Multifunctional near-infrared light-triggered biodegradable micelles for chemo- and photo-thermal combination therapy

Abstract

A combination of chemo- and photo-thermal therapy (PTT) has provided a promising efficient approach for cancer therapy. To achieve the superior synergistic chemotherapeutic effect with PTT, the development of a simple theranostic nanoplatform that can provide both cancer imaging and a spatial-temporal synchronism of both therapeutic approaches are highly desired. Our previous study has demonstrated that near-infrared (NIR) light-triggered biodegradable chitosan-based amphiphilic block copolymer micelles (SNSC) containing light-sensitive 2-nitrobenzyl alcohol and NIR dye cypate on the hydrophobic block could be used for fast light-triggered drug release. In this study, we conjugated the SNSC micelles with tumor targeting ligand c(RGDyK) and also encapsulated antitumor drug Paclitaxel (PTX). The results show that c(RGDyK)-modified micelles could enhance the targeting and residence time in tumor site, as well as be capable performing high temperature response for PTT on cancer cells and two-photon photolysis for fast release of anticancer drugs under NIR irradiation. In vitro release profiles show a significant controlled release effort that the release concentration of PTX from micelles was significantly increased with the exposure of NIR light. In vitro and in vivo antitumor studies demonstrate that, compared with chemo or PTT treatment alone, the combined treatment with the local exposure of NIR light exhibited significantly enhanced anti-tumor efficiency. These findings indicate that this system exhibited great potential in tumor-targeting imaging and synchronous chemo- and photo-thermal therapy.

Keywords: chemotherapy; near-infrared light-triggered nanomicelles; paclitaxel; photo-thermal therapy; tumor targeting.

Figure 1. Synthetic scheme and structures of targeted NIR light sensitive nanoplatform c(RGDyK)-SNSC and α_vβ₃-mediated binding of tumor cells

Figure 2. Size distribution and optical properties of c(RGDyK)-SNSC-cypate-PTX

(A) Size distribution of c(RGDyK)-SNSC-cypate-PTX micelles; (B) Transmission electron microscope pictures of c(RGDyK)-SNSC-cypate-PTX; TEM images of c(RGDyK)-SNSC-cypate-PTX after NIR illumination (765 nm, 400 mW/cm²) for 10 min (C) and 1 h (D). (E) Absorption spectra of PTX, c(RGDyK)-SNSC-PTX, cypate and c(RGDyK)-SNSC-cypate-PTX; (F) Photoluminescence spectra of cypate and c(RGDyK)-SNSC -cypate-PTX; (G) Temperature change curves of 500 μL water (black), free cypate in DMSO (red), c(RGDyK)-SNSC-PTX (blue) and c(RGDyK)-SNSC-cypate-PTX (green) exposed to NIR light (765 nm, 400 mW/cm²) for 20 min; (H) Photostability of cypate and c(RGDyK)-SNSC-cypate-PTX, which was obtained by irradiation of cypate or c(RGDyK)-SNSC-cypate-PTX under NIR light (765 nm, 400 mW/cm²).

Figure 3. The laser confocal fluorescence microscopy images of α_vβ₃-positive MDA-MB-231 and α_vβ₃-negative MCF-7 cells incubated with SNSC-cypate

(A, D), c(RGDyK)-SNSC-cypate (B, E) at 37°C for 8 h; (C) Blocking experiments of c(RGDyK)-SNSC-cypate in MDA-MB-231 cells in the presence of c(RGDyK) (50 mmol/L); Scale bars correspond to 50 μm. (F) Mean fluorescence intensity of MDA-MB-231 and MCF-7 cells, incubated with SNSC-cypate or c(RGDyK)-SNSC-cypate in the absence or presence of c(RGDyK). Data are given as mean ± SD (n = 5). *P < 0.05.

Figure 4

(A) Cumulative release rate of paclitaxel (PTX) from c(RGDyK)-SNSC-PTX and c(RGDyK)-SNSC-cypate-PTX micelles with and without NIR illumination (Ex: 765 nm, 800 mW/cm²). The cell viability of (B) α_vβ₃-positive MDA-MB-231 cells and (C) α_vβ₃-negative MCF-7 cells were determined following the incubation with free PTX (as control), and SNSC micelles loaded with various PTX concentration (0.01, 0.1, 1, 10, and 100 μg/ml) with or without NIR irradiation (Ex: 765 nm, 800 mW/cm², 20 min). (D) Dynamic temperature profile of MDA-MB-231 cells transfected with c(RGDyK)-SNSC-cypate-PTX and exposed to NIR laser light (Ex: 765 nm, 800 mW/cm²). Non-treated cells exposed to NIR light were as control. The arrows indicate when the laser turned on and off, respectively. (E) Cell uptake and morphology after incubating PTX or c(RGDyK)-SNSC-cypate-PTX with or without NIR irradiation (Ex: 765 nm). n = 5 for all groups.

Figure 5. In vivo targeting behavior of c(RGDyK)-SNSC micelles in tumor bearing animals for 1 to 96 hours

Fluorescence images of α_vβ₃-positive MDA-MB-231 tumor-bearing mice after systemic injection of (A) SNSC-cypate and (B) c(RGDyK)-SNSC-cypate micelles; (C) c(RGDyK)-SNSC-cypate micelles with blocking dose of c(RGDyK). In comparison, Fluorescence images of αvβ3-negative MCF-7 tumor-bearing mice after injection of (D) SNSC-cypate and (E) c(RGDyK)-SNSC-cypate micelles. (F) Tumor/normal tissue ratio (T/N ratio = [tumor signal – background signal]/[normal signal (muscle) – background signal] × 100%) calculated from the ROIs at 24 h post-injection of SNSC-cypate and c(RGDyK)-SNSC-cypate micelles with or without blocking dose of c(RGDyK). Data are given as mean ± SD (n = 5). *P < 0.05.

Figure 6. Combinatorial PTT and chemotherapy efficacy of various samples in eradicating MDA-MB-231 tumors implanted in nude mice was determined

Test samples include saline (as negative control), PTX (as control), SNSC-cypate-PTX (with or without NIR light irradiation), c(RGDyK)-SNSC-cypate-PTX (with or without NIR light irradiation). Various treated micelles and controls were administered via intravenous injection and the therapeutic effect of various samples was determined by measuring (A) tumor volumes, (B) animal weight, (C) 14-day survival rates of MDA-MB-231 tumors-bearing mice, and (E) tumor size of the mice with different samples sacrificed after 14-day post-injection. (D) Temperature changes inside the tumor treated with c(RGDyK)-SNSC-cypate-PTX and exposed to NIR laser light (Ex: 765 nm, 800 mW/cm²). Untreated tumor exposed to NIR light were as control. The arrows indicate when the laser turned on and off, respectively. (F) H&E stained tissue samples excised from animals treated with saline (F1), PTX (F2), c(RGDyK)-SNSC-cypate-PTX (without (F3) or with NIR irradiation (F4)) micelles. Image magnification is 200× and n = 8 in all groups.

Table of Contents Graphic

A multifunctional micellar drug delivery system with the capability of active tumor targeting, imaging and light-triggered release for combination photo-thermal and chemo-therapy was developed.