Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice - PubMed (original) (raw)

Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice

Kanyi Pu et al. Nat Nanotechnol. 2014 Mar.

Abstract

Photoacoustic imaging holds great promise for the visualization of physiology and pathology at the molecular level with deep tissue penetration and fine spatial resolution. To fully utilize this potential, photoacoustic molecular imaging probes have to be developed. Here, we introduce near-infrared light absorbing semiconducting polymer nanoparticles as a new class of contrast agents for photoacoustic molecular imaging. These nanoparticles can produce a stronger signal than the commonly used single-walled carbon nanotubes and gold nanorods on a per mass basis, permitting whole-body lymph-node photoacoustic mapping in living mice at a low systemic injection mass. Furthermore, the semiconducting polymer nanoparticles possess high structural flexibility, narrow photoacoustic spectral profiles and strong resistance to photodegradation and oxidation, enabling the development of the first near-infrared ratiometric photoacoustic probe for in vivo real-time imaging of reactive oxygen species--vital chemical mediators of many diseases. These results demonstrate semiconducting polymer nanoparticles to be an ideal nanoplatform for developing photoacoustic molecular probes.

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Conflict of interest statement

Competing financial interests: Dr. Gambhir serves on the board of Endra Inc. and serves as a consultant for Visualsonics Inc., both manufacturers of small animal PA imaging equipment.

Figures

Figure 1

Figure 1. Synthesis and characterization of SPNs

(a) Molecular structures of SP1 and SP2 used for the preparation of SPN1 and SPN2, respectively. (b) Schematic of the preparation of SPNs through nanoprecipitation. SP is represented as a long chain of chromophore units illustrated as red oval beads. DPPC contains a short hydrophobic tail and a charged head, and is illustrated as a string with a dark green ball at its end. (c) Photographic images of the SPN solutions (10 μg/mL). (d) Representative TEM images of SPN1 (left) and SPN2 (right). (e) Representative DLS profiles of SPNs. (f) UV-Vis absorption (dash lines) and PA spectra (solid lines) of SPNs. (g) PA amplitudes of SPNs at 700 nm in an agar phantom as a function of mass concentration. R2 = 0.998 and 0.997 for SPN1 and SPN2, respectively. Error bars represent standard deviation of three separate measurements.

Figure 2

Figure 2. Comparison of PA properties of SPN1 with SWNT (1.2 × 150 nm) and GNR (15 × 40 nm)

(a) PA amplitudes of the nanoparticles based on the same mass (25 μg/mL) (top) and molar (48 nM) (bottom) concentrations in an agar phantom. (b) PA amplitudes of indicated nanoparticles in agar phantoms versus number of laser pulses; the total laser exposure time was 120 μs for 24,000 pulses. (c) PA amplitudes of the nanoparticle-matrigel inclusions (30 μL) in the subcutaneous dorsal space of living mice as a function of nanoparticle mass concentration. The tissue background signal calculated as the average PA signal in areas where no nanoparticles were injected was 0.21 ± 0.03 a.u. R2 = 0.992, 0.990 and 0.992 for SPN1, SWNT, and GNR, respectively. (d) PA/US coregistered images of the nanoparticle-matrigel inclusions at a concentration of 8 μg/mL. The images represent transverse slices through the subcutaneous inclusions (dotted circles). A single laser pulse at 700 nm with a laser fluence of 9 mJ cm−2 and a pulse repetition rate of 20 Hz were used for all experiments. All data represent standard deviation of three separate measurements.

Figure 3

Figure 3. In vivo and ex vivo PA and FL imaging of lymph nodes using SPN1

(a) US (upper) and PA/US coregistered (lower) images of mouse lymph nodes following tail vain injection of SPN1 (50 μg/mouse). The images represent transverse slices through the lymph nodes. BLN: brachial lymph node; ILN: inguinal lymph node; SCLN: superficial cervical lymph node. (b) FL/bright-field images of the corresponding mouse. (c) Ex vivo PA/US coregistered (top) and FL/bright-field (bottom) images of resected lymph nodes from the mouse in (a) (left) or a control mouse without SPN1 injection (right) in an agar phantom. (d) Ex vivo quantification of PA and FL signals of the lymph nodes from SPN1-administrated mice (n=4) and control mice (n=4). *Statistically significant difference in both PA and FL signals between the lymph nodes from SPN1-administrated and control mice (p < 0.05).

Figure 4

Figure 4. In vitro characterization of RSPN for ROS sensing

(a) Proposed ROS sensing mechanism. (b) Representative PA spectra of RSPN in the absence and presence of ROS. [RSPN] = 5 μg/mL, [ROS] = 5 μM. (c) The ratio of PA amplitude at 700 nm to that at 820 nm (PA700/PA820) after treatment with indicated ROS (5 μM). (d) PA images of macrophage RAW264.7 cell pellets (1.5 × 106 cells) without (upper) or with (middle) stimulation with LPS/INF-γ, and with NAC protection (lower). Cell pellets were inserted into an agar phantom and imaged with pulsed laser tuned to (i) 700 nm or (ii) 820 nm, (iii) overlays of images from columns i and ii. The cells were incubated with RSPN (6 μg/mL) for 3 h before trypsinization. (e) Quantification of the absorption ratio (PA700/PA820) for macrophage cell pellets with and without LPS/INF-γor LPS/INF-γ/NAC treatment. The error bars represent the standard deviation from four separate measurements. *Statistically significant difference in PA700/PA820 between LPS/INF-γ treated and untreated or NAC-protected cell pellets (p < 0.05).

Figure 5

Figure 5. In vivo PA imaging of ROS generation from a mouse model of acute edema using RSPN (n=3 each)

(a) PA/US overlaid images of saline-treated (i) and zymosan-treated (ii) regions in the thigh of living mice (n=3). RSPN (3 μg in 50 μL) was intramuscularly injected into the thigh 20 min after zymosan treatment. (b) The ratio of PA amplitude at 700 nm to that at 820 nm (PA700/PA820) as a function of time post-injection of RSPN. *Statistically significant difference in PA700/PA820 between zymosan-treated and saline-treated mice at all time points starting from 10 min (p<0.05).

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