WO2020047824A1 - 一种基于胆绿素的光热制剂、其制备方法及应用 - Google Patents

一种基于胆绿素的光热制剂、其制备方法及应用 Download PDF

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WO2020047824A1
WO2020047824A1 PCT/CN2018/104555 CN2018104555W WO2020047824A1 WO 2020047824 A1 WO2020047824 A1 WO 2020047824A1 CN 2018104555 W CN2018104555 W CN 2018104555W WO 2020047824 A1 WO2020047824 A1 WO 2020047824A1
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bilirubin
concentration
photothermal
solution
metal
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French (fr)
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闫学海
邢蕊蕊
邹千里
李淑坤
赵鲁阳
沈桂芝
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中国科学院过程工程研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • 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; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

  • the invention belongs to the field of photothermal nanomedicine, and particularly relates to a photothermal formulation based on bilirubin and having near-infrared absorption, and a preparation method and application thereof.
  • Photothermal therapy is a new type of disease (such as tumor, skin, vascular disease, etc.) treatment. This method is easy to operate, highly targeted, non-invasive to normal tissues, and can effectively inhibit recurrence.
  • the photothermal preparation can capture light energy and convert it into heat.
  • the local high fever causes irreversible apoptosis of cells, thereby achieving the effect of photothermal treatment.
  • a pulsed laser is irradiated to the diseased tissue, a photoacoustic signal is generated in the light absorption region of the tissue, thereby realizing photoacoustic imaging of the diseased part. It is a non-invasive, radiation-free, high-resolution imaging method for the disease Early detection and treatment monitoring provide new methods. Nanophotothermal formulations have attracted much attention due to their targeting and high conversion efficiency.
  • Nano preparations used for photothermal treatment should have the following properties: 1) high tissue penetration; 2) high photothermal conversion efficiency; 3) biological safety and degradability.
  • a variety of photothermal preparations and their preparation methods have been disclosed so far: a graphene-based photothermal preparation has been disclosed (patent publication number CN107080844A). When co-incubated with cells and irradiated with red light, cancer cells are realized Effective killing.
  • the biosafety and degradation properties of graphene have greatly limited the development of such inorganic photothermal preparations.
  • a photothermal preparation based on metal phthalocyanine has been disclosed (Patent Publication No.
  • Bilirubin is an endogenous metabolite of hemoglobin and various hemoglobin-containing proteins in animals. It is a very important biological substance. At the same time, bilirubin will be further metabolized in the body to produce bilirubin and bilirubin. The binding of vegetin and proteins in the body is further excreted through the hepato-intestinal circulation and has a clear metabolic mechanism. It is generally believed that bilirubin is a metabolic waste, but bilirubin can effectively alleviate vascular injury, rejection of organ transplantation, and has anti-inflammatory and antioxidant effects.
  • bilirubin is a linear tetrapyrrole structure, non-toxic, and has an obvious absorption peak in the near-infrared region, and bilirubin has a low fluorescence quantum yield, so it has a large photothermal conversion. potential.
  • bilirubin has high protein affinity and metal ion chelating ability. Therefore, the development of bilirubin-based photothermal preparations has far-reaching significance in the field of photothermal therapy. At present, there have been no public reports.
  • the present invention provides photobilirubin based photothermal preparations, preparation methods and applications thereof.
  • the prepared photothermal preparation has the following advantages: 1) near infrared absorption and high tissue penetration depth; 2) high photothermal conversion efficiency; 3) clear metabolic mechanism and high safety; 4) simple preparation.
  • the preparation of this series of bilirubin-based photothermal preparations is of great significance for the development of multifunctional phototherapeutic drugs, and has great application potential in tumors, skin diseases (such as pigment diseases), and vascular diseases.
  • the present invention provides a bilirubin-based photothermal formulation and its use in preparing a photothermal formulation.
  • the bilirubin-based photothermal formulation includes bilirubin and bilirubin gel Bilirubin-metal chelate, bilirubin-biomolecule complex, bilirubin-biomolecule-metal functional assembly;
  • the metal is Fe 3+ , Fe 2+ , Co 2+ , Ni 2+ , Mn 2+ , Cu 2+ , Zn 2+ , Gd 2+ , Se 2+ , Pt 2+ , Ti 2 + , Eu 2+ , Tb 3+, etc., or a combination of at least two of them;
  • the biomolecule includes any one or a combination of at least two of amino acids, polypeptides, proteins, and the like;
  • the amino acid refers to phenylalanine, histidine, tyrosine, glutamic acid, lysine, cysteine, aspartic acid, and tryptophan modified at the N-terminus by a protecting group. Any one or a combination of at least two of leucine, leucine, asparagine, glutamine, etc., preferably, the amino acid is histidine having metal coordination energy;
  • the polypeptide refers to any one or a combination of at least two of amino acid sequences such as dipeptide, tripeptide, and tetrapeptide modified by a protective group at the N-terminus.
  • the polypeptide is a histidine-containing peptide. Dipeptide
  • the N-terminal protecting group includes acetyl (Ac-), phenyl, N-benzyloxycarbonyl, N-tert-butoxycarbonyl, ⁇ -naphthylamide, N- (3-indoleacetyl ), Any one or a mixture of at least two of ferrocenylcarbonyl, cysteine or N-fluorenylmethoxycarbonyl;
  • the protein includes any one or a combination of at least two of albumin, hemoglobin, myoglobin, collagen, lipoprotein, insulin, etc., preferably, the protein is albumin.
  • the present invention provides the photobilirubin-based photothermal preparation according to the first aspect, which is characterized in that:
  • the bilirubin is a single molecule bilirubin solution obtained by completely dissolving bilirubin in an alkaline solution;
  • the bilirubin gel is a shear-thinning, recoverable gel formed by a high concentration of bilirubin through weak intermolecular interactions
  • the bilirubin-metal chelate is a complex composed of bilirubin and metal ions, including a solution of a gelling agent complex, and the addition of metal ions causes quenching of the energy in the bilirubin molecule;
  • the bilirubin-biomolecule complex is a conjugate formed by the assembly of bilirubin and biomolecules, which is mainly formed due to an amidation reaction between the two or based on a weak interaction between molecules;
  • the bilirubin-biomolecule-metal functional assembly is a complex formed by the assembly of bilirubin, biomolecules, and metal ions. Non-covalent bonding and metal coordination contribute to the complex at the same time. Formation.
  • the present invention provides a bilirubin-based photothermal formulation according to the second aspect, characterized in that the prepared photothermal formulation has a property of near-infrared absorption.
  • the present invention provides a bilirubin-based photothermal formulation according to the third aspect, which is characterized in that the prepared photothermal formulation can achieve near-infrared light absorption and thermal energy conversion, and has high photothermal Conversion efficiency.
  • the present invention provides a bilirubin-based photothermal preparation according to the fourth aspect, characterized in that the prepared photothermal preparation has high biological safety and a clear metabolic pathway.
  • the present invention provides a method for preparing bilirubin based on a bilirubin-based photothermal preparation according to the first to fifth aspects, which includes the following steps:
  • bilirubin powder Weigh a certain amount of bilirubin powder and directly dissolve it in a 0.1M NaOH solution, and accelerate the dissolution by sonication or stirring.
  • concentration of said bilirubin is 0.1-100 mM, preferably, said concentration is 0.5 -20mM;
  • the dissolved bilirubin solution is placed in a refrigerator at 4 ° C and aged for 4 to 72 hours in the dark, preferably, the aging time is 12-24 hours to obtain a photothermal formulation bilirubin solution.
  • the present invention provides a method for preparing a bilirubin gel based on a bilirubin-based photothermal formulation according to the first to fifth aspects, which includes the following steps:
  • bilirubin powder Weigh a certain amount of bilirubin powder and directly dissolve it in a small amount of DMSO solution. After the bilirubin is completely dissolved, add an appropriate amount of water and stir well.
  • concentration of the bilirubin is 5-100 mM, preferably, the concentration is 8-50 mM;
  • the solution is placed in a refrigerator at 4 ° C. and left to stand for 4 to 72 hours in the dark, preferably, the aging time is 12 to 24 hours to obtain a photothermal formulation bilirubin gel.
  • the present invention provides a method for preparing a bilirubin-metal chelate based on a bilirubin-based photothermal preparation according to the first to fifth aspects, which includes the following steps:
  • bilirubin powder Weigh a certain amount of bilirubin powder and directly dissolve it in a 0.1M NaOH solution or a small amount of dimethyl sulfoxide (DMSO), and sonicate or stir to accelerate its dissolution.
  • concentration of bilirubin is 0.1 -100 mM, preferably, the concentration is 0.5-20 mM;
  • the concentration of the metal ion is 0.01M-1.0M, preferably, the concentration is 0.1-5M;
  • the concentration ratio of bilirubin to metal ions is 0.01-1000, and preferably, the concentration ratio is 0.1-100;
  • step (3) Adjusts the pH of the mixed solution obtained in step (3) to 6.8-7.3.
  • the pH value is adjusted by adding a basic substance or an acidic substance:
  • the alkaline substance is any one or a mixture of two or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and the like;
  • the acidic substance is any one or a mixture of two or more of hydrochloric acid, sulfuric acid, nitric acid, and the like.
  • step (4) The mixed solution obtained in step (4) is aged at 4 ° C and protected from light for 4 to 72 hours, and preferably, the aging time is 12 to 24 hours to obtain a photothermal preparation-bilelin-metal chelate .
  • the present invention provides a method for preparing a bilirubin-biomolecular complex based on a bilirubin-based photothermal formulation according to the first to fifth aspects, which includes the following steps:
  • bilirubin powder Weigh a certain amount of bilirubin powder and directly dissolve it in a 0.1M NaOH solution or a small amount of dimethyl sulfoxide (DMSO), and sonicate or stir to accelerate its dissolution.
  • concentration of bilirubin is 0.1 -100 mM, preferably, the concentration is 0.5-20 mM;
  • a biomolecular solution is configured, and the biomolecules are weighed and dissolved in an appropriate solvent (such as water, acid, alkali, DMSO, etc.).
  • concentration of the biomolecules is 0.1-1000Mm, preferably, the concentration is 1 -500mM;
  • step (3) Mix the bilirubin solution obtained in step (1) and the biomolecule solution obtained in step (2), and stir well.
  • concentration ratio of bilirubin to biomolecules is 0.01-100, preferably, the concentration ratio is 0.01-1;
  • step (3) Adjusts the pH of the mixed solution obtained in step (3) to 6.8-7.3.
  • the pH value is adjusted by adding a basic substance or an acidic substance:
  • the alkaline substance is any one or a mixture of two or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and the like;
  • the acidic substance is any one or a mixture of two or more of hydrochloric acid, sulfuric acid, nitric acid, and the like.
  • step (4) The mixed solution obtained in step (4) is aged at 4 ° C and protected from light for 4 to 72 hours.
  • the aging time is 12 to 24 hours to obtain a photothermal formulation bilirubin-biomolecule complex.
  • the present invention provides a method for preparing a bilirubin-biomolecule-metal functional assembly based on a bilirubin-based photothermal formulation according to the first to fifth aspects, which includes the following steps:
  • bilirubin powder Weigh a certain amount of bilirubin powder and directly dissolve it in a 0.1M NaOH solution or a small amount of dimethyl sulfoxide (DMSO), and sonicate or stir to accelerate its dissolution.
  • concentration of bilirubin is 0.1 -100 mM, preferably, the concentration is 0.5-20 mM;
  • a biomolecular solution is configured, and the biomolecules are weighed and dissolved in an appropriate solvent (such as water, acid, alkali, DMSO, etc.).
  • concentration of the biomolecules is 0.1-1000Mm, preferably, the concentration is 1 -500mM;
  • the concentration of the metal ion is 0.01M-1.0M, preferably, the concentration is 0.1-5M;
  • the concentration ratio of bilirubin, biomolecules and metal ions is 0.1: 1: 0.01-1000: 100: 1, and preferably, the concentration ratio is 0.2: 1: 0.2;
  • step (4) Adjust the pH of the mixed solution obtained in step (4) to 6.8-7.3.
  • the pH value is adjusted by adding a basic substance or an acidic substance:
  • the alkaline substance is any one or a mixture of two or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and the like;
  • the acidic substance is any one or a mixture of two or more of hydrochloric acid, sulfuric acid, nitric acid, and the like.
  • step (5) The mixed solution obtained in step (5) is aged at 4 ° C and protected from light for 4 to 72 hours.
  • the aging time is 12 to 24 hours to obtain a bilirubin-biomolecule-metal functional assembly.
  • the present invention provides the bilirubin-based photothermal preparation according to the first to tenth aspects for developing a multifunctional phototherapeutic drug, which is applied to tumors, skin diseases (such as pigment diseases), and vascular diseases. treatment.
  • the present invention provides the bilirubin-based photothermal preparation according to the first to eleventh aspects for use in photoacoustic imaging, magnetic resonance imaging (MRI), dynamic enhanced scanning (DMRI), and positron emission computer One or a combination of at least two of tomography (PET), computed tomography (CT), and PET-CT imaging.
  • MRI magnetic resonance imaging
  • DMRI dynamic enhanced scanning
  • PET positron emission computer
  • PET computed tomography
  • PET-CT imaging PET-CT imaging
  • the present invention has the following beneficial effects:
  • Bilirubin has the advantages of non-toxicity, near-infrared absorption, and clear metabolic mechanism. At present, there are no reports of photothermal preparations based on bilirubin.
  • the present invention is to develop a multifunctional photothermal therapeutic drug to achieve tumor Laid the foundation for the treatment of skin diseases (such as pigment diseases) and vascular diseases;
  • Bilirubin has high protein affinity and metal ion chelating ability. Based on the weak interaction between molecules and the intramolecular ability quenching caused by metal complexes, the developed photothermal formulation has higher Stability and better photothermal conversion effect;
  • Photothermal formulations based on bilirubin can realize photoacoustic imaging while achieving photothermal treatment for diseases.
  • the chelation of functional metal ions has further expanded its imaging applications (such as magnetic resonance imaging (MRI), dynamic enhanced scanning (DMRI), positron emission computed tomography (PET), and electronic computed tomography (CT) ), PET-CT), and then achieve multi-modal visualization of lesions (such as tumors, blood vessels, etc.).
  • imaging applications such as magnetic resonance imaging (MRI), dynamic enhanced scanning (DMRI), positron emission computed tomography (PET), and electronic computed tomography (CT)
  • PET-CT electronic computed tomography
  • FIG. 1 is an ultraviolet absorption spectrum of an aqueous solution of a single molecule of bilirubin prepared in Example 1, showing a broad absorption peak in the near infrared region (600-900 nm);
  • FIG. 2 is a result of a photothermal conversion experiment of an aqueous solution of bilirubin single molecule prepared in Example 2.
  • FIG. 2 Under the irradiation of a laser with a specific wavelength, the temperature of the system was increased from room temperature 24 ° C to 40 ° C within 10 minutes, and the temperature was increased to nearly 16 ° C.
  • the simple aqueous solution only raises 2-4 ° C, indicating that this single-molecule aqueous solution can be used as a photothermal preparation for local temperature increase;
  • FIG. 3 is a physical diagram of a bile green single molecule gel prepared in Example 3. After being inverted, the system does not flow, indicating that the system is in a gel state;
  • FIG. 4 shows the rheological properties of the bilirubin-metal copper ion gel prepared in Example 4.
  • the results show that (a) the gel is gradually strengthened and the viscoelastic modulus of the gel is reversed, indicating the gel Under the action of large shear force, the rheology changes to change from the gel state to the sol state; (b) a mild shear force is applied to the prepared gel, so that the gel state does not change, and An enhanced angular frequency, the results show that the gel has a wide viscoelastic interval, which can maintain a good gel morphology; (c) after applying a large shear force, it changes to a milder shear force, and its viscosity The elastic modulus results show that the secondary gel has better recovery;
  • Example 5 is a transmission electron microscope image of a bilirubin hydrogel prepared in Example 5. It can be seen that the gel exhibits a uniform fiber network, and the fibers are slender and soft;
  • FIG. 6 is a photothermal conversion result of the bilirubin-titanium metal ion gel prepared in Example 6.
  • the gel was irradiated with a laser of 1 W cm -2 for 10 minutes, and the composite gel could achieve rapid and continuous temperature rise. Due to the slow heat dissipation of the gel from the temperature, the in situ temperature can reach nearly 55 ° C, which shows a good photothermal conversion. Under the same laser conditions, the temperature of pure water is only 3-5 ° C;
  • Example 7 is a transmission electron microscope image of a bilirubin-protein (collagen) complex prepared in Example 7, showing that the particle size distribution of the complex is 100-200 nm and is spherical;
  • FIG. 8 is a scanning electron microscope picture of the bilirubin-peptide complex prepared in Example 8, showing that the particle size distribution of the complex is within a range of 100-150 nm;
  • FIG. 9 is a physical picture and particle size distribution of the bilirubin-albumin complex prepared in Example 9, showing an emerald green uniform solution with a narrow particle size distribution, with an average of 110 nm;
  • FIG. 10 is a result of a photothermal conversion cycle of a bilirubin-amino acid-metal ion complex prepared in Example 10, showing that the composite maintained a good photothermal conversion in three cycles of irradiation experiments;
  • Example 11 is a (a) particle size distribution and (b) a scanning electron microscope picture of a bilirubin-peptide-metal ion assembly prepared in Example 11;
  • FIG. 12 shows the temperature rise of tumors in mice under the irradiation of a specific wavelength (730nm, 10min, 0.3W cm -2 ) of the bilirubin-peptide-metal ion assembly prepared in Example 12;
  • FIG. 13 is a photothermal temperature rise curve of an aqueous solution of bilirubin-metal ion chelate prepared in Example 13, showing that in-situ temperature increase can be achieved under specific laser irradiation.
  • the ultraviolet absorption chart of the photothermal preparation bilirubin single molecule solution is shown in FIG. 1. It shows a wide absorption peak in the near-infrared region of 600-900nm.
  • a bilirubin DMSO solution at a concentration of 10 mg mL -1 and a dipeptide Fmoc-His-Phe- ⁇ Na aqueous solution 50 mg mL -1 , mix the two at room temperature, and the bilirubin concentration is 0.5 mg mL -1 .
  • the peptide concentration was 1 mg mL -1 .
  • Adjust the pH value of the mixed solution to 7.2, and place at 4 ° C in the dark for 48 hours to obtain a photothermal formulation of bilirubin-peptide complex. As shown in the scanning electron microscope of FIG. 8, the resulting complex is spherical particles.
  • DMSO solution of bilirubin at a concentration of 20mg mL -1 and 200mg mL -1 of bovine serum albumin in water. Mix the two evenly at room temperature. The concentration of bilirubin is 0.5mg mL -1 and the concentration of bovine serum albumin. It is 5 mg mL -1 . Adjust the pH value of the mixed solution to 7.5, and place it at 4 ° C for 48 hours in the dark to obtain a photothermal formulation bilirubin-albumin complex.
  • the solution physical diagram and particle size distribution are shown in Figure 9, showing emerald green Homogeneous solution with a particle size of about 100 nm and a narrow distribution.
  • the present invention illustrates the detailed method of the present invention through the foregoing embodiments, but the present invention is not limited to the detailed method, which does not mean that the present invention must rely on the detailed method to be implemented.
  • Those skilled in the art should know that any improvement to the present invention, equivalent replacement of the raw materials of the products of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

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Abstract

本发明涉及基于胆绿素的光热制剂，包括：胆绿素、胆绿素凝胶、胆绿素-金属螯合物、胆绿素-生物分子复合物、胆绿素-生物分子-金属功能组装体。

Description

一种基于胆绿素的光热制剂、其制备方法及应用 技术领域
本发明属于光热纳米医药领域，尤其涉及一种基于胆绿素、具有近红外吸收的光热制剂、其制备方法及应用。
背景技术
光热治疗是一种新型的疾病(如肿瘤、皮肤、血管疾病等)治疗方法。该方法操作简便、针对性强、对正常组织无侵袭性，且能够有效抑制复发。在光热治疗过程中，光热制剂能够捕获光能并转化成热量，进而，局部的高热造成细胞的不可逆凋亡，从而达到光热治疗的效果。同时，当脉冲激光照射到病变组织时，组织的光吸收区域会产生光声信号，进而实现了病变部位的光声成像，是一种无损伤、无辐射、高分辨率的成像手段，为病变的早期检测和治疗监测提供了新方法。纳米光热制剂因其靶向性、高转化效率受到了诸多的关注。
用于光热治疗的纳米制剂应具备以下性能：1)高的组织穿透性；2)高的光热转化效率；3)生物安全性及降解性。目前已公开了多种光热制剂及其制备方法：已公开了一种基于石墨烯的光热制剂(专利公开号CN 107080844 A)，当与细胞共孵育且红光照射后，实现了癌细胞的有效杀伤。但石墨烯的生物安全性能及降解性能极大的限制了此类无机光热制剂的发展；再如，已公开一种基于金属酞菁的光热制剂(专利公开号CN 107915739 A)，指出在近红外激光照射下，具有显著的光致升温作用。但酞菁分子具有较长的代谢周期，且具有较高的光毒性，同样限制了它的后续应用。
胆绿素，是动物体内血红蛋白及各种含血红素的蛋白质的内源性代谢产物，是一种非常重要的生物物质，同时，胆绿素在体内将进一步的代谢生成胆红素，胆红素与体内的蛋白质结合进一步通过肝肠循环，排出体外，具有明确的代谢机制。一般认为，胆绿素是一种代谢废物，但是胆绿素能够有效的缓解血管损伤、器官移植的排异、具有抗炎、抗氧化等作用。同时，胆绿素是一种线性的四吡咯结构，无毒、且在近红外区有明显的吸收峰，而胆绿素自身荧光量子产率很低，因此在光热转化方面具有较大的潜力。同时，胆绿素具有较高的蛋白质亲和性及金属离子螯合能力。因此，开发基于胆绿素的光热制剂在光热治疗领域具有深远的意义，目前，还尚未有公开的报道。
发明内容
针对现有光热制剂面临的应用难题，本发明提供了基于胆绿素的光热制剂、其制备方法及应用。所制备的光热制剂具有以下优势：1)近红外吸收，组织穿透深度高；2)光热转化效率高；3)代谢机制明确，安全性高；4)制备简便。此系列基于胆绿素的光热制剂的制备对于开发多功能光治疗药物具有重要意义，在肿瘤、皮肤病(如色素类疾病)、血管疾病等方面应用潜力巨大。
为达到此发明的目的，本发明采用以下技术方案：
第一方面，本发明提供了一种基于胆绿素的光热制剂及其在制备光热制剂中的应用，所述的基于胆绿素的光热制剂包括胆绿素、胆绿素凝胶、胆绿素-金属螯合物、胆绿素-生物分子复合物、胆绿素-生物分子-金属功能组装体；
优选地，所述的金属为Fe 3+、Fe 2+、Co 2+、Ni 2+、Mn 2+、Cu 2+、Zn 2+、Gd 2+、Se 2+、Pt 2+、Ti 2+、Eu 2+、Tb 3+等中的任意一种或至少两种的组合；
优选地，所述的生物分子包括氨基酸、多肽、蛋白质等中的任意一种或至少两种的组合；
优选地，所述的氨基酸指N末端被保护基团修饰的苯丙氨酸、组氨酸、酪氨酸、谷氨酸、赖氨酸、半胱氨酸、天冬氨酸、色氨酸、亮氨酸、天冬酰胺、谷氨酰胺等中的任意一种或至少两种的组合，优选地，所述氨基酸为具有金属配位能量的组氨酸；
优选地，所述的多肽指N末端被保护基团修饰的二肽、三肽、四肽等氨基酸序列中的任意一种或至少两种的组合，优选地，所述多肽为含组氨酸的二肽；
优选地，所述的N末端保护基团包括乙酰基(Ac-)、苯基、N-苄氧羰基、N-叔丁氧羰基、β-萘酰胺基、N-(3-吲哚乙酰基)、二茂铁羰基、半胱氨酸或N-芴甲氧羰基中的任意一种或至少两种的混合；
优选地，所述蛋白质包括白蛋白、血红蛋白、肌红蛋白、胶原蛋白、脂蛋白、胰岛素等中的任意一种或至少两种的组合，优选地，所述蛋白质为白蛋白。
第二方面，本发明提供第一方面所述的基于胆绿素的光热制剂，其特征在于：
所述的胆绿素为将胆绿素完全溶解于碱溶液得到的单分子胆绿素溶液；
所述的胆绿素凝胶为高浓度的胆绿素通过分子间弱相互作用形成的可剪切变稀、可恢复的凝胶；
所述的胆绿素-金属螯合物，为胆绿素与金属离子组成的配合物，包括凝胶剂配合物溶液，金属离子的添加造成胆绿素分子内能量的淬灭；
所述的胆绿素-生物分子复合物，为胆绿素与生物分子组装形成的缀合物，主要由于二者之间的酰胺化反应形成，或基于分子间的弱相互作用形成；
所述的胆绿素-生物分子-金属功能组装体，为胆绿素、生物分子及金属离子三者共同组装形成的复合物，非共价键作用及金属的配位作用同时贡献于复合物的形成。
第三方面，本发明提供第二方面所述的基于胆绿素的光热制剂，其特征在于，所制备的光热制剂具有近红外吸收的特性。
第四方面，本发明提供第三方面所述的基于胆绿素的光热制剂，其特征在于，所制备的光热制剂能够实现近红外光的吸收及热能转化，且具有较高的光热转化效率。
第五方面，本发明提供第四方面所述的基于胆绿素的光热制剂，其特征在于，所制备的光热制剂具有较高的生物安全性及较为明确的代谢途径。
第六方面，本发明提供第一至五方面所述的基于胆绿素的光热制剂胆绿素的制备方法，其特征在于，包括以下步骤：
(1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
(2)将溶解的胆绿素溶液置于4℃冰箱，避光老化4-72h，优选地，所述老化时间为12-24h，即可得到光热制剂胆绿素溶液。
第七方面，本发明提供第一至五方面所述的基于胆绿素的光热制剂胆绿素凝胶的制备方法，其特征在于，包括以下步骤：
(1)称取一定量的胆绿素粉末，直接溶解于少量的DMSO溶液中，待胆绿素完全溶解后，加入适量的水，搅拌均匀。所述胆绿素的浓度为5-100mM，优选地，所述浓度为8-50mM；
(2)将上述溶液置于4℃冰箱，静置、避光老化4-72h，优选地，所述老化时间为12-24h，即可得到光热制剂胆绿素凝胶。
第八方面，本发明提供第一至五方面所述的基于胆绿素的光热制剂胆绿素-金属螯合物的制备方法，其特征在于，包括以下步骤：
(1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液或少量的二甲基亚砜(DMSO)中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
(2)配置金属离子水溶液，称取一定量的金属盐于干净容器，定容。所 述金属离子的浓度为0.01M-1.0M，优选地，所述浓度为0.1-5M；
(3)将步骤(1)所得的胆绿素溶液与步骤(2)所得的金属离子溶液混合，搅拌均匀。胆绿素与金属离子的浓度比为0.01-1000，优选地，所述浓度比为0.1-100；
(4)将步骤(3)所得混合溶液的pH值调节为6.8-7.3。所述pH值通过加入碱性物质或酸性物质进行调节：
优选地，所述碱性物质为氢氧化钠、氢氧化钾、碳酸钠等的任意一种或两种以上的混合物；
优选地，所述酸性物质为盐酸、硫酸、硝酸等中的任意一种或两种以上的混合物。
(5)将步骤(4)得到的混合溶液置于4℃避光老化4-72h，优选地，所述老化时间为12-24h，即可得到光热制剂-胆绿素-金属螯合物。
第九方面，本发明提供第一至五方面所述的基于胆绿素的光热制剂胆绿素-生物分子复合物的制备方法，其特征在于，包括以下步骤：
(1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液或少量的二甲基亚砜(DMSO)中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
(2)配置生物分子溶解液，将生物分子称量后溶解于适当的溶剂(如水、酸、碱、DMSO等)，所述生物分子的浓度为0.1-1000Mm,优选地，所述浓度为1-500mM；
(3)将步骤(1)所得的胆绿素溶液与步骤(2)所得的生物分子溶液混合，搅拌均匀。胆绿素与生物分子的浓度比为0.01-100，优选地，所述浓度比为0.01-1；
(4)将步骤(3)所得混合溶液的pH值调节为6.8-7.3。所述pH值通过加入碱性物质或酸性物质进行调节：
优选地，所述碱性物质为氢氧化钠、氢氧化钾、碳酸钠等的任意一种或两种以上的混合物；
优选地，所述酸性物质为盐酸、硫酸、硝酸等中的任意一种或两种以上的混合物。
(5)将步骤(4)得到的混合溶液置于4℃避光老化4-72h，优选地，所述老化时间为12-24h，即可得到光热制剂胆绿素-生物分子复合物。
第十方面，本发明提供第一至五方面所述的基于胆绿素的光热制剂胆绿素 -生物分子-金属功能组装体的制备方法，其特征在于，包括以下步骤：
(1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液或少量的二甲基亚砜(DMSO)中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
(2)配置生物分子溶解液，将生物分子称量后溶解于适当的溶剂(如水、酸、碱、DMSO等)，所述生物分子的浓度为0.1-1000Mm,优选地，所述浓度为1-500mM；
(3)配置配置金属离子水溶液，称取一定量的金属盐于干净容器，定容。所述金属离子的浓度为0.01M-1.0M，优选地，所述浓度为0.1-5M；
(4)将步骤(1)所得的胆绿素溶液与步骤(2)所得的生物分子溶液混合，搅拌均匀后迅速将步骤(3)所配置的金属离子加入上述混合溶液。胆绿素、生物分子与金属离子的浓度比为0.1:1:0.01-1000:100:1，优选地，所述浓度比为0.2:1:0.2；
(5)将步骤(4)所得混合溶液的pH值调节为6.8-7.3。所述pH值通过加入碱性物质或酸性物质进行调节：
优选地，所述碱性物质为氢氧化钠、氢氧化钾、碳酸钠等的任意一种或两种以上的混合物；
优选地，所述酸性物质为盐酸、硫酸、硝酸等中的任意一种或两种以上的混合物。
(6)将步骤(5)得到的混合溶液置于4℃避光老化4-72h，优选地，所述老化时间为12-24h，即可得到胆绿素-生物分子-金属功能组装体。
第十一方面，本发明提供第一至十方面所述的基于胆绿素的光热制剂用于开发多功能光治疗药物，应用于肿瘤、皮肤病(如色素类疾病)、血管疾病等的治疗。
第十二方面，本发明提供第一至十一方面所述的基于胆绿素的光热制剂用于光声成像、磁共振成像(MRI)、动态增强扫描(DMRI)、正电子发射型计算机断层显像(PET)、电子计算机断层扫描(CT)、PET-CT的成像中的一种或至少两种的联合。
与现有技术相比，本发明具有如下有益效果：
(1)胆绿素具有无毒、近红外吸收、代谢机制明确等优点，目前，尚未有基于胆绿素的光热制剂的报道，本发明为开发多功能的光热治疗药物，实现对肿瘤、皮肤病(如色素类疾病)、血管疾病等的治疗奠定了基础；
(2)胆绿素具有较高的蛋白质亲和性及金属离子的螯合能力，基于分子间的弱相互作用以及金属配合物导致的分子内能力淬灭，所开发的光热制剂具有较高的稳定性及较好的光热转化效果；
(3)基于胆绿素的光热制剂能够在实现对疾病光热治疗的同时，实现光声成像。同时，功能性金属离子的螯合，进一步拓展了其成像应用(如磁共振成像(MRI)、动态增强扫描(DMRI)、正电子发射型计算机断层显像(PET)、电子计算机断层扫描(CT)、PET-CT)，进而实现了病灶(如肿瘤、血管等)的多模式可视化。
附图说明
图1为实例1所制备胆绿素单分子水溶液的紫外吸收光谱，在近红外区(600-900nm)表现出一个宽的吸收峰；
图2为实例2所制备的胆绿素单分子水溶液的光热转化实验结果。在特定波长的激光的照射下，体系的温度在10min内由室温24℃升高至40℃，升温将近16℃。单纯的水溶液仅升高2-4℃，说明此单分子水溶液可作为光热制剂，用于局部温度的升高；
图3为实例3所制备的胆绿素单分子凝胶的实物图，倒置后，体系不流动，说明此体系为凝胶状态；
图4为实例4所制备的胆绿素-金属铜离子凝胶的流变性为结果，结果显示(a)给予凝胶逐渐增强的剪切力，凝胶粘弹模量发生翻转，说明凝胶可在较大的剪切力作用下流变性为发生变化，由凝胶状态转变为溶胶状态；(b)对所制备的凝胶施加一个温和的剪切力，使得凝胶状态不发生变化，给予一个增强的角频率，结果说明凝胶有较宽的粘弹区间，能够保持较好的凝胶形态；(c)施加一个较大的剪切力后转变为施加较温和剪切力，其粘弹模量结果表明，次凝胶具有较好的恢复性；
图5为实例5所制备的胆绿素水凝胶的透射电镜图片，可以看出凝胶呈现均一的纤维网络，纤维细长且较为柔软；
图6为实例6所制备的胆绿素-猛金属离子凝胶的光热转化结果，给予凝胶以1W cm -2的激光照射10min，此复合凝胶能够实现快速且持续的升温。因凝胶对温度的散热较慢，原位温度可达将近55℃，表现出较好的光热转化。同等激光条件下，纯水的升温仅为3-5℃；
图7为实例7所制备的胆绿素-蛋白质(胶原蛋白)复合物的透射电镜图 片，显示此复合物的粒径分布于100-200nm，为球形；
图8为实例8所制备的胆绿素-肽复合物的扫描电镜图片，显示此复合物的粒径分布于100-150nm范围内；
图9为实例9所制备的胆绿素-白蛋白复合物的实物图及粒径分布，呈现翠绿色均一溶液，其粒径分布较窄，平均为110nm；
图10为实例10所制备的胆绿素-氨基酸-金属离子复合物的光热转化循环结果，显示此复合物在三个循环的照射实验中，维持了较好的光热转换；
图11为实例11所制备的胆绿素-肽-金属离子组装体的(a)粒径分布及(b)扫描电镜图片；
图12为实例12所制备的胆绿素-肽-金属离子组装体在特定波长激光照射下(730nm，10min，0.3W cm -2)，小鼠体内肿瘤的升温情况；
图13为实例13所制备的胆绿素-金属离子螯合物水溶液的光热升温曲线，显示在特定激光照射下，能够实现原位温度的升高。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合本发明的优选实施例来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行，或者按照本领域的常规技术手段进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
称量胆绿素粉末10mg，溶解于0.1M的NaOH溶液中，最终浓度为0.2mg mL -1。即光热制剂胆绿素单分子溶液，其紫外吸收图如图1所示。在近红外区域600-900nm范围内显示宽的吸收峰。
实施例2
称量胆绿素粉末10mg，溶解于0.1M的NaOH溶液中，最终得到浓度为0.2mg mL -1的胆绿素单分子溶液，在730nm激光器下进行升温测试，激光功率设定为0.3W cm -2。其升温曲线如图2所示。
实施例3
称取胆绿素粉末8mg溶解于50uL的DMSO溶液中，超声辅助混合均匀，加入950uL超纯水，混合均匀。新配置的混合溶液调节pH值至6.9，静置于4℃避光老化8h，可得到浓度为8mg mL -1的胆绿素凝胶，所得到的凝胶实物 图如图3所示，倒置后为不流动的均一状态。
实施例4
称取胆绿素粉末5mg溶解于20uL的DMSO溶液中，超声辅助混合均匀；配置铜离子水溶液，浓度为0.1M。将980uL的铜离子水溶液加入到上述胆绿素的DMSO溶液中，混合均匀。新配置的混合溶液调节pH值至7.2，静置于4℃避光老化6h，可得到胆绿素-金属离子凝胶，如图4所示，所得到的凝胶具有较好的剪切变稀性质及恢复性质。
实施例5
称取胆绿素粉末10mg溶解于50uL的DMSO溶液中，超声辅助混合均匀，加入940uL超纯水，混合均匀。新配置的混合溶液调节pH值至6.8，静置于4℃避光老化24h，可得到胆绿素凝胶，如图5透射电镜结果所示，所得到的凝胶呈现均一的纤维网络。
实施例6
称取胆绿素粉末10mg溶解于50uL的DMSO溶液中，超声辅助混合均匀；配置锰离子水溶液，浓度为0.1M。将950uL的锰离子水溶液加入到上述胆绿素的DMSO溶液中，混合均匀。新配置的混合溶液调节pH值至7.0，静置于4℃避光老化12h，可得到胆绿素-金属离子凝胶，所得到的凝胶能够实现原位温度的上升。给予1mL凝胶一定的激光照射(激光强度为1.0W cm -2，照射时间为10min，激光波长选用730nm)，升温曲线如图6所示。
实施例7
准备胆绿素的DMSO溶液，浓度为5mg mL -1，胶原蛋白醋酸溶液100mg mL -1，将二者在室温下混合均匀，胆绿素浓度为0.2mg mL -1，胶原蛋白浓度为2mg mL -1。调节混合溶液的pH值为7.0，放置在4℃避光老化24h，即可得到光热制剂胆绿素-蛋白质复合物，如图7所示，尺寸约为100-200nm的颗粒生成。
实施例8
准备胆绿素的DMSO溶液，浓度为10mg mL -1，二肽Fmoc-His-Phe-βNa水溶液50mg mL -1，将二者在室温下混合均匀，胆绿素浓度为0.5mg mL -1，肽浓度为1mg mL -1。调节混合溶液的pH值为7.2，放置在4℃避光老化48h，即可得到光热制剂胆绿素-肽复合物，如图8扫描电镜所示，生成的复合物为球形颗粒。
实施例9
准备胆绿素的DMSO溶液，浓度为20mg mL -1，牛血清白蛋白水溶液200mg  mL -1，将二者在室温下混合均匀，胆绿素浓度为0.5mg mL -1，牛血清白蛋白浓度为5mg mL -1。调节混合溶液的pH值为7.5，放置在4℃避光老化48h，即可得到光热制剂胆绿素-白蛋白复合物，其溶液实物图及粒径分布如图9所示，呈现翠绿色均一溶液，粒径约为100nm，分布较窄。
实施例10
准备胆绿素的DMSO溶液，浓度为10mg mL -1，氨基酸Fmoc-His-OH溶液50mg mL -1，将二者在室温下混合均匀，胆绿素浓度为0.2mg mL -1，氨基酸浓度为1mg mL -1。调节混合溶液的pH值为7.1，放置在4℃避光老化36h，即可得到光热制剂胆绿素-氨基酸复合物，如图10所示，此复合颗粒能够进行高效的光热转化，给予此体系一定的激光照射，体系能够实现原位升温。三个循环照射，该体系能够维持较好的光热转换。
实施例11
准备胆绿素的DMSO溶液10mg mL -1，苯环保护的氨基酸Z-His-Obzl的DMSO溶液100mg mL -1，锌离子溶液0.1M。取20uL的上述胆绿素溶液与10uL的上述肽溶液混合均匀，向上述混合液中加入950uL的双重水，混合均匀后，加入上述锌离子水溶液5uL。将得到的混合溶液搅拌均匀，调节pH至7.0，置于4℃避光老化48h。所得到的组装体的粒径分布及扫描电镜结构如图11所示。
实施例12
准备胆绿素的DMSO溶液10mg mL -1，苯环保护的氨基酸Z-His-Obzl的DMSO溶液100mg mL -1，锰离子溶液0.1M。取20uL的上述胆绿素溶液与10uL的上述肽溶液混合均匀，向上述混合液中加入950uL的双重水，混合均匀后，加入上述锰离子水溶液5uL。将得到的混合溶液搅拌均匀，调节pH至7.0，置于4℃避光老化48h。所得到的组装体在730nm激光的照射下的升温情况如图12所示。
实施例13
称取胆绿素粉末1mg，溶解于20uL DMSO溶液中，超声溶解均匀。后加入930uL水混合均匀。配置0.1M的铜离子水溶液，迅速取50uL加入上述胆绿素溶液中，混合均匀得到胆绿素-金属螯合物水溶液。测量此螯合物的光热转化效果。取1mL置于完全透光的石英池子中，调整激光器激光光斑，使光斑完全辐照于螯合物溶液。调整激光参数为0.3W cm -2，照射12min。使用热电偶测量器记录照射过程的温度变化，如图13所示。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 胆绿素在制备光热制剂中的应用，其特征在于所述的光热制剂选自胆绿素、胆绿素凝胶、胆绿素-金属螯合物、胆绿素-生物分子复合物、胆绿素-生物分子-金属功能组装体；
    所述的金属为Fe 3+、Fe 2+、Co 2+、Ni 2+、Mn 2+、Cu 2+、Zn 2+、Gd 2+、Se 2+、Pt 2+、Ti 2+、Eu 2+、Tb 3+中的任意一种或至少两种的组合；
    所述的生物分子包括氨基酸、多肽、蛋白质中的任意一种或至少两种的组合；
    所述的氨基酸指N末端被保护基团修饰的苯丙氨酸、组氨酸、酪氨酸、谷氨酸、赖氨酸、半胱氨酸、天冬氨酸、色氨酸、亮氨酸、天冬酰胺、谷氨酰胺等中的任意一种或至少两种的组合，优选地，所述氨基酸为具有金属配位能力的组氨酸；
    所述的多肽指N末端被保护基团修饰的二肽、三肽、四肽等氨基酸序列中的任意一种或至少两种的组合，优选地，所述多肽为含组氨酸的二肽；
    所述的N末端保护基团包括乙酰基(Ac-)、苯基、N-苄氧羰基、N-叔丁氧羰基、β-萘酰胺基、N-(3-吲哚乙酰基)、二茂铁羰基、半胱氨酸或N-芴甲氧羰基中的任意一种或至少两种的混合；
    所述蛋白质包括白蛋白、血红蛋白、肌红蛋白、胶原蛋白、脂蛋白、胰岛素等中的任意一种或至少两种的组合；优选地，所述蛋白质为白蛋白。
  2. 根据权利要求1所述的应用，所述的胆绿素为将胆绿素完全溶解于碱溶液得到的单分子胆绿素溶液；
    所述的胆绿素凝胶为5～15mg/mL的胆绿素水溶液通过分子间弱相互作用形成的可剪切变稀、可恢复的凝胶；
    所述的胆绿素-金属螯合物，为胆绿素与金属离子组成的配合物；
    所述的胆绿素-生物分子复合物，为胆绿素与生物分子组装形成的缀合物，由二者之间的酰胺化反应形成，或基于分子间的弱相互作用形成；
    所述的胆绿素-生物分子-金属功能组装体，为胆绿素、生物分子及金属离子三者共同组装或分级组装形成的复合物。
  3. 根据权利要求2所述的应用，其特征在于，所述的胆绿素-生物分子复合物为纳米颗粒，其平均粒径为100～200nm。
  4. 根据权利要求1-3任意一项所述的应用，所述光热制剂应用于肿瘤、 皮肤病、血管疾病等的治疗。
  5. 根据权利要求1-3任意一项所述的应用，所述光热制剂用于光声成像、磁共振成像(MRI)、动态增强扫描(DMRI)、正电子发射型计算机断层显像(PET)、电子计算机断层扫描(CT)、PET-CT的成像中的一种或至少两种的联合。
  6. 根据权利要求1所述的应用，所述光热制剂通过如下方法制备得到：
    (1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
    (2)将溶解的胆绿素溶液置于4℃冰箱，避光老化4-72h，优选地，所述老化时间为12-24h，即可得到光热制剂胆绿素溶液。
  7. 根据权利要求1所述的应用，所述光热制剂通过如下方法制备得到:
    (1)称取一定量的胆绿素粉末，直接溶解于少量的二甲基亚砜(DMSO)溶液中，待胆绿素完全溶解后，加入适量的水，搅拌均匀，所述胆绿素的浓度为5-100mM，优选地，所述浓度为8-50mM；
    (2)将上述溶液置于4℃冰箱，静置、避光老化4-72h，优选地，所述老化时间为12-24h，即可得到光热制剂胆绿素凝胶。
  8. 根据权利要求1所述的应用，所述光热制剂通过如下方法制备得到：
    (1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液或少量的DMSO中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
    (2)配置金属离子水溶液，称取一定量的金属盐于干净容器，定容。所述金属离子的浓度为0.01M-1.0M，优选地，所述浓度为0.1-5M；
    (3)将步骤(1)所得的胆绿素溶液与步骤(2)所得的金属离子溶液混合，搅拌均匀。胆绿素与金属离子的浓度比为0.01-1000，优选地，所述浓度比为0.1-100；
    (4)将步骤(3)所得混合溶液的pH值调节为6.8-7.3。所述pH值通过加入碱性物质或酸性物质进行调节：
    优选地，所述碱性物质为氢氧化钠、氢氧化钾、碳酸钠等的任意一种或两种以上的混合物；
    优选地，所述酸性物质为盐酸、硫酸、硝酸等中的任意一种或两种以上的混合物。
    (5)将步骤(4)得到的混合溶液置于4℃避光老化4-72h，优选地，所 述老化时间为12-24h，即可得到光热制剂-胆绿素-金属螯合物。
  9. 根据权利要求1所述的应用，所述光热制剂通过如下方法制备得到：
    (1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液或少量的二甲基亚砜(DMSO)中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
    (2)配置生物分子溶解液，将生物分子称量后溶解于适当的溶剂(如水、酸、碱、DMSO等)，所述生物分子的浓度为0.1-1000Mm,优选地，所述浓度为1-500mM；
    (3)将步骤(1)所得的胆绿素溶液与步骤(2)所得的生物分子溶液混合，搅拌均匀。胆绿素与生物分子的浓度比为0.01-100，优选地，所述浓度比为0.01-1；
    (4)将步骤(3)所得混合溶液的pH值调节为6.8-7.3。所述pH值通过加入碱性物质或酸性物质进行调节：
    优选地，所述碱性物质为氢氧化钠、氢氧化钾、碳酸钠等的任意一种或两种以上的混合物；
    优选地，所述酸性物质为盐酸、硫酸、硝酸等中的任意一种或两种以上的混合物。
    (5)将步骤(4)得到的混合溶液置于4℃避光老化4-72h，优选地，所述老化时间为12-24h，即可得到光热制剂胆绿素-生物分子复合物。
  10. 根据权利要求1所述的应用，所述光热制剂通过如下方法制备得到：
    (1)称取一定量的胆绿素粉末，直接溶解于0.1M的NaOH溶液或少量的二甲基亚砜(DMSO)中，超声或搅拌加速其溶解，所述胆绿素的浓度为0.1-100mM，优选地，所述浓度为0.5-20mM；
    (2)配置生物分子溶解液，将生物分子称量后溶解于适当的溶剂(如水、酸、碱、DMSO等)，所述生物分子的浓度为0.1-1000Mm,优选地，所述浓度为1-500mM；
    (3)配置配置金属离子水溶液，称取一定量的金属盐于干净容器，定容。所述金属离子的浓度为0.01M-1.0M，优选地，所述浓度为0.1-5M；
    (4)将步骤(1)所得的胆绿素溶液与步骤(2)所得的生物分子溶液混合，搅拌均匀后迅速将步骤(3)所配置的金属离子加入上述混合溶液。胆绿素、生物分子与金属离子的浓度比为0.1:1:0.01-1000:100:1，优选地，所述浓度比为0.2:1:0.2；
    (5)将步骤(4)所得混合溶液的pH值调节为6.8-7.3。所述pH值通过加入碱性物质或酸性物质进行调节：
    优选地，所述碱性物质为氢氧化钠、氢氧化钾、碳酸钠等的任意一种或两种以上的混合物；
    优选地，所述酸性物质为盐酸、硫酸、硝酸等中的任意一种或两种以上的混合物。
    (6)将步骤(5)得到的混合溶液置于4℃避光老化4-72h，优选地，所述老化时间为12-24h，即可得到胆绿素-生物分子-金属功能组装体。
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