Fabrication of self-assembling nanofibers with optimal cell uptake and therapeutic delivery efficacy

Abstract

Effective strategies to fabricate finite organic nanoparticles and understanding their structure-dependent cell interaction is highly important for the development of long circulating nanocarriers in cancer therapy. In this contribution, we will capitalize on our recent development of finite supramolecular nanofibers based on the self-assembly of modularly designed cationic multidomain peptides (MDPs) and use them as a model system to investigate structure-dependent cell penetrating activity. MDPs self-assembled into nanofibers with high density of cationic charges at the fiber-solvent interface to interact with the cell membrane. However, despite the multivalent charge presentation, not all fibers led to high levels of membrane activity and cellular uptake. The flexibility of the cationic charge domains on self-assembled nanofibers plays a key role in effective membrane perturbation. Nanofibers were found to sacrifice their dimension, thermodynamic and kinetic stability for a more flexible charge domain in order to achieve effective membrane interaction. The increased membrane activity led to improved cell uptake of membrane-impermeable chemotherapeutics through membrane pore formation. In vitro cytotoxicity study showed co-administering of water-soluble doxorubicin with membrane-active peptide nanofibers dramatically reduced the IC50 by eight folds compared to drug alone. Through these detailed structure and activity studies, the acquired knowledge will provide important guidelines for the design of a variety of supramolecular cell penetrating nanomaterials not limited to peptide assembly which can be used to probe various complex biological processes.

Keywords: Cell penetrating peptide; Cell uptake; Drug delivery; Membrane activity; Supramolecular assembly.

Graphical abstract

Fig. 1

TEM images of the nanofibers formed by (a) K10 and (b) K6 and statistical measurements of fiber length and length distribution based on a total number of 200 fibers. Peptide concentration: 100 μM in Tris buffer (20 mM, pH = 7.4). Scale bar: 100 nm.

Fig. 2

CAC determination through fluorescence measurements of peptides as a function of concentration in Tris buffer (20 mM, pH 7.4) (a) K10 and (b) K6.

Fig. 3

(a) Cell membrane current change upon cell exposure to 16 μM of K10 or 26 μM of K6 for duration of 95 s. (b) Statistical measurements of the onset of current leakage. Data are presented as the mean ± SEM. Statistically significant differences are indicated by *p ≤ 0.05.

Fig. 4

Cell uptake of FITC-labeled K10 and K6 upon incubation with HeLa cells. (a) MDP localization after incubation with HeLa cells, Scale bar: 10 μm (b) Time-dependent cell uptake of MDPs monitored by flow cytometry after 2 h and 24 h of incubation with HeLa cell. Data are presented as the mean ± SD, n = 4. Statistically significant differences are indicated by **p ≤ 0.01, *p ≤ 0.05. K10: 16 μM. K6: 16 μM.

Fig. 5

Hela cell viability upon incubation with DOX at different concentrations in the presence and absence of peptide nanofibers for (a) 1 h, (b) 8 h and (c) 24 h. Data are presented as the mean ± SD, n = 4. Statistically significant differences are indicated by **p ≤ 0.01, *p ≤ 0.05. K10: 16 μM. K6: 26 μM.

Fig. 6

Hela cell viability upon incubation with DOX in the presence and absence of peptide nanofibers with different stereochemistry for 24 h. Data are presented as the mean ± SD, n = 4. The final concentrations of both peptides are 16 μM.