1.

Introduction

Photoacoustic imaging (PAI) is a hybrid imaging modality that combines rich optical contrast and high ultrasonic resolution at depth.^1,2 In PAI, short laser pulses are used to irradiate the sample. The absorption of light causes a small temperature rise and subsequently, due to thermoelastic expansion, pressure waves are generated in the form of acoustic waves [also known as photoacoustic (PA) waves]. By acquiring the PA waves using a wideband ultrasound detector at the tissue surface, images can be formed to reveal the internal structures and function of the tissue. PAI has a potential to image biological features from organelle to organs.¹ The advantages of PAI include deeper penetration depth, good spatial resolution, and high soft tissue contrast.³ PAI provides anatomical, functional, metabolic, molecular, and genetic contrasts of vasculature, hemodynamics, oxygen metabolism, biomarkers, and gene expression.^1,4 These unique advantages made PAI a potential tool for many applications in vascular biology, oncology, neurology, ophthalmology, dermatology, gastroenterology, cardiology, and so on.^1,3

The PA image contrast is based on the optical absorption by endogenous molecules (hemoglobin, melanin, and so on); however, these molecules show relatively weaker light absorption in the near-infrared (NIR) region. Nevertheless, image contrast can be improved with the aid of exogenous contrast agents having strong optical absorption in the NIR wavelength region. Several metallic, inorganic, organic nanoparticles, quantum dots, carbon nanotubes, and so on were demonstrated for deep-tissue PAI.^5–10 Contrast agents not only help to enhance the PA signals but are also useful for targeted molecular imaging, drug delivery, therapy, and so on.^11–14 Research is in progress to find a stable, nontoxic, biocompatible, and yet powerful exogenous contrast agent for PAI.^5,15

Among various nanocarriers, liposomes exhibit stable encapsulation of therapeutics and long circulation half-time,^16,17 and these agents are clinically successful.^18,19 The most conventional composition of liposomes contains dipalmitoylphosphatidyl choline (DPPC) as its main constituent. DPPC with a phase transition (transition from gel to liquid–crystalline state) temperature at around 41°C enables release of the encapsulated therapeutic from liposome in response to temperature.²⁰ However, one of the major constraints of these liposomes is their inability to initiate the drug release at the target site due to inadequate temperature control in vivo.²¹ One of the various attempts to modulate the temperature in physiological conditions has resulted in stimuli responsive liposomes that can be externally manipulated by light. Simplicity, clinical adaptability, and unparalleled spatiotemporal control on the drug release being the key aspects, gold nanostructures are coupled with liposomes to make them better responsive to light.^22,23 Earlier, a photoresponsive system encapsulated by gold nanoparticles (AuNPs) and the model drug calcein within temperature-sensitive liposomes by a simple thin film hydration method was developed. However, this method suffers from the low encapsulation of nanoparticles and the drug and is limited by penetration depth of visible light for clinical applications.²⁴

In this work, we describe the synthesis and characterization of temperature-sensitive liposome coated with gold nanostars (AuNSs) with a strong absorption peak at 690 nm. The strong absorption in the NIR window makes it suitable for PAI, making it an ideal dual functional imaging agent and drug delivery carrier for therapeutics. Coating the liposomes surface with AuNSs facilitates optimal encapsulation of the therapeutics within the liposomes, and the optical properties of AuNSs at NIR wavelength facilitate deep-tissue drug delivery.^22,25 In addition, the proximity of the AuNSs to the lipid bilayer is more effective even in low intensities of light to cause the disruption of liposomes and subsequent drug release. Despite the advantages, till date, limited studies have been reported on gold-coated liposomes. Troutman et al. and Leung et al. reported the synthesis of gold-coated liposomes using ascorbic acid as the reducing agent. Although they were successful in demonstrating wavelength-selective drug release, the efficacy of the system was not tested on in vitro or in vivo systems.²⁵ Gold-coated liposomes reported by Rengan et al. demonstrate light-triggered drug release by a photothermal effect, requiring a prolonged exposure of laser.²⁶ Along with drug release applications, liposome–gold hybrid systems are being actively investigated for their applications in photothermal therapy and multimodal imaging.^26,27 However, for PAI, gold-coated liposomes has not been reported. In this work, AuNS-coated liposomes as a potential exogenous contrast agent for deep-tissue PAT imaging is demonstrated. The near instantaneous controlled drug delivery from AuNSs-coated liposomes using PA effect is also explored.

2.

Materials and Methods

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-hydroxy-sn-glycero-3 phosphocholine (MPPC), and 1, 2-distearoyl-sn-glycero-3-phosphoethanol-amine-$N$-[carboxy (polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000) were purchased from Avanti Polar Lipids (Alabama). ${\mathrm{HAuCl}}_4$ and Paclitaxel were purchased from ACROS Organics. Fresh mouse blood was collected from the animal facility center, Nanyang Technological University. All other chemicals unless otherwise mentioned were purchased from Sigma-Aldrich. Deionized (DI) water was purified by a Millipore Milli-DI water purification system.

3.

Results and Discussion

Temperature-sensitive liposomes were prepared using the conventional thin film hydration method and coated with AuNSs by reducing the gold precursor, ${\mathrm{HAuCl}}_4$ using HEPES buffer. HEPES was found to reduce the ${\mathrm{Au}}^{3+}\text{ions}$ in ${\mathrm{HAuCl}}_4$ to ${\mathrm{Au}}^0$, thus forming AuNSs.³¹ This zerovalent state leads to the deposition of AuNSs on the surface of the liposomes.³² The liposome was characterized as shown in Fig. 1. The presence of AuNSs on the surface of the liposomes imparts characteristic optical properties to the AuNSs-coated liposomes making it responsive to light. The maximum absorbance of light occurred at 690 nm measured from the UV–vis spectroscopy as shown in Fig. 1(a). The PLs and AuNSs-coated liposomes were characterized for their size, surface potential, and morphology. The hydrodynamic diameter measured by the DLS method was found to be $141.4\pm 0.5$ and $113.2\pm 0.7\mathrm{nm}$ as shown in Fig. 1(b), while the surface charge was $-28.38\pm 1.25$ and $-22.37\pm 0.23\mathrm{mV}$ for PLs and AuNSs-coated liposomes, respectively. The TEM images indicated the presence of AuNSs on the surface of the liposomes and the size of the AuNSs liposomes from the TEM images was found to be 96.77 nm [Fig. 1(c)], which is comparable to the hydrodynamic diameter measured. The HR-TEM images clearly revealed the branched star-shaped morphology of the AuNSs as shown in Fig. 1(d). From the ICP results, the concentration of gold in AuNSs-coated liposomes was found to be $160\mu \mathrm{M}$. The loading efficiencies of calcein and PTX were found to be 24% and 25%, respectively.

Fig. 1

Characterization profile of AuNSs-coated liposomes represented by the (a) UV–vis absorption spectrum of AuNSs-coated liposomes, (b) hydrodynamic diameter of AuNSs-coated liposomes and PLs, (c) TEM image of AuNSs-coated liposomes, and (d) HR-TEM image of AuNSs. Scale bar: 50 nm.

The PA spectrum revealed that there is a strong PA signal at 690 nm as shown in Fig. 2(a), indicating high optical absorbance at this wavelength. Based on this observation, the laser excitation was fixed at 690 nm for further experiments. At 690 nm, it was found that the PA signal from $2\mathrm{mg}/\mathrm{mL}$ AuNSs-coated liposomes was two times stronger than that from the animal blood as shown in Fig. 2(b). The peak–to-peak PA signal from blood and AuNSs-coated liposomes were found to be 0.21 and 0.45 V, respectively. The potential use of AuNSs-coated liposomes as PA contrast agent and their effective imaging depth at a wavelength of 690 nm were tested. As shown in Fig. 2(d), two LDPE tube phantoms were filled with fresh mouse blood and AuNSs-coated liposomes ($2\mathrm{mg}/\mathrm{mL}$), respectively. They were sandwiched between chicken breast tissue of varying depths (1 and 2 cm). Figures 2(e) and 2(f) show the cross-sectional PAT images of the tubes. The signal-to-noise ratio (SNR) values of blood and AuNSs-coated liposomes at 1 cm depth was 13 and 25, respectively, as shown in Fig. 2(e). Similarly, SNR values of blood and AuNSs-coated liposomes at 2 cm depth were 6 and 14, respectively, as shown in Fig. 2(f). Our results indicate that AuNSs-coated liposomes are promising contrast agents for PAT, providing good PA signal enhancement (approximately two times) and image contrast in biological tissues even at 2-cm depth.

Fig. 2

PA spectrum and imaging: (a) PA spectrum of AuNSs-coated liposomes (AuL) in the NIR wavelength range, (b) PA signals collected from blood and AuNSs-coated liposomes, (c) photograph of the LDPE tubes filled with $2\mathrm{mg}/\mathrm{mL}$ AuNSs-coated liposomes and animal blood placed inside (sandwiched) chicken breast tissue (the top layer was removed to take the picture), (d, e) cross-sectional PAT images of the tubes at (d) 1 cm and (e) 2 cm imaging depth.

The tube containing the B16F10 melanoma cells in the media is shown in Fig. 3(a). The combined ultrasound and B-scan PA image (beam-formed image) of the tubes containing the unlabeled and the labeled melanoma cells are shown in Figs. 3(b) and 3(c), respectively. The grayscale image represents the ultrasound image and the colors represent the PA image. Melanin has an intrinsic PA signal. It has a good contrast against blood in a wide range of wavelengths (600 to 800 nm). The bright red spots on the image show the melanoma cells. It can be seen that in both the labeled and the unlabeled samples, the cells can be imaged. The difference in the signal intensity might not be so obvious from the PA/US image because the system normalizes the images by default. If the PA images alone are extracted from it, PA signal would be more pronounced and obvious. This can be noted from Figs. 3(d) and 3(e), which show the normalized PA images of both the labeled and the unlabeled melanoma cells. Also, for clarity the SNR from both the images is reported in Fig. 3(f). The SNR was defined as the amplitude of the PA signal from the melanoma cells divided by the standard deviation of the background noise, $\mathrm{SNR}=V/n$, where $V$ is the PA signal amplitude and $n$ is the standard deviation of the background noise. As shown in Fig. 3(f), the SNR of the unlabeled B16F10 mouse melanoma cells is 45, while that of the AuNSs-coated liposomes labeled B16F10 mouse melanoma cells is 174. This 3.8 time better signal intensity of the labeled cells further confirms that AuNSs-coated liposomes can be used as a PA contrast agent for cell labeling and tracking.

Fig. 3

(a) Photograph of a tube containing B16F10 mouse melanoma cells in media, (b) combined ultrasound and B-scan PA image unlabeled B16F10 mouse melanoma cells, (c) combined ultrasound and B-scan PA image of AuNSs-coated liposomes labeled B16F10 mouse melanoma cells, (d) B-scan PA image of unlabeled B16F10 mouse melanoma cells, (e) B-scan PA image of AuNSs-coated liposomes labeled B16F10 mouse melanoma cells, and (f) graph comparing the SNR values of unlabeled B16F10 mouse melanoma cells (UNL) and AuNSs-coated liposomes labeled B16F10 mouse melanoma cells (L).

Next, the drug release capability of the liposomes was tested. Cal-AuL and PTX-AuL solutions were exposed to the NIR pulsed laser for varying time points (0 to 60 s) with an energy density of $10{\mathrm{mJ}/\mathrm{cm}}^2$. The percentage of drug release from liposomes and AuNS-coated liposomes is shown in Figs. 4(a) and 4(b), respectively. From the figures, it can be observed that the presence of AuNS enabled $72.09\pm 0.88\%$ and $76.08\pm 3.28\%$ release of encapsulated calcein and PTX, respectively, within 60 s under the laser exposure. While the liposomes without AuNSs had only $\sim 10\%$ drug release under the same experimental conditions.

Fig. 4

Percentage of drug release (a) release of calcein from Cal-AuL and Cal-L. (b) Release of PTX from PTX-AuL and PTX-L upon laser irradiation at 690 nm with an energy density of $10\mathrm{mJ}/{\mathrm{cm}}^2$ at a pulse repetition rate of 10 Hz.

Fluorescence imaging using B16F10 melanoma cells was done to demonstrate the release of calcein from the liposomes triggered by light-induced pressure waves (PA waves). B16F10 melanoma cells were incubated with free calcein solution ($120\mu \mathrm{M}$), liposomes containing calcein, and AuNSs-coated liposomes containing calcein overnight. After washing the cells with PBS to remove the particles that were not taken up by the cells, each of the treated groups was exposed to a laser at 690 nm at $10\mathrm{mJ}/{\mathrm{cm}}^2$ for 30 s. When subject to a laser source at 690 nm, the absorbed light causes a temperature rise, which generates pressure waves (PA waves). These pressure waves lead to the disruption of liposomes, releasing the encapsulated drug.³³ Laser-untreated groups were compared against the laser-treated groups. This is shown in Fig. 5. It was observed that free calcein stained the cells in both laser-treated and -untreated groups; no fluorescence was observed in both groups in the case of cells incubated with liposomes containing calcein. However, in the cells incubated with AuNSs-coated liposomes containing calcein, a significant increase in fluorescence was observed in laser-treated groups while no fluorescence was observed in groups unexposed to laser. As reported earlier, the local temperature increase in the nanoparticle microenvironment generates microbubble cavitation that disrupts the liposomal membrane to release the encapsulated drug.^22,24 Likewise, the presence of AuNSs in the liposomes results in calcein release due to microbubble cavitation. It is also to be noted that the rise in temperature of the bulk solution containing the same concentration of AuNSs-coated liposomes was found to be less than 4°C upon laser exposure of about 10 min. This further validates that the encapsulated drug is released by the liposome disruption due to the pressure wave generation and eliminates the possibility of drug release due to a rise in temperature of the bulk liposomal solution. At a concentration of 60 mM, calcein self-quenches within the liposomes and the cells are nonfluorescent. As the laser triggers the AuNSs-coated liposomes containing calcein, the released calcein stains the cells and can be detected as their fluorescence recovers.

Fig. 5

In vitro release of calcein from Cal-AuL into the cytoplasm of B16F10 melanoma cells with and without laser exposure. The cells were irradiated with a laser for 30 s at an energy density of $10\mathrm{mJ}/{\mathrm{cm}}^2$. (a–d) The DAPI-stained cell nuclei of control group without any laser exposure. (e–h) The calcein-stained cell cytoplasm of control group without any laser exposure. (i–l) The DAPI-stained cell nuclei of laser-treated group. (m–p) The calcein-stained cell cytoplasm of laser-treated group. (p) The cells stained with calcein released from Cal-AuL after laser exposure. Blue, nuclei; green, cell cytoplasm. Scale bar: $100\mu \mathrm{m}$.

To test the biocompatibility of the AuNSs-coated liposomes, B16F10 mouse melanoma cells were incubated with AuNSs, free drug paclitaxel, liposomes loaded with paclitaxel, AuNSs-coated liposomes loaded with paclitaxel for 24 h. The cells were then washed with PBS, and the cell viability was assessed using Alamar blue assay against the untreated cells. Similarly, cells treated with laser at 690 nm for 30 s were tested for viability 24 h post laser treatment to see the efficacy of the drug release. These results are shown in Fig. 6. It was found that the cells that were not exposed to laser did not show any significant changes in the viability. While the cells treated with PTX showed significant decreases in cell viability in both control and laser-treated groups, about 20% cell death was observed from cells treated with AuNSs-coated liposomes without PTX. This clearly indicates that the microbubble cavitation generated due to the presence of AuNSs in the liposomes has led to the release of PTX into the cell cytoplasm, which in turn resulted in cell death.³⁴ However, the highest percentage of cell death was observed from cells treated with AuNSs-coated liposomes containing paclitaxel upon laser exposure. Cells treated with liposomes with paclitaxel did not show significant changes in the cell viability ensure stable encapsulation of PTX within liposomes. This shows that the AuNSs cause significant drug release from the liposomes.

Fig. 6

Cell viability study of B16F10 melanoma cells treated with PTX Au-liposomes with and without laser exposure. The sample was irradiated with laser for 30 s at $15\mathrm{mJ}/{\mathrm{cm}}^2$ energy density. (PL, plain liposomes, AuL, AuNS-coated liposomes, PTX, free drug paclitaxel, PTX L, liposomes loaded with paclitaxel, PTX-L AuL, AuNS-coated liposomes loaded with paclitaxel).

PAI can be used for theranostic applications when used with a suitable contrast agent. Here, we explore the theranostic potential of NIR light-sensitive liposomes coated with AuNSs for both imaging and drug release applications using PAI system. The liposome being amphiphilic can encapsulate both hydrophobic and hydrophilic molecules. Irradiation by laser at a particular wavelength triggers the rapid release of the contents encapsulated in the liposomes due to local heating and pressure wave formation (PA wave) by the AuNSs. The pressure waves can also be used for PA imaging as well. However, further in vivo studies on small animals need to be done to prove the efficacy of this system for clinical applications.

4.

Conclusion

In this work, we have demonstrated that AuNSs-coated liposomes show strong absorption in the NIR region and can be used as contrast agent for PAI of deep tissues. The drug release study promises that it can be used to deliver both hydrophilic and hydrophobic drugs effectively. Thus we present AuNSs-coated liposomes as a dual modal therapeutic and contrast agent responsive to NIR light. Further use of these particles for in vivo small animal models is under investigation for its potential use in the clinics.