Biomaterial-coated nanostructures for photoacoustic imaging and photothermal therapy of tumor lesions

Abstract

Metal-based nanoparticles coated with a polymer functionalized with thiol and -NH groups, said polymer being preferably selected from among thiolated chitosan, thiolated and aminated alginate, thiolated and aminated hyaluronic acid, or proteins, preferably selected from the group consisting of albumin and gelatin, or synthetic thiolated and aminated polymers, preferably α-thio-ω-amino polyethylene glycol, said groups being linked to ligands of integrin family receptors, preferably peptides containing integrin binding motifs, and antibodies or antibody parts, peptidomimetics or aptamers via heterobifunctional crosslinkers, said crosslinkers preferably containing N-hydrocarbonyl groups. Metal-based nanoparticles bearing a functional group capable of binding to an amino group, selected from the group consisting of N-hydroxysuccinimidyl ester groups (NHS esters), isocyanate groups (-NCO), isothiocyanate groups (-NCS), sulfo-N-hydroxysuccinimidyl ester groups (sulfo-NHS esters), or carboxylic acid groups linked by activation with a carbodiimide coupling agent, and / or a functional group capable of binding to a thiol group, preferably selected from the group consisting of a maleimide group, a terminal vinyl group, or a terminal alkyne group; and / or a functional group capable of binding to an alkyne, such as an azide; and / or a functional group capable of binding to an azide, such as an alkyne.

Description

[Technical Field]

[0001] The present invention relates to a technological platform for diagnostic, therapeutic or diagnostic-therapeutic applications.The present invention relates to metallic nanostructures carrying functionalizing agents, preferably ligands capable of recognizing tumor- or inflammation-associated components, associated to the nanoparticles via heterobifunctional crosslinkers.

[0002] In particular, the present invention provides metal-based nanoparticles comprising: - coated with a polymer functionalized with thiol and -NH groups, said polymer being preferably selected from among thiolated chitosan, thiolated and aminated alginic acid, thiolated and aminated hyaluronic acid, or a protein, preferably selected from the group consisting of albumin and gelatin, or a synthetic thiolated and aminated polymer, preferably α-thio-ω-amino polyethylene glycol; - said group is linked via a heterobifunctional crosslinker to a ligand of an integrin family receptor, preferably a peptide containing an integrin-binding motif, and to an antibody or antibody part, peptidomimetic or aptamer, - the crosslinking agent preferably comprises a functional group capable of binding to an amino group, selected from the group consisting of an N-hydroxysuccinimidyl ester group (NHS ester), an isocyanate group (-NCO), an isothiocyanate group (-NCS), a sulfo-N-hydroxysuccinimidyl ester group (sulfo-NHS ester) or a carboxylic acid group which is linked by activation with a carbodiimide coupling agent; and / or functional groups capable of binding to thiol groups, preferably selected from the group consisting of maleimide groups, terminal vinyl groups or terminal alkyne groups; and / or functional groups capable of binding to alkynes, such as azides; and / or metal-based nanoparticles carrying functional groups capable of binding to azides, such as alkynes. Regarding. [Background technology]

[0003] Early detection and treatment of tumors are fundamental for improving oncological outcomes, survival, and patient quality of life. Bladder cancer (BC) limited to the mucosa and BC that invade the lamina propria are classified as stage Ta and stage T1, respectively, according to the tumor, node, and metastasis (TNM) classification system [1]. High-grade intraepithelial tumors limited to the mucosa are classified as carcinoma in situ (CIS). Approximately 75% of BC patients present with disease limited to the mucosa (stage Ta, CIS) or the submucosa (stage T1) [2]. All of these tumors can be treated with transurethral resection of the bladder (TURB), which is ultimately combined with intravesical instillation and classified as non-muscle-invasive bladder cancer (NMIBC) for curative purposes.

[0004] Bladder CIS is characterized by a small number of high-grade neoplastic cells, forming reddish areas indistinguishable from inflammation and having a flat appearance in the urothelium. Without biopsy, it can be missed during cystoscopy or mistaken for an inflammatory lesion. Management of patients with bladder CIS remains a challenge in the field of oncourological medicine [3, 4].

[0005] Several imaging techniques have been used to diagnose bladder cancer, including computed tomography urography (CT urography), intravenous urography (IVU), ultrasound (US), multiparametric magnetic resonance imaging (mpMRI), and cystoscopy. However, the main limitation of imaging remains tumor size and the detectable size limit of each technique. US and CT have very low detection rates for bladder cancers smaller than 5 mm [5]. Cystoscopy remains the gold standard diagnostic method for patients with suspected bladder cancer [6]. In fact, even cystoscopy, including photodynamic diagnosis using violet light after intravesical instillation of 5-ALA or hexaminolevulinic acid, has limited diagnostic value for CIS. In fact, cystoscopy and TURB usually require multiple biopsies from the suspected urothelium to detect and diagnose CIS from surgical tissue specimens [3]. Nevertheless, 40% of patients still have residual high-grade disease after initial TURB.[7] Due to these technical limitations, patients with bladder CIS frequently relapse after initial diagnosis, resulting in frequent and endless follow-up with ineffective treatments, resulting in a reduced quality of life and the highest cost per patient of all cancers.[8]

[0006] To overcome the limitations of current clinical imaging methods for bladder CIS, there is a need in the art to develop approaches and technologies for the noninvasive early diagnosis of in vivo orthotopic bladder cancer by utilizing imaging modalities based on the photoacoustic (PA) imaging (PAI) approach. PAI is a hybrid imaging modality that combines the high contrast of optical absorption generated by chromophores after nonionizing pulsed laser irradiation with the high spatial resolution of US. Because acoustic waves generally exhibit less scattering and tissue attenuation than light, PAI can provide higher-resolution images than conventional US and achieve deeper penetration than purely optical imaging systems [9, 10]. PAI also enables the collection of functional and molecular information in real time by using nonionizing radiation to reach clinically relevant depths

[11] .

[0007] Pulsed laser light in the near-infrared (NIR) spectral region has been used to target endogenous contrast agents, such as melanin, oxyhemoglobin, deoxyhemoglobin, lipids, collagen, and water

[12] , and to target PAI in melanoma

[13] , tumor microenvironment

[14] , atherosclerotic plaque

[15] , and lesions

[16] , respectively. Exogenous contrast agents can also be used to enhance the sensitivity and spectroscopic specificity of PA signals. The use of targeted contrast agents can also extend the application of PAI to molecular imaging [17, 18]. Among the various contrast agents developed to date, gold nanoparticles have attracted particular attention due to their versatility, unique optical and physicochemical properties, relative inertness, and success in many biomedical applications. Gold nanorods (GNRs), in particular, exhibit the highest extinction coefficient and high PA conversion efficiency in the near-infrared region. Furthermore, tailoring the shape of GNRs allows for the selection of the optimal wavelength of optical stimulation, thereby enabling the use of these nanoparticles for desired PAI applications

[17] .

[0008] Integrins are involved in almost every stage of cancer progression, from primary tumor formation to late-stage metastasis, making integrins a potential target for human bladder cancer

[19] . Among the various integrins involved in cancer progression

[20] , we investigated the expression of α5β1 integrin. Overexpression of α5β1 integrin has been reported as a marker of high-grade bladder cancer [21, 22] and poor prognosis in BC patients [23, 24]. Therefore, a new technological platform for in situ diagnosis of non-muscle-invasive bladder cancer is needed. Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to overcome the drawbacks of the known art.

[0010] In particular, it is an object of the present invention to solve the problems and limitations presented by the limitations of imaging methods currently used clinically for the detection of solid tumors, preferably bladder cancer.

[0011] In particular, the object of the present invention are metal-based nanoparticles functionalized with ligands capable of recognizing tumor- or inflammation-associated components. [Means for solving the problem]

[0012] Various embodiments of the invention are set forth in the following detailed description, and preferred embodiments are set forth in the claims, which form an integral part of this specification.

[0013] In one embodiment, the present invention provides a metal-based nanoparticle comprising: - coated with a polymer functionalized with thiol and -NH groups, said polymer being preferably selected from among thiolated chitosan, thiolated and aminated alginic acid, thiolated and aminated hyaluronic acid, or proteins, preferably selected from the group consisting of albumin and gelatin, or synthetic thiolated and aminated polymers, preferably α-thio-ω-amino polyethylene glycol; - said thiol group and -NH group are linked via a heterobifunctional crosslinker to a ligand of an integrin family receptor, preferably a peptide containing an integrin-binding motif, and to an antibody or antibody part, peptidomimetic or aptamer, - the crosslinking agent preferably comprises a functional group capable of binding to an amino group, selected from the group consisting of an N-hydroxysuccinimidyl ester group (NHS ester), an isocyanate group (-NCO), an isothiocyanate group (-NCS), a sulfo-N-hydroxysuccinimidyl ester group (sulfo-NHS ester) or a carboxylic acid group which is linked by activation with a carbodiimide coupling agent; and / or functional groups capable of binding to thiol groups, preferably selected from the group consisting of maleimide groups, terminal vinyl groups or terminal alkyne groups; and / or functional groups capable of binding to alkynes, such as azides; and / or metal-based nanoparticles carrying functional groups capable of binding to azides, such as alkynes. Regarding.

[0014] In a preferred embodiment, the nanoparticles are metal-based, gold, silver, or hybrid gold / silver nanoparticles, preferably gold, and the nanoparticles have photoacoustic properties, and in a further preferred embodiment, the nanoparticles are selected from among nanospheres, nanorods, nanostars, nanocages, nanoprisms, or nanoshells, or nanowires, nanoplates, hollow shells, preferably nanorods with a length in the range of 10-200 nm and a width in the range of 2-50 nm, more preferably a length in the range of 20-100 nm and a width in the range of 10-25 nm, and a preferred aspect ratio in the range of 1.2-15, more preferably a range of 3-7.

[0015] Preferably, the functionalized polymer is a thiolated chitosan having an average molecular weight preferably in the range of 0.5 kD to 1000 kDa, more preferably in the range of 50 kDa to 200 kDa, and an average degree of deacetylation in the range of 75% to 100%, more preferably in the range of 85% to 95%.

[0016] Preferably, in the above metal-based nanoparticles, the ligand for the integrin family receptor is [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, [CphgisoDGRG] peptide of SEQ ID NO: 7, XFETLRGDERILSILRHQNLLKELQD of SEQ ID NO: 8, XFETLRGDLRILSILRHQNLLKEL of SEQ ID NO: 9, SEQ ID NO: 10 XFETLRGDLRILSILRX1QNLX2KELQD is a peptide selected from wherein "X" is selected from cysteine, lysine, or an unnatural amino acid containing an alkyne or azide group, preferably selected from propargylglycine or azidolysine; X1 and X2 are propargylglycine and azidolysine, respectively, linked via a triazole bridge.

[0017] In a further preferred embodiment, the ligand for the integrin family receptor is a peptide containing an RGD or isoDGR motif, more preferably the peptide is the cyclic head-to-tail [CphgisoDGRG] peptide of SEQ ID NO: 7.

[0018] More preferably, for metal-based nanoparticles, the crosslinker is maleimide-PEG 12 -NHS ester.

[0019] In a preferred embodiment, the present invention comprises: - The nanoparticles are gold nanorods, - the functionalized polymer is thiolated chitosan, - the peptide is the [CphgisoDGRG] peptide of SEQ ID NO: 7, - Crosslinker is maleimide-PEG 12 -NHS ester, Metal-based nanoparticles are provided.

[0020] A further object of the present invention is a composition comprising metal-based nanoparticles as defined above and at least one of the following solvents: water; physiological solution; Dulbecco's Modified Eagle's Medium (DMEM); Dulbecco's Phosphate Buffered Saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer, each containing divalent metal ions such as Ca2+, Mg2+; preferably, said composition also comprises at least one or more anti-tumor agents, preferably selected from among chemotherapeutic agents, immunomodulatory agents, immune cells.

[0021] A further object of the present invention is a kit comprising a single-use vial containing metal-based nanoparticles, a solvent for resuspending the nanoparticles, preferably water, or a physiological solution, or Dulbecco's Modified Eagle's Medium (DMEM), or a buffer solution such as Dulbecco's Phosphate Buffered Saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer, etc., containing divalent metal ions such as Ca2+, Mg2+, respectively, optionally a syringe, and instructions for use.

[0022] Preferably, the metal-based nanoparticles or composition or 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, preferably the tumors are selected from among urothelial, bladder, gastroesophageal, colorectal, pancreatic, ovarian, lung, cervical, breast and renal cancers, brain tumors and hepatocellular carcinoma.

[0023] The present invention also provides the metal-based nanoparticles or the composition or the kit described above for use in the photothermal therapy of solid tumors, preferentially bladder cancer.

[0024] A further object of the present invention is to provide a method for performing in vitro ultrasound and photoacoustic imaging, comprising at least the following steps: a) applying the metal-based nanoparticles or compositions of the present invention to the target tissue to be imaged; b) photoacoustic visualization of the target tissue; and c) evaluating the visualized target tissue The method includes:

[0025] Further features and objects of the present invention will become apparent from the following description. [Brief explanation of the drawings]

[0026] [Figure 1]Expression of α5 and β1 integrins in human bladder CIS and mouse noninvasive orthotopic bladder cancer. A) Representative immunohistochemical micrographs of human bladder sections with non-neoplastic tissue and CIS obtained by TURB. One representative image of normal tissue and two representative images of CIS are shown. Tissues were immunostained with the indicated anti-integrin antibodies. Ur; urothelium; Lp; lamina propria. Arrows indicate membrane staining for α5 integrin. B) Representative immunohistochemical micrographs of mouse orthotopic bladder cancer 11 days after intravesical instillation of MB49-Luc cells. Immunostained with the indicated anti-integrin antibodies. Mouse bladders from one of three mice analyzed are shown. The top row shows low magnification (scale bar 500 μm), and the bottom row shows high magnification (scale bar 50 μm) of non-neoplastic and neoplastic tissues. [Figure 2] Figure 1. Binding of Iso4 peptide to MB49-Luc and 5637 cell lines. A) Binding of Iso4-Qdot or ARA-Qdot (control) to MB49-Luc and 5637 cells measured by FACS. A representative FACS experiment (left) and quantification of Qdot binding (right) are shown. Circles: mean ± SEM of duplicates. B) Binding of Iso4-Qdot or ARA-Qdot to viable 5637 cells. Cells were cultured in 96-well plates and incubated with the indicated doses of Qdot (2 h, 37 °C, 5% CO). After washing and fixation, cell-associated fluorescence was acquired using a Cellomics ArrayScan XTI Studio Scan (Thermo Fischer Scientific) system. Magnification: 20x; scale bar: 10 μm; red, Qdot. C) Adhesion of MB49-Luc and 5637 cells to solid phases coated with Iso4-HSA or *HSA (control) and stained with crystal violet. Representative images (left) and quantification of cell adhesion (right) of wells coated with 30 μg / mL Iso4-HSA or *HSA. Images were acquired with a scanner. Bars represent mean ± SE, n = 4. [Figure 3]Synthesis and characterization of GNR@CTAB, GNR@Chit, and GNR@Chit-Iso4. A) Synthesis process of GNR@Chit-Iso4. Chemical modification of chitosan by EDC-coupled amidation of thioglycolic acid with chitosan's amino groups; removal of CTAB by thiolated chitosan attached to GNRs; attachment of the NHS ester end of a bifunctional PEG linker to free amino groups on chitosan; and conjugation of Iso4 using a cysteine ​​residue reactive to the maleimide end of the linker. B) VIS-NIR spectrum of GNR@Chit-Iso4 compared with those of GNR@CTAB and GNR@Chit. C) Morphology of GNR@Chit-Iso4 by TEM analysis (scale bar = 50 nm). D) Width and length distribution of nanorods present in the nanosystem GNR@Chit-Iso4 and the corresponding Gaussian fit (length mean ± SD and R2, 90.2 ± 7.2 nm and 0.86; width mean ± SD and R2, 24.9 ± 2.6 nm and 0.93; n = 300, TEM). E) Aliquot of GNR@Chit-Iso4 during the freeze-drying process. F) VIS-NIR spectra and absorption intensity evolution over time of GNR@Chit-Iso4 in the presence of human urine. [Figure 4]GNR@Chit-Iso4 promotes the adhesion and spreading of MB49-Luc and 5637 cells, but not GNR@Chit. A) Adhesion of MB49-Luc and 5637 cells to microtiter plates coated with various amounts of GNR@Chit-Iso4 or GNR@Chit-Cys. Representative images (by dry matter content) of cell adhesion to 250 μg / ml gold nanorods and quantification of cell adhesion are shown (circles, mean ± SE, n = 3). EC50 values ​​reported in each plot are the results of three to four independent experiments (mean ± SE). B) Effect of free Iso4 on the adhesion of MB49-Luc and 5637 cells to microtiter plates coated with 250 μg / ml GNR@Chit-Iso4 or GNR@Chit-Cys. MB49-Luc and 5637 cells were mixed with the indicated amount of free Iso4 and allowed to adhere to the GNR-coated microtiter plates. A representative experiment out of three independent experiments is shown (circles, mean ± SE, n = 2). C) Binding of various amounts of Iso4-HRP or ARA-HRP to α5β1-coated or non-coated microtiter plates detected with OPD chromogenic substrate (circles, mean ± SE, n = 2). D) Effect of human urine on the binding of Iso4-HRP to α5β1-coated or non-coated microtiter plates. Iso4-HRP (300 mM) was mixed with the indicated amount of urine, and the mixture was added to the plate. After washing, bound peroxidase was detected with OPD chromogenic substrate (bars, mean ± SE, n = 2). E) Effect of human urine on the adhesion of MB49-Luc cells to solid phases coated with GNR@Chit-Iso4 or GNR@Chit-Cys. Cells were suspended in DMEM containing 0.1% BSA (top row) or 25 mM Hepes buffer, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, and 0.1% BSA (bottom row), and the indicated amount of human urine was added. The mixture was added to a microtiter plate coated with 250 μg / ml GNRs and incubated at 37°C and 5% CO for 1–2 hours. After washing, the adherent cells were fixed and stained with crystal violet.Representative images of cell adhesion to wells coated with 250 μg / ml gold nanorods and quantification of cell adhesion are shown (bars, mean ± SE, n = 3). *P < 0.05, ***P < 0.01, ***P < 0.001, determined by two-tailed t-test using GraphPadPrism software. [Figure 5]In vitro and in vivo PAI of GNR@Chit-Iso4. A) PAI of an agar droplet containing GNR@Chit-Iso4 (15 nmol Au) and its corresponding PA spectrum using a 0.6% Intralipid (IL) optical attenuator. The echogenic signal (gray) originates from the mucus in which the agar droplet is embedded. The purple ROI indicates the PA signal of the GNR from which the PA spectrum was derived. PA images were acquired and the spectra were decomposed using VevoLab software to separate the contributions of the mucus and GNR (green signal corresponds to the PA signal of the GNR). B) Normalized PA spectra of GNR@Chit-Cys and GNR@Chit-Iso4 (15 nmol Au) embedded in agar droplets (representative one of five experiments). C) 3D distribution of the GNR@Chit-Iso4 signal in the agar droplet acquired using a 0.6% IL optical attenuator. D) Dose-response plot of the PA signal of GNR@Chit-Iso4 in agar droplets analyzed using optical attenuators prepared with the indicated concentrations of IL. The dynamic range is linear from 0 to 3.75 nmol Au, with a plateau trend from 3.75 to 15 nmol Au. E) Overlay of PA spectra of GNR@Chit-Iso4 (3.75 nmol Au) acquired using optical attenuators prepared with the indicated concentrations of IL. F) TEM analysis of GNR@Chit-Iso4 recovered from agar droplets after PA analysis with optical attenuators prepared without IL. G) Energy fluence at 750 nm, 800 nm, and 850 nm, in the presence and absence of optical attenuators containing the indicated amounts of IL (mean ± SEM of triplicates). H) Average energy distribution along depth obtained from a Monte Carlo model of the reported simulation region. I) In vivo PAI of mouse bladder after intravesical instillation of 100 μl of vehicle (saline) or GNR@Chit-Iso4 (3 nmol Au). A representative frame (one representative of five experiments) taken at the center of the bladder was obtained. The green signal corresponds to the PA signal of GNR after decomposition of the PA signals of melanin, deoxygenated blood, oxygenated blood, and GNR. J) PA spectra of saline and GNR@Chit-Iso4 (3 nmol Au) in mouse bladder.K) 3D distribution of PA signal from GNR@Chit-Iso4 (3 nmol Au) in a mouse bladder, imaged at the indicated time points (representative experiment out of 5). L) 3D distribution of PA signal from GNR@Chit-Iso4 in the upper and lower halves of a mouse bladder 30 min after injection, followed by quantification of the volume occupied by PA signal (% PA signal of GNR) in the upper and lower halves of the bladder at different time points (data shown as mean ± SEM, each dot represents one animal). *, **; p-values ​​by two-tailed Mann-Whitney test between the upper and lower halves of the bladder at each time point. M) PAI in a mouse bladder with tumor colonization on the left side (red asterisk). Absence of PA signal after intravesical injection of GNR@Chit-Iso4 (3 nmol Au) and two vesical lavages to remove unbound GNRs is shown; axial diameter of the bladder lumen = 3.7 mm. Vol: volume. [Figure 6] Illustration of early diagnosis of bladder cancer. A) Axial frame of PAI from an animal with established bladder tumor after three cycles of intravesical instillation of GNR@Chit-Iso4 (10 nmol Au), followed by manual urine processing, GNR removal, and saline rinsing (representative of six experiments). B) Axial frame of PAI from an animal with a low-volume bladder tumor after three cycles of intravesical instillation of GNR@Chit-Iso4 (10 nmol Au), followed by manual urine processing, GNR removal, and saline rinsing (representative of six experiments). C) US scan of PAI from the same animal reported in panel C after 13 days with GNR. D) Axial frame of PAI from an animal with a US-undetectable bladder tumor (bladder stone) (representative of six experiments) after intravesical instillation of GNR@Chit-Iso4 (10 nmol Au), followed by three cycles of manual urine processing, GNR removal, and saline irrigation. E) US scan of PAI of GNRs from the same animal reported in panel E after 13 days. [Figure 7]Figure 1. Expression of α5 and β1 integrins in Von Brunn's nests and human bladder CIS. A) One representative immunohistochemical micrograph of two examined human bladder sections with normal urothelium present in Von Brunn's nests. B) Five of six examined human bladder CIS showed positive α5 integrin staining at the membrane level. All tissues were obtained from TURB. [Figure 8] Figure 1. Expression of α5 integrin and β1 integrin in the human bladder according to tumor stage in one bladder following radical cystectomy. Representative immunohistochemical micrographs of human bladder sections from one cancer patient who underwent radical cystectomy for non-neoplastic urothelium, NMIBC (pTa and Cis), and paired MIBC (pT2-pT4) were immunostained with the indicated anti-integrin antibodies. *Non-neoplastic urothelium; #Stroma; **Tumor tissue. Scale bar: 100 μm. [Figure 9] Illustration of synthesis and characterization of GNR@CTAB and GNR@Chit. A) 2-liter jacketed reactor equipped with a mechanical stirrer for large-scale synthesis of GNRs. B) Representative VIS-NIR spectrum (normalized absorbance) of GNR@CTAB showing the presence of two distinct absorption bands for transverse (left) and longitudinal (right) surface plasmon resonance. C) Quantification of gold in GNR@CTAB by EDX analysis. D) Morphology of GNR@Chit by TEM analysis (scale bar = 50 nm). E) VIS-NIR spectrum of GNR@Chit. [Figure 10] TEM and SAED analysis of GNR@CTAB. A) Representative TEM image of GNR@CTAB. B) and C) Selected area electron diffraction of a single GNR, showing two distinct reflections assigned to the crystal plane sets

[0200] and

[0222] of the Au face-centered cubic structure. Two diffraction spots were present, and by calculating the corresponding interplanar distances in the electron diffraction of the GNR, 2.04 and 1.19 Å were determined. These correspond to the distances between specific planes, the

[0200] and

[0222] planes, of the gold FCC crystal structure (space group Fm3m), respectively. [Figure 11]H-NMR spectra of chitosan, thiolated chitosan, and GNR@Chit at 600 MHz. A) H-NMR of chitosan (600 MHz, 1% CH3COOH in DO). B) H-NMR of thiolated chitosan (600 MHz, DO). Spectral features were fully assigned by evaluating peak positions and intensities. Peaks corresponding to the bound thioglycol residue (7) are located at 3.2 and 3.5 ppm. Unambiguous peak assignments were possible by comparing the obtained spectra with those reported in the literature. [6] Upon conjugation with thioglycolic acid, sharp singlet peaks appear at 3.20 and 3.50 ppm. This is related to the splitting of the CH2 residue of the thioglycol moiety bound to the amino group of chitosan due to partial deprotonation of the thiol group in a neutral aqueous environment. C) H-NMR spectrum of GNR@Chit (600 MHz, DO). The obtained spectrum resembles all the features of that of chitosan. The absence of signals from CTAB indicates a successful ligand exchange reaction. Furthermore, it is clear that the sharp peak corresponding to the CH2 of the thioglycol moiety does not show any splitting due to the deprotonation equilibrium of the thiol. This may be directly linked to the efficient attachment of chitosan to the GNR surface, as the thiol group is bound to the gold atom, disallowing the protonation equilibrium. [Figure 12]Characterization of GNR@Chit. A) Thermogram of GNR@Chit decomposition. The dashed line corresponds to the switch from N2 atmosphere to air. The TGA profile shows an initial loss corresponding to a -10% mass loss due to desorption of residual moisture from the freeze-drying process. After the sample was held in air at 600 °C for 15 min, the residual mass corresponding to the total inorganic content was as low as 2.1 wt%. B) Shear stress-shear rate and C) viscosity-shear rate rotational rheological analysis of GNR@Chit at 20 °C and 37 °C. At low shear rates (below 300 Hz), the shear stress-shear rate curve is linear, suggesting Newtonian behavior of the fluid and confirming constant viscosity in this range. At higher shear rates, the viscosity decreases, indicating shear-thinning behavior at higher stresses, which is even more pronounced at 37 °C. D) Temperature dependence of viscosity measured at a constant shear rate of 50 Hz. [Figure 13] TEM analysis of GNR@Chit-Iso4. A) High-angle-angle dark-field (HAADF) image of GNR@Chit-Iso4 and energy-dispersive X-ray spectroscopy (EDX) analysis of two isolated GNRs. Gold was detected as the only atomic component of the GNR core, and bromine from the cytotoxic CTAB was not detected. Unlabeled peaks are associated with copper and carbon from the sample supporting the TEM grid. B) TEM image of GNR@Chit-Iso4 and its corresponding selected-area electron diffraction (SAED) pattern confirmed the preservation of the crystallinity of the gold core. Reflections corresponding to 1.20 Å, 1.41 Å, 2.03 Å, and 2.31 Å were assigned to the

[0311] ,

[0220] ,

[0200] , and

[0111] planes, respectively, by comparing the interplanar distances with compiled literature data [7]. [Figure 14]Representative HPLC chromatograms of hydrolysis products obtained from GNR@Chit-Iso4 (A) and GNR@Chit-Cys (B). Lyophilized GNRs (0.5–1 mM Au, 0.5–1 × 10 NPs / ml) were suspended in water at 5 mg / ml (based on dry matter content), and an aliquot was subjected to acidic hydrolysis (6 M hydrochloric acid, 0.1% phenol, and 0.1% thioglycolic acid, at 110 °C for 20 h under reduced pressure in a nitrogen atmosphere). The amino acid content in the hydrolysis products was then quantified by ion-exchange chromatography and post-column derivatization with ninhydrin. Arrows indicate the types of amino acids (single-letter codes) that were clearly detected and quantified above background (compare panel B). The amino acids (three-letter codes) in gray correspond to the elution times of the standard amino acid mixture used to calibrate the column. Amino acids (three-letter codes) in parentheses are below the detection limit. Sarcosine (Sar) was added to the samples as an internal control standard. There is no standard for d-PHG, and in this case d-PHG elutes as methionine. Cys cannot be quantified by this method. [Figure 15] Diagram of the materials and setup used for in vitro visualization of GNRs by PAI. A) A representative optical attenuator consisting of 1% agar and IL in a disposable mold cassette. B) Example of a 1% agar droplet containing GNRs. C) Setup of the setup for PAI of agar droplets containing GNRs. D) US imaging and PA signal of one representative agar droplet containing GNR@Chit-Iso4 (15 nmol) embedded in mucus and acquired using an optical attenuator consisting of 1% agar and 0.6% IL. The echogenic signal (gray) is generated by the mucus in which the agar droplet is embedded. E) Laser beam on photographic paper placed 8 mm from the laser fiber and exposed to the indicated wavelength for 5 seconds. Each spot was 13 mm long and 3 mm wide. [Figure 16]Illustration of the Monte Carlo model of light transport. A) Light fluence distribution within a simulated region consisting of a skin line and standard tissue. This simulated image shows a very good light fluence distribution, with more fluence observed at the skin line and then decreasing along the depth direction. The color bar shows the fluence distribution, with red indicating high fluence and black indicating very low fluence. B) Average energy distribution along the depth, taking into account the presence of different IL layers. After transmitting through the tissue interface, the light is slightly collimated, and at slightly deeper depths, both incident and backscattered light contribute, causing a fluence peak that then decays exponentially. [Figure 17] Figure 1 shows that GNR@Chit-Iso4 binding to tumorous urothelium is specific. Three representative axial frames and 3D PAIs of GNR@Chit-Iso4 (containing 5.6 μg of Iso4, 1.5 nmol Au) (A), 296 μg of ARA peptide (B), or 296 μg of Iso4 peptide (C) were intravesically instilled, followed by manual urine processing and two washes. One representative mouse out of two is shown for each condition, and animals with comparable bioluminescence were used (total flux [p / s] of 7.2 × 107 for panel A, 1.22 × 107 for panel B, and 2 × 107 for panel C). DETAILED DESCRIPTION OF THE INVENTION

[0027] The object of the present invention is to provide a technological platform for diagnostic, therapeutic, or diagnostic-therapeutic applications. While the present invention is susceptible to various alternative modifications, some preferred embodiments are detailed below. The embodiments are exemplary and should not be intended as limiting the scope of the invention to any particular alternative.

[0028] Therefore, the object of the present invention is the development of functionalized agents, preferably metallic nanostructures or nanoparticles, carrying ligands capable of recognizing tumor- or inflammation-associated components.

[0029] According to the present invention, nanostructure means a chemical substance or material having a particle size of 1 to 100 nanometers in at least one dimension.

[0030] In a preferred embodiment, the agents according to the invention are designed to recognize tumor-associated components and are used for the early diagnosis and / or treatment of solid tumors.

[0031] To overcome the obstacles of available diagnostic tools, the inventors have developed a new technological platform based on the use of metal nanostructures coated with polymers carrying thiol and -NH groups conjugated with ligands capable of recognizing tumor- or inflammation-associated components. In a preferred embodiment, the nanoparticles described in the present invention have photoacoustic properties, more preferably in the near-infrared region I and II.

[0032] The ligands according to the present invention are attached to the metal nanostructures via heterobifunctional crosslinkers.

[0033] In a preferred embodiment, the nanostructures are nanoparticles composed of gold, silver, or hybrid gold / silver, with the preferred metal being gold.

[0034] The nanoparticles according to the present invention can be designed to have different shapes, including but not limited to spheres, rods, stars, cages, prisms, shells, hollow shells, wires, plates, etc.

[0035] In a preferred embodiment, the nanoparticles are nanorods with a length of 10-200 nm and a width of 2-50 nm, more preferably a length of 20-100 nm and a width of 10-25 nm, and an aspect ratio of 1.2-15, preferably 3-7.

[0036] In a preferred embodiment, the nanoparticles according to the present invention are gold nanorods (GNRs) with an aspect ratio of 3.6.

[0037] According to the present invention, nanoparticles are coated with polymers bearing thiol and -NH groups, including, but not limited to, thiolated chitosan (thiol-modified chitosan), thiolated and aminated alginic acid, thiolated and aminated hyaluronic acid, and other thiolated polysaccharides, or preferably proteins such as albumin and gelatin, or synthetic thiolated and aminated polymers such as α-thio-ω-amino polyethylene glycol. In a preferred embodiment, the nanoparticles according to the present invention are coated with thiolated chitosan, and in a more preferred embodiment, the nanoparticles are gold nanoparticles coated with thiolated chitosan.

[0038] The thiolated chitosan described in the present invention preferably has an average molecular weight in the range of 0.5 kD to 1000 kDa, more preferably in the range of 50 kD to 200 kDa, and an average degree of deacetylation in the range of 75% to 100%, more preferably in the range of 85% to 95%. To introduce thiol moieties, chitosan is chemically modified with carboxylic acids bearing free thiol groups, such as 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobenzoic acid, cysteine, homocysteine, cysteine-containing peptides and proteins, and α-thio-ω-aminopolyethylene glycol. Thiol functionalization involves less than 90% of the chitosan's free amino groups, more preferably in the range of 75% to 85%.

[0039] According to the present invention, the coated metal nanoparticles are linked to the aforementioned ligands via heterobifunctional crosslinkers, which possess functional groups capable of binding with amino groups and / or functional groups capable of binding with thiol groups.

[0040] In a preferred embodiment, the functional group capable of binding to an amino group is one of an N-hydroxysuccinimidyl ester group (NHS ester), an isocyanate group (-NCO), an isothiocyanate group (-NCS), a sulfo-N-hydroxysuccinimidyl ester group (sulfo-NHS ester), or a carboxylic acid group that is linked by activation with a carbodiimide coupling agent.

[0041] In a preferred embodiment, the functional group capable of binding to a thiol group is one of a maleimide group, a terminal vinyl group, or a terminal alkyne group; and / or a functional group capable of binding to an alkyne, such as an azide; and / or a functional group capable of binding to an azide, such as an alkyne.

[0042] In a preferred embodiment, the heterobifunctional linker is maleimide-PEG 12 -NHS ester.

[0043] Thus, a crosslinker is a bifunctional moiety that possesses at least two functional groups capable of binding or reacting with specific groups, where the terms "binding" and "reacting" have the same meaning. The at least two functional groups may be the same or different, and thus the crosslinker may be homofunctional or heterofunctional. Preferably, the at least two functional groups are functional groups capable of binding or reacting with amino groups and / or thiol groups, and / or alkyne groups, and / or azide groups. Preferably, the crosslinker according to the present invention is a bifunctional moiety that possesses functional groups capable of binding with amino groups and sulfhydryl groups, and / or functional groups capable of binding with amino groups and azide / alkyne groups, and / or functional groups containing lipoamide or lipoic acid moieties, or sulfhydryl or disulfide-containing compounds.

[0044] Non-limiting examples of cross-linking agents according to the invention are reported below.

[0045] Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is a non-cleavable, membrane-permeable crosslinker with the following structure:

[0046] [ka]

[0047] SMCC contains an amine-reactive N-hydroxysuccinimide (NHS ester) and a sulfhydryl-reactive maleimide group. The NHS ester reacts with primary amines at pH 7-9 to form stable amide bonds. The maleimide reacts with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds.

[0048] Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) corresponds to the compound having the chemical structure shown below:

[0049] [ka]

[0050] The maleimide group of SMCC and sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is remarkably stable up to pH 7.5 due to the presence of a cyclohexane bridge in the spacer arm.

[0051] The cross-functional linkers of the present invention can have the general structures reported below.

[0052] [ka]

[0053] In the formula, A and B contain different reactive groups, x is an integer of 2 to 10 (such as 2, 3, or 4), and y is an integer of 2 to 30, such as 1 to 50, for example 3 to 20 or 4 to 12.

[0054] Non-limiting examples of crosslinkers 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):

[0055] [ka]

[0056] Mal-amide-PEG-TFP ester refers to a PEG linker containing a maleimide group and a TFP ester end group. The maleimide group is reactive to thiols between pH 6.5 and 7.5. TFP esters can react with primary amine groups and are less susceptible to hydrolysis than NHS esters. The hydrophilic PEG chains enhance the solubility of compounds in aqueous media. Longer PEG chains improve water solubility compared to shorter PEG chains. PEG linkers vary in the number of glycol units, such as MAL-dPEG®8-TFP ester and MAL-dPEG®4-TFP ester.

[0057] [ka]

[0058] Propargyl-PEG-NHS ester is an amine-reactive reagent that can be used to derivatize peptides, antibodies, and amine-coated surfaces. This alkyne group reacts with azide-bearing compounds and biomolecules in copper-catalyzed click chemistry reactions. Examples include propargyl-PEG1-NHS ester, which contains one glycol unit, and propargyl-PEG4-NHS ester, a four-unit amine-reactive PEG linker.

[0059] [ka]

[0060] Azido-PEG-TFP ester is a click reagent containing an azide group and a TFP moiety. The azide group enables click chemistry. TFP esters can be used to label primary amines (-NH2) on proteins, amine-modified oligonucleotides, and other amine-containing molecules. The chemical structure of a representative compound (2,3,5,6-tetrafluorophenyl 3-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethoxy]propanoic acid) is shown below.

[0061] [ka]

[0062] A similar PFP derivative (perfluorophenyl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate; N3-PEG4-PFP) has the following structure:

[0063] [ka]

[0064] Azido-PEG-NHS ester is a PEG reagent containing an azide group and an NHS ester. Non-limiting examples include 2,5-dioxo-1-pyrrolidinyl 3-[(23-azido-3,6,9,12,15,18,21-heptaoxatricos-1-yl)oxy]propanoate.

[0065] [ka]

[0066] Thiol PEG

[0067] [ka]

[0068] Thiol-PEG-acid

[0069] [ka]

[0070] Thiol-PEG-amine H2N-(CH2CH2O) n -CH2CH2-SH Thiol-PEG-azide N3-(CH2CH2O) n -CH2CH2-SH Propargyl-PEG-MAL

[0071] [ka]

[0072] Azide-PEG-MAL

[0073] [ka]

[0074] In the above compounds, the polyethylene glycol chain (PEG) has a molecular weight between 0.05 and 40 KDa, preferably lipoamide / lipoic acid-PEG-MAL, in which the PEG has a molecular weight of 5 KDa.

[0075] According to the present invention, nanostructures coated with a polymer bearing thiol and -NH groups are bound to a ligand capable of recognizing tumor- or inflammation-associated components via the aforementioned heterobifunctional crosslinker. According to the present invention, ligands of integrin family receptors, proteins (more preferably antibodies or antibody parts), peptides, peptidomimetics, or aptamers are preferred. Integrins are chosen because they are important regulators of cell structure and behavior, affecting cell morphology, proliferation, survival, and differentiation.

[0076] In a preferred embodiment, the integrin family receptor is selected from the group consisting of αvβ1, α8β1, α5β1, αvβ3, αvβ5, αvβ6, αvβ8, α3β1, α6β1, α7β1, α6β4, α1β1, α2β1, α10β1, α11β1, α4β1 and α9β1, with α5β1 being preferred.

[0077] According to the present invention, the ligand may be a peptide containing an integrin binding motif as RDG (Arg-Gly-Asp) or isoDGR. In a preferred embodiment, the peptide is [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, SEQ ID NO: 6 [XisoDGRGphg] and a cyclic isoDGR peptide selected from the group consisting of: wherein "X" is preferably cysteine, lysine, or any alkyne or azide functionalized amino acid such as propargylglycine or azidolysine.

[0078] In another preferred embodiment, the ligand is XFETLRGDERILSILRHQNLLKELQD of SEQ ID NO: 8, XFETLRGDLRILSILRHQNLLKEL of SEQ ID NO: 9, SEQ ID NO: 10 XFETLRGDLRILSILRX1QNLX2KELQD is a linear peptide selected from the group wherein "X" is preferably cysteine, lysine, or any alkyne- or azide-functionalized amino acid such as propargylglycine or azido-lysine, while X1 and X2 form a triazole bridge via propargylglycine (X1) and azido-lysine (X2).

[0079] In a preferred embodiment, the ligand is SEQ ID NO: 7 [CphgisoDGRG] and the following structure:

[0080] [ka]

[0081] This compound can exist as a mixture of isomers corresponding to the cyclic head-to-tail configuration with D-phenylglycine (D-phg) and L-phenylglycine (L-phg).

[0082] As used herein, "phg" refers to D-phenylglycine (D-phg) and "Phg" refers to L-phenylglycine (L-Phg). The ratio of D-phenylglycine (D-phg) to L-phenylglycine (L-Phg) can be between 50:50 and 99:1, between 60:40 and 90:10, or between 65:35 and 80:20. In a representative example, the compound contains about 70% D-phenylglycine (D-phg) and about 30% L-phenylglycine (L-phg).

[0083] As used herein, "isoD" refers to isoaspartic acid (isoaspartic acid, isoaspartyl, β-aspartic acid), which is an aspartic acid residue isomeric to the typical α-peptide bond. It is a β-amino acid in which the carboxyl side chain has been transferred to the backbone.

[0084] Head-to-tail cyclized peptides are peptides with a cyclic structure. Head-to-tail backbone (homodetic) cyclization is a peptide modification that results in a rigid structure, a biologically relevant turn conformation, increased proteolytic stability, and improved membrane permeability.

[0085] An object of the present invention is therefore metal-based nanoparticles, preferably gold nanoparticles, more preferably gold nanorods, coated with a polymer functionalized with thiol and -NH groups, said polymer being preferably selected from among thiolated chitosan, thiolated and aminated alginate, thiolated and aminated hyaluronic acid, or proteins, preferably selected from the group consisting of albumin and gelatin, or synthetic thiolated and aminated polymers, preferably α-thio-ω-amino polyethylene glycol, said groups being linked via a heterobifunctional crosslinker to ligands of integrin family receptors, preferably peptides containing an integrin binding motif, and antibodies or parts of antibodies, peptidomimetics or The crosslinker linked to the aptamer preferably possesses a functional group capable of binding to an amino group, selected from the group consisting of an N-hydroxysuccinimidyl ester group (NHS ester), an isocyanate group (-NCO), an isothiocyanate group (-NCS), a sulfo-N-hydroxysuccinimidyl ester group (sulfo-NHS ester), or a carboxylic acid group linked by activation with a carbodiimide coupling agent; and / or a functional group capable of binding to a thiol group, preferably selected from the group consisting of a maleimide group, a terminal vinyl group, or a terminal alkyne group; and / or a functional group capable of binding to an alkyne such as an azide; and / or a functional group capable of binding to an azide such as an alkyne.

[0086] According to the present invention, preferred agents are those in which the heterobifunctional linker is maleimide-PEG. 12 -NHS ester and gold nanorods coated with thiolated chitosan whose ligand is the [CphgisoDGRG] peptide.

[0087] Any of the agents of the described embodiments can be prepared in solution or in lyophilized form. The term "lyophilization" also refers to drying, freeze-drying, or precipitation with a water-miscible organic solvent such as acetone.

[0088] The present invention also provides a composition comprising the diagnostic and therapeutic agent described above, which can be prepared in a variety of solutions, including water, physiological solution / saline, Dulbecco's Modified Eagle Medium (DMEM), Ca 2+ , Mg 2+ The solution contains at least one of Dulbecco's phosphate buffered saline (DPBS), HEPES buffer, TRIS buffer, and PIPES buffer containing divalent metal ions such as phosphate buffer, ...

[0089] In particular, for use in treatment, compositions may be prepared which contain, in addition to the components mentioned above, one or more pharmaceutical agents, particularly at least one of a chemotherapeutic agent, an immunomodulatory agent, and immune cells.

[0090] In a preferred embodiment, the chemotherapeutic agent is selected from the group consisting of mitomycin C, Bacillus Calmette-Guerin (BCG), doxorubicin, melphalan, gemcitabine, taxol, cisplatin, vincristine, or vinorelbine, more preferably the immunomodulatory agent is an anti-cancer vaccine and / or an immune checkpoint blocker such as an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody, and even more preferably the immune cell is a lymphocyte or a genetically modified T lymphocyte such as a CAR-T cell, a TCR-redirected T cell, or an NK cell.

[0091] In a preferred embodiment, the present invention encompasses a composition comprising thiolated chitosan-coated gold nanoparticles, preferably nanorods as described in the present invention, and Dulbecco's Modified Eagle's Medium (DMEM) or Dulbecco's Phosphate Buffered Saline (DPBS) containing calcium and magnesium, wherein the heterobifunctional linker is maleimide-PEG. 12 -NHS ester and the ligand is the [CphgisoDGRG] peptide.

[0092] The drug as described above, a solvent for resuspending the nanoparticles, preferably physiological solution / saline and Dulbecco's Modified Eagle Medium (DMEM), respectively, 2+, Mg 2+ Also an object of the present invention is a kit comprising a single-use vial containing Dulbecco's Phosphate Buffered Saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer containing a divalent metal ion such as thiazolinone, optionally a syringe, and instructions for use.

[0093] It is also an object of the present invention to apply the described agents, compositions and kits to medical and diagnostic fields.

[0094] In particular, the nanoparticle objects of the present invention having photoacoustic properties are developed for use in the diagnosis of solid tumors or inflammation, and may be particularly used for the early detection of small cancer lesions, especially urothelial carcinoma, bladder cancer, gastroesophageal cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, breast cancer and renal cancer, brain tumor and hepatocellular carcinoma. The nanoparticles disclosed by the present invention are particularly suitable for detecting urothelial carcinoma, bladder cancer, chronic actinic cystitis by photoacoustic imaging.

[0095] In a preferred embodiment, the nanoparticles according to the present invention can be used for the early diagnosis of bladder lesions, in particular bladder cancer.Accordingly, the disclosed nanoparticles for use in an in vivo method for the diagnosis of bladder cancer, in particular small, flat urothelial lesions of high-grade bladder cancer in situ (CIS), are an object of the present invention.

[0096] Indeed, it is possible to proceed with intravesical instillation of urine-stable nanoparticles designed according to the invention, which have photoacoustic properties and which process urine containing said nanoparticles in the bladder to avoid precipitation, and then use multimodal imaging of the target lesions using PAIs.

[0097] In a preferred embodiment, the target area is subjected to thermal ablation and the bound nanoparticles are irradiated to the target area by continuous light irradiation. Assisted photothermal therapy is produced by exciting the particles with a selected wavelength.

[0098] It is therefore also an object of the present invention to apply the described agents, compositions and kits in the medical and diagnostic fields, in particular in diagnostic and therapeutic applications.

[0099] In a preferred embodiment, the nanoparticles or compositions according to the invention may be used in combination with pharmaceuticals, in particular chemotherapeutic agents, immunomodulatory agents, immune cells, in a method of combination therapy in which the administration of nanoparticles and pharmaceuticals may be simultaneous, contemporaneous or sequential.

[0100] In a preferred embodiment, the chemotherapeutic agent is selected from the group consisting of mitomycin C, bacillus Calmette-Guerin (BCG), doxorubicin, melphalan, gemcitabine, taxol, cisplatin, vincristine, or vinorelbine; more preferably, the immunomodulatory agent is an anti-cancer vaccine and / or an immune checkpoint blocker such as an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody; and even more preferably, the immune cells are lymphocytes or genetically modified T lymphocytes such as CAR-T cells, TCR-redirected T cells, or NK cells.

[0101] To demonstrate the efficacy of the targeting agents described in this invention, particularly the nanoparticles of interest, we designed chemically engineered gold nanorods (GNRs) with chitosan (Chit) and peptide Iso4 (a head-to-tail cyclized c(CphgisoDGRG) peptide with selectivity for the α5β1 integrin (Ki = 15 nM)

[25] ) to enable tumor targeting. These particles were developed for use in a method for the in vivo diagnosis and treatment of bladder cancer based on intravesical instillation of the urine-stable targeted GNRs (referred to as GNR@Chit-Iso4); in displacing the GNR@Chit-Iso4 suspension to prevent nanoparticle precipitation within the bladder; and in multimodal imaging of cancer lesions using PAIs.

[0102] We developed a combination of strategies that allowed us to detect these tumors with unprecedented sensitivity. In an orthotopic model of bladder cancer, we demonstrated that the combination of PAI and intravesical GNR@Chit-Iso4 transfer revealed the presence of lesions undetectable by US imaging or bioluminescence. Our technological platform was able to detect tumor lesions smaller than 0.5 mm with a sensitivity far exceeding that of US and CT urography for bladder cancer [5]. We demonstrated GNRs that can be used to detect orthotopic mouse bladder cancer lesions smaller than 0.5 mm, which are undetectable by US imaging or bioluminescence. We were able to detect tumor lesions up to 250 μm in size in the upper bladder of experimental mice. According to a preferred embodiment, the targeted nanoparticles of the present invention are gold nanorods designed with a diameter of 10 nm and an aspect ratio of 3.6 to exploit an optical window with a peak optical absorption at 808 nm, allowing deeper tissue penetration [40, 41] and to overcome different endogenous contrast molecules present in tissues

[18] . The PA dynamic range of the above nanoparticles was investigated and established to identify the maximum fluence and energy of pulsed laser light to avoid nanostructure remodeling and obtain diagnostic imaging using targeted nanoparticles.

[0103] The bladder environment in particular offers the possibility of utilizing intravesical instillation of targeted nanoparticles, which is characterized by advantages and disadvantages compared to systemic administration. Intravesical instillation avoids the off-target effects and off-target accumulation observed with systemic instillation, such as accumulation of gold in the liver, spleen, kidneys, testes, and brain [42, 43]. However, some issues that remain to be resolved are that intravesical delivery of therapeutics i) requires enduring urine containing a wide variety of by-products resulting from the metabolism of endogenous and exogenous substances

[37] , bacteria [38, 44], bacterial mucus, and suspended urothelial cells; ii) is characterized by transient retention; and iii) it is not possible to utilize the enhanced permeability of tumor vasculature and the retention effect of tumor blood vessels to allow intravenously injected targeted nanoparticles to accumulate in the tumor environment.

[0104] Therefore, the first step in the development of nanoparticles was to identify targets that are expressed only in tumor cells and not in non-neoplastic bladder epithelium, and direct them exclusively to tumor tissue.

[0105] Integrins are involved in almost every stage of cancer progression, from primary tumors to late-stage metastasis, making integrins a potential tumor target in human bladder cancer. Among the various integrins that play a role in cancer progression, we investigated the expression of α5β1 integrin 7, a cellular receptor for fibronectin whose expression level increases in the tumor stroma in association with tumor stage. α5β1 integrin is a marker of poor prognosis in bladder cancer patients and has been reported to be overexpressed in high-grade invasive bladder cancer. To understand whether α5β1 integrin could be a targeting receptor for non-invasive tumors, we evaluated its expression in non-neoplastic and neoplastic bladder tissues by immunohistochemical analysis of tissue sections obtained from TURB specimens histologically diagnosed with CIS. The α5 subunit was not expressed in non-neoplastic urothelial cells, but membranous staining was observed in CIS. Stromal cells within the lamina propria of non-neoplastic and neoplastic bladder tissues showed similar expression (Figure 1A). The β1 subunit was strongly expressed in non-neoplastic urothelial and stromal cells, as well as in CIS (Figure 1A). The lack of α5 subunit expression in normal urothelium was also observed in Von Brunn's nests. Expression of the α5 subunit in CIS was confirmed in five of six TURBs (Figure 7). The β1 subunit was consistently expressed in normal urothelium and stromal cells. In one of two radical cystectomies, α5 subunit expression was confirmed in CIS, and band-like expression was observed in non-invasive papillary tumors, whereas almost no expression was observed in invasive tumors pT1, pT2, and pT4 (Figure 8). Therefore, α5β1 may be a potential receptor targeting non-invasive tumors such as non-invasive papillae and CIS.

[0106] Next, we confirmed the expression of α5β1 integrin in two non-neoplastic human primary urothelial cells (PCS-420-010 and HBLAK) and four bladder cancer cell lines (RT4, RT112, 5637, and HT1376) derived from human tumors of different stages and grades by flow cytometry analysis using different sets of anti-integrin antibodies. The results, shown in Table 1 below, indicated that human bladder cancer cell lines expressed more α5 and β1 than human primary urothelial cells.

[0107] [Table 1]

[0108] Taken together, these results suggest that expression of α5β1 by human bladder CIS may be a potential target for the development of new tumor-targeting diagnostic tools based on α5β1 targeting.

[0109] We also investigated whether MB49-Luc tumor-bearing mice, a widely used syngeneic model of orthotopic bladder cancer

[35] , could recapitulate the α5β1 expression pattern observed in human bladder CIS. Results showed that the α5 subunit was expressed in MB49-Luc tumor cells but not in adjacent non-neoplastic epithelial cells. In contrast, stromal cells in both neoplastic and non-neoplastic tissues showed comparable expression (Figure 1B). The β1 subunit was expressed in basal urothelial cells in both non-neoplastic and neoplastic tissues (Figure 1B). Therefore, as shown in Table 2 below, flow cytometry analysis of MB49-Luc bladder cancer cells stained with the indicated rabbit anti-integrin antibodies followed by a goat anti-hamster Alexa Fluor 488-conjugated secondary antibody confirmed that MB49-Luc cells express the α5β1 integrin.

[0110] [Table 2]

[0111] Therefore, mice bearing MB49-Luc-derived orthotopic tumors can be used as a preclinical model for the development of new tumor-targeting strategies based on targeting α5β1. These experiments confirmed that integrin α5β1 is a truly excellent target, as it is expressed in human bladder CIS and human bladder cancer cell lines, the mouse MB49-luciferase (MB49-Luc) cell line, and MB49-Luc-derived orthotopic syngeneic mouse bladder cancer

[51] , but not in non-neoplastic human or mouse urothelium.

[0112] We decided to investigate the use of the Iso4 peptide, which has previously been reported to selectively recognize the α5β1 integrin.

[25] We needed to assess whether Iso4 could recognize α5β1-positive bladder cancer cells. We also needed to investigate and demonstrate whether the peptide Iso4 could maintain its functional properties (i.e., α5β1 integrin recognition) after coupling to GNR@Chit, especially in the presence of urine, and whether coupling to GNRs would alter the PA spectrum of the nanoparticles.

[0113] To this end, we conjugated Iso4, with the sequence [CphgisoDGRG], to fluorescent nanoparticles (quantum dots, Qdots) via the sulfhydryl groups of the cysteines and evaluated the binding of this conjugate (Iso4-Qdot) to MB49-Luc and 5637 cells. An unrelated head-to-tail cyclized c(CGARAG) peptide (ARA-Qdot) was also conjugated to Qdots and used as a negative control. Flow cytometry and fluorescence microscopy experiments demonstrated that Iso4-Qdot, but not ARA-Qdot, bound to these cells with a potency that correlated with the expression level of α5β1 on these cells (Figure 2A and B).

[0114] Next, since a peptide containing isoDGR conjugated to human serum albumin (HSA) promotes and supports endothelial cell adhesion [25, 36], we conjugated Iso4 to HSA and tested the conjugate (Iso4-HSA) in cell adhesion assays using MB49-Luc and 5637 cells. Iso4-HSA, but not activated HSA lacking the peptide (*HSA), promoted and supported cell adhesion (Figure 2C), suggesting that both cell lines express functional Iso4 receptors, likely α5β1. Taken together, these results suggest that Iso4 can functionally bind various compounds and target α5β1-positive bladder cancer cells. To obtain GNRs that can be used for PAI of α5β1-positive tumors, we functionalized chitosan-coated GNRs (GNR@Chit) prepared from CTAB-coated GNRs (GNR@CTAB) with a longitudinal surface plasmon resonance peak centered at 800 nm (Figures 9-12). GNR@Chit was functionalized with cycloCphgisoDGRG (Iso4), a selective ligand for α5β1 integrin. To this end, we used an ethylene oxide spacer (PEG) bearing an N-hydroxysuccinimidyl (NHS) ester at the terminus. 12 ) and maleimide functional group (NHS-PEG 12We utilized a heterobifunctional crosslinker reagent consisting of a hydroxyl group (OH) and a hydroxyl group (H-maleimide). The NHS ester terminus reacts with the free amino groups of chitosan, ensuring the attachment of the linker to chitosan. Meanwhile, the maleimide group can react with the sulfhydryl groups of Iso4 in a further step. Iso4 was then conjugated with activated GNR@Chit. Control GNRs were also prepared using cysteine ​​(Cys) instead of Iso4. The resulting products were designated GNR@Chit-Iso4 and GNR@Chit-Cys (Figure 3A). Next, we evaluated the physicochemical properties of the metal core of GNR@Chit-Iso4, determining its longitudinal LSPR peak at 802 nm (Figure 3B), its shape and dispersity (Figure 3C), and its aspect ratio of 3.62 ± 0.67 (length 90.2 ± 7.2 nm, width 24.9 ± 2.6 nm, Figure 3D). These findings indicate that the conjugation of Iso4 does not alter the physicochemical properties of GNRs, and the slight gradual changes in the longitudinal LSPR peaks for GNR@Chit (800 nm) and GNR@CTAB (798 nm) represent changes in the chemical environment surrounding the GNRs. Due to the presence of the PEG linker, the amount of free amino groups on chitosan decreased, and both Cys- and Iso4-conjugated nanosystems showed a decrease in Z potential (+10.3 mV and +10.6 mV for GNR@Chit-Iso4 and GNR@Chit-Cys, respectively, and +40 mV for GNR@Chit).

[0115] Furthermore, the crystallinity and composition of GNRs@Chit-Iso4 were proven to be preserved after successive conjugation steps, and no detectable Br atoms were found from CTAB (Figure 13). Therefore, the GNRs@Chit-Iso4 was aliquoted into vials, lyophilized, and vacuum-sealed, yielding 50 sterile, single-use vials containing the lyophilized product with a concentration of 1 mM GNRs@Chit-Iso4 when dissolved in 500 μL of water (Figure 3E).

[0116] We then investigated the stability of GNR@Chit-Iso4 in human urine by incubating it at 37 °C for up to 2 h. Although incubation in urine for up to 2 h did not significantly change the shape of the LSPR spectrum (Figure 3F), the absorbance at approximately 800 nm decreased by 14% over the first 40 min, followed by a further 7% decrease over the subsequent 80 min of incubation (Figure 3F). These results suggest that GNR@Chit-Iso4 does not dramatically degrade in its optical properties in the NIR even after incubation in urine at 37 °C for at least 2 h. We also characterized GNR@Chit-Cys using the same method, but no obvious differences were observed compared to GNR@Chit-Iso4 (results reported in Table 3).

[0117] [Table 3]

[0118] To quantify the amount of peptide loaded onto GNR@Chit-Iso4, we measured the total amino acid composition present in the supernatant of GNR@Chit-Iso4 after acidic hydrolysis. Hydrolyzed GNR@Chit-Cys was used as a negative control because it is expected to be an "amino acid-free" compound (Figure 14). The quantitative results for GNR@Chit-Iso4 shown in Table 4 indicate that approximately 1 × 10 11 The nanoparticles were found to be loaded with approximately 0.5 mg of peptide, which corresponds to approximately 6 × 10 per GNR. 6 It corresponds to a peptide.

[0119] [Table 4]

[0120] Next, to demonstrate the presence of functional Iso4 on GNRs, we measured the cell adhesion-promoting ability of GNR@Chit-Iso4 and GNR@Chit-Cys. To this end, microtiter plates were coated with various amounts of GNR@Chit-Iso4 or GNR@Chit-Cys and seeded with MB49-Luc and 5637 cells. GNR@Chit-Iso4, but not GNR@Chit-Cys, induced cell adhesion and spreading in both cell lines (Figure 4A), suggesting that the conjugation of Iso4 to GNR@Chit does not affect the function of the peptide. Notably, the effective concentration of GNR@Chit-Iso4 for both cell lines was 50 (EC 50 ) were similar, 0.73 ± 0.05 μg / ml for MB49-Luc cells and 1.3 ± 0.6 μg / ml for 5637 cells (Figure 4A). Addition of excess free Iso4 peptide completely inhibited cell adhesion, suggesting that adhesion was specific and dependent on the binding of Iso4 peptide to GNRs (Figure 4B).

[0121] We next investigated the effect of urine on the binding of GNR@Chit-Iso4 to α5β1 integrin and MB49-Luc cells. Intravesically administered GNR@Chit-Iso4 is expected to target bladder tumor cells in a harsh environment characterized by the presence of urine, a wide variety of metabolic products that may impair the binding ability of GNR@Chit-Iso4 to α5β1

[37] , bacteria

[38] , bacterial mucus, and floating urothelial cells. Since we intended to use targeted GNRs intravesically using a protocol similar to that used for photodynamic diagnosis using Hexvix

[39] , we assumed that the bladder would be emptied before instilling GNRs into the bladder. Therefore, the instilled GNRs were expected to mix with increasing amounts of urine over time.

[0122] Therefore, we first investigated the effect of human urine on the binding of Iso4 to purified α5β1. To this end, Iso4 was conjugated with maleimide-activated horseradish peroxidase (HRP) to create an Iso4-HRP conjugate, which was used as a probe in a direct binding assay using a microtiter plate coated with α5β1. An HRP control conjugate using the ARA peptide instead of Iso4 (ARA-HRP) was also prepared. As expected, Iso4-HRP was able to bind to α5β1 in a dose-dependent manner, whereas ARA-HRP was unable to bind (Figure 4C), suggesting that the probe was functional. Of note, to mimic the urinary environment, we omitted the addition of detergent to the binding and washing buffers. We then tested the effect of varying amounts of human urine (range 11.5–90%). As a result, urine partially inhibited the binding of Iso4-HRP, and the overall binding recovery was approximately 55% with 90% urine (Fig. 4D), demonstrating that, unexpectedly, Iso4 was able to bind to α5β1 even under these unfavorable conditions.

[0123] Furthermore, we investigated the effect of human urine on the binding ability of GNR@Chit-Iso4 to MB49-Luc cells. To this end, we tested the effect of various amounts of urine on the adhesion of MB49-Luc cells to plates coated with GNR@Chit-Iso4. In parallel, plates coated with GNR@Chit-Cys were used as negative controls to monitor nonspecific cell adhesion. Surprisingly, when MB49-Luc cells were seeded in DMEM medium containing 25–75% urine, a significant increase (approximately 20%) in cell adhesion to GNR@Chit-Iso4 was observed, but no adhesion to GNR@Chit-Cys or control plates (without nanoparticles) was observed (Figure 4E). The divalent ion (Mg 2+ / Mn 2+ When the assay was performed in Hepes buffer supplemented with urea, the increase in cell adhesion to GNR@Chit-Iso4 caused by low concentrations of urine was even more pronounced (Figure 4F).

[0124] These results unexpectedly demonstrated that urine did not interfere with the ability of Iso4 grafted onto GNRs to bind to bladder cancer cells. Because GNRs are prone to undergo shape changes and consequently lose their photoacoustic properties when stimulated with pulsed light above a certain energy threshold, we reduced the laser fluence using an optical attenuator to avoid GNR reshaping. We investigated the in vitro PA properties of GNRs using agar droplets containing GNR@Chit-Iso4 or GNR@Chit-Cys (15 nmol Au) and a 0.6% Intralipid (IL)-based optical attenuator attached to the tip of a Vevo LAZR-X optical fiber (Figure 15). The PA signals and spectra of GNR@Chit-Iso4 and GNR@Chit-Cys were similar, exhibiting a maximum peak at 810 nm (Figures 5A and B), and approximately 0.2 nmol Au was detected when 0.6% IL was used (Figure 5C). Next, we evaluated the minimum amount of IL required to detect the highest PA signal and correct spectrum of GNRs in vitro. Using optical attenuators containing 0.2, 0.4, or 0.6% IL, we found that the PA signal intensity of GNRs@Chit-Iso4 was inversely proportional to the amount of IL (Figure 5D). As expected, GNRs@Chit-Iso4 analyzed using optical attenuators without IL underwent reshaping, as observed by a change in the shape of the PA spectrum (Figure 5E), further supported by a transition from rod-like (Figure 3) to spherical (Figure 5F).

[0125] Without an optical attenuator, the energy fluence at wavelengths of 750 nm, 800 nm, and 850 nm (i.e., optical absorption of deoxygenated blood, GNR@Chit-Iso4, and oxygenated blood) was 30–35 mJ / cm. 2 In the presence of 0.2% IL, the energy fluence was 9 mJ / cm 2 and further decreased with increasing IL concentration (Figure 5G). These findings suggest that the maximum PA signal from GNRs was 9 mJ / cm 2 This shows that the energy fluence can be obtained by irradiation with

[0126] Considering the in vivo use of GNRs, we considered using an optical attenuator with a small amount of IL because of the presence of tissue surrounding the mouse bladder. Using Monte Carlo simulation, we estimated the optical energy of 800 nm wavelength reaching the mouse bladder, assuming a distance from the skin to the base of the bladder of 0.3 cm.

[0127] Considering the measured light energy to estimate the light fluence in Figure 5G, at a depth of 0.3 cm, the energy value without a light attenuator was estimated to be 5.3 mJ, while the energy value with a light attenuator made of 0.1% IL was 2.458 mJ (Figure 5H, Figure 16).

[0128] We then proceeded to develop conditions for detecting the PA signal of GNRs in the mouse bladder. An optical attenuator containing 0.1% IL was selected to image GNR@Chit-Iso4 nanoparticles instilled into the mouse bladder. In the mouse bladder instilled with GNR@Chit-Iso4 (10 nmol of Au), "dot-like" PA signals were clearly visible, whereas in the mouse bladder instilled with 100 μl of vehicle (saline) they were not (Figure 5I). The PA spectral shape of these "dot-like" structures was similar to that observed in vitro, i.e., with a peak at 810 nm (Figure 5J). This result suggests that GNR@Chit-Iso4 nanoparticles can be easily detected in vivo without significantly altering the PA properties of the nanoparticles.

[0129] We identified 2.458 mJ as the energy value required to obtain the maximum PA signal of GNRs in the mouse bladder without reconstitution. Time-course analysis of PAUS at 0, 5, 10, 20, and 30 min after implantation of GNR@Chit-Iso4 (10 nmol) showed a gradual accumulation of signal toward the bladder base (Figure 5K). Notably, after 5 min of implantation, only approximately 25% of the PA signal was detectable in the upper half of the bladder, while the majority of the signal remained accumulated in the lower half of the bladder (Figure 5L). Under these conditions, tumors located in the upper / lateral parts of the bladder could not be detected (Figure 5M). Notably, no sedimentation of GNRs was observed even after storage at 37 °C for up to 2 h, suggesting that GNRs maintain their colloidal properties even outside the bladder. With the intention of providing a technological platform for the early diagnosis of cancer present anywhere in the bladder, we attempted to keep GNRs suspended in the bladder by aspirating the urine contents with a syringe connected to a catheter and manually handling the obtained urine, which was then re-injected three times every four minutes.

[0130] The particles can be well maintained in suspension and moved by a variety of means: manual, physical, magnetic, and ultrasonically assisted movement.

[0131] Manual processing of urine-stable GNR@Chit-Iso4 inhibits colonized orthotopic α5β1 + The diagnostic utility of GNR@Chit-Iso4 for bladder tumors (i.e., tumors derived from mice MB49-Luc) was verified. For this purpose, GNR@Chit-Iso4 or GNR@Chit-Cys was instilled into the bladder, followed by PAI. To remove unbound GNRs, three cycles of manual urine processing and two bladder lavages were performed. Binding of GNR@Chit-Iso4 was observed in three established tumor lesions at the base, side, and top of the bladder, but not in non-tumorous tissues (Figure 6A). Therefore, the absence of GNR@Chit-Iso4 binding in healthy mice and GNR@Chit-Cys binding in tumor-bearing mice suggests that the Iso4 peptide binds to the α5β1 ...+ Interestingly, the binding of GNR@Chit-Iso4 to MB49-Luc tumors was reduced by coadministration of excess free Iso4 peptide, but not ARA peptide (Figure 19), suggesting that α5β1 + We confirmed that the binding of GNR@Chit-Iso4 to cells was mediated by the Iso4 peptide.

[0132] Next, we verified whether our technological platform can be utilized for the detection of small superficial tumors. To this end, we analyzed the binding of GNR@Chit-Iso4 in mice bearing small orthotopic tumors (i.e., 4 days after MB49-Luc cell implantation). Two representative animals are reported, showing that GNR@Chit-Iso4: i) 7.9 mm in volume; 3 (equivalent to 79 μl) that later developed into tumors (Figures 6B and 6C), and i) a neoplastic area in the upper region of the bladder that was not detectable by US scanning (Figure 6D) but later developed into a tumor (Figure 6E). These data suggest that the developed technology platform can recognize small, flat bladder cancers that cannot be detected by bioluminescence or US imaging.

[0133] Finally, we demonstrated that the nanoconstructs were reproducible and stable, with aliquots successfully tested for over 3 years stored at +4°C.

[0134] Therefore, we demonstrated the safety and feasibility of our platform for detecting bladder tumor lesions. By designing nanoparticles, specifically gold nanorods linked to peptides targeting α5β1 integrin, we were able to accurately identify α5β1 integrin-positive bladder tumors, such as human bladder CIS.

[0135] The main clinical limitations of this tumor imaging method relate to the heterogeneity of tumor marker expression among bladder cancer patients and the depth of imaging with PAIs. For example, expression of α5β1 was observed in 6 of 8 (75%) specimens from patients diagnosed with bladder CIS, similar to previously reported findings for cytokeratin 20 in bladder CIS

[52] or EPCAM and μPAR in MIBC

[53] . Combining two or more targeting ligands conjugated to the same or different GNRs may potentially be utilized to reach all bladder CIS. In this preclinical study, a 40 MHz central frequency transducer was used, which achieved a spatial resolution of 40 μm and an imaging depth of 15 mm. To move to clinical studies, lower frequency transducers will be investigated to achieve the imaging depth required for human studies. In animal models of bladder cancer, the GNRs described in this invention were able to detect bladder lesions less than 1 mm in size early in patients. Due to the heat-emitting properties of GNRs, these particles may also open new avenues for the early detection and treatment of bladder cancer [ 33 , 54 ].

[0136] The results of this study demonstrate the feasibility of using the α5β1-targeted GNR@Chit-Iso4 conjugate as a photoacoustic imaging agent for the early diagnosis of bladder cancer. Given that the α5β1 integrin is also expressed by endometrial tumors

[55] , gastric cancer

[56] , breast cancer

[57] , and tumor neovasculature [58, 59], the photoacoustic imaging method described herein may also be applicable to the diagnosis of other solid neoplasms.

[0137] The particles according to the present invention are used for the photothermal ablation of solid tumors.

[0138] The cystoscope injects the GNRs and shines light near the urothelium. Under ultrasound guidance, the optical probe (on the cystoscope) is moved along the entire bladder to identify small lesions revealed by photoacoustic imaging of the tumor-bound GNRs. Continuous light irradiation irradiates the bound GNRs to the target area. Assisted photothermal therapy is produced by exciting the particles with an 808 nm wavelength. [Example]

[0139] Reagents: Bovine serum albumin (BSA) fraction V, lipopolysaccharide from Escherichia coli O111:B4, and other reagents were all obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Medical-grade, endotoxin-free chitosan prepared from snow crab (87.6% deacetylation, 66 mPa.s viscosity at 20°C) was obtained from ChitoLytic (St. John's, Newfoundland, Canada). Maleimide-PEG 12 -NHS (1-maleimido-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-4-azatritetracontan-43-oic acid succinimidyl ester, 99%) was obtained from Iris Biotech GmbH (Marktredwitz, Germany). Wild-type human integrin α5β1 (octyl β-D-glucopyranoside preparation) was obtained from Immunological Sciences (Rome, Italy). The head-to-tail cyclized peptides c(CGARAG) and c(CGARAG), designated Iso4 and ARA, respectively, were obtained from Biomatik (Delaware, USA). The identity and purity of Iso4 and ARA were confirmed by mass spectrometry and HPLC analysis (expected / observed monoisotopic mass, MH). +The Da / Da values ​​were 622.35 / 622.40 and 516.23 / 561.23, respectively; purity was >95% for both. The peptide was dissolved in sterile water and stored in aliquots at -80°C. Human serum albumin (HSA) was obtained from Baxter (Deerfield, IL). Iso4-HSA conjugate (Iso4 chemically coupled to HSA via 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC)) and *HSA (HSA activated with sulfo-SMCC and quenched with β-mercaptoethanol instead of Iso4) were prepared as described

[26] .

[0140] Synthesis of GNR@CTAB with maximum absorption wavelength at 800 nm. Cetyltrimethylammonium bromide (CTAB)-coated GNRs (GNR@CTAB) with an absorption maximum of 800 nm were prepared using a previously published procedure [1] with minor modifications, using a volume of approximately 2 L. The initial solution for GNR growth was prepared by dissolving CTAB (31.14 g, 85.4 mmol) and sodium oleate (4.28 g, 14.0 mmol) in 1.8 L of warm water (approximately 50 °C) in a thermostatically controlled 2 L jacketed reactor equipped with mechanical stirring. When the solution reached 30 °C, 810 μL of AgNO3 solution (0.4 M in ultrapure water) was added, and the mixture was then incubated for 15 min without stirring. Next, 8.652 mL of HAuCl4 solution (0.1 M in ultrapure water) was added under continuous stirring (700 rpm). Au(III) was reduced to Au(I) using sodium oleate for 90 min. Then, 3.56 mL of hydrochloric acid (37%) and 3.6 mL of ascorbic acid (0.079 M) were added to adjust the pH to 1.0 to ensure complete reduction of the gold precursor. Separately, a seed solution was prepared by dissolving 364 mg of CTAB (1.0 mmol) in 10 mL of warm water in a 50 mL round-bottom flask. After cooling to room temperature, 25 μL of HAuCl4 solution (0.1 M) was added with stirring. Seeds were formed by quickly injecting 600 μL of ice-cold sodium borohydride (0.01 M) into the solution. The solution color changed from yellow to brown, indicating the formation of ultrasmall gold seeds. Finally, after aging the seed solution at room temperature for 30 min, 690 μL of the solution was added to the growth solution, vigorously stirred for 30 s, and then left at 30 °C overnight to promote GNR growth. The GNR@CTAB was purified by: i) centrifugation in a 50 mL Falcon tube (6,000 rpm, 100 min), ii) removal of 40 mL of the supernatant, and iii) redispersion in 40 mL of ultrapure water. This process was repeated three times. The final product was then recovered in 200 mL of ultrapure water. The gold concentration in the GNR@CTAB was determined by atomic absorption spectroscopy to be 2.09 mM.

[0141] Coating of GNRs with chitosan (GNR@Chit). Medical-grade chitosan (500 mg, 3.1 mmol) was dissolved in 50 mL of 1% acetic acid (volume) and mixed with 0.5 mL of thioglycolic acid (7.2 mmol) while stirring at medium speed. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (500 mg, 2.6 mmol) was then added to activate the carboxyl group of the thioglycolic acid and promote coupling to the amino group of chitosan. The reaction was incubated at room temperature for 6 hours under continuous stirring. The mixture was then dialyzed overnight against ultrapure water using 3.5 kDa cutoff dialysis tubing, and the resulting thiolated chitosan was diluted to 500 mL with water. At this point, 30 mL of GNR@CTAB was added dropwise with gentle stirring, and the resulting solution was incubated (48 hours at room temperature) to couple the thiolated chitosan to the GNRs. The product was then concentrated using an Amicon® stirred cell equipped with a PES membrane (100 kDa cutoff, using 4 bar nitrogen pressure) to remove CTAB, and the final product (60 mL), designated GNR@Chit, was stored at +4 °C until the next step.

[0142] Functionalization of GNR@Chit with Iso4 or Cys GNR@Chit was prepared from cetyltrimethylammonium bromide (CTAB)-coated GNRs (GNR@CTAB; synthesis and characterization are detailed in the Supporting Information). To functionalize GNR@Chit with Iso4, 30 mL of maleimide-PEG was added. 12 Mix 30 mL of GNRs@Chit (1 mg / mL in ultrapure water, 35 μmol = 29.7 mg) with 30 mL of GNRs@Chit (1 mM Au, 30 μmol = 5.909 mg Au) under stirring until the final mass ratio reaches maleimide-PEG 12 The ratio of NHS to Au was adjusted to 5:1. The mixture was then incubated overnight at room temperature and dialyzed against ultrapure water using a 3.5 kDa cutoff dialysis tube for 24 hours at room temperature to remove excess crosslinker. The activated GNR@Chit-PEG 12The product (65 mL) consisting of GNR@Chit-Iso4 was then mixed with Iso4 peptide (21 mg, 34 μmol, 10 mg / mL in ultrapure water) and allowed to react for 24 hours at room temperature. Excess cysteine ​​hydrochloride (18 mg, 102 μmol) was then added to quench any unreacted maleimide groups. The product, designated GNR@Chit-Iso4, was then dialyzed against ultrapure water, its Au content quantified, and aliquoted into vials containing 50 μg of Au. The product was lyophilized and stored at -80 °C. In parallel, control nanoparticles with cysteine ​​instead of Iso4 were prepared as above, but with excess cysteine ​​(36 mg, 204 μmol) instead of the Iso4 peptide. This product was designated GNR@Chit-Cys. Approximately 50 vials of both products were prepared.

[0143] Characterization of the physicochemical properties of GNR@CTAB, GNR@Chit, and functionalized GNR@Chit-Iso4 Gold concentrations were measured by flame atomic absorption spectrometry (FAAS) using a SpectraAA 100 Varian photometer (Agilent Technologies, Santa Clara, USA). Gold nanorods (100 μL) were dissolved in aqua regia (3 mL) and diluted to 10 mL with ultrapure water before analysis. For calibration of the FAAS analysis, Au standard solutions of 1, 2, 5, and 10 mg / L were prepared by diluting the appropriate volume of a 1000 mg / mL TraceCERT® solution with 30% aqua regia.

[0144] VIS-NIR absorption spectra (range 400–1100 nm) were recorded using a Cary 3500 UV-VIS-NIR modular spectrophotometer (Agilent Technologies, Santa Clara, USA) using plastic cuvettes with a 1 cm path length.

[0145] Transmission electron microscopy (TEM) was performed using a TEM / STEM FEITECNAI F20 operating at 200 keV and equipped with probes for energy dispersive X-ray spectroscopy (EDX), selected area electron diffraction (SAED), and a high-angle annular dark-field detector (HAADF). Prior to analysis, samples were placed on a continuous carbon film, supported on a copper grid, and dried at 120 °C.

[0146] 1 H-NMR spectra were obtained using a Varian Inova NMR spectrometer (14.09 T, 600 MHz). Chemical shifts are reported in ppm of frequency relative to the residual solvent signal (H-NMR: 4.80 ppm relative to deuterium oxide).

[0147] Viscosity measurements were performed using an MCR102 (Anton-Parr, Graz, Austria) modular compact rheometer with DPP25-SN0 geometry, i.e., a double plate geometry with a diameter of 25 mm.

[0148] Zeta potential measurements were performed at 25°C using a Zetasizer-nano-S (Malvern Panalytical, Malvern, UK) with a DTS1060C-Clear disposable zeta cell.

[0149] Thermogravimetric analysis (TGA) was performed using a Q600 Thermoscale (TA Instruments, New Castle, USA) in a nitrogen atmosphere with a heating gradient of 20°C / min from room temperature to 600°C, followed by switching to air and holding at 600°C for 15 min.

[0150] Stability of GNR@Chit and GNR@Chit-Iso4 in human urine The stability of GNR@Chit-Iso4 in human urine was evaluated by diluting a GNR solution (0.725 mM Au in 10 mL of water) with 10 mL of human urine samples collected from 10 healthy adult volunteers (4 males, 6 females) and placing them under stirring in a water bath at 37 °C. At various time points, 1 mL of the mixture was removed, diluted with 10 mL of cold water (+4 °C), and subjected to VIS-NIR. Urine solution (50% in water) was used as a blank standard for spectroscopic analysis.

[0151] cell line Two types of human primary bladder epithelial cells (ATCC catalog no. PCS-420-010; CELLnTEC catalog no. HBLAK) were cultured according to the manufacturer's instructions and used at passage four. Human bladder cancer cell lines RT4, 5637, and HT-1376 were obtained from ATCC (catalog nos. HTB-2™, HTB-9™, HTB-4™, and CRL-1472™, respectively), and the RT112 cell line was obtained from Merck (catalog no. 85061106). These cell lines were cultured in RPMI medium (Gibco; Thermo Fisher Scientific) supplemented with standard supplements. Mouse bioluminescent MB49-Luc cells were kindly provided by Professor Carla Molthoff (VU University Medical Center, The Netherlands) and were cultured in DMEM medium (Gibco; Thermo Fisher Scientific) supplemented with standard supplements. For this cell line, a cell bank was prepared and the absence of cross-contamination was confirmed by analysis of nine short tandem repeat DNA

[27] (IDEXX Bioanalytics, Ludwigsburg, Germany). Vials from the cell bank were used to initiate new experiments. Cells were routinely tested for mycoplasma contamination and cultured for no more than 4 weeks before use.

[0152] Human data collection Collection of human data followed the principles outlined in the Declaration of Helsinki. Patients signed informed consent and agreed to provide their anonymized information and tissue samples for future research. This study was approved by the Institutional Review Board (Ethic Committee IRCCS Ospedale San Raffaele, Milan, Italy). All methods were carried out in accordance with approved guidelines.

[0153] Surgical specimens were staged according to the TNM classification

[28] . Paired non-tumorous and tumorous bladder areas were collected from the same bladders of individuals who underwent TURB or radical cystectomy for bladder cancer; six TURBs with histologically diagnosed CIS and two bladders with non-muscle-invasive bladder cancer (NMIBC; CIS, pTa, pT1) and muscle-invasive bladder cancer (MIBC; pT2-pT4) were used.

[0154] Immunohistochemical analysis Tissue samples were fixed in 10% buffered formalin at room temperature for 24–48 h, embedded in paraffin, and stained as previously described

[29] . Briefly, 3 μm tissue sections were dehydrated according to standard procedures and boiled twice for 5 min in 0.25 mM EDTA pH 8.0 in a microwave oven (780 W). After washing with PBS and quenching endogenous peroxidase with 3% H2O2, the tissue sections were incubated with anti-α5 or anti-β1 rabbit monoclonal antibodies (see Table 5 below) for 2 h at room temperature.

[0155] [Table 5]

[0156] After washing, the binding of the rabbit primary antibody was detected using the Universal HRP-Polymer Biotin-free Detection System (MACH4, BioCare Medical, USA) and 3,3-diaminobenzidine-free base (DAB) as the color developer, and the tissue samples were then counterstained with Harris' hematoxylin.

[0157] FACS analysis Staining of cell surface-expressed α5 and β1 integrins was performed as described

[30] using 5 μg / ml of the monoclonal antibodies listed in Table 6. Isotype-matched antibodies were used as negative controls. Primary antibody binding was detected using Alexa Fluor 488-conjugated goat anti-mouse or hamster secondary antibodies, depending on the animal species.

[0158] [Table 6]

[0159] Binding assay of Iso4-Qdot to MB49-Luc and 5637 cell lines Iso4 and ARA were conjugated to amino-functionalized quantum dot nanoparticles, Qdot605 ITK Amino PEG (Thermo Fischer), as previously described

[30] . The binding of Iso4-Qdot and ARA-Qdot to MB49-Luc and 5637 cells was assessed by FACS and fluorescence microscopy. FACS analysis was performed as follows: cells were detached with DPBS containing 5 mM EDTA pH 8.0 (DPBS-E), washed with DPBS, and then incubated in 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, 1% BSA (binding buffer 1) and Iso4-Qdot or ARA-Qdot (range 30–0 nM, 5 × 10 5 The cells were suspended in 25 mM Hepes buffer, pH 7.4, containing 100 μl of 5637 cells / 100 μl tube. After 2 h of incubation at 37°C, the cells were washed with binding buffer 1 without BSA and fixed with 4% formaldehyde. Bound fluorescence was detected using a CytoFLEX S cytofluorimeter (Beckman Coulter). The binding of Iso4-Qdot and ARA-Qdot to live 5637 cells was analyzed as follows: 5637 cells were plated in a 96-well clear-bottom black plate (5 × 10 4The plates were incubated with 100 μg of Iso4-Qdot or ARA-Qdot solution (30 nM in Binding Buffer 1) for 48 hours at 37°C with 5% CO2. The plates were washed with Binding Buffer 1 and incubated with Iso4-Qdot or ARA-Qdot solution (30 nM in Binding Buffer 1) for 2 hours at 37°C with 5% CO2. The cells were then washed with Binding Buffer 1 and fixed with 3% paraformaldehyde and 2% sucrose for 20 minutes. Binding fluorescence was acquired using a Cellomics ArrayScan XTI Studio Scan (Thermo Fischer Scientific) system.

[0160] α5β1 integrin binding assay Iso4 and ARA were chemically conjugated via thiol groups to maleimide-activated HRP (InnovaBioscience, catalog no. 401-0002) as follows: A vial of lyophilized product was suspended in 1 ml of PBS (10 mM sodium phosphate buffer, pH 7.4, 138 mM sodium chloride, 2.7 mM potassium chloride, Sigma P-3813) containing 5 mM EDTA (PBS-E). This solution was divided into two aliquots and mixed with Iso4 or ARA peptide at a peptide:enzyme ratio of 3:1 (mol / mol). The final products were designated Iso4-HRP and ARA-HRP. The conjugate was diluted (ranging from 0 to 400 nM) in 25 mM Tris-HCl, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, and 1% BSA (binding buffer 2) and added to a 96-well polyvinyl chloride (PVC) microtiter plate (Carlo Erba, code: FA5280100) coated or uncoated with α5β1 (4 μg / ml). The plate was washed with 25 mM Tris-HCl, pH 7.4, containing 150 mM sodium chloride, 1 mM magnesium chloride, and 1 mM manganese chloride, and bound peroxidase was detected by adding a color development solution (o-phenylenediamine dihydrochloride, OPD). The color reaction was stopped by adding 1 N sulfuric acid. The absorbance at 490 nm was then measured using a microtiter plate reader. The effect of human urine (from healthy donors) on the binding of Iso4-HRP was investigated as described above by mixing Iso4-HRP (final concentration 300 nM) with various amounts of urine diluted in binding buffer 2.

[0161] Cell adhesion assay Iso4-HSA or GNR@Chit-Iso4 was coated onto 96-well PVC microtiter plates in 50 mM sodium phosphate (pH 7.3) containing 150 mM sodium chloride (incubated overnight at 4°C). The plates were washed and blocked with 2% BSA in DMEM or RPMI-1640 (200 μl / well, 1 h). MB49-Luc and 5637 cells were detached with DPBS-E, washed twice with DPBS, and then suspended in DMEM or RPMI-1640 containing 0.1% BSA (binding buffer 3) and added to the coated plates (1.5 × 10 5 After incubation at 37°C and 5% CO2 for 1–2 h, the plates were washed with binding buffer 3. Adherent cells were fixed and stained with 0.5% crystal violet in 20% methanol. After washing with water, the dye was extracted from the cells using a 10% acetic acid solution (140 μl / well), and the absorbance at 570 nm was measured using a microplate reader. The effect of urine on the adhesion-promoting properties of GNR@Chit-Iso4 was investigated as described above, except that cells were seeded in binding buffer 3 or binding buffer 1 plus various amounts of human urine (obtained from healthy volunteers).

[0162] Quantification of Iso4 peptide bound to GNR@Chit The amount of Iso4 loaded on GNR@Chit-Iso4 was quantified by measuring the total amino acid content after acidic hydrolysis of the nanoparticles by Alphalyse Inc., Denmark.

[0163] Mouse orthotopic bladder tumor model All procedures and studies involving mice were approved by the Institutional Animal Care and Use Committee of the San Raffaele Scientific Institute and were performed in accordance with the applicable guidelines (IACUC, approval number 942). Female albino C57BL / 6J mice (9 weeks old, weighing approximately 20 g, Charles River Laboratories, Italy) were anesthetized with ketamine (80 mg / kg) and xylazine (15 mg / kg) and kept in the supine position. Using a 24-gauge catheter, the bladder of each mouse was emptied and MB49-Luc cells (10 in DPBS) were inserted. 5 The mice were injected with or without 100 μl of 100 μg ...

[0164] US and PAI for GNR@Chit and GNR@Chit-Iso4 High-resolution US and PA images were acquired using the Vevo LAZR-X platform (FUJIFILM VisualSonics, Inc., Toronto, ON, Canada). This imaging platform combines a high-frequency US system (Vevo3100) with a 20 Hz repetition rate Nd:YAG nanosecond pulsed laser. The linear US transducer array, Mx 550D, consists of 256 elements with a nominal center frequency of 40 MHz (25–55 MHz bandwidth) and a spatial resolution of 40 μm with a maximum imaging depth of 15 mm. Light from the laser is directed into the tissue through optical fibers attached to either side of the transducer. For volumetric US-PA acquisition, a stepper motor is used to linearly move the US transducer and optical fiber along the sample. The linear stepper motor moves in minimum 0:1 mm steps while capturing 2D parallel images, with a maximum 3D coverage distance of 6.4 cm.

[0165] For in vitro (droplet) and in vivo (mouse bladder) studies, 3D B-mode scans were performed. Photoacoustic spectra were scanned between 680 nm and 970 nm with a 5 nm step size. For in vivo studies (mouse bladder), 3D multispectral PA scans were acquired, and PA spectral curves for tissue components melanin, deoxy- and oxy-generated blood, and GNRs were selected. The processing wavelengths (680; 722; 764; 810; 924; 970 nm) were automatically selected from spectral curves used to spectrally resolve GNR signals from other endogenous tissue chromophore signals, such as oxy- and deoxyhemoglobin. The algorithm reported by Luke et al.

[31] was used to select these wavelengths, ideal for separating the signal from GNRs from other endogenous absorbers. For in vitro studies (agar droplets embedded in mucus), 3D multispectral PA scans were acquired using the PA spectral curves for mucus and GNRs (processing wavelengths 680; 782; 810 nm).

[0166] Data analysis was performed using Vevo® Lab software. Volumes of interest (VOIs) were obtained by manually drawing volumes of interest (VOIs) on 3D B-mode images. The contents of GNRs, melanin, oxyhemoglobin, and deoxyhemoglobin were estimated by spectral unmixing analysis of the spectroscopic data.

[0167] Optical Attenuator Because GNRs are prone to shape changes at high laser thresholds, optical attenuators were prepared to reduce the laser fluence to avoid GNR reshaping. The optical attenuators were prepared as follows: Agar powder (catalog no. A9539, Sigma) was suspended in deionized and distilled water (final concentration 1%), melted at 95°C, and mixed with different concentrations of Intralipid (catalog no. I141, Sigma). The mixture (3 ml) was poured into disposable base molds (30 x 24 x 5 mm, Bio-Optica, Milan, Italy), allowed to solidify at room temperature for 2 minutes, and stored in a humidified chamber until use. The optical attenuators were then sliced ​​and attached to optical fibers.

[0168] In vitro PAI of GNRs was performed as follows: GNRs (30 μl in DPBS containing calcium and magnesium) were mixed with 1% agar solution (30 μl). The mixture was then poured onto Parafilm® M (Sigma) and left to solidify in a humidified chamber (Figure 15). The solidified product (referred to as an "agar droplet") was then placed on an ultrasound gel pad (Aquaflex, Parker) and embedded in mucus (mucus barrel) made of polyvinyl alcohol polymer cross-linked with sodium tetraborate (Figure 15). PAI was performed using two optical fiber-attached optical attenuators. The transducer was placed perpendicular to the investigation object, which had previously been coated with ultrasound transmission gel (Aquasonic100, Parker). Axial sections were acquired using the following settings: B-mode: 2D Power 100%, 2D Gain 13 dB; PA-mode: PA Power 100%, PA gain 44 dB; TGC and depth were kept the same for all droplets. PA and US data were analyzed using VevoLab 3.2.5 software (Figure 15).

[0169] Light fluence. The laser energy at wavelengths of 750, 800, and 850 nm was measured for 2 minutes using a laser energy meter (PE50BF-DIFH-C, P / N 7Z02943, Ophir, Germany). The laser beam size was evaluated by irradiating the laser for 5 seconds onto a piece of photographic paper (Kodak Linagraph Type 1895) placed 8 mm from the light source. The resulting burn area was then quantified with a ruler (Figure 15), and the light fluence was calculated by dividing the light energy by the light beam size.

[0170] Simulation of energy transport in the mouse urinary bladder. Numerical simulation of light fluence was performed to estimate the energy distribution within the tissue domain. The light energy distribution was obtained by implementing a Monte Carlo model of light transport based on MCXLAB computer simulation. The optical properties (absorption coefficient (μ)) of the skin, peribladder tissue, urine, and GNRs used in the simulation were a ), scattering coefficient (μ s ), scattering anisotropy coefficient (g), and refractive index (n) have been recently described

[33] and are reported in Table 7. Fluence simulations consider a Gaussian source within a tissue domain of 120 × 120 pixels, with a fluence of 10 at 800 nm. 9 I went with Photon.

[0171] [Table 7]

[0172] Ethical Approval and Consent to Participate. Human data collection followed the principles outlined in the Declaration of Helsinki. Patients signed informed consent and agreed to provide their anonymized information and tissue samples for future research. This study was approved by the Institutional Review Board (Ethic Committee IRCCS Ospedale San Raffaele, Milan, Italy). All methods were performed in accordance with the approved guidelines. All procedures and studies involving mice were approved by the Institutional Animal Care and Use Committee of the San Raffaele Scientific Institute and were performed in accordance with the prescribed guidelines (IACUC, approval number 942). (References) TIFF2025532274000022.tif246169TIFF2025532274000023.tif240165TIFF20255322740 00024.tif245165TIFF2025532274000025.tif245165TIFF2025532274000026.tif118165

Claims

1. 1. A metal-based nanoparticle, comprising: - coated with a polymer functionalized with thiol and -NH groups, said polymer being preferably selected from among thiolated chitosan, thiolated and aminated alginic acid, thiolated and aminated hyaluronic acid, or a protein, preferably selected from the group consisting of albumin and gelatin, or a synthetic thiolated and aminated polymer, preferably α-thio-ω-amino polyethylene glycol; - said group is linked via a heterobifunctional crosslinker to a ligand of an integrin family receptor, preferably a peptide containing an integrin-binding motif, and to an antibody or antibody part, peptidomimetic or aptamer, - the crosslinking agent preferably comprises a functional group capable of binding to an amino group, selected from the group consisting of an N-hydroxysuccinimidyl ester group (NHS ester), an isocyanate group (-NCO), an isothiocyanate group (-NCS), a sulfo-N-hydroxysuccinimidyl ester group (sulfo-NHS ester) or a carboxylic acid group which is linked by activation with a carbodiimide coupling agent; and / or a functional group capable of binding to a thiol group, preferably selected from the group consisting of a maleimide group, a terminal vinyl group or a terminal alkyne group; and / or a functional group capable of binding to an alkyne, such as an azide; and / or a metal-based nanoparticle carrying a functional group capable of binding to an azide, such as an alkyne.

2. 2. Metal-based nanoparticles according to claim 1, wherein the metal is selected from among gold, silver or hybrid gold / silver, preferably gold, and the nanoparticles have photoacoustic properties.

3. 3. Metal-based nanoparticles according to claim 1 or 2, wherein the nanoparticles are selected from among nanospheres, nanorods, nanostars, nanocages, nanoprisms or nanoshells or nanowires, nanoplates, hollow shells, preferably nanorods having a size in the range of 10-200 nm in length and 2-50 nm in width, more preferably in the range of 20-100 nm in length and 10-25 nm in width, with a preferred aspect ratio in the range of 1.2-15, more preferably in the range of 3-7.

4. 4. Metal-based nanoparticles according to any one of claims 1 to 3, wherein the functionalized polymer is a thiolated chitosan having an average molecular weight preferably in the range of 0.5 kD to 1000 kDa, more preferably in the range of 50 kDa to 200 kDa, and an average degree of deacetylation in the range of 75% to 100%, more preferably in the range of 85% to 95%.

5. 5. The metal-based nanoparticle of claim 1, wherein the ligand of the integrin family receptor is a peptide containing an RGD or isoDGR motif.

6. The ligands for integrin family receptors are [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, [CphgisoDGRG] peptide of SEQ ID NO: 7, XFETLRGDERILSILRHQNLLKELQD of SEQ ID NO: 8, XFETLRGDLRILSILRHQNLLKEL of SEQ ID NO: 9, XFETLRGDLRILSILRX of SEQ ID NO: 10 1 QNLX 2 KELQD is a peptide selected from wherein "X" is selected from cysteine, lysine, or an unnatural amino acid containing an alkyne or azide group, preferably selected from propargylglycine or azidolysine; X 1 and X 2 6. The metal-based nanoparticle of claim 1, wherein: are propargylglycine and azidolysine, respectively, linked via a triazole bridge.

7. 7. The metal-based nanoparticle of any one of claims 1 to 6, wherein the ligand of the integrin family receptor is the cyclic head-to-tail [CphgisoDGRG] peptide of SEQ ID NO:

7.

8. Crosslinker is maleimide-PEG 12 8. The metal-based nanoparticle of claim 1, wherein the metal-based nanoparticle is an -NHS ester.

9. - The nanoparticles are gold nanorods, - the functionalized polymer is thiolated chitosan, - the peptide is the [CphgisoDGRG] peptide of SEQ ID NO: 7, - Crosslinker is maleimide-PEG 12 -NHS ester, 9. Metal-based nanoparticles according to any one of claims 1 to 8.

10. A composition comprising the metal-based nanoparticles of any one of claims 1 to 9 and at least one of the following solvents: water; physiological solution; Dulbecco's Modified Eagle's Medium (DMEM); Dulbecco's Phosphate Buffered Saline (DPBS), HEPES buffer, TRIS buffer, and PIPES buffer, each containing a divalent metal ion such as Ca2+ or Mg2+.

11. The composition according to claim 10, preferably comprising at least one or more anti-tumor agents selected from among chemotherapeutic agents, immunomodulatory agents, and immune cells.

12. 10. A kit comprising the metal-based nanoparticles according to any one of claims 1 to 9, a single-use vial containing a solvent for resuspending the nanoparticles, preferably water, or a physiological solution, or Dulbecco's Modified Eagle Medium (DMEM), or a buffer solution such as Dulbecco's Phosphate Buffered Saline (DPBS), HEPES buffer, TRIS buffer, PIPES buffer, etc., containing divalent metal ions such as Ca2+, Mg2+, etc., optionally a syringe, and instructions for use.

13. 13. Metal-based nanoparticles according to any one of claims 1 to 9, or a composition according to claim 10 or 11, or a kit according to claim 12, for use in a method of diagnosis and / or treatment in vivo.

14. 14. The metal-based nanoparticle or composition or kit according to claim 13 for use in the in vivo diagnosis and / or treatment of solid tumors.

15. 15. The metal-based nanoparticle or composition or kit according to claim 14, wherein the tumor is selected from among urothelial cancer, bladder cancer, gastroesophageal cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, breast cancer and renal cancer, brain cancer and hepatocellular carcinoma.

16. 16. Metal-based nanoparticles or composition or kit according to any one of claims 1 to 15 for use in the photothermal therapy of solid tumors, preferentially bladder cancer.

17. 1. A method for performing in vitro ultrasound and photoacoustic imaging, comprising at least the following steps: a) applying metal-based nanoparticles according to any one of claims 1 to 9 or a 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 A method comprising:

Images