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Review ArticleState-of-the-Art Preclinical Photoacoustic Imaging in Oncology:Recent Advances in Cancer Theranostics

Sara Gargiulo , Sandra Albanese, and Marcello Mancini

Institute of Biostructure and Bioimaging of National Council of Research, Naples 80145, Italy

Correspondence should be addressed to Sara Gargiulo; [email protected]

Sara Gargiulo and Sandra Albanese contributed equally to this work.

Received 14 February 2019; Accepted 15 April 2019; Published 30 April 2019

Guest Editor: Luigi Aloj

Copyright © 2019 Sara Gargiulo et al.�is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

�e optical imaging plays an increasing role in preclinical studies, particularly in cancer biology. �e combined ultrasound andoptical imaging, named photoacoustic imaging (PAI), is an emerging hybrid technique for real-timemolecular imaging in preclinicalresearch and recently expanding into clinical setting. PAI can be performed using endogenous contrast, particularly from oxygenatedand deoxygenated hemoglobin and melanin, or exogenous contrast agents, sometimes targeted for speci�c biomarkers, providingcomprehensive morphofunctional and molecular information on tumor microenvironment. Overall, PAI has revealed notableopportunities to improve knowledge on tumor pathophysiology and on the biological mechanisms underlying therapy. �e aim ofthis review is to introduce the principles of PAI and to provide a brief overview of current PAI applications in preclinical research,highlighting also on recent advances in clinical translation for cancer diagnosis, staging, and therapy.

1. Introduction

�e �eld of cancer medicine requires robust approaches forearly detection, characterization, and monitoring of tumors.�e noninvasive study of key biological parameters in an-imal models represents a well-established tool for translationof positive results from biomedical research to the bedside.Imaging techniques provide a valuable contribution toimprove earlier diagnosis in oncology, as well as for studyingangiogenesis and measuring molecular factors implicated incancer progression and in the response to therapies. Pho-toacoustic imaging (PAI) has been extensively tested in vivoin preclinical studies during the past decade, in particular ononcological models, allowing to reduce the number ofsacri�ced animals at multiple time points. PAI performedover multiple wavelengths (spectroscopic imaging) candetect variations in the concentration of tissue componentsthat are hallmarks of cancer compared to the surroundingnoncancerous tissues.

Main advantages of PAI include imaging depths up tocentimeter and submillimetric resolution, high contrast-to-noise ratios and spectroscopic imaging, real-time acquisi-tion, lack of ionizing radiation, and integration with

ultrasound (US) scanners, as well as noninvasive imaging forlongitudinal studies, monitoring cancer progression, anddrug delivery. �erefore, biomedical community showsconsiderable interest in translating this methodology intothe clinical �eld. Depending on the biomedical requirement,the major types of PAI systems available can be brie�ycategorized as microscopy (PAM), endoscopy (PAE), andcomputed tomography (PACT, focused in this review).PAM and PAE scanners have been mainly used in mousemodels of human diseases to image super�cial areas andvascular and visceral tissues, respectively, with higher spatialresolution but limited imaging depth compared to PACTsystems. Moreover, PACT platforms may provide cross-sectional and/or three-dimensional PAI of living bi-ological structures. Hence, PACT technology appears to bethe most promising for PAI clinical implementation. Cur-rently, PAI instrumentation is commercially available onlyfor preclinical studies; few clinical applications are beingexplored in oncological trials on patients [1]. �e physicalfundamentals and the major technological implementationsof PAI in biomedicine have been summarized in detail by arecent manuscript and therefore are outside our purposes[1]. In this review, the general principles, current preclinical

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applications, and potential clinical translation of cancer PAIwill be primarily described.

2. Principles of PAI and PreliminaryClinical Applications

PAI is a hybrid technique based on the photoacoustic effect,a physical phenomenon in which the absorbed electro-magnetic energy is converted to acoustic waves. A shortpulsed (<10 ns) laser, comprising multiple wavelengths, isused to illuminate biological tissues, inducing ultrasonicwaves arising from several tissue constituents.

A typical PA system includes a short pulsed laser source,an US array transducer for signal detection, a component forsignal amplification and digitalization, a system for B modeUS and PA coregistration, data acquisition, and imagesrepresentation. *e imaging frame rate of the system isusually limited by the laser pulse repetition rate and the timerequired for multiwavelength data acquisition. In addition, arepeated wide field illumination is limited by the potentialtissue damage. Currently, the commercially available US-PAscanners operate at a repetition rate ranging from 5 to 20Hz[2].

*e process of PA signal generation can be described inseveral steps: (1) a target tissue is illuminated by a shortpulsed laser; (2) photons propagate unidirectionally intotissues and are absorbed by endogenous or exogenousmolecules with optical properties; (3) the absorbed opticalenergy is partially or completely converted into heat, leadingto a transient local temperature rise; (4) the heating inducesthermoelastic tissue expansion; (5) tissue thermal expansionchanges over time induce local pressure rise, that generatespressure acoustic waves; and (6) broadband acoustic wavesare detected by an ultrasound (US) transducer and processed(Figure 1).

Acoustic waves can be produced by employing either acontinuous wave laser with intensity modulation at a con-stant frequency or more commonly a pulsed laser thatprovides a higher signal-to-noise ratio (SNR).*e laser pulseduration is typically ∼10 ns, influencing the fractional vol-ume expansion during the illumination. Approximately each1mK temperature rise generates 800 Pa pressure incrementthat produces a pressure wave ultrasonically detectable. *egenerated pressure wave includes two components withequal magnitude traveling in opposite directions, as com-pression followed by rarefaction. Such PA wave propagatesthrough the tissue and is detected by an US multielement 2Dmatrix transducer array (up to 256 elements) [2, 3].

*e image formation is based on two major methods: (1)mechanical scanning using a focused single light beam and afocused single-element ultrasonic transducer (directmethod) or (2) using a wide-field light illumination andelectronic scanning by a multielement US array transducer(reconstruction method). Each US transducer element re-ceives PA signal over a large acceptance angle, and data areused to reconstruct a tomographic image.

*e PA signal is directly proportional to the light ab-sorption by either endogenous or exogenous contrast agents,as well as to the laser light fluence (mJ/cm2), generally

inducing a range of acoustic frequencies from 1 to 10MHz[2]. Different tissue components have unique optical scat-tering and absorption properties for each wavelength;therefore, PAI enables the detection of specific molecules ina complex tissue environment, allowing the study of mul-tiple biomarkers simultaneously. Moreover, the opticalabsorption coefficient and the amount of each chromophoredetermine the wavelength and amplitude of the producedacoustic waves. *erefore, the quality and concentration ofeach optical absorber can be derived through the spectro-scopic inversion technique.

*e main limitation of optical imaging is the low res-olution in deeper tissues, that is related to the physics of lightscattering. On the contrary, the acoustic scattering of tissuesis approximately one thousand times less than opticalscattering.*erefore, compared to pure optical imaging, PAIcombines the advantages of higher optical scattering (spatialresolution< 100 μm) with the deeper tissue penetration ofacoustic waves (up to 5 cm), with similar sensitivity [2–4].

Approximately, the ratio of imaging depth to the res-olution is ∼200, permitting higher resolution imaging acrossa wide range of imaging depth. Furthermore, PAI shows anabsolute sensitivity to relative small variation of opticalabsorption, hence providing an equivalent percentagechange in the PA signal intensity [1].

PAI can potentially improve cancer imaging by com-bining complementary information and instrumentationand providing morphological, functional, and moleculardata in a single image.

*e ideal molecular PA probe should have specificityfor the biological process under investigation, maximal

Pulsed laserillumination

Optical absorption

Light-absorbing targetheating

Optical path

Photoabsorber

Acoustic detection

Pressure waves

Thermal expansion

Sound path UltrasoundLaser

Tumor

Bloodvessel

Figure 1: Physical principles of cancer PAI. Short pulsed laser lightis used to irradiate the tumor area, inducing ultrasonic waves fromendogenous or exogenous photoabsorbers on the basis of ther-moelastic expansion. An US transducer is used to detect the PAsignal. Contrast obtained from PAI can be useful in character-ization and monitoring of tumors (adapted with permission fromValluru and Willmann [2], CC BY-NC-ND license, http://creativecommons.org/licenses/by-nc/3.0/).

2 Contrast Media & Molecular Imaging

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absorption capability in the NIR window to enable deeptissue imaging, biocompatibility and biosafety, and re-sistance to photobleaching.

Photons can be absorbed by endogenous or exogenousmolecules with various mechanisms, including electronicabsorption, vibrational absorption, stimulated Raman ab-sorption, and surface plasmon resonance (SPR) absorption [1].

*e PA signal is mainly provided by endogenous mol-ecules, such as hemoglobin (Hb), melanin, lipids, and col-lagen, or by molecularly targeted exogenous contrast agents,conjugated with antibodies or peptides, displaying opticalabsorption properties in the near-infrared (NIR) interval(700–1100 nm) [5, 6]. Several biocompatible small molecules(∼1 nm), such as indocyanine Green (ICG), IRDye800CW,Alexa Fluor 750, and methylene blue, show light absorptionin this convenient optical window, reducing backgroundsignal arising from surrounding tissues. Moreover, gold andsilver nanoparticles (NPs) have been widely used as PAcontrast agents, due to the SPR effect. *e charges on thesurface of noble metal NPs, related to oscillations accordingto the electromagnetic field, result in strong optical ab-sorption properties. Dyes and plasmonic NPs can be alsodirectly conjugated to molecular targeting moieties [6]. Inaddition, the spectroscopic technique allows to resolve thesignal contribution from multiple optical chromophores,thus enabling simultaneous molecular and functional im-aging [7] (Table 1). Technological analogies between PA andUS imaging systems allow easy implementation of dual-modality scanners, to expand their potential in morpho-functional and molecular imaging [98]. Nowadays, onlypreclinical PAI scanners are commercially available andhave been applied to study the tumor microenvironment,hypoxia, angiogenesis, and metastasis and for targetingspecific cancer receptors.

Although PAI appears highly attractive owing to itspotential for clinical translatability, it is still facing someoperating challenges, and at the moment, a limited numberof studies in human patients have been described, as wellsummarized in recent reviews [2, 99].

On the contrary, the interest in deepening PAI is en-couraged by several advantages over other imaging methodscurrently used in preclinical and clinical fields. Compared tofluorescent tomography, PAI provides deeper penetrationand higher spatial resolution across the entire field of view.Beyond functional magnetic resonance imaging (MRI), PAIallows noninvasive oxygenation imaging with high spatial-temporal resolution and relatively low costs. Furthermore,PAI can utilize endogenous contrast agents and it is freefrom artifacts of acoustic waves as multiple scattering signalinterference of returning waves (speeckle) that are found inUS imaging. Moreover, PAI can be considered a backgroundnoise-free method since nonabsorbing tissue componentsdo not generate a detectable signal, and through properwavelength selection, it is possible to discriminate the signalgenerated from endogenous photoabsorbers from the oneproduced by exogenous probes. Finally, differently fromX-ray and PETimaging, PAI does not use ionizing radiationsand provides higher spatial resolution. In the last decade,several pilot studies focusing on PAI clinical applications

have been underway. A promising research area for clinicalPAI is the examination of superficial tissues. *e feasibilityof breast PAI in patients has been explored as a potentialalternative to the standard screening techniques such asX-ray and US mammography, for earlier and more accuratediagnosis and staging. Some prototype clinical PAI mam-moscopes have been designed to investigate Hb oxygensaturation and microcalcifications and detect sentinel lymphnodes (SLNs) metastasis of breast tumors with limited datafrom patients or from bioptic specimens [100–107]. En-couraging results have been reported in differentiating be-nign and malignant breast masses, with low expense andpotentially high sensitivity and specificity, although furthertechnical refinements are needed in the future. In particular,Fakhrejahani and colleagues [105] first performed a pilotclinical study with a prototype PAI machine, in order tocompare the distribution pattern of the Hb signal and theoxygen saturation level in primary malignant breast tumorsand contralateral normal breasts as control. In this trial, aspecific microvascular pattern able to discriminate thenormal breast tissue from tumor lesions using PA signalmapping was evident in approximately 75% of patients.Moreover, oxygen saturation values resulted lower in tumorscompared to normal contralateral breast tissue, suggestinghypoxia in malignant lesions. In agreement, a significantcorrelation between histological profile of tumor micro-vasculature and of hypoxia with qualitative and quantitativeanalysis of the PA signal was found. Although furthertechnical improvements will be needed, Hb signal mappinghas proven promising for breast cancer diagnosis andprognosis, through noninvasive detection of tumor angio-genesis and of oxygenation decrease in malignant lesions.An upgraded prototype PAI system was used on patients byHeijblom et al. [106] to investigate the features of suspectedbreast lesions compared with X-ray mammography, ultra-sonography, and MRI. Using 1064 nm laser light wave-length, the authors demonstrated that PAI can visualizebreast malignancies with high contrast and sensitivity and isable to adequately estimate lesion size, showing goodcolocalization with conventional imaging. However, thepresence of high contrast areas around the malignant lesionsis indicative for the need of improved specificity, for ex-ample, implementing a multispectral technique. In addition,the potential of PAI in detecting skin lesions was based onpatients or on biopsies, for example, to differentiate a benignmelanocytic nevus from the surrounding blood vessels [108],as well as cutaneous melanoma metastasis in excised SLNs[10]. To date, it is well known that early detection of LNsmetastasis is crucial to increase survival in patients with skinmelanoma, but sensitivity of conventional cytology, biopsy,and imaging is relatively low. To overcome this issue,multispectral PAI was tested in melanoma patients thatunderwent lymphadenectomy, using a method with en-hanced specificity to differentiate the PA signal arising fromHb and melanin [10]. In accordance with the histologicalfindings, the PA signal from melanin was evident in theupper part of the metastatic SLNs, with different distributionpatterns among samples, while it has been not detectable indeeper areas, probably due to the strong melanin optical

Contrast Media & Molecular Imaging 3

absorption. Overall, these results appeared encouraging tocarry out further studies on the detection of SLNs metastaticfoci by PAI, to avoid invasive procedures. In the field ofendocrinology, PAI was able to identify malignant andnonmalignant prostate samples based on the deoxygenatedhemoglobin (HbR) and lipid signals [109]. A similar analysiswas performed on thyroid lesions harvested from cancerpatients, and an increase in HbR PA signal intensity wasreported in malignant thyroid biopsies compared to benigntissue [110]. Very recently, in vivo combined US-PAI ex-amination has added information to ex vivo studies onhuman ovaries extracted from patients, highlighting thepossibility to discriminate normal ovarian tissue versusmalignant lesions through the Hb signal [111–113]. On thecontrary, only biopsy samples from cancer of the cervix wereexamined by PAI, while to date, in vivo studies involvingpatients are not yet reported [114]. As contrast agents andmolecular targeting will become more advanced, the clinicalapplications of PAI could be improved. Clinical applicationsabove described highlight the relevant potential of PAItranslation in biomedical practice to synergically integrateother well-established tools in cancer theranostics.

3. Factors Influencing In Vivo PAI in MouseModels of Human Cancer

Besides technical limits of a given PAI system, severalvariables should be considered during in vivo preclinical

experiments, affecting image quality, inter- and intrasubjectreproducibility, and repeatability over time of PA signalquantification.

Animal handling and physiology for a particular mousestrain, age, and gender accounted for an experiment, animalpositioning, and anesthesia, as well as operator expertise andstandardization of operative procedures were found to be themain sources of variability for in vivo PAI measures.

Precision of PAI quantitative data have been evaluated inliving animal models of subcutaneous xenograft tumors, andat the moment, few data are available about total Hb (HbT),HbR, and oxygenated Hb (HbO2) measurement [115, 116].For example, body temperature may affect both peripheralperfusion and cardiorespiratory rates, hence influencingrecorded values of endogenous photoabsorbers like HbRand HbO2 or the kinetics of exogenous contrast agents intumors, and therefore, it should be stabilized in the normalmouse range (36–38°C) [115].

Similarly, respiration rate may impact PAI results due toboth the effects on oxygenation status and motion artifacts.*erefore, respiration rate in mouse models should becarefully monitored and maintained around 60–70 breathsper minute during PAI imaging [115] to minimize vari-ability, by implementing stable and standardized approachesfor anesthetic depth and oxygen supply (21% room air or100% pure oxygen breathing during general anesthesia). Inaddition, PA signal variation in subcutaneous xenografttumors, depending on the positioning of mouse, could be

Table 1: Summary of main endogenous and exogenous PA contrast agents.

Imaging agentAbsorptionwavelength

(nm)Cancer biomarker Cancer type Multimodality *eranostics Application Reference

Endogenous optical compounds

Hemoglobin 760–850 Angiogenesis Brain, breast,Prostate — — Preclinical/

clinical [7–9]

Melanin 700 αvβ3, acidicmicroenvironment Skin, ocular, lung PET-MRI-

PAI PTT Preclinical/clinical [10–22]

Exogenous optical compounds

Indocyanine green 600–900αvβ3, albumin, EGFR,B7-H3R, UPAR, acidicmicroenvironment, FR

Lung, liver, colon,brain MRI-PAI PTT Preclinical/

clinical [23–44]

Gold NPs 650–110

αvβ3, acidicmicroenvironment, FR,siRNA gene-silencing,MAGE, intratumoraltemperature, CXCR4

Lung, prostate,ovaries, stomach,

skin, brainCT-MRI-PAI PTT/PDT Preclinical [45–55]

Palladium NPs 826–1068 αvβ3 Breast — PTT Preclinical [56]Copper sulfide NPs 826–1068 αvβ3 Breast — PTT Preclinical [57–61]Bismuth NPs 1000–1700 EPR effect Breast, brain CT-PAI PTT Preclinical [62–64]Titanium NPs 808 EPR effect Breast, cervix — PTT/PDT Preclinical [65–67]

Iron oxide NPs 600–900 FR Breast, ovaries PET-MRI-PAI PTT Preclinical [68–70]

Superparamagneticiron oxide NPs 500–780 HER2 Breast — PTT Preclinical [71]

Carbon-based NPs 350–700 αvβ3, EGFR Breast, brain, skin,lung, stomach FL-PAI PTT/PDT Preclinical [72–84]

Graphdiyne NPs 690 FR Breast FL-PAI Preclinical [85]Semiconductingpolymer NPs 660–748 αvβ3, FR Breast, brain, cervix FL-PAI PTT/PDT Preclinical [86–97]


influenced by the pressure on the neoplasia exerted by theanimal’s body weight and/or animal holder, and/or by thetransducer, which in turn may alter tumor shape and he-modynamic. As reported by Costa et al. [116], the shape of asubcutaneous tumor may vary considerably as effect ofmouse repositioning, changing the size of an anatomicalregion of interest (ROI) used for lesion analysis, which inturn can modify the distribution maps of oxygenation andthe signal intensity. *erefore, Costa et al. [116] advise tokeep the position of the mouse body and of the tumor underexamination relatively constant throughout longitudinalstudies. Moreover, in serial investigations, it has been rec-ommended that both data acquisition and analysis should beperformed using a ROI standardized for size, form, andplacement across datasets and by operators with comparableexpertise [115]. Anesthesia has been found to be the majorexperimental source of PA signal variation over time inmouse xenograft models of human cancer [115].

Type and duration of anesthesia influence physiologicalparameters, such as hearth and breath rate or may induceperipheral vasoconstriction or vasodilation, producingchanges of tissue oxygenation, and affect contrast mediabiodistribution.*erefore, the anesthesia protocol should beconveniently adapted to the study aims, as well as to theimaging technique used [117, 118]. For these reasons, suchfactors may potentially affect also PA signal assessment intumors for different contrast agents, but at the moment, onlyfew studies have investigated the influence of anesthesia onPAI. Costa and colleagues [116] have found that Hb andoxygenation PA measures in mouse xenograft models ofhuman cancer significantly change under anesthesia over aperiod of 75minutes, so authors suggest to carefully plan inthe experimental protocol to image animals at the same timepoints. Overall, PAI in oncological mouse models should beperformed at a defined time after anesthesia induction, toproper inter- and intrasubject comparisons in longitudinalstudies, for example, to assess the effects of a new therapy[115]. Moreover, in studies involving contrast agent ad-ministration, it is suggested to set standard operating pro-cedures about route and volume of injection and on thebiodistribution time.

4. Biomedical PAI Applications Based onEndogenous Optical Compounds

Similar to other biomedical imaging techniques, PAI em-ploys endogenous or exogenous contrast agents to enhancesoft tissues discrimination for identifying pathologicalconditions.

PAI based on endogenous contrast is particularly at-tractive for oncology applications since it is not necessary toadminister exogenous compounds for monitoring tumorprogression or treatment response. Hb, melanin, lipids,collagen, elastin, and water are the major endogenous opticalabsorbers investigated, providing a molecular imaging ap-proach rapidly translatable [119]. Endogenous chromo-phores have different absorption spectra in the NIR rangebetween ∼700 and 1100 nm, and therefore, specific wave-lengths can be selected to obtain maximum contrast in PAI

from these compounds. Due to its valuable absorptionproperties, Hb of red blood cells represents the primarybiomarker for a label-free PAI of cancer, allowing the de-tection of the abnormal vascular network development intumor growing. Likewise, melanin is capable to generate astrong PA signal, and it has been effectively tested formelanoma detection. Even if different research fields havehighlighted the feasibility of endogenous contrast also forlipids, collagen, elastin, and water, they have limited ap-plications for cancer PAI. *ese biological constituents haveshown a lower absorption coefficient than Hb in the NIRregion and a signal-to-noise ratio not enough for a reliablecontribution to tumor visualization (Figure 2).

4.1. Oxygenated and Deoxygenated Hemoglobin. Angiogenesis,referring to the formation of irregular and hyperpermeableblood neovessels within a tumor, is one of the most relevantbiomarkers of cancer, which in turn affects the delivery ofnutrients and oxygen into neoplasia. Overall, the tumorgrowth is characterized by a high oxygen consumption ratebut at the same time to a decrease of oxygen saturation in thecentral region. *ese factors promote uncontrolled tumorcell proliferation, malignant progression, and metastasis,leading to the formation of hypoxic areas associated withchemotherapy (CHT) and radiotherapy (RT) resistance.Using Hb as the endogenous contrast agent, PAI mayrepresent a noninvasive, cheaper, and more accessible waythan conventional methods such as oxygen needle elec-trodes, PET, or BOLD MRI, to provide information aboutoxygenation status of tumors. Both HbR and HbO2 can beused for tissue characterization by in vivo PAI, since in theNIR region, they show distinct absorption peaks at 760 nmand 850 nm, respectively. Taking advantage from this dif-ference in absorption spectra, PAI may provide informationabout oxygenation status of tumors lesions, enabling thediscrimination between benign and malignant lesions, basedon a lower HbO2 PA signal in many of the most aggressivetypes of cancer. Nevertheless, PAI has certain limits fordetermination of blood oxygenation in deeper tissues pro-portional to the wavelength-dependent optical attenuation[120].

*e potential of PAI in oncological screening, focusingon angiogenesis, oxygen saturation, and drug responseevaluation, has been examined in different murine xenografttumor models [121] or in mice with brain neoplasia [7, 8].Breast cancer, the most frequently diagnosed neoplasiaamong women worldwide, has been the most widely ex-amined by PAI using xenograft or genetically modified(GEMs) mouse models. Based on oxygen saturation, HbT,and lipid content signals, Wilson and colleagues highlightedthe capability of PAI to differentiate the normal mammarygland from hyperplasia, ductal carcinoma in situ, and in-vasive breast carcinoma in a transgenic mouse model ofbreast cancer, as confirmed by the histological classification[9]. Spectroscopic PAI revealed a significant increase of theHbO2 signal and an analogue decrease of both HbTand lipidcontent during tumor progression compared to normalbreast tissue. *e authors hypothesized that the increased


oxygen saturation was due to a transition from a prevalentlyaerobic to anaerobic metabolism, while a low lipid contentwas related to the replacement of fat tissue by canceroustissue. *e unexpected lower levels of HbT in the mostaggressive breast cancer histologies have been confirmed byex vivo complementary analysis, which assessed a reductionof Hb concentrations in the same mouse model of invasivecarcinoma. Interestingly, in this peculiar experimentalsystem, lipids analysis was feasible, showing a distinct ab-sorption peak in the range of 680–900 nm, even if lipids havea markedly lower optical absorption coefficient compared toHb, probably due to the high proportion of fat in mammaryglands. Very recently, breast cancer PAI has achieved ef-fective translation from preclinical research on animalmodels to the clinical trial, and encouraging results havebeen described for improving specificity and sensitivity indifferentiation of benign and malignant breast masses usinga dual modality US-PAI [107]. Morphofunctional features,as well as Hb amount and intratumoral oxygenation status,were assessed in a perspective, multicenter study using aprototype of the hybrid US-PA device and correlated withhistopathological findings.

*e performance of the US-PAI approach in this studywas found favorably correlated with other morphofunc-tional breast imaging modalities such as nuclear medicine,X-ray mammography or grayscale, Doppler, and contrastenhanced US (CEUS) alone, with the potential advantage ofreducing the false-positive diagnosis and biopsies of benignmasses. Further improvement of knowledge about US-PAI-derived images analysis and interpretation of quantitativeresults is required to consolidate its use in clinical practice.Similarly, prostate cancer is an aggressive and commonlydiagnosed cancer in men, characterized by high morbidityand mortality rates. Hence, there is a growing interest todevelop relatively cheap, low time-consuming approacheswith minimal invasiveness for early diagnosis and having

improved sensitivity and specificity compared to conven-tional prostate-specific antigen (PSA) blood test andultrasonography-guided biopsy. In the last years, the abilityof PAI to identify tumoral lesions has been tested both exvivo and in vivo on canine prostate [122], as well as in axenograft mouse model of the prostate tumor [123], usingthe Hb signal to image tumor microenvironment and vas-cular growth. *e coregistration of PA and US images hasproven to provide complementary contrast and morpho-functional information. *e clinical feasibility of thesemethodologies needs technical improvements, for example,by implementing advanced US and light-delivering tran-surethral transducers. More recently, ex vivo multispectralPAI of HbR, HbO2, lipid, and water signal was performed onhuman prostatectomy samples, suggesting the potential todifferentiate malignant prostate tissue from benign prostatichyperplasia (BPH) and normal human prostate tissue, asvalidated by histopathology. *e HbR PA signal was foundto be significantly higher, and the lipid signal resultedmarkedly lower in malignant prostate than normal tissue,while only mean intensity of HbR showed a statisticallysignificant difference between malignant prostate and BPH[124].

*e potential utility of endogenous Hb and lipid com-plementary contrast has been investigated also for the as-sessment of lymph nodes, given their central role inmetastatic dissemination. Ex vivo pilot study on normalhuman mesenteric lymph nodes highlighted the ability ofPAI to reveal vascular structures using the Hb signal andexternal boundaries through the lipid signal, suggesting apotential application in future for the clinical detection ofextranodal metastasis in many types of cancer [125]. Ac-cordingly, in vivo spectroscopic PAI in an orthotopic mousemodel of squamous cell carcinoma in the oral cavitydemonstrated the feasibility to detect metastatic invasion bymonitoring changes in HbO2 in lymph nodes.

A good correlation of imaging findings with histologicconfirmation of the presence of micrometastases was found[126]. Moreover, PAI of the oxygenation status, in particularhypoxia, is receiving increasing attention for assessingmalignant progression and metastasis in clinically relevantorthotopic xenograft mouse models of glioblastoma andlung cancer.

Li et al. [7] demonstrated the feasibility of PAI fornoninvasive in vivo imaging of brains of immunocompro-mised nude mice to detect and characterize intracranialhuman U-87 glioblastoma xenografts. Functional parame-ters such as HbO2 and HbT of the brain tumors weremeasured without performing a craniotomy. Mean intensityHbO2 value was lower in tumor core compared to sur-rounding normal brain, suggesting hypoxia developmentwhich is correlated to cancer invasiveness, while HbTresulted higher, which is indicative of upregulated meta-bolism and angiogenesis.*is pioneering study aimed to putthe basis for an improvement of PAI strategies, useful toaccelerate the development of novel molecular anticancertherapies. Target probes at specific molecular markersexpressed on the endothelial and tumor cells surface andtechnical improvements will potentially give a strong

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10–2

10–4

Abs

orpt

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coef

ficie

nt (c

m–1

)

200 400 600 800 1000Wavelength (nm)

Water

Melanin

NIR-I NIR-II

Oxyhemoglobin

Deoxyhemoglobin

Lipids

1200 1400

Figure 2: Optical absorption spectra of major endogenous chro-mophores in tissues of living organisms. *e highlighted windows(NIR-I and NIR-II) indicate the wavelength ranges correspondingto minimized optical absorption (reprinted with permission fromDean-Ben et al. [119], CC BY-NC-ND license, https://creativecommons.org/licenses/by/3.0/).




contribution to brain PAI applications. Likewise, lungcancer is among the leading causes of cancer-related death,and therefore, more accurate and predictive preclinicalprotocols to study cancer biology and the response to newanticancer in animal models are needed. PAI, in associationwith CEUS, showed promising application to investigateprogression and to characterize relevant hallmarks such asoxygenation, perfusion status, and vascularization of tumorsin a orthotopic mouse model of lung cancer [127] (Figure 3).

In an integrate approach including US, bioluminescenceimaging (BLI), and X-ray computed tomography (CT), PAIcontributed to add crucial data about tumor hypoxia, amajor parameter influencing tumor resistance toward RTand CHT.

A multimodal imaging approach, combining US, BLI,and PAI, was also employed to longitudinally follow tumorgrowth in an orthotopic mouse model of bladder cancer,successfully mapping the variation over time in oxygensaturation from earlier to more advanced stages of tumordevelopment [128]. A reliable, real time, and comparativelyinexpensive imaging strategy using US and PAI is highlydesirable in clinic since therapeutic response of bladdercancer can vary widely among patients, and consequently,intensive monitoring is needed to predicting the personaloutcome. Overall, assessment of hypoxia levels and mi-crovascular network may be useful in planning and evalu-ation of the potential response to anticancer therapies. Inparticular, studies in preclinical models have demonstratedthe utility of PAI for monitoring tumor response to anti-angiogenic treatments.

It is well known that hypoxia may affect prognosis andCHTand RTefficacy. Rich and Seshadri [129] have describedthe utility of PAI, noninvasively measuring changes in HbTand HbO2 in patient-derived xenograft (PDX) models ofhead and neck cancer, for assessing tumor following radi-ation alone or associated with CHT. *e authors demon-strated that PAI is able to detect in vivo early changes inoxygenation of tumor as index of responsiveness to RT.

*ey found that 24 hours after treatment, with both CHTand RT, a variable tumor response was evident, showingfrom mild changes to significant increase of HbO2 mea-surement compared to baseline. Good correlation was foundamong PAI and other conventional methods of tumoroxygenation status measurement, like oxygen-enhancedMRI and the percentage of the viable tumor assessed byH&E histology. Moreover, a significant correlation wasreported between the percentual variation of HbO2 24 hoursafter treatment and of tumor volume assessed 2 weeks laterby US, suggesting that PAI may aid treatment planningearlier than conventional morphological outcomes. Com-plementarily, Costa et al. [130] highlighted in the samemouse model the potential utility of in vivo PAI to predictthe individual response to RT.

*is recent study found that tumors with higher HbO2baseline estimates are better responders to RT compared toones with inferior oxygenation levels, especially after lowradiation dose treatments. In comparison, tumors un-responsive to RT showed a raising trend of HbO2 over time.*ese results suggested that, using HbO2 readout, PAI could

be a promising tool to successfully guide personalizedtherapeutic protocols not only by rapidly assessing the ef-fectiveness of conventional anticancer treatments but also bypreliminarily predicting the responsiveness of the tumor toRT. Hysi et al. [131] investigated the feasibility of PAI formonitoring changes in the tumor vasculature of an ortho-topic mouse model of breast cancer after administration ofliposomes containing doxorubicin, showing a significantdrop in the HbO2 signal early in response of treatment.*eirwork highlighted the potential of PAI clinical translation forpersonalized medicine, helping the oncologist to assesstherapeutic response rapidly in the single patient.

4.2. Endogenous Melanin and Synthetic Melanin-LikeMolecules. Melanin is an endogenous pigment, normallypresents in hair, nails, skin, and other tissues of living or-ganisms, that is able to produce a PA signal, showing abroadband optical absorption depending on its polymorphstructure [11]. Such properties make natural melanin and itssynthetic analogues interesting molecules for biomedicalapplications, like imaging and therapeutic probes [11].

In this view, natural melanin may be potentially usefulfor PAI by a wide range of wavelength in the NIR interval,and in the last years, there was a growing impulse to thedevelopment of melanin-like biopolymers in order to en-hance fields of application [11, 12]. Endogenous melaninoffers the same advantages previously described for otherconstitutive chromophores. Moreover, synthetic com-pounds, such as melanin-like organic NPs, potentially offerhigh biosafety, promoting the clinical use of contrast agentswith low toxicity [11, 12]. Extensive overview on melanin-based contrast agents for PAI has been provided by recentpublications [11, 13] and therefore is outside the remit of thispaper. Moreover, melanin enables photothermal theranosticapproach, causing local hyperthermia after NIR irradiation,to treat several types of solid cancer [11, 12]. In first studies,endogenous melanin has been tested as target for inducingmalignant cells death of the heavily pigmented melanoticmelanoma. Afterwards, preliminary experiments in micehave demonstrated that melanin-like NPs offer better ab-sorption efficiency and energy conversion to heat after NIRlaser irradiation [14].

Given its high invasiveness and metastatic potential,early detection of melanoma is crucial to provide timely aneffective therapeutic strategy. Feasibility of PAI based onendogenous melanin has been demonstrated for in vivoevaluation of melanoma both in humans and animal models.In a pilot study, Swearingen et al. [15] applied multispectralPAI of Hb and melanin to improve differential diagnosis ofpigmented and vascular cutaneous lesion in patients. *eresults of this study suggested that PAI could providecomplementary information than conventional techniqueslike dermoscopy, contributing to a better in vivo classifi-cation of skin pathologies. More recently, Wang et al. [16]have demonstrated through in vitro and in vivo preclinicalexperiments that multimodal US-PAI is able to evaluate boththe thickness and angiogenesis of cutaneous melanoma,improving diagnosis, prognosis, and noninvasive tumor


biopsy. Similarly, SLN biopsy may improve staging andtherapeutic management of melanoma. Langhout et al. [10]evaluated the ability of PAI to identify metastasis in lymphnodes excised from patients with cutaneous melanoma. *eauthors highlighted the sensibility and specificity of themelanin signal to detect cancerous cells, as confirmed byhistopathology, encouraging future researches for in vivoclinical applications.

*e PA signal of endogenousmelanin has been proved tobe useful not only in examination of cutaneous melanomaand its nodal metastasis but also to detect melanoma cells inother sites of the body. Recently, Xu et al. [17] demonstratedthe possibility to characterize ocular tumors using Hb- andmelanin-based PAI both in vivo on the retinoblastomamouse model and ex vivo in enucleated human eyes bearinguveal melanoma. Another interesting preclinical applicationshowed the PAI ability to noninvasively detect melanomabrain metastases in an orthotopic mouse model [18]. Mel-anin, HbR, and HbO2 content were evaluated by PAIthrough intact skull using a brain-dedicated bimodal US-PAI probe, showing a clear discrimination of Hb andmelanoma cell signal, with higher intensity than the back-ground values assessed in control mice brain. *e authorshighlighted the possibility to in vivo detect intracranialmelanoma and the potential for angiogenesis and hypoxiaexamination using PAI, aiming at clinical translation in anear future. In recent years, functionalized NPs based on

melanin extraction from natural sources or synthetized bychemical oxidation of dopamine (MNPs) have been de-veloped in order to enhance PA contrast and to produce atherapeutic strategy by the photothermal effect [12]. Moreinterestingly, the MNPs are organic and biodegradable andare simply to modify the development of multimodal im-aging probes, highly promising for potential clinicaltranslation. Further improvements of MNPs are needed inorder to avoid their destruction by the endoplasmic re-ticulum, to enhance solubility and optical efficiency and toreduce toxicity in vivo [13]. PEGylation has been proven aconsolidated strategy to optimize stability, biodisponibility,and safety of MNPs through cell viability and proliferationtests and in mouse model, showing favorable in vivo bio-distribution for successful targeting of tumors [12]. *eMNPs offer the advantage to both visualize tumors in vivowith high sensitivity and to simplify synthesis of multimodalimaging probes with high theranostic and translationalpotential. To this aim, Fan et al. [19] developed a biopolymertargeting αvβ3 and performed multimodal PET-MRI-PAI inxenograft mouse models of melanoma. *is multifunctionalprobe allowed to take advantage of PET molecular in-formation, MRI anatomical reference, and high contrast ofPAI, improving simultaneously tumor detection and tar-geting [19]. Very recently, PEGylated melanin-basednanoliposomes for MRI-PAI have been developed to ob-tain the dual functionality of contrast and therapeutic effect

Figure 3: PAI of tumor oxygenation. (a) B mode image of an orthotopic xenograft of lung cancer with corresponding maximum intensityprojection (MIP) CEUS images at time points (t) 0, 2, and 10 s after microbubbles IV injection. (b) B mode image of a hypoxic lung tumorwith corresponding oxygenation map (OxyHemo mode), showing red areas representing the oxygenated part, while blue and dark areasrepresenting hypoxic parts of the tumor. In the following merged US-PA image, the 3D volumes of the whole tumor (green net) and of thehypoxic region of tumor (red net) are reconstructed. (c) Corresponding Bmode, OxyHemomode, merged US PA, and 3D volume images ofa more oxygenated lung tumor without hypoxic core (reprinted with permission from Raes et al. [127], CC BY-NC-ND license, http://creativecommons.org/licenses/by-nc-nd/4.0/).


http://creativecommons.org/licenses/by-nc-nd/4.0/


with the same agent. *e encapsulation of melanin inPEGylated liposomes increases the phagocytosis of thecontrast medium by the tumor cells, enhancing the pho-tothermal conversion of melanin after NIR irradiation andthe subsequent tumor cells destruction as demonstrated inbreast cancer-bearing mice [20]. Moreover, the melaninnanoliposomes showed improved biosafety in order to makethe product easily translatable for theranostic applications[20].

Despite the advantages, PEGylated MNPs have thelimitation of lower PA signal strength than inorganic PAcontrast agents. To overcome this issue and guarantee at thesame time a better biocompatibility, MNPs, modified bycitraconic amide surface conjugation, have been developedto induce selective aggregation in a mildly acidic environ-ment, typical of the tumor lesion [21].*is PA contrast agentwas tested in a xenograft mouse model of breast cancer,providing a greater and more persistent signal than ana-logues PEGylated NPs, with improved specific tumor ac-cumulation [21]. Another strategy recently tested to improvebiodisponibility, tumor targeting, and photothermal efficacyhas been to hide MNPs with red blood cell membrane,resulting in excellent biocompatibility and immune systemavoidance [22]. In a xenograft mouse model of lung cancer,such camouflaged MNPs showed excellent photothermalcapability, enhanced vascular retention, and improved ac-cumulation at tumor sites.

5. Biomedical PAI Applications Based onExogenous Optical Compounds

*e development of exogenous contrast agents is desirableto expand the potential clinical application of PAI, due totheir superior absorption coefficients in the NIR spectralregion compared to the intrinsic contrasts in biologicaltissue. Nontargeted PA agents allow to improve tumordetection mainly based on extravasation from the bloodstream due to enhanced vascular permeability and re-tention (EPR) in tumor lesions, while PA contrast mediabinding a specific biomarker can provide specific in-formation on molecular or cellular processes. Exogenouscontrast agents for PAI include mainly organic dyes,fluorescent proteins, metallic or organic NPs, and smallmolecules suitable for multimodal imaging. *e selectionof favorable exogenous PA probes for cancer applica-tions is based on the spectral differences of optical ab-sorption compared to endogenous absorbers, theirbiocompatibility, photostability, and targeting moieties.Few biocompatible dyes have already been approved forclinical use (ICG, Evans blue, and methylene blue), andnovel targeted contrast agents for PAI are currently underevaluation in preclinical and clinical settings. Overall,these imaging probes have the purpose to providetheranostic applications for clinical use, with the potentialadvantages, of circulation time extension, untargeted re-tention at tumor sites due to the enhanced permeability,specific targeting of tumor cells, reduced toxicity, andcapability of simultaneous multimodal imaging andtherapeutic activity.

5.1. Indocyanine Green. *e indocyanine green (ICG) is acontrast agent approved by US Food and Drug Adminis-tration (FDA) for human use that can be used for PAI. ICGabsorbs light primarily in the range of 600–900 nm and emitsfluorescence (FL) light from 750 to 950 nm [23]. After in-travenous (IV) injection, ICG rapidly binds albumin,establishing macromolecules that are unable to permeate theendothelium of many organs. In clinical field, ICG has beenused for angiography, liver functional evaluation, and SLNsdetection by FL and PA imaging, while in preclinical re-search, it has been proposed for monitoring tumor growthand therapeutic efficacy [24, 25]. PAI using ICG has beenproven to provide early detection of neoangiogenesis ingrowing neoplasia such as a Lewis lung carcinoma mousemodel, based on EPR effect exerted by ICG-albumin mac-romolecules within the tumor environment [26]. Moreover,this study highlighted that PAI of tumor vascular perme-ability has the potential to evaluate tumor response to novelantiangiogenic therapies. In addition to diagnostic prop-erties, it has been also proved that 88% of the ICG-absorbedlight is converted into heat, and therefore, this feature can beconveniently employed in oncology for photothermaltherapy (PTT), leading to effective tumor cell death based oncytotoxicity and reactive oxygen species (ROS) productionafter repeated irradiations [27]. Shirata and colleaguesdemonstrated that IV injected ICG was able to accumulateselectively in cells of hepatocellular carcinoma (HCC),among the major primary malignancy of the liver [27]. ICGwas able to induce HCC death due to the preserved ex-pression of the uptake transporter for ICG on HCC cells,while function of biliary excretion is altered. In vitro studiesshowed that the apoptotic effect of ICG after NIR irradiationis essentially mediated by heat and ROS generation, asconfirmed in vivo by detection of tumor growth suppressionin the Huh7 xenograft mousemodel. Although ICG has beenused to enhance the PA signal in tumor lesions, this simplecontrast agent shows limited applications due to its lacks ofspecificity, rapid clearance, low photo stability, low watersolubility, and tendency to form aggregates [24, 25]. Cir-culation time and photostability of ICG-based contrastagents have been improved through the encapsulationwithin NPs, for example, entrapping a high number of dyemolecules into bigger NPs using the PEBBLE (photonicexplorers for biomedical use by biologically localized em-bedding) technology. Using this method, Kim and col-leagues developed ICG NPs embedded in a biocompatibleormosil matrix [28], while Gupta et al. have encapsulatedmany ICG molecules into a protein shell derived from theplant-infecting brome mosaic virus (BMV) [29]. Improve-ment of ICG photostability has been also achieved by li-posomal formulation, resulting in enhancedbiocompatibility and photothermal efficiency, and in thepossibility to surface modification for targeted moleculesdevelopment [30]. Moreover, liposomal ICG is able toovercome the issue of ICG self-aggregation, which limits theICG fluorescence intensity and the absorbance ratio. Fur-thermore, liposomal ICG could potentially combine ther-apeutic effects based on photothermal heating withchemotherapeutics loading. *eranostic performance of


liposomal ICG has been tested in vivo after peritumoralinjection in a xenograft mouse model of 4T1 breast cancer.PA signal from tumors resulted a significant decrease afterlaser irradiation, suggesting that ICGmolecules, delivered bythe liposomal shell, exerted a successful phototherapeuticeffect [30]. In this perspective, liposomal ICG could bepromising for clinical translation in oncology due to the useof phospholipid and ICG already approved by FDA and inlight of the improvements of imaging and photothermalcapability compared to free ICG. Similarly, Liu and col-leagues tested in the same breast cancer mouse model abiocompatible nanosized ICG J-type aggregate (IJA), toenhance the in vivo imaging performances and photo-thermal conversion efficacy of ICG [31].*e ICG-J aggregateis produced starting from the ICG in aqueous solution, andboth in vitro and in vivo experiments confirmed that it issimply converted into free ICG after tumor cells retentionwith high biosafety. Moreover, using this dye assemblingpreparation method, the drawback of ICG photobleachingwas significantly reduced compared to plain ICG, withsubsequent improvement of photothermal capability. Fur-thermore, ICG-J is characterized by a favorable red shift ofthe absorption peak named “J-band,” and in vivo studieshighlighted that its narrow and intense PA peak was able toimprove resolution at deeper tissue. Overall, these advan-tages could be potentially useful for future theranostic ap-plications in oncology [31] (Figure 4).

ICG conjugation with nanomicelles based on the 30kbiocompatible hydrophilic polymer polysarcosine (PSar) hasbeen proposed as an alternative strategy to conventionalPEGylation, for improving ICG tumor uptake in a faster andhigher contrast way [32]. Both in vitro tests and in vivo PAIdemonstrated a rapid accumulation of ICG-labeled PSar30kin colon26 tumor cells and bearing mice, leading to highercancer-blood ratio in comparison to PEGylated ICG. *eauthors hypothesized that even if ICG-PSar could be mainlyretained by the tumor through a passive effect, its cellularuptake could be partially due to micropinocytosis, thusfavoring an early in vivo tumor accumulation after injection.Further studies are required to identify the target moleculeof PSar in order to select the most suitable cell lines for thisagent and to exploit its high contrast capability for guidingtumor resection.

Similarly, molybdenum disulfide (MoS2) nanosheets(NSs) have recently gained prominence in biomedicine dueto their high biocompatibility and capability to load opticaldyes on the surface for efficient tumor targeting. MoS2-ICGNSs has a wavelength absorption peak at 800 nm, allowinggreater penetration depth and sensitivity for in vivo PAI oforthotopic glioma [33]. In vivo PAI revealed MoS2-ICG NSsaccumulation at the tumor site from 3 to 5 hours afterinjection, likely due to both EPR effect and albuminreceptor-mediated mechanism. Moreover, an imaging depthof 3.5mm at the tumor site was reached, as demonstrated byMRI cross-sectional images. Overall, MoS2-ICG NSs haveshowed desirable features for in vivo PAI in orthotopicmouse model of deep brain glioma, with potential clinicaltranslation in future. In parallel, Chen and colleagues [34]have reported that molybdenum selenide- (sMoS2-) ICG

NSs show high photothermal capability and have validatedtheir use for both PAI and PTT in a xenograft mouse modelof 4T1 breast cancer, highlighting their potential fortheranostic clinical applications. Recent progresses in pre-clinical multimodal imaging has been reported by Swiderand colleagues [25] that developed polymeric NPs of D,L-lactic-co-glycolic acid (PLGA) entrapping perfluoro-15-crown-5-ether (PFCE) imaging agent for 19F MRI andICG, both approved for clinical use.*e ICG encapsulated inthese NPs results was characterized by increased half-life,slower protein bond, and improved photostability. More-over, PLGA-PFCE-ICG NPs allow to combine the highsensitivity and fast acquisition of PAI with the greater tissuepenetration and quantitative analysis capability of MRI [25].*e authors demonstrated the feasibility to use PLGA-PFCE-ICG NPs for in vitro primary human monocyte-derived dendritic cells (DCs) labeling and to detect invivo the cellular signal after their intramuscular adminis-tration in a mouse model. In addition, they were able tofollow labeled DCs in lymph nodes draining the injectionsite by dual-modality imaging, highlighting the potential forimplementing these NPs in cell tracking and cellular ther-apies. Furthermore, their biochemical features could alloweasily modifications for targeting imaging and drug delivery.*erefore, in the last years, ICG has been modified to im-prove both biochemical properties and molecular imagingcapability for theranostic purposes. Last-generation NPsexploit the biocompatibility and desirable optical propertiesof this ICG dye and have been functionalized with targetingmoieties and/or loaded with therapeutic agents [35]. Inseveral preclinical studies, monoclonal antibodies labeledwith ICG have been developed for tumor imaging. Forexample, specific binding of ICG PEBBLEs conjugated toHER-2 antibodies was tested on breast and prostatic cancercells, highlighting their potential for future in vivo appli-cations in cancer detection and treatment [28]. In followinginvestigations, ICG NPs with improved imaging perfor-mance have been tested in preclinical experimental systems.Sano et al. [36] have tested the FDA approved panitumumab(Pan), a antiepidermal growth factor receptor (EGFR)monoclonal antibody, labeled with ICG derivative (ICG-EG4), as a PAI probe to detect squamous cell carcinomalesions in a xenograft mouse model. In vitro, ICG-labeledPan has been applied as a fluorescent probe targeting theEGFR, a relevant biomarker of tumor cells proliferation,angiogenesis, and invasiveness. *e authors demonstratedby in vivo PA imaging and inhibition studies that Pan-EG4-ICG specifically targets EGFR-positive A431 cells, andtherefore, it could be potentially useful to discriminatemetastatic tumors and guide therapeutic approach. How-ever, some issues related to the injected dose of ICG-labeledmonoclonal antibodies, required to achieve adequate PAIsensitivity, have to be overcome for a safe clinical applica-tion. More recently, Wilson et al. [37] have identified the B7-H3 receptor, a potential target distinguishing tumoral le-sions from normal mammary tissue, on both the endothelialand epithelial cells in human samples of several subtypes ofmalignant breast cancer. Bimodal US-PAI with ICG con-jugated to a specific antibody against this biomarker was


performed in a relevant transgenic mouse model of breastcarcinoma. After binding to their molecular target, theseantibody-ICG conjugates undergo spectral shifts of opticalabsorption due to cellular internalization and cleavage ofantibody-ICG bond, with specific accumulation in breastcancer lesions. *e authors suggested that PAI with thistargeted contrast agent could be complementary to con-ventional mammography and ultrasonography to improveaccuracy in diagnosis and staging of breast cancer. More-over, the same authors have demonstrated in a furtherpreclinical study the potential utility of this B7-H3-ICGagent to intraoperatively guide breast tumors resection, bydiscriminating normal and pathological mammary glandtissue with high sensitivity and specificity [38]. Anotherattractive target for multimodal imaging is represented byintegrins, that are adhesion protein overexpressed in a va-riety of neoplasia in course of neoangiogenesis [39–41]. Veryrecently, Capozza and colleagues [24] have combined ICGwith the αvβ3-binding RGD peptide (ICG-RGD) to evaluatein vivo xenograft mouse models of glioblastoma and epi-dermoid carcinoma, with high and low levels of αvβ3 ex-pression, respectively. ICG-RGD showed improvedbiodistribution and imaging performances, and a selectiveintegrin-related retention in U-87MG, but not in A431tumor cells (Figure 5).

Similarly, EGFR is a relevant biomarker of aggressive-ness and resistance to therapy, highly expressed in manytypes of cancer, and therefore, it appears as a promisingtarget for in vivo imaging. Zhou et al. [42] have investigatedthe use of an EGFR-specific peptide bonded to Cy5.5, acyanine dye with spectral features similar to ICG and fa-vorable pharmacokinetic and labeling properties, to detectHCC in a preclinical model by multimodal US-MR-PAimaging. PAI added relevant in vivo molecular in-formation to the US and MRI characterization of tumorgrowth, as confirmed by immunofluorescence and

immunohistochemistry. Targeting strategy could enhancethe potential for precision medicine not only through im-provement in diagnostic accuracy, but also in therapeuticefficacy. Furthermore, even if the photothermal effect hasbeen the main mechanism exploited in ICG-NIR mediatedcancer therapy, more recently antitumoral activity of NPsloaded with ICG has been boosted with synergic drug de-livery strategy. For example, Gurka et al. [43] developed highbiosafety NPs based on mesoporous silica (MSNPs), loadedwith ICG and gemcitabine, improving their specificity forpancreatic cancer by the addition of both chitosan (COS)targeting acidic tumor microenvironment and urokinaseplasminogen activator (UPA) that binds to the UPA receptor(UPAR) on tumor cells. In vivo PAI of MSNPs bio-distribution showed their specific accumulation intohighly metastatic and chemoresistant human pancreaticcancers of orthotopic mouse models, due to the targeting of

–O

Heat

+NN

Na+O OS O

O–OS

Indocyanine green (ICG) ICG J-aggregates

NIR

Photoacoustic imaging

Fluorescence imaging

Photothermal therapy

Figure 4: Representative description of the IJA structure and potential use for multimodal imaging and PTT (reprinted with permissionfrom Liu et al. [31], CC BY-NC-ND license, http://creativecommons.org/licenses/by-nc-nd/4.0/).

ICG inU87MG

Pre

24h

ICG-RGD inU87MG

ICG-RGD inA431

Figure 5: Representative PA images of U-87MG and A431 xe-nografts acquired before and 24 hours after injection of ICG aloneand ICG-RGD. In contrast to epidermoid carcinoma, the PA signalintensity in glioblastoma was significantly higher after ICG-RGDinjection compared to measurements assessed at baseline and afterfree ICG administration (red region of interest) (adapted withpermission fromCapozza et al. [24], CC BY-NC-ND license, http://creativecommons.org/licenses/by-nc-nd/4.0/).





overexpressed UPAR, as confirmed by ex vivo analysis.Moreover, in vitro studies revealed that coating MSNPs withCOS enables the preferential drug release in an acidic en-vironment. *is feature could potentially allow to preciselydeliver the loaded chemotherapeutic agent into pancreatictumors, based on the difference in pH between normal andtumoral pancreatic tissues. *e authors hypothesized futuredevelopments for the MSNPs system such as preclinicalassessment of tumor response to the gemcitabine or otherchemotherapeutics, and their potential use for improvingclinical staging of pancreatic cancer combined with simul-taneous treatment. More recently, Liu and colleagues [44]employed a similar targeting strategy in xenograft mousemodels of breast cancer for theranostic purposes. *eydeveloped folate-receptor-targeted (FA) laser-activatablepoly(lactide-co-glycolic acid) (PLGA) NPs loaded withICG and paclitaxel (Ptx) in order to combine effectively PTTand drug delivery. *e folate receptor (FR) is overexpressedon the cell surface of breast, lung, prostate, ovarian, brain,and colorectal cancer, and therefore, it has emerged as thepromising molecular target for cancer therapy. Like ICG,PLGA has been approved by the FDA, and in the last years, ithas been extensively used for pharmaceuticals synthesis. *ePEG-functionalization of PLGA allows to avoid serumprotein binding and to enhance retention in targeted cells.*ese NPs were successfully applied in vivo as contrast agentfor dual US-PAI in mice-bearing breast cancer. Further-more, FA-PLGA-ICG-Ptx NPs showed the capability toselectively damage cancer cells both by the photothermaleffect and by Ptx release after laser irradiation, significantlyreducing the growth of tumors with high FR expression [44].

5.2.Metal-Based Contrast Agents. A lot of nanotechnologiesbased on golden, silver, ferromagnetic, and other metallicNPs have been developed in order to improve cancer di-agnosis and therapy. Taking advantage of strong absorptionin the NIR region, related to their peculiarity called the SPReffect (charges on the surface of noble metal NPs due tooscillations according to the electromagnetic field), thesenanoplatforms have been successfully implemented as PAcontrast agents. Moreover, these NPs have been function-alized allowing to combine a more precise diagnosis with aneffective PTT and CHT [132]. Gold NPs (AuNPs) havegained attention as promising PA contrast, due to theirgreater optical absorption compared to organic agents.AuNPs are characterized by various shapes, like sphere, rods,shell, or prism, and different sizes, which in turn confer thema wide range of absorption wavelengths (650–1100 nm) in awindow with high contrast compared to biological tissues[45]. Furthermore, AuNPs can be easily modified withdifferent surface moieties to increase targeting efficiency,enhancing the imaging contrast and PAI-based therapy. Inthis view, AuNPs conjugated with gadolinium (Gd3+) haveproven to be a useful multimodal contrast agent in the A549tumor bearing mice. Indeed, this nanoplatform showed asignificant signal amplification for CT imaging and rea-sonable r1 relaxivity forMRI, establishing a potential tool fortranslational cancer diagnosis [46]. Further improvements

of AuNPs applications have been achieved through advancesof their simultaneous diagnostic and therapeutic capability.Using the ligand-receptor pathway, the conjugation with FAenabled the internalization of AuNPs in HeLa cancer cells,allowing to visualize a high accumulation in tumor-bearingmice [47]. In addition, following laser irradiation triggeredstrong shock waves by FA-AuNPs, resulting in cancer cellablation. Recently, PA-guided PTT has been synergicallycombined to gene therapy, exploiting siRNAmediated gene-silencing effect on cancer cells. AuNPs, coupled with Zn(II)-dipicolylamine (Zn-DPA) were able to specifically bindsiRNA, leading to an alternative strategy for efficient and safesiRNA delivery into targeted cancer cells. *rough the use oftheranostic nanocomplexes, with both PAI and PTT activ-ities, it may be possible to overcome siRNA issues related totheir poor pharmacokinetic, cytotoxicity, and low cellularinternalization following systemic administration. More-over, the combination of siRNA agents and PTT couldpotentially provide enhanced antitumor efficacy comparedto conventional single treatments. In vivo PAI using Zn-DPA-AuNPs loaded with antipolo-like kinase 1 silencingsiRNA (siPLK1) showed favorable biodistribution andmaximum signal in the prostate carcinoma xenograft mousemodel at 24 hours after injection. *ereafter, to investigatetheir antitumoral efficacy, PTTwith 808 nm laser irradiationwas performed using both free Zn-DPA-AuNPs and thesiPLK1-Zn-DPA-AuNPs. *e combination of both siRNAgene silencing and PTT was effective to produce tumorregression, probably due to induced tumor cells apoptosis,compared to growth delay obtained by single therapy [48].

An alternative method to functionalize AuNPs forcancer theranostic is to create a pH-sensitive nanoplatform,exploiting the acidic tumor microenvironment in contrast tohealthy tissues. To this aim, the biocompatible, biodegrad-able, and pH-responsive copolymer PAsp(DIP)-b-PAsp(MEA) was synthesized and self-assembled intoPEGylated micelles, loaded with golden nanocages (GNCs)and doxorubicin (DOX) (D-PGNC) [49]. In vivo studieshave been performed by D-PGNC IV injection in a xeno-graft ovarian cancer mouse model, in order to evaluate theirpotential for PAI and to develop a chemotherapeutic systemtriggered by both NIR irradiation and the tumor pH. Astrong PA signal in the tumor was displayed at 8 hours afterD-PGNC IV injection compared to control mice receivingPBS. Moreover, although in this experiment PGNC withlaser irradiation and D-PGNC alone showed an evidenttherapeutic outcome on tumor growth, the DOX-loadedpH-sensitive micelles combined with PTT achieved tumorablation, presumably promoting DOX penetration intocancer cells without systemic side effects [49].

Another pH-sensitive drug delivery system was de-veloped using gold nanostars (GNSs) loaded with both ICGand calcium carbonate (CaCO3) (GNSs-CaCO3-ICG) [50].*e CaCO3 is able to self-dissociate in acidic microenvi-ronment, allowing the preferential release of ICG in specifictumor types, resulting in a more specific diagnostic PAsignal. Moreover, the GNSs photothermal capability and theICG photodynamic properties can be conveniently exploitedfor a boosted photodynamic (PDT) and PTT approach. In


vivo targeting specificity and antitumor efficacy of GNSs-CaCO3-ICG were tested in MGC 803 gastric carcinomabearing mice. Combination of ICG with GNSs-CaCO3promoted its selective accumulation in the tumor region,with a peak enhancement at 24 hours after IV injection,suggesting their potential use as the induced drug deliverysystem. In addition, dual PTT/PDTusing GNSs-CaCO3/ICGexhibited a strong synergic effect in inhibiting tumorgrowth, compared to the animal group injected with GNS orICG alone [50].

Similarly, smart AuNPs (SAuNPs) have been designed toform aggregate in mildly acidic microenvironment, resultingin redshift of the NIR absorption spectrum based on the SPReffect. SAuNPs can selectively accumulate in specific tumorsites without need of further structural modifications, andtherefore, they could be an interesting tool for both tumormonitoring and treatment by multimodal PAI-PTT [51].Furthermore, SAuNPs could be potentially incorporated inmicrobubbles (Mbs-SAuNPs) in order to favor their localrelease in tumor sites by US sonoporation. Such theranosticplatform has been tested in U-87MG bearing mice, high-lighting its usefulness for tumor detection by CEUS andachieving successful cancer cell ablation after laser irradi-ation [52].

Dual mode US-PAI has been used also by Li and co-workers, who developed a nanoplatform composed byAuNPs and liquid perfluorohexane (PFH) that provides amicrobubble contrast medium. Moreover, Au-PFH-NPshave been conjugated to a monoclonal antibody againstmelanoma-associated antigen (MAGE), to specifically targetmelanoma cells [53]. Preliminary results in B16 melanomatumor-bearing mice showed that after IV MAGE-Au-PFH-NPs injection, an early PA peak signal appeared in the tumorsite at 2 hours and persisted until 24 hours, highlighting afavorable kinetic for cancer imaging. After NIR irradiation,the AuNPs-mediated temperature increase triggered thePFH phase change from liquid to gaseous state, allowingCEUS imaging to improve tumor detection [53]. Furtherstudies are needed to implement the use of MAGE-Au-PFH-NPs as a promising approach for noninvasive melanomatargeting and treatment.

A different method inducing self-aggregation of AuNPsin tumor lesions is also represented by binding with ther-mosensitive peptides to promote tumor targeting [54]. Sunand colleagues developed a biocompatible and thermallysensitive elastin-like polypeptide (ELP) and applied AuNPsconjugated with this agent for in vivo multimodal CT-PAI.

*e authors hypothesized that ELP-AuNPs could formaggregates into neoplasia as the intratumoral temperature isassumed to be higher than ELP temperature cutoff (23°C) forphase transition. After intratumoral injection in a C8161melanoma xenograft mouse model, the ELP-AuNPs PAsignal resulted homogenous and increased over time, unlikecontrol mice injected with PEG-AuNPs or PBS, and inagreement with CT enhancement. Furthermore, upon laserirradiation, tumor lesions in the mice group injected withELP-AuNPs were eradicated without recurrence. *e au-thors suggested that ELP-AuNPs thermosensitivity couldrepresent an advantage over other tumor targeting strategies

since it is independent from the EPR effect and tumormicroenvironment [54].

Improvement of therapeutic efficacy has been reachedusing a multifunctional system including AuNPs associatedwith mesenchymal stem cells (MSC), exploiting MSC mi-gration into tumor site for targeted drug release and PTT.Stem cells tumor cell tropism, mainly exerted throughstromal cell-derived factor-1- (SDF-1-) CXCR4 receptorinteraction, have been used in combination with AuNPs, fortheranostic purposes [55].

To this aim, Xu et al. proposed a novel nanoplatformbased on MSC loaded with plasmonic-magnetic lipid NPs,DOX, AuNPs, and iron oxide (IO) NPs (MSCs-LDGI).

In vitro analysis demonstrated that LDGI are in-ternalized by MSCs with low cytotoxicity and that IONPswere able to upregulate the CXCR4 expression on the MSCs,improving tumor specificity. Based on encouraging resultsof cellular studies, the in vivo MSCs-LDGI antitumor effectwas tested in a xenograft mouse model of triple negativebreast cancer, with high metastasis and recurrence rate.

In vivo PAI after intratumoral injection revealed theability of MSCs-LDGI to migrate into the tumor areacompared to bare LDGI. Overall, MDA-MB-231 tumor-bearing mice treated with of MSCs-LDGI and subsequentlaser irradiation exhibited the greatest antitumor efficiencycompared to mice receiving single treatment, achievingtumor eradication without recurrence during the following2weeks. Similarly, after MSCs-LDGI IV administration, PAimages showed that mesenchymal stem cells are crucial foran optimal distribution of AuNPs in tumor site and to obtainthe best therapeutic results [55].

Despite their potential usefulness in PAI and PTT,AuNPs have some limits like cost and chemical instabilitydue to their complex nature, also affecting in vivopharmacokinetics.

Another noble metal recently proposed as theranosticagent alternative to AuNPs to overcome these issues ispalladium (Pd), thanks to its lower costs and stable plas-monic properties after repeated laser exposures. Only fewstudies have investigated its potential in vivo applications.For example, Pd combined with COS and RGD has beencurrently applied for theranostic purposes in MDA-MB-231xenograft mouse model [56]. After IV administration, Pd-COS-RGD accumulated in the tumor region, leading to asignificant PA signal compared to control mice. In-terestingly, the authors observed that photothermal effi-ciency of Pd was comparable to those of AuNPs, but withhigher stability. In vivo PTTusing Pd-COS-RGD induced anincrease of tumor temperature to 50°C over 2 minutes,leading to a complete tumor ablation [56] (Figure 6).

Further studies have been based on the research of othercost-effective metal-based nanomaterials with high thera-nostic performances and facile synthesis, such as copper-based semiconductors, bismuth-based NPs, transitionmetal-based nanomaterials, and magnetic IONPs [65].Among these, copper sulfide (CuS) NPs have acquired in-creasing interest in cancer diagnosis and therapy, due totheir advantageous properties, including good NIR opticalabsorption, its molar extinction coefficient, efficient


photothermal conversion, and good biodistribution, inaddition to relative economicity and low toxicity [57, 58].Moreover, CuSNPs are suitable to be functionalized fordual-modality imaging to improve theranostic specificityand sensitivity. In this regard, Gd3+ ions chelated dieth-ylenetriaminepentaacetic acid (DTPA-Gd3+) have beenconjugated with bovine serum albumin (BSA) and CuSNPs,to act as the contrast agent for tumor detection by PAI andMRI [57]. In vivo T1 weighted MRI in glioblastoma-bearingmice showed increasing signal during the 24 hours followingthe DTPA-Gd3+-BSA-CuSNPs IV injection, in agreementwith the PA enhancement, allowing a clear identification oftumor edges. Taking advantages from high sensitivity anddeep tissue penetration of PAI associated with high reso-lution of MRI, this dual nanoprobe could represent a usefultool for early detection of cancer and guidance therapy [57].

In spite of the PAI capability for deep tissues analysis, alimitation of imaging in oncology may be represented by thebackground signal produced by endogenous absorbers,which in turn could lead to reduced signal-to-noise ratio(SNR). In particular, hepatic neoplasias appear difficult todiagnose since the liver is a highly vascularized organ, and itis involved in nonspecific accumulation of several imagingprobes. *erefore, it could be advantageous to introducecontrast media with low background signal and deep pen-etration into tissues. To overcome the drawbacks, CuSNPswith excitation wavelengths in the range of 1000–1700 nm(NIR II), conjugated with BSA and RGD, have been testedfor the first time in a orthotopic mouse model of HCC. Invivo PAI showed high SNR and liver peak enhancement at24 hours after CuS-BSA-RGD NPs IV injection, allowingclear tumors detection, in contrast to CuS-BSA-NPs, andwith negligible toxicity.*ese results could be ascribed to thehigh PA signal from CuS-BSA-RGDNPs and to the low lighthepatic absorption of the liver upon 1064 nm wavelength, aswell as to the excellent selectivity of the RGD peptide forintegrin receptors, overexpressed in tumor vessels. Collec-tively, this study highlighted the potential utility of the NIRII contrast agent to investigate in vivo cancer disease in deep

organs, due to the low optical absorption of the biologicaltissues at these specific wavelengths [59]. Advances infunctionalization procedures may help to improve not onlytumor detection but also therapeutic efficacy. One of themajor challenges for cancer therapy is to prevent recurrencefrom residual cancer cells after surgery or therapy. Li et al.exploit photothermal conversion feature and bio-compatibility of CuSNPs, in order to develop a nuclear-targeted nanoplatform inducing cancer cell ablation. CuSenclosed into MSNPs has been modified with RGD tospecifically recognize angiogenic tumor vessels and with theTAT peptide for specific nuclear targeting [58]. After localand IV CuS-MSN-TAT-RGD injection in HeLa tumor-bearing mice, a 5-minute PTT was performed after 8hours at 980 nm wavelength, resulting in tumor eradication,without recurrence in a 14-day monitoring period. As-suming the significant increase of nuclear temperatureexerted by the irradiated CuSNPs, together with the selec-tivity of tumor and nucleus targeting by the RGD and theTAT, respectively, this nanoplatform could constitute apromising tool for cancer therapy avoiding recurrences [58].

Other novel copper- and sulfide-based theranosticnanoagents, like Cu-Ag2S and iridium (IrSx), have beenproposed, but the low photothermal conversion efficacy haslimited their use. In a recent study, the combination ofbiocompatible Cu2-xS and Ag2S into a multifunctionalnanoplatform for PAI-PTT has been described to overcomethe limitations of each single agent. Polyvinylpyrrolidone-(PVP-) stabilized Cu-Ag2S NPs were injected IV in a 4T1tumor xenograft mouse model, showing a maximum PAsignal after 6 hours after injection upon 808 nm laser ir-radiation. As expected on the basis of in vitro analysis, theCu-Ag2S-PVP after laser irradiation provided an effectiveinhibition of tumor growth. *e relevant efficiency of tumoreradication, in conjunction with their biocompatibility,makes Cu-Ag2S-PVP NPs interesting for advanced studiesin cancer therapy [60].

As a sulfide metal, Irs is able to provide excellent X-rayattenuation and, at the same time, an efficient photothermal

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Figure 6: In vivo PAI of MDA-MB231 xenograft mouse model before and 1 hour after IV injection of Pd-COS-RGD (adapted withpermission from Bharathiraja et al. [56], CC BY-NC-ND license, http://creativecommons.org/licenses/by-nc-nd/4.0/). (a) Before injection;(b) after injection.



conversion in the NIR region. Recently, IrSx-PEG-FA NPswere IV injected in the xenograft HeLa tumor mouse model[61], resulting in a strong PA signal in tumor site after 24hours, in agreement with CT enhancement, showing a goodcorrelation between Hounsfield units and IrSx concentra-tion. Furthermore, IrSx-PEG-FA NPs, conjugated with an-ticancer drug camptothecin, exhibited both photothermaleffect and pH-photothermal-responsive drug release prop-erties. In vivo combined PTT-CHT induced almost acomplete tumor eradication, more efficiently than the singletherapy administered in the control group. In summary,IrSx-based NPs would be further investigated to developfuture applications in integrated nanoplatform for tumormultimodal diagnosis and ablation [61].

Similarly, among semimetal elements, bismuth (Bi) ischaracterized by a high X-ray attenuation coefficient inassociation with high photothermal efficiency, due toplasmonic properties and biocompatibility.*erefore, Bi hasattracted more interest in multimodal cancer imaging andtherapy. In addition, Bi presents strong absorption in theNIR II, with the advantages of improved SNR and deeppenetration into tissues.

In recent years, ultrasmall Bi NPs, labeled with a peptideLyP-1, have showed high tumor uptake, offering a potentialtheranostic contrast agent for PAI, CT, and combined PTT-RT [62]. To corroborate this, Bi-LyP-1 NPs were IV injectedin a 4T1 breast cancer-bearing mice, showing significant PAsignal intensity at 8 hours after their administration. Furtherstudies using RT improvement and dual-modal imaging ofBi-LyP-1 NPs demonstrated the most relevant results fortumor growth inhibition in Bi-LyP-1 NPs treated mice withboth exposures to 1064 nm wavelength and 4Gy irradiation.In this regards, thanks to the ability to absorb both X-ray andNIR-II laser, the presented nanoplatform has gained interestin theranostics [62]. A similar strategy in the same breastcancer model was conducted by Yang and colleagues [63]which have drawn up the PEG-modified polypyrrole- (PPy-)coated Bi nanohybrids (Bi-PPy-PEG NPs) for multimodalCT-PAI and PTT with high biosafety. Both PAI and CTperformances have been explored after Bi-PPy-PEG NPsintratumoral injection, showing PA signal enhancementproportionally to the NPs injected concentration, and strongCT values with higher contrast than the iohexol conven-tionally used in clinic. In order to evaluate PTT efficacy, 24hours after Bi-PPy-PEG NPs IV injection, 4T1 tumor-bearing mice were irradiated by 808 nm NIR laser, result-ing in tumor growth inhibition without evident toxicitysigns. Importantly, Bi-PPy-PEG NPs offer improved pho-tothermal conversion upon short and repeated NIR irra-diation cycles and as the CT contrast agent showed superiorimaging features than currently used iodine-based agents[63]. Following the promising results in multimodal imagingand therapy obtained with Bi-based NPs, very recently Wuand coworkers [64] have explored the efficacy of Gd-PEG-BiNPs as novel nanoplatform for MRI-CT-PAI and PTT in aglioma mouse model after their IV injection. In vivo MRIevidenced a peak enhancement at 3 hours after adminis-tration, highlighting a much longer retention time in tumorthan the gadopentetate dimeglumine routinely used in the

clinic. Moreover, the potential use of these NPs as the CTcontrast agent has been confirmed in vivo, revealing sig-nificantly enhanced CT signal in tumor lesion 1-hour afterinjection. Finally, as for MRI, PA signal showed a peakenhancement at 3 hours after injection. Furthermore, theauthors demonstrated that upon 808 nm laser irradiation,tumor growth was effectively inhibited by PTT, and xeno-grafts were completely ablated. Overall, authors suggestedthat Gd-PEG-Bi NPs may potentially constitute an in-tegrated approach as the therapeutic agent whose efficacycan be conveniently monitored through trimodal imaging[64]. Among transition metals, titanium (Ti) has been al-ready widely used in tissue engineering due to its excellentbiocompatibility. Taking advantage of its strong NIR ab-sorption, Qian et al. [66] investigated for the first time ti-tanium disulfide (TiS2) conjugated with PEG as a newphotothermal agent for cancer therapy [66]. PAI in 4T1tumor-bearing mice, using IV TiS2-PEG NPs injection and808 nm NIR laser irradiation, resulted in evident signalenhancement at 24 hours, highlighting their distinct accu-mulation in lesion due to the EPR effect. Likewise, in vivoPTT exposing xenografts to the same NIR wavelengthproduced early tumor ablation 1 day after treatment,without recurrence after 10 days. *is study paved the wayfor the development of other Ti-based compounds fortheranostic applications in cancer research [66]. More re-cently, PAI-PTT performances of Ti-nitride NPs (TiN NPs)were confirmed in HeLa tumor-bearing mice following theirIV injection. An increased PA signal over time was observed,peaking at 24 hours after administration. *ereafter, PTTexerted an evident anticancer response within 15 days oftreatment, leading to complete tumor eradication withoutsystemic side effects [65].

In addition, Ti-based oxide nanomaterials (TiO2) haveshowed the potential for PDT after UV light or radio lu-minescence exposure, with the advantage of higher bio-compatibility over heavy metal-based compoundspreviously described. Nevertheless, some issues, related tolimited penetration depth of UV light in biological tissuesand to poor cost-yield ratio of radio luminescence, hasencouraged to develop TiO2 NPs with high NIR absorptionlike niobium-doped TiO2 NPs (Nb-PEG-TiO2) [67]. Inparticular, photothermal properties can be dynamicallymodulated by changing the molecular Nb doping levels,while PEGylation confers high stability and biocompatibilityto the nanosystem. 30 minutes after intratumoral injectionin HeLa tumor-bearing mice, Nb-PEG-TiO2 generated anevident PA signal. Moreover, subsequent exposure to1064 nm laser for 10 minutes was able to prove Nb-PEG-TiO2 efficiency for PTT, resulting in tumor ablation [67].Similar to Ti, magnetic IONPs have been already widely usedin biomedical research, especially in the field of MRI, and inlast decade have been optimized also for PAI and PTT inmouse models of cancer disease. Overall, several studiesusing magnetic IONPs for theranostic purposes have fo-cused on the synthesis of effective and biocompatible ma-terials at the same time for multimodal imaging, developingnew hybrid materials composed by both metal ions andorganic polymers. For example, Monaco et al. [68]


developed Fe3O4 NPs composed by a silica layer and a goldshell conjugated to FA for cancer cells targeting. In addition,Fe3O4-SiO2-Au NPs have been coated with an organic li-gand, in order to be included into biocompatible polymericmicelles, allowing to build targetable nanostructures fordual-modality imaging. MRI images, obtained followingFe3O4-SiO2-Au PMs-FA NPs IV injection in a xenograftmouse model of ovarian cancer overexpressing FR, showed arelevant T2 weighted contrast enhancement in tumor at 4hours after injection. In agreement, the PA peak signal wasfound at the same MRI time point, likely related to the FRtargeting in the lesion [68].

For advanced purposes, IONPs applications for imagingguided tumor treatment by PTT and synergistic CHT havebeen investigated. Jin et al. [69] developed coordination NPs(CPN) mixing a solution of FeCl3 and gallic acid modifiedwith PEG and 64Cu isotope for multimodal imaging andcancer therapy. In vivo PET imaging performed in 4T1tumor-bearing mice after 64Cu-Fe-GA-PEG CPNs IV ad-ministration showed a higher uptake of NPs compared to thecontrol group injected with 64Cu-Fe-GA CPNs, demon-strating that PEGylation promotes their tumoral tropism viaEPR. In parallel, the efficacy of such CPNs for PAI with808 nm NIR laser and T2 weighted MRI was also demon-strated, highlighting a clear contrast enhancement at thetumor site. Finally, the best therapeutic result was obtainedin tumor-bearing mice treated with Fe-GA-PEG CPNs andexposed at 808 nm NIR laser for 5 minutes, achieving thecomplete eradication of cancer lesions [69]. Another hybridNPs taking advantages of excellent biocompatibility andhigh photothermal conversion efficacy of eumelanin(euMel) in association with IONPs was developed for in vivoMRI-PAI and therapeutic purposes [70]. PAI and MRI wereperformed in U-87 tumor-bearing mice after interstitialinjection of euMel-Fe3O4 NPs, obtaining a significant signalenhancement of the tumor site for both imaging methods. Inaddition, in vivo cancer therapy efficacy was proved ex-posing tumor-bearing mice to 808 nm laser irradiation for 5minutes. Combining euMel-Fe3O4 NPs with laser irradia-tion, a 51.1°C final temperature of tumors was reached,followed by their eradication without surrounding tissuedamage. *ese hybrid NPs have combined the favorableparamagnetic properties of IONPs with the contrast efficacyand biodegradability of an endogenous chromophore,allowing a highly sensitive multimodal imaging withoutevident toxic effects [70]. Superparamagnetic IONPs(SPIONPs) are characterized by improved biocompatibilityand biosafety, which together with efficient photothermalconversion make them a promising tool for in vivo PAI andPTT.

To shedmore light on SPIO therapeutic capability, a NIRlight-controllable, targeted, and biocompatible drug deliverynanoplatform have been proposed.

PFH-PTX-PLGA-SPIO NPs targeting human epidermalgrowth factor receptor 2 (HER-2) have been tested as thetheranostic agent in a xenograft mouse model of SKBR3breast cancer [71]. In this nanoplatform, PLGA incorporatesSPIONPs, the chemotherapeutic, and it is conjugated withboth liquid PFH and the Herceptin HER-2 ligand, allowing

for selective accumulation in the tumor site. Furthermore,upon 808 nm NIR laser irradiation, SPIO converts NIR lightin thermal energy, resulting in cancer cell ablation. More-over, overheating triggers the optical droplet vaporization ofPFH, that exerts a double function: generating gas micro-bubbles for CEUS and contributes to the PTX release. *istheranostic strategy allowed to monitor tumor progressionwith high accuracy by US-PAI, reaching at the same time acomplete lesions eradication with improved specificity thanconventional chemotherapeutic protocols and without ev-ident side effects. Moreover, the PLGA, SPIO, and Herceptincomponents have been approved by the FDA, encouragingtheir clinical translation [71].

5.3. Carbon-Based Contrast Agents. In the last decade,carbon (C)-based NPs have gained growing attention inseveral field of biomedical research, including cancer di-agnosis and therapy, thanks to their marked photo-absorption, photostability, solubility, and biologicalcompatibility.

In particular, C nanotubes (CNTs) are able to be in-ternalized from cells without any functional group on theirsurface; nevertheless, they provide multiple sites for covalentor noncovalent attachment of different targeting moieties.Moreover, functionalized CNTs may be more efficientlyemployed to deliver a variety of molecules like peptides,antigens, nucleic acids, and drugs inside cancer cells fortheranostic purposes.

Based on their physical features, CNTs are mainlyclassified in single-walled CNTs (SWCNTs) consisting of acylinder with a unique sheet and diameter range of 0.2–2 nm, and multiwalled CNTs (MWCNTs) which are char-acterized by several layers and a diameter range of 2–10 nm[72, 73]. *e specific advantages of SWCNTs, includingoptical absorption of all the visible light spectrum, highthermal conversion, and maximum size of 2 nm, make themmore attractive for PAI. Instead, due to their larger size,MWCNTs are more suitable for delivery of large bio-molecules such as DNA plasmids. Furthermore, their PAIsignal can be enhanced through combination with othercontrast agents like AuNPs or ICG [72, 73].

In this perspective, several investigations have focusedon CNTs conjugation with optical dyes like ICG, improvingat the same time the PA signal and biosafety of contrastmolecules. Among first, Koo et al. [74] has enhanced PAsensitivity of SWCNTs by ICG attaching. In vivo PAI inhealthy rats has demonstrated the ICG-SWCNTs capabilityto map SLNs and to visualize urinary bladder based on renalexcretion. Moreover, ICG-SWCNTs reached a 4 time greaterPA signal intensity than plain SWNTs. *erefore, the au-thors suggested their potential utility to identify SLNs inbreast cancer patients and to perform cystography for cancerdiagnosis.

Similarly, Zanganeh et al. [75] have tested the feasibilityto assess tumormargins and size in a xenograft mousemodelof 4T1 breast cancer by ICG-SWCNTs, highlighting theirpotential application to guide surgical resection of neoplasia.In addition, de la Zerda and colleagues [76] have


functionalized ICG-SWCNTs with cyclic RGD peptides,allowing αvβ3 integrins targeting of U-87MG tumor xe-nografts in living mice, focusing on improved imagingperformances compared to simple SWCNTs-RGD.

Like metallic-based NPs, biodegradability is a relevantconcern for CNTs clinical translation, and different synthesisstrategies has been proposed to overcome this issue. Forexample, Lee et al. [77] demonstrated by in vitro UV-Visabsorbance test that the degradation of nitrogen-dopedcarbon nanodots (N-CNDs) was improved. In vivo exper-iments in a rat model revealed that N-CNDs allowed todetect SLNs 30minutes from intradermal injection, aidingfor metastatic cancer diagnosis with low toxicity, andthereafter, they are eliminated by renal clearance.

Another common issue of NPs is represented by theirrapid clearance after IV administration. To circumvent thislimit, Xie et al. [78] developed SWCNTs characterized bylong circulation time and capability of both FL-PA imagingand photothermal ablation of tumors. *e SWCNTs werecoated with Evans blue (EB), improving both their watersolubility and binding with serum albumin, which in turnprolong their circulation. Moreover, this SWCNTs-basedsystem was conjugated with an albumin-photosensitizerchlorin e6 (Ce6) complex, enabling dual-modality imag-ing of tumors and improving tumor treatment. *is mul-tifunctional SWCNTs allowed to detect tumor sites insquamous cells carcinoma-bearing mice, showing a fluo-rescent PA signal peak at 24 hours after IV administration.Moreover, albumin-Ce6-SWCNTs have demonstrated tu-mor ablation efficacy after irradiation, using a combinedPTT-PDTapproach. *e authors concluded that combiningFL and PA imaging could provide complementary in-formation for tumor monitoring and can optimize thera-peutic planning.

In the last years, theranostic applications of CNDs areemerging due to their high biosafety, photostability, andtargeting properties. Several novel synthesis CNDs, coupledwith other elements such as sulphur, phosphorus, and N tomodify their spectral emission, have showed high thermalefficiency and have been investigated in mouse models ofhuman cancer for both FL-PA imaging and PTT [79]. AfterIV administration, these innovative CNPs have showeddesirable biodistribution in tumoral tissues through EPRand renal clearance, making them promising for futureclinical translation [79].

In agreement, Parvin and Mandal [80] have demon-strated the utility of dual wavelengths emitting CNDscodoped with phosphorus and N (PN-CNDs) for FL-PAimaging of a xenograft mouse model of human gastriccarcinoma. After IV injection, the PN-CNDs revealed asignificant uptake in the tumor site at different time points,showing a rapid accumulation within 12 minutes, and aprogressive rise of the FL-PA signal, peaking at 3 and 6 hoursafter injection for green and red emitted light, respectively.

Nowadays, the combination of PDT and PTT has beenproved to give a better anticancer response compared tosingle strategy, allowing to use lower radiation dose.Moreover, this approach avoids toxic side effects and drugresistance compared to conventional CHT and RT.

Very recently, Yang and coworkers [81] investigated thepotential of CNPs doped with zeolitic imidazolateframework-8 (ZIF-8) as photothermal and photosensitizer,exploiting the simultaneous production of heat and ROSafter their NIR irradiation. Interestingly, ZIF-8-CNPs syn-thesis has proven to be relatively simple, economic, andefficient. Furthermore, in vivo experiments highlighted goodPAI contrast in a mouse model of human lung carcinomaand their suitability for oncotherapy, positively related to theincrease in NPs size. In light of their overall performances,the authors hypothesized that ZIF-8-CNPs are expected tohave wide clinical applications in a near future.

Analogous attractive results have been obtained by Xuet al. [82] in a xenograft mouse model of 4T1 breast cancerusing newly developed supra-CNDs. *is contrast agent isbased on an emerging technique to engineer CNDs throughelectrostatic interactions and hydrogen bonding, to obtain astrong absorption band from visible light to NIR and effi-cient photothermal conversion.

Using the same mouse model, Jia and colleagues havedescribed an innovative bimodal FL-PA imaging approachto guide synergistic PDT-PTTof cancer [83], employing theHypocrella bambusae (HB) biomaterial as the precursor forthe preparation of CNDs-based phototheranostic agents.HB-CNDs offer several advantages including a relativelysimple and cheap synthetic procedure, adequate photo-stability, good water solubility, broad absorption, red-lightemission spectra, and excellent biocompatibility. In vivoexperiments have demonstrated the feasibility of FL-PAimaging by HB-CNDs and encouraging results throughbifunctional treatment, with optimal time point for bothimaging and phototherapy at 8 hours after HB-CNDs IVinjection.

*e same research group proposed another type ofmultifunctional CNDs coupled with gold nanorods (GNRs)and a silica scaffold (SiO2). Globally, in GNRs-SiO2-CNDs,the GNRs exert both PAI contrast and photothermal effect,while the CNDs have been exploited for FL imaging andPDT. SiO2 aids improvements in both chemical and opticalstability of GNRs and CNDs. GNRs-SiO2-CNDs have beentested in a mouse model of skin melanoma, showing a fa-vorable in vivo biodistribution, high contrast, and spatialresolution to visualize the tumor for subsequent thermalablation. Overall, these pilot studies provided promisingperspectives of effective and safe application of these pho-totheranostic agents in biomedicine.

Other light-absorbing molecules have been combinedwith CNDs to develop new nanomaterials, providing mul-timodal imaging together with therapeutic strategies. Forexample, Wu and coworker [84] have recently preparedCNDs bonded with a porphyrin macrocycle (PCNDs),considering its desirable properties such as high radiationabsorption in the UV, visible, and NIR spectra, as well as itsfavorable photothermal efficiency. *ese biocompatible NPsnanomaterials are characterized by easy synthesis and couldbe helpful for PAI with enhanced performances comparedwith conventional optical imaging modalities. Furthermore,PCNDs have been functionalized with cetuximab (C225-PCNDs) for targeting cancer cells overexpressing EGFR.


Moreover, intratumoral injection of C225-PCNDs in amouse model of human breast cancer has demonstrated animproved capability for both tumor detection and ablation.Such in vivo results indicate that C225-PCNDs could rep-resent a promising platform for cancer theranostics. Finally,in the past decades, all carbon nanomaterials, includinggraphdiyne (GDY), have gained growing interest for ex-ploring their biomedical applications as potential photo-thermal agents, in light of their excellent ability of energyconversion. Li et al. [85] described the first in vivo use ofGDY-based PEGylated nanosheets (GDY-PEG) for simul-taneous PAI and PTT in a xenograft mouse model of 4T1breast cancer. After intratumoral injection of GDY-PEG andlaser irradiation, strong PA signal and efficient photothermalablation were observed. Moreover, the biodistribution ofGDYs-PEG has been studied, revealing a peak of tumorenhancement at 12 hours after IV administration, positivelycorrelated with the GDYs-PEG concentration. On the basisof these findings, the authors hypothesized that GDYs-PEGcould provide new insights for the development of noveltheranostic tools.

Nevertheless, in vivo applications of all carbon NPs arestill in infancy. Very recently, other all carbon NPs [133]have been synthetized, like N-doped graphene (N-G)CNDs, with the advantages of low cost, low cytotoxicity,and higher photothermal conversion efficiency than otherC-based nanomaterials currently available. In addition,N-G-CNDs conjugated with FA have enabled for activetargeting of several tumor cell types in vitro, aiming futurepreclinical employ for in vivo dual-mode FL-PA imagingand PTT.

5.4. Semiconducting Polymer Nanoparticles. Recent ad-vances have been made for cancer PAI and PTT usingsemiconducting polymer (SP) NPs, an emerging group ofcontrast agents characterized by optical features, bio-compatibility, photostability, and functionalization poten-tially more advantageous for in vivo applications comparedto the most widely used organic dyes, metallic NPs, andcarbon nanomaterials [86].

*e remarkable optical features of SPNPs are mainlyrelated to the presence of delocalized electrons, while PAsignal enhancement has been achieved by modifying theirpolymeric structure and designing activatable molecularprobes [87].

Initial investigations on the use of SPNPs for in vivo PAIwere conducted by Pu and colleagues [88] that highlightedthe advantages over SWNTs or GNRs, based on a wider andmore stable PA signal, and higher detection sensibility. In afollowing study, the same author and coworkers [89]highlighted the feasibility of diketopyrrolopyrrole- (DPP-)based SPNs for in vivo PAI on a HeLa xenograft mousemodel. After IV administration, DPP-SPNs exhibited asignificantly higher PA signal in tumor than surroundingtissues, presumably due to extravasation of SPNs throughcancer blood vessels.*ese studies provided the first insightsabout the potential of SPNs for high-resolution PAI inoncological research.

Further improvements in biodistribution of amphiphilicpolymers containing DPP-SPNs have been obtained throughPEGylation [90], overcoming also the potential issue of theirdissociation. In a proof of concept study, the ability of theseamphiphilic SPNs for in vivo tumor imaging was demon-strated at 24 hours after IV injection.

Attempts for advances in diagnostic specificity andsensitivity are also in progress through implementation ofmultispectral PAI. Zhang and colleagues [91] developedsemiconducting polymer dots (Pdots), which include intheir structure isoindigo (IID) and 4,7-dithien-2-yl-2,1,3-benzothiadiazole (DTBT) as electron acceptors. *ischemical modification, consisting in incorporation of anelectron-deficient element into the SPNs structure, isknown as the “self-quenched process,” and allows to en-hance both PA signal intensity and heat generation.SPNIID-DTBT-based Pdots are characterized by strongoptical absorption in the 500–700 nm spectral range,opening promising perspectives for better discriminationof tumoral tissue from blood vessels. In vivo US-PAIpreliminary results highlighted the possibility to detect aclear PA signal after injection of gelatin containing a Pdotssolution into the dorsal subcutaneous tissue of nude mice.Moreover, by applying such gelatin mixture onto miceears, the authors demonstrated the feasibility to dis-criminate between PA signal derived by Pdots solutionfrom that by surrounding vessels. In agreement, a newclass of indigoid π-conjugated SPNs have showed maxi-mum absorption in the optical window where endogenouschromophores have low absorption [92]. In vivo PAI aftersubcutaneous injection of such SPNs in mice has proventhe wavelength-dependent capability to distinguish the PAsignal generated by the contrast agent from that by vas-culature (Figure 7).

Despite enhanced sensitivity of last-generation PAIagents, the deep tissue penetration capability is a currentchallenge, depending on the excitation wavelength used, thelight scattering in different tissues, and the absorption fromendogenous Hb. In this perspective, Fan et al. [93] developedperylenediimide-based (PDI) NPs and demonstrated theirefficiency for specific and noninvasive brain imaging in anorthotopic glioblastoma mouse model, with better tumorpenetration than silica-coated AuNPs.

Moreover, SPNs are highly prone to synthetic modifi-cation for targeting specific receptors, through conjugationwith ligands such as antibodies, RGD peptide, FA, and biotinor for drug delivering [86].

In this perspective, self-quenched strategy has beenfurther improved for in vivo application by Xie and co-workers [94] through conjugation with a cyclic-RGD pep-tide, allowing evaluation of αvβ3 integrin receptorexpression in a xenograft mouse model of 4T1 breast cancer.

In addition, efforts for the development of more sensitivePAI agents have been made applying on DPP-SPNs a surfaceengineering method like coating with a silica layer (SiO2), toamplify the FL-PA signal and guarantee efficient photo-thermal conversion at the same time [89]. DPP-SPNs-SiO2,conjugated with PEG and cyclic RGD, have resulted suitablefor targeting of xenograft 4T1 breast tumors in living mice


after IV administration, showing a high PA signal tobackground ratio.

Chemical structure of different SPNs influences not onlyPA brightness but also photothermal conversion efficiencyfor cancer treatment, and recent preclinical studies haveexplored their theranostic performances to provide mo-lecular insights into the design of effective SNPs for bothPTT and PAI in oncology [95].

Chemophotothermal theranostic nanoplatforms areemerging as a promising tool for cancer therapy im-provements by a synergistic approach. To overcome thecurrent issue of cancer resistance to conventional CHT,amphiphilic PEGylated poly(cyclopentadithiophene-alt-benzothiadiazole) (PEG-PCB) SPNs have been recentlydeveloped as multifunctional nanocarrier integratingDOX delivery with thermal ablation [96]. After IV in-jection, PEG-PCB-SPNs were able to detect 4T1 breastcancer xenografts by FL-PA imaging, presumably en-hancing uptake by tumor lesions through the EPR effect,and to exert a boosted therapeutic effect after laser irra-diation compared to CHT alone. A relevant concern ofphototheranostic nanoagents is represented by their ef-fective delivery in tumor microenvironment. Very re-cently, Li and colleagues [97] have tested a biomimeticstrategy of camouflage, by coating SPNs with the cellmembranes of activated fibroblasts (AF). Such AF-SPNshave proven to be able for cancer-associated fibroblaststargeting after systemic administration, providing not onlyFL-PA signals for successful detection of xenograft 4T1breast tumors but also promoting a combined PDT/PTTapproach, leading to significant antitumor response.Further efforts are needed in future to overcome challengeslike long-term safety or fast renal clearance, bio-compatibility, stability, and tumor targeting, to expand the

SPNs applications in biomedical research from preclinicalto clinical field [97].

6. Perspectives and Conclusion

Over the last decade, PAI has attracted growing interest inoncology as it potentially offers relatively low cost, real time,and noninvasive in vivo imaging of tumor lesions com-plementarily to morphological B mode images provided byUS. Critical points to guarantee a successful clinical trans-lation of PAI are the demonstration of its feasibility, safety,and effectiveness. Preliminary preclinical studies haveexploited mainly endogenous chromophores such as mel-anin, HbR, and HbO2 to characterize tumor microenvi-ronment, in order to improve diagnosis and therapeuticoutcomes assessment. More recently, cancer nanomedicinehas rapidly evolved until to design novel PA nanoplatforms,characterized by high biocompatibility and photostability, aswell as excellent absorption coefficient extended in the NIRII region, enabling deep tissue analysis with good SNR.Further efforts allowed to functionalize these nanosystemswith a large number of targeting moieties and to carry si-multaneously multimodal imaging probes and therapeuticagents inside tumor lesions through a single multifunctionaltool. However, PAI translation for cancer detection andcharacterization in clinic is still in infancy, and there are stillshortcomings to overcome.

Even if PAI technology has reached preliminary appli-cations in clinical studies, improvements are needed rela-tively to spatial resolution for visualization of smaller lesionsin deeper tissues and temporal resolution for dynamicprocesses imaging [134]. Recent advances in laser technol-ogy, improving the pulse repetition rate and the opticalpenetration, have been proved highly effective to enhance

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(b)

Figure 7: Multiwavelength in vivo PAI (a) before and (b) after subcutaneous injection of indigoid π-conjugated SPNs, showing their abilityto distinguish PA signal generated by the contrast agent and vasculature for potential oncological applications (adapted with permissionfrom Stahl et al. [92], CC BY-NC-ND license, http://creativecommons.org/licenses/by-nc-nd/4.0/).



PAI systems performances for oncological applications,allowing for in vivo monitoring of the metastatic process in amouse model of melanoma and deep brain imaging inrodents [135]. Further engineering developments withregards to laser sources, detectors, data acquisition, andimage processing algorithms of PAI systems will open newperspectives for cancer disease detection and targetedtherapy, improving safety regarding laser exposure for pa-tients and operators, as well as SNR and motion artifacts. Toenhance PA image contrast, new exogenous molecularimaging agents are under investigation regarding theirability to produce an adequate PAI signal with a relativelylow injected dose, avoiding toxic effects, and will need FDAapproval for human use.

In addition, limitations in their molecular structure haveprompted for developing innovative strategies for a moreeffective drug delivery, and very recently, innovative smartactivatable theranostic nanoplatforms have been tested intoa glioma mouse model [136].

Similarly, progresses in the PA instrumentation will be akey factor for the approval regarding clinical adoption andmarketing of PAI devices by regulatory committees such asFDA in the United States and CE Mark in European Union.Very recently, a pilot study on healthy patients has provedthat PAI offers a good reproducibility for soft tissuescharacterization, encouraging PAI integration in clinicalsetting [137].

Conflicts of Interest

*e authors declare that there are no conflicts of interestregarding the publication of this article.

Authors’ Contributions

Sara Gargiulo and Sandra Albanese contributed equally tothis work.

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