WO2020029098A1 - 具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒及其制备方法和应用 - Google Patents

具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒及其制备方法和应用 Download PDF

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WO2020029098A1
WO2020029098A1 PCT/CN2018/099268 CN2018099268W WO2020029098A1 WO 2020029098 A1 WO2020029098 A1 WO 2020029098A1 CN 2018099268 W CN2018099268 W CN 2018099268W WO 2020029098 A1 WO2020029098 A1 WO 2020029098A1
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nanoparticles
platinum
solution
imaging function
infrared
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PCT/CN2018/099268
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French (fr)
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杨红
陈华兵
王雪
柯亨特
李明
徐涛
张米娅
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苏州大学张家港工业技术研究院
苏州大学
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Priority to PCT/CN2018/099268 priority Critical patent/WO2020029098A1/zh
Publication of WO2020029098A1 publication Critical patent/WO2020029098A1/zh
Priority to US17/169,455 priority patent/US11364312B2/en

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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; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/225—Microparticles, microcapsules
    • 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
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides

Definitions

  • the invention discloses an ultra-small platinum sulfide protein nanoparticle with a near-infrared photothermal effect and a multimodal imaging function and a high drug loading capacity, and a preparation method and application thereof.
  • the near-infrared light applied by it is different from ultraviolet light or visible light, and can penetrate into the tissue to a certain extent without causing abnormality and damage to the tissue at a relatively low intensity.
  • photothermal reagents play a key role in photothermal therapy. Its performance has a decisive influence on the effect of photothermal therapy, and accurate diagnosis is a prerequisite for effective treatment.
  • Nanomaterials play a vital role in improving the performance of photothermal agents.
  • Platinum element is a precious metal element, which is located next to “gold element” in the periodic table. Compared with gold, platinum drugs have been more successful in the field of tumor treatment. In addition to traditional platinum drugs, nanoparticles of platinum compounds also have potential anticancer activity and certain photothermal effects.
  • platinum nanoparticles Unfortunately, there are still some obvious defects in currently reported platinum nanoparticles, including: (1) platinum nanoparticle light Poor thermal effect, slow heating, and need to enhance the killing effect on cells; (2) In the process of synthesizing platinum nanoparticles, in order to control the growth of platinum particles or form stable platinum nanoparticles, some are not accepted by clinical injection Components (such as PVP or dendrimers), which will inevitably increase the toxicity of the product and reduce the possibility of clinical transformation. (3) The existing platinum nanoparticles do not have imaging capabilities and cannot provide a diagnosis and treatment solution for tumor treatment.
  • CT contrast agents currently in clinical use are likely to cause tumor targeting imaging and angiography failure due to pharmacokinetic limitations such as short half-life in the circulation and non-specific distribution.
  • CT imaging has some inherent limitations, especially because of the poor contrast between tumor tissue and soft tissue, which is not easy to distinguish, and has shortcomings such as radiation, which is the shortcoming of CT in clinical diagnosis.
  • Protein nanocarriers have attracted much attention due to their good biocompatibility.
  • albumin can be used as a protein nanoreactor, and the sulfhydryl group contained in the protein can be used as the source of sulfur element to prepare nanosulfide nanoparticle, or use the protein reactor for encapsulation.
  • Two compounds, bismuth sulfide and thorium oxide, are used in the diagnosis and treatment of tumors.
  • an object of the present invention is to provide a platinum sulfide protein nanoparticle having near-infrared photothermal effect and multi-modal imaging function and a preparation method thereof, which has good biological safety, tumor targeting and retention.
  • it has the ability to accurately identify tumors by near-infrared fluorescence imaging, photoacoustic imaging, CT imaging, and thermal imaging, and can generate efficient photothermal effects under the excitation of near-infrared light to kill tumor cells, achieving efficient, safe, and precise treatment of tumor Preparation and Application of Multifunctional Albumin Nanoparticles.
  • NIRF near-infrared fluorescence
  • PA photoacoustic
  • CT X-ray computed tomography
  • ⁇ thermal imaging
  • the present invention adopts the following technical scheme: a platinum sulfide protein nanoparticle with near-infrared photothermal effect and multi-modal imaging function, the nanoparticle is prepared in water at 0 to 55 ° C for 0 to 5 hours, and the particle size is 1 to 5 nm .
  • nanoparticles with different particle sizes have different behaviors in different organs due to the biofilm effect.
  • Ultra-small nanoparticles with a particle size smaller than 5 nm can be eliminated from the body by the kidney. Therefore, the ultra-small inorganic nanoparticles prepared under the mild conditions of the present invention constitute a more secure platform for multimodal imaging and tumor treatment, and the significance of the inorganic nanoparticles that are difficult to degrade in the body is particularly prominent.
  • the protein is albumin and serves as a skeleton of the nanoparticle.
  • the platinum source is platinum dichloride; and the sulfur source is sodium sulfide.
  • the invention also discloses a method for preparing platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function, including the following steps: mixing a platinum dichloride solution with a protein solution, adding sodium sulfide solution, and reacting The mixture is obtained; then the mixture is dialyzed and centrifuged by ultrafiltration to obtain platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function.
  • the invention also discloses a method for preparing a reagent with a near-infrared photothermal effect and a multi-modal imaging function, including the following steps: mixing a platinum dichloride solution with a protein solution, adding sodium sulfide solution, and reacting to obtain a mixture; and The mixture was dialyzed and centrifuged by ultrafiltration to obtain platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modality imaging function; the obtained platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modality imaging function were deionized Water is mixed and dispersed to obtain a reagent with near-infrared photothermal effect and multi-modal imaging function.
  • the concentration of the platinum dichloride solution is 2 to 8 mmol / L; the concentration of the protein solution is 1-9 mg / mL; the concentration of the sodium sulfide solution is 1-50 mmol / L; the volume ratio of the platinum dichloride solution, protein solution, and sodium sulfide solution is 1: 0.2: 0.05; and the dispersion is water.
  • the temperature of the reaction is 0 to 55 ° C and the time is 0 to 5 hours.
  • the molecular weight cut-off is 3500 kD during dialysis, and the time is 1 to 24 hours.
  • the deionized water is used as the medium and the dialysis medium is used during the dialysis.
  • the number of replacements is 6 to 8 times; the molecular weight cut-off is 100 kD during the ultrafiltration centrifugation, the rotation speed of the ultrafiltration centrifugation is 1500 to 4000 r / min, and the number of ultrafiltration centrifugation is at least 20 times.
  • the invention discloses a platinum sulfide protein nanoparticle having near-infrared photothermal effect and multi-modality imaging function or a reagent having near-infrared photothermal effect and multi-modality imaging function prepared according to the above-mentioned preparation method;
  • the diameter of the platinum sulfide protein nanoparticles with thermal effect and multi-modal imaging function is 1 ⁇ 5 nm, the protein is the skeleton of the nanoparticles, and the platinum sulfide is the core of the nanoparticles.
  • the invention discloses the above-mentioned platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modality imaging function or a reagent with near-infrared photothermal effect and multi-modality imaging function.
  • the multi-modality imaging includes near-infrared fluorescence imaging reagents, photoacoustic imaging, X-ray computed tomography (CT) imaging, and thermal imaging.
  • the invention discloses a method for preparing a platinum sulfide protein nanoparticle with near-infrared photothermal effect and multi-modal imaging function, including the following steps:
  • step (2) adding the sodium sulfide solution to the mixed solution in step (1), the concentration of the sodium sulfide solution is 1-50 mmol ⁇ L -1 , and then reacting the mixed solution at 0-55 °C for 0-5 hours;
  • step (3) Put the mixed solution after the reaction in step (2) into a dialysis bag (with a molecular weight cut-off of 3500) and dialyze for 1 to 24 hours to remove unreacted reaction raw materials to obtain dialysis nanoparticles, and then perform dialysis on the nanoparticles. Purification was performed by ultrafiltration (with a cut-off molecular weight of 100 kD in the ultrafiltration tube) to obtain platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function.
  • the dialysis uses deionized water as a receiving medium, and the number of dialysis medium replacements during the dialysis is 6 to 8 times; the speed of the ultrafiltration centrifugation is 1500 to 4000 r ⁇ min -1 , and the ultrafiltration centrifugation is performed. The number of times is at least 20 times.
  • the nanoparticles of the present invention have the following advantages: 1) strong X-ray attenuation ability, long in vivo circulation time, low toxicity, no residue, convenient preparation, low cost, small dosage and flexible use, etc., and can be used as an effective clinical CT contrast agent; 2) higher near-infrared absorption coefficient, based on near-infrared photoacoustic effect and heating effect, and 3) subsequent thermal expansion photoacoustic imaging function, which can provide higher spatial resolution distinguishing from soft tissue And used for real-time monitoring. Therefore, nanoparticles are very promising in photothermal therapy and near-infrared imaging, photoacoustic imaging, CT imaging, and thermal imaging applications.
  • the platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function disclosed by the present invention are composed of two components, the core is platinum sulfide, and the skeleton is albumin.
  • Protein as a nanoreactor can produce a variety of protein nanoparticles with different functions, which can form nanocomplexes with metal ions through electrostatic adsorption or special site binding. A precipitation reaction occurs in the swollen protein cavity and induces inorganic nanometers. Crystals nucleate and grow, showing good biocompatibility and tumor targeting, enabling early diagnosis and efficient treatment of tumors.
  • the platinum sulfide protein nanoparticles of the present invention are protein nanoparticles prepared under mild conditions: the size is extremely small (1 to 5 nm), and the drug load is high (15.6%).
  • the current drug load of existing nanoparticles is generally less than 10 %, Has good photothermal effect, and shows great application prospect in targeted multimodal imaging guided cancer treatment.
  • the platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function obtained by the present invention are used as near-infrared photothermal treatment preparations for tumors, near-infrared fluorescence imaging probes, photoacoustic imaging probes, CT imaging contrast agents, and Imaging probe applications have the following advantages:
  • the present invention uses albumin as a nanoreactor to prepare ultra-small-sized protein nanoparticles under mild conditions.
  • the reaction method is simple, the conditions are mild, and the time is short (reaction 0 to 5 h at 0 to 55 °C).
  • the existing protein nanoparticles are more convenient to prepare, the sample is well dispersed, and the size range can be excreted by the kidney to the outside, which is more effective and safe;
  • Nanoparticles of the invention high photothermal conversion efficiency (32.0%), high molar extinction coefficient (1.11 ⁇ 10 9 M -1 ⁇ cm -1 ), good photothermal stability (continuous light exposure for 15 min, absorption spectrum And the heating effect has no obvious attenuation); the light-to-heat conversion efficiency of the nanoparticles of the present invention is 32.0%, which is higher than the gold nanorods (13%) and the gold nanoparticle shells (21%), and is similar to the palladium nanosheets (27.6%) , Excellent performance in precious metal photothermal nano reagents;
  • the tumor is well targeted, can be effectively taken up by tumor cells, has good biocompatibility, and is basically non-toxic in the dark field; it can be accurately positioned and irradiated with excitable near-infrared light in vitro, and can It produces strong thermal effects in specific parts of the body, effectively eliminates tumors, and has near-infrared photothermal effects and near-infrared fluorescence, photoacoustic and X-ray computed tomography imaging, and multimodal imaging functions of thermal imaging.
  • Nano preparations are provided.
  • nanoparticles The ultra-small platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function are referred to as “nanoparticles" for short;
  • Figure 1 is a transmission electron microscope characterization diagram of the nanoparticles
  • Figure 2 is a further characterization map of the nanoparticles:
  • XPS X-ray photoelectron spectroscopy
  • FIG. 3 is a near-infrared heating curve of nanoparticles with different concentrations
  • FIG. 4 is a graph of investigation results of light-to-heat conversion efficiency of nanoparticles
  • FIG. 5 is a graph of investigation results of molar extinction coefficients of nanoparticles
  • FIG. 6 is a graph showing the effect of the illumination time of nanoparticles on heating and morphology
  • FIG. 7 is a diagram of the results of examining the photostability of the nanoparticles
  • FIG. 8 is a graph showing the results of investigations on the physical and chemical stability of the nanoparticles
  • FIG. 9 is a graph showing the results of cytotoxicity investigation of nanoparticles on 4T1 cells.
  • FIG. 10 is a diagram showing the results of examining the distribution of the nanoparticles
  • FIG. 11 is a graph of experimental results of tumor suppression of the tumor-bearing mice by nanoparticles
  • FIG. 12 is a near-infrared fluorescence image of a nanoparticle
  • FIG. 14 is an X-ray computed tomography image of nanoparticles
  • Figure 16 shows the preparation and working mechanism of nanoparticles.
  • the multi-modality imaging in the present invention includes near-infrared fluorescence imaging, photoacoustic imaging, X-ray computed tomography imaging, and thermal imaging, but is not limited thereto.
  • the ultra-small platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function are referred to as "nanoparticles" for short.
  • the molar ratio of Pt: S in the solution was 1: 4, and the volume ratio of the protein solution, platinum dichloride solution, and sodium sulfide solution was 1: 0.2: 0.05.
  • the solution was placed in a 55 ° C water bath and stirred vigorously for 4 h. After the reaction was completed, the reaction product was placed in a dialysis bag (cut-off molecular weight 3500), and the unreacted reaction raw materials were removed by dialysis for 24 h with ultrapure water, and the dialysis medium was replaced.
  • Platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function referred to as Nanoparticles (PtS-NDs).
  • the photosensitizer Cy 7.5 was protected from light, dissolved in a dimethyl sulfoxide solution, added to the prepared PtS-NDs aqueous solution, and stirred for 4 to 8 hours in the dark to obtain Cy 7.5-labeled PtS-NDs (Cy-labeled nanometers). Particles) for fluorescent tracer investigation.
  • the transmission electron microscope image of the above nanoparticles shows that the prepared nanoparticles are a kind of uniformly dispersed ultra-small particle diameter nanoparticles with an average particle diameter of 4.5 ⁇ 0.4 nm, as shown in FIG. 1.
  • the nanoparticles obtained by the present invention are: ultra-small platinum sulfide albumin nanoparticles with a surface hydration layer (size: 4.5 ⁇ 0.4 nm ).
  • h is the thermal conductivity coefficient
  • A is the surface area of the container
  • T max is the maximum temperature of the solution
  • T amb is the ambient temperature
  • I is the laser intensity (1.5 W cm -2 )
  • a ⁇ is the absorbance value at 785 nm).
  • the calculated photothermal conversion efficiency of the platinum sulfide nanoparticles is 32.0%, which is much higher than the photothermal conversion efficiency values of gold rods of photothermal materials reported in the literature, such as Au nanorods (21%) and Au nanoshells (13%). CuS nanocrystals (16.3%), indicating that the nanoparticles prepared by the present invention have more ideal light-to-heat conversion efficiency.
  • D is the particle size
  • M is the molar mass
  • N total is the molar concentration of the solution.
  • the molar extinction coefficient of the nanoparticles of the present invention is calculated to be 1.11 ⁇ 10 9 M -1 ⁇ cm -1 , as shown in FIG. 5, which is much higher than other photothermal materials.
  • the transmission electron microscopy image showed that the average particle size was 4.1 ⁇ 0.6 nm, which was not significantly different from the particle size of the nanoparticles before exposure to 4.5 ⁇ 0.4 nm. It was confirmed that PtS-NDs had good photostability.
  • FIG. 11A is a tumor growth curve of tumor-bearing mice in each group within 30 days
  • FIG. 11B is a tumor picture of mice at 30 days.
  • FIG. 12A shows: Cy7.5-labeled PtS-NDs fluorescence signal in vivo: 1) appeared in the liver at first, and then rapidly decayed; 2) fluorescence signal appeared at the tumor site 4 h after injection, and 8 h, 12 h , 24 h brightness gradually increased, and continued to 48 h, 72 h.
  • the fluorescence intensity of the tumor site was most obvious at 24 h, and it was not completely eliminated within 3 days.
  • Figure 12B shows the fluorescence intensity value of the tumor site automatically circled by the ROI. The values show the same result.
  • the fluorescence signal of the tumor site gradually increased in the first 24 h, reached a peak at 24 h, began to weaken at 48 h, and remained at 72 h. Fluorescent signal.
  • PtS-NDs can produce obvious photoacoustic signals at the tumor site under laser irradiation, and the signals gradually increase from 4 h to 24 h, and the photoacoustic signals cover the entire tumor, and the dispersion is more uniform, indicating that PtS-NDs After entering the tumor, it can penetrate into the entire tumor, providing information for the localization monitoring of deep tumors.
  • FIG. 15A shows a thermal image of a mouse.
  • FIG. 15B shows that as the size of the nanoparticles increases, the temperature of the tumor site increases. Under the same light conditions, the temperature increase of the tumor site was limited by the injection of PBS; the PtS-NDs at 2.1 nm further increased the temperature of the tumor site by 9 o C for 300 seconds (5 min); and 3.2 nm and 4.5 nm PtS-NDs can further increase the temperature of the tumor site by 13.0 ° C and 20.0 ° C, respectively.
  • the PtS-NDs of 4.5 nm in this paper make the temperature of the tumor site reach above 50 oC (can make the tumor thermal ablation), which has a very good photothermal treatment effect.
  • the ultra-small platinum sulfide protein nanoparticles with near-infrared photothermal effect and multi-modal imaging function of the present invention have good tumor treatment effect, and can be used for multi-infrared fluorescence imaging, photoacoustic imaging, CT imaging and thermal imaging Modal complementary tumor diagnosis, ultra-small particle size can be excreted by the kidney and is relatively safe. It has the potential to achieve clinically accurate integration of tumor diagnosis and treatment.
  • Example 2 When the platinum sulfide protein nanoparticles were prepared in Example 1, the human serum albumin concentration was adjusted to 4, 8 mg / mL (in Example 1, the protein concentration was 2 mg / mL), and other steps were the same as in Example 1.
  • Two kinds of nanoparticles with a size of 3.2 ⁇ 0.2 nm and 4.5 ⁇ 0.4 nm can be prepared.
  • PtS-NDs with a concentration of 1.0 mM are irradiated at (785 nm, 1.5 W cm -2 ) for 5 minutes, and the temperature of the solution can be made separately. At 16 ° C and 18.5 ° C, the light-to-heat conversion efficiency is 28.7% and 31.2%, respectively.
  • Example 1 The reaction time during the preparation of the platinum sulfide protein nanoparticles in Example 1 was adjusted to 1 h (in Example 1, the reaction time was 4 h), the absorption in the near infrared region was maximized and maintained stable, and a size of about 4.5 nm could be prepared.
  • PtS-NDs at a concentration of 1.0 mM can increase the solution temperature by 19.5 ° C within 5 min of irradiation at (785 nm, 1.5 W cm -2 ), and the light-to-heat conversion efficiency is 31.8%.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • Example 1 During the preparation of platinum sulfide protein nanoparticles in Example 1, the molar ratios of platinum element to sulfur element were adjusted to 1: 1 and 1: 8 (in the first embodiment, the molar ratio of Pt: S was 1: 4), and others The conditions are the same as in Example 1.
  • a platinum sulfide protein nanoparticle with good stability can be prepared with a size between 3.5 and 4.5 nm and a concentration of 1.0 mM PtS-NDs at (785 nm, 1.5 W cm -2 ) for 5 min. Within the irradiation, the temperature of the solution can be increased to 15.5 ° C and 17.3 ° C, respectively, and the light-to-heat conversion efficiency is 28.8% and 30.3%.
  • FIG. 16 illustrates that the present invention uses a protein as a nanoreactor and Pt 2+ and S 2- precipitation to prepare platinum sulfide protein nanoparticles, and utilizes the enhanced permeability and retention effect (EPR) of solid tumor cells Effect) into the cell, and under the irradiation of near-infrared light, the photothermal effect is good, and can be used for thermal imaging, photoacoustic imaging, near-infrared fluorescence imaging, and because of the large atomic number of platinum, with X-ray attenuation properties, CT imaging, That is, nanoparticles synthesized by biocompatible protein materials through a simple method are used for tumor photothermal treatment guided by multimodal imaging.
  • EPR enhanced permeability and retention effect
  • the present invention creatively designs and prepares an ultra-small platinum sulfide protein nanoparticle with near-infrared photothermal effect and multi-modal imaging function.
  • the visual diagnosis method using four modern diagnostic equipment greatly improves The accuracy and precision of tumor diagnosis, and can effectively play the role of photothermal treatment of tumors under external laser irradiation.
  • the ultra-small particle size it can be excreted through the kidney and has good biological safety.
  • the present invention has the advantages of accurate diagnosis of tumors, good curative effect, safety, and simple preparation of nanoparticles, and has achieved very prominent effects, and has the potential for further development and clinical application.

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Abstract

一种具有近红外光热效应的多模态成像功能的硫化铂蛋白纳米粒及其制备方法和应用。通过处方筛选及工艺限定，在水相中制备具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒，该纳米粒粒径超小，具有良好的稳定性、肿瘤靶向性和光热效果，并整合利用近红外成像、CT成像、热成像功能，实现了对肿瘤的高灵敏度、高分辨率精确定位，在近红外光激发下产生高效光热效应，热消融杀死肿瘤细胞，达到了高效安全、可视化精准治疗肿瘤的目的，具有进一步开发应用于临床的潜力。

Description

具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒及其制备方法和应用 技术领域
本发明公开了一种具有近红外光热效应和多模态成像功能高载药量的超小硫化铂蛋白纳米粒及其制备方法和应用。
 背景技术
恶性肿瘤是严重威胁人类健康的重大恶性疾病之一，其发病率和死亡率在国内外均呈明显的上升趋势。如何实现肿瘤的精确诊断与高效治疗是当前研究的重点及难点。由于温度是影响体内酶活性和各种生化反应速率的重要参数，体温升高通常意味着感染或者其他疾病。通过外部近红外光照射，使进入肿瘤细胞中的包载光热试剂的纳米材料，吸收光并转化为热量，可导致细胞凋亡甚至消融，因而成为了目前方兴未艾的光热治疗肿瘤的方法。与手术切除、放射治疗、化学药物治疗相比，光热治疗优点在于无创性、靶向性、高效性及非侵入性。它所应用的近红外光，与紫外光或可见光不同，可在相对低强度时，一定程度地穿透到组织中而不引起组织的异常和损伤。作为产热主体，光热试剂在光热治疗中扮演关键角色，其性能对光热治疗效果有决定性的影响，并且精准的诊断是有效治疗的前提。
由于传统光热试剂常存在各种各样的不足，严重制约了光热治疗的效果。纳米材料对改善光热剂的性能有至关重要的作用。“铂元素”是一种贵金属元素，在元素周期表中的位置紧邻“金元素”。与金相比，铂类药物在肿瘤治疗领域的应用更为成功。除了传统的铂类药物，铂化合物的纳米粒也具有潜在的抗癌活性和一定的光热效果，但是目前报道的铂纳米粒，还存在一些明显的缺陷，包括：（1）铂纳米粒子光热效果较差，升温缓慢，对细胞的杀伤作用有待增强；（2）在合成铂纳米粒的过程中，为了控制铂颗粒的生长或者形成稳定的铂纳米粒子，使用了一些不被临床注射接受的成分（例如PVP或者树状大分子），这必然会增加产物的毒性，降低临床转化的可能；（3）现有铂纳米粒子不具备成像能力，无法为肿瘤治疗提供诊疗一体的解决方案。因此，有必要进一步挖掘铂纳米粒在肿瘤的诊断和治疗方面的潜力，采用更为科学有效的制备方法，获得临床使用更加安全并兼具诊断和治疗功能的高性能铂纳米试剂，用于肿瘤的光热治疗（Photo-Thermal Therapy，PTT）。
目前临床使用的CT造影剂，如碘普罗胺等，由于体内循环半衰期短和非特异性分布等药代动力学限制，极可能导致肿瘤靶向成像和血管造影失败。并且CT成像还存在一些内在限制，特别是因为肿瘤组织与软组织对比差而不易区分，有辐射等缺点，是CT在临床诊断上的短板。
蛋白纳米载体因生物相容性好而备受关注。目前报道的白蛋白除了可负载药物分子外，将白蛋白既作为蛋白纳米反应器，又利用蛋白所含巯基而作为硫元素的来源，制备得到硫化纳蛋白纳米粒，或利用蛋白反应器包载硫化铋-氧化钆两种化合物，用于肿瘤的诊断和治疗。
然而，目前未见有硫化铂用于光热治疗的报导，现有技术还未见有包载一种铂类化合物的纳米试剂可达到同时发挥光热作用与四种模态的现代成像诊断的效果。
 技术问题
为解决上述技术问题，本发明的目的是提供一种具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒及其制备方法，它具有良好的生物安全性、肿瘤靶向性和滞留性，同时具有近红外荧光成像、光声成像、CT成像、热成像的精准识别肿瘤的能力，并能在近红外光激发下产生高效光热效应从而杀伤肿瘤细胞，实现了高效安全、精准治疗肿瘤的多功能白蛋白纳米粒的制备和应用。
因不同的成像技术各有优劣，四种模态成像的联合应用可更好地取长补短：近红外荧光（NIRF）成像灵敏度高，对比度强；光声（PA）成像可呈现显微结构，分辨率高；X射线计算机断层扫描（CT）成像穿透性好，三维空间定位准确；热（ΔΤ）成像能通过升温区域把握肿瘤的范围。它们联合后的优势互补为后续PTT提供更为精准的定位，也可对治疗效果进行更加有效的监控和评估。
 技术解决方案
本发明采用如下技术方案：一种具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒，所述纳米粒在水中0～55 ℃，0～5h制备，粒径为1～5 nm。一般而言，粒径不同的纳米粒，由于生物膜效应在不同器官有不同行为，粒径小于5 nm的超小纳米粒可经肾脏从体内消除。因此，本发明制备条件温和得到的超小无机纳米粒，构建了多模态成像和治疗肿瘤的更加安全的平台，对于体内难于降解的无机纳米粒的意义尤为突出。
上述技术方案中，所述蛋白为白蛋白，作为纳米粒的骨架。制备反应时，所述铂源为二氯化铂；所述硫源为硫化钠。
本发明还公开了一种具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒的制备方法，包括以下步骤：将二氯化铂溶液与蛋白溶液混合，再加入硫化钠溶液，反应得到混合物；然后将混合物透析、超滤离心，得到具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒。
本发明还公开了一种具有近红外光热效应和多模态成像功能的试剂的制备方法，包括以下步骤，将二氯化铂溶液与蛋白溶液混合，再加入硫化钠溶液，反应得到混合物；然后将混合物透析、超滤离心，得到具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒；将得到的具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒与去离子水混合分散，得到具有近红外光热效应和多模态成像功能的试剂。
本发明中，所述二氯化铂溶液的浓度为2～8 mmol/L；所述蛋白溶液的浓度为1～9 mg/mL；所述硫化钠溶液的浓度为1～50 mmol/L；所述二氯化铂溶液、蛋白溶液、硫化钠溶液的体积比为1:0.2:0.05；所述分散液为水。
本发明中，所述反应的温度为0～55 ℃，时间为0～5h；所述透析时截留分子量为3500 kD，透析时间为1～24 h，透析时以去离子水为介质，透析介质更换次数为6～8 次；所述超滤离心时截留分子量为100 kD，超滤离心的转速为1500～4000 r/min，超滤离心的次数为至少20次。
本发明公开了根据权利要求上述制备方法制备的具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒或者具有近红外光热效应和多模态成像功能的试剂；所述具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒的直径为1～5 nm，蛋白为纳米粒的骨架，硫化铂为纳米粒的核。
本发明公开了上述具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒或者具有近红外光热效应和多模态成像功能的试剂在制备肿瘤诊疗一体化用具有近红外光热效应和多模态成像的纳米制剂中的应用；所述多模态成像包括近红外荧光成像试剂、光声成像、X射线计算机断层扫描成像（CT）、热成像。
本发明公开了的具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒的制备方法，包括以下步骤：
（1）混合二氯化铂溶液与蛋白溶液，得到混合溶液，所述二氯化铂浓度为2～8 mmol ·L -1，所述蛋白溶液的浓度为1～9 mg·mL -1；
（2）将硫化钠溶液加入步骤（1）中的混合溶液中，所述硫化钠溶液的浓度为1～50 mmol·L -1，然后将混合溶液于0～55 ℃反应0～5 h；
（3）将步骤（2）反应后的混合溶液置于透析袋（截留分子量为3500）中透析1～24 h去除未反应的反应原料，得到透析后的纳米粒，然后对透析后的纳米粒进行超滤（超滤管截留分子量为100 kD）纯化，得到具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒。
本发明中，所述透析以去离子水为接收介质，所述透析时透析介质的更换次数为6～8 次；所述超滤离心的转速为1500～4000 r·min -1，超滤离心的次数为至少20次。
本发明纳米粒具有：1）较强的X射线衰减能力，较长的体内循环时间，毒性低、无残留、制备便捷、成本低、剂量较小且使用灵活等诸多优点，可作为有效的临床CT造影剂；2）较高的近红外吸收系数，基于近红外吸引起的光声效应和升温热效应，以及3）随后热膨胀的光声成像功能，可提供更高的与软组织区分的空间分辨率并用于实时监测。因此纳米粒在光热治疗及近红外成像、光声成像、CT成像、热成像应用方面极具发展前途。
本发明公开的具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒，由两种组分构成，内核为硫化铂，骨架为白蛋白。蛋白作为纳米反应器可制备出多种不同功能的蛋白纳米粒，可与金属离子通过静电吸附或特殊位点结合的方式形成纳米复合物，在溶胀的蛋白空腔中发生沉淀反应，诱导无机纳米晶成核并生长，显示出良好的生物相容性及肿瘤靶向性，实现肿瘤的早期诊断及高效治疗。本发明的硫化铂蛋白纳米粒是在温和条件下制备得到的蛋白纳米粒：尺寸超小（1～5 nm），载药量高（15.6 %），现有纳米粒载药量一般不到10%，具有良好的光热效果，在靶向多模态成像引导的癌症治疗中显示出极大的应用前景。
 有益效果
本发明所得具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒，作为肿瘤的近红外光热治疗制剂、近红外荧光成像探针、光声成像探针、CT成像造影剂和热成像探针应用，具有以下优点：
（1） 本发明以白蛋白为纳米反应器，在温和条件下制备得到超小尺寸蛋白纳米粒，反应方法简单、条件温和且时间短（于0～55 ℃反应0～5 h），相比现有蛋白纳米粒（体系复杂，成本高，时间长）制备更便捷，样品分散性良好，尺寸范围可经肾排泄至体外，更加有效而安全；
（2） 本发明纳米粒：具有光热转换效率高（32.0%）、摩尔消光系数高（1.11×10 9 M -1·cm -1）、光热稳定性好（持续光照15 min，吸收光谱和升温效果都无明显衰减）的特点；本发明纳米粒光热转换效率32.0%，比金纳米棒（13%）和金纳米粒壳（21%）高，与钯纳米片（27.6%）相近，在贵金属光热纳米试剂中表现出色；
（3）本发明纳米粒：肿瘤靶向性好，可有效被肿瘤细胞摄取，生物相容性好，在暗场基本无毒性；经体外可控的近红外光精确定位照射并激发后，能在体内特定部位产生强烈的热效应，有效消除肿瘤，并具有近红外光热效应和近红外荧光、光声和X射线计算机断层扫描成像、热成像的多模态成像功能，是安全有效的诊疗一体化的纳米制剂。
 附图说明
本发明具有近红外光热效应和多模态成像功能的超小硫化铂蛋白纳米粒，简称“纳米粒”；
图1为纳米粒的透射电镜表征图；
图2为纳米粒进一步表征图谱：
2A. 水合粒径；
2B. 紫外-可见分光光度计测定的近红外区光谱；
2C. 圆二色光谱（Circular Dichroism, CD）；
2D. X射线光电子能谱（X-ray photoelectron spectroscopy, XPS）；
2E. 场发射透射电子显微镜（Tecnai G 2F20 S-TWIN，ＦＥＩ）mapping图谱；
图3为不同浓度纳米粒的近红外升温曲线；
图4为纳米粒的光热转换效率考察结果图；
图5为纳米粒的摩尔消光消光系数考察结果图；
图6为纳米粒的光照时间对升温及形态的影响结果图；
图7为纳米粒的光稳定性考察结果图；
图8为纳米粒的物理化学稳定性考察结果图；
图9为纳米粒对4T1细胞的细胞毒性考察结果图；
图10为纳米粒的组织分布考察结果图；
图11为纳米粒对荷瘤老鼠的抑瘤实验考察结果图；
图12为纳米粒的近红外荧光成像图；
图13为纳米粒的光声成像图；
图14为纳米粒的X射线计算机断层扫描图；
图15为纳米粒的热成像图；
图16为纳米粒的制备和工作机制图。
 本发明的实施方式
下面结合附图和实施例，对本发明的具体实施方式进一步详细描述。其中，实施例用于说明本发明所述多模态成像包括近红外荧光成像、光声成像、X射线计算机断层扫描成像、热成像，但不局限于此。本发明具有近红外光热效应和多模态成像功能的超小硫化铂蛋白纳米粒，简称“纳米粒”。
实施例一 纳米粒的制备及应用
1.纳米粒的制备：称取20.0 mg人血清白蛋白（HAS，分子量66KD）溶解于10.0 mL去离子水中，称取2.7 mg二氯化铂（PtCl 2，分子量265.99）溶于2 mL去离子水中向溶液中，称取9.6 mg硫化钠（Na 2S·9H 2O，分子量240.18）溶于0.5 mL去离子水中。向蛋白溶液中缓慢加入二氯化铂水溶液并剧烈搅拌使二者充分混合，随后向溶液中加入硫化钠水溶液。溶液中Pt:S摩尔比为1:4，蛋白溶液、二氯化铂溶液、硫化钠溶液的体积比为1:0.2:0.05。将溶液放置于55°C水浴锅中剧烈搅拌4 h，待反应结束后将反应产物放置于透析袋（截留分子量3500）中，用超纯水透析24 h去除未反应的反应原料，透析介质更换次数为7 次，随后用超滤离子能管2000 r/min离心5 min，20次超滤水洗后离心浓缩得到纯化产品：具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒，简称纳米粒（PtS-NDs）。
另外，避光取光敏剂Cy 7.5溶于二甲亚砜溶液，加入上述制备得到的纳米粒PtS-NDs水溶液中，避光搅拌4～8 h，得到Cy 7.5 标记的PtS-NDs（Cy标记纳米粒），作荧光示踪考察用。
2.纳米粒载药量的考察：将制备得纳米粒冷冻干燥后称取一定质量冻干粉末，用水溶液复溶，采用ICP测得溶液中Pt含量，通过以下公式，计算得到载药量（LE）为15.6 %。
LE（%）= W e÷W m×100%   （其中W e为Pt含量，W m为纳米粒质量。）
3.纳米粒的透射电镜表征图：
上述纳米粒的透射电镜图显示，制得的纳米粒是一种均匀分散的超小粒径纳米颗粒，平均粒径为4.5±0.4 nm，如图1。
4.纳米粒PtS-NDs的进一步表征，结果如图2：
（1）纳米粒PtS-NDs的水合粒径测定：以动态光散射（Dynamic Light Scattering，DLS）测得所制备的纳米粒的水合粒径为40.2±0.5 nm，如图2A；
（2）纳米粒PtS-NDs的紫外可见光谱，呈衰减趋势，在785 nm处仍有较小吸收，如图2B；
（3）纳米粒PtS-NDs的圆二色谱图，如图2C。结果显示，纳米粒与HSA蛋白质溶液曲线对照无明显差异，证明纳米粒制备过程中并未破坏蛋白质二级结构；
（4）纳米粒的X射线能谱图，如图2D。结果显示，纳米粒中铂元素的价态主要为正2价；
（5）纳米粒的场发射透射电子显微镜（Tecnai G 2F20 S-TWIN，FEI）面扫分析图，如图2E，结果显示，纳米粒中含有Pt元素及S元素。
因此，通过以上透射电镜、X射线光电子能谱、圆二色谱等方法表征，证明本发明所得的纳米粒是：具有表面水化层的超小硫化铂白蛋白纳米粒（尺寸为4.5±0.4 nm）。
5. 纳米粒的体外升温效应考察：按铂元素含量计，将PtS-NDs溶液分别制备成浓度为0.5，0.75，1.0，1.5，2.0 mmol·L -1的水溶液，采用785 nm激光器 按1.5 W cm -2功率照射5 min，每隔30 s记录溶液的温度。结果如图3，可见纳米粒的光热效应具有浓度依赖性，浓度增加，升温效果明显增加，当纳米粒的浓度在1.0 mM时温度升高17.5℃，当纳米粒的浓度在2.0 mM时温度升高31.2℃，预示纳米粒有着良好的光热治疗前景。
6. 纳米粒的光热转换效率考察：取浓度为1.0 mM的PtS-NDs溶液500 微升，以785 nm激光器（1.5 W cm -2）光照10 min，随后关闭激光器，使溶液自然降温至室温，期间每隔30 s记录一次溶液温度，结果如图4。光热转换效率计算公式为：
Figure 953198dest_path_image002
其中， h为热传导系数， A为容器表面积， T max 为溶液最高温度， T amb 为环境温度， I为激光强度（1.5 W cm -2）， A λ 为785 nm处的吸光度值）。
计算得到硫化铂纳米粒的光热转换效率为32.0%，远高于文献报道的光热材料金棒等的光热转换效率值，如：Au 纳米棒（21%），Au 纳米壳（13%），CuS 纳米晶（16.3%），表明本发明所制备的纳米粒具有更为理想的光热转换效率。
7. 纳米粒的摩尔消光系数考察：选取1、2、3、4、5 mmol·L -1的PtS-NDs水溶液各2 mL，并对其进行紫外光谱的扫描，然后根据样品在785 nm处的吸光度值与其对应的摩尔浓度绘制曲线，结果如图5。其中摩尔浓度计算公式为：
Figure 135917dest_path_image004
其中，
Figure dest_path_image005
为密度， D为粒子粒径， M为摩尔质量， N total为溶液摩尔浓度。
计算得到本发明纳米粒的摩尔消光系数为1.11×10 9 M -1·cm -1，如图5，远高于其他光热材料。
8. 纳米粒的稳定性考察
（1）光照时间对纳米粒的升温及形态的影响考察：取1.0 mM的PtS-NDs溶液0.5 mL，以785 nm激光器（1.5 W cm -2）光照5 min后关闭激光器，待溶液自然冷却至室温后，以相同条件再次光照5 min，随后再次关闭激光器使溶液自然冷却。如此光照、去光照反复5次，过程中每隔30 s记录样品溶液温度。结果如图6，显示每次升温过程所至最高温度均维持在恒定的水平。另外，透射电镜图显示其平均粒径为4.1±0.6 nm，与光照前纳米粒的粒径4.5±0.4 nm无显著差异。证实了PtS-NDs具有良好的光稳定性。
（2）纳米粒光稳定性的进一步考察：各取1.0 mM的PtS-NDs（4.5 nm）溶液2 mL，分别放置在6个2 mL EP管中，然后这6个样品分别在785 nm激光器（1.5 W cm -2）下光照0、1、2、4、8、15 min，光照完成后对其紫外吸收进行扫描。结果如图7，显示在光照长达15 min内PtS-NDs在近红外区域的吸收并未发生明显变化，进一步证实了纳米粒的光稳定性良好。
（3）纳米粒的物理和化学稳定性考察：先配制pH 6.2、pH 7.4、pH 8.0的缓冲溶液。再以三种不同pH的缓冲液、去离子水以及血清为溶剂配制不同的纳米粒溶液，分别在0、1、2、4、8、12、24、48 h对其在785nm进行紫外-可见吸收扫描，测定PtS-NDs在不同介质中的吸光度值。结果如图8，显示48h PtS-NDs在不同介质中均表现出良好的稳定性。
以上结果均表明，本发明纳米粒具有良好的稳定性，为后期应用奠定了基础。
9. MTT实验考察纳米粒的细胞毒性：取对数生长期的小鼠乳腺癌细胞4T1，以每孔5000个的密度，接种于96孔细胞培养板中，37 oC细胞培养箱中培养24 h。然后向孔中加入不同浓度的PtS-NDs水溶液20 µL，使最终药物浓度分别为0.1、0.5、1、1.5、2 mM（以铂元素定量），每个浓度设立4个复孔，同时设置不给药的为对照组。在细胞培养箱中培养24 h后，每孔用PBS清洗3 遍，洗去残余药液，加入新鲜培养基，用785 nm激光器（1.5 W cm -2）每孔光照5 min，同时设置同样加药但不光照的为对照组。继续培养24 h后，每孔中加入20 µL MTT（0.5 mg·mL -1）溶液，仍继续培养4 h后小心弃去细胞板各孔内溶液，每孔加入100 µL 二甲亚砜（DMSO），震荡10 min后用酶标仪对490 nm处每孔的吸光度值（OD）进行检测。结果如图9，显示：1）在未光照条件下，PtS-NDs浓度在4 mM及以下时细胞存活率均大于80 %，说明未表现出明显的细胞毒性，相对安全；2）在785 nm光照后，PtS-NDs对肿瘤细胞的杀伤效果大大增强，且具有浓度依赖性，算得纳米粒IC 50为1.13 mM。
10. 纳米粒的体内分布和抑瘤作用考察
（1）构建肿瘤模型：每只小鼠在其右后背处皮下注射2×10 6个对数期小鼠乳腺癌4T1细胞。待小鼠肿瘤块体积达到60 mm 3时，即可使用。肿瘤体积的计算公式为V=a*b 2/2（a为肿瘤长径，b为肿瘤宽径）。
（2）纳米粒的组织分布考察：对荷瘤小白鼠尾静脉注射PtS-NDs（80 µmol·kg -1），设置3只1组。注射24 h之后，将小鼠颈椎脱臼法处死并解剖，分别取出心、肝、脾、肺、肾、肿瘤，称重并记录，随后置于锥形瓶中，加入王水及高氯酸对样品进行高温硝解，最后用ICP-MS对样品中的Pt含量进行含量测定，结果如图10。显示，尽管PtS-NDs进入小鼠体内后在肝脏、肾脏及肿瘤部位的富集量都明显高于其他组织器官，但由于在非光照条件下PtS-NDs毒性低（暗毒性低），完全可以人为地操控激光器，只照射肿瘤部位，产生光热效应杀死肿瘤细胞，从而避免对肝肾造成影响。
（3）纳米粒对荷瘤鼠的抑瘤作用考察：为考察不同尺寸（平均粒径分别为4.5 nm、3.2 nm、2.1 nm）的PtS-NDs对肿瘤的治疗作用。对荷瘤小鼠随机分组，每组设置5只。以PBS为阴性对照，给药4.5 nm、3.2 nm、2.1 nm PtS-NDs为实验组，均设定非光照组和光照组。尾静脉注射200 μL PBS或80 µmol kg -1 PtS-NDs水溶液（以铂元素定量），24 h后采用785 nm激光器（1.5 W cm -2）对光照组小鼠的肿瘤部位照射5 min，随后每天用游标卡尺测量肿瘤体积，记录并计算肿瘤生长情况，持续监测30天。30天后颈椎脱臼法处死小鼠，取出肿瘤并拍摄照片。结果如图11 。其中，图11A是30 d内各组荷瘤小鼠的肿瘤生长曲线，图11B是30 d时小鼠肿瘤图片。结果显示：1）PBS组在光照和非光照条件下，小鼠肿瘤生长相似，说明单独光照对肿瘤无抑制作用；2）注射2.1 nm 、3.2 nm、4.5 nm PtS-NDs（80.0 μmol·kg -1）的未光照组，同样也未表现出明显的抑瘤效果，最终肿瘤大小均生长为原来的30倍左右，说明单纯注射PtS-NDs无抑瘤效果；但是，3）当2.1 nm PtS-NDs组注射24 h后加以激光照射（785 nm，1.5 W cm -2，5 min）后，小鼠的肿瘤先后出现结痂、脱落，但在第7天开始出现复发；4）当3.2 nm PtS-NDs注射并光照后，小鼠肿瘤先后出现结痂、脱落，但在第16天有两只出现复发，其余三只的肿瘤完全消除未见复发；5）当4.5 nm PtS-NDs组注射24 h后以激光照射，小鼠肿瘤均结痂脱落且30天内未见复发，说明4.5 nm PtS-NDs完全可以达到消除小鼠肿瘤的效果。因此确定以4.5 nm PtS-NDs开展后续实验。
11. 纳米粒的多模态成像效果考察：
（1）纳米粒的近红外荧光成像效果考察：对荷瘤小鼠尾静脉注射200 µL浓度为80 µmol·kg -1的Cy7.5标记的PtS-NDs溶液（4.5 nm），每组3只，分别在0、2、4、8、12、24、48及72 h时用小动物活体成像系统对小鼠进行全身荧光扫描，体内食物及组织自身的荧光采用软件波普分离进行处理扣除，结果如图12。其中，图12A显示：Cy7.5标记的PtS-NDs在体内的荧光信号：1）开始时出现在肝脏，随后迅速衰减；2）注射后4 h肿瘤部位出现荧光信号，且8 h，12 h，24 h亮度逐渐增加，并一直持续到48 h、72 h。24 h时肿瘤部位荧光亮度最为明显，且在3天内未完全消除。图12B显示：ROI自动圈出的肿瘤部位的荧光强度值，数值显示出相同结果，肿瘤部位荧光信号在前24 h逐渐增强，在24 h达到峰值，48 h开始减弱，到72 h都仍有荧光信号。说明Cy7.5标记PtS-NDs能有效地靶向肿瘤并在肿瘤部位滞留较长时间。另外从小鼠荧光图像可以看出，近红外荧光成像灵敏度高，能清晰地标记肿瘤区域，显示边界，可有效指导PTT。
（2）纳米粒的光声成像效果考察：对荷瘤小鼠尾静脉注射200 µL PtS-NDs（80 µmol·kg -1），并由785 nm激光激发下在0, 2, 4, 8, 12, 24, 48, 72 h采集肿瘤部位光声信号并由软件计算荧光强度值。结果如图13，PtS-NDs在激光照射下可在肿瘤部位产生明显光声信号，从4 h~24 h内信号逐渐增强，且光声信号覆盖整个肿瘤，且分散较均匀，说明PtS-NDs进入肿瘤后可渗透进入整个肿瘤，为深部肿瘤的定位监测提供信息。
（3）纳米粒的CT成像效果考察：取荷瘤小鼠采用瘤内注射，注射剂量为150.0 μmol·kg -1的PtS-NDs，在注射后0，5，10，30，60，120 min使用小动物CT机采集小鼠全身CT信号，并进行三维重建，结果如图14A，以信号值作图，如图14B。可见：1）0 min时，肿瘤部位的亮度与附近的肌肉组织的差异不大，不易分辨肿瘤边界。2）注射PtS-NDs后肿瘤部位显著变亮，与周围正常组织有明显区别，肿瘤边界明显。与临床使用的对比剂碘海醇相比，注射PtS-NDs的肿瘤亮度更高、更明显。说明PtS-NDs能显著增强肿瘤部位的CT值，是一种很有潜力的CT造影剂。
（4）纳米粒的热成像考察：对荷瘤小鼠尾静脉注射200 µL不同尺寸PtS-NDs（80 µmol kg -1），在注射24 h后，对小鼠腹腔注射200 μL浓度为35 mg·mL -1水合氯醛进行麻醉，随后使用785 nm激光器，以1.5 W cm -2功率照射小鼠肿瘤部位5 min，使用近红外热成像仪对小鼠全身温度进行监测，结果如图15。图15A小鼠热成像图显示，相较于注射PBS的隐性对照组，注射PtS-NDs后，小鼠肿瘤部位亮度程度更大。图15B显示，随纳米粒尺寸增大，肿瘤部位的升温越高。同样的光照条件下，注射PBS的使肿瘤部位的温度升高有限；光照300秒（5 min），2.1 nm的PtS-NDs使肿瘤部位温度进一步升高9 oC；而3.2 nm和4.5 nm的PtS-NDs可分别使肿瘤部位温度进一步升高13.0°C、20.0°C。本文4.5 nm的PtS-NDs使肿瘤部位的温度到达50 oC以上（可使肿瘤热消融），具有非常好的光热治疗效果。
因此，本发明具有近红外光热效应和多模态成像功能的超小硫化铂蛋白纳米粒，具有良好的肿瘤治疗效果，并且可用于近红外荧光成像、光声成像、CT成像和热成像的多模态互补肿瘤诊断，粒径超小可肾脏排出而相对安全，具有实现临床精准的肿瘤诊疗一体化的潜力。同时，应当指出，基于本发明的技术原理，还可进行若干改进和变型，这些改进和变型也应该视为本发明的保护范围。
实施例二：
在实施例一中制备硫化铂蛋白纳米粒时，分别将人血清白蛋白浓度调节为4、8 mg/mL时（实施例一中，蛋白浓度为2 mg/mL），其他步骤同实施例一，可制得尺寸分别为3.2 ± 0.2 nm，4.5 ± 0.4 nm的两种纳米粒，浓度为1.0 mM的PtS-NDs在（785 nm，1.5 W cm -2）照射5 min，可分别使溶液温度升高16°C、18.5°C，光热转换效率分别为28.7%、31.2%。
实施例三：
将实施例一中硫化铂蛋白纳米粒的制备过程中反应时间调整为1 h（实施例一中，反应时间为4h），近红外区吸收达到最大且维持稳定，可制备得到尺寸为4.5 nm左右，浓度为1.0 mM的PtS-NDs在（785 nm，1.5 W cm -2）5 min照射内可使溶液温度升高19.5°C，光热转换效率为31.8%。
实施例四：
将实施例一中硫化铂蛋白纳米粒制备过程中，分别调节铂元素与硫元素的摩尔比例为1:1、1:8（实施例一中，Pt:S摩尔比为1:4），其他条件与实施例一相同，可制得稳定性良好的硫化铂蛋白纳米粒，尺寸在3.5～4.5 nm之间，浓度为1.0 mM的PtS-NDs在（785 nm，1.5 W cm -2）5 min照射内可使溶液温度升高分别为15.5°C、17.3°C，光热转换效率为28.8%、30.3%。
图16说明：本发明以蛋白为纳米反应器，以Pt 2+与S 2-沉淀反应，制得硫化铂蛋白纳米粒，利用实体瘤细胞的通透和滞留效应(enhanced permeability and retention effect，EPR效应)进入细胞，并在近红外光照射下，光热效应良好，并可用于热成像、光声成像、近红外荧光成像，同时因铂的原子序数大，具有X射线衰减性质，可CT成像，即以生物相容蛋白材料，经简单方法合成的纳米粒，用于多模态成像指导的肿瘤光热治疗。由上可知，本发明创新性地设计和制备了一种具有近红外光热效应和多模态成像功能的超小硫化铂蛋白纳米粒，借用四种现代诊断设备的可视化诊断方法，极大提高了肿瘤诊断的准确性和精确度，并能在体外激光照射下有效发挥光热治疗肿瘤的作用，同时因其粒径超小，可经肾脏排出体外而生物安全性好，解决了现有技术长期存在的无机药物无法从体内消除的安全性难题。因此，本发明对肿瘤诊断精准，疗效好，安全且制备简便的纳米粒，取得了非常突出的效果，具有进一步开发应用于临床的潜力。

Claims (10)

  1. 一种具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒的制备方法，其特征在于，包括以下步骤，将二氯化铂溶液与蛋白溶液混合，再加入硫化钠溶液，反应得到混合物；然后将混合物透析、超滤离心，得到具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒。
  2. 一种具有近红外光热效应和多模态成像功能的试剂的制备方法，其特征在于，包括以下步骤，将二氯化铂溶液与蛋白溶液混合，再加入硫化钠溶液，反应得到混合物；然后将混合物透析、超滤离心，得到具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒；然后将具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒用去离子水分散，得到具有近红外光热效应和多模态成像功能的试剂。
  3. 根据权利要求1或者2所述的制备方法，其特征在于，所述二氯化铂溶液的浓度为2～8 mmol/L；所述蛋白溶液的浓度为1～9 mg/mL；所述硫化钠溶液的浓度为1～50 mmol/L；所述二氯化铂溶液、蛋白溶液、硫化钠溶液的体积比为1:0.2:0.05。
  4. 根据权利要求1或者2所述的制备方法，其特征在于，所述反应的温度为0～55 ℃，时间为0～5h。
  5. 根据权利要求1或者2所述的制备方法，其特征在于，所述透析时截留分子量为3500 kD，透析时间为1～24 h，透析时以去离子水为接收介质，透析介质更换次数为6～8 次。
  6. 根据权利要求1或者2所述的制备方法，其特征在于，所述超滤离心时截留分子量为100 kD，超滤离心的转速为1500～4000 r/min，超滤离心的次数为至少20次。
  7. 根据权利要求1或者2所述的制备方法制备的具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒或者具有近红外光热效应和多模态成像功能的试剂；所述具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒的直径为1～5 nm。
  8. 根据权利要求7所述具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒，其特征在于，蛋白为纳米粒的骨架，硫化铂为纳米粒的核。
  9. 权利要求7所述具有近红外光热效应和多模态成像功能的硫化铂蛋白纳米粒或者具有近红外光热效应和多模态成像功能的试剂，在制备具有近红外光热效应和多模态成像功能的肿瘤诊疗一体化纳米制剂中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述多模态成像包括近红外荧光成像、光声成像、X射线计算机断层扫描成像、热成像。
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Publication number Priority date Publication date Assignee Title
CN113713169B (zh) * 2021-07-23 2023-07-11 温州医科大学 一种近红外光敏感的zif8功能化明胶纳米纤维支架体系及其应用
CN114306597B (zh) * 2021-12-21 2022-08-30 北京化工大学 一种多功能含氟硫化铜纳米探针及其制备方法
CN115054689B (zh) * 2022-06-13 2023-06-30 哈尔滨工程大学 一种近红外光增强催化性能的铂锡双金属纳米粒子的制备方法
CN115887644B (zh) * 2022-09-20 2024-06-14 山东大学 一种铂掺杂叶酸碳化聚合物点及其制备方法与应用
CN115569147A (zh) * 2022-09-29 2023-01-06 浙江瑞邦药业股份有限公司 铂单原子负载MXene纳米片的制备方法及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104587493A (zh) * 2015-02-05 2015-05-06 东南大学 应用于心脑血管相关疾病早期、快速、实时动态监测与多模态成像的检测试剂
CN105056233A (zh) * 2015-08-12 2015-11-18 苏州大学 具有近红外光热和体内荧光成像特性的多功能介孔二氧化硅纳米粒及其制备方法和应用
CN105106958A (zh) * 2015-09-08 2015-12-02 苏州大学 具有近红外光热效应的铜基人血白蛋白纳米复合物及其制备方法和应用
CN106267198A (zh) * 2016-04-07 2017-01-04 苏州大学 靶向光热治疗联合免疫治疗抗肿瘤复合制剂及其制备方法与应用
CN107551279A (zh) * 2017-09-14 2018-01-09 苏州大学 具有近红外光热效应和多模态成像功能的超小蛋白复合纳米粒及其制备方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100469688C (zh) * 2006-05-19 2009-03-18 中国科学院理化技术研究所 一系列椭球形金属硫化物颗粒及其制备方法和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104587493A (zh) * 2015-02-05 2015-05-06 东南大学 应用于心脑血管相关疾病早期、快速、实时动态监测与多模态成像的检测试剂
CN105056233A (zh) * 2015-08-12 2015-11-18 苏州大学 具有近红外光热和体内荧光成像特性的多功能介孔二氧化硅纳米粒及其制备方法和应用
CN105106958A (zh) * 2015-09-08 2015-12-02 苏州大学 具有近红外光热效应的铜基人血白蛋白纳米复合物及其制备方法和应用
CN106267198A (zh) * 2016-04-07 2017-01-04 苏州大学 靶向光热治疗联合免疫治疗抗肿瘤复合制剂及其制备方法与应用
CN107551279A (zh) * 2017-09-14 2018-01-09 苏州大学 具有近红外光热效应和多模态成像功能的超小蛋白复合纳米粒及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TANG, Y. ET AL.: "Albumin-Coordinated Assembly of Clearable Platinum Nanodots for Photo- Induced Cancer Theranostics", BIOMATERIALS, vol. 154, 20 October 2017 (2017-10-20), pages 248 - 260, XP055682636 *

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