Synthesis and in vitro evaluation of cyclic NGR peptide targeted thermally sensitive liposome

Abstract

The Asn-Gly-Arg (NGR) motif in both cyclic and linear form has previously been shown to specifically bind to CD13/aminopeptidase N that is selectively overexpressed in tumor vasculature and some tumor cells. However, previous versions of cyclic NGR used a liable disulfide bridge between cysteine residues that may be problematic for liposome targeting due to disulfide bond formation between adjacent peptides on the liposomal surface. In this study, we report the design, synthesis, and characterization of a novel cyclic NGR-containing peptide, cKNGRE, which does not contain a disulfide bridge. cKNGRE was synthesized in good yield and purity and attached to the fluorescent reporter Oregon Green (cKNGRE-OG) and lysolipid-containing temperature sensitive liposomes (LTSLs). The identity of cKNGRE was verified with NMR and mass spectral techniques. In vitro fluorescence microscopy evaluation of cKNGRE-OG demonstrated binding and active uptake by CD13(+) cancer cells and minimal binding to CD13(-) cancer cells. The cKNGRE-OG ligand displayed 3.6-fold greater affinity for CD13(+) cancer cells than a linear NGR-containing peptide. Affinity for CD13(+) cancer cells was similarly improved 10-fold for both the cyclic and linear NGR when presented in a multivalent fashion on the surface of an LTSL. cKNGRE-targeted LTSLs rapidly released (>75% in <4s) doxorubicin at 41.3 degrees C with minimal release at 37 degrees C. These results demonstrate the ability to synthesize a cKNGRE-targeted temperature sensitive liposome that lacks a disulfide bridge and has sufficient binding affinity for biological applications.

Figure 1

MALDI-TOF-MS Spectra for compounds 6a (A) & b (B).

Figure 2

Fluorescence microscopy to evaluate in vitro binding of anti-CD13 antibody WM15 (top row: A, B, and C) and cKNGRE-OG (bottom row: D, E, and F) to CD13⁺ HT-1080 cells (B, C, E, and F) and CD13⁻ MCF7 cells (A and D). Green, signal from cKNGRE-OG or WM15 antibody. Blue, signal from the nuclear staining agent DAPI. Images AB and DE were acquired with identical exposure times and displayed consistently to compare binding between MCF7 and HT-1080 cells. Bar equals 100 µm for A, B, D, and E and 50 µm for C and F

Figure 3

Epifluorescence microscopy to evaluate in vitro binding of linear KNGRG-OG (A) and cKNGRE-OG (B) by HT-1080 cells at 37 °C. Green signal from KNGRG-OG or cKNGRE-OG and Blue signal from the nuclear staining agent DAPI. Images were acquired with identical exposure times and displayed consistent window and level. Bar equals 100 µm.

Figure 4

Epifluorescence microscopy to evaluate in vitro internalization of cKNGRE-OG by HT-1080 cells at 4 °C (A) or 37 °C (B). Green signal from cKNGRE-OG and Blue signal from the nuclear staining agent DAPI. Images were acquired with identical exposure times and displayed consistent window and level. Bar equals 100 µm.

Figure 5

Confocal microscopy to evaluate internalization of cKNGRE-OG in HT-1080 cells at 37°C. Blue signal from the nuclear staining agent DAPI (A), Green signal from cKNGRE-OG (B), merged image of A and B (C), and merged image of A and B with Dodt contrast to visualize cell boundaries (D). Bar equals 50 µm.

Figure 6

In vitro binding expressed as % positive HT-1080 cells (CD13⁺) versus concentration of cKNGRE and linear KNGRG conjugated to OG (A) and LTSL (B). The half maximal effective concentration (EC₅₀) was determined from the Hill equation (shown as a solid line).

Figure 7

Release of Dox from cKNGRE-targeted LTSLs. Percent release is calculated by assuming 100% release with Triton® X-100 and 0% release at 25 °C in a HEPES buffer.

Scheme 1

Reagents and conditions: a) N-Fmoc-Glu-(OAll)-OH (2.5 equiv), HATU (2 equiv), DIPEA (4 equiv), NMP, rt, 30 min.; b) Piperidine-DMF (1:4), rt, 5 min (X 2); c) N-Fmoc-Arg-(Pbf)-OH (2 equiv), HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; d) N-Fmoc-Gly-OH, HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; e) N-Fmoc-Asn-(Trt)-OH, HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; f) N-Fmoc-Lys-(Boc)-OH, HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; g) Pd(PPh3)4 (2 equiv), CHCl3-AcOH-N-Methylmorpholine (37:2:1), rt, 2 h; h) HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min;17 h; i) TFA-CH2Cl2 (1:1), rt, 2 h.

Scheme 2

Reagents and conditions: a) Piperidine-DMF (1:4), rt, 5 min (X 2); b) N-Fmoc-Arg-(Pbf)-OH (2 equiv), HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; c) N-Fmoc-Gly-OH, HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; d) N-Fmoc-Asn-(Trt)-OH, HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; e) N-Fmoc-Lys-(Boc)-OH, HATU, (2 equiv), DIPEA (4 equiv), rt, 30 min; f) CH2Cl2: Ac2O: DIPEA (20: 40: 1); g) TFA-CH2Cl2 (1:1), rt, 2 h.

Scheme 3

Reagents and conditions: a) Oregon Green 488 carboxylic acid, succinimidyl ester, 6-isomer (1.1 equiv), DIPEA (2 equiv), NMP (1 mL), rt, 5 hr.

Scheme 4

Reagents and conditions: a) HOBt (1.1 equiv), DCC (1.1 equiv.), DIPEA (3 equiv), NMP (1 mL), 30 min.), 3 or 4 (1.2 equiv), rt, overnight.