Thermoresponsive Collagen/Cell Penetrating Hybrid Peptide as Nanocarrier in Targeting-Free Cell Selection and Uptake

Abstract

The effective delivery of therapeutics and imaging agents to a selected group of cells has been at the forefront of biomedical research. Unfortunately, the identification of the unique cell surface targets for cell selection remains a major challenge, particularly if cells within the selected group are not identical. Here we demonstrate a novel approach to cell section relying on a thermoresponsive peptide-based nanocarrier. The hybrid peptide containing cell-penetrating peptide (CPP) and collagen (COLL) domains is designed to undergo coil-to-helix transition (folding) below physiological temperature. Because only the helical form undergoes effective internalization by the cells, this approach allows effective temperature-discriminate cellular uptake. The cells selected for uptake are locally cooled, thus enabling the carrier to fold and subsequently internalize. Our approach demonstrates a generic method as selected cells could differ from the adjacent cells or could belong to the same cell population. The method is fast (<15 min) and selective; over 99.6% of cells in vitro internalized the peptide carrier at low temperatures (15 °C), while less than 0.2% internalized at 37 °C. In vivo results confirm the high selectivity of the method. The potential clinical applications in mixed cell differentiation carcinoma, most frequently encountered in breast and ovarian cancer, are envisioned.

Figure 1

Molar ellipticity at 224 nm (solid line) of (a) FL8V1, (b) FL7V1, and (c) FL6V1, and first derivative (dotted curve). Thermal unfolding shows that the peak (224nm) is eliminated in a cooperative transition what indicates the helix-to-coil transition.

Figure 2

Confocal microscopy images of 3T3 mouse fibroblasts incubated for 15 min with FL6V1, FL7V1 and FL8V1 peptide nanocarrier (15μM) at 37 °C (panel 1) and 15 °C (panel 2); bar represents 20 μm.

Figure 3

Flow cytometry of NIH 3T3 Swiss mouse fibroblasts incubated for 15 min with FL6V1 at 15 °C and 37 °C.

Figure 4

Fluorescence (a–d) and bright-field (e–h) images of NIH 3T3 fibroblasts subjected to temperature gradient and incubated with FL6V1 for 15 min; bar represents 50 μm.

Figure 5

Confocal microscopy images of Pristina leidyi incubated for 15 min with FL7V1 peptide at 37°C (a–f) and 19 °C (g–l); (k, l) arrows indicate FL7V1internalization; bar represents 100 μm; control experiments were performed with no peptide present.

Figure 6

Fluorescent microscopy image of Pristina leidyi (a) incubated with FL7V1 for 15 min at 19 °C and (b) control, no peptide present; bar represents 200 μm.

Figure 7

Fluorescence and bright field images of Pristina leidyi subjected to temperature gradient (indicated in color) and (a,b) incubated with FL6V1 for 15 min, (c,d) control, no peptide present; bar represents 200μm.

Scheme 1

Scheme of hybrid peptide domains and reversible thermal folding. FITC (fluorescein tag), COLL (collagen folding domain), CPP (cell penetrating peptide domain).