WO2025213625A1 - 基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用 - Google Patents

基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用

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WO2025213625A1
WO2025213625A1 PCT/CN2024/107939 CN2024107939W WO2025213625A1 WO 2025213625 A1 WO2025213625 A1 WO 2025213625A1 CN 2024107939 W CN2024107939 W CN 2024107939W WO 2025213625 A1 WO2025213625 A1 WO 2025213625A1
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fusion protein
contrast agent
collagen
agent based
photoacoustic
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French (fr)
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黄鹏
陈帅
陈欣
林静
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Shenzhen University
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Shenzhen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasonic imaging preparations
    • A61K49/221—Echographic preparations; Ultrasonic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the technical field of contrast agents, and in particular to a photoacoustic contrast agent based on a reversible photoswitch fusion protein, and a preparation method and application thereof.
  • Photoacoustic imaging is a nonionizing hybrid technique that combines the high optical contrast of optical imaging with the excellent spatial resolution of ultrasound in deep tissue. It overcomes the limitations of both pure optical and ultrasound imaging and enables label-free imaging at multiple scales, setting it apart from other imaging modalities.
  • Recent research advances have focused on developing exogenous contrast agents to enhance image contrast and signal-to-noise ratio for disease monitoring.
  • exogenous contrast agents to enhance image contrast and signal-to-noise ratio for disease monitoring.
  • several inherent challenges particularly the absorption of endogenous chromatin such as hemoglobin and melanin, introduce significant background noise, thereby reducing the sensitivity of PAI. Overcoming these limitations is crucial to maximizing the potential of PAI in clinical applications.
  • fibrotic diseases including but not limited to liver fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis and skin fibrosis. It not only affects tissue structure and function but is also a significant marker of disease progression. Although some progress has been made in studying tumors and various fibrotic diseases using advanced imaging techniques such as photoacoustic imaging, there is currently no probe that can be used simultaneously for in vitro tissue section staining and in vivo photoacoustic imaging to monitor changes in collagen in these fibrotic diseases.
  • the purpose of the present invention is to provide a photoacoustic contrast agent based on a reversible photoswitch fusion protein and its preparation method and application, aiming to solve the problems of the existing photoacoustic contrast agents lacking tumor-specific binding sites and poor photoacoustic imaging contrast.
  • a photoacoustic contrast agent based on a reversible photoswitch fusion protein 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.
  • the photoacoustic contrast agent based on the reversible photoswitch fusion protein wherein the amino acid sequence of the photoacoustic contrast agent based on the reversible photoswitch fusion protein is shown as SEQ ID No.1.
  • the photoacoustic contrast agent based on the reversible photoswitch fusion protein wherein the photosensitive pigment protein includes an N-terminal photosensitive module, the cysteine residue of the N-terminal photosensitive module is bound to biliverdin; and the collagen binding domain is used to target collagen.
  • a method for preparing a photoacoustic contrast agent based on a reversible photoswitchable fusion protein comprises the following steps:
  • DrBphP-CBD gene was amplified by PCR and cloned into a vector for cultivation to obtain bacterial culture;
  • 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.
  • the preparation method of the photoacoustic contrast agent based on the reversible light switch fusion protein wherein the vector is one or more of pET28a vector, pET28b, and pET28c; the restriction enzyme site used in the cloning into the vector is NdeI and XhoI.
  • the method for preparing a photoacoustic contrast agent based on a reversible photoswitch fusion protein wherein the incubation temperature is 15°C-17°C, and the incubation time is 18h-22h; the first centrifugal treatment speed is 7000rpm-8500rpm, and the first centrifugal treatment time is 25min-35min; the second centrifugal treatment speed is 18000rpm-22000rpm, and the second centrifugal treatment time is 45min-60min.
  • the method for preparing a photoacoustic contrast agent based on a reversible photoswitch fusion protein wherein the buffer comprises Tris-HCl, NaCl, imidazole, dipyrrolidine chloride, Tris (2-carboxyethyl) phosphate hydrochloride, and protease inhibitors.
  • the photoacoustic contrast agent based on the reversible photoswitch fusion protein, wherein the fibrotic disease includes one or more of liver fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, and skin fibrosis.
  • the present invention provides a photoacoustic contrast agent based on a reversible photoswitch fusion protein, as well as its preparation method and application.
  • the photoacoustic contrast agent based on the reversible photoswitch fusion protein comprises a photochrome protein derived from Deinococcus radiodurans and a collagen-binding domain fused to the C-terminus of the photochrome protein.
  • the photochrome protein is derived from Deinococcus radiodurans and is fused to the C-terminus of the photochrome protein.
  • the present invention utilizes fusion protein technology to construct a highly efficient delivery system for the treatment of tumors and various fibrotic diseases.
  • the present invention designs and prepares a DrBphP-CBD fusion protein, which combines the photochrome protein (DrBphP) from Deinococcus radiodurans with a collagen-binding domain (CBD), cleverly integrating the photoswitch function of DrBphP with the high affinity of CBD for collagen.
  • the DrBphP-CBD fusion protein maintains high affinity for type I and type III collagen and its photoswitchable properties; moreover, this photoacoustic contrast agent utilizes the atypical vascular structure of tumors and the transitional deposition of collagen in fibrotic diseases, enabling it to specifically attach to exposed collagen, facilitating sustained delivery and retention in the affected tissue microenvironment, and providing enhanced photoswitchable photoacoustic differential imaging.
  • FIG1 is a design diagram of the DrBphP-CBD fusion protein in Example 1 of the present invention.
  • FIG2 is a diagram showing the purification of the DrBphP-CBD fusion protein in Example 1 of the present invention
  • FIG3 is a graph showing the binding affinity test of the DrBphP-CBD fusion protein to recombinant type I and type III collagen in Example 1 of the present invention
  • FIG4 is an absorption spectrum of the DrBphP-CBD fusion protein in Example 1 of the present invention.
  • FIG5 is an in vitro photoacoustic imaging image of the DrBphP-CBD fusion protein in Example 1 of the present invention.
  • FIG6 is a graph showing the in vitro reversible photoswitching characteristics of the DrBphP-CBD fusion protein in Example 1 of the present invention.
  • FIG7 is a diagram showing the differential photoacoustic imaging effect of the DrBphP-CBD fusion protein in vivo in Example 1 of the present invention.
  • FIG8 is a diagram showing the in vivo reversible photoswitching characteristics of the DrBphP-CBD fusion protein in Example 1 of the present invention.
  • FIG9 is a time-resolved PA differential imaging image of the DrBphP-CBD fusion protein in Example 1 of the present invention in the MC38 tumor model;
  • FIG10 is a time-resolved PA differential imaging image of the DrBphP-CBD fusion protein in the MDA-MB-231 tumor model in Example 1 of the present invention.
  • FIG11 is a time-resolved PA differential imaging image of the DrBphP-CBD fusion protein in Example 1 of the present invention in a 4T1 tumor model;
  • FIG12 is a data diagram showing the tumor targeting activity of the DrBphP-CBD fusion protein in Example 1 of the present invention.
  • the present invention provides a photoacoustic contrast agent based on a reversibly photoswitchable fusion protein, as well as its preparation method and application.
  • a photoacoustic contrast agent based on a reversibly photoswitchable fusion protein, as well as its preparation method and application.
  • Collagen as the main component of mammalian extracellular matrix, plays an important role in healthy tissues. However, its abnormal overexpression and accumulation become problems in tumor development and fibrotic diseases. Collagen is difficult to access due to the low permeability of the intact vasculature; however, the abnormal vascular structure in tumor tissue increases its permeability, exposing collagen to the systemic circulation. This abnormal exposure, combined with collagen's overexpression in various cancers, makes it an ideal target for medical imaging. Previous studies have demonstrated that engineered cytokines and monoclonal antibodies with affinity for collagen can effectively mediate the selective localization and retention of therapeutic agents in the tumor stroma.
  • the collagen-binding domain (CBD) of von Willebrand factor exhibits significant affinity for type I and type III collagens, which are abundant in the tumor stroma and fibrotic lesions.
  • CBD collagen-binding domain
  • most of these studies have focused on the tumor microenvironment rather than on widespread fibrotic lesions. Therefore, the development of a probe that can monitor collagen changes in vivo via photoacoustic imaging and can also be used for in vitro staining of collagen tissue sections is of great significance for the diagnosis and treatment of tumors and fibrotic diseases.
  • Such probes must not only possess high specificity and sensitivity to distinguish collagen in healthy and diseased tissues, but also possess stable photochemical and biochemical properties in complex biological environments to enable accurate imaging and disease monitoring. Fusion of CBD to BphPs may endow BphPs with the ability to specifically target and accumulate in tumor sites, thereby enhancing their local bioavailability and optimizing their potential in diagnostic imaging.
  • the present invention provides a photoacoustic contrast agent based on a reversible photoswitch fusion protein, comprising 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.
  • a collagen-binding domain with the ability to target collagen is fused to the C-terminus of a bacterio-phytochrome protein (DrBphP) from Deinococcus radiodurans by genetic recombination, thereby obtaining a fusion protein DrBphP-CBD capable of targeting tumor tissues.
  • DrBphP bacterio-phytochrome protein
  • This fusion protein can efficiently and specifically target tissues with high collagen expression, such as tumors or fibrotic disease sites.
  • the background noise in the lesion area can be reduced, thereby enhancing the contrast of photoacoustic imaging and achieving accurate disease diagnosis.
  • the present invention constructs an efficient delivery system for the treatment of tumors and various fibrotic diseases through fusion protein technology, specifically through the design and preparation of 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), cleverly integrating the photoswitching function of BphPs and the high affinity of CBD for collagen.
  • DrBphP-CBD fusion protein maintains a high affinity for type I and type III collagen and its photoswitching properties, confirming its success in design, expression and purification.
  • DrBphP-CBD fusion protein photoacoustic contrast agent
  • the amino acid sequence of the photoacoustic contrast agent based on the reversible photoswitch fusion protein is shown in SEQ ID No. 1.
  • the photoacoustic contrast agent is designed to have the amino acid sequence shown in SEQ ID No. 1, which has the characteristics of efficiently and specifically targeting tissues with high collagen expression. Furthermore, by leveraging the reversible light conversion ability of the reversible photoswitch protein DrBphP, it can reduce background noise in the lesion area, thereby enhancing the contrast of photoacoustic imaging and achieving accurate disease diagnosis.
  • 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.
  • the full-length DrBphP1 consists of 755 amino acids and has an N-terminal light-sensing core module (PSM, residues 1-594), which autocatalytically binds biliverdin (BV plastid) to a conserved cysteine residue; in addition, DrBphP1 also contains a C-terminal variable output module (OM, residues 595-755), which is responsible for triggering downstream cellular responses; the present invention retains the PSM domain of DrBphP1 (called DrBphP) and replaces the OM domain with the CBD domain to give DrBphP specific tumor-targeting properties; at the same time, a flexible loop structure is introduced between the PSM and CBD domains to promote the correct folding of the two structures.
  • PSM N-terminal light-sensing core module
  • OM C-terminal variable output module
  • loop structure is the amino acid sequence connecting PSM and CBD.
  • the present invention also provides a method for preparing a photoacoustic contrast agent based on a reversible photoswitch fusion protein, comprising the steps of:
  • Step S10 After codon optimization of the DrBphP-CBD gene, the gene was amplified by PCR and cloned into a vector for cultivation to obtain a bacterial culture;
  • Step S20 using an inducer to induce the bacterial culture to express protein, incubating the culture and performing a first centrifugation to obtain cells;
  • Step S30 resuspending the cells in a buffer solution for cell lysis, and performing a second centrifugation to obtain a lysate;
  • Step S40 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.
  • the preparation method can be used to prepare a photoacoustic contrast agent that efficiently and specifically targets collagen-highly expressed tissues.
  • the photoacoustic contrast agent can reduce the background noise in the lesion area, thereby enhancing the contrast of photoacoustic imaging and achieving accurate disease diagnosis.
  • the vector is one or more of pET28a, pET28b, and pET28c; the restriction enzyme sites used for cloning into the vector are NdeI and XhoI.
  • the pET28a vector provides an N-terminal His6 tag to facilitate protein purification.
  • the inducer is isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG).
  • the concentration of the inducer is 0.6 mM-1.0 mM.
  • the incubation temperature is 15°C-17°C, and the incubation time is 18h-22h;
  • the first centrifugal rotation speed is 7000rpm-8500rpm, and the first centrifugal time is 25min-35min;
  • the second centrifugal rotation speed is 18000rpm-22000rpm, and the second centrifugal time is 45min-60min.
  • the buffer comprises Tris-HCl, NaCl, imidazole, dipyrrolidine chloride, Tris (2-carboxyethyl) phosphate hydrochloride, and protease inhibitors.
  • the culture was incubated at 16°C for 20 hours, and the cells were harvested by centrifugation at 8000 rpm for 30 minutes.
  • the cell pellet was resuspended in a binding buffer containing 30 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, 0.1 mM bipyrrolidine chloride (BV), 1 mM Tris (2-carboxyethyl) phosphate hydrochloride (TCEP), and 0.5 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF), and the cells were lysed using an ultra-high pressure homogenizer.
  • Tris-HCl 500 mM NaCl
  • 5 mM imidazole 0.1 mM bipyrrolidine chloride (BV)
  • TCEP 1 mM Tris (2-carboxyethyl) phosphate hydrochloride
  • PMSF protease inhibitor phenylmethylsulfonyl flu
  • the supernatant was purified by centrifugation at 20,000 rpm for 50 minutes to obtain a clarified lysate (supernatant).
  • the supernatant was subjected to immobilized metal affinity chromatography (IMAC) using a HisTrapTM HP column (Cytiva, USA).
  • IMAC immobilized metal affinity chromatography
  • 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 photoacoustic contrast agent based on the reversible photoswitchable fusion protein was obtained.
  • the present invention also provides an 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.
  • Fibrosis is not limited to tumors, but also covers liver fibrosis. Abnormal collagen accumulation is a common pathological feature of various diseases, including fibrosis, renal fibrosis, and skin fibrosis. Therefore, leveraging the unique properties of the DrBphP-CBD fusion protein can enable specific imaging of these fibrotic diseases and tumors, providing new strategies for early diagnosis and treatment while also broadening the medical application of photoacoustic imaging.
  • This photoacoustic contrast agent not only enhances tumor diagnosis and treatment but also provides important support for the diagnosis of fibrotic diseases, demonstrating the broad potential of photoacoustic imaging technology in modern medicine.
  • the fibrotic disease includes but is not limited to one or more of liver fibrosis, lung fibrosis, kidney fibrosis, cardiac fibrosis, and skin fibrosis.
  • the photoacoustic contrast agent is used to prepare a drug for treating tumors or fibrotic diseases, and its administration method includes intravenous, intramuscular, intradermal or subcutaneous injection.
  • the drug has a wide range of applications and is intended to be used for a variety of diseases including tumors, liver fibrosis, renal fibrosis and skin fibrosis, as well as other related fibrotic diseases that may be caused by these conditions.
  • liver fibrosis may be caused by a variety of factors, such as viral hepatitis, alcoholic hepatitis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure, drug reactions, etc., including some unexplained conditions and other diseases that may be caused by liver fibrosis.
  • causes of renal fibrosis include but are not limited to hypertension, glomerulonephritis, systemic lupus erythematosus, scleroderma, renal transplant rejection, pyelonephritis, kidney stones, hyperlipidemia, diabetes, hyperuricemia, hypercalciuria, etc., as well as other unexplained renal fibrosis and diseases induced by renal fibrosis.
  • Skin fibrosis may be caused by drug reactions, idiopathic, genetic, or other factors, including those with unknown causes, and other diseases that may be associated with skin fibrosis.
  • the fusion protein photoacoustic contrast agent provided by the present invention can be applied to a variety of fibrotic diseases caused by different reasons and can be administered through a variety of routes.
  • This embodiment provides a photoacoustic contrast agent based on a reversible photoswitchable fusion protein and characterizes it, specifically including the following:
  • the full-length DrBphP1 consists of 755 amino acids and has an N-terminal light-sensing core module (PSM, residues 1-594). This module autocatalytically binds the BV plastid to a conserved cysteine residue. In addition, it also contains a C-terminal variable output module (OM, residues 595-755), which is responsible for triggering downstream cellular responses.
  • PSM N-terminal light-sensing core module
  • OM C-terminal variable output module
  • the PSM domain of DrBphP1 (called DrBphP) is retained, and the OM domain is replaced with the CBD domain to give DrBphP specific tumor targeting properties.
  • a flexible loop structure is introduced between the PSM and CBD domains to promote the correct folding of the two domains (as shown in Figure 1).
  • the amino acid sequence of the DrBphP-CBD fusion protein is shown in SEQ ID No.1.
  • the DrBphP-CBD gene was codon-optimized for expression in E. coli, then amplified by PCR and cloned into the pET28a vector using the restriction enzyme sites NdeI and XhoI.
  • the pET28a vector provides an N-terminal His6 tag to facilitate protein purification.
  • the cell pellet was resuspended in a binding buffer containing 30 mM Tris-HCl (pH 7.6), 500 mM NaCl, 5 mM imidazole, 0.1 mM bipyrrolidine chloride (BV), 1 mM Tris (2-carboxyethyl) phosphate hydrochloride (TCEP) and 0.5 mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF).
  • the cells were lysed using an ultra-high pressure homogenizer and the clarified lysate was obtained by centrifugation at 20,000 rpm for 50 minutes.
  • the supernatant was processed by immobilized metal affinity chromatography (IMAC) using a HisTrapTM HP column (Cytiva, USA). Protein fractions eluted from IMAC were pooled and further purified by size exclusion chromatography on a Superdex 200 16/600 column (Cytiva, USA) using an AKTA Pure FPLC system (Cytiva, USA). The homogeneity of the purified DrBphP-CBD protein was confirmed by SDS-PAGE analysis ( Figure 2).
  • DrBphP-CBD in vitro binding affinity of DrBphP-CBD to recombinant collagen was evaluated by indirect enzyme-linked immunosorbent assay (ELISA).
  • Recombinant collagen I or III (10 ⁇ g/mL each in PBS) was coated on an ELISA plate, incubated at 37°C overnight, and then blocked with 2% BSAPBS containing 0.05% Tween 20 (PBS-T) for 1 hour and then washed.
  • DrBphP-CBD fusion protein at different concentrations was then added and incubated for 2 hours.
  • DrBphP-CBD fusion protein was incubated with anti-His antibody (ab18184, Abcam) and goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (ab205719, Abcam) and detected using tetramethylbenzidine substrate.
  • HRP horseradish peroxidase
  • the binding affinity test graph of DrBphP-CBD fusion protein to recombinant type I and type III collagen is shown in Figure 3, and the results show that DrBphP-CBD has a strong affinity for type I and type III collagen. and force enhancement, with dissociation constants (Kd) of 0.48 nM and 26.45 nM, respectively.
  • this example verifies the photochromic properties of DrBphP-CBD.
  • the fusion protein converts from the Pr (on) state to the Pfr (off) state, and reversibly converts from the Pfr state to the Pr state under 808nm light irradiation.
  • the contrast generated by the photoswitch is crucial to improving imaging specificity and suppressing background interference.
  • This contrast is quantified by the ratio of the absorption coefficients of the on and off states. A higher ratio indicates that after removing the background signal, the intrinsic signal of the pigment protein is better maintained, thereby optimizing the image contrast in differential photoacoustic imaging.
  • the absorption spectrum of the DrBphP-CBD fusion protein is shown in Figure 4.
  • the spectral absorption characteristics of DrBphP-CBD between its two states change significantly.
  • the absorption coefficient ratio (Pfr/Pr) at 760nm is approximately 11, indicating that it has excellent photoswitch contrast.
  • the in vitro photoacoustic imaging image of the DrBphP-CBD fusion protein is shown in Figure 5.
  • the result of subtracting the off state from the on-state PA image is a difference image with significantly enhanced contrast.
  • this example evaluated the reversible photoswitching properties of the DrBphP-CBD fusion protein in vitro (as shown in Figure 6).
  • This example evaluated the in vivo differential photoacoustic imaging effect of the DrBphP-CBD fusion protein. As shown in Figure 7, the photoacoustic image in the on state was subtracted from the result in the off state, resulting in a difference image with significantly enhanced contrast inside the tumor.
  • the reversible photoswitching properties of the DrBphP-CBD fusion protein were further evaluated (as shown in Figure 8). By alternating irradiation at wavelengths of 760 nm and 635 nm, repeated photoactivation cycles were successfully induced, verifying the reversible photoswitching ability of the DrBphP-CBD fusion protein in vivo and the repeatability of its imaging.
  • DrBphP-CBD fusion protein in different subcutaneous tumor models and its effectiveness in photoacoustic imaging.
  • DrBphP fusion proteins containing CBD and without CBD were injected into mice carrying MC38, MDA-MB-231 and 4T1 tumors via the tail vein, respectively.
  • the signal accumulation of DrBphP-CBD and DrBphP in tumor tissue was observed by photoacoustic imaging, and tumor-bearing mice were imaged at different time intervals before and after injection (2, 4, 6, 8, 10, 12, 24, 48 and 72 hours) to comprehensively analyze the targeting efficiency of DrBphP-CBD and its retention in tumor tissue.
  • DrBphP-CBD showed significantly improved tumor-specific targeting and retention properties in tumor tissue in the three evaluated tumor types.
  • 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 purer difference images.
  • extra-organ photoacoustic imaging performed 10 hours after injection showed that the photoacoustic signal of the DrBphP-CBD fusion protein mainly accumulated in the tumor, followed by localization in other organs, further confirming its specificity for tumor targeting.
  • the present invention provides a photoacoustic contrast agent based on a reversible photoswitch fusion protein, as well as its preparation method and application.
  • the photoacoustic contrast agent based on a reversible photoswitch fusion protein comprises a photochrome protein derived from Deinococcus radiodurans and a collagen-binding domain fused to the C-terminus of the photochrome protein.
  • the photochrome protein is derived from Deinococcus radiodurans and is fused to the C-terminus of the photochrome protein.
  • the present invention utilizes fusion protein technology to construct a highly efficient delivery system for the treatment of tumors and various fibrotic diseases.
  • DrBphP-CBD fusion protein which combines the photochrome protein (DrBphP) from Deinococcus radiodurans with the collagen-binding domain (CBD), cleverly integrating the photoswitch function of DrBphP with the high affinity of CBD for collagen.
  • the DrBphP-CBD fusion protein maintains high affinity for type I and type III collagen and its photoswitchable properties; moreover, this photoacoustic contrast agent utilizes the atypical vascular structure of tumors and the transitional deposition of collagen in fibrotic diseases, enabling it to specifically attach to exposed collagen, facilitating sustained delivery and retention in the affected tissue microenvironment, and providing enhanced photoswitchable photoacoustic differential imaging.

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Abstract

一种基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用，光声造影剂包括光敏色素蛋白，以及融合于光敏色素蛋白C端的胶原结合域；光敏色素蛋白来自于耐辐射奇球菌。该光声造影剂将来自于耐辐射奇球菌的光敏色素蛋白与胶原结合域结合，巧妙集成了DrBphP的光开关功能和CBD对胶原蛋白的高度亲和性。DrBphP-CBD融合蛋白保持了对I型和III型胶原蛋白的高亲和性以及其光开关特性；该光声造影剂利用肿瘤的非典型血管结构和纤维化疾病中胶原蛋白的过渡沉积，使其能特异性地附着于暴露的胶原蛋白，有助于在受影响组织微环境中实现持续输送和保留，并提供增强的光开关光声差分成像，用于疾病的病理切片染色和活体成像检测。

Description

基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用 技术领域
本发明涉及造影剂技术领域，尤其涉及一种基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用。
背景技术
光声成像是一种结合了高光学对比度的光学成像和深层组织中卓越空间分辨率的超声波的非电离混合技术，它克服了纯光学和超声成像各自的局限性，并且能够在多种尺度上进行无需标记的成像，这使其与其他成像方式截然不同。近期的研究进展主要聚焦于开发外源性对比剂以提高图像对比度和增强疾病监测的信噪比。然而，一些内在挑战，特别是像血红蛋白和黑色素这样的内源性色素体的吸收，引入了显著的背景噪声，从而削弱了光声成像的灵敏度，克服这些限制对于最大化光声成像在临床应用中的潜力至关重要。
在多尺度光声成像的最新进展中，可逆光开关色素蛋白的潜力被强调，尤其是细菌型植物色素蛋白(Bacteriophytochrome photoreceptors,BphPs)。这些由基因编码的光吸收蛋白结合胆绿素(BV)，能够在红光和近红外光吸收状态之间切换，并具有抗光漂白性，使其成为纵向成像的理想选择。通过减去两种不同状态下捕获的光声图像，这种光致变色特性有助于消除外来背景噪声。这种被称为背景抑制的光声成像技术，有望用于探测深部疾病。
目前的方法，包括利用光开关色素蛋白进行光声成像的技术，虽然在研究肿瘤方面取得了一定的成功，但这些方法主要关注肿瘤特异性的探针设计和递送系统的开发，且在实际的临床应用中遇到了局限。特别是，BphPs色素蛋白以其独特的光化学特性而受到关注，包括其在深层组织中保持的高效率、具有的双重吸收状态、无细胞毒性、以及对内源性BV的选择性结合能力，但它们缺乏特定于纤维化疾病胶原蛋白的结合位点，限制了其在非肿瘤性纤维化疾病成像中的应用。
胶原蛋白的异常积累在肿瘤发生发展及多种纤维化疾病中扮演着关键角色，这些疾病包括但不限于肝纤维化、肺纤维化、肾纤维化、心脏纤维化和皮肤纤维化。这种异常积累 不仅影响组织结构和功能，还是疾病进展的一个显著标志。尽管利用先进的成像技术如光声成像来研究肿瘤及多种纤维化疾病已经取得一定进展，目前还没有一种探针能同时适用于体外组织切片染色和活体光声成像，以监测胶原蛋白在这些纤维化疾病中的变化。
因此，现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足，本发明的目的在于提供一种基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用，旨在解决现有光声造影剂缺乏肿瘤特定结合位点，光声成像对比度较差等问题。
本发明的技术方案如下：
一种基于可逆光开关融合蛋白的光声造影剂，包括光敏色素蛋白，以及融合于所述光敏色素蛋白C端的胶原结合域；所述光敏色素蛋白来自于耐辐射奇球菌。
所述的基于可逆光开关融合蛋白的光声造影剂，其中，所述基于可逆光开关融合蛋白的光声造影剂的氨基酸序列如SEQ ID No.1所示。
所述的基于可逆光开关融合蛋白的光声造影剂，其中，所述光敏色素蛋白包括一个N端感光模块，所述N端感光模块的半胱氨酸残基结合有胆绿素；所述胶原结合域用于靶向胶原蛋白。
一种基于可逆光开关融合蛋白的光声造影剂的制备方法，包括步骤：
对DrBphP-CBD基因进行密码子优化后，通过PCR扩增并克隆到载体中进行培养，得到细菌培养物；
使用诱导剂诱导所述细菌培养物进行蛋白表达，经孵育处理，第一离心处理后得到细胞；
将所述细胞重悬于缓冲液中进行细胞裂解，经第二离心处理后，得到裂解液；
所述裂解液通过固定金属亲和层析处理，并进行尺寸排阻层析进行纯化，得到所述基于可逆光开关融合蛋白的光声造影剂。
所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其中，所述载体为pET28a载体、pET28b、pET28c中的一种或多种；所述克隆到载体中使用的限制性酶切位点为Ndel 和XhoI。
所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其中，所述诱导剂为异丙基β-D-1-硫代半乳糖苷。
所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其中，所述孵育处理的温度为15℃-17℃，所述孵育处理的时间为18h-22h；所述第一离心处理的转速为7000rpm-8500rpm，所述第一离心处理的时间为25min-35min；所述第二离心处理的转速为18000rpm-22000rpm，所述第二离心处理的时间为45min-60min。
所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其中，所述缓冲液包括Tris-HCl、NaCl、咪唑、双吡咯绿胆碱氯化物、Tris(2-羧乙基)磷酸盐氢氯化物、蛋白酶抑制剂。
一种基于可逆光开关融合蛋白的光声造影剂在肿瘤和纤维化疾病中胶原蛋白含量检测中的应用。
所述的基于可逆光开关融合蛋白的光声造影剂的应用，其中，所述纤维化疾病包括肝纤维化、肺纤维化、肾纤维化、心脏纤维化、皮肤纤维化中的一种或多种。
有益效果：本发明提供一种基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用，基于可逆光开关融合蛋白的光声造影剂包括光敏色素蛋白，以及融合于所述光敏色素蛋白C端的胶原结合域；所述光敏色素蛋白来自于耐辐射奇球菌。本发明通过融合蛋白技术构建一种旨在治疗肿瘤及多种纤维化疾病的高效递送系统，具体是通过DrBphP-CBD融合蛋白的设计与制备，该融合蛋白将来自于耐辐射奇球菌的光敏色素蛋白(DrBphP)与胶原结合域(CBD)结合，巧妙集成了DrBphP的光开关功能和CBD对胶原蛋白的高度亲和性。DrBphP-CBD融合蛋白保持了对一型和三型胶原蛋白的高亲和性以及其光开关特性；并且，该光声造影剂利用肿瘤的非典型血管结构和纤维化疾病中胶原蛋白的过渡沉积，使其能特异性地附着于暴露的胶原蛋白，有助于在受影响组织微环境中实现持续输送和保留，并提供增强的光开关光声差分成像。
附图说明
图1为本发明实施例1中DrBphP-CBD融合蛋白的设计图；
图2为本发明实施例1中DrBphP-CBD融合蛋白的纯化图；
图3为本发明实施例1中DrBphP-CBD融合蛋白与重组I型和III型胶原蛋白的结合亲和力测试图；
图4为本发明实施例1中DrBphP-CBD融合蛋白的吸收光谱；
图5为本发明实施例1中DrBphP-CBD融合蛋白体外光声成像图；
图6为本发明实施例1中DrBphP-CBD融合蛋白的体外可逆光开关特性图；
图7为本发明实施例1中DrBphP-CBD融合蛋白在体内的差分光声成像效果图；
图8为本发明实施例1中DrBphP-CBD融合蛋白的体内可逆光开关特性图；
图9为本发明实施例1中DrBphP-CBD融合蛋白在MC38肿瘤模型中的时间分辨PA差分成像图；
图10为本发明实施例1中DrBphP-CBD融合蛋白在MDA-MB-231肿瘤模型中的时间分辨PA差分成像图；
图11为本发明实施例1中DrBphP-CBD融合蛋白在4T1肿瘤模型中的时间分辨PA差分成像图；
图12为本发明实施例1中DrBphP-CBD融合蛋白对肿瘤的靶向性数据图。
具体实施方式
本发明提供一种基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本技术领域技术人员可以理解，除非另外定义，这里使用的所有术语(包括技术术语和科学术语)，具有与本发明所属领域中的普通技术人员的一般理解相同的意义。还应该理解的是，诸如通用字典中定义的那些术语，应该被理解为具有与现有技术的上下文中的意义一致的意义，并且除非像这里一样被特定定义，否则不会用理想化或过于正式的含义来解释。
胶原蛋白作为哺乳动物细胞外基质的主要成分，在健康组织中扮演着重要角色，但在肿瘤发生发展和纤维化疾病中，其异常过度表达和积累成为了问题所在。当前研究中，由 于完整血管系统的低渗透性难以接触胶原；肿瘤组织中的异常血管结构增加了其渗透性，使胶原蛋白暴露于全身循环中，这种异常暴露，结合胶原蛋白在多种癌症中的过度表达，使其成为医学成像的理想标靶。在以往研究已证实，工程化细胞因子和单克隆抗体对胶原蛋白具有亲和力，能有效地介导治疗剂在肿瘤基质中的选择性定位和保留。特别是血管性血友病因子的胶原蛋白结合域(CBD)对I型和III型胶原蛋白表现出显著亲和力，这两种胶原蛋白在肿瘤基质中以及纤维化病灶处大量存在。然而，这些成果大多聚焦于肿瘤微环境，而不是广泛的纤维化病变。因此，开发一种既能在活体中通过光声成像技术监测胶原蛋白变化，又可以用于体外胶原组织切片染色的探针，对于肿瘤及纤维化疾病的诊断和治疗具有重要意义。这样的探针不仅需要具备高度的特异性和敏感性，以区分健康和病变组织中的胶原蛋白，还需具备在复杂生物环境中稳定的光化学和生物化学性质，以实现准确的成像和疾病监测。将CBD与BphPs融合，可能赋予BphPs特异性地靶向肿瘤部位并蓄积的能力，从而增强其局部生物可用性，优化其在诊断成像中的潜力。
基于此，本发明提供一种基于可逆光开关融合蛋白的光声造影剂，包括光敏色素蛋白，以及融合于所述光敏色素蛋白C端的胶原结合域；所述光敏色素蛋白来自于耐辐射奇球菌。
在本实施方式中，通过基因重组方法将具有靶向胶原蛋白能力的胶原结合域(CBD)融合到来自耐辐射奇球菌(Deinococcus radiodurans)的光敏色素蛋白(bacterio-phytochrome,DrBphP)的C端，得到了能够靶向肿瘤组织的融合蛋白DrBphP-CBD；该融合蛋白可以高效且特异性地靶向胶原高表达组织，如肿瘤或纤维化疾病部位；并且，借助于可逆光开关蛋白DrBphP的可逆光转换能力，可以减少病灶区背景噪声，从而增强光声成像的对比度，实现精确的疾病诊断。
具体地，本发明通过融合蛋白技术构建了一种旨在治疗肿瘤及多种纤维化疾病的高效递送系统，具体是通过DrBphP-CBD融合蛋白的设计与制备。这一融合蛋白将来自Deinococcus radiodurans细菌的光敏色素(DrBphP)的N端感光模块与胶原蛋白结合域(CBD)结合，巧妙集成了BphPs的光开关功能和CBD对胶原蛋白的高度亲和性。并且通过实验表明，DrBphP-CBD融合蛋白保持了对I型和III型胶原蛋白的高亲和性以及其光开关特性，证实了在设计、表达和纯化方面的成功。同时，利用DrBphP-CBD融合蛋白(光声造影剂)开展了体内肿瘤和纤维化疾病的光声差分成像。其独特设计利用肿瘤的非典型 血管结构和纤维化疾病中胶原蛋白的过度沉积，使其能特异性地附着于暴露的胶原蛋白，有助于在受影响组织微环境中实现持续输送和保留，并提供增强的光开关光声差分成像。在各种小鼠疾病模型中的体内展示了DrBphP-CBD在病变组织中的优先靶向和富集，通过显著降低背景信号以提高成像的对比度。
在一些实施方式中，所述基于可逆光开关融合蛋白的光声造影剂的氨基酸序列如SEQ ID No.1所示。将所述光声造影剂的氨基酸序列设计成如SEQ ID No.1所示，其具有高效且特异性地靶向胶原高表达组织的特点，并可借助于可逆光开关蛋白DrBphP的可逆光转换能力，可以减少病灶区背景噪声，从而增强光声成像的对比度，实现精确的疾病诊断。
在一些实施方式中，所述光敏色素蛋白包括一个N端感光模块，所述N端感光模块的半胱氨酸残基结合有胆绿素；所述胶原结合域用于靶向胶原蛋白。
具体地，全长DrBphP1由755个氨基酸组成，具备一个N端光感核心模块(PSM，1-594残基)，该模块自催化地将胆绿素(BV色素体)结合到一个保守的半胱氨酸残基；此外，DrBphP1还包含一个C端可变输出模块(OM，残基595-755)，负责触发下游细胞反应；而本发明保留了DrBphP1的PSM结构域(称为DrBphP)，而将OM域替换为CBD结构域，以赋予DrBphP特定的肿瘤靶向属性；同时PSM和CBD结构域之间引入一个灵活的loop结构，以促进两个结构的正确折叠。
需要说明的是，loop结构是连接PSM和CBD之间的氨基酸序列。
除此之外，本发明还提供一种基于可逆光开关融合蛋白的光声造影剂的制备方法，包括步骤：
步骤S10：对DrBphP-CBD基因进行密码子优化后，通过PCR扩增并克隆到载体中进行培养，得到细菌培养物；
步骤S20：使用诱导剂诱导所述细菌培养物进行蛋白表达，经孵育处理，第一离心处理后得到细胞；
步骤S30：将所述细胞重悬于缓冲液中进行细胞裂解，经第二离心处理后，得到裂解液；
步骤S40：所述裂解液通过固定金属亲和层析处理，并进行尺寸排阻层析进行纯化，得到所述基于可逆光开关融合蛋白的光声造影剂。
本实施方式中，利用该制备方法可制得高效且特异性地靶向胶原高表达组织的光声造影剂，其可借助于可逆光开关蛋白DrBphP的可逆光转换能力，可以减少病灶区背景噪声，从而增强光声成像的对比度，实现精确的疾病诊断。
在一些实施方式中，所述载体为pET28a载体、pET28b、pET28c中的一种或多种；所述克隆到载体中使用的限制性酶切位点为Ndel和XhoI。所述pET28a载体提供了N端His6标签以便于蛋白纯化。
在一些实施方式中，所述诱导剂为异丙基β-D-1-硫代半乳糖苷(IPTG)。
在一些实施方式中，所述诱导剂的浓度为0.6mM-1.0mM。
具体地，当所述细菌培养物的光密度600nm(OD600)达到0.8时，使用0.8mM异丙基β-D-1-硫代半乳糖苷诱导蛋白表达。
在一些实施方式中，所述孵育处理的温度为15℃-17℃，所述孵育处理的时间为18h-22h；所述第一离心处理的转速为7000rpm-8500rpm，所述第一离心处理的时间为25min-35min；所述第二离心处理的转速为18000rpm-22000rpm，所述第二离心处理的时间为45min-60min。
在一些实施方式中，所述缓冲液包括Tris-HCl、NaCl、咪唑、双吡咯绿胆碱氯化物、Tris(2-羧乙基)磷酸盐氢氯化物、蛋白酶抑制剂。
具体地，诱导后，将培养物在16℃下孵育20小时，通过8000rpm离心30分钟收获细胞，细胞沉淀物重悬于含30mM Tris-HCl、500mM NaCl、5mM咪唑、0.1mM双吡咯绿胆碱氯化物(BV)、1mM Tris(2-羧乙基)磷酸盐氢氯化物(TCEP)、0.5mM蛋白酶抑制剂苯甲基磺酰氟化物(PMSF)的结合缓冲液中，使用超高压均质机进行细胞裂解，并经20000rpm离心50分钟后得到澄清的裂解液(上清液)，上清液使用HisTrapTM HP柱(Cytiva，USA)进行固定金属亲和层析(IMAC)处理，从IMAC洗脱的蛋白质部分汇集后，使用AKTA Pure FPLC系统(Cytiva,USA)通过Superdex 200 16/600柱(Cytiva,USA)进行尺寸排阻层析以进一步纯化。得到所述基于可逆光开关融合蛋白的光声造影剂。
另外，本发明还提供一种基于可逆光开关融合蛋白的光声造影剂在肿瘤和纤维化疾病中胶原蛋白含量检测中的应用。
在扩大该光声造影剂的应用范围至纤维化疾病方面，纤维化的发展过程中胶原蛋白的过度沉积为该光声造影剂提供了新的应用前景，纤维化不仅限于肿瘤，而且涵盖了肝纤维 化、肾纤维化和皮肤纤维化等多种疾病，其中胶原蛋白的异常积累是共通的病理特征。因此，利用DrBphP-CBD融合蛋白的独特性能，可以实现对这些纤维化疾病和肿瘤的特异性成像，为早期诊断和治疗提供新策略，同时也拓宽了光声成像技术在医学领域的应用。该光声造影剂不仅能增强对肿瘤的诊断和治疗，还能对纤维化疾病的诊断提供重要支持，展示了光声成像技术在现代医学中的广泛应用潜力。
在一些实施方式中，所述纤维化疾病包括但不限于肝纤维化、肺纤维化、肾纤维化、心脏纤维化、皮肤纤维化中的一种或多种。
在一些实施方式中，利用所述光声造影剂制备治疗肿瘤或纤维化疾病药物，其给药方法包括通过静脉、肌肉注射、皮内注射或皮下注射等方式。此外，该药物的应用范围广泛，旨在应用于包括肿瘤，肝纤维化、肾纤维化和皮肤纤维化在内的多种疾病，以及这些病症可能引发的其他相关纤维化疾病。具体而言，肝纤维化可能由多种因素引起，如病毒性肝炎、酒精性肝炎、自身免疫性疾病、脂肪肝、营养不良、慢性充血性心力衰竭、药物反应等，包括一些原因不明的情况以及肝纤维化可能导致的其他疾病。肾纤维化的成因包括但不限于高血压、肾小球肾炎、系统性红斑狼疮、硬皮病、肾移植排斥反应、肾盂肾炎、肾结石、高血脂症、糖尿病、高尿酸血症、高钙尿症等，以及其他原因不明的肾脏纤维化和由肾纤维化诱发的疾病。皮肤纤维化可能是由药物反应、特发性、遗传等因素引起的，包括那些原因不明的情况，以及皮肤纤维化可能引发的其他疾病。
具体地，本发明所提供的融合蛋白光声造影剂可应用于由不同原因引起的多种纤维化疾病，并可通过多种给药途径。
下面进一步举实施例以详细说明本发明。同样应理解，以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。
实施例1
本实施例提供一种基于可逆光开关融合蛋白的光声造影剂，并对其进行表征，具体包括如下：
1.设计DrBphP-CBD融合蛋白
全长DrBphP1由755个氨基酸组成，具备一个N端光感核心模块(PSM，1-594残基)， 该模块自催化地将BV色素体结合到一个保守的半胱氨酸残基。此外，还包含一个C端可变输出模块(OM，残基595-755)，负责触发下游细胞反应。在本实施例中，保留了DrBphP1的PSM结构域(称为DrBphP)，而将OM域替换为CBD结构域，以赋予DrBphP特定的肿瘤靶向属性。同时，PSM和CBD结构域之间引入了一个灵活的loop结构，以促进两个结构域的正确折叠(如图1所示)。DrBphP-CBD融合蛋白的氨基酸序列如SEQ ID No.1所示。
2.DrBphP-CBD融合蛋白的表达和纯化
本实施例中，DrBphP-CBD基因经过密码子优化以便于在大肠杆菌中表达，随后通过PCR扩增并克隆到pET28a载体中，使用的限制性酶切位点为NdeI和XhoI。pET28a载体提供了N端His6标签以便于蛋白纯化。当细菌培养物的光密度600nm(OD600)达到0.8时，使用0.8mM异丙基β-D-1-硫代半乳糖苷(IPTG)诱导蛋白表达。诱导后，将培养物在16℃下孵育20小时，通过8000rpm离心30分钟收获细胞，细胞沉淀物重悬于含30mM Tris-HCl(pH 7.6)、500mM NaCl、5mM咪唑、0.1mM双吡咯绿胆碱氯化物(BV)、1mM Tris(2-羧乙基)磷酸盐氢氯化物(TCEP)和0.5mM蛋白酶抑制剂苯甲基磺酰氟化物(PMSF)的结合缓冲液中，使用超高压均质机进行细胞裂解，并经20000rpm离心50分钟后得到澄清的裂解液。上清液通过固定金属亲和层析(IMAC)处理，使用HisTrapTM HP柱(Cytiva,USA)；从IMAC洗脱的蛋白质部分汇集后，通过Superdex 200 16/600柱(Cytiva,USA)进行尺寸排阻层析以进一步纯化，并使用AKTA Pure FPLC系统(Cytiva,USA)。纯化的DrBphP-CBD蛋白通过SDS-PAGE分析确认其均一性(如图2所示)。
3.DrBphP-CBD融合蛋白的体外表征
本实施例通过间接酶联免疫吸附测定(ELISA)评估了DrBphP-CBD与重组胶原蛋白的体外结合亲和力。重组胶原蛋白I或III(每种10μg/mL在PBS中)涂覆在ELISA板上，于37℃下过夜，随后用含0.05％Tween 20的2％BSAPBS(PBS-T)封闭1小时，然后清洗。随后加入不同浓度的DrBphP-CBD融合蛋白并孵育2小时。清洗后，板子与抗His抗体(ab18184,Abcam)和山羊抗小鼠辣根过氧化物酶(HRP)偶联抗体(ab205719,Abcam)孵育，使用四甲基联苯胺底物进行检测。DrBphP-CBD融合蛋白与重组I型和III型胶原蛋白的结合亲和力测试图如图3所示，结果显示DrBphP-CBD对I型和III型胶原蛋白的亲 和力增强，解离常数(Kd)分别为0.48nM和26.45nM。
此外，本实施例验证了DrBphP-CBD的光致变色特性。在635nm光照射下，融合蛋白从Pr(开)状态转化为Pfr(关)状态，并在808nm光照射下可逆地从Pfr状态转变为Pr状态。通过光开关产生的对比度对提高成像特异性和抑制背景干扰至关重要。这种对比度由开状态和关状态的吸收系数之比来量化，更高的比值表明去除背景信号后，色素蛋白本征信号的维持更好，从而在差异光声成像中优化图像对比度。DrBphP-CBD融合蛋白的吸收光谱如图4所示，DrBphP-CBD在其两种状态之间的光谱吸收特性有明显变化，760nm处的吸收系数比率(Pfr/Pr)约为11，表明其具有卓越的光开关对比度。DrBphP-CBD融合蛋白体外光声成像图如图5所示，从开状态PA图像中减去关状态的结果是一个具有显著增强对比度的差异图像。此外，本实施例评估了DrBphP-CBD融合蛋白在体外的可逆光开关特性(如图6所示)，通过交替暴露于808nm和635nm波长，成功诱导了重复的光激活周期，从而验证了DrBphP-CBD融合蛋白在体外的可逆光开关能力及成像技术的可重复性。
4.DrBphP-CBD融合蛋白的体内表征
本实施例评估了DrBphP-CBD融合蛋白在体内的差分光声成像效果。如图7所示，开启状态下的光声图像与关闭状态下的结果相减，得到的是肿瘤内部具有显著增强对比度的差异图像。
并且进一步评估了DrBphP-CBD融合蛋白的可逆光开关特性(如图8所示)，通过在760nm和635nm波长下交替照射，成功诱导了重复的光激活周期，验证了DrBphP-CBD融合蛋白在体内的可逆光开关能力及其成像的重复性。
此外，本实施例还验证了DrBphP-CBD融合蛋白在不同皮下肿瘤模型中的积累及其在光声成像中的有效性。含CBD和不含CBD的DrBphP融合蛋白分别通过尾静脉注射到携带MC38、MDA-MB-231和4T1肿瘤的小鼠中。通过光声成像观察了DrBphP-CBD和DrBphP在肿瘤组织内的信号积累，并对肿瘤携带小鼠在注射前后不同时间间隔(2、4、6、8、10、12、24、48和72小时)进行了成像，以全面分析DrBphP-CBD的靶向效率和在肿瘤组织中的保留情况。如图9至图11所示，在三种肿瘤模型中，注射后2至4小时内观察到DrBphP-CBD的初始积累。这些融合蛋白在肿瘤组织内的光声信号显著增强，于10至 12小时达到高峰，并持续至72小时。相比之下，只含DrBphP的光声信号在注射后4至6小时内在肿瘤组织中可监测到，但随后逐渐减弱并在8小时后消失。与仅含DrBphP相比，DrBphP-CBD在三种评估肿瘤类型中显示出显著提高的肿瘤特异性靶向和在肿瘤组织中的保留特性。同时，DrBphP-CBD介导的差分光声成像模式有效减少了非靶向信号(如来自肿瘤相关血管、皮肤层和黑色素的信号)的干扰，生成更纯净的差异图像。此外，注射后10小时进行的器官外光声成像(如图12所示)显示，DrBphP-CBD融合蛋白的光声信号主要在肿瘤中积累，其次是在其他器官的定位，进一步证实了其对肿瘤靶向的特异性。
综上所述，本发明提供的一种基于可逆光开关融合蛋白的光声造影剂及其制备方法与应用，基于可逆光开关融合蛋白的光声造影剂包括光敏色素蛋白，以及融合于所述光敏色素蛋白C端的胶原结合域；所述光敏色素蛋白来自于耐辐射奇球菌。本发明通过融合蛋白技术构建一种旨在治疗肿瘤及多种纤维化疾病的高效递送系统，具体是通过DrBphP-CBD融合蛋白的设计与制备，该融合蛋白将来自于耐辐射奇球菌的光敏色素蛋白(DrBphP)与胶原结合域(CBD)结合，巧妙集成了DrBphP的光开关功能和CBD对胶原蛋白的高度亲和性。DrBphP-CBD融合蛋白保持了对一型和三型胶原蛋白的高亲和性以及其光开关特性；并且，该光声造影剂利用肿瘤的非典型血管结构和纤维化疾病中胶原蛋白的过渡沉积，使其能特异性地附着于暴露的胶原蛋白，有助于在受影响组织微环境中实现持续输送和保留，并提供增强的光开关光声差分成像。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (10)

  1. 一种基于可逆光开关融合蛋白的光声造影剂，其特征在于，包括光敏色素蛋白，以及融合于所述光敏色素蛋白C端的胶原结合域；所述光敏色素蛋白来自于耐辐射奇球菌。
  2. 根据权利要求1所述的基于可逆光开关融合蛋白的光声造影剂，其特征在于，所述基于可逆光开关融合蛋白的光声造影剂的氨基酸序列如SEQ ID No.1所示。
  3. 根据权利要求1所述的基于可逆光开关融合蛋白的光声造影剂，其特征在于，所述光敏色素蛋白包括一个N端感光模块，所述N端感光模块的半胱氨酸残基结合有胆绿素；所述胶原结合域用于靶向胶原蛋白。
  4. 一种如1-3任一项所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其特征在于，包括步骤：
    对DrBphP-CBD基因进行密码子优化后，通过PCR扩增并克隆到载体中进行培养，得到细菌培养物；
    使用诱导剂诱导所述细菌培养物进行蛋白表达，经孵育处理，第一离心处理后得到细胞；
    将所述细胞重悬于缓冲液中进行细胞裂解，经第二离心处理后，得到裂解液；
    所述裂解液通过固定金属亲和层析处理，并进行尺寸排阻层析进行纯化，得到所述基于可逆光开关融合蛋白的光声造影剂。
  5. 根据权利要求4所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其特征在于，所述载体为pET28a载体、pET28b、pET28c中的一种或多种；所述克隆到载体中使用的限制性酶切位点为Ndel和XhoI。
  6. 根据权利要求4所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其特征在于，所述诱导剂为异丙基β-D-1-硫代半乳糖苷。
  7. 根据权利要求4所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其特征在于，所述孵育处理的温度为15℃-17℃，所述孵育处理的时间为18h-22h；所述第一离心处理的转速为7000rpm-8500rpm，所述第一离心处理的时间为25min-35min；所述第二离心处理的转速为18000rpm-22000rpm，所述第二离心处理的时间为45min-60min。
  8. 根据权利要求4所述的基于可逆光开关融合蛋白的光声造影剂的制备方法，其特征在于，所述缓冲液包括Tris-HCl、NaCl、咪唑、双吡咯绿胆碱氯化物、Tris(2-羧乙基)磷 酸盐氢氯化物、蛋白酶抑制剂。
  9. 一种如权利要求1-3任一项所述的基于可逆光开关融合蛋白的光声造影剂在肿瘤和纤维化疾病中胶原蛋白含量检测中的应用。
  10. 根据权利要求9所述的基于可逆光开关融合蛋白的光声造影剂的应用，其特征在于，所述纤维化疾病包括肝纤维化、肺纤维化、肾纤维化、心脏纤维化、皮肤纤维化中的一种或多种。
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LEOPOLD ANNA V.; PLETNEV SERGEI; VERKHUSHA VLADISLAV V.: "Bacterial Phytochrome as a Scaffold for Engineering of Receptor Tyrosine Kinases Controlled with Near-Infrared Light", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 432, no. 13, 14 April 2020 (2020-04-14), United Kingdom , pages 3749 - 3760, XP086192685, ISSN: 0022-2836, DOI: 10.1016/j.jmb.2020.04.005 *
LI LEI, SHEMETOV ANTON A., BALOBAN MIKHAIL, HU PENG, ZHU LIREN, SHCHERBAKOVA DARIA M., ZHANG RUIYING, SHI JUNHUI, YAO JUNJIE, WANG: "Small near-infrared photochromic protein for photoacoustic multi-contrast imaging and detection of protein interactions in vivo", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, UK, vol. 9, no. 1, UK, XP093362848, ISSN: 2041-1723, DOI: 10.1038/s41467-018-05231-3 *

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