Generation of Self‐Assembled Structures Composed of Amphipathic, Charged Tripeptides for Intracellular Delivery of Pro‐Apoptotic Chemotherapeutics

Abstract

Chemotherapeutic drugs remain the most efficacious treatment options for a many human cancers. However, the inability to deliver these drugs directly to cancerous cells often results in dose limiting and sometimes life-threatening adverse effects. Rather than developing new chemical moieties, researchers have begun focusing on the development of drug carriers, which are specifically designed to shuttle chemotherapeutics into malignant cells while sparing healthy cells. Charged nanoparticles have emerged as effective delivery platforms for several xenobiotic classes including anticancer agents, oligonucleotides and antibodies. Notably, peptide-based self-assembled nanostructures are of particular interest due to their biocompatibility, high drug loading capacity, and potential for customization for cellspecific targeting. We synthesized and studied the selfassembling properties of two charged, cell penetrating tri-peptides: H 2 NÀ ArgÀ LeuÀ PheÀ OMe (S1) with Arg as cationic amino acid and BocÀ PheÀ LeuÀ GluÀ OH (S2) with Glu as negatively charged amino acid. The fibrils and spherical selfassembled structures formed by S1 and S2, respectively, can encapsulate the chemotherapeutic drug Doxorubicin and facilitate intracellular drug delivery via endocytosis. Furthermore, S1-and S2-encapsulated Doxorubicin (Dox-S1, Dox-S2), like the unencapsulated drug, induced oxidative stress and mitochondrial dysfunction, activated the ATM/p53 signaling cascades, and triggered apoptosis in cancer cells. Thus, while the surface charge of molecular building blocks used to generate supramolecular assemblies influences the morphology of generated nanostructures, both cationic and anionic peptide-based assemblies are capable of functioning as drug delivery vehicles.

Functionalized Fluorescent Nanostructures Generated from Self-Assembly of a Cationic Tripeptide Direct Cell-Selective Chemotherapeutic Drug Delivery

“…Furthermore, we checked the drug release capability of PA1−Dox by monitoring steady-state fluorescence, as described in our earlier work. 19 We observed a gradual increase in the fluorescence intensity of the PBS buffer solution located outdoor of the dialysis bag due to the release of the encapsulated Dox molecule from the PA1−Dox-based spherical structures (Figure 4C). A steady rise of the fluorescence intensity was observed up to 45 h, after that there was no noticeable change in the fluorescence intensity with time (up to 70 h).…”

“…The calculated EE and LC were 61.29 and 16.6%, respectively (see Materials and Methods). Furthermore, we checked the drug release capability of PA1 – Dox by monitoring steady-state fluorescence, as described in our earlier work . We observed a gradual increase in the fluorescence intensity of the PBS buffer solution located outdoor of the dialysis bag due to the release of the encapsulated Dox molecule from the PA1 – Dox- based spherical structures (Figure C).…”

“…For example, inorganic nanoparticles having toxic elements and non-biodegradable metal ions are inappropriate for administration in vivo . In contrast, short peptide-based supramolecular nanostructures are based on biocompatible materials and have displayed a remarkable degree of customization. , Short peptides can self-assemble into various supramolecular nanostructures with unique morphology by modifying the self-assembly parameters such as the solvent medium, ionic strength, concentration, pH, temperature, and structural modifications all of which significantly influence non-covalent interactions between the building blocks. − Notably, the synthesized peptides can resemble endogenous ligands, allowing for the targeting of receptors overexpressed on the surface of most of the malignant cells. , The facile synthesis and diversity of potential structural modifications can allow for further optimization of peptide-based nanoparticles for drug delivery. − …”

Liposomes Containing Zinc-Based Chemotherapeutic Drug Block Proliferation and Trigger Apoptosis in Breast Cancer Cells

“…The results are reported as the fold raise in the enrichment factor of cytoplasmic nucleosomes. 58 Nuclear Fragmentation Assay. MCF-7 cells were seeded at a density of 1 × 10 5 cells/well on sterile cover glasses placed in the 6 well plates and incubated for 24 h in the incubator.…”

“…The cell lysates were incubated at 37 °C for approximately 30−35 min with JC-1 (2.5 μg/mL) in a PBS solution, with continuous shaking. 58 Subsequently, the cells were washed three times with cold PBS for 5 min each and then resuspended in PBS. The ratio of fluorescence intensity at 590 to 530 nm was assessed to measure the mitochondrial membrane potential (MMP ψM).…”