as the mean ± standard deviation. Comparisons between two groups were performed using t-test, whereas comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). A p-value less than 0.05 was considered statistically significant.

3. Results

3.1. Characterization of the physical and chemical properties of nanoliposomes

To enhance the bioavailability and targeting of atorvastatin while reducing its side effects, polymer materials DSPE-PEG-CHP and DSPE-PEG were prepared. The infrared spectrum (Figure 1(A)) showed characteristic peaks corresponding to the O-H bond, C = O bond, and amide bond at 3436.40 cm⁻¹, 1729.35 cm⁻¹, and 1640.04 cm⁻¹, respectively, which confirmed the existence of PEG and DSPE and the successful coupling of CHP with DSPE-PEG2000. Next, atorvastatin was encapsulated using the thin-film dispersion method to prepare two types of nanoliposomes: Ato@DSPE-PEG and Ato@DSPE-PEG-CHP. The encapsulation efficiencies were 33.6% and 50.8%, respectively, with drug loading capacities of 2.2% and 2.8%, respectively. TEM revealed that both types of liposomes exhibited regular morphology and uniform dispersion (Figure 1(B)). DLS results showed that the average particle size of the nanomaterials increased slightly but without significant change before and after surface modification with CHP (Figure 1(C)), indicating successful loading of CHP and maintenance of liposome morphological stability. The absolute value of the zeta potential of Ato@DSPE-PEG-CHP was higher than that of Ato@DSPE-PEG (Figure 1(D)), indicating that the CHP-functionalized nanoliposome dispersion system was more stable. Additionally, release (Figure 1(E)) and stability experiments (Figure 1(F)) in vitro demonstrated that both liposomes exhibited sustained release characteristics and good stability, with a slight increase in the cumulative release of CHP-functionalized nanoliposomes. In summary, we successfully developed a novel atorvastatin nanoliposome loaded with a cardiac homing peptide, offering an improved delivery system for targeted therapy.

Figure 1.

Characterization of the physicochemical properties of nanoliposomes. (A) Infrared spectroscopy reveals O–H bonds at 3436.40 cm⁻¹, C = O bonds at 1729.35 cm⁻¹, and amide bonds at 1640.04 cm⁻¹; (B) Typical TEM images showing the morphological structures of Ato@DSPE-PEG and Ato@DSPE-PEG-CHP nanoliposomes at 20,000× magnification (scale bar: 200 nm); (C–D) DLS measurements of the average particle size and zeta potential of both nanoliposomes; (E) Comparison of in vitro drug release profiles for the two nanoliposomes; (F) Stability assessment of the two nanoliposomes.

3.2. Cell uptake, targeting, and safety evaluation of nanoliposomes

RhB fluorescent probes were used to evaluate the uptake of two types of liposomes – RhB@DSPE-PEG (Figure 2(A)) and RhB@DSPE-PEG-CHP (Figure 2(B)) by HL-1 cells at different time points (0.5, 4, and 24 h) in real-time. The fluorescence intensity of RhB showed a time-dependent increase (Figure 2(C)), indicating enhanced uptake of both liposomes by HL-1 cells. Interestingly, RhB@DSPE-PEG-CHP exhibited minimal fluctuations in fluorescence intensity over time compared to RhB@DSPE-PEG, indicating that the presence of CHP did not confer any discernible advantage in uptake by normal cardiomyocytes.

Figure 2.

Cellular uptake, targeting efficiency, and safety assessment of nanoliposomes. Confocal fluorescence microscopy was used to observe the uptake of RhB@DSPE-PEG (A) and RhB@DSPE-PEG-CHP (B) by HL-1 cells at 0.5, 4, and 24 h. RhB exhibited red fluorescence; phalloidin labeled the cardiomyocyte cytoskeleton, appearing green; and DAPI stained the nuclei, appearing blue. Magnification: 200×; scale bar: 50 μm. (C) Quantitative analysis of average RhB fluorescence intensity using ImageJ software. (D) The CCK8 assay was used to assess differences in cell viability between HL-1 cells and OGD/R model cells. (E) Flow cytometry was employed to detect differences in ROS levels between HL-1 cells and the OGD/R model. (F) Biochemical test kits detected the SOD and MDA levels in HL-1 cells and OGD/R model. (G) Confocal fluorescence microscopy was used to observe the targeted distribution characteristics of RhB@DSPE-PEG-CHP in HL-1 and OGD/R cells 24 h after drug treatment. ImageJ software was used to quantitatively analyze the average fluorescence intensity of RhB. Magnification: 200×; Scale bar: 50 μm. (H) Cell viability was assessed using the CCK8 assay to evaluate drug cytotoxicity after co-incubation of CHP and blank liposomes (DSPE-PEG and DSPE-PEG-CHP) with normal mouse cardiomyocytes (HL-1) and hypoxic cardiomyocytes (OGD/R) for 24 h. (I) CCK8 assay was used to evaluate cytotoxicity after 24 h of treatment with Ato and drug-loaded liposomes (Ato@DSPE-PEG and Ato@DSPE-PEG-CHP). N = 3.

Based on the experimental results above, the targeting properties of RhB@DSPE-PEG-CHP were further validated using HL-1 and OGD/R cell models. First, the OGD/R cell model was successfully established. Its validity was confirmed using CCK8 assay (Figure 2(D)), flow cytometry (Figure 2(E)), and ELISA (Figure 2(F)). Results demonstrated that, compared to untreated HL-1 cells, OGD/R cells exhibited significantly reduced viability, markedly elevated levels of ROS and MDA, and substantially decreased SOD activity – characteristics consistent with a typical OGD cell model. Fluorescence confocal microscopy observations (Figure 2(G)) demonstrated that the fluorescence intensity of RhB was significantly higher in OGD/R cells compared to normal HL-1 cells, confirming that CHP modification enhanced the targeting of liposomes to ischemic myocardial cells.

Finally, the CCK8 assay was used to evaluate the effects of CHP, blank liposomes (DSPE-PEG and DSPE-PEG-CHP), Ato, and drug-loaded liposomes (Ato@DSPE-PEG and Ato@DSPE-PEG-CHP) on the viability of HL-1 and OGD/R cells. The results were shown in Figure 2(H,I). The results showed that CHP and the two blank liposomes exhibited no significant cytotoxicity, whereas Ato demonstrated significant cytotoxic effects. However, after formulation into liposomes (Ato@DSPE-PEG), the cell viability of both cell types significantly increased, indicating that the nano-drug delivery system can reduce drug toxicity. Additionally, in normal cardiomyocytes, there was no significant difference in cell viability between Ato@DSPE-PEG and Ato@DSPE-PEG-CHP. However, in OGD/R cells, the recovery of cell viability was more pronounced following treatment with CHP, further supporting that CHP modification enhanced the drug’s targeting to ischemic myocardial regions.

3.3. Targeted distribution and myocardial protective effects of Ato@DSPE-PEG-CHP in MI mice

To investigate the targeted therapeutic effect of Ato@DSPE-PEG-CHP on MI, a mouse model of MI was established using the LAD method. During the modeling process, whitening was observed below the ligation site. Compared with the sham group, the model group exhibited an increased myocardial infarction area (Figure 3(A)), disorganized myocardial cell arrangement (Figure 3(B)), myocardial necrosis (Figure 3(B)), and macrophage infiltration (Figure 3(C)), indicating the successful establishment of the myocardial infarction mouse model.

Figure 3.

Establishment of a MI mouse model and validation of targeted distribution of RhB@DSPE-PEG-CHP. A MI mouse model was established by ligating the left anterior descending (LAD) coronary artery. Mice were euthanized with carbon dioxide 24 h after surgery, and cardiac tissue was collected. Myocardial infarction areas were visualized using TTC staining (A, N = 3), hematoxylin and eosin (HE) staining (B, N = 3), and flow cytometry (C, N = 3) to assess infarct size, pathological damage, and inflammatory cell infiltration in cardiac tissue. For in vivo targeted validation, we prepared two RhB-labeled samples: RhB@DSPE-PEG and RhB@DSPE-PEG-CHP. Six mice received intravenous injections of these samples at a concentration of 15 mg/mL. Twenty-four hours after administration, mice were processed for ex vivo tissue imaging of the heart, liver, spleen, lung, and kidney (D, N = 3), followed by quantitative analysis of fluorescence intensity. Confocal microscopy was used to visualize RhB distribution in mouse tissues, including the heart, liver, and kidney (E, N = 3). Optical density (OD) values were quantified using a microplate reader (F, N = 3).

To investigate the in vivo targeted distribution of nanoparticles, RhB-fluorescently labeled nanoparticles, RhB@DSPE-PEG and RhB@DSPE-PEG-CHP, were administered via intravenous injection. In vitro tissue imaging results (Figure 3(D)) revealed a sparse distribution of RhB@DSPE-PEG in the heart and liver, whereas RhB@DSPE-PEG-CHP predominantly accumulated in the hearts of myocardial infarction mice, with significantly reduced distribution in organs such as the liver and kidneys. Furthermore, fluorescence confocal microscopy analysis of RhB fluorescence distribution in the heart, liver, and kidneys revealed a markedly elevated RhB fluorescence intensity and OD values in myocardial tissue following injection of RhB@DSPE-PEG-CHP, with comparatively weaker signals observed in liver and kidney tissues (Figure 3(E,F)). This indicates that CHP-functionalized liposomes specifically target myocardial ischemic sites, consistent with the results of in vitro targeting experiments.

To evaluate the pharmacological effects of Ato@DSPE-PEG-CHP and to demonstrate the advantages of this nanocarrier system, a series of histopathological examinations were conducted. TTC (Figure 4(A)), HE (Figure 4(B)), Masson (Figure 4(C)), and TUNEL staining (Figure 4(D,E)) results demonstrated that MI mice exhibited increased myocardial infarction area, irregular myocardial cell morphology, disorganized cell arrangement, extensive myocardial necrosis, inflammatory cell infiltration, and collagen deposition. Compared to the Model group, both Ato monotherapy and liposome treatment alleviated myocardial damage. However, compared to the Ato group and the Ato@DSPE-PEG group, the Ato@DSPE-PEG-CHP group exhibited well-organized myocardial cells with no obvious necrotic areas, significantly reduced inflammatory cell infiltration, collagen fibrosis, and apoptosis, demonstrating a more pronounced therapeutic effect. Additionally, Ato@DSPE-PEG-CHP significantly reduced serum levels of inflammatory factors (TNF-α and IL-6, Figure 4(F,G)) and oxidative stress markers (MDA and SOD, Figure 4(H,I)) in MI mice, further confirming the targeted therapeutic effect of Ato@DSPE-PEG-CHP on myocardial infarction.

Figure 4.

Cardioprotective effects of Ato@DSPE-PEG-CHP in MI mice. Cardiac tissue was collected four weeks post-surgery and examined using TTC staining (A, N = 3), HE staining (B, 200×, 100 μm, N = 6), Masson staining (C, 200×, 100 μm, N = 6), and TUNEL staining (D, 400×, 50 μm, N = 6) to assess infarct size, histopathological changes, fibrosis, and apoptosis in mouse cardiac tissue. (E) The proportion of TUNEL-positive cells was quantified using ImageJ software. TNF-α (F, N = 6), IL-6 (G, N = 6), MDA (H, N = 3), and SOD (I, N = 3) levels in each treatment group.

3.4. Safety evaluation of Ato@DSPE-PEG-CHP

Preliminary in vitro and in vivo experiments demonstrated that Ato@DSPE-PEG-CHP exhibited highly effective targeted therapeutic effects. To further assess the safety of Ato@DSPE-PEG-CHP, hemolysis assays and single-dose toxicity studies were conducted in normal mice. The results showed that no significant hemolysis occurred in the tube containing Ato@DSPE-PEG-CHP (Figure 5(A)), indicating that the newly constructed nanoliposome exhibits excellent blood compatibility. Additionally, no significant toxic reactions were observed in normal mice after administration of Ato@DSPE-PEG-CHP, and no obvious pathological damage was detected in kidney and liver tissues following HE staining (Figure 5(B,C)).

Figure 5.

Safety evaluation of Ato@DSPE-PEG-CHP. (A) Collect fresh anticoagulated rat blood and isolate the red blood cells. Add the following sequentially: physiological saline (negative control), 2% Triton X-100 (100% hemolysis, positive control), Ato, DSPE-PEG, Ato@DSPE-PEG, and Ato@DSPE-PEG-CHP. Incubate at room temperature for 3 h, then visually inspect for hemolysis. If the solution in the centrifuge tube appeared clear red with no cellular residue or only a small amount of red blood cells at the bottom, hemolysis has occurred. Conversely, if all red blood cells have settled and the supernatant was colorless and clear, no hemolysis has occurred. (B-C) At the experimental endpoint, collect kidney and liver tissues from each group of mice. HE staining was used to observe histopathological changes in liver and kidney tissues and to assess the in vivo safety of the drug. Magnification: 200×; Scale bar: 100 μm.

In summary, the Ato@DSPE-PEG-CHP nanoliposomes successfully prepared in this study demonstrated significant targeted therapeutic effects, excellent blood compatibility, and in vivo safety, providing robust experimental evidence to support subsequent clinical applications.

4. Discussion

This study successfully developed a novel functionalized atorvastatin nanoliposome delivery system (Ato@DSPE-PEG-CHP). Compared to existing treatment strategies for ischemic myocardial injury – such as miR302-loaded, myocardial cell-specific peptide-modified bone marrow mesenchymal stem cell-derived exosomes [26] and protein-mimetic polymer systems targeting the Keap1/Nrf2 pathway [27]—this nanoliposome system offered significant technical advantages. These advantages included a simplified and controllable preparation process, reduced production costs, excellent biocompatibility, and the elimination of risks associated with inflammation caused by degradation products. Research findings confirmed that Ato@DSPE-PEG-CHP achieved highly efficient targeted accumulation in ischemic myocardial regions, effectively suppressing inflammatory cascades and oxidative stress damage within the ischemic microenvironment. This reduced tissue injury following myocardial ischemia-reperfusion and promoted the repair of cardiac function. Concurrently, systems biology assessments confirm its favorable biocompatibility and in vivo safety. This research presented an innovative solution to the critical challenges associated with traditional atorvastatin administration, including low bioavailability, inadequate tissue targeting, and significant systemic adverse effects. It established a technological foundation for the precision treatment of ischemic heart disease.

The core breakthrough of this study was the achievement of active, targeted drug delivery to ischemic myocardium, based on the high affinity of the CHP sequence for the myocardial ischemic microenvironment [28]. The molecular mechanism by which CHP targeted ischemic myocardium may involve proteins associated with ischemia. Studies have revealed partial sequence homology between the CSTSMLKAC peptide and proteins such as titin, optic atrophy 1 (OPA1), and dynamin-like protein 1 (DLP1), all of which were closely implicated in myocardial ischemic injury [29]. Previous studies indicated that ischemia promoted the binding of alpha-B-crystallin to titin in cardiomyocytes, thereby counteracting titin protein denaturation caused by ischemic injury [30]. The mechanism underlying CSTSMLKAC enrichment in ischemic cardiomyocytes may involve a direct interaction with alpha-B-crystallin. Research indicates that OPA1 played a crucial role in mitochondrial fusion. Ischemia significantly reduces myocardial OPA-1 levels, thereby inducing apoptosis [31]. DLP-1 was an essential protein involved in mitochondrial fission [32]. Both proteins regulated mitochondrial function during ischemia-reperfusion injury. The CSTSMLKAC peptide may target ischemic cardiomyocytes exhibiting impaired mitochondrial function by interacting with specific proteins. However, this study has not yet validated the underlying molecular mechanisms involved. Future identification of receptors targeted by ischemia-homing peptides could provide new insights into the molecular mechanisms underlying ischemic injury. This study’s in vitro experiments demonstrated that in normal cardiomyocytes (HL-1), CHP modification did not significantly enhance uptake. However, in the OGD/R cell model simulating ischemic injury, the uptake capacity of Ato@DSPE-PEG-CHP was superior to that of the blank liposome group, strongly confirming the specificity of its targeting effect. In vivo imaging experiments further confirmed this finding, CHP-functionalized liposomes exhibited highly concentrated fluorescent signals in the cardiac region of MI mice, while their distribution in non-target organs such as the liver and kidneys was significantly reduced following intravenous injection. This effect may be attributed to the PEG modification on the surface of the nanoliposomes. The hydration layer formed by PEG modification effectively reduces plasma protein adsorption, thereby conferring immune evasion capabilities to nanoparticles and prolonging their systemic circulation time [33]. This prolonged circulation property establishes an optimal time window for CHP-mediated active targeting, allowing more nanoparticles to reach the ischemic myocardium before being cleared by the liver and kidneys. This approach achieves a synergistic effect by enhancing cardiac-targeted accumulation while reducing accumulation in off-target organs. This precise “navigation” capability effectively overcame the major obstacle of insufficient local drug concentration in the heart caused by the first-pass effect of traditional oral atorvastatin [15].

Precise drug delivery led to superior therapeutic outcomes. Histopathological analysis clearly demonstrated the advantages of the Ato@DSPE-PEG-CHP treatment group in preserving myocardial structural integrity, inhibiting fibrosis, and reducing cellular apoptosis. This advantage arose from the multifunctional properties of atorvastatin itself, including its anti-inflammatory [34], antioxidant [35], and plaque-stabilizing effects [36], as well as from the targeted strategy employed in this study, which maximized the drug’s efficacy at the lesion site. Serological testing provided robust biochemical evidence to support this. Following treatment with Ato@DSPE-PEG-CHP, levels of pro-inflammatory factors (TNF-α, IL-6) and oxidative stress markers (MDA) were significantly reduced in MI mice, while the activity of the antioxidant enzyme (SOD) was markedly restored. These results indicated that by creating a high-concentration “action zone” of the drug in the ischemic myocardial region, the cascade of damaging reactions triggered by myocardial reperfusion injury can be effectively blocked, thereby providing robust protection for the heart.

Currently, significant research was focused on achieving targeted drug delivery using targeting peptides and enhancing bioavailability through nanocarrier systems. For example, VHPKQHR-targeting peptides and fluorescently labeled rapamycin magnetic liposomes were used for targeted therapy of early atherosclerosis [37]; similarly, CHP-modified cardiosphere-derived stem cell exosomes (CDCs) were employed for targeted therapy of myocardial infarction [12]. Previous studies have reported the widespread use of nanomaterials encapsulating atorvastatin in treating diseases such as ischemic stroke [21] and lung cancer [38]. The uniqueness of this study lied in its pioneering combination of the clinical first-line drug atorvastatin with the highly efficient CHP targeting peptide and a well-established liposome carrier. This innovative approach offered a new therapeutic strategy for effectively alleviating myocardial damage and inhibiting inflammation in patients with myocardial infarction.

Although this study has yielded preliminary results, its potential limitations must be objectively evaluated. First, this study employed an acute myocardial infarction mouse model, and its conclusions require validation in larger animal models (such as Beagle dogs) before they can be extrapolated to clinical patient applications. Second, acute toxicity experiments in mice demonstrated the short-term biocompatibility of Ato@DSPE-PEG-CHP, consistent with reports indicating the high safety of CHP peptides themselves [11]. However, the potential toxicity and immunogenicity associated with long-term use remain critical issues that must be addressed in future studies.

In summary, this study successfully designed and validated a myocardial-targeted atorvastatin nanodelivery system, Ato@DSPE-PEG-CHP, which demonstrated precise targeting capability, high therapeutic efficacy, and reliable biosafety. Future research will focus on evaluating the long-term efficacy and safety of this approach in large animal models, as well as exploring its potential applications in diseases such as chronic heart failure following myocardial infarction. The ultimate goal is to advance this innovative nanomedicine toward clinical translation.

5. Conclusion

This study successfully developed a novel nanoliposome delivery system for atorvastatin. Ato@DSPE-PEG-CHP targets ischemic myocardium, mitigating myocardial injury through its anti-inflammatory and antioxidant effects, while demonstrating excellent efficacy and safety. This approach addresses the challenges of low bioavailability and poor targeting associated with conventional atorvastatin, providing a safe and effective targeted therapeutic strategy for myocardial infarction with promising potential for clinical translation.

Funding Statement

This study was supported by grants from 2024 Yantai City Science and Technology Innovation Development Plan for Basic Research— “Study on the efficacy of a functionalized nanoscale system dual-supplying nitric oxide and hydrogen sulfide targeting cardiac homing peptides for treating myocardial infarction in mice [2024JCYJ054]”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article highlights

• Ato@DSPE-PEG-CHP nanoliposomes were successfully prepared using thin-film dispersion, exhibiting uniform morphology, high stability, and specific targeting of ischemic myocardium.

• In vitro and in vivo studies confirmed that the nanosystem enhanced atorvastatin accumulation at ischemic sites, thereby reducing infarct size, inflammation, fibrosis, and apoptosis.

• It modulated inflammatory markers (TNF-α, IL-6) and oxidative stress markers (SOD, MDA), exerting superior cardioprotective effects compared to free atorvastatin and non-targeted liposomes.

• Excellent biocompatibility was demonstrated, with no significant hemolysis or major organ toxicity observed, indicating a promising targeted therapy for myocardial infarction.

Author contributions statement

Hongqin Yu: data curation, investigation, writing – original draft; Shuai Li: methodology, visualization; Hongtao Niu: validation, investigation; Zhao Li: project administration; Yusheng Gai: formal analysis; Bei Sun: resources; Lan Zhao: writing – review & editing. All authors reviewed the manuscript.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Ethical disclosure

Our animal experiments comply with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and approved by the Ethics Committee of Binzhou Hospital (Ethics Approval Number: 2024-L068).

Data availability statement

Data available on request from the authors.