Photoacoustic contrast agent based on reversible optical switch fusion protein, preparation method therefor, and use thereof

Abstract

A photoacoustic contrast agent based on a reversible optical switch fusion protein, a preparation method therefor, and use thereof. The photoacoustic contrast agent comprises a phytochrome protein and a collagen-binding domain fused to the C-terminus of the phytochrome protein. The phytochrome protein is derived from Deinococcus radiodurans. The photoacoustic contrast agent combines the phytochrome protein derived from Deinococcus radiodurans and the collagen-binding domain, ingeniously integrating the optical switch function of DrBphP and the high affinity of the CBD for collagen. The DrBphP-CBD fusion protein retains the high affinity for type I collagen and type III collagen and its optical switch property. The photoacoustic contrast agent can be specifically attached to exposed collagen by utilizing the non-typical vascular structure of tumors and the excessive deposition of collagen in fibrotic diseases, thereby facilitating continuous delivery and retention in the affected tissue microenvironment and providing enhanced optical switch photoacoustic differential imaging for pathological section staining and in vivo imaging detection of diseases.

Description

Reversible light-switch fusion protein-based photoacoustic contrast agent, and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of contrast agents, and particularly relates to a reversible light-switch fusion protein-based photoacoustic contrast agent, and a preparation method and application thereof. BACKGROUND

[0002] Photoacoustic imaging is a non-ionizing hybrid technique that combines the high optical contrast of optical imaging and the excellent spatial resolution of ultrasound waves in deep tissues. It overcomes the limitations of pure optical and ultrasound imaging and enables label-free imaging at multiple scales, which makes it fundamentally different from other imaging modalities. Recent research progress has mainly focused on the development of exogenous contrast agents to improve image contrast and enhance the signal-to-noise ratio for disease monitoring. However, some intrinsic challenges, particularly the absorption of endogenous chromophores such as hemoglobin and melanin, introduce significant background noise, which weakens the sensitivity of photoacoustic imaging. Overcoming these limitations is crucial for maximizing the potential of photoacoustic imaging in clinical applications.

[0003] In the latest progress of multiscale photoacoustic imaging, the potential of reversible light-switch chromoprotein has been highlighted, especially the bacteriophytochrome photoreceptors (BphPs). These genetically encoded light-absorbing proteins bind biliverdin (BV) and can switch between red and near-infrared light absorption states, and have anti-photobleaching properties, making them an ideal choice for longitudinal imaging. This photochromic property helps to eliminate extraneous background noise by subtracting the photoacoustic images captured in two different states. This photoacoustic imaging technique, known as background suppression, has the potential to be used for deep disease detection.

[0004] Current methods, including the use of light-switch chromoproteins for photoacoustic imaging, have achieved some success in studying tumors, but these methods mainly focus on the design of tumor-specific probes and the development of delivery systems, and have encountered limitations in actual clinical applications. In particular, BphPs chromoproteins have attracted attention due to their unique photochemical properties, including their high efficiency in deep tissues, dual absorption states, non-cytotoxicity, and selective binding ability to endogenous BV, but they lack specific binding sites for collagen in fibrotic diseases, limiting their application in non-tumorous fibrotic disease imaging.

[0005] Abnormal accumulation of collagen plays a key role in tumor development and various fibrotic diseases, including but not limited to liver fibrosis, lung fibrosis, kidney fibrosis, heart fibrosis and skin fibrosis. This abnormal accumulation not only affects the structure and function of the tissue, but also is a significant marker of disease progression. Although some progress has been made in studying tumors and various fibrotic diseases by using advanced imaging techniques such as photoacoustic imaging, there is currently no probe that can be used for both in vitro tissue section staining and in vivo photoacoustic imaging to monitor changes in collagen in these fibrotic diseases.

[0006] Therefore, the prior art still needs to be improved and developed.

[0007] SUMMARY

[0008] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide a photoacoustic contrast agent based on a reversible light switch fusion protein and a preparation method and application thereof, aiming to solve the problems of the lack of tumor-specific binding sites of the existing photoacoustic contrast agent and poor photoacoustic imaging contrast.

[0009] The technical scheme of the present application is as follows:

[0010] A photoacoustic contrast agent based on a reversible light switch fusion protein, comprising a photosensitive pigment protein, and a collagen binding domain fused to the C-terminal of the photosensitive pigment protein; the photosensitive pigment protein is from Deinococcus radiodurans.

[0011] The photoacoustic contrast agent based on the reversible light switch fusion protein, wherein the amino acid sequence of the photoacoustic contrast agent based on the reversible light switch fusion protein is shown in SEQ ID No. 1.

[0012] The photoacoustic contrast agent based on the reversible light switch fusion protein, wherein the photosensitive pigment protein comprises an N-terminal photosensitive module, and the cysteine residue of the N-terminal photosensitive module is combined with chlorophyll; the collagen binding domain is used for targeting collagen.

[0013] A preparation method of a photoacoustic contrast agent based on a reversible light switch fusion protein, comprising the steps of:

[0014] After codon optimization of the DrBphP-CBD gene, the gene is amplified by PCR and cloned into a vector for culture to obtain a bacterial culture;

[0015] The bacterial culture is induced for protein expression using an inducer, and after incubation treatment, cells are obtained after first centrifugal treatment;

[0016] The cells are resuspended in a buffer for cell lysis, and after second centrifugal treatment, a lysate is obtained;

[0017] The lysate is treated by fixed metal affinity chromatography and size exclusion chromatography for purification to obtain the photoacoustic contrast agent based on the reversible light switch fusion protein.

[0018] The preparation method of the photoacoustic contrast agent based on the reversible light switch fusion protein, wherein the carrier is one or more of pET28a, pET28b and pET28c; the restriction enzyme cutting site used for cloning into the carrier is Ndel and XhoI.

[0019] The preparation method of the photoacoustic contrast agent based on the reversible light switch fusion protein, wherein the inducer is isopropyl β-D-1-thiogalactopyranoside.

[0020] The preparation method of the photoacoustic contrast agent based on the reversible light switch fusion protein, wherein the temperature of the incubation treatment is 15-17℃, the time of the incubation treatment is 18-22h; the speed of the first centrifugal treatment is 7000-8500rpm, the time of the first centrifugal treatment is 25-35min; the speed of the second centrifugal treatment is 18000-22000rpm, the time of the second centrifugal treatment is 45-60min.

[0021] The preparation method of the photoacoustic contrast agent based on the reversible light switch fusion protein, wherein the buffer solution comprises Tris-HCl, NaCl, imidazole, dipyrrometheneboron chloride, Tris(2-carboxyethyl)phosphonate hydrochloride, protease inhibitor.

[0022] The application of a photoacoustic contrast agent based on a reversible light switch fusion protein in the detection of collagen content in tumors and fibrotic diseases.

[0023] The application of the photoacoustic contrast agent based on the reversible light switch fusion protein, wherein the fibrotic disease includes one or more of liver fibrosis, lung fibrosis, kidney fibrosis, heart fibrosis and skin fibrosis.

[0024] Beneficial effects: the application provides a photoacoustic contrast agent based on a reversible light switch fusion protein and a preparation method and application thereof, the photoacoustic contrast agent based on the reversible light switch fusion protein comprises a photosensitive pigment protein, and a collagen binding domain fused to the C terminal of the photosensitive pigment protein; the photosensitive pigment protein is from Deinococcus radiodurans. The application constructs an efficient delivery system for treating tumors and various fibrosis diseases through a fusion protein technology, specifically through the design and preparation of DrBphP-CBD fusion protein, the fusion protein combines the photosensitive pigment protein (DrBphP) from Deinococcus radiodurans with the collagen binding domain (CBD), and ingeniously integrates the light switch function of DrBphP and the high affinity of CBD to collagen. The DrBphP-CBD fusion protein maintains high affinity to type I and type III collagen and its light switch characteristics; and the photoacoustic contrast agent utilizes the atypical vascular structure of tumors and the excessive deposition of collagen in fibrosis diseases, so that it can specifically adhere to the exposed collagen, helps to achieve continuous delivery and retention in the affected tissue microenvironment, and provides enhanced light switch photoacoustic differential imaging. BRIEF DESCRIPTION OF DRAWINGS

[0025] Fig. 1 is a design diagram of the DrBphP-CBD fusion protein in embodiment 1 of the application;

[0026] Fig. 2 is a purification diagram of the DrBphP-CBD fusion protein in embodiment 1 of the application;

[0027] Fig. 3 is a binding affinity test diagram of the DrBphP-CBD fusion protein and recombinant type I and III collagen in embodiment 1 of the application;

[0028] Fig. 4 is an absorption spectrum of the DrBphP-CBD fusion protein in embodiment 1 of the application;

[0029] Fig. 5 is an in vitro photoacoustic imaging diagram of the DrBphP-CBD fusion protein in embodiment 1 of the application;

[0030] Fig. 6 is a reversible light switch characteristic diagram of the DrBphP-CBD fusion protein in embodiment 1 of the application in vitro;

[0031] Fig. 7 is a differential photoacoustic imaging effect diagram of the DrBphP-CBD fusion protein in embodiment 1 of the application in vivo;

[0032] Fig. 8 is a reversible light switch characteristic diagram of the DrBphP-CBD fusion protein in embodiment 1 of the application in vivo;

[0033] Fig. 9 is a time-resolved PA differential imaging diagram of the DrBphP-CBD fusion protein in the MC38 tumor model in embodiment 1 of the application;

[0034] Figure 10 is a time-resolved PA differential imaging map of DrBphP-CBD fusion protein in MDA-MB-231 tumor model in Example 1 of the present application;

[0035] Figure 11 is a time-resolved PA differential imaging map of DrBphP-CBD fusion protein in 4T1 tumor model in Example 1 of the present application;

[0036] Figure 12 is a data map of tumor targeting of DrBphP-CBD fusion protein in Example 1 of the present application. DETAILED DESCRIPTION

[0037] The present application provides a photoacoustic contrast agent based on reversible light switch fusion protein and its preparation method and application. In order to make the purpose, technical scheme and effect of the present application more clear and definite, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0038] Those skilled in the art can understand that, unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs. It should also be understood that terms such as those defined in a general dictionary should be understood as having meanings consistent with those in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as such herein.

[0039] Collagen, as the main component of extracellular matrix in mammals, plays an important role in healthy tissues. However, its abnormal overexpression and accumulation in tumor development and fibrosis diseases become a problem. In the current research, due to the low permeability of the complete blood vessel system, it is difficult to contact collagen; the abnormal vascular structure in tumor tissue increases its permeability, so that collagen is exposed to the systemic circulation. This abnormal exposure, combined with the overexpression of collagen in various cancers, makes it an ideal target for medical imaging. Previous studies have shown that engineered cytokines and monoclonal antibodies have affinity for collagen, which can effectively mediate the selective positioning and retention of therapeutic agents in tumor stroma. In particular, the collagen-binding domain (CBD) of von Willebrand factor exhibits significant affinity for type I and III collagen, which are abundant in tumor stroma and fibrotic lesions. However, these achievements mostly focus on the tumor microenvironment rather than widespread fibrotic lesions. Therefore, developing a probe that can not only monitor collagen changes in vivo through photoacoustic imaging technology, but also be used for collagen tissue slice staining in vitro, is of great significance for the diagnosis and treatment of tumors and fibrosis diseases. Such a probe not only needs to have high specificity and sensitivity to distinguish collagen in healthy and diseased tissues, but also needs to have stable photochemical and biochemical properties in complex biological environments to achieve accurate imaging and disease monitoring. Fusion of CBD with BphPs may confer BphPs the ability to specifically target tumor sites and accumulate, thereby enhancing their local bioavailability and optimizing their potential in diagnostic imaging.

[0040] Based on this, the present application provides a kind of photoacoustic contrast agent based on reversible light switch fusion protein, including photosensitive pigment protein, and the collagen binding domain fused to the C terminal of the photosensitive pigment protein;The photosensitive pigment protein is from Deinococcus radiodurans.

[0041] In this embodiment, by gene recombination method, collagen binding domain (CBD) with collagen targeting ability is fused to the C terminal of photosensitive pigment protein (bacterio-phytochrome, DrBphP) from Deinococcus radiodurans, and a fusion protein DrBphP-CBD capable of targeting tumor tissue is obtained;The fusion protein can efficiently and specifically target collagen high expression tissues, such as tumor or fibrosis disease sites;And by virtue of the reversible light conversion ability of reversible light switch protein DrBphP, background noise in lesion area can be reduced, thereby enhancing the contrast of photoacoustic imaging and realizing accurate disease diagnosis.

[0042] Specifically, the present application constructs an efficient delivery system for treating tumors and various fibrosis diseases through a fusion protein technology, specifically through the design and preparation of a DrBphP-CBD fusion protein. This fusion protein combines the N-terminal photosensitive module of the photosensitive pigment (DrBphP) from Deinococcus radiodurans bacteria with the collagen binding domain (CBD), skillfully integrating the light switch function of BphPs and the high affinity of CBD for collagen. And experiments show that the DrBphP-CBD fusion protein maintains high affinity for type I and type III collagen and its light switch properties, confirming the success in design, expression and purification. At the same time, the DrBphP-CBD fusion protein (photoacoustic contrast agent) is used to carry out photoacoustic differential imaging of tumors and fibrosis diseases in vivo. Its unique design takes advantage of the atypical vascular structure of tumors and the excessive deposition of collagen in fibrosis diseases, allowing it to specifically adhere to exposed collagen, helping to achieve sustained delivery and retention in the affected tissue microenvironment, and providing enhanced light switch photoacoustic differential imaging. In vivo in various mouse disease models, DrBphP-CBD shows preferential targeting and enrichment in diseased tissues, significantly reducing background noise to improve the contrast of imaging.

[0043] In some embodiments, the amino acid sequence of the photoacoustic contrast agent based on the reversible light switch fusion protein is shown in SEQ ID No. 1. The amino acid sequence of the photoacoustic contrast agent is designed as shown in SEQ ID No. 1, which has the characteristics of efficiently and specifically targeting collagen-high-expressing tissues, and can reduce background noise in the lesion area by virtue of the reversible light conversion ability of the reversible light switch protein DrBphP, thereby enhancing the contrast of photoacoustic imaging and achieving accurate disease diagnosis.

[0044] In some embodiments, the photosensitive pigment protein comprises an N-terminal photosensitive module, and a cysteine residue of the N-terminal photosensitive module is combined with a chlorophyll; and the collagen binding domain is used for targeting collagen.

[0045] Specifically, full-length DrBphP1 consists of 755 amino acids, with an N-terminal photosensitive core module (PSM, 1-594 residues) that self-catalytically binds chlorophyll (BV chromophore) to a conserved cysteine residue; in addition, DrBphP1 also contains a C-terminal variable output module (OM, residues 595-755) responsible for triggering downstream cellular responses; and the present application retains the PSM domain of DrBphP1 (referred to as DrBphP), and replaces the OM domain with the CBD domain to endow DrBphP with specific tumor targeting properties; at the same time, a flexible loop structure is introduced between the PSM and CBD domains to facilitate the correct folding of the two structures.

[0046] It should be noted that the loop structure is the amino acid sequence connecting between the PSM and the CBD.

[0047] In addition, the application further provides a preparation method of the photoacoustic contrast agent based on the reversible light switch fusion protein, comprising the steps of:

[0048] Step S10: After codon optimization of the DrBphP-CBD gene, the gene is amplified by PCR and cloned into a vector for culture to obtain a bacterial culture;

[0049] Step S20: The bacterial culture is induced for protein expression using an inducer, and after incubation treatment, cells are obtained after first centrifugal treatment.

[0050] Step S30: The cells are resuspended in a buffer for cell lysis, and after second centrifugal treatment, a lysate is obtained.

[0051] Step S40: The lysate is treated by fixed metal affinity chromatography and purified by size exclusion chromatography to obtain the photoacoustic contrast agent based on the reversible light switch fusion protein.

[0052] In this embodiment, the preparation method can be used to prepare a photoacoustic contrast agent that is highly efficient and specifically targets collagen high-expression tissues. The reversible light switch protein DrBphP can reduce background noise in the lesion area by virtue of its reversible light conversion capability, thereby enhancing the contrast of photoacoustic imaging and achieving accurate disease diagnosis.

[0053] In some embodiments, the vector is one or more of pET28a vector, pET28b, pET28c; the restriction enzyme cutting site used for cloning into the vector is Ndel and XhoI. The pET28a vector provides an N-terminal His6 tag to facilitate protein purification.

[0054] In some embodiments, the inducer is isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0055] In some embodiments, the concentration of the inducer is 0.6mM-1.0mM.

[0056] Specifically, when the optical density 600nm (OD600) of the bacterial culture reaches 0.8, 0.8mM isopropyl β-D-1-thiogalactopyranoside is used to induce protein expression.

[0057] In some embodiments, the temperature of the incubation treatment is 15-17℃, the time of the incubation treatment is 18-22h; the speed of the first centrifugal treatment is 7000-8500rpm, the time of the first centrifugal treatment is 25-35min; the speed of the second centrifugal treatment is 18000-22000rpm, the time of the second centrifugal treatment is 45-60min.

[0058] In some embodiments, the buffer comprises Tris-HCl, NaCl, imidazole, BPTI-Cl, Tris(2-carboxyethyl)phosphatetate hydrochloride, protease inhibitors.

[0059] Specifically, after induction, the culture is incubated at 16℃ for 20 hours, the cells are harvested by centrifugation at 8000rpm for 30 minutes, the cell pellet is resuspended in a binding buffer containing 30mM Tris-HCl, 500mM NaCl, 5mM imidazole, 0.1mM BPTI-Cl, 1mM Tris(2-carboxyethyl)phosphatetate hydrochloride (TCEP), 0.5mM protease inhibitors phenylmethylsulfonyl fluoride (PMSF), the cells are lysed using an ultra-high pressure homogenizer, and the clarified lysate (supernatant) is obtained after centrifugation at 20000rpm for 50 minutes, the supernatant is subjected to immobilized metal affinity chromatography (IMAC) using a HisTrapTM HP column (Cytiva, USA), the protein fraction eluted from IMAC is pooled, and size exclusion chromatography is performed using an AKTA Pure FPLC system (Cytiva, USA) through a Superdex 200 16 / 600 column (Cytiva, USA) for further purification. The photoacoustic contrast agent based on the reversible photoswitching fusion protein is obtained.

[0060] In addition, the present application also provides a photoacoustic contrast agent based on the reversible photoswitching fusion protein for use in the detection of collagen content in tumors and fibrotic diseases.

[0061] In terms of expanding the application range of the photoacoustic contrast agent to fibrotic diseases, the excessive deposition of collagen during the development of fibrosis provides new application prospects for the photoacoustic contrast agent. Fibrosis is not only limited to tumors, but also covers a variety of diseases such as liver fibrosis, kidney fibrosis and skin fibrosis, in which abnormal accumulation of collagen is a common pathological feature. Therefore, by utilizing the unique performance of the DrBphP-CBD fusion protein, specific imaging of these fibrotic diseases and tumors can be achieved, providing new strategies for early diagnosis and treatment, and also broadening the application of photoacoustic imaging technology in the medical field. The photoacoustic contrast agent not only enhances the diagnosis and treatment of tumors, but also provides important support for the diagnosis of fibrotic diseases, demonstrating the broad application potential of photoacoustic imaging technology in modern medicine.

[0062] In some embodiments, the fibrotic disease includes, but is not limited to, one or more of liver fibrosis, lung fibrosis, kidney fibrosis, heart fibrosis, and skin fibrosis.

[0063] In some embodiments, the photoacoustic contrast agent is used to prepare a drug for treating tumors or fibrotic diseases, and the administration method includes intravenous, intramuscular, intradermal or subcutaneous injection, etc. In addition, the drug has a wide range of applications, and is intended to be applied to a variety of diseases including tumors, liver fibrosis, kidney fibrosis and skin fibrosis, as well as other related fibrotic diseases that may be caused by these conditions. Specifically, liver fibrosis can be caused by various factors such as viral hepatitis, alcoholic hepatitis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure, drug reactions, etc., including some cases where the cause is unknown, as well as other diseases that may be caused by liver fibrosis. The causes of kidney fibrosis include, but are not limited to, hypertension, glomerulonephritis, systemic lupus erythematosus, scleroderma, kidney transplant rejection, pyelonephritis, kidney stones, hyperlipidemia, diabetes, hyperuricemia, hypercalciuria, etc., as well as other unknown causes of kidney fibrosis and diseases induced by kidney fibrosis. Skin fibrosis can be caused by drug reactions, idiopathic, genetic factors, etc., including those cases where the cause is unknown, as well as other diseases that may be caused by skin fibrosis.

[0064] Specifically, the fusion protein photoacoustic contrast agent provided by the present application can be applied to a variety of fibrotic diseases caused by different causes, and can be administered through various routes.

[0065] The following examples are further provided to illustrate the present application in detail. It should also be understood that the following examples are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present application are within the scope of protection of the present application.

[0066] Example 1

[0067] The present embodiment provides a reversible light switch fusion protein-based photoacoustic contrast agent, and characterizes it, specifically including the following:

[0068] 1. Design of DrBphP-CBD fusion protein

[0069] Full-length DrBphP1 consists of 755 amino acids, with an N-terminal light sensing core module (PSM, residues 1-594) that autocatalytically binds BV chromophore to a conserved cysteine residue. In addition, it contains a C-terminal variable output module (OM, residues 595-755) responsible for triggering downstream cellular responses. In the present embodiment, the PSM domain of DrBphP1 (referred to as DrBphP) is retained, while the OM domain is replaced with a CBD domain to endow DrBphP with specific tumor targeting properties. At the same time, a flexible loop structure is introduced between the PSM and CBD domains to facilitate the correct folding of the two domains (as shown in Figure 1). The amino acid sequence of DrBphP-CBD fusion protein is shown in SEQ ID No. 1.

[0070] 2. Expression and purification of DrBphP-CBD fusion protein

[0071] In this example, the DrBphP-CBD gene was codon-optimized for expression in E. coli, followed by PCR amplification and cloning into pET28a vector using Ndel and Xhol restriction enzyme sites. The pET28a vector provides an N-terminal His6 tag for protein purification. Protein expression was induced using 0.8 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) when the optical density at 600 nm (OD600) of the bacterial culture reached 0.8. After induction, the culture was incubated at 16 °C for 20 h, and the cells were harvested by centrifugation at 8000 rpm for 30 min, the cell pellet was resuspended in binding buffer containing 30 mM Tris-HCl (pH 7.6), 500 mM NaCl, 5 mM imidazole, 0.1 mM bilipid green chlorin chloride (BV), 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 0.5 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF), cell lysis was performed using an ultra-high pressure homogenizer, and the clarified lysate was obtained after centrifugation at 20000 rpm for 50 min. The supernatant was processed by immobilized metal affinity chromatography (IMAC) using a HisTrap™ HP column (Cytiva, USA); the protein fractions eluted from IMAC were pooled and further purified by size exclusion chromatography using a Superdex 200 16 / 600 column (Cytiva, USA) using an AKTA Pure FPLC system (Cytiva, USA). The purified DrBphP-CBD protein was analyzed by SDS-PAGE to confirm its homogeneity (as shown in Figure 2).

[0072] 3. In vitro characterization of DrBphP-CBD fusion protein

[0073] The present example evaluated the in vitro binding affinity of DrBphP-CBD to recombinant collagen by indirect enzyme-linked immunosorbent assay (ELISA). Recombinant collagen I or III (10 pg / mL each in PBS) was coated on ELISA plates overnight at 37 °C, followed by blocking with 2% BSA PBS containing 0.05% Tween 20 (PBS-T) for 1 h, and then washing. Different concentrations of DrBphP-CBD fusion protein were then added and incubated for 2 h. After washing, the plates were incubated with anti-His antibody (ab18184, Abeam) and goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (ab205719, Abeam) and detected using tetramethylbenzidine substrate. The binding affinity of DrBphP-CBD fusion protein to recombinant collagen type I and III was tested as shown in Figure 3, and the results showed that DrBphP-CBD has enhanced affinity to collagen type I and III with dissociation constants (Kd) of 0.48 nM and 26.45 nM, respectively.

[0074] In addition, the present example verified the photochromic properties of DrBphP-CBD. Under 635 nm light irradiation, the fusion protein was converted from the Pr (open) state to the Pfr (closed) state, and reversibly from the Pfr state to the Pr state under 808 nm light irradiation. The contrast generated by the light switch is crucial for improving imaging specificity and suppressing background interference. This contrast is quantified by the ratio of the absorption coefficients of the open and closed states, and a higher ratio indicates better maintenance of the intrinsic signal of the pigment protein after removing the background signal, thereby optimizing image contrast in differential photoacoustic imaging. The absorption spectrum of DrBphP-CBD fusion protein is shown in Figure 4, and the spectral absorption properties of DrBphP-CBD between its two states have obvious changes, with an absorption coefficient ratio (Pfr / Pr) of about 11 at 760 nm, indicating that it has excellent light switch contrast. The in vitro photoacoustic imaging image of DrBphP-CBD fusion protein is shown in Figure 5, and the result of subtracting the closed state from the open state PA image is a differential image with significantly enhanced contrast. In addition, the present example evaluated the reversible light switch properties of DrBphP-CBD fusion protein in vitro (as shown in Figure 6), and successfully induced repeated light activation cycles by alternating exposure to 808 nm and 635 nm wavelengths, thereby verifying the reversible light switch capability of DrBphP-CBD fusion protein in vitro and the repeatability of the imaging technology.

[0075] 4. In vivo characterization of DrBphP-CBD fusion protein

[0076] The present embodiment evaluates the differential photoacoustic imaging effect of DrBphP-CBD fusion protein in vivo. As shown in Figure 7, the photoacoustic image in the open state is subtracted from the result in the closed state, and the resulting difference image has a significantly enhanced contrast inside the tumor.

[0077] And further evaluate the reversible light switch characteristics of DrBphP-CBD fusion protein (as shown in Figure 8), by alternating irradiation at 760 nm and 635 nm wavelength, successfully induced repeated light activation cycle, verified the reversible light switch ability of DrBphP-CBD fusion protein in vivo and the repeatability of its imaging.

[0078] In addition, the present embodiment also verifies the accumulation of DrBphP-CBD fusion protein in different subcutaneous tumor models and its effectiveness in photoacoustic imaging. DrBphP fusion protein containing CBD and not containing CBD was injected into mice carrying MC38, MDA-MB-231 and 4T1 tumors through the tail vein. The signal accumulation of DrBphP-CBD and DrBphP in tumor tissue was observed by photoacoustic imaging, and the tumor-bearing mice were imaged at different time intervals (2, 4, 6, 8, 10, 12, 24, 48 and 72 hours) before and after injection to comprehensively analyze the targeting efficiency of DrBphP-CBD and its retention in tumor tissue. As shown in Figures 9 to 11, in the three tumor models, the initial accumulation of DrBphP-CBD was observed within 2 to 4 hours after injection. The photoacoustic signal of these fusion proteins was significantly enhanced in the tumor tissue, peaked at 10 to 12 hours, and lasted up to 72 hours. In contrast, the photoacoustic signal containing only DrBphP was monitored in the tumor tissue within 4 to 6 hours after injection, but then gradually weakened and disappeared after 8 hours. Compared with DrBphP only, DrBphP-CBD showed significantly improved tumor-specific targeting and retention characteristics in the three types of tumors evaluated. At the same time, the DrBphP-CBD-mediated differential photoacoustic imaging mode effectively reduced the interference of non-targeted signals (such as signals from tumor-associated blood vessels, skin layers and melanin), generating a purer difference image. In addition, ex vivo photoacoustic imaging of organs performed 10 hours after injection (as shown in Figure 12) showed that the photoacoustic signal of DrBphP-CBD fusion protein was mainly accumulated in the tumor, followed by localization in other organs, further confirming its specificity for tumor targeting.

[0079] In summary, the present application provides a kind of photoacoustic contrast agent based on reversible light switch fusion protein and its preparation method and application, photoacoustic contrast agent based on reversible light switch fusion protein includes photosensitive pigment protein, and the collagen binding domain fused to the C terminal of the photosensitive pigment protein;The photosensitive pigment protein is from Deinococcus radiodurans.The present application constructs a kind of efficient delivery system for treating tumor and various fibrosis diseases by fusion protein technology, specifically by the design and preparation of DrBphP-CBD fusion protein, the fusion protein combines photosensitive pigment protein (DrBphP) from Deinococcus radiodurans with collagen binding domain (CBD), and the light switch function of DrBphP and the high affinity of CBD to collagen are ingeniously integrated.DrBphP-CBD fusion protein maintains the high affinity to type I and type III collagen and its light switch characteristics;And, the photoacoustic contrast agent utilizes the atypical vascular structure of tumor and the excessive deposition of collagen in fibrosis diseases, so that it can be specifically attached to exposed collagen, which helps to achieve sustained delivery and retention in affected tissue microenvironment, and provides enhanced light switch photoacoustic differential imaging.

[0080] It should be understood that the application of the present application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A photoacoustic contrast agent based on a reversible photoswitchable fusion protein, characterized in that: It comprises a photosensitive pigment protein and a collagen binding domain fused to the C-terminus of the photosensitive pigment protein; the photosensitive pigment protein is derived from Deinococcus radiodurans.

2. The photoacoustic contrast agent based on a reversible photoswitchable fusion protein according to claim 1, characterized in that: The amino acid sequence of the photoacoustic contrast agent based on the reversible photoswitch fusion protein is shown in SEQ ID No.

1.

3. The photoacoustic contrast agent based on a reversible photoswitchable fusion protein according to claim 1, characterized in that: The photosensitive pigment protein includes an N-terminal photosensitive module, and the cysteine ​​residue of the N-terminal photosensitive module is bound to biliverdin; the collagen binding domain is used to target collagen.

4. A method for preparing a photoacoustic contrast agent based on a reversible photoswitch fusion protein according to any one of items 1 to 3, characterized in that: Including steps: After codon optimization, the DrBphP-CBD gene was amplified by PCR and cloned into a vector for cultivation to obtain bacterial culture; Inducing the bacterial culture with an inducer to express protein, incubating the culture, and performing a first centrifugation to obtain cells; resuspending the cells in a buffer for cell lysis, and performing a second centrifugation to obtain a lysate; The lysate is treated by immobilized metal affinity chromatography and purified by size exclusion chromatography to obtain the photoacoustic contrast agent based on the reversible photoswitch fusion protein.

5. The method for preparing a photoacoustic contrast agent based on a reversible photoswitch fusion protein according to claim 4, characterized in that: The vector is one or more of pET28a, pET28b, and pET28c; the restriction enzyme sites used in cloning into the vector are NdeI and XhoI.

6. The method for preparing a photoacoustic contrast agent based on a reversible photoswitch fusion protein according to claim 4, characterized in that: The inducer is isopropyl β-D-1-thiogalactoside.

7. The method for preparing a photoacoustic contrast agent based on a reversible photoswitchable fusion protein according to claim 4, characterized in that: The incubation temperature is 15-17°C, and the incubation time is 18-22 hours; the first centrifugal rotation speed is 7000-8500 rpm, and the first centrifugal time is 25-35 minutes; the second centrifugal rotation speed is 18000-22000 rpm, and the second centrifugal time is 45-60 minutes.

8. The method for preparing a photoacoustic contrast agent based on a reversible photoswitch fusion protein according to claim 4, characterized in that: The buffer comprises Tris-HCl, NaCl, imidazole, dipyrrolidine chloride, Tris (2-carboxyethyl) phosphate hydrochloride, and protease inhibitors.

9. Use of the photoacoustic contrast agent based on the reversible photoswitchable fusion protein according to any one of claims 1 to 3 in detecting collagen content in tumors and fibrotic diseases.

10. The use of the photoacoustic contrast agent based on the reversible photoswitch fusion protein according to claim 9, characterized in that: The fibrotic disease includes one or more of liver fibrosis, lung fibrosis, kidney fibrosis, cardiac fibrosis, and skin fibrosis.

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