Water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex as contrast agent for photoacoustic imaging

Abstract

The present invention relates to water soluble chlorophyll binding protein (WSCP)-chlorophyll complexes as contrast agents for photoacoustic imaging. Disclosed is the use of a WSCP-chlorophyll complex in the preparation of a contrast agent for use in a method of photoacoustic imaging of a subject. Also disclosed are WSCP-chlorophyll complexes comprising Lepidium seu Pleurotus North, WSCP, and chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b. The WSCP-chlorophyll complexes of the present invention exhibit strong near infrared (NIR) absorption, unique and non-overlapping spectral characteristics, and concentration dependent photoacoustic (PA) signal generation. These properties make them suitable as contrast agents for PAIs in various applications, particularly in clinical contexts such as disease detection and monitoring.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511196824.5 (22) Application Date 2025.08.26 (30) Priority Data 63 / 686,861 2024.08.26 US (71) Applicant Hong Kong University of Science and Technology Address Clear Water Bay, Kowloon, Hong Kong, China (72) Inventors Huang Ziwei Zeng Zijun Jin Xiayan (74) Patent Agency Beijing Shifeng Intellectual Property Agency Co., Ltd. 11713 Patent Attorney Wang Jianxiu Liu Xiaoli (51) Int.Cl. A61K 49 / 22 (2006.01) (54) Invention Title Water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex as a contrast agent for photoacoustic imaging (57) Abstract This invention relates to water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex as a contrast agent for photoacoustic imaging. The use of WSCP-chlorophyll complexes in the preparation of contrast agents for use in methods of photoacoustic imaging of subjects is disclosed. WSCP-chlorophyll complexes comprising *Lysimachia christinae* WSCP, as well as chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b are also disclosed. The WSCP-chlorophyll complexes of the present invention exhibit strong near-infrared (NIR) absorption, unique and non-overlapping spectral characteristics, and concentration-dependent photoacoustic (PA) signal generation. These properties make them suitable as contrast agents for PAIs in a variety of applications, particularly in clinical settings such as disease detection and monitoring. Claims 2 pages, Description 16 pages, Drawings 14 pages, CN 121588253 A 2026.03.03 CN 1 21 58 82 53 A 1. Use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in the preparation of a contrast agent for use in a method of photoacoustic imaging of a subject, wherein the WSCP-chlorophyll complex comprises Lepidium virginicum WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a and bacterial chlorophyll b, the method comprising: (a) applying an effective amount of the WSCP-chlorophyll complex to a site of the subject; (b) irradiating the subject with light to induce a photoacoustic signal from the WSCP-chlorophyll complex; (c) detecting the photoacoustic signal; and (d) generating a photoacoustic image of the site based on the detected photoacoustic signal. 2. The use according to claim 1, wherein the light is pulsed light with a wavelength of 650 nm to 900 nm, optionally wherein the WSCP-chlorophyll complex exhibits a Qy absorption peak at a pulsed light wavelength of about 665 nm to 850 nm and a Qx absorption peak at a pulsed light wavelength of about 578 nm to 592 nm.3. The use according to claim 2, wherein the WSCP-chlorophyll complex: exhibits a Qy absorption peak at a pulsed light wavelength of about 665 nm; exhibits a Qy absorption peak at a pulsed light wavelength of about 765 nm, and exhibits a Qx absorption peak at a pulsed light wavelength of about 578 nm; or exhibits a Qy absorption peak at a pulsed light wavelength of about 850 nm, and exhibits a Qx absorption peak at a pulsed light wavelength of about 592 nm. 4. The use according to claim 1, wherein the chlorophyll a is spinach (Spinacia oleracea) chlorophyll a, the bacterial chlorophyll a is Rhodospirillum rubrum bacterial chlorophyll a, or the bacterial chlorophyll b is Blastochloris viridis bacterial chlorophyll b. 5. The use according to claim 2, wherein the method further comprises: (b1) irradiating the subject with a first pulse of light of a first wavelength to induce a first photoacoustic signal from the WSCP-chlorophyll complex; (b2) irradiating the subject with a second pulse of light of a second wavelength different from the first wavelength to induce a second photoacoustic signal from the WSCP-chlorophyll complex; (c1) detecting the first photoacoustic signal and the second photoacoustic signal; and (d1) generating a differential photoacoustic image of the site based on the detected first photoacoustic signal and the detected second photoacoustic signal. 6. The use according to claim 5, wherein the first and second wavelengths differ by about 20-235 nm, or about 20-50 nm; or wherein the WSCP-chlorophyll complex exhibits a Qy absorption peak at one of the first and second wavelengths, and exhibits weakened or negligible light absorption at the other of the first and second wavelengths. 7. The use according to claim 5, wherein the first and second wavelengths, or the second and first wavelengths: are about 650 nm to 850 nm and about 685 nm to 900 nm, respectively; about 665 nm and about 685 nm, respectively; about 765 nm and about 800 nm, respectively; or about 850 nm and about 890 nm, respectively, optionally wherein generating the differential photoacoustic image comprises subtracting the second photoacoustic signal from the first photoacoustic signal, or subtracting the first photoacoustic signal from the second photoacoustic signal. 8. The use according to claim 2, wherein the method further comprises: (b3) irradiating the subject with pulsed light prior to application of the WSCP-chlorophyll complex to induce a background photoacoustic signal from the endogenous chromophore at the site; (c3) detecting the background photoacoustic signal; and (d3) based on the detected background photoacoustic signal and the photoacoustic signal detected after application of the WSCP-chlorophyll complex.9. The use according to claim 8, wherein the pulsed light used to irradiate the subject before and after application of the WSCP-chlorophyll complex has the same wavelength; and wherein the wavelength is about 650-850 nm. 10. The use according to claim 8, wherein the wavelength is about 665 nm, about 765 nm, or about 850 nm, optionally wherein generating the differential photoacoustic image comprises subtracting the background photoacoustic signal from the photoacoustic signal detected after application of the WSCP-chlorophyll complex. 11. The use of claim 1, wherein the method comprises: (a4) applying effective amounts of two or more WSCP-chlorophyll complexes to two or more target sites of the subject, wherein each WSCP-chlorophyll complex comprises Lepidium apetalum WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b; (b4) irradiating the subject with pulsed light of two or more excitation wavelengths to induce two or more photoacoustic signals from the respective WSCP-chlorophyll complex; (c4) detecting the photoacoustic signals; and (d4) generating a composite photoacoustic image capable of spatial resolution of different target sites based on the detected photoacoustic signals. 12. The use of claim 11, wherein the two or more excitation wavelengths correspond to the maximum absorbance of each WSCP-chlorophyll complex and are from about 665 nm to 850 nm. 13. The use according to claim 11, wherein the chlorophyll comprises any two or all of spinach chlorophyll a, Rhodospirillum chlorophyll a, and Pleurotus ostreatus chlorophyll b; or the excitation wavelength comprises any two or all of the following: about 665 nm, about 765 nm, and about 850 nm, optionally wherein the subject is a human, a non-human primate, a rodent, a canine, a feline, a bovine, or an equine. 14. Use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in the preparation of a contrast agent for use in a photoacoustic imaging method, wherein the WSCP-chlorophyll complex comprises *Lysimachia christinae* WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b, the method comprising: providing an effective amount of the WSCP-chlorophyll complex; irradiating the WSCP-chlorophyll complex with pulsed light of a wavelength of 650 nm to 900 nm to induce a photoacoustic signal; detecting the photoacoustic signal; and generating a photoacoustic image based on the detected photoacoustic signal. 15. The use according to claim 14, wherein the chlorophyll a is spinach chlorophyll a, the bacterial chlorophyll a is *Rhodospirillum rubrum* bacterial chlorophyll a, or the bacterial chlorophyll b is *Vibrio vulgaris* bacterial chlorophyll b.16. A water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex comprising *Lysimachia christinae* WSCP (LvP); and *Rhodospirillum rubrum* bacterial chlorophyll a or *Phyllostachys glomeratus* bacterial chlorophyll b. Claims 2 / 2 Page 3 CN 121588253 A Water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex as a contrast agent for photoacoustic imaging

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 686,861, filed August 26, 2024. The contents of that application are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to the use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex as a contrast agent for photoacoustic imaging. Background Art

[0004] Photoacoustic (PA) imaging (PAI) has become a powerful non-invasive imaging technique that utilizes the photoacoustic effect to visualize anatomical structures and provide functional information at deeper imaging depths. This technique relies on the absorption of pulsed laser light by endogenous chromophores or exogenous contrast agents within biological tissues, resulting in transient thermoelastic expansion and the generation of ultrasound. These ultrasounds are then detected and analyzed to reconstruct images based on the distribution of absorbing molecules.

[0005] While PAI can function with the aid of endogenous absorbers such as hemoglobin and melanin, exogenous contrast agents can provide enhanced sensitivity and specificity for targeted imaging applications. Biocompatibility, stability, and effective clearance are crucial for the clinical translation of exogenous contrast agents.

[0006] Various materials, including organic dyes, nanoparticles, and gene-encoded proteins, have been explored as PA contrast agents. However, limitations such as photobleaching, toxicity, and complex synthesis processes hinder their widespread application.

[0007] The development and identification of novel substances that can serve as safe and effective contrast agents are of great significance for expanding the clinical application of PAI. Summary of the Invention

[0008] This disclosure provides the use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in the preparation of a contrast agent for use in a method of photoacoustic imaging of a subject, wherein the WSCP-chlorophyll complex comprises Lepidium virginicum WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b, the method comprising: (a) administering an effective amount of the WSCP-chlorophyll complex to a site of a subject; (b) irradiating the subject with light to induce a photoacoustic signal from the WSCP-chlorophyll complex; (c) detecting the photoacoustic signal; and (d) generating a photoacoustic image of the site based on the detected photoacoustic signal.

[0009] In some embodiments, the light is pulsed light with a wavelength of 650 nm to 900 nm.

[0010] In some embodiments, the WSCP-chlorophyll complex exhibits a Qy absorption peak at a pulsed light wavelength of about 665 nm to 850 nm and a Qx absorption peak at a pulsed light wavelength of about 578 nm to 592 nm.

[0011] In some embodiments, the WSCP-chlorophyll complex: exhibits a Qy absorption peak at a pulsed light wavelength of about 665 nm; exhibits a Qy absorption peak at a pulsed light wavelength of about 765 nm and a Qx absorption peak at a pulsed light wavelength of about 578 nm; or exhibits a Qy absorption peak at a pulsed light wavelength of about 850 nm and a Qx absorption peak at a pulsed light wavelength of about 592 nm. Instruction manual 1 / 16 page 4 CN 121588253 A

[0012] In some embodiments, chlorophyll a is spinach (Spinacia oleracea) chlorophyll a, bacterial chlorophyll a is Rhodospirillum rubrum bacterial chlorophyll a, or bacterial chlorophyll b is Blastochloris viridis bacterial chlorophyll b.

[0013] In some embodiments, the method further includes: (b1) irradiating the subject with a first pulse of light of a first wavelength to induce a first photoacoustic signal from the WSCP-chlorophyll complex; (b2) irradiating the subject with a second pulse of light of a second wavelength different from the first wavelength to induce a second photoacoustic signal from the WSCP-chlorophyll complex; (c1) detecting the first photoacoustic signal and the second photoacoustic signal; and (d1) generating a differential photoacoustic image of the site based on the detected first photoacoustic signal and the detected second photoacoustic signal.

[0014] In some embodiments, the first and second wavelengths differ by about 20-235 nm, or about 20-50 nm; or wherein the WSCP-chlorophyll complex exhibits a Qy absorption peak at one of the first and second wavelengths, and exhibits reduced or negligible light absorption at the other of the first and second wavelengths.

[0015] In some embodiments, the first and second wavelengths, or the second and first wavelengths, are about 650 nm to 850 nm and about 685 nm to 900 nm, respectively; about 665 nm and about 685 nm, respectively; about 765 nm and about 800 nm, respectively; or about 850 nm and about 890 nm, respectively.

[0016] In some embodiments, generating a differential photoacoustic image includes subtracting a second photoacoustic signal from a first photoacoustic signal, or subtracting a first photoacoustic signal from a second photoacoustic signal.

[0017] In some embodiments, the method further includes: (b3) irradiating the subject with pulsed light prior to the application of the WSCP-chlorophyll complex to induce a background photoacoustic signal from endogenous chromophores at the site; (c3) detecting the background photoacoustic signal; and(d3) A differential photoacoustic image of the site is generated based on the detected background photoacoustic signal and the photoacoustic signal detected after application of the WSCP-chlorophyll complex.

[0018] In some embodiments, the pulsed light used to irradiate the subject before and after application of the WSCP-chlorophyll complex has the same wavelength; and wherein the wavelength is about 650-850 nm.

[0019] In some embodiments, the wavelength is about 665 nm, about 765 nm, or about 850 nm.

[0020] In some embodiments, generating a differential photoacoustic image includes subtracting the background photoacoustic signal from the photoacoustic signal detected after application of the WSCP-chlorophyll complex.

[0021] In some embodiments, the method includes: (a4) administering effective amounts of two or more water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complexes to two or more target sites of a subject, wherein each WSCP-chlorophyll complex comprises *Lysimachia christinae* WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b; (b4) irradiating the subject with pulsed light of two or more excitation wavelengths to induce two or more photoacoustic signals from the respective WSCP-chlorophyll complex; (c4) detecting the photoacoustic signals; and (d4) generating a composite photoacoustic image capable of spatial resolution of different target sites based on the detected photoacoustic signals.

[0022] In some embodiments, the two or more excitation wavelengths correspond to the maximum absorbance of each WSCP-chlorophyll complex and are from about 665 nm to 850 nm.

[0023] In some embodiments, the chlorophyll comprises any two or all of spinach chlorophyll a, Rhodospirillum chlorophyll a, and Pleurotus ostreatus chlorophyll b; or the excitation wavelength comprises any two or all of the following: about 665 nm, about 765 nm, and about 850 nm.

[0024] In some embodiments, the subject is a human, a non-human primate, a rodent, a canine, a feline, a bovine, or an equine.

[0025] In another aspect, this disclosure provides the use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in a preparation specification (page 2 / 16, CN 121588253 A) as a contrast agent for use in a photoacoustic imaging method, wherein the WSCP-chlorophyll complex comprises *Lysimachia christinae* WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b, the method comprising: providing an effective amount of the WSCP-chlorophyll complex; irradiating the WSCP-chlorophyll complex with pulsed light of a wavelength of 650 nm to 900 nm to induce a photoacoustic signal; detecting the photoacoustic signal; and generating a photoacoustic image based on the detected photoacoustic signal.

[0026] In some embodiments, chlorophyll a is spinach chlorophyll a, bacterial chlorophyll a is Rhodospirillum chlorophyll a, or bacterial chlorophyll b is Green bud Bacterium chlorophyll b.

[0027] In another aspect, this disclosure provides a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex comprising Rhodospirillum rubrum WSCP (LvP); and Rhodospirillum rubrum chlorophyll a or Green bud Bacterium chlorophyll b. Brief Description of the Drawings

[0028] The above-mentioned objects and features of this disclosure, as well as other objects and features, will become apparent from the following description of this disclosure in conjunction with the accompanying drawings.

[0029] Figure 1 shows a PAI system based on a handheld linear array. (a) Schematic diagram of the PAI system. (b) Photograph of the handheld array and fiber bundle setup. (c) Photograph of the AcousticX DAQ system. OPO, Optical Parametric Oscillator; DAQ, Data Acquisition System.

[0030] Figure 2 shows the size exclusion chromatograms of (a) LvP-Chla, (b) LvP-Bchla, and (c) LvP-Bchlb solutions, where the absorption and corresponding Qy peaks of the protein at 280 nm have been normalized.

[0031] Figure 3 shows the absorption spectra of (a) LvP-chla, (b) LvP-bchla, and (c) LvP-bchlb measured using UV-Vis spectroscopy. (d) Superimposed spectra of all three complexes, used to compare their molar extinction coefficients with those of hemoglobin (dashed line) and oxyhemoglobin (solid line).

[0032] Figure 4 shows the spectral characterization results of LvP-chla. (a) UV-Vis absorption spectra of LvP-chla (solid line) and naked LvP (dashed line), showing the characteristic absorption peaks of chlorophyll a. (b) Molar extinction coefficient spectra of oxyhemoglobin (HbO2, solid line) and deoxyhemoglobin (Hb, dashed line) in the visible to near-infrared region. (c) Excitation spectrum of LvP-chla monitored at 672 nm emission, revealing excitation maxima at 437 nm, 619 nm and 663 nm. (d) Superposition of molar extinction coefficient spectra of LvP-chla, HbO2 and Hb, indicating that LvP-chla has better absorption at 665 nm compared to hemoglobin species.

[0033] Figure 5 shows PA phantom imaging at different excitation wavelengths in the NIR range (660 to 900 nm). (a) Normalized PA amplitude plots of LvP complex at different excitation wavelengths, with moving averages. (b) PA image collage of phantoms at different excitation wavelengths: (c) Images of PBS, (d) LvP-Chla, (e) LvP-Bchla and (f) LvP-Bchlb solutions in the corresponding glass capillaries.

[0034] Figure 6 shows the photoacoustic characterization results of LvP-chla and blood phantoms. (a) Linear regression analysis shows the normalized PA amplitude response of different concentrations of LvP-chla under excitation at 665 nm (top point) and 685 nm (bottom point), with blood as a control (first rhombus from top: whole blood 665 nm; second rhombus from top: whole blood 685 nm). (b) Bimodal imaging of phantom samples, showing the ultrasound image (top), PA image at 665 nm (middle), and PA image at 685 nm (bottom). The samples shown are (1) 0.5x blood, (2) whole blood, and LvP-chla at decreasing concentrations: (3) 10 mg / mL, (4) 7 mg / mL, (5) 5 mg / mL, (6) 3 mg / mL, and (7) 1 mg / mL. (c) Linear regression equations and R² values ​​of the normalized PA amplitude response of LvP-chla under excitation at 665 nm and 685 nm. (d) Depth penetration study, showing ultrasound images (left), PA images (middle) and overlays (right) of an LvP-chla phantom (5 mg / mL) obliquely embedded in chicken breast tissue. Dashed boxes indicate phantom locations, and white dashed lines mark the tissue surface. (e) Quantitative analysis of PA signal intensity relative to depth from tissue surface (vertical dashed lines in the right figure of Figure (d), showing imaging depth capability with a half-maximum signal at a depth of 6.30 mm.

[0035] Figure 7 shows the photoacoustic characterization results of the LvP-bchlb phantom. (a) Depth penetration study, showing ultrasound images (left), PA images (middle) and overlays (right) of an LvP-bchlb phantom (5 mg / mL) obliquely embedded in chicken breast tissue. The dashed box indicates the phantom location, and the white dashed line marks the tissue surface. (b) Quantitative analysis of PA signal intensity relative to depth from the tissue surface (vertical dashed line in the right figure of Figure (a)) shows imaging depth capability, with a half-maximum signal at a depth of 12.13 mm.

[0036] Figure 8 shows the cytotoxicity of LvP-chla in 4T1 cells as assessed by CyQUANT MTT cell viability assay. Cells were treated with 0, 1, 3, 5, 7, and 10 mg / mL LvP-chla for 12 hours. Cell viability was quantified as a percentage of untreated control cells (0 mg / mL LvP-chla) after subtracting background absorbance (negative control: medium containing MTT reagent, cell-free). Data are presented as mean ± standard deviation (n = 4). Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, relative to the positive control).

[0037] Figure 9 shows the experimental design and bimodal imaging of LvP-chla delivery in a 4T1 tumor model. (a) shows a schematic diagram of the experimental procedure: (1) subcutaneous injection of 4T1 cancer cells, (2) tumor development phase, (3) initial PAI scan, (4) intratumoral injection of LvP-chla, and (5) subsequent PAI experiment. Imaging data before injection (b-f): (b) gross photograph of tumor-bearing mice, (c) ultrasound image showing tumor morphology in three orthogonal views, (d) PA image at 665 nm, (e) PA image at 685 nm, and (f) differential PA image (665 nm-685 nm). Imaging data after injection (g-k): (g) gross photograph after injection of 200 μL LvP-chla (5 mg / mL in PBS), (h) ultrasound image, (i) PA image at 665 nm, (j) PA image at 685 nm, and (k) differential PA image showing protein distribution. (l) Bar chart showing the mean PA signal of the tumor region at 665 nm, 685 nm and differential imaging before and after LvP-chla injection (average of xy, xz and yz projections). Dashed circles indicate tumor boundaries. Scale bar: gross image is 10 mm.

[0038] Figure 10 shows the spatiotemporal analysis of differential PAI, showing the distribution and clearance of the protein over time. (a) xz projection images before injection and at indicated time points (0, 24, 48, 72 and 96 hours after injection). (b) The normalized PA signal within the target region (dashed circle outline at 0 hours) was fitted to an exponential decay model, yielding a signal half-life (t1 / 2) of 11.6 hours (R2 = 1.000). Scale bar: 10 mm.

[0039] Figure 11 shows H&E stained tissue sections of mice treated with LvP-chla. (a) Heart, showing the right atrial valve (arrow); (b) Spleen, highlighting the nominal white pulp (circle) and megakaryocytes (arrow); (c) 4T1 tumor, showing the necrotic area (circle); (d) Kidney, showing normal glomeruli; (e) Liver, showing normal histological morphology.

[0040] Figure 12 shows the photophysical characterization results of the LvP-Chla complex under different excitation conditions. (a-c) Fluorescence emission spectra after excitation at 437 nm, 619 nm and 663 nm, respectively; (d) Comparison and overlay of emission spectra at the three excitation wavelengths in (a-c); (e) Key photophysical parameters of LvP-Chla; (f) Normalized absorption and fluorescence spectra of LvP-Chla.

[0041] Figure 13 shows the fluorescence and absorption characteristics of the LvP-Chla complex under different excitation conditions. (a-c) Fluorescence and absorption characteristics of the LvP-Chla complex under different excitation conditions.Fluorescence emission spectra after excitation at 357nm, 577nm, and 761nm; (d) Comparison overlay of emission spectra at the three excitation wavelengths in (a-c); (e) Normalized absorption and fluorescence spectra of LvP-Bchla and LvP-chla, with Stokes shifts of 11nm and 28nm respectively. (f) Key photophysical parameters of LvP-Bchla. Detailed Implementation

[0042] Definitions

[0043] Throughout this specification, unless the context otherwise requires, the word "comprise" (comprises, components, etc.) shall be understood to mean including the integers, groups of integers, or elements stated herein, but not excluding any other integers, groups of integers, or elements. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, the term “comprising” can mean “including, included, including, etc.”; and terms such as “consisting substantially of…” allow for elements not expressly stated, but exclude elements found in the prior art or elements that affect the essential or novel features of the invention.

[0044] Furthermore, throughout this specification and claims, unless the context otherwise requires, the word “comprising, including, including, etc.” should be understood to mean including the said integers, groups of integers, or elements, but does not exclude any other said integers, groups of integers, or elements.

[0045] As used herein, the term “subject” refers to an animal, typically a mammal or human, that is about to be or has already been a subject of treatment, observation, and / or experimentation. When the term is used in conjunction with the PAI described herein, the subject is already a subject of observation, monitoring, and / or administration of the compound described herein.

[0046] As used herein, the term "effective amount" means the amount of a complex that generates a detectable acoustic signal after irradiation with pulsed light in a cell culture, tissue system, subject, animal, or human.

[0047] Unless otherwise expressly stated, the singular form used herein also includes the plural form (and vice versa). Furthermore, where the term "about" precedes a quantitative value, the technique also includes the specific quantitative value itself unless otherwise expressly stated.

[0048] As used herein, the term "about" means a variation of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0% compared to the stated value or range, unless otherwise stated or inferred. The range includes the stated value itself. For example, when the range "about 665 nm to 850 nm" is mentioned in the claims or specification, the range is intended to cover variations such as "665 nm ± 5% to 850 nm ± 5%", including variations such as 660 nm toRanges such as 855nm, 665nm to 855nm, 670nm to 855nm, 660nm to 845nm, 670nm to 845nm, and so on. As another example, when referring to "about 665nm," "about 765nm," or "about 850nm," the expression is intended to cover ranges such as "about 665nm ± 5% (including 660nm, 665nm, 670nm, 661nm, 664nm, 667nm, etc.)," ​​"about 765nm ± 5% (including 760nm, 765nm, 770nm, 761nm, 764nm, 767nm, etc.)," ​​or "about 850nm ± 5% (including 845nm, 850nm, 855nm, 847nm, 849nm, 852nm, 854nm, etc.)," ​​and so on.

[0049] As used herein, the term “water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex (or WSCP-chlorophyll complex)” refers to a complex comprising WSCP and chlorophyll (e.g., chlorophyll a, chlorophyll b, bacterial chlorophyll a, bacterial chlorophyll b, etc.), wherein WSCP and chlorophyll may have different sources (e.g., from different parts of the same plant, from different individuals of the same species, or from different species). Unless the context requires otherwise, WSCP and chlorophyll will be understood as being obtained independently, either directly from the organism or synthesized. For example, the WSCP-chlorophyll complex described herein may include *Lepidium apetalum* WSCP reconstructed with chlorophyll from different plants or bacteria, wherein the chlorophyll is obtained directly from the plant or bacteria or synthesized in vitro.

[0050] As used herein, “illuminating a subject with light (thereby inducing photoacoustic signals from the WSCP-chlorophyll complex)”, “illuminating a subject with pulsed light (e.g., first / second pulsed light)” or similar expressions refer to an irradiation process performed on or near the surface of a subject to bring light to a desired site, such as the site where the WSCP-chlorophyll complex is applied or distributed, or the site where the WSCP-chlorophyll complex is intended to be applied or distributed. In some cases where irradiation is performed near the subject's surface, the distance between the subject's surface and the internal imaging location can be approximately 0 mm to approximately 15 mm (e.g., approximately 1–13 mm, 2–12.5 mm, 3–9.5 mm, 4–9 mm, 4.5–8.5 mm, 5–8 mm, 5.5–7.5 mm, 6.3–13 mm, 6–12.5 mm, 6.2–12.2 mm, 6.5–11 mm, 7–11.5 mm, 7.5–10 mm, or any single value or subrange thereof), depending on factors such as the specific device used, the intensity and wavelength of the light, etc. In some cases, the irradiation site is relative to the application site (where the compound is applied locally).In the case of a light source, the distance can be from about 0 cm to about 15 cm (e.g., about 1–13 mm, 2–12.5 mm, 3–9.5 mm, 4–9 mm, 4.5–8.5 mm, 5–8 mm, 5.5–7.5 mm, 6.3–13 mm, 6–12.5 mm, 6.2–12.2 mm, 6.5–11 mm, 7–11.5 mm, 7.5–10 mm, or any single value or subrange thereof), depending on factors such as the specific device used, the intensity of the light, and the wavelength.

[0051] Similarly, “irradiating WSCP-chlorophyll complex with pulsed light” or similar expressions may include a distance of the light source from the WSCP-chlorophyll complex or a sample or container (e.g., a test tube) containing the WSCP-chlorophyll complex from approximately 0.1 cm to approximately 18 cm (e.g., approximately 0.1–5 cm, 0.5–3 cm, 2–3.5 cm, 1–4 cm, 1.5–2.5 cm, 1–12 cm, 2–10 cm, 3–8 cm, 4–7 cm, 5–15 cm, 6–13 cm, or any single value or subrange thereof).

[0052] As used herein, “inducing photoacoustic signals from WSCP-chlorophyll complex,” “inducing a first (or second) photoacoustic signal from WSCP-chlorophyll complex,” or similar expressions, in some cases cover the simultaneous induction of background photoacoustic signals (e.g., photoacoustic signals generated by endogenous absorbers (such as chromophores, such as hemoglobin and melanin)) during irradiation. Therefore, in this context, the expression "detection of photoacoustic signal" or similar terminology may include the simultaneous detection of a signal generated by an exogenous contrast agent (e.g., the WSCP-chlorophyll complex described herein) and a background signal generated by an endogenous absorber (if present). It should be understood that the influence of the background signal can be mitigated or eliminated, for example, by differential photoacoustic imaging, or, where the background signal is inherently weaker than the signal generated by the exogenous contrast agent, its influence may be negligible.

[0053] As used herein, "Qx" refers to the electronic transition dipole moment oriented along the short axis (x-diagonal) of the dihydroporphyrin macrocycle, corresponding to an absorption peak at a relatively short wavelength; "Qy" refers to the electronic transition dipole moment oriented along the long axis (y-diagonal) of the dihydroporphyrin macrocycle, corresponding to an absorption peak at a relatively long wavelength, unless otherwise stated.

[0054] As used herein, the term "differential photoacoustic imaging" refers to an image generated by comparing photoacoustic signals acquired under different conditions, such as (i) before and after the application of a contrast agent using pulsed light of the same wavelength, or (ii) after the application of a contrast agent using pulsed light of different wavelengths. Unless otherwise required, the term may also include other imaging methods that help reduce or eliminate the signal effects generated by endogenous absorbers.

[0055] In a first aspect, this document provides a method for photoacoustic imaging of a subject or the use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in the preparation of a contrast agent for use in a method for photoacoustic imaging of a subject, the method comprising: (a) applying an effective amount of the water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex to a site of the subject; (b) irradiating the subject with light to induce a photoacoustic signal from the WSCP-chlorophyll complex; (c) detecting the photoacoustic signal; and (d) generating a photoacoustic image of the site based on the detected photoacoustic signal.

[0056] In some embodiments, the light is pulsed light, for example, pulsed light with a wavelength of about 650 nm to 900 nm. In some embodiments, the wavelength is from about 665 nm to 890 nm, for example, about 665 nm, about 685 nm, about 700 nm, about 725 nm, about 750 nm, about 765 nm, about 764 nm, about 761 nm, about 800 nm, about 825 nm, about 850 nm, about 852 nm, about 890 nm, or any single value or subrange thereof.

[0057] In some embodiments, the WSCP-chlorophyll complex can be excited at about 650–900 nm or about 665–850 nm.

[0058] In some embodiments, the WSCP-chlorophyll complex exhibits a Qy absorption peak at a pulsed light wavelength of about 665 nm to 852 nm (e.g., about 665 nm to 852 nm, about 665 nm, about 761 nm, about 764 nm, about 765 nm, about 850 nm, or any single value or subrange thereof).

[0059] In some embodiments, the WSCP-chlorophyll complex exhibits a Qx absorption peak at a pulsed light wavelength of 578 nm to 592 nm (e.g., 578 nm, 592 nm, or any single value or subrange between these wavelengths, as specified in page 6 / 16 of CN 121588253 A).

[0060] In some embodiments, the WSCP-chlorophyll complex: exhibits a Qy absorption peak at a pulsed light wavelength of about 665 nm; exhibits a Qy absorption peak at a pulsed light wavelength of about 761 nm, about 764 nm, or about 765 nm, and exhibits a Qx absorption peak at a pulsed light wavelength of 578 nm; or exhibits a Qy absorption peak at a pulsed light wavelength of about 850 nm, and exhibits a Qx absorption peak at a pulsed light wavelength of 592 nm.

[0061] In some embodiments, the WSCP-chlorophyll complex comprises Lepidium apetalum WSCP (LvP) and chlorophyll, for example, LvP reconstructed with chlorophyll. In some embodiments, LvP is non-covalently conjugated to chlorophyll. In some embodiments, the chlorophyll is selected from chlorophyll a (e.g., spinach chlorophyll a) or bacterial chlorophyll a (e.g., Rhodospirillum rubrum bacterial chlorophyll a).The group consisting of bacterial chlorophyll b (e.g., chlorophyll b of *Phyllostachys edulis*).

[0062] In some embodiments, the method further includes: (b1) irradiating the subject with a first pulse of light of a first wavelength to induce a first photoacoustic signal from the WSCP-chlorophyll complex; (b2) irradiating the subject with a second pulse of light of a second wavelength different from the first wavelength to induce a second photoacoustic signal from the WSCP-chlorophyll complex; (c1) detecting the first photoacoustic signal and the second photoacoustic signal; and (d1) generating a differential photoacoustic image of the site based on the detected first photoacoustic signal and the detected second photoacoustic signal.

[0063] In some embodiments, the first and second wavelengths differ by about 20 nm to 235 nm, or about 20 nm to 50 nm. In some embodiments, the first and second wavelengths differ by about 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 100 nm, 150 nm, or greater.

[0064] In some embodiments, the WSCP-chlorophyll complex exhibits a Qy absorption peak at one of the first and second wavelengths, and weakened or negligible light absorption at the other of the first and second wavelengths.

[0065] In some embodiments, the first and second wavelengths, or the second and first wavelengths, are about 650 nm to 850 nm and about 685 nm to 900 nm, respectively. For example, the first and second wavelengths, or the second and first wavelengths, may be about 665 nm and about 685 nm (or 690 nm, 700 nm, 710 nm, 720 nm, 750 nm or greater); about 765 nm (or about 761 nm, about 764 nm) and about 800 nm (or 810 nm, 820 nm, 830 nm, 835 nm, 840 nm, 845 nm or greater); or about 850 nm (or about 852 nm) and about 890 nm (or 900 nm). For example, the first and second wavelengths, or the second and first wavelengths, may be: 665nm and 690nm; 665nm and 700nm; 665nm and 710nm; 665nm and 720nm; 665nm and 750nm, respectively.

[0066] In some embodiments, step (d1) (i.e., generating a differential photoacoustic image) includes subtracting a second photoacoustic signal from a first photoacoustic signal, or subtracting a first photoacoustic signal from a second photoacoustic signal.

[0067] In some embodiments, the method further includes: (b3) irradiating the subject with pulsed light before applying the WSCP-chlorophyll complex to induce a background photoacoustic signal from endogenous chromophores at that site; (c3) detecting the background photoacoustic signal; and (d3) generating the differential photoacoustic image based on the detected background photoacoustic signal and the photoacoustic signal detected after applying the WSCP-chlorophyll complex.Differential photoacoustic imaging of the site. In some embodiments, the pulsed light used to irradiate the subject before and after application of the WSCP-chlorophyll complex has the same wavelength. In some embodiments, the wavelength is 650-900 nm, for example, about 650-850 nm, or about 665-850 nm. In some embodiments, the wavelength is about 665 nm, about 765 nm, or about 850 nm.

[0068] In some embodiments, the endogenous chromophore comprises substances naturally present at the site, such as hemoglobin, melanin, or any combination thereof.

[0069] In some embodiments, step (d3) (i.e., generating a differential photoacoustic image) includes subtracting the background photoacoustic signal from the photoacoustic signal detected after application of the WSCP-chlorophyll complex.

[0070] In some embodiments, the method includes: (a4) applying an amount of two or more water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complexes to two or more target sites of a subject, wherein each WSCP-chlorophyll complex comprises *Lysimachia christinae* WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b; (b4) irradiating the subject with pulsed light of two or more excitation wavelengths to induce two or more photoacoustic signals from the respective WSCP-chlorophyll complexes; (c4) detecting the photoacoustic signals; and (d4) generating a composite photoacoustic image capable of spatial resolution of different target sites based on the detected photoacoustic signals.

[0071] In some embodiments, the chlorophyll comprises any two or all of spinach chlorophyll a, *Rhodospirillum rubrum* bacterial chlorophyll a, and *Phyllostachys pulmonale* bacterial chlorophyll b.

[0072] In some embodiments, the method includes applying two or three WSCP-chlorophyll complexes. In some embodiments, the chlorophyll is selected from the group consisting of chlorophyll a (e.g., spinach chlorophyll a), bacterial chlorophyll a (e.g., Rhodospirillum rubrum bacterial chlorophyll a), and bacterial chlorophyll b (e.g., Pteris viride bacterial chlorophyll b). When applying two WSCP-chlorophyll complexes, one WSCP-chlorophyll complex contains spinach chlorophyll a, and the other contains either Rhodospirillum rubrum bacterial chlorophyll a or Pteris viride bacterial chlorophyll b. When applying three or more WSCP-chlorophyll complexes, the three WSCP-chlorophyll complexes each contain spinach chlorophyll a, Rhodospirillum rubrum bacterial chlorophyll a, and Pteris viride bacterial chlorophyll b, respectively.

[0073] In some embodiments, two or more WSCP-chlorophyll complexes are applied to the same target site. Alternatively, each complex may be applied to a different target site.

[0074] In some embodiments, two or more excitation wavelengths correspond to the maximum excitation wavelength of each WSCP-chlorophyll complex.Absorbance value. In some embodiments, two or more excitation wavelengths are in the range of about 665 nm to 850 nm. In some embodiments, the two or more excitation wavelengths are spectrally different. In some embodiments, the two or more excitation wavelengths include any two of the following: about 665 nm, about 765 nm, and about 850 nm. When three or more WSCP-chlorophyll complexes are applied, the excitation wavelengths include all of the following: about 665 nm, about 765 nm, and about 850 nm.

[0075] In some embodiments, the site is selected from blood, tissue, urine, tumor, kidney, heart, spleen, liver, lung, pancreas, brain or lymph nodes, cancer site, any other diseased site in the subject's body, or any combination thereof. Examples of cancers or tumors may include superficial and subcutaneous cancers (e.g., cervical cancer, skin cancer, thyroid cancer, and malignant brain tumors), breast cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, lung cancer, colorectal cancer, melanoma, bladder cancer, non-Hodgkin lymphoma, endometrial cancer, pancreatic cancer, leukemia, liver cancer, or any combination thereof.

[0076] In some embodiments, the WSCP-chlorophyll complex is administered intravenously, subcutaneously, intraperitoneally, intramuscularly, intratumorally, intra-arterially, topically, or orally. In some embodiments, the WSCP-chlorophyll complex is administered subcutaneously.

[0077] In some embodiments, the WSCP-chlorophyll complex is included in or formulated into a composition (e.g., a pharmaceutical composition). In this case, the composition may be in the form of an aqueous solution, dispersion, or suspension, and may contain a pharmaceutically acceptable carrier. Examples of carriers include physiological saline, antibacterial aqueous solution, or phosphate-buffered saline (PBS).

[0078] In some embodiments, the WSCP-chlorophyll complex is used (e.g., applied) at a concentration of about 1-10 mg / mL (e.g., about 1-7 mg / mL, about 2-6 mg / mL, about 3-8 mg / mL, about 4-5 mg / mL, about 1-5 mg / mL, such as about 3 mg / mL, 4 mg / mL, 5 mg / mL, or any single value or subrange thereof). Where the WSCP-chlorophyll complex is included in or formulated into a composition, the composition may contain the WSCP-chlorophyll complex at a concentration of about 1-10 mg / mL (e.g., about 1-7 mg / mL, about 2-6 mg / mL, about 3-8 mg / mL, about 4-5 mg / mL, about 1-5 mg / mL, such as about 3 mg / mL, 4 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, or any single value or subrange thereof).

[0079] In some embodiments, the methods or uses described herein are applied in scattering media up to about 12.5 mm (e.g., as per the specification).Page 8 / 16, 11 CN 121588253 A A detectable photoacoustic signal is achieved at depths of approximately 12.5 mm, 12.13 mm, and 6.30 mm, where the signal intensity reaches half of its maximum value at these depths. In such embodiments, the scattering medium can refer to an optically opaque material that simulates the light scattering properties of biological tissue. The scattering medium can include ex vivo and in vivo biological tissues, as well as synthetic phantoms designed to simulate such environments. Examples of materials suitable for use as scattering media include intralipid solutions (e.g., 1-2%), agarose or gelatin-based phantoms, latex or polystyrene microspheres, titanium dioxide (TiO2), ex vivo biological tissues (e.g., chicken breast), in vivo tissues, custom-designed tissue-mimicking phantoms, etc.

[0080] In some embodiments, the methods or uses described herein are capable of detecting photoacoustic signals generated by the WSCP-chlorophyll complex at depths of up to about 12.5 mm (e.g., about 12.5 mm, 12.13 mm, 6.30 mm) below the surface of a subject.

[0081] In some embodiments, the WSCP-chlorophyll complex comprises an LvP having four subunits, each subunit being capable of binding up to one chlorophyll molecule. In some embodiments, each subunit binds to the same type of chlorophyll molecule (if present). In some embodiments, a single WSCP-chlorophyll complex comprises one, two, three, or four chlorophyll molecules of the same type.

[0082] In some embodiments, the subject is a human, a non-human primate, a rodent, a canine, a feline, a bovine, or an equine.

[0083] In a second aspect, this document provides a method for photoacoustic imaging or the use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in the preparation of a contrast agent for use in a photoacoustic imaging method, the method comprising: providing an effective amount of the water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex; irradiating the WSCP-chlorophyll complex with light to induce a photoacoustic signal; detecting the photoacoustic signal; and generating a photoacoustic image based on the detected photoacoustic signal. This method can be performed in vitro or in vivo.

[0084] In some embodiments, the light is pulsed light, for example, pulsed light with a wavelength of about 650 nm to 900 nm. In some embodiments, the wavelength is from about 665 nm to 890 nm, such as about 665 nm, about 685 nm, about 700 nm, about 725 nm, about 750 nm, about 765 nm, about 764 nm, about 761 nm, about 800 nm, about 825 nm, about 850 nm, about 852 nm, about 890 nm, or any single value or subrange therebetween.

[0085] In some embodiments, the WSCP-chlorophyll complex is as described in any of the embodiments disclosed herein.

[0086] In some embodiments, the WSCP-chlorophyll complex comprises *Lysimachia christinae* WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a (e.g., spinach chlorophyll a), bacterial chlorophyll a (e.g., *Rhodospirillum rubrum* bacterial chlorophyll a), and bacterial chlorophyll b (e.g., *Phyllostachys viride* bacterial chlorophyll b).

[0087] In some embodiments, the WSCP-chlorophyll complex is included in or formulated into a composition (e.g., a pharmaceutical composition). In this case, the composition may be in the form of an aqueous solution, dispersion, or suspension, and may contain a pharmaceutically acceptable carrier. Examples of carriers include physiological saline, antibacterial water, or phosphate-buffered saline (PBS). In some embodiments, the WSCP-chlorophyll complex is contained in a container (e.g., a test tube).

[0088] In some embodiments, the method further includes irradiating the WSCP-chlorophyll complex with a first pulse of light of a first wavelength to induce a first photoacoustic signal; irradiating the WSCP-chlorophyll complex with a second pulse of light of a second wavelength to induce a second photoacoustic signal; detecting the first and second photoacoustic signals; and generating a photoacoustic image based on the detected first and second photoacoustic signals. In some embodiments, generating a differential photoacoustic image includes subtracting the second photoacoustic signal from the first photoacoustic signal, or subtracting the first photoacoustic signal from the second photoacoustic signal. In some embodiments, the first and second wavelengths are as defined in the first aspect of this disclosure.

[0089] In a third aspect, a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex is provided herein.

[0090] In some embodiments, the WSCP-chlorophyll complex is as defined in any embodiment disclosed herein. In certain embodiments, as described on page 9 / 16 of CN 121588253 A, the WSCP-chlorophyll complex comprises *Lysimachia christinae* WSCP (LvP) and *Rhodospirillum rubrum* bacterial chlorophyll a, or *Phyllostachys glomeratus* bacterial chlorophyll b.

[0091] In a fourth aspect, the use of the WSCP-chlorophyll complex as defined in any of the embodiments disclosed herein is provided as a contrast agent. The contrast agent can be used to image a subject (e.g., to image a disease of the subject), monitor a subject's disease / condition, or diagnose whether a subject has a disease. In some embodiments, the disease includes cancer or a tumor. Examples of cancer or tumors may include superficial and subcutaneous cancers (e.g., cervical cancer, skin cancer, thyroid cancer, and malignant brain tumors), breast cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, lung cancer, colorectal cancer, melanoma, bladder cancer, non-Hodgkin's lymphoma, endometrial cancer, pancreatic cancer, leukemia, liver cancer, or any combination thereof.

[0092] In a fifth aspect, this document provides for the preparation of WSCP-chlorophyll complexes as defined in any aspect of this disclosure.Use in contrast agents. In some embodiments, the contrast agent is used in methods as defined in the first and / or second aspects of this disclosure.

[0093] In some embodiments of any aspect of this disclosure, the WSCP-chlorophyll complex further comprises a targeting portion to facilitate targeted delivery. In some embodiments, the targeting portion is selected from proteins, peptides, antibodies or antigen-binding fragments thereof, ligands, nucleic acid aptamers and any combination thereof. The targeting portion may be directly covalently conjugated to the WSCP-chlorophyll complex (e.g., to WSCP or chlorophyll) or linked to the WSCP-chlorophyll complex via a linker.

[0094] In some embodiments of any aspect of this disclosure, the pulsed light is generated by a laser, a light-emitting diode, a laser diode, a flash lamp or any combination thereof.

[0095] In some embodiments of any aspect of this disclosure, the method further includes displaying a photoacoustic image on a user interface (e.g., a computer).

[0096] This disclosure explores the potential of water-soluble chlorophyll-binding protein (WSCP) as a novel PA contrast agent. This disclosure successfully reconstructs a recombinant WSCP (LvP) derived from *Lepidium apetalum*, with chlorophyll a derived from spinach, bacterial chlorophyll a derived from *Rhodospirillum rubrum*, or bacterial chlorophyll b derived from *Phyllostachys viride*. The reconstructed WSCP (i.e., the complex of this disclosure) exhibits strong NIR absorption due to its ability to bind and stabilize chlorophyll and bacterial chlorophyll molecules.

[0097] As demonstrated in the following examples, the reconstructed complex exhibits strong near-infrared (NIR) absorption with a high molar extinction coefficient and a narrow Qy absorption band, enabling it to penetrate deep tissues and minimize interference from endogenous chromophores. The complex demonstrated concentration-dependent signal generation, enhanced imaging contrast, and effective visualization of tumor sites in both phantom and in vivo experiments. These results confirm that the complex is an effective and desirable contrast agent for PAI, with strong clinical translational potential in non-invasive anatomical and functional imaging.

[0098] Furthermore, toxicity evaluation of the WSCP-chlorophyll complex revealed its excellent biocompatibility, with no observable toxicity or side effects, and effective systemic clearance—highlighting its biosafety for in vivo application.

[0099] The WSCP-chlorophyll complex described herein is capable of detecting photoacoustic signals up to approximately 12.5 mm (e.g., approximately 12.5 mm, 12.13 mm, 6.30 mm) below the surface of a subject. In some embodiments, detectable depths include, for example, approximately 6.30 mm or 12.13 mm. Surprisingly, LvP-Bchlb achieved an imaging depth of 12.13 mm in a biomimetic tissue model, which is almost twice the 6.30 mm depth achieved by LvP-Chla under the same conditions. The improvement in penetration depth is significant, indicating a significant technical advantage for LvP-Bchlb.

[0100] Spectroscopic characterization revealed that LvP-Bchla exhibited a photoluminescence quantum yield (PLQY) of only 3.8%, representing a significant 49% reduction compared to the 7.5% PLQY measured for LvP-Chla. This lower quantum yield is particularly favorable for photoacoustic signal generation, as it indicates that a larger proportion of the absorbed photon energy is converted into heat, thereby enhancing photoacoustic signal generation. The protein scaffold appears to induce different quenching effects depending on the specific pigment, with bacterial chlorophyll undergoing enhanced nonradiative relaxation compared to chlorophyll a. This effect is even more pronounced considering that free bacterial chlorophyll typically exhibits a quantum yield of 16–20% in organic solvents, while free chlorophyll a exhibits approximately 29% PLQY in ethanol. In the complexes disclosed herein, the quantum yield of the bacterial chlorophyll variant decreases sharply, indicating an unexpected synergistic effect. This enhanced thermal conversion efficiency, combined with the redshift absorption curve, gives the bacterial chlorophyll complex excellent photoacoustic properties, enabling the generation of stronger signals at greater tissue depths.

[0101] In summary, these results highlight the potential of the complexes disclosed herein as safe, stable, effective, and reliable PA contrast agents with strong prospects for clinical translation.

[0102] The following are examples illustrating procedures for practicing the invention. These examples should not be considered limiting. Unless otherwise stated, all percentages are by weight and all solvent mixing ratios are by volume.

[0103] Examples

[0104] Materials and Methods

[0105] 1. Contrast Agent Synthesis

[0106] 1.1 Gene Construction and Expression of LvP

[0107] The gene encoding WSCP from LvP (PDB code: 6GIW) reported by Palm et al. (Palm, D.M.; Agostini, A.; Averesch, V.; Girr, P.; Werwie, M.; Takahashi, S.; Satoh, H.; Jaenicke, E.; Paulsen, H. Chlorophyll a / b binding-specificity in water-soluble chlorophyll protein. Nat. Plants 2018, 4, 920-929) was synthesized and cloned into the pET22b expression vector. Escherichia coli strain BL21(DE3) carrying the pET22b::WSCP construct was cultured at 37°C in Luria-Bertani (LB) medium supplemented with 100 mg / L ampicillin until the optical density (OD600) at 600 nm reached a certain value.0.6–1.2. Then, protein expression was induced by adding 400 μM isopropyl β-D-1-thiogalactoside (IPTG) and incubating at 37 °C for 3 hours.

[0108] 1.2. Reconstruction of LvP with photosynthetic pigments

[0109] Based on the method reported by Li et al. (Li, M.; Park, BM.; Dai, X.; Xu, Y.; Huang, J.; Sun, F. Controlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein. Nat. Commun. 2022, 13, 3197.), the photosynthetic pigment chlorophyll a was reconstructed into LvP. Fresh spinach leaves were used as the source of chlorophyll a (chla). The LvP-induced E. coli precipitate was homogenized with spinach leaves at a mass ratio of approximately 5:1 (spinach:E. coli precipitate) and lysis buffer (containing 300 mM sodium chloride, 20 mM Tris buffer, and 0.1 mM PMSF (phenylmethylsulfonyl fluoride), pH 7.5). The mixture was then sonicated using a Branson sonicator at 150 W for 30 seconds at 0.5 s intervals, followed by a 60 s rest period. Two rounds of sonication were performed, each lasting 5 minutes, for a total of 10 minutes.

[0110] For bacterial chlorophyll, phototropic bacteria *Rhodospirillum rubrum* and *Bacillus viridis* containing bacterial chlorophyll were cultured in sodium succinate medium, as previously described by Qian et al. (P. Qian, C.A. Siebert, P. Wang, DPCanniffe, and CNHunter, “Cryo-EM structure of the Blastochloris viridis LH1-RC complex at” Nature, vol. 556, no. 7700, pp. 203-208, Apr. 2018. doi: 10.1038 / s41586-018-0014-5). Then, the cell pellet and *E. coli* pellet expressing Lvp were sonicated in lysis buffer to ensure that Lvp was thoroughly mixed and reconstructed with bacterial chlorophyll (bacillus chlorophyll a, i.e., bchla; or bacillus chlorophyll b, i.e., bchlb). This simple method facilitates the preparation of desired contrast agents suitable for photoacoustic imaging, which can be customized according to the specific chlorophyll type used.

[0111] 1.3. Protein purification: Specification 11 / 16 pages 14 CN 121588253 A

[0112] After sonication, the resulting homogenate was centrifuged at 14,000 × g for 30 min at 4 °C to separate the supernatant containing the reconstituted proteins (i.e., Lvp-chla, Lvp-bchla, or Lvp-chlb) from the insoluble fraction. The soluble reconstituted proteins were then purified using standard nickel-nitrile triacetic acid (Ni-NTA) affinity chromatography on a HisTrap™ HP column (Cytiva, Marlborough, MA, USA). The purification process involved using wash buffer (300 mM NaCl, 20 mM Tris, 25 mM imidazole, pH 7.5) to remove impurities and elution buffer (300 mM NaCl, 20 mM Tris, 500 mM imidazole, pH 7.5) to elute the purified proteins.

[0113] Size exclusion chromatography (SEC) was performed as an additional purification step to further purify the protein formulation. Protein purity was assessed using SDS-PAGE analysis. Following SEC, the eluted protein fractions were analyzed by UV-vis spectroscopy to confirm successful reconstruction of the protein-pigment complex.

[0114] The obtained WSCP contrast agent can be used immediately or flash-frozen in liquid nitrogen and stored at -80°C for future photoacoustic applications. The final reconstructed protein solution was prepared in phosphate-buffered saline (PBS) for injection.

[0115] 1.4. Spectroscopic Measurements

[0116] Using a Varioskan LUX multimode microplate reader with PBS as a blank reference, the absorption spectra of the reconstructed protein solution, as well as oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), and naked LvP (without chla, bchla, or bchlb), were measured. Spectral scans were performed at a resolution of 1 nm in the wavelength range of 300 nm to 900 nm. The concentration of the protein solution was then determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, using Beer-Lambert's law, the molar extinction coefficient was calculated based on the normalized absorbance and concentration.

[0117] 1.5. Measurement of Photoluminescence Quantum Yield (PLQY)

[0118] PLQY was recorded at room temperature using a UV-NIR absolute PLQY spectrometer (Quantaurus-QY, Hamamatsu Photonics KK, Shizuoka, Japan). This instrument uses an integrating sphere with a fluorometer to measure the ratio of emitted photons to absorbed photons. The test procedure includes exciting the sample, measuring the absorbed light, and collecting all emitted photons for quantitative analysis. The quantum yield was then calculated directly, and the resulting value reflects the efficiency comparison between light conversion to fluorescence and conversion to heat.

[0119] 2. PAI Setup

[0120] The PAI system used in this study was based on the configuration described by Wang et al. (Wang, S.; Huang, B.; Chan, SC; Tsang, VT; Wong, TT). Tri-modality in vivo imaging for tumor detection with combined ultrasound, photoacoustic, and photoacoustic elastography. Photoacoustics 2024, 38, 100630. Minor modifications were made to the system to suit small animal imaging (Fig. 1a). The imaging device consisted of a linear array ultrasound transducer (UST), which was coaxially positioned within a water tank with a fiber bundle using a custom 3D-printed adapter (Fig. 1b). An optically transparent film was adhered to the bottom of the tank to facilitate effective coupling of the generated PA signal. During the procedure, tumor-bearing mice were placed in a supine position. Ultrasonic gel was applied directly to the skin of nude mice to enhance acoustic coupling between the tissue and the UT. The fiber bundle transmits pulsed laser excitation light from an optical parametric oscillator (OPO; NT352C-20-FWS-SH-H, EKSPLA Inc., Vilnius, Lithuania) to the imaging area. Data acquisition is managed by a commercial system (AcousticX, Cyberdyne Inc., Tsukuba, Japan) connected to a personal computer (PC), as shown in Figure 1c.

[0121] 3. PA phantom imaging

[0122] Chlorophyll-reconstructed WSCP protein was serially diluted to produce a concentration gradient from 1 mg / mL to 10 mg / mL and carefully loaded into a fine capillary via capillary action to produce a phantom sample for PAI so that phantom images can be obtained using PAI. In addition, 1 mL of mouse whole blood was collected by cardiac puncture and 0.1 mL of ethylenediaminetetraacetic acid solution was added to prevent clotting. A 0.5× diluted blood sample was prepared by mixing 0.5 mL of whole blood with 0.5 mL of PBS and loaded into a separate capillary. The loaded capillary was sealed with glue or an open flame to ensure the sample was sealed. The prepared capillary was placed on a support or platform to ensure stability during the imaging process. Next, the capillary containing the WSCP contrast agent was placed in the imaging field to acquire the photoacoustic signal generated by the contrast agent and the blood sample under laser excitation.

[0123] The imaging process was performed using the setup shown in Figure 1. The imaging system was configured to emit light pulses of a specific wavelength using an OPO laser and detect the photoacoustic signal using a 7 MHz linear array ultrasonic transducer in the AcousticX system.The AcousticX system's built-in software generates the final PA images.

[0124] 4. Animal Imaging

[0125] 4.1. Tumor Mouse Model

[0126] Allogeneic grafts of the 4T1 cell line were established using 4-5 week old Nu / J mice (The Jackson Laboratory, Bar Harbor, ME, USA). Each mouse was subcutaneously injected with 1×10⁶ 4T1 cells suspended in 100 μL PBS at two sites (right hind limb and left forelimb). Mice were housed under standard conditions (temperature: ~22℃, humidity: 40-70%, 12-hour light / dark cycle) with free access to sterile food and water. Tumor growth was monitored regularly, and imaging experiments were performed once the tumor volume reached approximately 2000 mm³. All experiments were conducted in accordance with the experimental animal protocol approved by the Office of Health, Safety and Environment of the Hong Kong University of Science and Technology (Approval No.: AEP-2022-0010).

[0127] 4.2. Image Acquisition

[0128] Tumor-bearing mice were sedated by intraperitoneal injection of a mixture of ketamine / xylazine / saline (KXS), consisting of 17.5% v / v ketamine, 2.5% v / v xylazine, and 80% v / v sterile saline, at a dose of 5 μL / g body weight. Anesthesia was confirmed by pinch reflex test. The mice were then placed supine on the imaging stage. A total of 200 μL of 5 mg / mL LvP-chla solution was injected intratumorally. Ultrasonic gel was applied to the tumor site and surrounding area to promote acoustic coupling. PAI was performed using a 665 nm laser wavelength (corresponding to the maximum absorption value of LvP-chla) to acquire volumetric PA images along the midline towards the tail. Subsequently, the same volumetric scan was repeated using 685 nm laser excitation, at which the molar extinction coefficient of LvP-chla was halved. The laser was operated at a repetition rate of 20 Hz and a pulse width of 3 to 5 ns. The laser fluence on the tissue surface was approximately 16.1 mJ / cm² at 665 nm and approximately 15.6 mJ / cm² at 685 nm, both below the American National Standards Institute (ANSI) safety limit of 20 mJ / cm².

[0129] 5. Toxicity Testing

[0130] 5.1. Cytotoxicity Assay

[0131] The cytotoxicity of LvP-chla was assessed using the CyQUANT MTT Cell Viability Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and 4T1 cells. 4T1 cells were seeded at a density of 3 × 10⁴ cells per well in 96-well plates and in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C, 5%Cells were cultured in a CO2 environment. After 24 hours, the cells were treated with fresh medium containing LvP-chla at concentrations of 0, 1, 3, 5, 7, and 10 mg / mL, repeated 4 times. This included a negative control (medium containing MTT reagent and cell-free medium) and a positive control (untreated cells, 0 mg / mL LvP-chla). After 12 hours of incubation, 10 μL of 12 mM MTT reagent was added to each well, and the plate was incubated at 37°C for 4 hours. The resulting formazan crystals were dissolved by adding 100 μL of the kit's SDS-HCl solution (0.1% SDS in 0.01 M HCl) to each well, followed by incubation at 37°C for 10 min with gentle mixing.

[0132] Absorbance was measured at 570 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Cell viability was calculated as the percentage of the positive control after subtracting the negative control absorbance. Data were analyzed using a one-way ANOVA combined with Dunnett's post-hoc test to compare the treatment group with the positive control group, with a significance level set at p<0.05. Statistical analysis was performed using GraphPad Prism (version 9.0).

[0133] 5.2. Histological Study Specification 13 / 16 pages 16 CN 121588253 A

[0134] To evaluate the potential cytotoxicity of LvP-chla in vivo, major organs (e.g., liver and kidney) and tumor tissues were harvested from euthanized mice 96 hours after injection of 5 mg / mL LvP-chla. Euthanasia was achieved by overdose of the KXS mixture as described above. The harvested tissues were immediately fixed in 4% neutral buffered formalin for 24 hours at room temperature. After fixation, the tissues were processed for 12 hours using a tissue processor (Revos, Thermo Fisher Scientific, Waltham, MA, USA) and then embedded in paraffin. Paraffin-embedded tissue was cut into 5 μm thick sections using a microtome (RM2235, Leica Microsystems, Wetzlar, Germany) and fixed onto glass slides. The tissue sections were stained with hematoxylin and eosin (H&E), and the stained slides were imaged using a digital slide scanner (NanoZoomer-SQ, Hamamatsu Photonics KK, Shizuoka, Japan).

[0135] Results

[0136] 1. Spectral characterization of the reconstructed LvP complexes

[0137] By detecting the characteristic chlorophyll absorption peaks, UV-vis spectroscopy confirmed the successful reconstruction of three LvP complexes (see Figure 1).2). LvP-chla tends to form homotetramers in which the pigment faces inward in the hydrophobic pocket, thus preventing oxidative photobleaching upon excitation by light absorption. As shown in Figure 2, similar homotetramer formation was observed in bacterial chlorophyll variants by size exclusion chromatography, therefore the properties reported in LvP-chla (e.g., resistance to photobleaching and stability at physiological temperatures) are likely to be retained in bacterial chlorophyll variants.

[0138] The absorption spectra of the reconstructed LvP complexes (LvP-chla, LvP-bchla, and LvP-bchlb) as well as HbO2, Hb, and naked LvP were measured using UV-Vis spectroscopy (Figures 3-4). LvP-chla exhibited a strong Qy peak at 665 nm, which is characteristic of chlorophyll a (Figures 3a, 4a), with a molar extinction coefficient (ε) of 59,716 M⁻¹ cm⁻¹. This value is significantly higher than that of Hb and HbO2 at the same wavelength, as shown in the comparative spectra (Fig. 4b, d). The absorbance at 653 nm and 685 nm is reduced by half, forming a narrow Qy band with a full width at half maximum (FWHM) of 20 nm. The excitation spectrum monitored at the emission wavelength of 672 nm (Fig. 4c) reveals multiple excitation peaks, with the maximum values ​​appearing at 437 nm, 619 nm, and 663 nm.

[0139] LvP-bchla shows a Qy peak at 765 nm and a Qx peak at 578 nm, while LvP-bchlb shows a Qy peak at 850 nm and a Qx peak at 592 nm (Fig. 3b-c). The large separation of the Qy and Qx peaks in bacterial chlorophyll reflects its structural differences, which leads to a more significant difference in excitation energy compared to chlorophyll a. These unique spectral properties, particularly for the complexes of bacterial chlorophyll reconstruction, demonstrate that LvP can successfully bind to pigments other than its natural counterparts and highlight their potential for multi-wavelength PAI.

[0140] Furthermore, it is noteworthy that these complexes exhibit higher absorption coefficients compared to abundant endogenous contrast agents (e.g., hemoglobin and deoxyhemoglobin) (Fig. 3d). The high extinction coefficient and Qy peak in the far-red to near-infrared range serve as a bio-optical window, indicating that these complexes have great potential as exogenous contrast agents for in vivo imaging.

[0141] The PLQY of LvP-chla was determined to be 7.5%, and that of LvP-bchla was 3.8%. This indicates that the bacterial chlorophyll complexes have higher thermal conversion efficiency, which is also the reason for their superior photoacoustic performance (see Figs. 12-13). However, due to the extreme redshift in their absorption, the corresponding fluorescence emission peaks are expected to be outside the detection range of existing spectrometers. Based on the consistency of the 28 nm Stokes shift observed for the LvP-bchla complex, the maximum fluorescence emission of LvP-bchlb can be reasonably inferred.The peak occurs at approximately 878 nm (850 nm + 28 nm). This hypothesis assumes a similar pigment-protein dynamic between the bchla and bchlb variants. Due to the enhanced nonradiative decay pathway in NIR, the PLQY of LvP-bchlb is expected to be lower (< 5%, close to 3.8% for LvP-bchla), thus making photoacoustic signal generation superior to fluorescence, which can be verified by NIR-optimized fluorescence spectrometry. 2. Photoacoustic response in the excitation wavelength range

[0142] The PA response of the three LvP complexes (LvP-chla, LvP-bchla, and LvP-bchlb) was evaluated at excitation wavelengths in the range of 660 nm to 900 nm. Figure 5 shows the PA amplitude of each complex as a function of excitation wavelength. Based on the maximum absorbance values ​​of each compound (pages 14 / 16 of the specification, CN 121588253 A), the experimental results confirmed the optimal excitation wavelength for each compound, further highlighting their versatility for multi-wavelength PA imaging. Furthermore, the efficiency of PA wave generation follows the extinction coefficient observed in Figure 3, where the efficiency of the bacterial chlorophyll complex is lower than that of chlorophyll a, with chlorophyll a having an extinction coefficient that is twice that of chlorophyll a. The full width at half maximum (FWHM) of bacterial chlorophyll a and b was measured at Qy peak ± 30 nm, and the full width at half maximum (FWHM) of chlorophyll a was measured at Qy peak ± 20 nm. The FWHM values ​​can be used for spectral PAI of all three contrast agent variants, and they are also orthogonal to each other, providing the possibility of achieving multi-target PAI.

[0143] 3. Concentration-dependent PA signal generation and spectral differentiation from endogenous absorbers

[0144] A phantom study was conducted to evaluate the PA signal generation efficiency of LvP-chla (Figure 6). LvP-chla solutions with increasing concentrations (1 mg / mL to 10 mg / mL) were prepared and imaged using a PAI system. LvP-chla successfully generated PA signals in the concentration range of 1 mg / mL to 10 mg / mL under 665 nm excitation. Figure 6a demonstrates the linear relationship between PA signal amplitude and LvP-chla concentration, indicating effective dissolution and prevention of aggregation.

[0145] In addition, the efficiency of generating PA signals at two close excitation wavelengths, 665 nm and 685 nm, was compared. The selection of 665 nm and 685 nm excitation wavelengths utilizes the maximum absorption peak of LvP-chla spectral characteristics while halving its absorption rate; at the same time, due to their narrow interval, differential imaging with minimal penetration depth variation and consistent laser energy output can be achieved. A linear relationship between PA signal amplitude and LvP-chla concentration was observed for 665 nm and 685 nm excitation, including whole blood and 0.5 × 10⁻⁶ blood as controls (Figures 6a-b). Linear regression analysis yielded high coefficients of determination (R² for 665 nm and 685 nm excitation).=0.9905 and 0.9079), the regression equations are y = 17.40*x - 5.818 and y = 6.104*x - 14.29, demonstrating robust concentration-dependent PA signal generation (Figure 6c).

[0146] Despite a difference of only 20 nm, the signal amplitude at 685 nm decreased to one-third of that at 665 nm (i.e., the maximum absorbance). This significant reduction highlights the sensitivity of PA signal generation to the excitation wavelength. As the concentration of the contrast agent increases, its viscosity also increases, thereby impairing the injectability of exogenous contrast agents. A slight change in the excitation wavelength leads to a rapid decrease in PA signal generation, which addresses the challenge associated with high protein concentrations. By utilizing spectral PAI, the challenge associated with high protein concentrations can be overcome. This technique distinguishes the contrast agent from the background signal even at low concentrations by utilizing the specific spectral characteristics of LvP-chla.

[0147] For the penetration depth study, 5 mg / mL LvP-chla was chosen because it provides sufficient signal intensity while maintaining low viscosity and cytotoxicity, making it suitable for subsequent intratumoral injection. Chicken breast tissue was used as a bioscattering medium to evaluate the penetration depth characteristics (Fig. 6d). Quantitative signal analysis revealed a half-maximum amplitude at a depth of 6.30 mm (Fig. 6e), indicating its good potential for deep tissue imaging applications.

[0148] The penetration depth of LvP-bchlb was studied under essentially the same experimental conditions as LvP-chla (Fig. 7).

[0149] These findings highlight the potential of these three LvP complexes as effective multifunctional PA contrast agents, capable of delivering strong signals at optimal wavelengths while mitigating the problems associated with high-concentration formulations.

[0150] 4. In vitro evaluation of suitable LvP-chla concentrations for PAI

[0151] To evaluate the tolerable dose for in vivo imaging, the cytotoxic effects of LvP-chla on 4T1 cells were evaluated using an MTT assay. A significant concentration-dependent cytotoxic response was observed 12 hours after exposure to LvP-chla (Figure 8). Treatment with lower concentrations (1, 3, and 5 mg / mL) of LvP-chla did not show significant cytotoxicity, maintaining cell viability close to that of the untreated control. Conversely, higher concentrations of LvP-chla (7 and 10 mg / mL) significantly reduced cell viability compared to the untreated control (p<0.05 and p<0.01, respectively). These results indicate that LvP-chla concentrations of 7 mg / mL or higher have significant cytotoxic effects on 4T1 cells, while 5 mg / mL is a safe and effective dose for in vivo studies.

[0152] 5. In vivo application of LvP-chla as a PA contrast agent

[0153] After successful in vitro validation, the efficacy of LvP-chla as an in vivo PA contrast agent was evaluated using a nude mouse 4T1 allogeneic transplantation model. 200 μL of LvP-chla was administered intratumorally at a concentration of 5 mg / mL optimized in the cytotoxicity test, followed by dual-wavelength spectral PAI at 665 nm and 685 nm (Fig. 9). The experimental workflow is shown in Fig. 9a. Baseline imaging prior to LvP-chla administration revealed typical endogenous PA signals at both wavelengths (Fig. 9d, e), with negligible contrast in differential images of the tumor site (Fig. 9f).

[0154] Simultaneous ultrasound imaging (Fig. 9c) provided the necessary anatomical background, enabling precise tumor localization and providing structural information, while also demonstrating the system's dual-modal capability. Post-administration imaging (Fig. 9g-k) demonstrated significant PA signal enhancement within the tumor region, and ultrasound images (Fig. 9h) continued to provide anatomical guidance for precise spatial registration of the PA images. Differential imaging (Fig. 9k) enhances the visualization of contrast agent distribution by effectively eliminating background signals while preserving the structural information provided by ultrasound imaging. As shown in the differential image of Fig. 9l, the average PA signal intensity in the tumor region increased by 6.52 times, due to the strong absorption (59,716 M⁻¹ cm⁻¹) of LvP-chla at 665 nm and effective background subtraction, compared to an increase of 1.54 times at 665 nm and 1.24 times at 685 nm.

[0155] These results highlight the ability of LvP-chla combined with dual-wavelength differential PAI to superimpose high-contrast molecular information onto ultrasound images, representing a significant advancement in the field of dual-modal molecular imaging that combines the specificity of PAI with the anatomical information of ultrasound.

[0156] 6. In vivo clearance of LvP-chla

[0157] To assess in vivo clearance, longitudinal PAI was performed in nude mice bearing 4T1 tumors after injection of LvP-chla, using the pre-injection PA signal as a baseline reference. Differential PA images acquired within 96 hours clearly showed a gradual decrease in signal intensity (Fig. 10). Exponential decay analysis of the normalized PA signal revealed a signal half-life (t1 / 2) of approximately 11.6 hours (R2 = 1.000), with the signal reaching steady state 24 hours post-injection. Importantly, the PA signal recovered to baseline within 96 hours post-injection, indicating that LvP-chla has a very short long-term retention time in tumor tissue.

[0158] 7. In vivo toxicity evaluation

[0159] For the toxicity test of LvP-chla, the mice did not show any adverse symptoms throughout the observation period, and the tumor size remained unchanged. Histological analysis of vital organs (Fig. 11) revealed that no cell damage or abnormalities were observed in the heart (Fig. 11a), kidney (Fig. 11d), or liver (Fig. 11e) after LvP-chla administration.

[0160] Although splenomegaly and an increase in leukocytes in the spleen were observed, this was likely due to an immune response to tumor growth rather than to the LvP-chla injection, as normal white pulp was observed in Figure 11b.

[0161] In summary, these results indicate that LvP-chla did not exhibit observable toxicity and can safely monitor targeted biomarkers in deep tissues longitudinally. Instruction manual page 16 / 16, page 19, CN 121588253 A, Figure 1; Instruction manual figure 1 / 14 page, page 20, CN 121588253 A, Figure 2; Instruction manual figure 2 / 14 page, page 21, CN 121588253 A, Figure 3; Instruction manual figure 3 / 14 page, page 22, CN 121588253 A, Figure 4; Instruction manual figure 4 / 14 page, page 23, CN 121588253 A, Figure 5; Instruction manual figure 5 / 14 page, page 24, CN 121588253 A, Figure 5 (continued); Instruction manual figure 6 / 14 page, page 25, CN 121588253 A, Figure 6; Instruction manual figure 7 / 14 page, page 26, CN 121588253 A, Figure 7; Instruction manual figure 8 / 14 page, page 27, CN 121588253 A, Figure 8; Instruction manual figure 9 / 14 page, page 28, CN 121588253 A, Figure 9. Figure 10, Figure 11, Figure 11, Figure 12, Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17, Figure 18, Figure 19, Figure 10, Figure 10, Figure 11, Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17, Figure 18, Figure 19, Figure 10, Figure 19, Figure 10, Figure 11, Figure 12, Figure 12, Figure 13, Figure 14, Figure 18, Figure 19, Figure 19, Figure 10 ...of performing photoacoustic imaging on a subject. Also disclosed are WSCP- chlorophyll complexes comprising Lepidium virginicum WSCP and chlorophyll a, bacteriochlorophyll a, and bacteriochlorophyll b. The WSCP-chlorophyll complexes of the present invention exhibit strong near-infrared (NIR) absorption, distinct and non-overlapping spectral profiles, and concentration-dependent photoacoustic (PA) signal generation. These properties make them suitable as contrast agents for PAI in various applications, particularly in clinical settings such as disease detection and monitoring.

Claims

1. Use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in the preparation of a contrast agent for use in a method of photoacoustic imaging of a subject, wherein the WSCP-chlorophyll complex comprises Lepidium virginicum WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b, the method comprising: (a) Apply an effective amount of WSCP-chlorophyll complex to the site of the subject; (b) Illuminate the subject with light to induce photoacoustic signals from the WSCP-chlorophyll complex; (c) Detect the photoacoustic signal; as well as (d) Generate a photoacoustic image of the region based on the detected photoacoustic signal.

2. The use according to claim 1, wherein the light is pulsed light with a wavelength of 650 nm to 900 nm, optionally The WSCP-chlorophyll complex exhibits a Qy absorption peak at pulsed light wavelengths of approximately 665 nm to 850 nm and a Qx absorption peak at pulsed light wavelengths of approximately 578 nm to 592 nm.

3. The use according to claim 2, wherein the WSCP-chlorophyll complex: exhibits a Qy absorption peak at a pulsed light wavelength of about 665 nm; exhibits a Qy absorption peak at a pulsed light wavelength of about 765 nm and a Qx absorption peak at a pulsed light wavelength of about 578 nm; or exhibits a Qy absorption peak at a pulsed light wavelength of about 850 nm and a Qx absorption peak at a pulsed light wavelength of about 592 nm.

4. The use according to claim 1, wherein the chlorophyll a is spinach (Spinacia oleracea) chlorophyll a, the bacterial chlorophyll a is Rhodospirillum rubrum bacterial chlorophyll a, or the bacterial chlorophyll b is Blastochloris viridis bacterial chlorophyll b.

5. The use according to claim 2, wherein the method further comprises: (b1) Irradiate the subject with a first pulse of light of a first wavelength to induce a first photoacoustic signal from the WSCP-chlorophyll complex; (b2) Irradiate the subject with a second pulse of light of a second wavelength different from the first wavelength to induce a second photoacoustic signal from the WSCP-chlorophyll complex; (c1) Detect the first photoacoustic signal and the second photoacoustic signal; and (d1) Generate a differential photoacoustic image of the region based on the detected first photoacoustic signal and the detected second photoacoustic signal.

6. The use according to claim 5, wherein the first and second wavelengths differ by about 20-235 nm, or about 20-50 nm; or The WSCP-chlorophyll complex exhibits a Qy absorption peak at one of the first and second wavelengths, and weakened or negligible light absorption at the other of the first and second wavelengths.

7. The use according to claim 5, wherein the first and second wavelengths, or the second and first wavelengths: These are approximately 650nm to 850nm and approximately 685nm to 900nm, respectively; They are approximately 665nm and 685nm, respectively; They are approximately 765nm and approximately 800nm ​​respectively; or They are approximately 850nm and approximately 890nm respectively, optionally Generating the differential photoacoustic image includes subtracting the second photoacoustic signal from the first photoacoustic signal, or subtracting the first photoacoustic signal from the second photoacoustic signal.

8. The use according to claim 2, wherein the method further comprises: (b3) The subject was irradiated with pulsed light prior to the application of the WSCP-chlorophyll complex to induce background photoacoustic signals from the endogenous chromophores at the site. (c3) Detect the background photoacoustic signal; as well as (d3) A differential photoacoustic image of the site is generated based on the detected background photoacoustic signal and the photoacoustic signal detected after the application of the WSCP-chlorophyll complex.

9. The use according to claim 8, wherein the pulsed light used to irradiate the subject before and after application of the WSCP-chlorophyll complex has the same wavelength; and The wavelength mentioned therein is approximately 650-850 nm.

10. The use according to claim 8, wherein the wavelength is about 665 nm, about 765 nm, or about 850 nm, optionally. Generating the differential photoacoustic image involves subtracting the background photoacoustic signal from the photoacoustic signal detected after the application of the WSCP-chlorophyll complex.

11. The use according to claim 1, wherein the method comprises: (a4) Apply effective amounts of two or more WSCP-chlorophyll complexes to two or more target sites of the subject, wherein each WSCP-chlorophyll complex contains Lepidium apetalum WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a and bacterial chlorophyll b. (b4) Irradiate the subject with pulsed light of two or more excitation wavelengths to induce two or more photoacoustic signals from the corresponding WSCP-chlorophyll complex; (c4) Detect photoacoustic signals; as well as (d4) Based on the detected photoacoustic signals, generate composite photoacoustic images that can spatially resolve different target parts.

12. The use according to claim 11, wherein the two or more excitation wavelengths correspond to the maximum absorbance of each WSCP-chlorophyll complex and are from about 665 nm to 850 nm.

13. The use according to claim 11, wherein the chlorophyll comprises any two or all of spinach chlorophyll a, Rhodospirillum chlorophyll a, and Pleurotus ostreatus chlorophyll b; or The excitation wavelength includes any two or all of the following: approximately 665 nm, approximately 765 nm, and approximately 850 nm, optionally. The subjects mentioned therein are humans, non-human primates, rodents, canines, felines, bovines, or equines.

14. Use of a water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex in the preparation of a contrast agent for use in a photoacoustic imaging method, wherein the WSCP-chlorophyll complex comprises *Lysimachia christinae* WSCP (LvP) and chlorophyll selected from the group consisting of chlorophyll a, bacterial chlorophyll a, and bacterial chlorophyll b, the method comprising: Provides an effective amount of WSCP-chlorophyll complex; The WSCP-chlorophyll complex was irradiated with pulsed light with a wavelength of 650 nm to 900 nm to induce photoacoustic signals. Detect the photoacoustic signal; as well as Photoacoustic images are generated based on the detected photoacoustic signals.

15. The use according to claim 14, wherein the chlorophyll a is spinach chlorophyll a, the bacterial chlorophyll a is Rhodospirillum erythrospira bacterial chlorophyll a, or the bacterial chlorophyll b is Green buds bacterial chlorophyll b.

16. A water-soluble chlorophyll-binding protein (WSCP)-chlorophyll complex comprising Lysimachia christinae WSCP (LvP); and Rhodospirillum rubrum bacterial chlorophyll a or Rhodospirillum chlorophyll b.

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