Biodegradable Contrast Agents for Photoacoustic Imaging

: Over the past twenty years, photoacoustics—also called optoacoustics—have been widely investigated and, in particular, extensively applied in biomedical imaging as an emerging modality. Photoacoustic imaging (PAI) detects an ultrasound wave that is generated via photoexcitation and thermoelastic expansion by a short nanosecond laser pulse, which signiﬁcantly reduces light and acoustic scattering, more than in other typical optical imaging and renders high-resolution tomographic images with preserving high absorption contrast with deep penetration depth. In addition, PAI provides anatomical and physiological parameters in non-invasive manner. Over the past two decades, this technique has been remarkably developed in the sense of instrumentation and contrast agent materials. In this review, we brieﬂy introduce state-of-the-art multiscale imaging systems and summarize recent progress on exogenous bio-compatible and -degradable agents that address biomedical application and clinical practice.


Introduction
Photoacoustic imaging (PAI) is considered a promising volumetric imaging technique for biomedical applications based on hybrid contrast mechanism under a light illumination and emitted ultrasound capturing scheme which enables to provide spectroscopic optical contrast and ultrasonic high-resolution. Additionally, PAI can achieve deep tissue imaging (several centimeters) by overcoming the bottleneck of the typical optical penetration depth (theoretically~1 mm) [1][2][3][4][5][6][7][8]. Furthermore, using the intrinsic optical absorption contrast between various components, including hemoglobin, melanoma, collagen and fat, PAI can visualize anatomical information such as vasculature distributions, melanin location, tendons and plaques, as well as physiological information such as the total amount of hemoglobin, oxygen saturation ratio, blood velocity and metabolic ratio [9][10][11]. In parallel, diverse PAI systems have been consistently developed with multiscale imaging ability [12]. Therefore, photoacoustic microscopy (PAM) achieves high spatial resolution with a focused excitation laser or a focused ultrasound transducer by scanning using a mechanical scanning stage or a high-speed optical scanner [13][14][15]. Also, photoacoustic computed tomography (PACT) delivers two-and (D) Three-dimensional PA images and (E) depth-resolved two-dimensional PA images of sentinel lymph nodes. Reproduced with permission from [43]. Copyright American Chemical Society, 2016.

Nitrogen-Doped Carbon Nanodots as Biodegradable PA Agents
Carbon-based nanoparticles have been investigated as therapeutic agents for PAI and photothermal therapy owing to their strong photoabsorbtion ability, biocompatibility, water solubility and photostability [44][45][46][47]. However, biodegradability is the main challenge of these materials. Lee et al. synthesized nitrogen-doped carbon nanodots (N-CNDs) to overcome this issue [48]. N-CNDs were produced by the solvothermal carbonization of citric acid in nitric acid ( Figure 2A). First, a heated mixture of citric acid and nitric acid formed polymer-like intermediates by dehydration. Then, the intermediates underwent carbonization to generate carbogenic cores. Simultaneously, oleylamine was implanted on the surface of the core to inhibit undesired intergranular aggregation during the carbonization process. Finally, oleylamine was replaced with ethanolamine to improve the water solubility. The N-CNDs were determined to have a size of approximately 3 nm ( Figure 2B). The amplitude of the measured PA signals of N-CNDs (2.2 ± 0.2) was better than those of gold nanorods (1.0 ± 0.1) and MB (1.3 ± 0.1) ( Figure 2C). As photothermal agents, aqueous solutions of N-CNDs showed temperature changes when irradiated with an 808 nm laser ( Figure 2D). Time-course PA images of SLNs in a rat model showed that the signal increased 30 min (D) Three-dimensional PA images and (E) depth-resolved two-dimensional PA images of sentinel lymph nodes. Reproduced with permission from [43]. Copyright American Chemical Society, 2016.

Nitrogen-Doped Carbon Nanodots as Biodegradable PA Agents
Carbon-based nanoparticles have been investigated as therapeutic agents for PAI and photothermal therapy owing to their strong photoabsorbtion ability, biocompatibility, water solubility and photostability [44][45][46][47]. However, biodegradability is the main challenge of these materials. Lee et al. synthesized nitrogen-doped carbon nanodots (N-CNDs) to overcome this issue [48]. N-CNDs were produced by the solvothermal carbonization of citric acid in nitric acid ( Figure 2A). First, a heated mixture of citric acid and nitric acid formed polymer-like intermediates by dehydration. Then, the intermediates underwent carbonization to generate carbogenic cores. Simultaneously, oleylamine was implanted on the surface of the core to inhibit undesired intergranular aggregation during the carbonization process. Finally, oleylamine was replaced with ethanolamine to improve the water solubility. The N-CNDs were determined to have a size of approximately 3 nm ( Figure 2B). The amplitude of the measured PA signals of N-CNDs (2.2 ± 0.2) was better than those of gold nanorods (1.0 ± 0.1) and MB (1.3 ± 0.1) ( Figure 2C). As photothermal agents, aqueous solutions of N-CNDs showed temperature changes when irradiated with an 808 nm laser ( Figure 2D). Time-course PA images of SLNs in a rat model showed that the signal increased 30 min after intradermal injection of the N-CNDs ( Figure 2E). Subsequently, the PA signal of the SLNs gradually decreased until 180 min ( Figure 2F). after intradermal injection of the N-CNDs ( Figure 2E). Subsequently, the PA signal of the SLNs gradually decreased until 180 min ( Figure 2F). The PA signal of the bladder increased 100 min after the time of injection and an enhanced signal was observed until 450 min, indicating that renal clearance occurred in the rat body. Therefore, these biodegradable carbon nanodots are a promising PA contrast agent and photothermal therapeutic agent.

Gold Nanoparticles as Biodegradable PA Agents
The characteristics of gold nanoparticles (AuNPs) are inert and biocompatible and can be varied through ligand attachment and shape modification [49][50][51]. Although various AuNPs has been demonstrated as molecular PAI contrast agents [52][53][54], there are no FDA-approved AuNPs owing to concerns related to hepatic retention. To achieve efficient renal clearance, the particles should be smaller than approximately 6 nm [55]. Therefore, several attempts have been made to make biodegradable AuNPs platforms that can be degraded under specific conditions.

Polymer-Stabilized AuNP Clusters for PAI
Polymer-stabilized AuNPs have been applied to PAI. Tam et al. assembled sub-5 nm AuNPs with the triblock copolymer of polylactic acid and polyethylene glycol, PLA(2K)-b-PEG(10K)-b-PLA(2K) [56]. The primary AuNPs were aggregated in the presence of the polymer ( Figure 3A). The average diameter of the primary AuNPs was 4.1 ± 0.8 nm ( Figure 3B). Changing the polymer-to- The PA signal of the bladder increased 100 min after the time of injection and an enhanced signal was observed until 450 min, indicating that renal clearance occurred in the rat body. Therefore, these biodegradable carbon nanodots are a promising PA contrast agent and photothermal therapeutic agent.

Gold Nanoparticles as Biodegradable PA Agents
The characteristics of gold nanoparticles (AuNPs) are inert and biocompatible and can be varied through ligand attachment and shape modification [49][50][51]. Although various AuNPs has been demonstrated as molecular PAI contrast agents [52][53][54], there are no FDA-approved AuNPs owing to concerns related to hepatic retention. To achieve efficient renal clearance, the particles should be smaller than approximately 6 nm [55]. Therefore, several attempts have been made to make biodegradable AuNPs platforms that can be degraded under specific conditions.

Polymer-Stabilized AuNP Clusters for PAI
Polymer-stabilized AuNPs have been applied to PAI. Tam et al. assembled sub-5 nm AuNPs with the triblock copolymer of polylactic acid and polyethylene glycol, PLA(2K)-b-PEG(10K)-b-PLA(2K) [56]. The primary AuNPs were aggregated in the presence of the polymer ( Figure 3A). The average diameter of the primary AuNPs was 4.1 ± 0.8 nm ( Figure 3B). Changing the polymer-to-AuNP ratio, the AuNP concentration and the surface ligands on the primary AuNPs made it possible to control the size of the clustered AuNPs. Deaggregation of the AuNP clusters occurred within live cells ( Figure 3C). The 40-nm sized AuNP clusters, which showed a higher PA signal intensity than gold nanorods ( Figure 3D), are another promising PA contrast agent [36]. AuNP ratio, the AuNP concentration and the surface ligands on the primary AuNPs made it possible to control the size of the clustered AuNPs. Deaggregation of the AuNP clusters occurred within live cells ( Figure 3C). The 40-nm sized AuNP clusters, which showed a higher PA signal intensity than gold nanorods ( Figure 3D), are another promising PA contrast agent [36].

Biodegradable Gold Nanovesicles as Theranostic Agents
In addition to PAI, AuNPs can simultaneously be used as photothermal therapy agents. Huang et al. produced biodegradable gold nanovesicles (BGVs) consisting of poly(ethylene glycol)-b-poly(εcaprolactone) (PEG-b-PCL)-tethered AuNPs [34]. Each BGV had a single layer of AuNPs with a hollow interior cavity ( Figure 4A). The average PA signal intensity of the BGVs was approximately ten times higher than the background and two times higher than that of GVs, which composed of nonbiodegradable PEG-b-polystyrene polymer-tethered AuNPs ( Figure 4B). The intense PA signal was monitored in the tumoral area after intratumoral injection of the BGVs in a mouse model with an MDA-MB-435 tumor ( Figure 4C). Owing to strong near-infrared absorption properties, the BGVs AuNP ratio, the AuNP concentration and the surface ligands on the primary AuNPs made it possible to control the size of the clustered AuNPs. Deaggregation of the AuNP clusters occurred within live cells ( Figure 3C). The 40-nm sized AuNP clusters, which showed a higher PA signal intensity than gold nanorods ( Figure 3D), are another promising PA contrast agent [36].

Biodegradable Gold Nanovesicles as Theranostic Agents
In addition to PAI, AuNPs can simultaneously be used as photothermal therapy agents. Huang et al. produced biodegradable gold nanovesicles (BGVs) consisting of poly(ethylene glycol)-b-poly(εcaprolactone) (PEG-b-PCL)-tethered AuNPs [34]. Each BGV had a single layer of AuNPs with a hollow interior cavity ( Figure 4A). The average PA signal intensity of the BGVs was approximately ten times higher than the background and two times higher than that of GVs, which composed of nonbiodegradable PEG-b-polystyrene polymer-tethered AuNPs ( Figure 4B). The intense PA signal was monitored in the tumoral area after intratumoral injection of the BGVs in a mouse model with an MDA-MB-435 tumor ( Figure 4C). Owing to strong near-infrared absorption properties, the BGVs showed a higher photothermal conversion efficiency (37%) than GVs (18%), gold nanorods (22%) and

Biodegradable Gold Nanovesicles as Theranostic Agents
In addition to PAI, AuNPs can simultaneously be used as photothermal therapy agents. Huang et al. produced biodegradable gold nanovesicles (BGVs) consisting of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL)-tethered AuNPs [34]. Each BGV had a single layer of AuNPs with a hollow interior cavity ( Figure 4A). The average PA signal intensity of the BGVs was approximately ten times higher than the background and two times higher than that of GVs, which composed of nonbiodegradable PEG-b-polystyrene polymer-tethered AuNPs ( Figure 4B). The intense PA signal was monitored in the tumoral area after intratumoral injection of the BGVs in a mouse model with an MDA-MB-435 tumor ( Figure 4C). Owing to strong near-infrared absorption properties, the BGVs showed a higher photothermal conversion efficiency (37%) than GVs (18%), gold nanorods (22%) and gold nanoshells (13%). The BGVs gradually began to break down when the system temperature exceeded 70 °C. Because PCL has a melting point of approximately 60 °C, most of the BGVs were degraded after 10 min. Under irradiation with an 808 nm laser, tumors injected with the BGVs showed retarded growth and tumor regression ( Figure 4D). Therefore, these BGVs are promising multifunctional theranostic platforms for PAI and photothermal therapeutic applications. More recent works on BGVs used smaller primary AuNPs to increase the biocompatibility and enhance the renal excretion of the degraded AuNPs [35].

Polymeric Materials as Biodegradable PA Agents
Benefiting from the development of polymer science and nanotechnology, polymeric materials have attracted great attention as a new class of PA agents that offers considerable structural diversity and designed material properties via chemical functionalization [57][58][59][60][61][62][63][64]. Notably, polymer nanoparticles have been successfully demonstrated as molecular imaging agents [65]. Such nanoparticles principally consist of π-conjugated polymer cores and hydrophilic polymer shells, which are generally formed via nanoprecipitation or nanoemulsion techniques. Therefore, the photophysical properties of a semi-conducting polymer can be translated into detectable ultrasonic signals in response to laser pulses, while a peripheral surfactant polymer stabilizes the overall structure under physiological conditions. Seminal review papers have covered semi-conductingpolymer-based PA agents [66,67]. In contrast, this section focuses on polymeric materials for biodegradable contrast agents. For this purpose, the agents are expected to be biocompatible, or at least amphiphilic, possess cleavable bonds, or exhibit the desired optical properties [68][69][70]. As an example, owing to their intrinsic properties, natural polymeric materials such as polysaccharides, phospholipids and deoxyribonucleic acid (DNA) can be used as biodegradable agents ( Figure 5). In addition, conventional hydrophilic polymers play a key role as an essential component for the The BGVs gradually began to break down when the system temperature exceeded 70 • C. Because PCL has a melting point of approximately 60 • C, most of the BGVs were degraded after 10 min. Under irradiation with an 808 nm laser, tumors injected with the BGVs showed retarded growth and tumor regression ( Figure 4D). Therefore, these BGVs are promising multifunctional theranostic platforms for PAI and photothermal therapeutic applications. More recent works on BGVs used smaller primary AuNPs to increase the biocompatibility and enhance the renal excretion of the degraded AuNPs [35].

Polymeric Materials as Biodegradable PA Agents
Benefiting from the development of polymer science and nanotechnology, polymeric materials have attracted great attention as a new class of PA agents that offers considerable structural diversity and designed material properties via chemical functionalization [57][58][59][60][61][62][63][64]. Notably, polymer nanoparticles have been successfully demonstrated as molecular imaging agents [65]. Such nanoparticles principally consist of π-conjugated polymer cores and hydrophilic polymer shells, which are generally formed via nanoprecipitation or nanoemulsion techniques. Therefore, the photophysical properties of a semi-conducting polymer can be translated into detectable ultrasonic signals in response to laser pulses, while a peripheral surfactant polymer stabilizes the overall structure under physiological conditions. Seminal review papers have covered semi-conducting-polymer-based PA agents [66,67]. In contrast, this section focuses on polymeric materials for biodegradable contrast agents. For this purpose, the agents are expected to be biocompatible, or at least amphiphilic, possess cleavable bonds, or exhibit the desired optical properties [68][69][70]. As an example, owing to their intrinsic properties, natural polymeric materials such as polysaccharides, phospholipids and deoxyribonucleic acid (DNA) can be used as biodegradable agents ( Figure 5). In addition, conventional hydrophilic polymers play a key role as an essential component for the biocompatibility of organic nanostructures including particles, hydrogels, micelles, vesicles and polymersomes [68,[71][72][73][74][75]. Theses polymers have been incorporated into contrast agents through controlled polymerization, copolymerization, or postpolymerization modification ( Figure 6). More interestingly, functional polymers that have chemically removable groups (e.g., labile groups, stimuli-responsive groups, or self-immolative groups) have shown biodegradability when exposed to bio-related signals such as pH, enzymes, temperature and biogenic chemicals.

Natural Polymers as PA Agents
The major advantages of using natural products include not only good dispersibility under physiological conditions, but also the availability of metabolic clearance pathways, which prevents nonspecific, long-term accumulation in biological systems. Cellulose, a renewable biomass, has been used as a biodegradable contrast agent, as demonstrated by the Gambhir group [76]. Highly crystalline cellulose nanocrystals were prepared by acid treatment. Rayleigh scattering from the material was observed over a wide spectral range, enabling PAI when excited at 700 nm. The resulting PA signal was comparable to or even better than that of gold nanorods (GNRs) (Figure 7). The crystals were degradable in the presence of cellulase, an enzyme capable of decomposing polysaccharides such as cellulose which humans lack. However, it was claimed that the agent can be used as a smart probe triggered by the external enzyme stimulus, as revealed by detecting the biocompatibility of organic nanostructures including particles, hydrogels, micelles, vesicles and polymersomes [68,[71][72][73][74][75]. Theses polymers have been incorporated into contrast agents through controlled polymerization, copolymerization, or postpolymerization modification ( Figure 6). More interestingly, functional polymers that have chemically removable groups (e.g., labile groups, stimuli-responsive groups, or self-immolative groups) have shown biodegradability when exposed to bio-related signals such as pH, enzymes, temperature and biogenic chemicals.

Natural Polymers as PA Agents
The major advantages of using natural products include not only good dispersibility under physiological conditions, but also the availability of metabolic clearance pathways, which prevents nonspecific, long-term accumulation in biological systems. Cellulose, a renewable biomass, has been used as a biodegradable contrast agent, as demonstrated by the Gambhir group [76]. Highly crystalline cellulose nanocrystals were prepared by acid treatment. Rayleigh scattering from the material was observed over a wide spectral range, enabling PAI when excited at 700 nm. The resulting PA signal was comparable to or even better than that of gold nanorods (GNRs) (Figure 7). The crystals were degradable in the presence of cellulase, an enzyme capable of decomposing polysaccharides such as cellulose which humans lack. However, it was claimed that the agent can be used as a smart probe triggered by the external enzyme stimulus, as revealed by detecting the concentration of glucose (the repeating unit of cellulose) after decomposition and by transmission A B C Figure 6. Typical synthetic polymeric components incorporated into various nanostructures for PA agents via controlled polymerization, copolymerization, or postpolymerization modification.

Natural Polymers as PA Agents
The major advantages of using natural products include not only good dispersibility under physiological conditions, but also the availability of metabolic clearance pathways, which prevents nonspecific, long-term accumulation in biological systems. Cellulose, a renewable biomass, has been used as a biodegradable contrast agent, as demonstrated by the Gambhir group [76]. Highly crystalline cellulose nanocrystals were prepared by acid treatment. Rayleigh scattering from the material was observed over a wide spectral range, enabling PAI when excited at 700 nm. The resulting PA signal was comparable to or even better than that of gold nanorods (GNRs) (Figure 7). The crystals were degradable in the presence of cellulase, an enzyme capable of decomposing polysaccharides such as cellulose which humans lack. However, it was claimed that the agent can be used as a smart probe triggered by the external enzyme stimulus, as revealed by detecting the concentration of glucose (the repeating unit of cellulose) after decomposition and by transmission electron microscopy.

Biodegradable Polymers as PA Agents
Conventional biodegradable synthetic polymers have been extensively applied as contrast agents for PAI, as mentioned above. As a representative example, poly(lactic-co-glycolic acid) (PLGA) can be used for biodegradable agents. The polymer is typically obtained from cyclic monomers of lactic acid and glycolic acid through statistical copolymerization or block copolymerization ( Figure  8A). Because of the ester linkages in the backbone, PLGA is subject to hydrolysis under aqueous conditions. Furthermore, the composition ratio between lactic acid and glycolic acid determines the rate of hydrolysis. The higher the content of glycolide repeating units, the faster the polymer hydrolyzes. In contrast, the incorporation of lactide units tends to retard the degradation. Wang et al. used PLGA to fabricate a biodegradable nanoparticle agent through the double emulsion method [77]. Herein, PLGA positioned in a polymer shell and imparted biodegradability as well as sufficient mechanical properties to the nanoparticles. Therefore, the resulting hierarchical PLGA-based agent was multimodal: (i) the agent was able to generate PA signals when stimulated by a laser pulse owing to AuNPs incorporated in the core, (ii) further incorporation of a dye in the core enabled fluorescent imaging and (iii) the agent showed potential for delivering paclitaxel, a cancer therapeutic drug, as demonstrated by in vitro experiments. Thus, theragnosis-a combined concept of remedial therapy

Biodegradable Polymers as PA Agents
Conventional biodegradable synthetic polymers have been extensively applied as contrast agents for PAI, as mentioned above. As a representative example, poly(lactic-co-glycolic acid) (PLGA) can be used for biodegradable agents. The polymer is typically obtained from cyclic monomers of lactic acid and glycolic acid through statistical copolymerization or block copolymerization ( Figure 8A). Because of the ester linkages in the backbone, PLGA is subject to hydrolysis under aqueous conditions. Furthermore, the composition ratio between lactic acid and glycolic acid determines the rate of hydrolysis. The higher the content of glycolide repeating units, the faster the polymer hydrolyzes. In contrast, the incorporation of lactide units tends to retard the degradation. Wang et al. used PLGA to fabricate a biodegradable nanoparticle agent through the double emulsion method [77]. Herein, PLGA positioned in a polymer shell and imparted biodegradability as well as sufficient mechanical properties to the nanoparticles. Therefore, the resulting hierarchical PLGA-based agent was multimodal: (i) the agent was able to generate PA signals when stimulated by a laser pulse owing to AuNPs incorporated in the core, (ii) further incorporation of a dye in the core enabled fluorescent imaging and (iii) the agent showed potential for delivering paclitaxel, a cancer therapeutic drug, as demonstrated by in vitro experiments. Thus, theragnosis-a combined concept of remedial therapy and diagnostic imaging-was achieved.
In this work, an organic-inorganic hybrid contrast agent was designed using functional polymers. First, the polymerization of equimolar amounts of 4-aminomethyl piperidine (AMPD) and equimolar N,N′-bis(acryloyl)cystamine (BAC) via a Michael addition reaction afforded the poly(AMBD-BAC) backbone. The disulfide bond in BAC provided redox-responsive behavior and was cleaved when exposed to glutathione (GSH). Postpolymerization modification introduced poly(ethylene glycol) moieties into the backbone, which facilitated drug delivery and reduced nonspecific adsorption during circulation in physiological environments. Furthermore, diethylenetriaminepentaacetic acid (DTPA) and Cisplatin were added sequentially to form Pt(II) complexes that generated PA signals ( Figure 8B). The functional polymers were developed into nanoparticles under aqueous conditions and the resulting particles allowed in vivo imaging of rat cerebral vasculature, taking advantage of the PA signal. Cytotoxicity tests further confirmed that the polymeric material was biocompatible.

Functional Polymers as PA Agents
Cleavable chemical groups give rise to biodegradability in polymeric contrast agents. Recently, Lyu et al. have designed functional nanoparticle agents for PAI ( Figure 9) [79]. The semi-conducting polymer core of these particles generated not only PA signals but also heat for photothermal therapy. The core polymer was further tethered with poly(ethylene glycol) methyl ether-b-poly(lactide-coglycolide) (PLGA-co-PEG), which endowed biocompatibility and water dispersibility ( Figure 9C). In particular, the semiconducting polymer, DPPV ( Figure 9A), had vinylene groups in the backbone. Thus, the whole chain was fully π-conjugated but the oxidizable nature of the vinylene group allowed degradation of the chain, which is in contrast to DPPT ( Figure 9B). As a result, the particles, SPNV (DPPV-based polymer nanoparticle) and SPNT (DPPT-based polymer nanoparticle), showed comparable PAI and photothermal therapy simultaneously ( Figure 9D-I) but SPNV was only degraded biologically to monomeric species in the presence of a peroxidase enzyme (myeloperoxidase) reducing the signal. Notwithstanding, the examples of well-designed, biodegradable, functional polymers for PAI are still rare as far as we know. The use of other chemistry, such as continuous depolymerization [80][81][82] and dynamic physicochemical bonds or the formation of reversible and hierarchical structures of polymeric materials [83][84][85][86], would further provide new, biocompatible, degradation pathways of polymer structures without losing PA properties. In another example, a synthetic block copolymer that contains Pt(II) complexes was used [78]. In this work, an organic-inorganic hybrid contrast agent was designed using functional polymers. First, the polymerization of equimolar amounts of 4-aminomethyl piperidine (AMPD) and equimolar N,N -bis(acryloyl)cystamine (BAC) via a Michael addition reaction afforded the poly(AMBD-BAC) backbone. The disulfide bond in BAC provided redox-responsive behavior and was cleaved when exposed to glutathione (GSH). Postpolymerization modification introduced poly(ethylene glycol) moieties into the backbone, which facilitated drug delivery and reduced nonspecific adsorption during circulation in physiological environments. Furthermore, diethylenetriaminepentaacetic acid (DTPA) and Cisplatin were added sequentially to form Pt(II) complexes that generated PA signals ( Figure 8B). The functional polymers were developed into nanoparticles under aqueous conditions and the resulting particles allowed in vivo imaging of rat cerebral vasculature, taking advantage of the PA signal. Cytotoxicity tests further confirmed that the polymeric material was biocompatible.

Functional Polymers as PA Agents
Cleavable chemical groups give rise to biodegradability in polymeric contrast agents. Recently, Lyu et al. have designed functional nanoparticle agents for PAI (Figure 9) [79]. The semi-conducting polymer core of these particles generated not only PA signals but also heat for photothermal therapy. The core polymer was further tethered with poly(ethylene glycol) methyl ether-b-poly(lactide-co-glycolide) (PLGA-co-PEG), which endowed biocompatibility and water dispersibility ( Figure 9C). In particular, the semiconducting polymer, DPPV ( Figure 9A), had vinylene groups in the backbone. Thus, the whole chain was fully π-conjugated but the oxidizable nature of the vinylene group allowed degradation of the chain, which is in contrast to DPPT ( Figure 9B). As a result, the particles, SPNV (DPPV-based polymer nanoparticle) and SPNT (DPPT-based polymer nanoparticle), showed comparable PAI and photothermal therapy simultaneously ( Figure 9D-I) but SPNV was only degraded biologically to monomeric species in the presence of a peroxidase enzyme (myeloperoxidase) reducing the signal. Notwithstanding, the examples of well-designed, biodegradable, functional polymers for PAI are still rare as far as we know. The use of other chemistry, such as continuous depolymerization [80][81][82] and dynamic physicochemical bonds or the formation of reversible and hierarchical structures of polymeric materials [83][84][85][86], would further provide new, biocompatible, degradation pathways of polymer structures without losing PA properties. Reprinted with permission from [79]. Copyright American Chemical Society, 2018.

Conclusions
PAI is a new class of preclinical and clinical biomedical imaging techniques owing to its unique ability to generate multicontrast images, as well as its relatively deep penetration depth and high spatial resolution. Various types of contrast agents have been investigated to obtain significant contrast and sensitivity in invisible biological tissues and the deep biological tissue region. Although many PA contrast agents demonstrated their ability at the preclinical level, clinical applications have been limited by biodegradability issues. Recently, several studies have been devoted to the development of biocompatible and biodegradable PA materials, including melanoidin, N-CNDs, polymer-stabilized AuNP clusters, GVs and polymeric agents. However, for clinical and commercial applications, focused systemic investigations are necessary to (i) enhance renal clearance efficiency, . The SPN solutions were prepared in PBS buffer (pH 7.4). The 60 µg·mL −1 solutions were used for photography and the PA spectral measurements. A single laser pulse (energy, 100 mJ pulse −1 ; duration, 5 ns; pulse repetition rate, 10 Hz) was used for the PA intensity measurements. The error bars are based on the standard deviations of three parallel samples. * Statistically significant differences in the PA intensities of SPNV and SPNT in the concentration range from 0 to 80 µg·mL −1 (p < 0.01, n = 3). Reprinted with permission from [79]. Copyright American Chemical Society, 2018.

Conclusions
PAI is a new class of preclinical and clinical biomedical imaging techniques owing to its unique ability to generate multicontrast images, as well as its relatively deep penetration depth and high spatial resolution. Various types of contrast agents have been investigated to obtain significant contrast and sensitivity in invisible biological tissues and the deep biological tissue region. Although many PA contrast agents demonstrated their ability at the preclinical level, clinical applications have been limited by biodegradability issues. Recently, several studies have been devoted to the development of biocompatible and biodegradable PA materials, including melanoidin, N-CNDs, polymer-stabilized AuNP clusters, GVs and polymeric agents. However, for clinical and commercial applications, focused systemic investigations are necessary to (i) enhance renal clearance efficiency, (ii) achieve high photostability, (iii) decrease phototoxicity and (iv) develop large-scale production methods. In addition, to demonstrate the biodistribution and pharmacokinetics of nanoparticles for clinical translation, rigorous experiments and analyses should be conducted with careful consideration of the chemical composition, structure, size and surface modification of the contrast agents. Moreover, as contrast agent studies have been conducted primarily in the field of cancer imaging and therapy, there is a need to expand the research to identify the potential of this technique in a variety of areas such as heart, brain and neurological diseases.