WO2024068099A1

WO2024068099A1 - Pegylated nanostructures for photoacoustic imaging and photothermal therapy of tumor lesions

Info

Publication number: WO2024068099A1
Application number: PCT/EP2023/070923
Authority: WO
Inventors: Mauro Comes Franchini; Mirko MATURI; Erica LOCATELLI; Massimo Alfano; Elisa ALCHERA; Irene LOCATELLI; Flavio Curnis; Angelo Corti
Original assignee: Ospedale San Raffaele S.R.L.
Priority date: 2022-09-28
Filing date: 2023-07-27
Publication date: 2024-04-04

Abstract

A photoacoustic agent chosen among the group consisting of a metal-based nanoparticle made of gold, silver, or hybrid gold/silver, an organic photoacoustic dye, cyanine dyes, phthalein and xanthene dyes, squaraine and croconaine dyes, tetrapyrrole, BODIPY dyes, curcumin dyes, and IRDye800 linked to a ligand of the integrin family receptors, preferably a peptide containing an integrin binding motif, and antibody or part of an antibody, a peptidomimetic or an aptamer, via a crosslinker selected from the group consisting of a crosslinker bearing amino and sulfhydryl reactive groups, bearing amino and azide/alkyne reactive groups, bearing lipoamide/lipoic acid (LA) or sulfhydryl or disulphide containing compounds.

Description

PEGYLATED NANOSTRUCTURES FOR PHOTOACOUSTIC IMAGING AND PHOTOTHERMAL THERAPY OF TUMOR LESIONS TECHNICAL FIELD The present invention refers to a simple and robust theragnostic agent for photoacoustic imaging and photothermal therapy of tumor lesions in particular bladder cancer lesions, consisting of an agent, preferably a photoacoustic dye or a metal nanoparticle functionalized via a crosslinker to a ligand designed to recognize a tumor associated component and to be used in the early diagnosis and/or treatment of solid tumors, in particular of bladder cancer. BACKGROUND ART Bladder cancers (BC) confined to the mucosa and invading the lamina propria are classified as stage Ta and T1, respectively, according to the Tumour, Node, Metastasis (TNM) classification system [1]. Intra-epithelial, high-grade tumors confined to the mucosa are classified as carcinoma in situ (CIS). Approximately 75% of patients with BC present with a disease confined to the mucosa (stage Ta, CIS) or submucosa (stage T1) [2]. All these tumors can be treated by transurethral resection of the bladder (TURB), eventually in combination with intravesical instillations and are grouped as non-muscle invasive bladder cancer (NMIBC) for therapeutic purposes. Bladder CIS is characterized by a small number of high-grade neoplastic cells that create a reddish area, indistinguishable from inflammation, and have a flat appearance in the urothelium. It can be missed or misinterpreted as an inflammatory lesion during cystoscopy if not biopsied. The management of patients with bladder CIS still represents a challenge in the onco-urological field [3, 4]. Several imaging methods such as computed tomography urography (CT urography), intravenous urography (IVU), ultrasound (US), multiparametric magnetic resonance imaging (mpMRI) and cystoscopy have been used to attempt the diagnosis of bladder cancer. However, major remaining limitations of diagnostic imaging involve the size of the tumor and the dimensional limit of detectability of any method, with US and CT ‐1‐ resulting in very poor detection rates for bladder cancers < 5 mm in size [5]. Cystoscopy remains the gold standard diagnostic method for patients with the suspicion of bladder cancer [6]. Indeed, even cystoscopy, including photodynamic diagnosis performed using violet light after intravesical instillation of 5-ALA or hexaminolaevulinic acid, has limited diagnostic utility for CIS. In fact, during cystoscopy and TURB several biopsies from suspicious urothelium should be usually taken to detect and diagnose CIS on surgical tissue specimens [3]. Still, residual high- grade lesions are found in 40% of patients after the first TURB [7]. Due to these technological limitations, patients with bladder CIS experience very high frequencies of relapse following the first diagnosis, thus undergoing frequent and endless follow- up with poorly effective treatments, resulting in a poor quality of life and the highest cost per patient among all cancers [8]. To overcome the limitations of the imaging methods currently used in clinics for bladder CIS, authors of the present invention aimed to develop approaches and technologies for a non-invasive early diagnosis of in vivo orthotopic bladder cancer, by exploiting an imaging modality based on the photoacoustic (PA) imaging (PAI) approach. PAI is a hybrid imaging modality that combines the high contrast of optical absorption and the high spatial resolution of US generated by chromophores after irradiation by a non-ionizing pulsed laser. As acoustic waves generally undergo less scattering and tissue attenuation compared to light, PAI can provide higher resolution images than traditional US, and achieve deeper penetration than purely optical imaging systems [9, 10]. PAI also allows for the collection of functional and molecular information in real time by employing non-ionizing radiation to reach clinically relevant imaging depths [11]. Endogenous contrast agents, such as melanin, oxy and deoxy hemoglobin, lipids, collagen and water [12], and pulsed laser light in the near-infrared (NIR) spectral range have been exploited in PAI of melanoma [13], the tumor microenvironment [14], atherosclerotic plaque [15] and injuries [16], respectively. Exogenous contrast agents can also be used to enhance the sensitivity and spectroscopic specificity of PA signals. Targeted contrast agents can also be exploited to extend the range of applications of ‐2‐ PAI to molecular imaging [17, 18]. Among the various contrast agents developed so far, gold nanoparticles are of particular interest for their versatility, unique optical and physicochemical properties, relatively inert nature, and successful use in many biomedical applications. In particular, gold nanorods (GNRs) show the highest extinction coefficient in the NIR range and high PA conversion efficiency. Furthermore, tuning the shape of GNRs allows the best wavelength of light stimulation to be selected, thereby enabling the use of these nanoparticles for the needed PAI application [17]. Integrins represent a potential neoplastic target for human bladder cancer, as they are implicated in almost every step of cancer progression from the primary tumor to late stage metastasis development [19]. SUMMARY The purpose of the present invention is to overcome the drawbacks of the known art. In particular, the object of the present invention solves the problems and the limitation presented by the limitations of the imaging methods currently used in clinics for the detection of solid tumors and preferably bladder cancer. In particular, the object of the present invention is photoacoustic agent functionalized with a ligand capable of recognizing a tumor-associated or inflammation associated component. The various embodiments of the invention are presented in the detailed description below and the preferred embodiment are presented in the claims which form an integral part of the present description. In one embodiment, the invention relates to a photoacoustic agent chosen among the group consisting of a metal-based nanoparticle made of gold, silver, or hybrid gold/silver or hybrid gold/iron, an organic photoacoustic dye, cyanine dyes, phthalein and xanthene dyes, squaraine and croconaine dyes, tetrapyrrole, BODIPY dyes, curcumin dyes, and IRDye800 linked to ‐3‐ - a ligand of the integrin family receptors, preferably a peptide containing an integrin binding motif, and antibody or part of an antibody, a peptidomimetic or an aptamer, - via a crosslinker selected from the group consisting of a crosslinker bearing amino and sulfhydryl reactive groups, bearing amino and azide/alkyne reactive groups, bearing lipoamide/lipoic acid (LA) or sulfhydryl or disulphide containing compounds. The invention also provides ‐ the photoacoustic agent as defined above for use in ultrasound and photoacoustic imaging; ‐ the photoacoustic agent as defined above for use as a carrier of drugs and for use in photodynamic therapy; ‐ the use of the photoacoustic agent as defined above for tissue imaging ex vivo It is therefore an object of the invention a photoacoustic agent chosen among the group consisting of a metal-based nanoparticle made of gold, silver, or hybrid gold/silver, an organic photoacoustic dye, cyanine dyes, phthalein and xanthene dyes, squaraine and croconaine dyes, tetrapyrrole, BODIPY dyes, curcumin dyes, and IRDye800 linked to - a ligand of the integrin family receptors, preferably a peptide containing an integrin binding motif, and antibody or part of an antibody, a peptidomimetic or an aptamer, - via a crosslinker selected from the group consisting of a crosslinker bearing amino and sulfhydryl reactive groups, bearing amino and azide/alkyne reactive groups, bearing lipoamide/lipoic acid (LA) or sulfhydryl or disulphide containing compounds. Preferably said crosslinker is selected among SMCC, sulfoSMCC, MAL-PEG-NHS ester, MAL-PEG-TFP ester, propargyl-PEG-NHS ester, azido-PEG-TFP ester, azido- PEG-NHS ester, LA-PEG-MAL, LA-PEG-TFP ester, LA-PEG-biotin, LA-PEG-acid, LA-PEG-NHS ester, LA-PEG, LA-PEG-amine, LA-PEG-azide, Thiol-PEG, Thiol-PEG- acid, Thiol-PEG-amine, Thiol-PEG-azide, propargyl-PEG-MAL, azido-PEG-MAL ‐4‐ wherein the polyethylene glycol chain (PEG) has a molecular weight comprised between 0.05-40 KDa, preferably lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa. Still preferably the photoacoustic agent is a metal-based nanoparticle, preferably a gold nanoparticle, more preferably with a shape selected from the group of sphere, rod, star, cage, prism, shell, hallow shell, wire, plates, preferably nanorod with size ranging from 10 to 200 nm in length, and 2 to 50 nm in width, more preferably ranging from 10 to 100 nm in length and from 5 to 25 nm in width and the preferred aspect ratio (length/width) ranges between 1.2 and 15, more preferably between from 3 and 7. In a preferred embodiment of the invention, the photoacoustic agent according to any one of previous claims comprises a peptide comprising the RGD or isoDGR motif as the ligand of the integrin family receptors. More preferably, said ligand of the integrin family receptors is a peptide selected from: [XGisoDGRG], of SEQ ID No:1 [XisoDGRGG], of SEQ ID No:2 [XphgisoDGRG], of SEQ ID No:3 [XGisoDGRphg], of SEQ ID No:4 [XisoDGRphgG], of SEQ ID No:5 [XisoDGRGphg] of SEQ ID No:6 XFETLRGDERILSILRHQNLLKELQD, of SEQ ID No:8 XFETLRGDLRILSILRHQNLLKEL, of SEQ ID No:9 XFETLRGDLRILSILRX₁QNLX₂KELQD, of SEQ ID No:10 wherein “X” is selected from cysteine, lysine or a non-natural amino acid comprising an alkyne or azide group preferably selected from propargylglycine or azidolysine; X₁ and X₂ are respectively propargyl glycine and azidolysine joined via a triazole bridge. Even more preferably, said ligand of the integrin family receptors is cyclic head-to-tail peptide [CphgisoDGRG] of SEQ ID No:7. ‐5‐ According to a preferred embodiment, it is an object of the invention a photoacoustic agent wherein - the photoacoustic agent is a gold nanoparticle, preferably with shape of nanorods - the crosslinker is lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa - and the ligand of the integrin family receptors is the cyclic head-to-tail [CphgisoDGRG]peptide of SEQ ID NO: 7. Preferably the cross-linker is bound to the gold nanoparticle via the lipoic acid and the ligand is covalently bound to the maleimide moiety of the cross-linker. It is a further object of the invention a composition comprising the photoacoustic agent as defined above and at least one of the following solvents: water, physiological solution, Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate- buffered saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer each containing divalent metal ions such as Ca²⁺and Mg²⁺; said composition preferably further comprised one or more antitumor agents preferably chosen among a chemotherapeutic agent, an immunomodulator, an immune cell. It is a further object of the invention a kit comprising single use vials containing the photoacoustic agent of the invention, a solvent, preferably water, or physiological solution, or Dulbecco's Modified Eagle Medium (DMEM), or a buffer such as Dulbecco's phosphate-buffered saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer each containing divalent metal ions such as Ca²⁺ and Mg²⁺, for resuspending the photoacoustic agent, optionally syringes and instruction for use. Preferably, the photoacoustic agent or the composition or the kit are for use in a method of diagnosis and/or treatment in vivo, preferably for use in the in vivo diagnosis and/or treatment of solid tumors; for use in photoacoustic imaging and/or for use as a carrier of drugs preferably chosen among a chemotherapeutic agent, an immunomodulator, an immune cell. ‐6‐ In a preferred embodiment of the invention, the tumor is selected among urothelial, bladder, gastroesophageal, colorectal, pancreatic, ovarian, lung, cervix, breast and renal cancer, brain tumors and hepatocellular carcinoma. It is a further object of the invention, the photoacoustic agent or compositions or kit as defined above for use in the photothermal therapy of solid tumors, preferentially bladder cancer. The invention also provides the use of the photoacoustic agent of the invention for tissue imaging ex vivo. The invention also provides a method for ultrasound and photoacoustic imaging ex vivo which comprises at least the following steps: a) applying the photoacoustic agent or the composition to the target tissue to be imaged b) photoacoustic visualization of the target tissue, and c) evaluating the visualized target tissue. Further characteristics and objects of the present invention will become clearer from the following description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 – Zoomed one-dimensional NMR spectrum of peptide Iso4 centered on the Hα signals of phenylglycine. NMR analysis shows that peptide Iso4 consists of two isomers corresponding to a cyclic head-to-tail peptide with D-phenylglycine (D-phg) and L-phenylglycine (L- Phg), the latter accounting for about 30%. The HαD and HαL signals of phenylglycine (each give rise to a doublet) are indicate. Figure 2 – Schematic representation and characterization of GNRs₈₀₀@PEG5K-Iso4 and GNRs₈₀₀@PEG5K-Cys. Representation of the structures of head-to-tail cyclized peptide Iso4 (A), lipoic acid-PEG-maleimide heterobifunctional cross-linker (LA-PEG- MAL) containing a PEG chain of 5KDa (B), and gold nanorods (GNRs₈₀₀) functionalized with peptide Iso4 (GNRs₈₀₀@PEG5K-Iso4) or Cys (GNRs₈₀₀@PEG5K- Cys) via LA-PEG-MAL (C). UV-IR absorption spectra of the GNRs₈₀₀@PEG5K-Iso4 ‐7‐ and GNRs₈₀₀@PEG5K-Cys. The dotted line corresponds to the uncoated gold nanorods (GNRs₈₀₀) (D). Representative microphotographs of GNRs₈₀₀@PEG5K-Iso4 and GNRs₈₀₀@PEG5K-Cys, as determined by transmission electron microscopy (TEM) (E). Stability of GNRs₈₀₀@PEG5K-Iso4, as determined by UV-IR absorption analysis of: a) the product stored at +4 °C and after one cycle of freezing (-80 °C) and thawing (F), b) the product mixed with or without sodium chloride (5% NaCl, final concentration) (G), the product before and after addition of synthetic urine (90 % urine, final concentration) (H). Note that the addition of sodium chloride caused a dramatic change in the UV-IR absorption spectrum of GNRs₈₀₀, but not of GNRs₈₀₀@PEG5K- Iso4, suggesting that the latter compound is protected from aggregation induced by high salt concentration. Figure 3 - In vitro PAUS imaging of GNRs₈₀₀@PEG5K-Iso4. A) Representative 2D PAUS image of agar drops containing GNRs₈₀₀@PEG5K-Iso4 (30 µl nanogold, 30 nmol Au, ~ 1.16x10¹¹ NPs) (left) and lacking GNRs₈₀₀@PEG5K-Iso4 (right) both embedded in slime gel. The dotted line delineates the agar drop surrounded by the slime gel (white signal). The specific PA signal of the GNRs (green signal) was obtained by spectral unmixing of the slime and GNRs signals. The GNRs accumulated mainly at the periphery of the drop owing to the uneven polymerization of the agar/GNRs mixture. Bar, 1 mm. B) 3D visualization of the PAUS signal of the agar drops shown in panel A. C) PA spectra obtained with the indicated amounts of gold (nmol) added to the agar drops. Figure 4 - Binding of GNRs₈₀₀@PEG5K-Iso4 and GNRs₈₀₀@ PEG5K-Cys to α5β1-coated plates. A) Binding curve of GNRs₈₀₀@PEG5K-Iso4 and GNRs₈₀₀@ PEG5K-Cys to plates coated with or without α5β1-integrin. The binding was detected with an anti-PEG mAb followed by HRP-labelled goat anti-rat antiserum. Mean±SE of technical duplicates. Representative data of 3 independent experiments. B) Binding curves of GNRs₈₀₀@PEG5K-Iso4 to plates coated with or without α5β1 integrin before (+4 °C) and after freezing at -80 °C and thawing. Mean±SE of technical ‐8‐ duplicates. Note that freezing and thawing of GNRs₈₀₀@PEG5K-Iso4 does not affect its α5β1 binding properties. Figure 5 - Expression of ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^-integrin subunits, and ^ ^ ^6 integrin on murine bladder MB49-Luc carcinoma cells. Integrin expression by MB49-Luc cells was analyzed by FACS using the indicated anti- integrin antibodies (5 µg/ml), and appropriate species-specific Alexa Fluor 488- labeled secondary antibodies (5 µg/ml). Binding of an isotype control antibody is also shown (see Table 3 for antibody description). Figure 6 - Binding of GNRs₈₀₀@PEG5K-Iso4 and GNRs₈₀₀@PEG5K-Cys to bladder cancer MB49-Luc cells as determined by FACS. Detached MB49-Luc cells were incubated with the indicated amounts of nanoparticles for 1 h on ice. After washing, the cells were incubated with an anti-PEG antibody (0.5 h on ice) followed by a FITC-labelled secondary antibody (0.5 h in ice). The bound fluorescence was quantified by flow cytometry analysis. FACS plots (A) and dose- dependent binding curves (dots, mean±SE of technical triplicates) (B) are shown. Figure 7 - TEM analysis of MB49-Luc cells incubated with GNRs₈₀₀@PEG5K-Iso4 or GNRs₈₀₀@PEG5K-Cys. MB49-Luc cells, cultured in a 12-well plate (cell confluency >90%), were washed twice with 0.9% sodium chloride and then incubated for 5 min with 25 mM Hepes buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, 1% w/v BSA. The cells were then incubated for 2 h at 37 °C, 5% CO₂, with GNRs₈₀₀@PEG5K-Iso4 or GNRs₈₀₀@PEG5K-Cys (1x10¹¹ NPs/ml, 500 μl well). The cells were then washed with the same buffer (3 times, 5 min each), fixed, and analyzed by TEM as described in [23]. Figure 8 - Expression of α5- and β1-integrin subunit in the MB49-Luc bladder cancer model. Representative immunohistochemistry photomicrographs of the expression of α5- and β1-integrin subunit in different areas of the bladder of a tumor-bearing mouse, 15 days after intravesical instillation of MB49-Luc cells. T, MB49-Luc tumor; arrows indicate the healthy bladder epithelium. ‐9‐ Figure 9 - In vivo 2D PA and US images of an orthotopic MB49-Luc tumor before and after administration of GNRs₈₀₀@PEG5K-Iso4. MB49-Luc cells were implanted orthotopically in the bladder of a mouse. After 14 days, the bladder was imaged by PA and US analysis before and immediately after intravesical instillation of GNRs₈₀₀@PEG5K-Iso4 (26 nmol Au, ~1x10¹¹ NPs), and after 15 min of incubation and bladder washing. A) A representative axial 2D PA and US images of the entire bladder according to the indicated time of analysis. Bar, 2 mm. B) Zoomed images of Panel A and PA spectra in the selected ROIs according to the indicated time of analysis. Asterisk, Tumor; Dashed and solid features delineate the ROIs drawn on the apical part of the tumor (ROI 1) and outside the bladder (ROI 2), respectively. Note that the PA spectrum detected in the tumor after installation of GNRs₈₀₀@PEG5K-Iso4, but not that of the PA signal observed outside the bladder, is very similar to the spectrum obtained with the same nanoparticles dispersed in agar drops (inset). Arrows, ^ ^max: ^830 nm Figure 10 - GNRs₈₀₀@PEG5K-Iso4 binds to orthotopic MB49-Luc bladder tumors but not to healthy bladder tissue. Representative axial 2D PAUS images of the bladder from tumor-bearing mice (n=4) or from healthy control mice (n=2) before and after instillation of GNRs₈₀₀@PEG5K- Iso4 (26 nmol of Au in 100 µl of DPBS/Ca/Mg, ~ 1x10¹¹ NPs, see Methods). Tumor- bearing mice were PAUS imaged 11-14 days after MB49-Luc cell implantation. Arrows, PA signal generated by GNRs₈₀₀@PEG5K-Iso4. Arrowheads , PA signal independent of GNRs₈₀₀@PEG5K-Iso4. Bar, 2 mm. Figure 11 - 3D PAUS images of bladders from MB49-Luc tumor-bearing mice or healthy control mice before and after instillation of GNRs₈₀₀@PEG5K-Iso4 Representative 3D visualization (left and middle panels) and 3D reconstruction (right panels) of the PAUS signal of mice depicted in Figure 10. Arrows, specific signal of GNRs₈₀₀@PEG5K-Iso4. Arrowheads, nonspecific signal recorded outside the bladder present before administration of GNRs₈₀₀@PEG5K-Iso4. Note the complete absence of PA signal inside the bladder in control animals. Thin arrows indicate small spots of PA ‐10‐ signal likely corresponding to small bladder cancer lesions (<0.5 mm) that are undetectable by standard US echography. Figure 12 - GNRs₈₀₀@PEG5K-Cys does not bind to orthotopic MB49-Luc tumor lesions. A) Schematic representation of the experimental procedure. A mouse with orthotopic MB49-Luc tumor lesions underwent PAUS imaging of the bladder before (Step 1) and after (Step 2) intravesical instillation of GNRs₈₀₀@PEG5K-Cys (26 nmol Au). After bladder washing, additional PAUS imaging of the bladder was then performed before (Step 3) and after (Step 4) instillation of GNRs₈₀₀@PEG5K-Iso4 (26 nmol Au). B) Representative PAUS images (axial 2D) of the bladder after step 1, 2, 3, and 4. Arrows, specific PA signal (generated by GNRs₈₀₀@PEG5K-Iso4). Arrowheads, unspecific PA signal (independent from nanoparticles). Bar, 2 mm. Specific PA signal on tumor lesions was observed only after step 4, suggesting that peptide Iso4 is crucial for tumor recognition by nanoparticles. Figure 13. Effect of a neutralizing anti- ^5 ^1 mAb on the uptake of GNRs₈₀₀@PEG5K- Iso4 to MB49-Luc bladder tumors. Mice bearing orthotopic MB49-Luc tumors were intravescically administered with a control isotype mAb or a neutralizing anti- ^5 ^1 mAb (clone: RTK2758 and 5H10- 27(MFR5), respectively, 20 µg/mouse). After 15 min, the bladders were emptied and subsequently filled with GNRs₈₀₀@PEG5K-Iso4 (26 nmol Au in 100 µl, ~ 1x10¹¹ NPs). After 15 min, the bladders were washed, and PA and US imaged. PA signals associated with whole tumors or adjacent healthy tissues (background) were quantified using VevoLab 5.6.1 software. Box-plots with median, interquartile and 5-95 percentile, in which dots represent well-established tumors. N=4-5 mice, with one or two tumors per bladder. P, by Mann–Whitney nonparametric test. DETAILED DESCRIPTION OF THE INVENTION The aim of the invention is to provide a technological platform for diagnostic, therapeutic or theragnostic applications. While the invention is susceptible to various alternative modifications, some preferred embodiments are described below in detail; said embodiments are example and shouldn’t be intended as limiting of the scope of ‐11‐ the invention to the specific alternative. To overcome the obstacles of the available diagnostic tools the inventors have developed a new technological platform based on the use of a photoacoustic agent functionalized with a ligand capable of recognizing a tumor-associated or inflammation associated component. In a preferred embodiment the agents according to the invention are designed to recognize a tumor associated component and to be used in the early diagnosis and/or treatment of solid tumors, in particular of bladder cancer. It is therefore object of the present invention the development of functionalized agents, preferably metal nanostructures or nanoparticles or an organic photoacoustic dye, cyanine dyes, phthalein and xanthene dyes, squaraine and croconaine dyes, tetrapyrrole, BODIPY dyes, curcumin dyes, and IRDye800 bearing a ligand capable of recognizing a tumor-associated or inflammation- associated component. As used herein, the term “photoacoustic agent” refers to an agent comprising: a) a probe selected from the group consisting of: ‐ a metal-based nanoparticle made of gold, silver, or hybrid gold/silver; ‐ an organic photoacoustic dye selected from the group comprising cyanine dyes, phthalein and xanthene dyes, squaraine and croconaine dyes, tetrapyrrole, BODIPY dyes, curcumin dyes, and IRDye800; b) a ligand of the integrin family receptors, preferably selected from a peptide, an antibody or part of an antibody, a peptidomimetic or an aptamer; and a c) a crosslinker bearing functional groups able to bind to amino groups and sulfhydryl groups and/or functional groups able to bind to amino groups and azide/alkyne groups and/or functional groups comprising lipoamide or lipoic acid moiety or sulphydryl or disulphide containing compounds; wherein said probe and said ligand are joined via said cross-linker. In the above photoacoustic agent the probe is preferably a metal-based nanoparticle, preferably a gold nanoparticle. According to the present invention by nanoparticles or nanostructures is meant a chemical substances or materials with particle sizes between 1 to 100 nanometers in at least one dimension. ‐12‐ According to the invention the organic photoacoustic dyes are photoacoustic probes based on organic dyes. More specifically cyanine dyes, also referred to as tetramethylindo(di)-carbocyanines, are a synthetic dye family belonging to the polymethine group Chemically, cyanines are a conjugated system between two nitrogen atoms; in each resonance structure, exactly one nitrogen atom is oxidized to an iminium. Typically, they form part of a nitrogenous heterocyclic system. Xanthene dyes are fluorescent dyes containing a xanthene, i.e. the three-membered ring structure below:

Fluorescein and rhodamine belong to the most known xanthene fluorophores. Phthalein dyes are a class of dyes mainly used as pH indicators, due to their ability to change colors depending on pH. They are formed by the reaction of phthalic anhydride with various phenols. They are a subclass of triarylmethane dyes. Squaraine dyes are a class of organic dyes showing intense fluorescence, typically in the red and near infrared region (absorption maxima are found between 630 and 670 nm and their emission maxima are between 650–700 nm). They are characterized by their unique aromatic four membered ring system derived from squaric acid. Croconaine dyes are organic dyes with croconaine backbone, as described for example in Liu et al, Photoacoustics Volume 22, June 2021, 100263. Structures of squaraine and croconaine are reported below:

Tetrapyrrole and bodipy dyes are described for example in Frenette M, Hatamimoslehabadi M, Bellinger-Buckley S, Laoui S, Bag S, Dantiste O, Rochford J, Yelleswarapu C. Nonlinear optical properties of multipyrrole dyes. Chem Phys Lett. 2014 Jul 21;608:303-307. Doi: 10.1016/j.cplett.2014.06.002. PMID: 25242819; PMCID: PMC4166509. ‐13‐ Curcumin is an organic molecule defined as (1,7-bis [4-hydroxy-3-methoxy-phenyl]- 1,6-heptadiene-3,5-dione), which constitutes the major pigment component in the yellow Indian spice turmeric. Derivatives of curcumin are known, featuring structural modification on the phenyl rings. IRDye^® 800 is as an infrared dye used for in vivo fluorescence imaging applications. In a preferred embodiment the photoacoustic agent according to the invention comprises the probe as above defined and the ligand capable of recognizing a tumor- associated or inflammation-associated component, and said probe is linked to the aforementioned ligand via a crosslinker. In a preferred embodiment the crosslinker is selected from the group consisting of a crosslinker bearing amino and sulfhydryl reactive groups, bearing amino and azide/alkyne reactive groups, bearing lipoamide/lipoic acid (LA) or sulfhydryl and/or disulphide containing compounds. Said crosslinker is preferably chosen from the group consisting of SMCC, sulfoSMCC, MAL-PEG-NHS ester, MAL-PEG-TFP ester, propargyl-PEG-NHS ester, azido-PEG- TFP ester, azido-PEG-NHS ester, Lipoicacid/lipoamide(LA)-PEG-MAL, LA-PEG-TFP ester, LA-PEG-biotin, LA-PEG-acid, LA-PEG-NHS ester, LA-PEG, LA-PEG-amine, LA-PEG-azide, Thiol-PEG, Thiol-PEG-acid, Thiol-PEG-amine, Thiol-PEG-azide, propargyl-PEG-MAL, azido-PEG-MAL, wherein the polyethylene glycol chain (PEG) has a molecular weight comprise between 0.05-40 KDa, preferably lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa. According to the invention, a cross-linker is a bifunctional moiety bearing at least two functional groups able to bind to or react with specific groups, wherein the terms bind to or react with have the same meaning. Said at least two functional groups can be the same or different, so that the cress-linker can be homo-functional or heterofunctional. Preferably said at least two functional groups are amino groups and/or functional groups able to bind to or react with thiol groups and/or able to bind or react with alkyne groups and/or able to bind to or react with azide groups. Preferably, the crosslinker according to the invention is a bifunctional moiety bearing functional groups able to bind to amino groups and sulfhydryl groups and/or functional groups ‐14‐ able to bind to amino groups and azide/alkyne groups and/or functional groups comprising lipoamide or lipoic acid moiety or sulfhydryl or disulfide containing compounds. Non limitative examples of the cross-linker according to the invention are reported below. Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is a non- cleavable and membrane permeable crosslinker, with the following structure:

SMCC contains an amine-reactive N -hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. NHS esters react with primary amines at pH 7- 9 to form stable amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) corresponds to the compound having the chemical structure indicated below:

The maleimide groups of SMCC and Sulfo-SMCC (sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate) and are unusually stable up to pH 7.5 because of the cyclohexane bridge in the spacer arm. Cross-fuctional linker of the invention may have the general structure reported below wherein A and B include different reactive groups, x is an integer from 2 to 10 (such as 2, 3 or 4), and y is an integer from 1 to 50, for example, from 2 to 30 such as from 3 to 20 or from 4 to 12. Non limitative examples of cross-linkers of this structural class are reported below. Poly(ethylene glycol) (N-hydroxysuccinimide 5-pentanoate) ether N′-(3- maleimidopropionyl)aminoethane (Cas No. 851040-94-3; MAL-PEG-NHS ester): ‐15‐ Mal-amido-PEG-TFP ester indicates a PEG linker containing a maleimide and TFP ester end group. Maleimide groups are reactive with thiols between pH 6.5 and 7.5. The TFP ester can react with primary amine groups and is also less susceptible to undergo hydrolysis compared to NHS ester. The hydrophilic PEG chains increase the water solubility of a compound in aqueous media. Longer PEG chains have improved water solubility relative to shorter PEG chains. The PEG linker has a variable number of glycol units, such as the MAL-dPEG®8-TFP ester, MAL-dPEG®4-TFP ester

Propargyl-PEG-NHS ester is an amine reactive reagent that can be used for derivatizing peptides, antibodies, amine coated surfaces etc. The alkyne group reacts with azide-bearing compounds or biomolecules in copper catalyzed Click Chemistry reactions. It comprises for example propargyl-PEG1-NHS ester with one glycol unit and propargyl-PEG4-NHS ester which is a 4-unit amine reactive PEG linker

Azido-PEG-TFP ester is a click reagent containing an azide group and a TFP moiety. The azide group enables Click Chemistry. The TFP ester is can be used to label the primary amines (-NH2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. Chemical structure of representative compound (2,3,5,6- Tetrafluorophenyl 3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]propanoic acid) is depicted below:

Similar PFP derivative (perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate; ‐16‐ N3-PEG4-PFP) has the structure below:

Azido-PEG-NHS ester is a PEG reagent which contain an azide group and an NHS ester. A non limitative example is the 2,5-Dioxo-1-pyrrolidinyl 3-[(23-azido- 3,6,9,12,15,18,21-heptaoxatricos-1-yl)oxy]propanoate:

α-lipoic acid (Lipoic acid, or thioctic acid, LA, ALA) is an organosulfur compound derived from caprylic acid (octanoic acid)

PEG-Lipoic acid derivatives contain the PolyEthyleGlycol arm (PEG) in different lengths (from 400da to 40KDa) that imparts hydrophilicity and other physicochemical properties. PEG-Lipoic acid derivatives contain a functional group, such as NHS, Maleimide, Carboxyl, Amine, Azide, Hydroxyl, Thiol, that can be used by conventional chemistry to create conjugates Lipoic acid PEG amine (LA-PEG-amine) is

Lipoid acid – PEG – Succinimide (LA-PEG-NHS ester)

Lipoid acid – PEG – Maleimide (LA-PEG-MAL): ‐17‐ Lipoid acid – PEG – Thiol (LA-PEG-Thiol):

Lipoid acid – PEG (homobifunctional LA-PEG):

Thiol-PEG-acid ‐18‐ In the above compounds the polyethylene glycol chain (PEG) has a molecular weight comprise between 0.05-40 KDa, preferably lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa. In a particular preferred embodiment said crosslinker is lipoamide-PEG(5KDa)-MAL. In a preferred embodiment the nanoparticles according to the invention have photoacoustic properties, more preferably the photoacoustic properties are preferably in the near infrared region I and II. In a preferred embodiment the agent according to the invention, is a metal nanoparticle having photoacoustic properties. In a preferred embodiment the nanostructures are nanoparticles made of gold, silver, or hybrid gold/silver, the preferred metal being gold. ‐19‐ The nanoparticles according to the invention can be designed having a different shapes; by way of example but non-exclusively they can be designed in the shapes of sphere, rod, star, cage, prism, shell, hallow shell, wire, plates. In a preferred embodiment the metal nanoparticles are nanorods, with a size ranging from 10 to 200 nm in length, and 2 to 50 nm in width, more preferably ranging from 10 to 100 nm in length and from 5 to 25 nm in width. In a preferred embodiment the aspect ratio (length/width) ranges between 1.2 and 15 and preferably between 3 and 7. In a preferred embodiment the nanoparticles according to the invention are gold nanorods (GNRs) having and aspect ratio of 4.3 ± 0.53. When the agent is a gold nanoparticle, in a preferred embodiment said nanoparticle can be functionalized with a protein (preferably serum albumin), a sugar (preferably chitosan) or a polymer (preferably a polyethyleneglycol-derivative). According to the present invention the photoacoustic agent is linked to ligand capable of recognizing a tumor-associated or inflammation-associated component. According to the invention it is preferred a ligand of the integrin family receptors, a protein (more preferably and antibody or part of an antibody), a peptide, a peptidomimetic or an aptamer. The integrins are chosen as they are key regulators of cell structure and behavior, affecting cell morphology, proliferation, survival and differentiation. In a preferred embodiment the integrin family receptor is selected from the group consisting of ^v ^1, ^ ^8 ^1, ^ ^5 ^1, ^ ^v ^3, ^ ^v ^5, ^ ^v ^6, ^ ^v ^8, α3β1, α6β1, α7β1, α6β4, α1β1, α2β1, α10β1, α11β1, α4β1 and α9β1, ^ ^5 ^1 being preferred. In an alternative embodiment the ligand is able to bind an extracellular matrix component preferably selected from the group consisting of fibronectins, laminins and collagens. According to the invention the ligand can be a peptide comprising an integrin binding motif as RDG (Arg-Gly-Asp) or isoDGR; in a preferred embodiment the peptide consists of a cyclic isoDGR peptide, selected from the group of [XGisoDGRG], of SEQ ID No:1 [XisoDGRGG], of SEQ ID No:2 ‐20‐ [XphgisoDGRG], of SEQ ID No:3 [XGisoDGRphg], of SEQ ID No:4 [XisoDGRphgG], of SEQ ID No:5 [XisoDGRGphg] of SEQ ID No:6 wherein “X” is preferably a cysteine, a lysine or any alkyne- or -azide functionalized amino acid such as propargylglycine or azidolysine In another preferred embodiment the ligand is a linear peptide selected from the group of XFETLRGDERILSILRHQNLLKELQD, of SEQ ID No:8 XFETLRGDLRILSILRHQNLLKEL, of SEQ ID No:9 XFETLRGDLRILSILRX₁QNLX₂KELQD, of SEQ ID No:10 wherein “X” is preferably a cysteine, a lysine or any alkyne- or -azide functionalized amino acid such as propargylglycine or azidolysine, whereas X₁ and X₂ in form a triazole bridge via a propargylglycine (X₁) and azidolysine (X₂). In a preferred embodiment the ligand is [CphgisoDGRG] of SEQ ID No:7 and structure below:

The compound can exists as a mixture of isomers corresponding to a cyclic head-to- tail peptide with D-phenylglycine (D-phg) and L-phenylglycine (L-Phg). As used herein “phg” indicates D-phenylglycine (D-phg) and “Phg” indicates L-phenylglycine (L-Phg). The ratio of D-phenylglycine (D-phg) and L-phenylglycine (L-Phg) can be comprised between 50:50 and 99:1, 60:40 and 90:10, 65:35 and 80:20. In a representative example the compound comprises about 70% of D-phenylglycine (D-phg) and about 30% of L-phenylglycine (L-Phg). ‐21‐ As used herein, “isoD” indicates isoaspartic acid (isoaspartate, isoaspartyl, β- aspartate), which is an aspartic acid residue isomeric to the typical α peptide linkage. It is a β-amino acid, with the side chain carboxyl moved to the backbone. Head-to-tail cyclized peptides are peptide with a cyclic structure. Head-to-tail backbone (homodetic) cyclization represents a peptide modification that imparts rigidified structure, biorelevant turn conformations, increased proteolytic stability, and improved membrane permeability. In a preferred embodiment the ligand is coupled to albumin, or chitosan, or to a bifunctional cross-linking reagent to generate a ligand-albumin conjugate, or ligand- chitosan conjugate, or ligand-liker conjugate, useful for the functionalization of nanogold. Therefore are object of the present invention photoacoustic agents, preferably gold nanoparticles, more preferably gold nanorods, linked to a ligand of the integrin family receptors, preferably a peptide containing an integrin binding motif, and antibody or part of an antibody, a peptidomimetic or an aptamer, via a crosslinker, the crosslinker selected from the group consisting of a crosslinker bearing amino and sulfhydryl reactive groups, bearing amino and azide/alkyne reactive groups, bearing lipoamide/lipoic acid (LA) or sulfhydryl or disulphide containing compounds. According to the present invention the preferred agents are gold nanorods, linked to cyclic head-to-tail [CphgisoDGRG] peptide via a crosslinker, the crosslinker being lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa. Any of the agents of the described embodiment can be prepared in solution or can be prepared in a lyophilized form. With the term “lyophilized” is meant also dried or freeze-dried. It also object of the present invention a composition comprising the described theragnostic agents; the composition according to the invention comprises at least one of water, physiologic solution/saline, Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer each containing metal divalent ions such as Ca²⁺, Mg²⁺, a pharmaceutical acceptable excipient, a pH regulator and albumin. ‐22‐ In particular, for use in treatment, the composition can be prepared comprising, in addition to the above described components, one or more medicament, in particular at least one of a chemotherapeutic agent, a immunomodulator, an immune cell. In a preferred embodiment chemotherapeutic agent is selected from the group of: mitomycin-C, Bacillus Calmette Guerin (BCG), doxorubicin, melphalan, gemcitabine, taxol, cisplatin, vincristine, or vinorelbine; more preferably the immunomodulator is an anticancer vaccine and/or an immune check point blocker, such as anti-PD1 or anti- PDL1 or anti-CTLA4 antibodies, and more preferably the immune cell is a lymphocyte or a genetically modified T-lymphocyte, such as CAR-T cells, or TCR redirected T-cells or NK cells. In a preferred embodiment, the invention encompasses a composition comprising gold nanorods, gold nanorods linked to cyclic head-to-tail [CphgisoDGRG] peptide via a crosslinker, the crosslinker being lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa and Dulbecco's Modified Eagle Medium (DMEM) or Dulbecco's phosphate-buffered saline (DPBS) containing divalent metal ions such as Ca²⁺ and Mg²⁺. It is also object of the present invention a kit comprising single use vials containing the agent as described above, a solvent, preferably physiologic solution/saline, Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer each containing metal divalent ions such as Ca²⁺ and Mg²⁺ for resuspending the nanoparticles, optionally syringes and instruction for use. It is also object of the present invention the application of the described agents, compositions and kits in the medical and diagnostic field. In particular the nanoparticles object of the present invention with photoacoustic properties are developed for use in the diagnosis of solid tumors or inflammation and can be used in particular for the early detection of small cancer lesion, in particular urothelial, bladder gastroesophageal, colorectal, pancreatic, ovarian, lung, cervix, breast and renal cancer, brain tumors and hepatocellular carcinoma. The ‐23‐ nanoparticles, as disclosed by the present invention, are particularly suitable to detect urothelial and bladder cancer and actinic cystitis chronic by photoacoustic imaging. In a preferred embodiment nanoparticles according to the present inventions can be used in the early diagnosis of bladder lesions, in particular of bladder cancer. Are therefore object of the present invention the disclosed nanoparticles for use in a method of in vivo diagnosis of bladder cancer in particular of small and flat urothelial lesions of high-grade bladder carcinoma in situ (CIS). It is in fact possible to proceed with intravesical instillation of urine-stable nanoparticles designed according to the invention, having photoacoustic properties, then use them in a multimodal imaging of the targeted lesions with PAI. In a preferred embodiment the targeted area can be subject to thermal ablation; the delivery of the laser light will irradiate the targeted area with the bound nanoparticles. Assisted photothermal therapy is generated by the excitation of particles at a chosen wavelength. It is therefore object of the present invention the application of the described agents, composition and kits in the medical and diagnostic field, in particular for theragnostic application. In a preferred embodiment nanoparticles or compositions according to the present inventions can be used in combination with a medicament, in particular chemotherapeutic agent, a immunomodulator, an immune cell in a method of combination therapy wherein the administration of the nanoparticles and of the medicament can be simultaneous, contemporaneous or sequential. In a preferred embodiment chemotherapeutic agent is selected from the group of: mitomycin-C, Bacillus Calmette Guerin (BCG), doxorubicin, melphalan, gemcitabine, taxol, cisplatin, vincristine, or vinorelbine; more preferably the immunomodulator is an anticancer vaccine and/or an immune check point blocker, such as anti-PD1 or anti- PDL1 or anti-CTLA4 antibodies, and more preferably the immune cell is a lymphocyte or a genetically modified T-lymphocyte, such as CAR-T cells, or TCR redirected T-cells or NK cells. ‐24‐ In order to demonstrate the effectiveness of the targeted agents according to the invention the inventors proceeded to design Gold nanorods (GNRs) that have been functionalized with the cyclic head-to-tail [CphgisoDGRG] peptide (ISO4) via crosslinker, the crosslinker being the lipoamide/lipoic acid-PEG-MAL and the PEG having a molecular weight of 5 KDa. The ligand is selective for the α5β1 integrin (ki=15 nM) and is selected and characterized to enable bladder tumor targeting; said particles are developed for use in a method of in vivo diagnosis and treatment of bladder cancer based on the intravesical instillation of said urine-stable targeted GNRs. The particles have been designed selected and characterized to allow early detection of small lesions in the bladder and have been developed for use in a method of in vivo diagnosis and treatment of bladder cancer based on the intravesical instillation of said urine-stable targeted GNRs (called GNRs₈₀₀@PEG5K-Iso4) followed by multimodal imaging of cancer lesions with PAI. The inventors developed a combination of strategies that allow tumor detection with an unprecedented sensibility. The results show that the combination of PAI of intravesical instilled GNRs in an orthotopic model of bladder cancer can reveal the presence of lesions undetectable with US imaging. The technological platform, according to the invention, could detect neoplastic lesions smaller than a half millimeter, with a sensitivity that far exceeds that of the US and CT urography for bladder carcinoma. The inventors were able to realize agents that can be used to detect orthotopic murine bladder cancer lesions <0.5 mm, undetectable by US imaging. According to a preferred embodiment the targeted nanoparticles of the invention are gold nanorods designed with an average aspect ratio of 4.3 ± 0.53 to have a peak light absorption at ^830 nm, to leverage the optical window that allows for deeper tissue penetration and to overcome the different endogenous contrast molecules present in tissues. It was investigated and established the PA dynamic range of the above nanoparticles and identified the maximum fluence and energy of the pulsed laser light to obtain diagnostic imaging using targeted nanoparticles avoiding reshaping of the nanostructure. ‐25‐ The urinary bladder environment in particular offers the possibility to exploit the intravesical instillation of the targeted nanoparticles, which is characterized by pro and cons compared to systemic delivery. Intravesical instillation allows for the avoidance of off target effects and off-target accumulations, such as the accumulation of gold in the liver, spleen, kidney, testis and brain, as has been observed in cases of systemic instillation. On the other hand, one of the problem to be solved is that intravesical delivery of the treatment i) must content with urine, which contains a broad variety of byproducts from the metabolism of endogenous and exogenous substances, bacteria, bacteria-derived mucus and floating urothelial cells, ii) is characterized by temporary retention, and iii) cannot exploit the enhanced permeability of the tumor vasculature and retention effects of the neoplastic vasculature to accumulate the intravenously injected target nanoparticles in the neoplastic environment. The first step in developing the nanoparticle was therefore the identification of a target expressed only in the tumor cells and not expressed in the non-neoplastic bladder epithelium to direct them only on the tumoral tissue. Integrins represent a potential neoplastic target for human bladder cancers, as they are implicated in almost every step of cancer progression from the primary tumor to late stage metastasis development. The α5β1 integrin was selected as the appropriate target, said integrin has in fact been reported to be both a marker of unfavorable prognosis for bladder cancer patients and also overexpressed by high-grade invasive bladder cancer; the cyclic head-to-tail [CphgisoDGRG] peptide (Iso4) was chosen as the ligand able to correctly bind and identify the α5β1 integrin. The particles according to the invention were prepared using as a crosslinker lipoamide/lipoic acid (LA)- 5KDa polyethylene glycol (PEG)-maleimide (MAL). The ability of the nanoparticles realized to recognize recombinant α5β1 was confirmed using α5β1-coated plates and the stability was tested under different condition; the nanoparticles were resistant to freezing- or salt-induced aggregation and even in a 90% urine solution no significant change in its absorption spectrum was observed. Finally, the ability of the nanoparticles to bind and therefore identify the target was tested both in vivo and in vitro. In vitro, the particles according to the invention were ‐26‐ able to bind and therefore recognize the surface of bladder cancer cells MB49-Luc; the same properties were confirmed in vivo using an orthotopic syngeneic model of bladder cancer based on the MB49-Luc cells implanted intravesically into mice and the particles were able to selectively recognize bladder cancer cells in vivo, thereby enabling the PA imaging of small cancer lesions. The nanoparticles according to the invention can be therefore successfully used in an in vivo method of diagnosis of solid tumors and in particular of bladder cancer, allowing early detection of small lesion and overcoming the limits of the currently available diagnostic method. The nanoparticles can be, in addition to the diagnosis, used in the photothermal ablation of solid tumors. Cystoscope will instill the nanoparticles and deliver light close to the urothelium. With ultrasound guidance an optical probe (on a Cystoscope) will be moved along the entire bladder to identify small lesions revealed by photoacoustic imaging of the nanoparticles bound to the tumors. The delivery of the laser light will irradiate the targeted area with the bound GNR. Assisted photothermal therapy is generated by the excitation of particles at a wavelength of ~830 nm. EXAMPLES Reagents Human serum albumin (HSA) (20% w/v Flexbumin, Baxalta, catalog #07-19-76-995); recombinant human α5β1 integrin (R&D System, catalog #3230-A5-050); Dulbecco's phosphate buffered saline (DPBS) with calcium and magnesium (DPBS/Ca/Mg) (Thermofischer, catalog #14080); gold nanorods NanoXactTM (NanoComposix, #GRCN800) in citrate buffer (GNRs₈₀₀); lipoic acid-polyethyleneglycol (PEG, 5KDa)- maleimide (CD Bioparticles, catalog #CDN1712); anti-polyethyleneglycol (PEG) rat monoclonal antibody (mAb) clone 26A04 (Abcam, catalog #ab94764); anti-β1 integrin antibody (clone HMβ1-1, Biolegend, catalog #1022019); anti- α5 integrin antibody (clone HMα5-1, Biolegend, catalog # 103902); armenian hamster IgG Isotype Control (eBioscience™; catalog # 14-4888-81); Alexa Fluor™ 488-labeled goat anti-mouse secondary antibody (ThermoFisher, Catalog # A-11001); Alexa Fluor™ 488-labeled goat anti-hamster secondary antibody (ThermoFisher); Alexa Fluor 488-labeled goat ‐27‐ anti-rat secondary antibody (ThermoFisher, catalog #A-11006); horseradish peroxidase (HRP)-labeled goat anti-rat-IgM antiserum (Sigma, catalog #SAB3700672); FITC-labeled mouse anti-rat IgM antibody (clone MRM-47, Biolegend, catalog #408905); bovine serum albumin (BSA) fraction-V (Sigma); normal goat serum (NGS, Sigma); Sigmacote® (a siliconizing reagent, Sigma, catalog #SL-100 ml). Neutralizing anti-α5 integrin antibody (clone 5H10-27(MFR5), rat IgG2a, k, Biolegend, catalog # 103817). Synthetic urine consisting of 128 mM sodium chloride, 60 mM potassium chloride, 40 mM sodium phosphate, 303 mM urea, 50 µg/ml bovine serum albumin and 2 mg/ml creatinine, pH 6.0, was prepared as described [20]. Agar powder (catalog #A9539) and intralipid (20% v/v, catalog #I141) were form Sigma. Peptide synthesis and characterization The head-to-tail cyclized peptide [CphgisoDGRG], called Iso4, was synthesized in- house. Briefly, the resin-bound linear precursor (CphgisoDGRG-resin) was assembled by standard stepwise solid-phase peptide synthesis (SPPS) protocols on a 2- chlorotrityl chloride resin using HBTU/DIEA as activators. The fully protected peptide was then detached from the resin by treatment with a 25% hexafluoropropanol solution in dichloromethane (4 x 5 mL). The solvent was removed under vacuum and the resulting crude linear peptide was dissolved in N,N-dimethylformamide (100 mM) and treated with HBTU/DIEA (1 eq./2 eq) to perform the cyclization step. The reaction was allowed to proceed overnight at room temperature and then the solvent was evaporated. The resulting product was then treated with a TFA-based cleavage mixture to obtain the unprotected peptide, which was recovered by precipitation in cold diethyl ether. Finally, the peptide was purified by reverse-phase (RP)-HPLC and lyophilized (final yield: about 100 mg as gross weight). The peptide was dissolved in sterile water and stored in aliquots at -80°C until use. The concentration of Iso4 was determined by Ellman's assay using 5,5-dithio-bis-2- nitrobenzoic acid (DTNB, Ellman's Reagent, Thermo Fisher Catalog #22582). The identity and purity of Iso4 were assessed by mass spectrometry and HPLC analysis. The structure of Iso4 was characterized by nuclear magnetic resonance (NMR) spectroscopy using a 600 MHz spectrometer (Bruker Avance600 Ultra Shield Plus) equipped with a triple-resonance TCI cryoprobe with a z-shielded pulsed-field ‐28‐ gradient coil. The percentage of [CphgisoDGRG] and of [CPhgisoDGRG] (where phg and Phg correspond to D-phenylglycine and L-phenylglycine, respectively) was estimated integrating the corresponding Hα resonances (respectively at 5.59 ppm and 5.55 ppm) in the 1H monodimensional nuclear magnetic resonance (NMR) spectrum. The following experimental conditions were used: 1 mM peptide in 20 sodium phosphate buffer, pH 6.5, containing 150 sodium chloride, 2.0 mM tris(2- carboxyethyl)phosphine (TCEP) and 10% D2O; temperature, 300 K/27 °C. Functionalization of gold nanorods with peptide Iso4 Eighty ml of GNRs800 in citrate buffer, pH 6.4 (with a longitudinal surface plasmon resonance (LSPR) peak maximum at ∼820 nm and ∼1 unit of optical density (OD)), were poured into a 150 ml silanized beaker, placed under stirring (500 rpm), and mixed with 8 ml of lipoic acid-polyethyleneglycol (PEG, 5KDa)-maleimide (1 mg/ml in 50 mM sodium phosphate buffer, pH 7.3, added dropwise over 2 min). The mixture was incubated at room temperature for 1 h under stirring, and transferred to two silanized 50 ml polypropylene tubes and centrifuged (9000 x g, 45 min at 4°C). The supernatants were discarded; the pellets were resuspended with 5 mM sodium phosphate buffer, pH 7.3, and the resulting products were pooled and transferred to a 20 ml silanized beaker. The product was mixed with 10 ml of peptide Iso4 (0.160 mg/ml, by Ellman’s assay, in 5 mM sodium phosphate buffer, pH 7.3, added dropwise over 2 min, under stirring) and incubated for 2 h at room temperature. To saturate the gold nanorods we added 0.5% HSA (in 0.5 ml aliquots every 2 min, four times) and incubated for 10 min at room temperature under stirring. The product was then transferred to two 50 ml silanized polypropylene tubes and centrifuged as described above. Each pellet was resuspended in 0.05% HSA (40 ml) and centrifuged again (three rounds of centrifugation). The pellets were then resuspended in 8 ml of 0.05% HSA (final volume). The resulting product (called GNRs₈₀₀@PEG5K-Iso4) was dispensed in aliquots (0.5 ml) and stored at −80°C. Control nanoparticles bearing a cysteine in place of Iso4 (called GNRs₈₀₀@PEG5K-Cys) were prepared following the same procedure, except that 0.390 mg of cysteine was used in place of the peptide. Physicochemical characterization of the functionalized nanoparticles ‐29‐ Absorption spectra of bare- and functionalized-GNRs (hereinafter called uncoated and coated, respectively) were recorded using an UltroSpec 2100 spectrophotometer (Amersham Biosciences) and 1 cm path-length quartz cuvette. HSA (0.05% w/v) or 5 mM sodium citrate buffer, pH 6.0, respectively, were used as “blanks”. The concentration of coated-GNRs was calculated by interpolating the absorbance values at 820 nm on a calibration curve obtained using uncoated nanogold (stock solution: 4.3×10¹¹ nanoparticles (NPs)/ml, λ_max820 nm: ∼1.0 OD, 27 µg/ml). Transmission electron microscopy (TEM) analysis was performed using a TALOS L120C microscope (ThermoScientific) and undiluted samples. Morphometric analysis of GNRs was performed on TEM images using the ImageJ software. Table 1 summarizes the physicochemical characterization of GNRs. Table 1. Characterization of coated and uncoated gold nanorods (GNRs800) by ultraviolet, visible, and infrared spectroscopy (UV–IR), transmission electron microscopy (TEM), and ^5 ^1 integrin binding assay Nanogold UV–IR ^a TEM ^5 ^1 binding assay (Mean ^ SD) (Mean ^ SD) ^(Mean ^ SE) TSPR LSPR PW‐ A₅₁₀ Length Width EC₅₀ (nm) (nm) 85% /A₈₂₀ (nm) (nm) (NPs/ml) (nm) Uncoated GNRs₈₀₀ 509 818 83.3 0.28 43.9 10.2 NA^g ^1.1 ^ 2.3 ^1.3 ±0.01 ± 5.4^e ± 0.7^e (n=4) ^b (n=4)^b (n=4)^b (n=4)^b Uncoated GNRs₈₀₀ ^d _in 5% NaCl ^{0.517 >900} >100 0.59 ND ^f ND ND Coated GNRs GNRs₈₀₀@PEG5K‐Cys 511 827 99.8 0.33 41.0 12.9 >>10¹¹ ^h ^ 1.4 ^ 1.4 ± 10.4 ± 3.6 (n=2)^c (n=2)^c (n=100)^b (n=100)^b (n=3)^c GNRs₈₀₀@PEG5K‐Iso4 511 832 92.1 0.33 42.1 11.9 1.59x10¹⁰ ^ 1.0 ^ 2.0 ^ 3.4 ^ 0.02 ± 9.4 ± 3.3 ± 0.33 (n=3)^c (n=3)^c (n=3)^c (n=3)^c (n=100)^b (n=100)^b (n=3)^c GNRs₈₀₀@PEG5K‐Iso4 i_n 5% NaCl ^{512 832 94.3 0.33 ND ND ND} ‐30‐ a) TSPR, transverse surface plasmon resonance (peak λ_max); LSPR, longitudinal surface plasmon resonance (peak λ_max); PW-85%, peak-width at 85% of height; A₅₁₀/A₈₂₀: absorbance ratio at 510 and 820 nm. b) Number of independent analyses on the same batch. c) Number of independent preparations analyzed. d) Maximum wavelength that can be recorded by the UltroSpec spectrophotometer. e) As reported on the datasheet provided by the supplier (Nanocompoxis). f) ND, not done. g) NA, not applicable. h) Maximum concentration tested that gave no binding. . Stability of GNRs₈₀₀@PEG5K-Iso4 in synthetic urine GNRs₈₀₀@PEG5K-Iso4 (0.1 ml, about 8 units of optical density at 820 nm) was added to synthetic urine (1 ml) and the resulting mixture was analyzed by spectrophotometry. Synthetic urine containing 0.05% HSA (i.e., the diluent of nanogold) was used as “blank” reference. α5β1 integrin binding assay The binding properties of GNRs₈₀₀@PEG5K-Iso4 were investigated using a sandwich assay based on the use of α5β1-coated plates in the capture step and an anti-PEG monoclonal antibody (mAb) in the detection step, essentially as described [21]. Briefly various amounts of nanoparticles in 25 mM Tris-HCl buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, 1% w/v BSA (binding buffer), were added to microtiter plates coated with or without human recombinant α5β1 (1-2 µg/ml, 50 µl/well) and incubated for 1.5 h. After washing, plates were incubated with a rat anti-PEG monoclonal antibody in binding buffer containing 1% v/v NGS (5 µg/ml, 50 µl/well, 1.5 h), followed by a goat anti-rat HRP- labelled polyclonal antibody (1:2000, 50 µl/well, 1 h). Bound peroxidase was detected by adding the chromogenic substrate o-phenylenediamine. FACS analysis The expression of α5β1 on MB49-Luc cell surface was assessed by FACS analysis using an anti- α 5 integrin subunit mAb (clone HMα5-1) and an anti-

integrin subunit mAb (clone HMα5-1), as described previously [22]. Isotype-matched antibodies were used as negative controls. The binding of primary antibodies was detected using Alexa ‐31‐ Fluor 488-labeled goat anti-hamster secondary antibodies according to their animal species. The binding of GNR₈₀₀@PEG5K-Iso4 to MB49-Luc cells was assessed by FACS analysis as follows: MB49-Luc cells were detached with DPBS containing 5 mM EDTA, pH 8.0, washed with DPBS, and suspended in 25 mM Hepes buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, 1% w/v BSA, 2% v/v NGS (binding buffer-1) and GNRs₈₀₀@PEG5K-Iso4 or GNRs800@PEG5K- Cys (range 0-1x10¹¹ NPs/ml, 5x10⁵ cells/100 μl tube). After 1 h incubation on ice, the cells were washed with binding buffer-1 (without BSA and NGS) and resuspended in binding buffer-1 containing the anti-PEG mAb 26A04 (1 µg/ml, 0.5 h on ice) followed by a FITC-labelled mouse anti-rat mAb MRM-47 (2.5 µg/ml, 0.5 h on ice). After washing, with DPBS/Ca/Mg, cells were fixed with 4% formaldehyde, and bound fluorescence was detected using a CytoFLEX S cytofluorimeter (Beckman Coulter). Orthotopic bladder cancer model Studies in animal models were approved by the Ospedale San Raffaele Animal Care Use Committee and by the Minister of Health (approval number 942). The tumor- binding properties of GNRs₈₀₀@PEG5K-Iso4 were investigated using an orthotopic mouse model of bladder cancer based on intravesical instillation of MB49-Luc cells. Briefly, female albino C57BL/6J mice (9 weeks old, weighing about 20 g, Charles River Laboratories, Italy) were anesthetized and intravesically instilled with MB49-Luc cells (10⁵ cells/100 µl in DPBS) using 24-gauge catheter. Tumor engraftment was assessed 4 days later by measuring the tumor bioluminescence using an IVIS-Spectrum imaging system (PerkinElmer). The tumor growth was monitored by ultrasound (US) imaging using a Vevo 3100 LAZR-X system (Fujifilm, Visualsonics Inc). After 8 days from tumor cells implantation mice were subjected to US and photoacoustic (PA) (PAUS) imaging studies. All imaging experiments on mice were conducted under gaseous anesthesia (isoflurane/air 4% for induction and 1.5% thereafter). In vitro and in vivo PAUS imaging High-resolution PAUS analysis was performed using a Vevo 3100 LAZR-X system equipped with the MX550D transducer (256 elements linear array; 40 MHz center ‐32‐ frequency, 40 μm axial resolution) integrated with 2 optical fibers (pulsed laser, 680– 970 nm) and connected to a stepper motor. In vitro PAUS imaging of GNRs₈₀₀@PEG5K-Iso4 was performed as follows: various amounts of NPs (0-32 nmol of Au, 30 µl in DPBS/Ca/Mg) were mixed with 1% w/v agar (30 µl); the mixture was then placed on Parafilm ^{^} M (Sigma) and left to solidify. The resulting product (called “agar drop”) was then placed on an ultrasound gel pad (Aquaflex, Parker), embedded with a slime gel (Barrel-O-Slime^TM) and covered with an ultrasound transmission gel (Aquasonic 100, Parker). PAUS imaging was then performed using light attenuators (consisting of 1% w/v agar and 0.6% v/v intralipids) placed in contact with the optical fibers to prevent the reshaping of gold nanorods. Ultrasound imaging of the agar drop was acquired in brightness (B) mode in the axial orientation (2D Power, 100% and 2D Gain, 13 dB). PA imagining was performed in PA Mode Spectro (acquisition range; 680-970 nm with a step size 5 nm; PA Power, 100%; PA gain 44 dB), and in PA Mode 3D multi-wavelengths (3D step size and 200 nm). The signal corresponding to GNRs₈₀₀@PEG5K-Iso4 was identified by spectral unmixing using the PA signal derived from agar drops lacking nanoparticles. In vivo PAUS imaging was performed as follows: GNRs₈₀₀@PEG5K-Iso4 (26 nmol Au in 100 µl DPBS/Ca/Mg) were intravesically instilled into the bladder of mice via a catheter and incubated for 15 min (every 5 min the bladder content was mixed with 3 cycles of aspiration/injection with a syringe connected to the catheter). The bladder content was then aspirated, and the excess of unbound NPs was removed by washing the bladder twice with DPBS/Ca/Mg. US imaging of the bladder was performed using the transducer placed perpendicular to the mouse abdomen (B-mode: 2D Power, 50% and 2D Gain, 23 dB). PA imaging of the bladder was performed essentially as described above, except that PA gain was set to 39 dB. PA analysis was performed by spectral unmixing using the spectral reference curves obtained from the tissue components (i.e., melanin and deoxygenated/oxygenated blood) and the GNRs₈₀₀@PEG5K-Iso4 spectral curve (generated as described above), using the build-in VevoLab 5.6.1 software. Characterization of peptide Iso4 ‐33‐ Mass spectrometry analysis of Iso4 showed a molecular weight consistent with the expected value as represented in Table 2. Table 2. Biochemical characterization of peptide Iso4 by electrospray ionization mass spectrometry (ESI-MS) analysis, Ellman’s assay, RP-HPLC, NMR and integrin binding. Peptide Molecular Peptide Peptide D‐phg Binding affinity for integrin code mass concentration purity purity by by by by by competitive binding assay^c ESI‐MS Ellman’s assay RP‐HPLC NMR

I^{so4 622.24 a} _{8.380 ^ 0.156} ^b ^> 95 _^ 70 ¹⁵ 46 51 1121 1493 a) Expected (Da, MH⁺): 622.32. b) The lyophilized peptide was resuspended at 10 mg/ml, based on the gross weight. Mean ^SD of two independent quantification. c) Ki, inhibitory constant values. Reverse-phase HPLC analysis showed that its purity was >95% (not shown). NMR analysis revealed the presence of D-phenylglycine (D-phg) (70%) and L-phenylglycine (L-Phg) (30%), as judged from the peak height of the resonances corresponding to the HαD and HαL of D-Phg and L-Phg, respectively (Fig.1 and Table 2). Preparation and characterization of GNRs₈₀₀@PEG5K-Iso4 GNRs₈₀₀@PEG5K-Iso4 were prepared by a two-step procedure. The first step included GNRs₈₀₀ activation with lipoic acid (LA)-5KDa polyethylene glycol (PEG)-maleimide (MAL) (LA-PEG-MAL, see Fig.2B), a heterobifunctional crosslinking reagent. The LA moiety of this reagent can react with the nanogold surface forming dative bonds. The second step included the GNRs functionalization with Iso4 via reaction of peptide sulfhydryl group with MAL and formation of a thioether bond (Fig. 2B-C). Optimization studies showed that 90-120 µg of LA-PEG-MAL per ml of nanogolds (1 OD_800nm) was sufficient to protect nanogold from peptide-induced aggregation. Thus, a larger batch of GNRs₈₀₀@PEG5K-Iso4 was prepared using 80 ml of GNRs₈₀₀, 8 mg of LA-PEG-MAL, and 8 mg of Iso4 peptide. In parallel, control nanoparticles bearing cysteine in place of Iso4 were also prepared (GNRs₈₀₀@PEG5K-Cys). ‐34‐ UV-IR spectrophotometric analysis of both products showed absorption spectra similar to that of uncoated GNRs₈₀₀, indicating that both products contained low or undetectable amounts of aggregates (Figure 2D and Table 1). Morphometric analysis of GNRs₈₀₀@PEG5K-Iso4 and GNRs₈₀₀@PEG5K-Cys by TEM revealed gold nanorods with similar longitudinal and transversal length of 42 nm and 12 nm, respectively, with an average aspect ratio (length/width) of about 3.53 (Fig.2E and Table 1). Stability of GNRs₈₀₀@PEG5K-Iso4 To assess the stability of GNRs₈₀₀@PEG5K-Iso4, this product was frozen at -80 °C or spiked with a large amount of sodium chloride (5% final concentration), i.e., two treatments known to induce nanoparticle aggregation. The UV-IR spectrophotometric analysis showed that these treatments did not change the absorption spectrum of GNRs₈₀₀@PEG5K-Iso4 (Fig. 2F-G), indicating that the nanoparticles were resistant to freezing- or salt-induced aggregation. Notably, incubation of GNRs₈₀₀@PEG5K-Iso4 in ~90% synthetic urine did not cause significant change in its absorption spectrum, suggesting that this product is stable also in urine (Fig.2H). In vitro photoacoustic properties of GNRs₈₀₀@PEG5K-Iso4 The photoacoustic (PA) properties of the GNRs₈₀₀@PEG5K-Iso4 were investigated in vitro using agar drops containing GNRs₈₀₀@PEG5K-Iso4 (range 0-30 nmol of Au) embedded in a slime gel (as support). Using a Vevo 3100 LAZR-X photoacoustic system, a wide PA peak with a maximum signal at ~ 820-840 nm, corresponding to GNRs, was recorded in the region 680-970 nm (Figure 3), data suggesting that GNRs₈₀₀@PEG5K-Iso4 is endowed of PA properties. The resulting spectrum was used as reference for the next in vivo experiment. GNRs₈₀₀@PEG5K-Iso4 binds to recombinant human ^5 ^1 The capability of GNRs₈₀₀@PEG5K-Iso4 and GNRs₈₀₀@PEG5K-Cys to recognize recombinant α5β1 was then investigated using α5β1-coated plates. The binding of nanoparticles to microtiter plates was detected with an anti-PEG mAb. The results showed that GNRs₈₀₀@PEG5K-Iso4, but not GNRs₈₀₀@PEG5K-Cys, could bind to α5β1 in a dose-dependent manner, with an effective concentration 50 (EC₅₀) of (1.59 ± 0.33) ‐35‐ x 10¹⁰ NPs/ml (Fig. 4A). Notably, the binding of GNRs₈₀₀@PEG5K-Iso4 to α5β1 was not affected by one cycle of freezing and thawing (Fig.4B). GNRs₈₀₀@PEG5K-Iso4 binds to MB49-Luc bladder cancer cells in vitro To assess the capability of GNRs₈₀₀@PEG5K-Iso4 of recognizing bladder cancer cells we then analyzed the binding of these nanoparticles to MB49-Luc cells, a murine bladder cancer cell line that expresses α5β1 (Fig. 5 and Table 3). Flow cytometry analysis of MB49-Luc cell suspensions, pre-incubated with various amount of GNRs₈₀₀@PEG5K-Iso4, showed that these cells were recognized by nanoparticles in a dose-dependent manner (Fig. 6). In contrast, little or no binding at all was observed with GNRs₈₀₀@PEG5K-Cys, suggesting that the binding of GNRs₈₀₀@PEG5K-Iso4 was mediated by Iso4. TEM analysis of adherent cells pre-incubated with 1x10¹¹ NPs/ml showed that GNRs₈₀₀@PEG5K-Iso4, but not GNRs₈₀₀@PEG5K-Cys could bind to the MB49-Luc cell surface (Fig. 7). This result suggests that GNRs₈₀₀@PEG5K-Iso4 can recognize the surface of bladder cancer cells, likely by binding to α5β1. Table 3: Expression of cell surface integrins on murine MB49-Luc bladder carcinoma cells as determined by FACS analysis. Anti-integrin mAb Binding of anti-integrin mAb to MB49-Luc cells Antigen Clone Host ^a Isotype Species reactivity ^b n ^c Fold increase ^d ^5 subunit HM ^5- ^Ha _IgG ^{M, R} _{2 3.79 ^ 0.18} _{^1 subunit HM} 1 _^1-1 ^{Ha IgG M, R 2} _{8.02 ^ 1.04} _{^3 subunit HM ^3-1} ^{Ha IgG M, R 2} _{1.02 ^ 0.01} _{^5 subunit} ^{KN52 M IgG} ₁ ^{Hu, M, R, Ha 2} _{1.04 ^ 0.28} _{^v subunit} ^{RMV-7 R IgG} ₁ ^{M 1 3.48} ^_{v ^6} ^{10D5 M IgG} _2a ^{Hu, M, R 2} _{1.61 ^ 0.78} a) R, rat; M, mouse; Ha, Armenian hamster; Hu, human. b) According to the technical data sheet or published data. c) n, number of independent experiments, each in duplicate. d) Fold increase corresponds to the ratio of the mean fluorescence intensity of a given anti-integrin antibody over the mean fluorescence intensity of an isotype control matched antibody (mean ± SEM). e) Na, not applicable. GNRs₈₀₀@PEG5K-Iso4 binds to orthotopic MB49-Luc bladder tumors in vivo ‐36‐ The ability of GNRs₈₀₀@PEG5K-Iso4 to bind bladder cancer cells in vivo was investigated using an orthotopic syngeneic model of bladder cancer based on murine MB49-Luc cells implanted intravesically into mice. In this model, neoplastic cells, but not non-neoplastic epithelial cells of the bladder, express α5β1 as determined by immunohistochemical analysis (Fig. 8). To assess whether GNRs₈₀₀@PEG5K-Iso4 can accumulate on tumor lesions inventors performed photoacoustic imaging studies (PAUS) imaging studies on an MB49-Luc tumor-bearing mouse before and after intravesical instillation of the NPs. The results showed that before administration of GNRs₈₀₀@PEG5K-Iso4 no PA signals were detected on tumor and inner bladder surface (Fig.9A, left and central panel), while some scattered PA signals were found outside the bladder. The PA spectrum of these signals showed a pattern different from that of GNRs₈₀₀@PEG5K-Iso4 (Fig. 9B, upper panel), indicating that they were not related to gold nanoparticles. In contrast, after intravesical instillation, incubation and washing of GNRs₈₀₀@PEG5K-Iso4, additional PA signals were detected on the apical part of the tumor (i.e., on the luminal side of the bladder) (Fig. 9A, right panel). Notably, the PA spectrum of the signal associated to the tumor showed a pattern very similar to the that expected for gold nanoparticles (Fig. 9B, lower panel), suggesting that, in this case, the signal was related to GNRs₈₀₀@PEG5K-Iso4 accumulation on the cancer lesion. Inventors next investigated the specificity and capability of GNRs₈₀₀@PEG5K-Iso4 to detect tumor lesion of different size. To this aim GNRs₈₀₀@PEG5K-Iso4 was administered in 3 additional tumor-bearing mice and in 2 healthy mice. The result showed that GNRs₈₀₀@PEG5K-Iso4 could recognize tumor lesions in all tumor-bearing mice, including small tumor lesions (diameter ~0.5 mm), but not the adjacent normal bladder tissue (Fig.10 and 11, upper panels). No binding of GNRs₈₀₀@PEG5K-Iso4 to the bladder of healthy mice was observed Fig.10 and 11, lower panels). As expected, GNRs₈₀₀@PEG5K-Cys (a non-targeted nanoformulation having a cysteine residue in place of the Iso4 peptide) failed to detect tumor lesions, suggesting that peptide Iso4 is crucial for tumor recognition by nanoparticles (Fig.12). Taken together these results ‐37‐ suggest that GNRs₈₀₀@PEG5K-Iso4 can selectively recognize bladder cancer cells in vivo, thereby enabling the PA imaging of small cancer lesions. Role of α5β1 integrin as a target receptor of GNRs₈₀₀@PEG5K-Iso4 in MB49-Luc bladder tumors To investigate the role of α5β1 on the binding of GNRs₈₀₀@PEG5K-Iso4, mice bearing orthotopic MB49-Luc tumors were intravesical injected with an α5β1-blocking antibody, or with an isotype-matched control antibody, followed, 15 min later by GNRs₈₀₀@PEG5K-Iso4. The bladders were washed again and the uptake of GNRs₈₀₀@PEG5K-Iso4 was quantified by photoacoustic imaging. A reduced photoacoustic signal was observed in mice treated with the α5β1-blocking antibody, suggesting that α5β1 is an important receptor for GNRs₈₀₀@PEG5K-Iso4 (Fig.13). DISCUSSION The present invention demonstrates that pegylated nanostructures, such as the GNRs₈₀₀@PEG5K-Iso4, represent a stable and robust nanosystem for photoacoustic imaging of small α5β1-positive bladder cancer lesions. GNRs₈₀₀@PEG5K-Iso4 consists of PEGylated gold nanorods that absorb light in the near-infrared region of the electromagnetic spectrum (peak maximum at ^ ^820 nm), functionalized with the cyclic peptide [CphgisoDGRG] (Iso4), a ligand of α5β1 integrin. Notably, these nanoparticles can be prepared by a simple two-step procedure. In the first step, GNRs are activated with a heterobifunctional reagent consisting of a) lipoic acid (which can form stable sulfur-gold bonds with the nanoparticle surface), b) a PEG 5kDa linker, and c) a maleimide group (which can react with the thiol group of Iso4). In the second step, maleimide activated-GNRs are coupled to Iso4 via its thiol group (see Fig. 2). The results of biochemical and biological studies on these nanoparticles show that Iso4 preserves its functional properties after coupling to GNRs, as suggested by the observation that GNRs₈₀₀@PEG5K-Iso4 could specifically bind purified α5β1 and α5β1-positive bladder cancer cells (MB49-Luc). Furthermore, the results of stability studies show that GNRs₈₀₀@PEG5K-Iso4 is stable and does not aggregate in urine or in 5% sodium chloride, and even after one freeze/thaw cycle. ‐38‐ The results of in vivo photoacoustic imaging studies, performed in mice bearing orthotopic MB49-Luc bladder tumors, show that GNRs₈₀₀@PEG5K-Iso4 can bind the surface of well-established cancer lesions (α5β1-positive, detectable by standard US echography), but not to the bladder of heathy mice (α5β1-negative). Notably, small spots of photoacoustic signals were detected also at sites in which standard US echography failed to detect tumor lesions: considering that no uptake of nanoparticles was observed by the urothelium in healthy mice, these sites likely correspond to small bladder cancer lesions <0.5 mm, undetectable by standard US echography. At variance, the photoacoustic signals observed in tissues around the bladder of both tumor-bearing and normal mice were likely related to artifacts dependent on the acoustic inhomogeneity of the tissue, which may cause signal reflection inside the imaging plane (26-28). This hypothesis is supported by the observation that a) these signals were present also before nanoparticle administration, and b) these signals exhibited a photoacoustic spectrum completely different from that of GNRs₈₀₀@PEG5K-Iso4. The tumor-binding properties of GNRs₈₀₀@PEG5K-Iso4 depend on a targeting mechanism mediated by α5β1-integrin expressed by the tumor cells, as suggested by the observation that a) no binding occurred to α5β1-negative healthy bladder and b) nanoparticle accumulation on tumor lesions was partially inhibited by pre- administration of a neutralizing anti-α5β1 monoclonal antibody. However, because the inhibition was not complete, we cannot exclude the possibility that other receptors are involved. Notably, immunohistochemical studies performed by other investigators have shown that healthy bladder tissues of mice and humans can express high levels of αvβ6 and αvβ8 (28, 29), i.e., two potential additional receptors of peptide Iso4 (Ki for α5β1, αvβ6 and αvβ8: 15, 46 and 51 nm, respectively). However, no binding of GNRs₈₀₀@PEG5K-Iso4 to the healthy mouse bladder was detected, suggesting that these receptors, at least those exposed to the luminal side, are somehow not "accessible" to GNRs₈₀₀@PEG5K-Iso4, possibly because they are already engaged by other ECM ligands or because they have a conformation corresponding to the inactive “resting” state of the integrin (31). The photoacoustic signal of GNRs₈₀₀@PEG5K-Iso4, detected ‐39‐ in MB49-Luc bladder cancer model, was characterized by a "patchy pattern" that was particularly evident in reconstructed 3D photoacoustic images. This peculiar pattern may be related to a) heterogeneous expression of α5β1 molecules characterized by differential accessibility of the ligand (32), and b) presence of discontinuous urothelial cell layers (α5β1-negative) above the tumor, which may prevent or reduce the binding of nanoparticles to the underlying tumor cells (α5β1-positive). At this regard, it is important to highlight the fact that the immunohistochemical characterization of in situ carcinomas-tissue sections obtained from patients showed that α5β1-positive cells are exposed to the bladder lumen, which are, therefore, accessible to intravesically administered nanoparticles (15). Intravesical administration of NPs may represent an important advantage comparted to intravenous administration as local delivery may reduce potential systemic toxicological effects. In summary, the results of PAI studies performed in the MB49-Luc model show that GNRs₈₀₀@PEG5K-Iso4 can specifically accumulate on α5β1-positive tumors, but not on the normal urothelium. GNRs₈₀₀@PEG5K-Iso4 may have different applications in bladder cancer patients, such as diagnostic imaging and image-guided surgery of small lesions. GNRs₈₀₀@PEG5K-Iso4 is in principle also applicable for photothermal therapy of bladder cancer. In addition, considering that α5β1 is expressed also by other tumor types, e.g. gastric tumors, endometrial cancer (33, 34), bone metastases of breast cancer, (35) and by the neovasculature of several solid tumors (31), the present results may stimulate other studies aimed at assessing the utility of GNRs₈₀₀@PEG5K-Iso4 for imaging and therapy of other tumors. 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Claims

CLAIMS 1. A photoacoustic agent chosen among the group consisting of a metal-based nanoparticle made of gold, silver, or hybrid gold/silver, an organic photoacoustic dye, cyanine dyes, phthalein and xanthene dyes, squaraine and croconaine dyes, tetrapyrrole, BODIPY dyes, curcumin dyes, and IRDye800 linked to - a ligand of the integrin family receptors, preferably a peptide containing an integrin binding motif, and antibody or part of an antibody, a peptidomimetic or an aptamer, - via a crosslinker selected from the group consisting of a crosslinker bearing amino and sulfhydryl reactive groups, bearing amino and azide/alkyne reactive groups, bearing lipoamide/lipoic acid (LA) or sulfhydryl or disulphide containing compounds.

2. The photoacoustic agent according to claim 1, wherein the crosslinker is selected among SMCC, sulfoSMCC, MAL-PEG-NHS ester, MAL-PEG-TFP ester, propargyl-PEG-NHS ester, azido-PEG-TFP ester, azido-PEG-NHS ester, LA-PEG- MAL, LA-PEG-TFP ester, LA-PEG-biotin, LA-PEG-acid, LA-PEG-NHS ester, LA-PEG, LA-PEG-amine, LA-PEG-azide, Thiol-PEG, Thiol-PEG-acid, Thiol-PEG-amine, Thiol- PEG-azide, propargyl-PEG-MAL, azido-PEG-MAL wherein the polyethylene glycol chain (PEG) has a molecular weight comprised between 0.05-40 KDa, preferably lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa.

3. The photoacoustic agent according to any one of previous claims, wherein the photoacoustic agent is a metal-based nanoparticle, preferably a gold nanoparticle.

4. The photoacoustic agent according to claim 3 wherein the gold nanoparticle has a shape selected from the group of sphere, rod, star, cage, prism, shell, hallow shell, wire, plates, preferably nanorod with size ranging from 10 to 200 nm in length, and 2 to 50 nm in width, more preferably ranging from 10 to 100 nm in length and from 5 to ‐45‐ 25 nm in width and the preferred aspect ratio (length/width) ranges between 1.2 and 15, more preferably between from 3 and 7.

5. The photoacoustic agent according to any one of previous claims, wherein the ligand of the integrin family receptors is a peptide comprising the RGD or isoDGR motif.

6. The photoacoustic agent according to claim 5 wherein the ligand of the integrin family receptors is a peptide selected from: [XGisoDGRG] of SEQ ID No:1 [XisoDGRGG] of SEQ ID No:2 [XphgisoDGRG] of SEQ ID No:3 [XGisoDGRphg] of SEQ ID No:4 [XisoDGRphgG] of SEQ ID No:5 [XisoDGRGphg] of SEQ ID No:6 XFETLRGDERILSILRHQNLLKELQD of SEQ ID No:8 XFETLRGDLRILSILRHQNLLKEL of SEQ ID No:9 XFETLRGDLRILSILRX₁QNLX₂KELQD of SEQ ID No:10 wherein “X” is selected from cysteine, lysine or a non-natural amino acid comprising an alkyne or azide group preferably selected from propargylglycine or azidolysine; X₁ and X₂ are respectively propargyl glycine and azidolysine joined via a triazole bridge.

7. The photoacoustic agent according to any one of previous claims wherein the ligand of the integrin family receptors is cyclic head-to-tail peptide [CphgisoDGRG] of SEQ ID No:7.

8. The photoacoustic agent according to any of previous claims wherein - the photoacoustic agent are gold nanoparticles, preferably with shape of nanorods ‐46‐ - the crosslinker is lipoamide/lipoic acid-PEG-MAL and the PEG has a molecular weight of 5 KDa - and the ligand of the integrin family receptors is the cyclic head-to-tail [CphgisoDGRG]peptide of SEQ ID NO: 7.

9. The photoacoustic agent according to claim 8 wherein the cross-linker is bound to the gold nanoparticle via the lipoic acid and the ligand is covalently bound to the maleimide mojety of the cross-linker.

10. A composition comprising the photoacoustic agent according to any of the previous claims and at least one of the following solvents: water, physiological solution, Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate- buffered saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer each containing divalent metal ions such as Ca²⁺and Mg²⁺.

11. The composition according to claim 10 additionally comprising one or more antitumor agents preferably chosen among a chemotherapeutic agent, an immunomodulator, an immune cell.

12. A kit comprising single use vials containing the photoacoustic agent according to any one of claims 1-9, a solvent, preferably water, or physiological solution, or Dulbecco's Modified Eagle Medium (DMEM), or a buffer such as Dulbecco's phosphate-buffered saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer each containing divalent metal ions such as Ca²⁺ and Mg²⁺, for resuspending the photoacoustic agent, optionally syringes and instruction for use.

13. The photoacoustic agent according to any one of claims 1-9 or the composition according to claim 10 or 11 or the kit according to claim 12 for use in a method of diagnosis and/or treatment in vivo. ‐47‐

14. The photoacoustic agent or compositions or kit according to claim 13 for use in the in vivo diagnosis and/or treatment of solid tumors.

15. The photoacoustic agent according to any one of claims 13 and 14 for use in photoacoustic imaging and/or for use as a carrier of drugs preferably chosen among a chemotherapeutic agent, an immunomodulator, an immune cell.

16. The photoacoustic agent or compositions or kit according to any one of claims 14 and 15 wherein the tumor is selected among urothelial, bladder, gastroesophageal, colorectal, pancreatic, ovarian, lung, cervix, breast and renal cancer, brain tumors and hepatocellular carcinoma.

17. The photoacoustic agent or compositions or kit according to claims 13 and 14 for use in the photothermal therapy of solid tumors, preferentially bladder cancer.

18. Use of the photoacoustic agent according to any one of claims 1-9 for tissue imaging ex vivo.

19. A method for ultrasound and photoacoustic imaging ex vivo which comprises at least the following steps: a) applying the photoacoustic agent according to any one of claims 1-9 or the composition according to claim 10 to the target tissue to be imaged b) photoacoustic visualization of the target tissue, and c) evaluating the visualized target tissue. ‐48‐