Heart-targeting agents for therapeutic delivery in failing cardiomyocytes – CardioTarget

Cell biology

Cell dissociation and culture of freshly isolated cardiomyocytes. Functional studies were performed on cardiomyocytes. Thereafter, our hypotheses were validated in an ex vivo model.

Flow cytometry: Cardiomyocytes were subjected to LIVE/DEAD cell viability staining according to the manufacturer’s protocol, fixed with PFA, and analyzed by flow cytometry, with a minimum of 10,000 events analyzed per sample. Gating was performed to monitor the number of live cells, the number of positively stained cells, and the mean fluorescence intensity (MFI) per cell.

Cellular imaging: Immunohistochemistry (IHC) and immunocytochemistry (ICC) were performed on heart slices (< 80 μm for IHC) at UMS IPSIT. For ICC, all acquisitions were performed on an SP8 confocal microscope at the MIPSIT microscopy facility (UMS IPSIT) or on a spinning disk at UMR144.

Physiological studies

Langendorff perfusion: The rat heart aorta was cannulated and perfused with oxygenated Krebs-Henseleit solution at a constant flow to achieve a stable 80 mmHg aortic pressure. Electrical mapping was performed to measure heart rate and action potential propagation. Peptides were infused via a recirculating circuit, and isoproterenol (100 nM) was subsequently infused to assess cardiac function.

Formulation and physicochemical characterization

Liposome formulation: Liposomes were prepared from a well-established composition similar to clinically approved liposomes (i.e., N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPEPEG), dipalmitoylphosphatidylcholine (DPPC), or fully hydrogenated soy phosphatidylcholine (HSPC) and cholesterol).

They were prepared by the thin lipid film hydration method, followed by repeated extrusion through 100 nm pore filters. The functionalization of liposomes with targeting ligand conjugates was achieved primarily by post-insertion of phospholipid-ligand conjugates to fine-tune the rate and composition of functionalization.

Physicochemical characterization: Liposome formulations were characterized for colloidal properties, including hydrodynamic size by dynamic light scattering and zeta potential measurements in controlled electrolytes. Centrifugal filtration enabled separation of liposomes from non-associated peptides, thereby allowing sequential quantification of encapsulation and functionalization.

1. In vitro and ex vivo study of SCHoP-PKI cardiac targeting. Studies SCHoP1 and SCHoP2 deliver the PKI 6-22 peptide to cardiomyocytes, with 70% of NRVM cells remaining viable and 50% internalizing the conjugates. SCHoP1-PKI localizes mainly in the cytoskeleton, while SCHoP2-PKI distributes across all compartments, and strongly the nucleus. In hiPSC-CM, SCHoP1-PKI reduces spontaneous beating frequency without affecting conduction velocity, whereas SCHoP2-PKI reduces conduction velocity but not frequency. In rat hearts, these peptides block the isoproterenol-induced increase in conduction velocity. SCHoP1-PKI partially reduces PKA target phosphorylation, while SCHoP2-PKI fully inhibits it. These findings confirm the effectiveness of SCHoPs in targeting cardiomyocytes.

2. The functionalization of liposomes was achieved through post-insertion of targeting molecules, coupled to a PEGylated phospholipid, onto the surface of pre-formed liposomes. The liposomes used (140 nm) were stably labeled with rhodamine fluorescence. Post-insertion was initially validated using a phospholipid-PEG conjugated with carboxyfluorescein (CF). This technique was successfully adapted for the cardiac-targeting peptide SCHoP, after optimizing micelles based on DSPE-PEG-SCHoP. The cell-penetrating peptide TAT was also incorporated into the liposomes.

The targeting efficiency of the functionalized liposomes was evaluated using primary cultures of neonatal rat ventricular cardiomyocytes. The results showed that only SCHoP-functionalized liposomes exhibited significantly higher uptake by cardiomyocytes compared to non-targeted cells (fibroblasts and macrophages), thereby validating the targeting strategy of the project.

3. Kinetics and Biodistribution of SCHoP-DiD Liposomes. FLIT in vivo imaging shows that SCHoP1-DiD and SCHoP2-DiD liposomes mainly accumulate in the liver (362–432 pmol) and lungs (200–226 pmol) within just 20 minutes, with retention at 3 and 24 hours. Heart targeting remains low (13–48 pmol). Ex vivo analysis confirms these results: high fluorescence levels in the liver (up to 2.8×10⁹ µW/cm²), spleen, lungs, and kidneys, but very little in the heart (1.78×10⁸ µW/cm² for SCHoP1-DiD; 5.9×10⁷ for SCHoP2-DiD). Liposomes effectively target the liver and lungs, but cardiac targeting needs further optimization.

We have identified two novel cardiac ligands, SCHoP1 and SCHoP2, that can deliver functional cargo to cardiomyocytes in vitro and ex vivo. These findings on precise drug delivery not only offer transformative therapies to improve patient outcomes but may also reduce the global burden of cardiovascular diseases. Based on these data, two patents have been filed (INPI FR2506612 and FR2506614 in June 2025) for each SChoP peptide. Additionally, two original papers are ready for submission—one validating the proof of concept for delivering a functional therapeutic to cardiomyocytes in vitro and ex vivo, and the other characterizing the liposomal platform functionalized with SChoP. The publication of these data has been delayed due to patent validation. Despite significant progress, much work remains to fully realize the potential of SCHoP1 and SCHoP2. While our results demonstrate proof of principle in small animal models, both in vitro and ex vivo, studies in larger animals are still needed. Moreover, pharmacokinetic (PK), pharmacodynamic (PD), and biodistribution studies of SCHoP1 and SCHoP2 in vivo must be conducted in rodent models with proper controls. Furthermore, full in vivo toxicity studies of SCHoP1 and SCHoP2 with the various cargos, as well as multi-dose and longitudinal studies, would be necessary before human trials leading to clinical applications could be conducted. A market study has been conducted by the University of Paris-Saclay (UPSay) to identify potential industrial partnerships. In the meantime, we will apply for a pre-clinical call with SATT UPSay to validate PKPD and biodistributionin vivo. Once tested and validated, we will be able to consider submitting a request to SATT for a more mature project to assess the feasibility of treating heart disease using our cardiac-targeting platforms. Future research directions to improve targeted drug delivery to cardiomyocytes should include the development of advanced nanocarriers, the exploration of innovative ligand conjugation techniques and cargo delivery mechanisms, and the integration of emerging technologies such as gene editing or RNA-based therapeutics. This will usher in a new era in cardiovascular therapeutics, offering patients safer and more effective treatment options.

Heart failure (HF) is one of the main cardiovascular diseases and is defined as the inability of the heart to meet the body’s circulatory demand. HF constitutes a major health problem, affecting about 1-2% of the adult industrialized-countries population. In spite of huge progress in its treatment over the last decades, no definite cure exists for HF and 5-years mortality remains as high as ~50%. Regardless the causes of HF, a common feature is the persistent activation of the ß-adrenergic/cAMP signaling cascade that leads to chronic Protein Kinase-A (PKA) activation responsible for adverse cardiac remodeling, cardiac myocyte death and fibrosis replacement. Therefore, ß-blockers are a cornerstone therapy for HF. However, they cause severe side effects and are ineffective in ~50% of HF patients. Thus, it becomes imperative to improve and/or to propose novel therapeutics to treat HF. Ideally, these medicines should deliver therapeutic molecules efficiently and rapidly directly to the myocardium in a tissue-specific manner. Such strategy would improve the treatment of cardiovascular diseases (i.e. hypertrophic cardiomyopathies, acute or chronic HF, rhythm disorders) and abolish or minimize undesirable side effects.
Heart homing peptides CTP and PCM have been previously described to preferentially bind to and to internalize cardiomyocytes. However, the characterization of these targeting agents is limited and the targeting efficacy remains modest. Therefore, we have designed, engineered over 100 optimized variants from original peptide sequences and selected new optimized-preferential cardiac targeting peptides (PCTP). We believe that our PCTP will offer a new class of therapeutic when coupled to a medicine-cargo to treat HF.
CardioTarget aims at developing an efficient heart-targeted drug delivery platform. For that, liposomal platforms will encapsulate PKA-inhibitor (i.e. PKI) as therapeutic and will be grafted with our optimized-PCTP, thus allowing rapid and efficient delivery of PKI specifically to cardiomyocytes for the treatment of HF. Therefore, in the course of this project, we propose: 1/ To characterize efficacy and specificity of PKI-coupled PCTP in pharmacological models (i.e. in vitro & ex vivo); 2/ To develop original delivery systems conjugated with PCPT to improve their plasmatic stability and cardiac delivery in vivo; 3/ To assess the efficiency of specific cardiac therapeutic vectors (i.e. PCTP targeted-liposomes) in the delivery of PKI as therapeutic in a pathophysiological model of HF.
CardioTarget will identify new specific cardiac targeting platforms to favor effective and preferential delivery of medicines. These plaftforms will promote development of new therapeutic approaches (peptides or nucleic acids) for the treatment of cardiac diseases with an improvement of the therapeutic index.