2.1.

Materials

10 × phosphate buffer saline (PBS) buffer with $\mathrm{pH}=7.4$ (ultrapure grade) is a commercial product of first BASE Singapore. MilliQ water (18.2 MQ) was used to prepare the buffer solution from the 10 × PBS stock buffer. 1 × PBS consists of NaCl (137 mM), KCl (2.7 mM), ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ (10 mM), and ${\mathrm{KH}}_2{\mathrm{PO}}_4$ (1.8 mM). Chloroform-D (99%) was purchased from Cambridge Isotope Laboratories, Inc. All other chemicals and reagents were purchased from Aldrich or Merck and used as received unless otherwise specified. Conjugated polymer PIGD was synthesized according to our previous report.

2.2.

Characterization

UV–vis-NIR absorption spectra of the samples were measured on a SHIMADZU UV-2450 spectrophotometer. TEM measurements were performed with a TEM Carl Zeiss Libra 120 Plus at an acceleration voltage of 120 kV. A $5\text{-}\mu \mathrm{L}$ droplet of diluted samples was directly dropped onto a copper grid (300 mesh) coated with a carbon film, followed by drying at room temperature. The size distribution of resulting nanoparticles was determined by dynamic light scattering (DLS) using a BI-200SM (Brookhaven) with angle detection at 90 deg.

2.3.

Preparation of Nanoparticles

Conjugated polymer PIGD (5 mg) and macromolecular surfactant Pluronic F-127 (50 mg) were co-dissolved in 0.5 mL of THF. The mixture was rapidly injected into 2.5 mL of 1 × PBS buffer under ultrasonication (Alstron ultrasonic cleaner, model: ALD-40100-38H). After evaporation of excess THF, a well-dispersed solution of PIGD NPs with a concentration of $2\mathrm{mg}/\mathrm{mL}$ was obtained. Specifically, a mixture of PIGD polymers and a macromolecular surfactant Pluronic F-127 (The mass ratio of PIGD and F127 is $1:10$) codissolved in 1 mL of THF was rapidly injected into 10 mL of water under sonication [Fig. 1(b)], followed by exposure in air at room temperature overnight to remove THF. The resulting SPNs were well-dispersed in PBS buffer.

2.4.

PAT System for Imaging at 1064-nm Wavelength

A home-made PAT system was used for PA measurements. The excitation source is 1064 nm Nd:YAG laser (Continuum, Surelite Ex) that can generate 5-ns pulses at 10-Hz repetition rate. The 1064-nm beam was guided to the circular scanner, and homogenized using an optical ground glass (GG). The test sample and the ultrasound transducer (UST) were immersed in water for coupling the PA signal to the transducer. The PA signal generated by the sample was received by a nonfocused transducer (V323-SU/2.25 MHz, Olympus NDT) with a 13-mm active area and 70% nominal bandwidth. The PA signals were subsequently amplified, bandpass filtered by an ultrasound pulser/receiver unit (AU), (Olympus NDT, 5072PR), and then digitized and recorded by the PC with a DAQ (data acquisition) card ($25\mathrm{Ms}/\mathrm{s}$, GaGe, compuscope 4227). For solution-level testing, we performed experiments on fresh rat blood and PIGD NPs inside a low-density polyethylene tube [$\text{inner diameter}(\mathrm{ID})=0.59\mathrm{mm}$, $\text{outer diameter}(\mathrm{OD})=0.78\mathrm{mm}$]. For two-dimensional (2-D) imaging, the transducer was driven by a computer-controlled stepper motor (SM) to continuously move in a circular motion. The acquired A-lines were used to reconstruct the PA image of the sample using a delay-and-sum back projection algorithm.^38,39 The imaging system has a spatial resolution of $\sim 380\mu \mathrm{m}$ using 2.25-MHz UST.

2.5.

Agar Gel Phantom for Deep-Tissue Imaging

To demonstrate the deep-tissue imaging capability of the system, three agar gel cylinders containing PIGD NPs with different concentrations were embedded inside agar gel of 2.5 cm in diameter. The three cylinders s1, s2, and s3 [Fig. 3(a)] contain PIGD NPs with concentrations 1.0, 0.5, and $0.25\mathrm{mg}/\mathrm{mL}$, respectively. Fresh chicken-breast tissue slices were cut and placed on the phantom to determine the maximum imaging depth. The chicken slice fully covered the phantom to avoid leakage of light to the sample. Several slices of chicken-breast tissue were sequentially placed to make the objects at different depths from the laser-illuminated tissue surface.

2.6.

Imaging Rat Brain Vasculature In Vivo

For brain vascular imaging, NTac:Sprague Dawley^®SD^® healthy female rats of body weight $\sim 95\pm 3\mathrm{gm}$, procured from InVivos Pte. Ltd., Singapore, were used. All the in vivo experiments were performed according to the guidelines and regulations approved by the institutional Animal Care and Use committee of Nanyang Technological University, Singapore (Animal Protocol Number ARF-SBS/NIE-A0263). Before going for brain vascular imaging, the rat was anaesthetized for short period of time to depilate the hair on the scalp and to mount it inside the scanner. This anaesthesia mixture was prepared with 2 mL of Ketamine ($100\mathrm{mg}/\mathrm{mL}$), 2 mL of xylazine ($20\mathrm{mg}/\mathrm{mL}$), and 1 mL of saline. 0.2 mL per 100 g rat weight was injected intraperitoneally. This ketamine/xylazine cocktail can make the animal anesthetized for 30 to 40 min. For brain vascular imaging, the hair on the scalp was depilated. The animal was mounted in the system as shown in Fig. 2(a). While acquiring images in post-injection time period (about 2 h), the anaesthesia was achieved by the continuous inhalation of a mixture of $1.0\mathrm{L}/\min$ oxygen and 0.75% isoflurane. PAT imaging was performed before the injection of the contrast agent. Afterward, PIGD NPs (0.25 mL, $2\mathrm{mg}/\mathrm{mL}$) was injected into the rat tail vein, then PAT images were acquired for about 2 hours. After collecting the data, the animal was euthanized by the intraperitoneal injection of pentobarbital ($300\mathrm{mg}/\mathrm{mL}$).

Fig. 2

(a) Schematic diagram of NIR-II PAT system. Here, M, mirror; CSP, circular scanning plate; MPS, motor pulley system, SM, stepper motor; DAQ, data acquisition card; AU, ultrasound pulser/receiver unit; UST, ultrasound transducer; GG, ground glass; PF, $100\text{-}\mu \mathrm{m}$ transparent polythene membrane; (b) PA signals of PIGD NPs compared with fresh rat blood, and (c) PA amplitude as a function of concentration of PIGD nanoparticles.

3.1.

Preparation and Characterization of NIR-II Light-Absorbing Polymeric Nanoparticles

The synthesis of PIGD semiconducting polymers is described in Fig. 1(a). Direct arylation polymerization (DAP) was utilized to synthesize the target semiconducting polymers by coupling between the brominated DPP and nonbrominated TIIG monomers under our optimized reaction conditions.^40–42 In contrast to conventional cross-coupling techniques, such as Suzuki⁴³ coupling or Stille⁴⁴ coupling, which requires preactivation of C-H bonds, DAP technique enables the synthesis of semiconducting polymers in fewer steps. Moreover, the absence of flammable organometallic reagents and toxic byproducts makes DAP as a better choice to afford more synthetically scalable and biocompatible materials for biological applications.

PIGD SPNs were prepared by a traditional method of nanoprecipitation.^45,46 The representative transmission electron microscopy (TEM) image [Fig. 1(c)] shows that the PIGD SPNs appear spherical, with an average diameter of $21\pm 5\mathrm{nm}$ in the dry state, which is smaller than that (28.8 nm) in the hydrated state in water as measured with DLS [Fig. 1(d)]. This dispersion displayed a broad absorption band, mainly covering the range from 800 to 1200 nm [Fig. 1(e)] with a maximum (${\lambda }_{\mathrm{abs},\max }$) at 960 nm. The presentation of the absorption spectrum of PIGD in toluene, in comparison with its colloidal nanoparticle in water, is to understand the aggregation of PIGD inside the core of the nanoparticles. As compared with that of PIGD in dilute toluene (${\lambda }_{\mathrm{abs},\max }=1100\mathrm{nm}$), a significant blue shift of absorption band was observed. This behavior could be attributed to the formation of H-aggregates of PIGD macromolecules due to their high planarity and strong $\pi -\pi$ stacking in the aggregating state.⁴⁷ The measured absorption coefficient of the PIGD SNPs ($40\mu \mathrm{g}/\mathrm{mL}$) at 1064 nm is $\sim 1.5{\mathrm{cm}}^{-1}$. The absorption coefficients of $40\mu \mathrm{g}/\mathrm{mL}$ SNPs at 1064 nm reported are $\sim 1.96{\mathrm{cm}}^{-1}$,²³ $2.2{\mathrm{cm}}^{-1}$,²² and that for 0.5 mM copper sulfide is $1.5{\mathrm{cm}}^{-1}$.¹⁷

3.2.

PA Signals from Blood and PIGD Samples at 1064 nm

The strong light absorption of PIGD NPs in the second NIR region [Fig. 1(e)] suggests that the potential of these NPs as a second window contrast agent. To determine the suitable concentration of NPs for deep-tissue and in vivo imaging, the PA signals of PIGD were compared with the signal of rat blood at 1064 nm. The PA signal amplitude from $2\mathrm{mg}/\mathrm{mL}$ PIGD NPs is $\sim 10$ times stronger than that from the blood. Figure 2(b) shows the PA signals from rat blood and PIGD NPs with 0.5, 1.0, and $2\mathrm{mg}/\mathrm{mL}$ concentrations. Figure 2(c) shows that the PA signal intensities increased linearly with the concentration of PIGD NPs.

3.3.

Deep-Tissue Photoacoustic Imaging at 1064 nm

To validate the advantage of PIGD for PA imaging in the NIR II window, deep-tissue imaging was conducted using the system shown in Fig. 2(a). The solutions of PIGD NPs with three different concentrations were embedded in an agar gel phantom [Fig. 3(a)]. The concentrations of three spots, s1, s2, and s3 are 1.0, 0.5, and $0.25\mathrm{mg}/\mathrm{mL}$, respectively. The phantom was placed under chicken-breast tissues with different thicknesses [Fig. 3(b)]. PA cross-sectional images were acquired with laser energy density $\sim 25\mathrm{mJ}/{\mathrm{cm}}^2$ on the tissue surface. PA images at different depths are shown in Figs. 3(c)–3(g). The PA signals from all three spots (s1 to s3) detectable at the tissue depth up to 5 cm [Fig. 3(g)]. The signal-to-noise ratio (SNR) of these images were calculated using the relation, $\mathrm{SNR}(\mathrm{dB})=20\times {\log }_{10}(\frac{V}{n})$, where $V$ is the average of PA signal amplitudes, and $n$ is the average of background noise amplitude. Figure 3(h) shows that with decreased NPs concentration, the SNR gradually decreased. The decrease in SNR is due to the fact that the 1064 nm beam was weakened as the depth of the chicken-breast tissue increased due to the light scattering and absorption by tissue. According to ANSI safety limit, the maximum permissible exposure (MPE) for skin is $100\mathrm{mJ}/{\mathrm{cm}}^2$ for 1064-nm laser.¹⁶ Due to the limitation of laser power, we could use $25\mathrm{mJ}/{\mathrm{cm}}^2$ for deep-tissue imaging. But additional improvement in imaging depth can be achieved using higher energy lasers. These deep imaging results demonstrated that PIGD NPs could act as an excellent PAT agent to enhance the PA contrast for potential use in noninvasive deep-tissue imaging.

Fig. 3

Deep-tissue PA imaging of PIGD NPs embedded inside chicken-breast tissue at 1064 nm. Photograph of the (a) agar gel phantom containing PIGD NPs dots with different concentrations (s1 to s3: 1.0, 0.5, and $0.25\mathrm{mg}/\mathrm{mL}$, respectively), (b) stack of chicken tissue layers on the agar phantom, (c–g) PA images of the agar gel phantom containing PIGD NPs solutions acquired at different depths, (h) SNR with different PIGD NPs concentration as a function of penetration depth inside chicken-breast tissue. Laser energy density used on tissue surface is $\sim 25\mathrm{mJ}/{\mathrm{cm}}^2$.

3.4.

In Vivo Brain Vascular Imaging on Rats at 1064 nm

To evaluate the PA imaging capability of PIGD NPs, in vivo imaging of rat brain was performed. PAT imaging was done noninvasively, i.e., with skin and skull intact. As shown in Fig. 4, the cerebral cortex of a rat was imaged by PAT system [Fig. 2(a)] at 1064 nm before and after a single injection of PIGD NPs (0.25 mL per rat, $2\mathrm{mg}/\mathrm{mL}$). The cross-sectional images of the rat brain before injecting NPs are shown in Fig. 4(a). Although the brain vasculature is visible, the contrast of the image is poor due to little absorption of blood at 1064 nm. The SNR of this image is $\sim 18\mathrm{dB}$. After intravenous injection of PIGD NPs (0.25 mL per rat, $2\mathrm{mg}/\mathrm{mL}$), the PA signals from the blood vessels were increased. Brain vascular images were collected for about 2 h after injection. From the postinjection images, we observed that compared with the vascular image based on intrinsic contrast agent [Fig. 4(a)], the images acquired after the administration of NPs show the brain vasculature with better contrast. The NPs circulating in the blood stream enhanced the contrast between the blood vessels and the brain parenchyma, showing the ability of PIGD NPs to generate strong PA signal. Brain cortex images of the rat acquired at 40- and 70-min postinjection time are shown in Figs. 4(b) and 4(c), respectively. The SNR of these images is $\sim 29$ and $\sim 37\mathrm{dB}$, respectively. Figure 4(d) shows the PA signal in the blood vessel area and image SNR as a function of postinjection time. From the graph, it is clear that the PIGD in the blood vessels helped to enhance the PA contrast. After injecting the NPs in to the blood stream, the concentration of NPs increased within the cortex vessels and reached maximum $\sim 70\min$ as shown in Fig. 4(d). The SNR of the image collected at 70 min is $\sim 2$ times higher than that of image collected at 0 min (preinjection image). The increase SNR achieved after the injection of NPs indicates that PIGD NPs could act as an excellent PA contrast agent. After 70-min postinjection, the PA signal remained strong, indicating a sufficient number of NPs circulating in the blood. The NPs circulation was monitored for a relatively short period ($\sim 120\min$). But, the actual circulation time of the NPs in the vasculature could be longer. Long-circulation of NPs could be due to their suitable size of $\sim 25\mathrm{nm}$ and the PEGylated surfaces of the NPs. The photographs of the brain taken before and after opening the scalp are shown in Figs. 4(e) and 4(f), respectively. An open scalp anatomical photograph of the cortex vasculature was taken after removing the scalp for comparison. This in vivo brain studies proved that the distribution of long-circulating NPs in the vasculature can provide contrast-enhanced PA imaging of brain vessels of rats with intact skin and skull.

Fig. 4

In vivo PA imaging of rat brain vasculature: cross-sectional brain vascular images at (a) 0 min (before injection), (b) 40 min-, (c) 70 min postinjection of PIGD NPs. PIGD NP solution ($2\mathrm{mg}/\mathrm{mL}$) was administered via tail vein injection with a dose of 0.25 mL per 100-gm body weight, (d) PA amplitude and SNR (dB) as a function of postinjection time. The blue arrow indicates injection point. Photograph of rat brain before (a) and after (b) removing the scalp. Laser energy density used for brain vascular imaging is $\sim 5\mathrm{mJ}/{\mathrm{cm}}^2$.