Drug-mimic Peptide Hydrogelators for Treatment of Hypertension

Introduction: Obesity, a recognized precursor to hypertension, significantly escalates the risk of cardiovascular ailments such as stroke, and heart failure. While pharmaceutical interventions such as diuretics and calcium channel blockers for hypertension treatment exist, approximately one-third of patients, identified as having resistant hypertension, exhibit inadequate responses to conventional treatments. This underscores the imperative for the development of novel therapeutic formulations tailored to address this unmet clinical need.
Obesity-induced elevation in leptin levels underscores its role in hypertension pathogenesis through the activation of TRPM7 receptor within the carotid body (CB), thereby instigating the sympathetic nervous system activation. FTY720, a TRPM7 blocker, demonstrates potential in mitigating leptin-induced hypertension by competitively binding to the TRPM7 receptor, thereby attenuating leptin levels. However, the effects only last two weeks, necessitating strategies to prolong its therapeutic effects.
To achieved hypertension treatment strategies with improved efficacy, we leverage the unique materials and biological properties of peptide-based hydrogels to achieve controlled and long-acting release of therapeutic agents. Our approach entails the development of drug-mimic hydrogelators to improve drug loading capacity while enabling effective control over the sustained release rate. Specifically, we engineered peptide amphiphiles (PAs) mimicking the chemical structure of FTY720, an antihypertensive leptin inhibitor. The similarity in molecular structures promotes co-assembly of the FTY720 drug with the designed PA with enhanced interactions and allows for a controlled drug release over a long period of time. This innovative formulation holds promise in addressing the shortcomings of current hypertension treatments and warrants further investigation for its therapeutic potential.

Materials and

Methods: Self-assembling peptide and peptide amphiphile (PA)-based hydrogels offer versatile platforms for localized drug delivery, owing to their tunable mechanical and biological properties. Leveraging the hydrophobicity and alkyl tail of FTY720, we designed a drug-mimetic PA structurally akin to FTY720, incorporating an octyl benzoic acid segment. The alternating sequence of aspartic acid and phenylalanine facilitated PA self-assembly, synthesized via solid-phase peptide synthesis employing Fmoc chemistry. Purification was achieved through reverse phase high-performance liquid chromatography (RP-HPLC), with mass verification conducted via electrospray ionization-mass spectrometry (ESI).
PAs spontaneously associate in aqueous environments, forming filamentous nanostructures such as nanofibers or nano-bands through non-covalent interactions. Co-assembly of FTY720 within the PA matrix was characterized using transmission electron microscopy (TEM), demonstrating filamentous morphology conducive to physiological entanglement, facilitating the formation of self-supporting, supramolecular hydrogels via charge screening.
Drug release kinetics were evaluated through HPLC analysis of gel elution profiles, determining FTY720 concentrations over 30 days. In vitro and in vivo biocompatibility assessments affirmed the non-toxic nature of the FTY720-loaded hydrogel. Animal studies involving diet-induced obesity C57BL/6J mice monitored mean arterial pressure (MAP) via telemetry following bilateral administration of FTY720 hydrogel or control hydrogel (PA without drug) directly into the CB area. Concurrent measurements of heart rate, body weight, and food intake provided insight into potential treatment-associated side effects.

Results, Conclusions, and Discussions: We synthesized five distinct molecules, namely OBA-2DF, OBA-2FD, C16-2DF, OBA-4DF, and OBA-4FD. Employing a co-assembly method, FTY720 and each peptide amphiphile (PA) were prepared. Briefly, PAs were dissolved in HFIP to achieve a final FTY720 concentration of 5 mM. Following HFIP evaporation and water addition, resulting in a final peptide concentration of 10 mM, the solution was heated to 80°C for an hour, annealed at room temperature, and aged overnight to promote co-assembly. Purification of all three molecules was conducted via HPLC, with nanostructure investigation performed through TEM imaging.
OBA-2DF PA exhibited a twisted ribbon-like structure capable of forming rope-like assemblies via entanglement. Co-assembly with FTY720 yielded a similar structure, indicating unaltered morphology and complete drug encapsulation. Gelation studies with 5 mM FTY720 co-assembled with 10 mM OBA-2DF demonstrated gel formation within minutes upon dilution to 1x DPBS. In contrast, OBA-2FD formed a ribbon-like structure, and co-assembly with FTY720 mirrored this pattern, suggesting the positional arrangement of aspartic acid and phenylalanine influenced molecular nanostructure.
Among the five molecules, OBA-2DF exhibited the highest drug loading capacity at 31.89%, releasing 3.7% of free FTY720 within 30 days. In vitro cytotoxicity studies via MTT assay revealed non-toxicity across all molecules. Injection of OBA-2DF hydrogel into C57BL6J mice, with body weight measurement every other day, demonstrated biocompatibility. Subsequent measurement of mean arterial pressure (MAP) post-treatment with OBA-2DF hydrogel compared to controls revealed a significant reduction in blood pressure, with effects lasting up to 6 weeks.
Collectively, these findings underscore the potential of our drug-mimetic hydrogelators, showcasing high drug loading and sustained release properties, thereby presenting promising avenues for therapeutic cargo design.

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