WO2022179307A1 - 一种多功能肠道诊疗制剂制备方法及其产品 - Google Patents

一种多功能肠道诊疗制剂制备方法及其产品 Download PDF

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WO2022179307A1
WO2022179307A1 PCT/CN2021/143578 CN2021143578W WO2022179307A1 WO 2022179307 A1 WO2022179307 A1 WO 2022179307A1 CN 2021143578 W CN2021143578 W CN 2021143578W WO 2022179307 A1 WO2022179307 A1 WO 2022179307A1
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preparation
intestinal
fluorine
containing oxygen
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French (fr)
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崔大祥
朱君
朱竞尧
徐艳
杨迪诚
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上海纳米技术及应用国家工程研究中心有限公司
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Priority claimed from CN202110205888.2A external-priority patent/CN112957483A/zh
Priority claimed from CN202111610209.6A external-priority patent/CN114306653A/zh
Priority claimed from CN202111620713.4A external-priority patent/CN114306276B/zh
Application filed by 上海纳米技术及应用国家工程研究中心有限公司 filed Critical 上海纳米技术及应用国家工程研究中心有限公司
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    • 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
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast 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
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a preparation method of a multifunctional intestinal diagnosis and treatment preparation and a product thereof, including a photoacoustic imaging contrast agent targeting the intestine and a preparation method thereof, a PET/ultrasound multifunctional intestinal contrast agent and a preparation method thereof, and a 18 F-fluorocarbon, M2 macrophage probe of gold nanorod and preparation method thereof.
  • Tumor has become an important factor affecting human health, and tumor metastasis is an important cause of death of tumor patients.
  • the 1-year recurrence rate of newly diagnosed tumor patients in my country is 60%, and more than 80% of patients die from tumor recurrence and metastasis.
  • "Early detection, early diagnosis, early treatment” has become an important way to prevent tumor development and reduce postoperative recurrence and metastasis. Therefore, it is of great significance and social value to study the methods of early detection, early diagnosis and early treatment of tumors.
  • TAMs tumor-associated macrophages
  • TAMs are macrophages infiltrating tumor tissues, and they are the most abundant immune cells in the tumor microenvironment. They can secrete a variety of cytokines. In the early stage of tumorigenesis, TAMs can recognize and remove tumor cells. It also plays a key role in tumor growth, invasion and metastasis. Since TAM comes from the autologous body, it is inextricably linked with the occurrence and development of tumors. Therefore, TAM is considered to be a substance with high affinity to tumors, which can not only kill tumors well, but also deliver drugs to tumors efficiently.
  • TAMs play a "double-edged sword" role in the occurrence and development of tumors, and they can be differentiated into M1 or M2 macrophages.
  • M1 type has an anti-tumor effect
  • this type of macrophages highly express cytokines such as interleukin 1 (IL-1) and interleukin 6 (IL-6), which can assist in the anti-tumor function
  • IL-1 interleukin 1
  • IL-6 interleukin 6
  • M2 type macrophages Cells mainly promote tumor cell invasion and metastasis and peripheral inflammatory response, and participate in tumor invasion, growth, angiogenesis, metastasis, and immunosuppression. Therefore, more and more studies have used M2 macrophages to achieve efficient diagnosis and treatment of tumors.
  • Chitosan is a natural polysaccharide with good biocompatibility and degradability, no biological toxicity, and rich in amino and hydroxyl groups, which are easy to be modified and modified. Therefore, chitosan nanomaterials are widely used in the field of nano-drug delivery, for example, Cheng J., Zhou X., Zhou W., Wu H., Zhang X., Lu Q., A Novel Magnetic Contrast Agent for Gastrointestinal Mucosa -Targeted Imaging Through Oral Administration.
  • Gold nanorods are a representative gold nanomaterial with high-intensity light absorption properties in the near-infrared region, and have always been a research hotspot in the fields of photoacoustic imaging and photothermal therapy.
  • Jokerst J., Cole J., Van de Sompel D., Gambhir S.S. Gold nanorods for ovarian cancer detection with photoacoustic imaging and resection guidance via Raman imaging in living mice.
  • ACS nano., 6(11), 10366-77 In this study, gold nanorods with different aspect ratios were prepared to achieve photoacoustic imaging of subcutaneous ovarian cancer in mice.
  • the purpose of the present invention is to provide a preparation method of a photoacoustic imaging contrast agent targeting the intestinal tract.
  • Another object of the present invention is to provide a photoacoustic imaging contrast agent product targeting the intestinal tract prepared by the above method.
  • Another object of the present invention is to provide a preparation method of a PET/ultrasound multifunctional intestinal contrast agent.
  • Another object of the present invention is to provide a PET/ultrasound multifunctional intestinal contrast agent product prepared by the above method.
  • Another object of the present invention is to provide an application of the above-mentioned PET/ultrasound multifunctional intestinal contrast agent product.
  • Another object of the present invention is to provide a preparation method of a multifunctional intestinal diagnosis and treatment preparation.
  • Another object of the present invention is to provide a multifunctional intestinal diagnosis and treatment preparation prepared by the above method.
  • the invention provides a preparation method of a photoacoustic imaging contrast agent targeting the intestine.
  • Gold nanorods are first coated in chitosan nanospheres, and then targeting antibodies are coupled to the nanospheres through EDC/NHS reaction
  • Obtaining a gut-targeted photoacoustic imaging contrast agent on the surface at least includes the following preparation steps:
  • step d Add 10-15 mg of the nanospheres obtained in step b to the reaction system, stir for 4 hours, and then collect by centrifugation. Use deionized water to wash three times to remove unreacted antibodies to obtain the product.
  • the diameter of the gold nanorods is 40-100 nm.
  • the aspect ratio of the gold nanorods is 2-5.
  • the targeting antibodies are serotonin receptor 3 antibody (Anti-5-HT3R), vascular endothelial growth factor antibody (Anti-VEGF), histamine receptor H1 antibody (Anti-HRH1), tryptase polyclonal One of the antibodies (Polyclonal Antibody to Tryptase).
  • the invention provides a photoacoustic imaging contrast agent targeting the intestinal tract, which is prepared according to any one of the above-mentioned methods.
  • the average particle size of the obtained product is 173-228 nm.
  • the product of 200 ⁇ g/mL is The temperature was raised to 45°C-52°C within 1 min, and the targeting antibodies were targeting intestinal serotonin receptor 3, targeting intestinal histamine receptor H1, and targeting vascular endothelial growth factor.
  • the photoacoustic imaging contrast agent with targeted intestinal tract formed by the method of the present invention has the advantages of simple method, good water dispersibility of the obtained product and high imaging contrast.
  • the use of this product is enema.
  • the nanospheres are attached to the surface of the intestinal mucosa through the principle of antigen-antibody binding.
  • the gold nanorods wrapped in the nanospheres can significantly increase the intensity of the photoacoustic signal under the irradiation of the near-infrared laser.
  • Targeted photoacoustic imaging of the gut Taking advantage of the safe and non-toxic carrier properties of chitosan nanospheres, the near-infrared light absorption properties of gold nanorods, and the active targeting properties of antibodies, targeted photoacoustic imaging of the intestine was achieved.
  • the preparation method is simple, the reaction is easy to control, the stability is good, and it has a broad application prospect.
  • the obtained product has excellent water dispersibility and biocompatibility, and high imaging contrast.
  • the use method is an enema type, which is easy to operate and has little toxicity to living organisms.
  • the invention provides a preparation method of a PET/ultrasound multifunctional intestinal contrast agent:
  • Intestinal M2 macrophages (cell density of 5000 cells/well plate) were incubated with the fluorine-containing oxygen-carrying emulsion prepared by the above steps for 12-72 hours to obtain PET/ultrasound multifunctional intestinal angiography with targeting function. agent.
  • Described fluorocarbon compound is perfluorocyclic ether, perfluorodecalin, perfluoromethylcyclohexylpiperidine, perfluorobromooctane, 1-bromopentadecafluoroheptane, 1-bromotridecafluorohexane one of the.
  • the salt solution is physiological saline, phosphate buffer, acetate buffer, trimethylaminomethane buffer, barbiturate buffer, sodium formate buffer, acetate-lithium salt buffer, acetic acid-sodium acetate buffer It is a kind of inorganic salt solution such as acetic acid-potassium acetate buffer.
  • the emulsifier is phospholipid, including soybean lecithin, egg yolk lecithin, hydrogenated lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated egg phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidyl Choline, distearoyl phosphatidyl choline, 1-myristoyl-2-palmitoyl phosphatidyl choline, 1-palmitoyl-2-stearoyl phosphatidyl choline, 1-stearoyl-2 - Palmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-linoleoylphosphatidylcholine or dioleoylphosphatidylcholine, dioleoylphospholipid Acylcholine, hydrogenated dipalmitoyl phosphatidyl choline,
  • auxiliary agent is Tu-80 (polyoxyethylene sorbitan monooleate), 6501 (coconut oil fatty acid diethanolamide), AEO-9 (fatty alcohol polyoxyethylene ether), Brij-35 (lauryl alcohol) Polyoxyethylene ether) or Triton X-100 (polyethylene glycol octyl phenyl ether) one or more mixtures.
  • the intermittent stirring is 1 minute of stirring and 1 minute of termination, and the stirring time is 2-4 hours.
  • the present invention provides a PET/ultrasound multifunctional intestinal contrast agent, which is prepared according to any one of the above-mentioned methods.
  • the invention provides the application of a PET/ultrasonic multifunctional intestinal contrast agent in preparing a multifunctional probe material with positron emission tomography (PET) and ultrasonic imaging.
  • PET positron emission tomography
  • the multifunctional probe for electron emission tomography (PET) and ultrasound imaging can simultaneously meet the requirements of precise tumor localization, high sensitivity and long-term imaging, and improve the targeting imaging efficiency of the probe to the tumor.
  • PET electron emission tomography
  • Its advantage lies in the use of macrophages as a "reservoir" for drug carriers.
  • 18F nuclides are used as imaging agents for PET.
  • the tumor location and metastases are determined by PET imaging to achieve precise localization of tumors throughout the body.
  • Fluorocarbons as ultrasound contrast agents. Improve the diagnostic ability of local tumors through ultrasound imaging.
  • the invention provides a preparation method of a multifunctional intestinal diagnosis and treatment preparation, comprising the following steps:
  • Intestinal M2 macrophages (cell density of 5000 cells/well plate) were incubated with the prepared nanoparticles and fluorine-containing oxygen-carrying emulsion for 12-72 hours to obtain macrophage-encapsulated chitosan nanoparticles and The fluorine-containing oxygen-carrying emulsion realizes multifunctional intestinal diagnosis and treatment preparations.
  • the diameter of the gold nanorod is 40-100 nm, and the aspect ratio is 2-5.
  • the targeting antibodies are serotonin receptor 3 antibody (Anti-5-HT3R), vascular endothelial growth factor antibody (Anti-VEGF), histamine receptor H1 antibody (Anti-HRH1), tryptase polyclonal One of the antibodies (Polyclonal Antibody to Tryptase).
  • Described fluorocarbon compound is perfluorocyclic ether, perfluorodecalin, perfluoromethylcyclohexylpiperidine, perfluorobromooctane, 1-bromopentadecafluoroheptane, 1-bromotridecafluorohexane one of the.
  • the salt solution is physiological saline, phosphate buffer, acetate buffer, trimethylaminomethane buffer, barbiturate buffer, sodium formate buffer, acetate-lithium salt buffer, acetic acid-sodium acetate buffer It is a kind of inorganic salt solution such as acetic acid-potassium acetate buffer.
  • the emulsifier is phospholipid, including soybean lecithin, egg yolk lecithin, hydrogenated lecithin, hydrogenated soybean phosphatidylcholine, hydrogenated egg phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidyl Choline, distearoyl phosphatidyl choline, 1-myristoyl-2-palmitoyl phosphatidyl choline, 1-palmitoyl-2-stearoyl phosphatidyl choline, 1-stearoyl-2 - Palmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1-stearoyl-2-linoleoylphosphatidylcholine or dioleoylphosphatidylcholine, dioleoylphospholipid Acylcholine, hydrogenated dipalmitoyl phosphatidyl choline,
  • auxiliary agent is Tu-80 (polyoxyethylene sorbitan monooleate), 6501 (coconut oil fatty acid diethanolamide), AEO-9 (fatty alcohol polyoxyethylene ether), Brij-35 (lauryl alcohol) Polyoxyethylene ether) or Triton X-100 (polyethylene glycol octyl phenyl ether) one or more mixtures.
  • the intermittent stirring is 1 minute of stirring and 1 minute of termination, and the stirring time is 2-4 hours.
  • the present invention also provides a multifunctional intestinal diagnosis and treatment preparation prepared by the above method, which has a multifunctional probe for positron emission tomography (PET), ultrasonic imaging and photothermal therapy, which can simultaneously satisfy precise tumor localization, high sensitivity and long-term stability.
  • Periodic imaging improves the targeting imaging efficiency of probes to tumors.
  • a multifunctional preparation for intestinal diagnosis and treatment which is characterized in that it has a multifunctional probe for PET imaging, ultrasonic imaging and photothermal therapy, which can simultaneously satisfy precise tumor localization, high sensitivity and long-term imaging, and improve the target of the probe to the tumor. towards imaging efficiency.
  • Macrophages are used as the "reservoir" of drug carriers, and the transport of macrophages can enhance the enrichment of carriers in tumor sites, while chitosan can be effectively adsorbed on the surface of intestinal mucosa; 18F nuclides are used as PET Imaging agent, to determine tumor location and metastases through PET imaging, to achieve precise localization of tumors in the whole body; fluorocarbons are used as ultrasound contrast agents. Improve the diagnostic ability of local tumors through ultrasound imaging. Gold nanorods have photothermal effects and have application prospects for photothermal therapy of tumor sites.
  • the invention includes a photoacoustic imaging contrast agent targeting the intestine and a preparation method thereof, a PET/ultrasound multifunctional intestinal contrast agent and a preparation method thereof, and M2 type macrophages containing 18 F-fluorocarbon and gold nanorods Probes and methods of making the same.
  • the multi-functional intestinal diagnosis and treatment preparation products also have multi-functional probes for PET imaging, ultrasonic imaging and photothermal therapy, which can simultaneously meet the requirements of accurate tumor localization, high-sensitivity and long-term imaging, and improve the targeting imaging efficiency of the probe to the tumor.
  • Macrophages are used as drug carrier "reservoirs". The enhanced carrier was enriched at the tumor site, while chitosan could be effectively adsorbed on the intestinal mucosal surface.
  • 18F nuclides are used as imaging agents for PET. Fluorocarbons as ultrasound contrast agents.
  • Gold nanorods have photothermal effects and can treat tumor sites.
  • An intestinal-targeting photoacoustic imaging contrast agent which firstly coats gold nanorods in chitosan nanospheres, and then couples the targeting antibody to the surface of the nanospheres through EDC/NHS reaction to obtain an intestinal
  • the targeted photoacoustic imaging contrast agent is prepared according to the following preparation steps:
  • step d Add 10 mg of the nanospheres obtained in step b to the reaction system, stir for 4 hours, and then collect by centrifugation. Use deionized water to wash three times to remove unreacted antibodies to obtain the product.
  • the average particle size of the product was 228 nm, and under the irradiation of 808 nm laser, the product of 200 ⁇ g/mL was heated to 45 °C within 1 min, targeting intestinal serotonin receptor 3.
  • a photoacoustic imaging contrast agent targeting the intestinal tract is similar to the steps of the embodiment, and is prepared according to the following steps:
  • step d Add 10 mg of the nanospheres obtained in step b to the reaction system, stir for 4 hours, and then collect by centrifugation. Use deionized water to wash three times to remove unreacted antibodies to obtain a product.
  • the average particle size of the product was 173 nm. Under the laser irradiation of 808 nm, the product of 200 ⁇ g/mL was heated to 48 °C within 1 min, targeting the intestinal histamine receptor H1.
  • a photoacoustic imaging contrast agent targeting the intestinal tract is similar to the steps in Example 1, and is prepared as follows:
  • the average particle size of the product is 215 nm. Under the laser irradiation of 808 nm, the product of 200 ⁇ g/mL is heated to 52 °C within 1 min, targeting vascular endothelial growth factor.
  • a PET/ultrasound multifunctional intestinal contrast agent is prepared according to the following steps:
  • fluorine-containing oxygen-carrying emulsion in 10 ml of a 1:1 volume ratio of physiological saline/ethanol, add 10% fluorocarbon perfluorocyclic ether, 18F 1,2-difluorocarbon Palmitic acid glyceride 18 F-DP and 5% mannose-modified distearoyl phosphatidylethanolamine Man-DSPE, in addition, add 5% emulsifier soybean lecithin, and use a homogenizer at room temperature to Carry out intermittent stirring at a certain rate (stir for 1 minute, stop for 1 minute, and stir for 2 hours), centrifuge after cooling, and take the supernatant to obtain a fluorine-containing oxygen-carrying emulsion;
  • Intestinal M2 macrophages with a cell density of 5000 cells/well plate were incubated with the fluorine-containing oxygen-carrying emulsion prepared in the above steps for 24 hours to obtain a PET/ultrasound multifunctional intestinal contrast agent with targeting function.
  • the prepared contrast agent has a particle size of 230 nanometers and a dissolved oxygen content of 15%.
  • a PET/ultrasound multifunctional intestinal contrast agent similar to Example 4, is prepared according to the following steps:
  • fluorine-containing oxygen-carrying emulsion in 10 ml of sodium formate buffer/ethanol solvent with a volume ratio of 1:2, add 50% fluorocarbon perfluorodecalin, 20% 18F 1, 2-Dipalmitoglyceride 18 F-DP and 10% mannose-modified distearoyl phosphatidylethanolamine Man-DSPE, in addition, 10% emulsifier dioleoylphosphatidylcholine and 2 %Tu-80, at room temperature, use a homogenizer to carry out intermittent stirring at a certain rate (stir for 1 minute, stop for 1 minute, and stir for 4 hours), centrifuge after cooling, and take the supernatant to obtain the fluorine-containing carrier.
  • Oxygen emulsion in 10 ml of sodium formate buffer/ethanol solvent with a volume ratio of 1:2, add 50% fluorocarbon perfluorodecalin, 20% 18F 1, 2-Dipalmitoglyceride 18 F-DP and 10% mannose-modified di
  • Intestinal M2 macrophages with a cell density of 5000 cells/well plate were incubated with the fluorine-containing oxygen-carrying emulsion prepared in the above steps for 48 hours to obtain a PET/ultrasound multifunctional intestinal contrast agent with targeting function.
  • the prepared contrast agent has a particle size of 75 nanometers and a dissolved oxygen content of 35%.
  • a PET/ultrasound multifunctional intestinal contrast agent similar to Example 4, is prepared according to the following steps:
  • fluorine-containing oxygen-carrying emulsion in 10 ml of acetic acid-sodium acetate buffer/ethanol solvent with a volume ratio of 1:2, add 50% fluorocarbon perfluorooctane, 30% 18 F's 1,2-dipalmitoglyceride 18 F-DP and 20% mannose-modified distearoyl phosphatidyl ethanolamine Man-DSPE, in addition, add 10% emulsifier dioleoyl phosphatidyl Choline and 2% Tu-80 were stirred intermittently with a homogenizer at a certain rate at room temperature (stir for 1 minute, stop for 1 minute, and the stirring time was 4 hours), centrifuge after cooling, and take the supernatant. Obtain fluorine-containing oxygen-carrying emulsion;
  • Intestinal M2 macrophages with a cell density of 5000 cells/well plate were incubated with the fluorine-containing oxygen-carrying emulsion prepared in the above steps for 72 hours to obtain a PET/ultrasound multifunctional intestinal contrast agent with targeting function.
  • the prepared contrast agent has a particle size of 25 nanometers and a dissolved oxygen content of 50%.
  • a multifunctional intestinal diagnosis and treatment preparation is prepared according to the following steps:
  • Intestinal M2 macrophages with a cell density of 5000/well plate were incubated with the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 24 hours to obtain macrophage-encapsulated chitosan Sugar nanoparticles and fluorine-containing oxygen-carrying emulsion to obtain PET/ultrasound multifunctional intestinal contrast agent with targeting function.
  • the prepared multifunctional intestinal diagnosis and treatment preparation has a particle size of 430 nanometers and a dissolved oxygen content of 15%. Under laser irradiation at 808 nm, the product at 200 ⁇ g/mL was heated to 45 °C within 1 min.
  • a multifunctional intestinal diagnosis and treatment preparation similar to the steps of Example 7, is prepared according to the following steps:
  • Intestinal M2 macrophages with a cell density of 5000/well plate were incubated with the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 48 hours to obtain macrophage-encapsulated chitosan Sugar nanoparticles and fluorine-containing oxygen-carrying emulsion can obtain a PET/ultrasound multifunctional intestinal contrast agent with targeting function.
  • the prepared multifunctional intestinal diagnosis and treatment preparation has a particle size of 310 nanometers and a dissolved oxygen content of 35%. Under laser irradiation at 808 nm, the product at 200 ⁇ g/mL was heated to 48 °C within 1 min.
  • a multifunctional intestinal diagnosis and treatment preparation similar to the steps of Example 7, is prepared according to the following steps:
  • Intestinal M2 macrophages with a cell density of 5000/well plate were incubated with the chitosan nanoparticles and/or the fluorine-containing oxygen-carrying emulsion for 72 hours to obtain macrophage-encapsulated chitosan Sugar nanoparticles and fluorine-containing oxygen-carrying emulsion can obtain a PET/ultrasound multifunctional intestinal contrast agent with targeting function.
  • the prepared contrast agent has a particle size of 460 nanometers and a dissolved oxygen content of 50%. Under laser irradiation at 808 nm, the product at 200 ⁇ g/mL was heated to 52 °C within 1 min.

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Abstract

提出了多功能肠道诊疗制剂制备方法及其产品，包括靶向肠道的光声成像对比剂及其制备方法，PET／超声多功能肠道造影剂及其制备方法，以及含 18F－氟碳化合物、金纳米棒的M2型巨噬细胞探针及其制备方法。尤其是多功能肠道诊疗制剂产品同时具有PET成像、超声成像和光热治疗的多功能探针，可以同时满足肿瘤精准定位、高灵敏长周期成像，提高了探针对肿瘤的靶向成像效率。采用巨噬细胞作为药物载体"蓄水池"。增强载体在肿瘤部位富集，而壳聚糖能有效地吸附在肠道粘膜表面。 18F类核素作为PET的显像剂。氟碳化合物作为超声造影剂。金纳米棒具有光热作用，具有对肿瘤部位进行治疗的作用。

Description

一种多功能肠道诊疗制剂制备方法及其产品 技术领域
本发明涉及一种多功能肠道诊疗制剂制备方法及其产品，包括靶向肠道的光声成像对比剂及其制备方法，PET/超声多功能肠道造影剂及其制备方法，以及含 18F-氟碳化合物、金纳米棒的M2型巨噬细胞探针及其制备方法。
背景技术
肿瘤已经成为影响人类健康的重要因素，肿瘤转移更是导致肿瘤患者死亡的重要原因。据目前统计显示，我国新诊断的肿瘤患者中1年复发率为60％，死于肿瘤复发和转移的患者超过80％。“早发现、早诊断、早治疗”成为防止肿瘤发展、减少术后复发和转移的重要途径。因此，研究肿瘤的早发现、早诊断、早治疗的方法具有重要的意义和社会价值。
近年来，以肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)为目标的肿瘤研究成为了热点。TAM是浸润在肿瘤组织中的巨噬细胞，是肿瘤微环境中最多的免疫细胞，可以分泌多种细胞因子，在肿瘤发生的初期，能够识别并清除肿瘤细胞，但随着肿瘤的发生发展，又对肿瘤的生长、侵袭、转移起着关键作用。由于TAM来自于自体，且与肿瘤的发生、发展有密不可分的联系。因此，TAM被认为是与肿瘤高度亲和的物质，其不仅能很好地杀灭肿瘤，还能向肿瘤高效地输送药物。在肿瘤的发生发展中TAM发挥着“双刃剑”的作用，它们可以被分化为M1型或M2型巨噬细胞。其中，M1型具有抗肿瘤作用，该型巨噬细胞高表达白介素1(IL-1)、白介素6(IL-6)等细胞因子，这些细胞因子可协助行使抗肿瘤功能；但是M2型巨噬细胞则主要促进肿瘤细胞侵袭转移与周围炎症反应，参与肿瘤的侵袭、生长、血管发生、转移、免疫抑制等。因此，越来越多的研究利用M2型巨噬细胞实现了对肿瘤的高效诊断和治疗。
壳聚糖是一种天然的多糖，具有良好的生物相容性和可降解性，无生物毒性，且富含氨基和羟基易于进行修饰和改性。因此壳聚糖纳米材料被广泛使用于纳米递药领域中，例如，Cheng J.,Zhou X.,Zhou W.,Wu H.,Zhang X.,Lu Q.,A Novel Magnetic Contrast Agent for Gastrointestinal Mucosa-Targeted Imaging Through Oral Administration.J Biomed Nanotechnol.,2019,15(6),1162-71，报道了一种新型的核磁共振造影剂，通过将Gd负载于壳聚糖纳米颗粒中并通过口服给药实现了对肠道的多功能评估，其关键技术在于壳聚糖能有效地粘附与肠道粘膜表面，被肠道粘膜吸收。
金纳米棒是一种代表性的金纳米材料，具有近红外区高强度的光吸收特性，一直以来都是光声成像、光热治疗等领域的研究热点。例如，Jokerst J.,Cole J.,Van de Sompel D.,Gambhir S.S.,Gold nanorods for ovarian cancer detection with photoacoustic imaging and resection guidance via Raman imaging in living mice. ACS nano.,6(11),10366-77，该研究制备不同长径比的金纳米棒，实现了对小鼠皮下卵巢癌的光声成像。
目前，精准的诊疗技术不断出现，而这些诊疗技术研究往往集中于高灵敏成像、高效率治疗和高精准靶向3个方面。其中，靶向技术是肿瘤诊疗的关键；肿瘤定位和成像是肿瘤诊断的核心；提高传统治疗手段的疗效是肿瘤临床治疗的重点。但是，现有技术通常很难同时满足以上三个方面，由于肝脏首过效应等因素靶向效率仍有待提高。
发明内容
针对现有技术中的不足，本发明目的在于提供一种靶向肠道的光声成像对比剂的制备方法。
本发明的再一目的在于：提供一种上述方法制备的靶向肠道的光声成像对比剂产品。
本发明的又一目的在于：提供一种PET/超声多功能肠道造影剂的制备方法。
本发明的又一目的在于：提供一种上述方法制备的PET/超声多功能肠道造影剂产品。
本发明的又一目的在于：提供一种上述PET/超声多功能肠道造影剂产品的应用。
本发明的又一目的在于：提出一种多功能肠道诊疗制剂的制备方法。
本发明的又一目的在于：提供上述方法制备的多功能肠道诊疗制剂。
本发明提供了一种靶向肠道的光声成像对比剂的制备方法，先将金纳米棒包覆于壳聚糖纳米球中，接着通过EDC/NHS反应将靶向抗体偶联至纳米球表面获得一种肠道靶向的光声成像对比剂，至少包括如下制备步骤：
a.取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入2-5mg金纳米棒，超声分散均匀；
b.在磁力搅拌下逐滴添加无水乙醇直至体系变色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球，得包覆了金纳米棒壳的聚糖纳米球；
c.取100μL靶向抗体溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体羧基2-4h；
d.向反应体系加入10-15mg步骤b所得的纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得产物。
所述的金纳米棒的直径为40-100nm。
所述的金纳米棒的长径比为2-5。
所述的靶向抗体为5-羟色胺受体3抗体(Anti-5-HT3R)、血管内皮生长因子抗体(Anti-VEGF)、组胺受体H1抗体(Anti-HRH1)、类胰蛋白酶多克隆抗体(Polyclonal  Antibody to Tryptase)中的一种。
本发明提供一种靶向肠道的光声成像对比剂，根据上述任一所述方法制备得到，所得产物的平均粒径为173-228nm，在808nm的激光照射下，200μg/mL的产物在1min内升温到45℃-52℃，靶向抗体为靶向肠道5-羟色胺受体3、靶向肠道组胺受体H1、靶向血管内皮生长因子。
分别称取壳寡糖、乙二酸四乙胺加入去离子水中，充分搅拌溶解后，再将一定量的金纳米棒加入上述溶液中并超声分散，之后逐滴加入无水乙醇直至反应体系变为乳光色，加入戊二醛溶液交联。将产物先离心洗净，再连接靶向抗体，得到靶向肠道的光声成像对比剂，实现肠道靶向光声成像。
本发明采用上述方法形成的具有靶向肠道的光声成像对比剂具有方法简单，所得的产物水分散性好，成像对比度高等优点。本产品的使用方法为灌肠，通过抗原抗体结合原理使纳米球附着在肠粘膜表面，同时包裹在纳米球内的金纳米棒在近红外激光的辐照下能显著提升光声信号的强度，实现对肠道的靶向光声成像。利用壳聚糖纳米球安全无毒性的载体特性、金纳米棒的近红外光吸收特性以及抗体的主动靶向特性，实现对肠道的靶向光声成像。
与现有技术相比，具有如下的优点：
(1)制备方法简单，反应易于控制，稳定性好，具有广阔的应用前景。
(2)所得的产物具有优良的水分散性和生物相容性，成像对比度高。
(3)使用方式为灌肠式，易于操作，对生物体的毒性小。
本发明提供了一种PET/超声多功能肠道造影剂的制备方法：
在10毫升体积比为1:0.5-1:2的盐溶液/乙醇的溶剂中，加入质量浓度分别为1％-50％的氟碳化合物、 18F的1,2-二棕榈酸甘油酯( 18F-DP)和甘露糖修饰的二硬脂酰基磷脂酰乙醇胺(Man-DSPE)，另外再加入乳化剂和其他助剂(质量浓度分别为0％-10％)，在室温下采用匀浆机以一定的速率进行间歇性搅拌，冷却后离心，取上清液即可得到含氟载氧乳剂；
将肠道M2型巨噬细胞(细胞密度为5000个/孔板)与上述述步骤制备的含氟载氧乳剂共孵育12-72小时，得到具有靶向功能的PET/超声多功能肠道造影剂。
所述的氟碳化合物为全氟环醚、全氟萘烷、全氟甲基环已基哌啶、全氟溴辛烷、1-溴十五氟庚烷、1-溴十三氟己烷中的一种。
所述的盐溶液为生理盐水、磷酸盐缓冲液、醋酸盐缓冲液、三强甲氨基甲烷缓冲液、巴比妥缓冲液、甲酸钠缓冲液、醋酸-锂盐缓冲液、醋酸-醋酸钠缓冲液、醋酸-醋酸钾缓冲液等无机盐溶液的一种。
所述的乳化剂为磷脂，包括大豆卵磷脂、蛋黄卵磷脂、氢化卵磷脂、氢化大豆磷脂 酰胆碱、氢化蛋磷脂酰胆碱、二月桂酰磷脂酰胆碱、二肉豆寇酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-肉豆寇酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-硬脂酰磷脂酰胆碱、1-硬脂酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-油酰磷脂酰胆碱、1-硬脂酰-2-亚油酰磷脂酰胆碱或二油酰磷脂酰胆碱、二油酰磷脂酰胆碱、氢化二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷脂酸、二棕榈酰磷脂酸、二硬脂酰磷脂酸、儿肉豆莲酰磷脂酰乙二醇胺、二棕榈酰磷脂耽乙二醇胺、脑磷脂酰丝氨酸、二肉豆蔻县磷脂酰丝氨酸、儿棕榈酰磷脂酰丝氨酸、蛋磷脂酰甘油、二月桂酰磷脂酰甘油、二肉豆酰磷脂酰甘油、二棕榈酰磷脂酰甘油、脑鞘磷脂中的至少一种。
所述的助剂为Tu-80(聚氧乙烯山梨醇酐单油酸酯)，6501(椰油脂肪酸二乙醇酰胺)，AEO-9(脂肪醇聚氧乙烯醚)，Brij-35(月桂醇聚氧乙烯醚)或Triton X-100(聚乙二醇辛基苯基醚)的一种或几种混合物。
所述的间歇性搅拌为搅拌1分钟，终止1分钟，搅拌时间为2-4小时。
本发明提供一种PET/超声多功能肠道造影剂，根据上述任一所述方法制备得到。
本发明提供一种PET/超声多功能肠道造影剂在制备具有正电子发射断层扫描(PET)和超声成像的多功能探针材料中的应用。
电子发射断层扫描(PET)和超声成像的多功能探针，可以同时满足肿瘤精准定位、高灵敏长周期成像，提高了探针对肿瘤的靶向成像效率。其优势在于：采用巨噬细胞作为药物载体“蓄水池”。通过巨噬细胞转运作用，增强载体在肿瘤部位富集。18F类核素作为PET的显像剂。通过PET成像确定肿瘤位置和转移灶，实现全身肿瘤的精准定位。氟碳化合物作为超声造影剂。通过超声成像，提高局部肿瘤的诊断能力。
本发明提供一种多功能肠道诊疗制剂的制备方法，包括以下步骤：
(1)壳聚糖纳米粒的制备
取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入2-5mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体羧基2-4h；向反应体系加入10-15mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得产物；
(2)含氟载氧乳剂的制备
在10毫升体积比为1:0.5-1:2的盐溶液/乙醇的溶剂中，加入质量浓度分别为1％-50％的氟碳化合物、 18F的1,2-二棕榈酸甘油酯( 18F-DP)和甘露糖修饰的二硬脂酰基磷脂酰乙醇胺(Man-DSPE)，另外再加入乳化剂和其他助剂(质量浓度分别为0％-10％)，在室温下采用匀浆机以一定的速率进行间歇性搅拌，冷却后离心，取上清 液即可得到含氟载氧乳剂；
(3)巨噬细胞包载壳聚糖纳米粒和含氟载氧乳剂
将肠道M2型巨噬细胞(细胞密度为5000个/孔板)与所制备的纳米颗粒和含氟载氧乳剂共孵育12-72小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，实现多功能肠道诊疗制剂。
所述的金纳米棒的直径为40-100nm，长径比为2-5。
所述的靶向抗体为5-羟色胺受体3抗体(Anti-5-HT3R)、血管内皮生长因子抗体(Anti-VEGF)、组胺受体H1抗体(Anti-HRH1)、类胰蛋白酶多克隆抗体(Polyclonal Antibody to Tryptase)中的一种。
所述的氟碳化合物为全氟环醚、全氟萘烷、全氟甲基环已基哌啶、全氟溴辛烷、1-溴十五氟庚烷、1-溴十三氟己烷中的一种。
所述的盐溶液为生理盐水、磷酸盐缓冲液、醋酸盐缓冲液、三强甲氨基甲烷缓冲液、巴比妥缓冲液、甲酸钠缓冲液、醋酸-锂盐缓冲液、醋酸-醋酸钠缓冲液、醋酸-醋酸钾缓冲液等无机盐溶液的一种。
所述的乳化剂为磷脂，包括大豆卵磷脂、蛋黄卵磷脂、氢化卵磷脂、氢化大豆磷脂酰胆碱、氢化蛋磷脂酰胆碱、二月桂酰磷脂酰胆碱、二肉豆寇酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-肉豆寇酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-硬脂酰磷脂酰胆碱、1-硬脂酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-油酰磷脂酰胆碱、1-硬脂酰-2-亚油酰磷脂酰胆碱或二油酰磷脂酰胆碱、二油酰磷脂酰胆碱、氢化二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷脂酸、二棕榈酰磷脂酸、二硬脂酰磷脂酸、儿肉豆莲酰磷脂酰乙二醇胺、二棕榈酰磷脂耽乙二醇胺、脑磷脂酰丝氨酸、二肉豆蔻县磷脂酰丝氨酸、儿棕榈酰磷脂酰丝氨酸、蛋磷脂酰甘油、二月桂酰磷脂酰甘油、二肉豆酰磷脂酰甘油、二棕榈酰磷脂酰甘油、脑鞘磷脂中的至少一种。
所述的助剂为Tu-80(聚氧乙烯山梨醇酐单油酸酯)，6501(椰油脂肪酸二乙醇酰胺)，AEO-9(脂肪醇聚氧乙烯醚)，Brij-35(月桂醇聚氧乙烯醚)或Triton X-100(聚乙二醇辛基苯基醚)的一种或几种混合物。
所述的间歇性搅拌为搅拌1分钟，终止1分钟，搅拌时间为2-4小时。
本发明还提供了上述方法制备的一种多功能肠道诊疗制剂，具有正电子发射断层扫描(PET)、超声成像和光热治疗的多功能探针，可以同时满足肿瘤精准定位、高灵敏长周期成像，提高了探针对肿瘤的靶向成像效率。
一种多功能肠道诊疗制剂，其特征在于共同具有PET成像、超声成像和光热治疗的多功能探针，可以同时满足肿瘤精准定位、高灵敏长周期成像，提高了探针对肿瘤的靶向成像效率。
采用巨噬细胞作为药物载体“蓄水池”，通过巨噬细胞转运作用，增强载体在肿瘤部位富集，而壳聚糖能有效地吸附在肠道粘膜表面； 18F类核素作为PET的显像剂，通过PET成像确定肿瘤位置和转移灶，实现全身肿瘤的精准定位；氟碳化合物作为超声造影剂。通过超声成像，提高局部肿瘤的诊断能力。金纳米棒具有光热作用，具有对肿瘤部位进行光热治疗的应用前景。
本发明包括靶向肠道的光声成像对比剂及其制备方法，PET/超声多功能肠道造影剂及其制备方法，以及含 18F-氟碳化合物、金纳米棒的M2型巨噬细胞探针及其制备方法。尤其是多功能肠道诊疗制剂产品同时具有PET成像、超声成像和光热治疗的多功能探针，可以同时满足肿瘤精准定位、高灵敏长周期成像，提高了探针对肿瘤的靶向成像效率。采用巨噬细胞作为药物载体“蓄水池”。增强载体在肿瘤部位富集，而壳聚糖能有效地吸附在肠道粘膜表面。 18F类核素作为PET的显像剂。氟碳化合物作为超声造影剂。金纳米棒具有光热作用，具有对肿瘤部位进行治疗的作用。
具体实施方式
以下通过具体的实施例对本发明的技术方案作进一步描述。以下的实施例是对本发明的进一步说明，而不限制本发明的范围。
实施例1
一种靶向肠道的光声成像对比剂的，先将金纳米棒包覆于壳聚糖纳米球中，接着通过EDC/NHS反应将靶向抗体偶联至纳米球表面获得一种肠道靶向的光声成像对比剂，按如下制备步骤制备：
a.取30mg壳寡糖、17mg EDTA溶于10mL去离子水中，再加入2mg金纳米棒，超声分散均匀；
b.在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL 25％的戊二醛溶液交联4h，离心收集纳米球，得包覆了金纳米棒壳的聚糖纳米球；
c.取100μL靶向抗体Anti-5-HT3R溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体羧基2-4h；
d.向反应体系加入10mg步骤b所得的纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得产物。
产物的平均粒径为228nm，在808nm的激光照射下，200μg/mL的产物在1min内升温到45℃，靶向肠道5-羟色胺受体3。
实施例2：
一种靶向肠道的光声成像对比剂，与实施例步骤近似，按如下步骤制备：
a、取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入5 mg金纳米棒，超声分散均匀；
b、在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL 25％的戊二醛溶液交联4h，离心收集纳米球，得包覆了金纳米棒壳的聚糖纳米球；
c、取100μL Anti-HRH1溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体羧基2-4h；
d、向反应体系加入10mg步骤b所得的纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得产物。
产物的平均粒径为173nm，在808nm的激光照射下，200μg/mL的产物在1min内升温到48℃，靶向肠道组胺受体H1。
实施例3：
一种靶向肠道的光声成像对比剂，与实施例1步骤近似，按如下步骤制备：
a、取30mg壳寡糖、17mg EDTA溶于10mL去离子水中，再加入10mg金纳米棒，超声分散均匀；
b、在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL 25％的戊二醛溶液交联4h，离心收集纳米球；
c、取100μL Anti-VEGF溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体羧基2-4h；
d、向反应体系加入15mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得产物。
产物的平均粒径为215nm，在808nm的激光照射下，200μg/mL的产物在1min内升温到52℃，靶向血管内皮生长因子。
实施例4
一种PET/超声多功能肠道造影剂，按下述步骤制备：
含氟载氧乳剂的制备:在10毫升体积比为1:1的生理盐水/乙醇的溶剂中，加入质量浓度分别为10％的氟碳化合物全氟环醚、 18F的1,2-二棕榈酸甘油酯 18F-DP和5％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外,再加入质量浓度为5％的乳化剂大豆卵磷脂，在室温下采用匀浆机以一定的速率进行间歇性搅拌(搅拌1分钟，终止1分钟，搅拌时间为2小时)，冷却后离心，取上清液即可得到含氟载氧乳剂；
将细胞密度为5000个/孔板的肠道M2型巨噬细胞与上述步骤制备的含氟载氧乳剂共孵育24小时，得到具有靶向功能的PET/超声多功能肠道造影剂。
制备的造影剂粒径为230纳米，溶氧量为15％。
实施例5
一种PET/超声多功能肠道造影剂，与实施例4近似，按下述步骤制备：
含氟载氧乳剂的制备:在10毫升体积比为1:2的甲酸钠缓冲液/乙醇的溶剂中，加入质量浓度为50％的氟碳化合物全氟萘烷、20％的 18F的1,2-二棕榈酸甘油酯 18F-DP和10％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外,再加入质量浓度为10％的乳化剂二油酰磷脂酰胆碱和2％Tu-80，在室温下采用匀浆机以一定的速率进行间歇性搅拌(搅拌1分钟，终止1分钟，搅拌时间为4小时)，冷却后离心，取上清液即可得到含氟载氧乳剂；
将细胞密度为5000个/孔板的肠道M2型巨噬细胞与上述步骤制备的含氟载氧乳剂共孵育48小时，得到具有靶向功能的PET/超声多功能肠道造影剂。
制备的造影剂粒径为75纳米，溶氧量为35％。
实施例6
一种PET/超声多功能肠道造影剂，与实施例4近似，按下述步骤制备：
含氟载氧乳剂的制备:在10毫升体积比为1:2的醋酸-醋酸钠缓冲液/乙醇的溶剂中，加入质量浓度为50％的氟碳化合物全氟溴辛烷、30％的 18F的1,2-二棕榈酸甘油酯 18F-DP和20％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外,再加入质量浓度为10％的乳化剂二油酰磷脂酰胆碱和2％Tu-80，在室温下采用匀浆机以一定的速率进行间歇性搅拌(搅拌1分钟，终止1分钟，搅拌时间为4小时)，冷却后离心，取上清液即可得到含氟载氧乳剂；
将细胞密度为5000个/孔板的肠道M2型巨噬细胞与上述步骤制备的含氟载氧乳剂共孵育72小时，得到具有靶向功能的PET/超声多功能肠道造影剂。
制备的造影剂粒径为25纳米，溶氧量为50％。
实施例7
一种多功能肠道诊疗制剂，按下述步骤制备：
(1)壳聚糖纳米粒的制备
取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入2mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体Anti-5-HT3R溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体5-羟色胺受体3羧基2-4h；向反应体系加入10-15mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得壳聚糖纳米粒产物；
(2)含氟载氧乳剂的制备
在10毫升体积比为1:1的盐溶液生理盐水/乙醇的溶剂中，加入质量浓度分别为10％的氟碳化合物全氟环醚、10％ 18F的1,2-二棕榈酸甘油酯 18F-DP和5％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外，再加入质量浓度为5％的乳化剂大豆卵磷脂，在室温下采用匀浆机进行间歇性搅拌，即搅拌1分钟、终止1分钟，搅拌时间为2小时，冷却后离心，取上清液即可得到含氟载氧乳剂；
(3)巨噬细胞包载壳聚糖纳米粒和/或含氟载氧乳剂
将细胞密度为5000个/孔板肠道M2型巨噬细胞与所述的壳聚糖纳米粒和/或所述的含氟载氧乳剂共孵育24小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，得到具有靶向功能的PET/超声多功能肠道造影剂。
制备的多功能肠道诊疗制剂粒径为430纳米，溶氧量为15％。在808nm的激光照射下，200μg/mL的产物在1min内升温到45℃。
实施例8
一种多功能肠道诊疗制剂，与实施例7步骤近似，按下述步骤制备：
(1)壳聚糖纳米粒的制备
取30mg壳寡糖、10mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入5mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体Anti-HRH1溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体组胺受体H1羧基2-4h；向反应体系加入10mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得壳聚糖纳米粒产物；
(2)含氟载氧乳剂的制备
在10毫升体积比为1:2的盐溶液甲酸钠缓冲液/乙醇的溶剂中，加入质量浓度分别为50％的氟碳化合物全氟萘烷、20％ 18F的1,2-二棕榈酸甘油酯 18F-DP和10％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外，再加入质量浓度为10％的二油酰磷脂酰胆碱和2％Tu-80，在室温下采用匀浆机进行间歇性搅拌，即搅拌1分钟、终止1分钟，搅拌时间为4小时，冷却后离心，取上清液即可得到含氟载氧乳剂；
(3)巨噬细胞包载壳聚糖纳米粒和/或含氟载氧乳剂
将细胞密度为5000个/孔板肠道M2型巨噬细胞与所述的壳聚糖纳米粒和/或所述的含氟载氧乳剂共孵育48小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，即得到具有靶向功能的PET/超声多功能肠道造影剂。
制备的多功能肠道诊疗制剂粒径为310纳米，溶氧量为35％。在808nm的激光照射下，200μg/mL的产物在1min内升温到48℃。
实施例9
一种多功能肠道诊疗制剂，与实施例7步骤近似，按下述步骤制备：
(1)壳聚糖纳米粒的制备
取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入10mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体Anti-VEGF溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体血管内皮生长因子羧基2-4h；向反应体系加入15mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得壳聚糖纳米粒产物；
(2)含氟载氧乳剂的制备
在10毫升体积比为1:2的盐溶液醋酸-醋酸钠缓冲液/乙醇的溶剂中，加入质量浓度分别为50％的氟碳化合物全氟溴辛烷、30％ 18F的1,2-二棕榈酸甘油酯 18F-DP和20％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外，再加入质量浓度为10％的二肉豆酰磷脂酰甘油和5％Brij-35，在室温下采用匀浆机进行间歇性搅拌，即搅拌1分钟、终止1分钟，搅拌时间为4小时，冷却后离心，取上清液即可得到含氟载氧乳剂；
(3)巨噬细胞包载壳聚糖纳米粒和/或含氟载氧乳剂
将细胞密度为5000个/孔板肠道M2型巨噬细胞与所述的壳聚糖纳米粒和/或所述的含氟载氧乳剂共孵育72小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，即得到具有靶向功能的PET/超声多功能肠道造影剂。
制备的造影剂粒径为460纳米，溶氧量为50％。在808nm的激光照射下，200μg/mL的产物在1min内升温到52℃。

Claims (19)

  1. 一种靶向肠道的光声成像对比剂的制备方法，其特征在于，先将金纳米棒包覆于壳聚糖纳米球中，接着通过EDC/NHS反应将靶向抗体偶联至纳米球表面获得一种肠道靶向的光声成像对比剂，至少包括如下制备步骤：
    a、取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入2-5mg金纳米棒，超声分散均匀；
    b、在磁力搅拌下逐滴添加无水乙醇直至体系变色，加入30μL 25％的戊二醛溶液交联4h，离心收集纳米球，得包覆了金纳米棒壳的聚糖纳米球；
    c、取100μL靶向抗体溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体羧基2-4h；
    d、向反应体系加入10-15mg步骤b所得的纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得产物。
  2. 根据权利要求1所述靶向肠道的光声成像对比剂的制备方法，其特征在于所述的金纳米棒的直径为40-100nm。
  3. 根据权利要求1所述靶向肠道的光声成像对比剂的制备方法，其特征在于所述的金纳米棒的长径比为2-5。
  4. 根据权利要求1所述靶向肠道的光声成像对比剂的制备方法，其特征在于所述的靶向抗体为5-羟色胺受体3抗体(Anti-5-HT3R)、血管内皮生长因子抗体(Anti-VEGF)、组胺受体H1抗体(Anti-HRH1)、类胰蛋白酶多克隆抗体(Polyclonal Antibody to Tryptase)中的一种。
  5. 一种PET/超声多功能肠道造影剂的制备方法，其特征在于，包括下述步骤：
    含氟载氧乳剂的制备:在10毫升体积比为1:0.5-1:2的盐溶液/乙醇的溶剂中，加入质量浓度分别为1％-50％的氟碳化合物、 18F的1,2-二棕榈酸甘油酯 18F-DP和甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外,再加入质量浓度分别为0％-10％的乳化剂和助剂，在室温下采用匀浆机以一定的速率进行间歇性搅拌，冷却后离心，取上清液即可得到含氟载氧乳剂；
    将细胞密度为5000个/孔板的肠道M2型巨噬细胞与上述步骤制备的含氟载氧乳剂共孵育12-72小时，得到具有靶向功能的PET/超声多功能肠道造影剂。
  6. 根据权利要求5所述PET/超声多功能肠道造影剂的制备方法，其特征在于,所述的氟碳化合物为全氟环醚、全氟萘烷、全氟甲基环已基哌啶、全氟溴辛烷、1-溴十五氟庚烷、1-溴十三氟己烷中的一种。
  7. 根据权利要求5所述PET/超声多功能肠道造影剂的制备方法，其特征在于,所述的盐溶液为生理盐水、磷酸盐缓冲液、醋酸盐缓冲液、三强甲氨基甲烷缓冲液、巴比妥缓冲液、甲酸钠缓冲液、醋酸-锂盐缓冲液、醋酸-醋酸钠缓冲液、醋酸-醋酸钾缓冲液等无机盐溶液的一种。
  8. 根据权利要求5所述PET/超声多功能肠道造影剂的制备方法，其特征在于,所述的乳化剂为磷脂，包括大豆卵磷脂、蛋黄卵磷脂、氢化卵磷脂、氢化大豆磷脂酰胆碱、氢化蛋磷脂酰胆碱、二月桂酰磷脂酰胆碱、二肉豆寇酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-肉豆寇酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-硬脂酰磷脂酰胆碱、1-硬脂酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-油酰磷脂酰胆碱、1-硬脂酰-2-亚油酰磷脂酰胆碱或二油酰磷脂酰胆碱、二油酰磷脂酰胆碱、氢化二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷脂酸、二棕榈酰磷脂酸、二硬脂酰磷脂酸、儿肉豆莲酰磷脂酰乙二醇胺、二棕榈酰磷脂耽乙二醇胺、脑磷脂酰丝氨酸、二肉豆蔻县磷脂酰丝氨酸、儿棕榈酰磷脂酰丝氨酸、蛋磷脂酰甘油、二月桂酰磷脂酰甘油、二肉豆酰磷脂酰甘油、二棕榈酰磷脂酰甘油、脑鞘磷脂中的至少一种；
    所述的助剂为聚氧乙烯山梨醇酐单油酸酯Tu-80、椰油脂肪酸二乙醇酰胺6501、脂肪醇聚氧乙烯醚AEO-9、月桂醇聚氧乙烯醚Brij-35或聚乙二醇辛基苯基醚Triton X-100中的一种或几种的混合物。
  9. 根据权利要求5所述PET/超声多功能肠道造影剂的制备方法，其特征在于，所述的间歇性搅拌为搅拌1分钟，终止1分钟，搅拌时间为2-4小时。
  10. 一种多功能肠道诊疗制剂的制备方法，其特征在于，包括下述步骤：
    (1)壳聚糖纳米粒的制备
    取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入2-5mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体羧基2-4h；向反应体系加入10-15mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得壳聚糖纳米粒产物；
    (2)含氟载氧乳剂的制备
    在10毫升体积比为1:0.5-1:2的盐溶液/乙醇的溶剂中，加入质量浓度分别为1％-50％的氟碳化合物、 18F的1,2-二棕榈酸甘油酯 18F-DP和甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外，再加入质量浓度分别为0％-10％的乳化剂 和/或其他助剂，在室温下采用匀浆机以一定的速率进行间歇性搅拌，冷却后离心，取上清液即可得到含氟载氧乳剂；
    (3)巨噬细胞包载壳聚糖纳米粒和/或含氟载氧乳剂
    将细胞密度为5000个/孔板肠道M2型巨噬细胞与所述的壳聚糖纳米粒和/或所述的含氟载氧乳剂共孵育12-72小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，实现多功能肠道诊疗制剂。
  11. 根据权利要求10所述多功能肠道诊疗制剂的制备方法，其特征在于，所述的金纳米棒的直径为40-100nm，长径比为2-5。
  12. 根据权利要求10所述多功能肠道诊疗制剂的制备方法，其特征在于，所述的靶向抗体为5-羟色胺受体3抗体Anti-5-HT3R、血管内皮生长因子抗体Anti-VEGF、组胺受体H1抗体Anti-HRH1、类胰蛋白酶多克隆抗体Polyclonal Antibody to Tryptase中的一种。
  13. 根据权利要求10所述多功能肠道诊疗制剂的制备方法，其特征在于，所述的氟碳化合物为全氟环醚、全氟萘烷、全氟甲基环已基哌啶、全氟溴辛烷、1-溴十五氟庚烷、1-溴十三氟己烷中的一种；
    所述的盐溶液为生理盐水、磷酸盐缓冲液、醋酸盐缓冲液、三强甲氨基甲烷缓冲液、巴比妥缓冲液、甲酸钠缓冲液、醋酸-锂盐缓冲液、醋酸-醋酸钠缓冲液、醋酸-醋酸钾缓冲液等无机盐溶液的一种。
  14. 根据权利要求10所述多功能肠道诊疗制剂的制备方法，其特征在于，所述的乳化剂为磷脂，包括大豆卵磷脂、蛋黄卵磷脂、氢化卵磷脂、氢化大豆磷脂酰胆碱、氢化蛋磷脂酰胆碱、二月桂酰磷脂酰胆碱、二肉豆寇酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、1-肉豆寇酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-硬脂酰磷脂酰胆碱、1-硬脂酰-2-棕榈酰磷脂酰胆碱、1-棕榈酰-2-油酰磷脂酰胆碱、1-硬脂酰-2-亚油酰磷脂酰胆碱或二油酰磷脂酰胆碱、二油酰磷脂酰胆碱、氢化二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二肉豆蔻酰磷脂酸、二棕榈酰磷脂酸、二硬脂酰磷脂酸、儿肉豆莲酰磷脂酰乙二醇胺、二棕榈酰磷脂耽乙二醇胺、脑磷脂酰丝氨酸、二肉豆蔻县磷脂酰丝氨酸、儿棕榈酰磷脂酰丝氨酸、蛋磷脂酰甘油、二月桂酰磷脂酰甘油、二肉豆酰磷脂酰甘油、二棕榈酰磷脂酰甘油、脑鞘磷脂中的至少一种；
    所述的助剂为聚氧乙烯山梨醇酐单油酸酯Tu-80、椰油脂肪酸二乙醇酰胺6501、脂肪醇聚氧乙烯醚AEO-9、月桂醇聚氧乙烯醚Brij-35或聚乙二醇辛基苯基醚Triton X-100中的一种或几种混合物。
  15. 根据权利要求10所述多功能肠道诊疗制剂的制备方法，其特征在于， 所述的间歇性搅拌为搅拌1分钟，终止1分钟，搅拌时间为2-4小时。
  16. 根据权利要求10至15任一项所述多功能肠道诊疗制剂的制备方法，其特征在于，按下述步骤制备：
    (1)壳聚糖纳米粒的制备
    取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入2mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体Anti-5-HT3R溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体5-羟色胺受体3羧基2-4h；向反应体系加入10-15mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得壳聚糖纳米粒产物；
    (2)含氟载氧乳剂的制备
    在10毫升体积比为1:1的盐溶液生理盐水/乙醇的溶剂中，加入质量浓度分别为10％的氟碳化合物全氟环醚、10％ 18F的1,2-二棕榈酸甘油酯 18F-DP和5％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外，再加入质量浓度为5％的乳化剂大豆卵磷脂，在室温下采用匀浆机进行间歇性搅拌，即搅拌1分钟、终止1分钟，搅拌时间为2小时，冷却后离心，取上清液即可得到含氟载氧乳剂；
    (3)巨噬细胞包载壳聚糖纳米粒和/或含氟载氧乳剂
    将细胞密度为5000个/孔板肠道M2型巨噬细胞与所述的壳聚糖纳米粒和/或所述的含氟载氧乳剂共孵育24小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，得到具有靶向功能的PET/超声多功能肠道造影剂。
  17. 根据权利要求10至15任一项所述多功能肠道诊疗制剂的制备方法，其特征在于，按下述步骤制备：
    (1)壳聚糖纳米粒的制备
    取30mg壳寡糖、10mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入5mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体Anti-HRH1溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体组胺受体H1羧基2-4h；向反应体系加入10mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得壳聚糖纳米粒产物；
    (2)含氟载氧乳剂的制备
    在10毫升体积比为1:2的盐溶液甲酸钠缓冲液/乙醇的溶剂中，加入质量浓 度分别为50％的氟碳化合物全氟环醚、20％ 18F的1,2-二棕榈酸甘油酯 18F-DP和10％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外，再加入质量浓度为10％的二油酰磷脂酰胆碱和2％Tu-80，在室温下采用匀浆机进行间歇性搅拌，即搅拌1分钟、终止1分钟，搅拌时间为4小时，冷却后离心，取上清液即可得到含氟载氧乳剂；
    (3)巨噬细胞包载壳聚糖纳米粒和/或含氟载氧乳剂
    将细胞密度为5000个/孔板肠道M2型巨噬细胞与所述的壳聚糖纳米粒和/或所述的含氟载氧乳剂共孵育48小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，即得到具有靶向功能的PET/超声多功能肠道造影剂。
  18. 根据权利要求10至15任一项所述多功能肠道诊疗制剂的制备方法，其特征在于，按下述步骤制备：
    (1)壳聚糖纳米粒的制备
    取30mg壳寡糖、17mg乙二酸四乙胺(EDTA)溶于10mL去离子水中，再加入10mg金纳米棒，超声分散均匀；在磁力搅拌下逐滴添加无水乙醇直至体系颜色变为乳光色，加入30μL25％的戊二醛溶液交联4h，离心收集纳米球；取100μL靶向抗体Anti-VEGF溶于10mL去离子水中，加入12mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和8mg N-羟基琥珀酰亚胺(NHS)于室温下活化抗体血管内皮生长因子羧基2-4h；向反应体系加入15mg纳米球，搅拌4h后离心收集，使用去离子水清洗三次除去未反应的抗体，获得壳聚糖纳米粒产物；
    (2)含氟载氧乳剂的制备
    在10毫升体积比为1:2的盐溶液醋酸-醋酸钠缓冲液/乙醇的溶剂中，加入质量浓度分别为50％的氟碳化合物全氟溴辛烷、30％ 18F的1,2-二棕榈酸甘油酯 18F-DP和20％甘露糖修饰的二硬脂酰基磷脂酰乙醇胺Man-DSPE，另外，再加入质量浓度为10％的二肉豆酰磷脂酰甘油和5％Brij-35，在室温下采用匀浆机进行间歇性搅拌，即搅拌1分钟、终止1分钟，搅拌时间为4小时，冷却后离心，取上清液即可得到含氟载氧乳剂；
    (3)巨噬细胞包载壳聚糖纳米粒和/或含氟载氧乳剂
    将细胞密度为5000个/孔板肠道M2型巨噬细胞与所述的壳聚糖纳米粒和/或所述的含氟载氧乳剂共孵育72小时，得到巨噬细胞包载的壳聚糖纳米粒和含氟载氧乳剂，即得到具有靶向功能的PET/超声多功能肠道造影剂。
    制备的造影剂粒径为460纳米，溶氧量为50％。在808nm的激光照射下，200μg/mL的产物在1min内升温到52℃。
  19. 一种多功能肠道诊疗制剂，根据权利要求10至18任一项所述方法制备 得到的，同时具有PET成像、超声成像和光热治疗的多功能探针，可以同时满足肿瘤精准定位、高灵敏长周期成像。
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CN106267241A (zh) * 2015-06-26 2017-01-04 重庆医科大学 一种多功能多模态肿瘤特异性靶向相变型纳米微球光声造影剂及其应用
CN112957483A (zh) * 2021-02-24 2021-06-15 上海纳米技术及应用国家工程研究中心有限公司 一种靶向肠道的光声成像对比剂的制备方法及其产品

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