Polymeric dual-modal imaging nanoprobe with two-photon aggregation-induced emission for fluorescence imaging and gadolinium-chelation for magnetic resonance imaging

Abstract

Nanoprobes that offer both fluorescence imaging (FI) and magnetic resonance imaging (MRI) can provide supplementary information and hold synergistic advantages. However, synthesis of such dual-modality imaging probes that simultaneously exhibit tunability of functional groups, high stability, great biocompatibility and desired dual-modality imaging results remains challenging. In this study, we used an amphiphilic block polymer from (ethylene glycol) methyl ether methacrylate (OEGMA) and N-(2-hydroxypropyl) methacrylamide (HPMA) derivatives as a carrier to conjugate a MR contrast agent, Gd-DOTA, and a two-photon fluorophore with an aggregation-induced emission (AIE) effect, TPBP, to construct a MR/two-photon fluorescence dual-modality contrast agent, Gd-DOTA-TPBP. Incorporation of gadolinium in the hydrophilic chain segment of the OEGMA-based carrier resulted in a high r ₁ value for Gd-DOTA-TPBP, revealing a great MR imaging resolution. The contrast agent specifically accumulated in the tumor region, allowing a long enhancement duration for vascular and tumor contrast-enhanced MR imaging. Meanwhile, coupling TPBP with AIE properties to the hydrophobic chain segment of the carrier not only improved its water solubility and reduced its cytotoxicity, but also significantly enhanced its imaging performance in an aqueous phase. Gd-DOTA-TPBP was also demonstrated to act as an excellent fluorescence probe for two-photon-excited bioimaging with higher resolution and greater sensitivity than MRI. Since high-resolution, complementary MRI/FI dual-modal images were acquired at both cellular and tissue levels in tumor-bearing mice after application of Gd-DOTA-TPBP, it has great potential in the early phase of disease diagnosis.

Keywords: Amphiphilic block polymers; Magnetic resonance/fluorescence dual-modal imaging; RAFT polymerization; Tumor/vascular imaging; Two-photon AIE fluorescent contrast agent.

Graphical abstract

Scheme 1

Schematic diagram for synthesis of an imaging probe, Gd-DOTA-TPBP, for magnetic resonance imaging (MRI) and two-photon fluorescent imaging (TPFI) after stepwise copper-free click chemistry and chelation. The amphiphilic block conjugate Gd-DOTA-TPBP could self-assemble to form stable nanoparticles for dual-modality imaging of mice with high sensitivity.

Fig. 1

Physicochemical characterizations of Gd-DOTA-TPBP. (A) ¹H NMR spectrum of p(DOTA-OEG)-b-p(TPBP) in CD₃OD. (B) FTIR spectra of p(DOTA-OEG)-CTA, p(DOTA-OEG)-b-p(BzN₃) and p(DOTA-OEG)-b-p(TPBP). (C) Absorbance spectra of TPBP, Gd-DOTA-TPBP, and p(DOTA-OEG)-b-p(BzN₃). (D) EDX spectrum of Gd-DOTA-TPBP. The peak for each element is assigned from the standard references. (E) Critical micelle concentration (CMC) of Gd-DOTA-TPBP using pyrene as a fluorescence probe. (F) Fluorescent spectra of Gd-DOTA-TPBP at different ratios of DMSO/water. (G) Typical TEM image and hydrodynamic size of Gd-DOTA-TPBP nanoparticles measured by dynamic light scattering (DLS). (H) The stability results of Gd-DOTA-TPBP in PBS (pH 7.4) for 36 h. (I) T₁-weighted MR images of Gd-DOTA-TPBP in PBS at different Gd(III) concentrations. (J) Water proton longitudinal relaxation rate (1/T₁) versus the Gd(III) concentration of Gd-DTPA and Gd-DOTA-TPBP conjugates in PBS in a field of 3.0 T.

Fig. 2

Cellular uptake, cell viability and toxicity assessment of Gd-DOTA-TPBP. (A) Two-photon CLSM images of 4T1 cells cultured with Gd-DOTA-TPBP for different durations excited at 800 nm. (B) Two-photon CLSM images of HUVECs cultured with Gd-DOTA-TPBP for different durations excited at 800 nm. The scale bar is 50 μm. (C) Viability of 4T1 cells cultured with Gd-DOTA-TPBP, Gd-DTPA or TPBP at different Gd(III) or TPBP concentrations for 24 h (n = 5). (D) Viability of HUVECs cultured with Gd-DOTA-TPBP, Gd-DTPA or TPBP at different Gd(III) or TPBP concentrations for 24 h (n = 5). (E) Photos of red blood cells incubated with different solutions for 24 h after centrifugation. (F) Hemolysis rates of Gd-DOTA-TPBP at different concentrations (n = 3). (G) H&E analysis of different organs at 24 h post-injection of Gd-DOTA-TPBP or saline. The scale bar is 100 μm.

Fig. 3

Vascular images after injection of Gd-DOTA-TPBP. (A) Cerebrovascular enhanced MR images at different time points post injection of Gd-DOTA-TPBP or Gd-DTPA (n = 3). White arrows point to cerebral blood vessels. 1. superior sagittal sinus vein; 2. middle cerebral artery; 3. anterior cerebral artery; 4. internal carotid artery; 5. basilar artery. (B) Enhanced MRI scans of peritumor blood vessels at different time points post injection of Gd-DOTA-TPBP or Gd-DTPA. Red arrows point to the enhanced blood supply vessel of the tumor (n = 3). (C) Two-photon excitation confocal images of mouse brain blood vessels at 90 min post injection of Gd-DOTA-TPBP (n = 3). (D) Two-photon excitation confocal images of mouse tumor blood vessels at 90 min post injection of Gd-DOTA-TPBP (n = 3).

Fig. 4

Enhanced MRI/FI scans of the mouse tumor after injection of Gd-DOTA-TPBP and its pharmacokinetics. (A) In vivo MRI scan images of tumors after injection of Gd-DOTA-TPBP. The red dotted circles refer to the tumors. (B) Semi-quantitative analysis of relative MRI signal enhancement (n = 3). (C) Semi-quantitative analysis of fluorescence signal intensity in tumors after injection of Gd-DOTA-TPBP or TPBP (n = 3). (D) Pharmacokinetics of Gd-DOTA-TPBP and Gd-DTPA (n = 3).

Fig. 5

Enhanced MR images of main organs of mice (n = 3). (A) MR images of the bladder. (B) MR images of the kidney. (C) MR images of the liver. (D) Semi-quantitative analysis of relative signal enhancement in the bladder. (E) Semi-quantitative analysis of relative signal enhancement in the kidney. (F) Semi-quantitative analysis of relative signal enhancement in the liver. The red dashed circles in (A–C) indicate the corresponding organs. *p < 0.05 for Gd-DOTA-TPBP vs Gd-DTPA.