CN109481701B - Brain glioma image nano probe and preparation method and application thereof - Google Patents
Brain glioma image nano probe and preparation method and application thereof Download PDFInfo
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- CN109481701B CN109481701B CN201711440558.1A CN201711440558A CN109481701B CN 109481701 B CN109481701 B CN 109481701B CN 201711440558 A CN201711440558 A CN 201711440558A CN 109481701 B CN109481701 B CN 109481701B
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Classifications
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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
- A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
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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
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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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
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
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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
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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- 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
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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
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
- A61K49/0084—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion liposome, i.e. bilayered vesicular structure
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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
- A61K49/227—Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric
Abstract
The invention provides a brain glioma image nano probe and a preparation method and application thereof, and relates to the technical field of nano probes.
Description
Technical Field
The invention relates to the technical field of nano probes, in particular to a brain glioma image nano probe and a preparation method and application thereof.
Background
Gliomas are intracranial malignant tumors derived from glial cells, also a typical gene mutant central nervous system disorder, accounting for about 40-50% of craniocerebral tumors. World Health Organization (WHO) divides brain gliomas into four grades of capillary astrocytomas, low-grade malignant gliomas, anaplastic astrocytomas and glioblastoma multiforme according to the malignancy degree of the brain gliomas, wherein the glioblastoma multiforme has the highest malignancy degree, has the characteristics of high mortality rate, easy recurrence and difficult healing, the survival rate of 2 years after treatment by means of surgical excision, radiotherapy, chemotherapy and the like is still lower than 30 percent, the survival rate of 5 years is less than 10 percent, the median survival time is only 12 to 15 months, and the three-position death cause of patients with tumors of 34 to 54 years has been established.
At present, the clinical treatment of brain glioma is mainly surgical excision treatment, and because the characteristics of brain glioma and other craniocerebral tumors are very different, the brain glioma is in crab foot-like local infiltration and grows in normal brain group infiltration towards the periphery of a primary disease focus, so that the tumor boundary is unclear, the surgery is difficult to thoroughly excision, and the postoperative recurrence is very easy. Usually, glioma easily occurs in important brain functional areas, such as nerve center control areas of cognition, movement, language and the like of people, so that great sequelae is brought to patients after operation, and the quality of life after operation is seriously influenced. Therefore, it is highly desirable to provide an image probe capable of accurately recognizing the brain glioma boundary to improve the recognition capability of the tumor boundary.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a brain glioma image nano probe capable of accurately identifying the boundary of brain glioma, so as to solve the technical problem that the existing brain glioma is unclear in boundary and difficult to completely cut off in operation.
The brain glioma image nano probe provided by the invention comprises carboxylated modified liposome, a contrast agent and lactoferrin, wherein a hydrophobic layer of the carboxylated modified liposome encapsulates the contrast agent, a hydrophilic layer of the carboxylated modified liposome is connected with the lactoferrin through a chemical bond, and the contrast agent comprises at least one of fluorescent contrast agent, photo-acoustic contrast agent and fluorescent photo-acoustic bimodal contrast agent, preferably fluorescent photo-acoustic bimodal contrast agent.
Further, the carboxylated modified liposome is a carboxylated polyethylene glycol modified liposome;
preferably, in the carboxylated polyethylene glycol modified liposome, the polymerization degree of polyethylene glycol is 1000-10000, preferably 2000-5000.
Further, the carboxylated polyethylene glycol modified liposome is at least one selected from carboxylated polyethylene glycol modified phosphatidylcholine, carboxylated polyethylene glycol modified dipalmitoyl phosphatidylcholine, carboxylated polyethylene glycol modified phosphatidylethanolamine and carboxylated polyethylene glycol modified distearoyl phosphatidylcholine.
Further, the fluorescent contrast agent is selected from at least one of Cy3, cy5 or Cy 5.5;
preferably, the photoacoustic contrast agent is selected from the group consisting of lamellar molybdenum disulfide or tungsten disulfide;
preferably, the fluorescent photoacoustic bimodal contrast agent is selected from indocyanine dyes and/or heptamethine cyanine dyes;
preferably, the indocyanine dye is indocyanine green;
preferably; the heptamethine cyanine dye is selected from at least one of IR-780, IR-775, IR-797, IR-792, IR-806 or IR-808.
The second purpose of the invention is to provide a preparation method of the brain glioma image nanoprobe, which comprises the following steps:
(a) Adding the carboxylated modified liposome and the contrast agent into an organic solvent, uniformly mixing, and then adding into water, uniformly mixing, so that the contrast agent is coated in a hydrophobic layer of the carboxylated modified liposome, and obtaining a liposome probe;
(b) And uniformly mixing the liposome probe with a carboxyl activating agent, and then adding lactoferrin to uniformly mix, thus obtaining the brain glioma image nano probe.
Preferably, the organic solvent is a volatile organic solvent, preferably at least one of ethanol, chloroform, dichloromethane and acetone, more preferably ethanol.
Preferably, the carboxyl activator is N-hydroxysuccinimide and/or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, preferably N-hydroxysuccinimide.
Further, the mass ratio of the carboxylated modified liposome to the contrast agent is (1-10): 1, preferably (1-2): 1.
Further, the mass ratio of the carboxylated modified liposome to the lactoferrin is 1 (1-3), preferably 1 (1.5-2).
Further, in step (a), further comprising purification of the liposome probe, said purification comprising removal of organic solvent and unencapsulated contrast agent;
preferably, the organic solvent is volatilized and removed by adopting an inert gas purge;
preferably, centrifugation is used to remove unencapsulated contrast agent.
Further, in step (b), further comprising purification of the brain glioma imaging nanoprobe, said purification comprising removal of the carboxyl activator and unreacted lactoferrin;
preferably, the carboxy activator and unreacted lactoferrin are removed by dialysis or ultrafiltration.
The invention further aims to provide application of the brain glioma image nano probe in preparation of brain glioma diagnosis and/or treatment medicines.
The brain glioma image nano probe provided by the invention is biodegradable, good in water solubility, capable of penetrating through a blood brain barrier, actively targeting brain glioma cells, and capable of realizing tumor imaging in the microenvironment of the brain glioma, so that the boundary of the brain glioma is accurately identified, the diagnosis accuracy of the brain glioma is improved, references are provided for surgical excision of the brain glioma, the brain glioma is cleanly excised, the occurrence of sequelae is reduced, and the postoperative life quality is improved.
The preparation method of the brain glioma image nano probe provided by the invention has the advantages of simple process and convenience in operation, and can be suitable for large-scale production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a preparation process of a brain glioma-affecting nanoprobe provided by the invention;
FIG. 2 is a near infrared fluorescence image of a nude mouse injected with the brain glioma imaging nanoprobe solution provided in example 2 of the present invention;
fig. 3 is a photo-acoustic image of a nude mouse injected with the brain glioma image nanoprobe provided in example 2 of the present invention.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
According to one aspect of the present invention, there is provided a brain glioma imaging nanoprobe, which is mainly prepared from carboxylated modified liposome, a contrast agent and lactoferrin, wherein the contrast agent is encapsulated in the carboxylated modified liposome, the lactoferrin and the carboxylated modified liposome are connected through a chemical bond, and the contrast agent comprises at least one of a fluorescent contrast agent, a photoacoustic contrast agent and a fluorescent photoacoustic bimodal contrast agent, preferably a fluorescent photoacoustic bimodal contrast agent.
Lactoferrin is an important non-heme iron-binding glycoprotein in humans, which is involved not only in iron transport, antioxidant, regulating immune system and other powerful biological functions, but also in its passage through the blood brain barrier function. Along with the development of the brain glioma, the surface of the brain glioma highly expresses a lactoferrin receptor, so that the lactoferrin is an active targeting molecule of the brain glioma.
According to the brain glioma image nano probe provided by the invention, the lactoferrin is connected with the carboxylated modified liposome through the chemical bond, so that the targeting property of the brain glioma image nano probe is enhanced, the brain glioma image nano probe can image the blood brain barrier, the boundary of the brain glioma is monitored, and the diagnosis accuracy of the brain glioma is improved.
In the brain glioma image nano probe provided by the invention, the carboxylation modified liposome hydrophobic layer is coated with the contrast agent, so that the contrast agent is protected, and the contrast agent is carried into the brain glioma microenvironment for imaging.
The brain glioma image nano probe provided by the invention is biodegradable, good in water solubility, capable of penetrating through a blood brain barrier, actively targeting brain glioma cells, and capable of realizing tumor imaging in the microenvironment of the brain glioma, so that the boundary of the brain glioma is accurately identified, the diagnosis accuracy of the brain glioma is improved, references are provided for surgical excision of the brain glioma, the brain glioma is cleanly excised, the occurrence of sequelae is reduced, and the postoperative life quality is improved.
In a preferred embodiment of the invention, the carboxylated modified liposomes are carboxylated polyethylene glycol modified liposomes.
In this preferred embodiment of the invention, carboxylated polyethylene glycol modified liposomes are used to provide more effective protection of the contrast agent from quenching of the contrast agent.
In a further preferred embodiment of the present invention, the degree of polymerization of the polyethylene glycol in the carboxylated polyethylene glycol modified liposomes is in the range of 1000 to 10000, preferably 2000 to 5000.
If the polymerization degree of polyethylene glycol is too small, the carboxylated modified liposome cannot provide comprehensive protection for the contrast agent, if the polymerization degree of polyethylene glycol is too large, the difficulty of liposome modification is increased, the preparation efficiency of the carboxylated polyethylene glycol modified liposome is too low, and multiple experiments prove that the carboxylated modified polyethylene glycol liposome can provide comprehensive protection for the contrast agent when the polymerization degree of polyethylene glycol is 1000-10000, and particularly, when the polymerization degree of polyethylene glycol is 2000-5000, the protection effect of the carboxylated polyethylene glycol modified liposome on the contrast agent is better.
In this preferred embodiment of the invention, the typical, but non-limiting, degree of polymerization of polyethylene glycol is 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000.
In a further preferred embodiment of the present invention, the carboxylated polyethylene glycol modified liposomes are selected from at least one of carboxylated polyethylene glycol modified phosphatidylcholine (PC-PEG-COOH), carboxylated polyethylene glycol modified dipalmitoyl phosphatidylcholine (DPPC-PEG-COOH), carboxylated polyethylene glycol modified phosphatidylethanolamine (PE-PEG-COOH) and carboxylated polyethylene glycol modified distearoyl phosphatidylcholine (DSPE-PEG-COOH).
The carboxylated polyethylene glycol modified liposome can be carboxylated polyethylene glycol modified phosphatidylcholine, carboxylated polyethylene glycol modified dipalmitoyl phosphatidylcholine, carboxylated polyethylene glycol modified phosphatidylethanolamine or carboxylated polyethylene glycol modified distearoyl phosphatidylcholine, or a mixture of any two of the above, such as a mixture of carboxylated polyethylene glycol modified phosphatidylcholine and carboxylated polyethylene glycol modified dipalmitoyl phosphatidylcholine, a mixture of carboxylated polyethylene glycol modified phosphatidylethanolamine and carboxylated polyethylene glycol modified distearoyl phosphatidylcholine, or a mixture of any three of the above, such as carboxylated polyethylene glycol modified phosphatidylcholine, a mixture of carboxylated polyethylene glycol modified dipalmitoyl phosphatidylcholine and carboxylated polyethylene glycol modified phosphatidylethanolamine, carboxylated polyethylene glycol modified dipalmitoyl phosphatidylcholine, carboxylated polyethylene glycol modified phosphatidylethanolamine and carboxylated polyethylene glycol modified distearoyl phosphatidylcholine, or a mixture of the above four of the above: carboxylated polyethylene glycol modified phosphatidylcholine, carboxylated polyethylene glycol modified dipalmitoyl phosphatidylcholine, carboxylated polyethylene glycol modified phosphatidylethanolamine and carboxylated polyethylene glycol modified distearoyl phosphatidylcholine.
In a preferred embodiment of the invention, the fluorescent contrast agent is selected from at least one of Cy3, cy5 or Cy 5.5.
In a preferred embodiment of the invention, the photoacoustic contrast agent is selected from the group consisting of lamellar molybdenum disulfide or tungsten disulfide.
In a preferred embodiment of the invention, the fluorescent photoacoustic bimodal contrast agent is selected from the group consisting of indocyanine dyes and/or heptamethine cyanine dyes.
In a further preferred embodiment of the invention, the indocyanine dye is indocyanine green (ICG); the heptamethine cyanine dye is at least one selected from IR-780, IR-775, IR-797, IR-792, IR-806 or IR-808, and more preferably indocyanine green (ICG).
ICG is a dye of near infrared fluorescence and photoacoustic, which has good absorption in the near infrared region, can rapidly generate a large amount of heat to emit photoacoustic signals under 808nm excitation, is suitable for photoacoustic imaging, and has near infrared fluorescence emission property in the near infrared region, so that ICG can be used for near infrared fluorescence/photoacoustic bimodal imaging. In addition, ICG is photosensitive and can also be used for tumor phototherapy.
When ICG is selected as a contrast agent, the glioma image nano probe provided by the invention can not only perform near infrared fluorescence imaging in a glioma microenvironment, but also perform photoacoustic imaging, and can also perform light treatment on glioma.
According to a second aspect of the present invention, the present invention provides a method for preparing the brain glioma imaging nanoprobe, comprising the following steps:
(a) Adding the carboxylated modified liposome and the contrast agent into an organic solvent, uniformly mixing, and then adding the mixture into water, uniformly mixing, so that the contrast agent is coated in the modified liposome, and obtaining a liposome probe;
(b) And uniformly mixing the liposome probe with a carboxyl activating agent, and then adding lactoferrin to uniformly mix, thus obtaining the brain glioma image nano probe.
FIG. 1 is a schematic diagram of a preparation process of a brain glioma imaging nanoprobe provided by the invention; as can be seen from fig. 1, according to the brain glioma image nano probe provided by the invention, in the mixing process of carboxylated modified liposome and contrast agent, the contrast agent is coated inside the carboxylated modified liposome, and then carboxyl reacts with lactoferrin under the action of a carboxyl activating agent, so that the carboxylated modified liposome and the lactoferrin are connected through chemical bonds, and the brain glioma image nano probe is prepared.
The preparation method of the brain glioma image nano probe provided by the invention has the advantages of simple process and convenience in operation, and can be suitable for large-scale production.
In a preferred embodiment of the present invention, in step (a), the organic solvent is a volatile organic solvent, preferably at least one of ethanol, chloroform, dichloromethane and acetone, more preferably ethanol.
The carboxylation modified liposome and the contrast agent are dissolved by selecting a volatile organic solvent, so that the organic solvent is conveniently removed by volatilization in the follow-up process, and the damage to organisms caused by the existence of the organic solvent is avoided.
In a preferred embodiment of the invention, the carboxyl activator is N-hydroxysuccinimide (EDC) and/or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, preferably N-hydroxysuccinimide (NHS).
In the step (b), the liposome probe and the carboxyl activating agent are uniformly mixed so as to promote the connection of the carboxyl modified liposome and the lactoferrin through chemical bonds, so that the capability of the brain glioma image probe provided by the invention for penetrating through the blood brain barrier is enhanced, and the targeting of the brain glioma is enhanced.
In a preferred embodiment of the present invention, the mass ratio of carboxylated modified liposomes to contrast agent is (1-10): 1, preferably (1-2): 1.
In this preferred embodiment of the invention, typical but non-limiting mass ratios of carboxylated modified liposomes to contrast agent are 1:1, 1.25:1, 1.5: 1. 1.75:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.
The mass ratio of the carboxylated modified liposome to the contrast agent is set to be (1-10): 1, so that the carboxylated modified liposome is ensured to be excessive in contrast agent, and the loss of the contrast agent is avoided; especially when the mass ratio of the carboxylated modified liposome to the contrast agent is (1-2): 1, the encapsulation rate of the carboxylated modified liposome to the contrast agent is higher, and the loss of raw materials can be effectively reduced.
In a preferred embodiment of the invention, the mass ratio of carboxylated modified liposomes to lactoferrin is 1 (1-3), preferably 1 (1.5-2).
In this preferred embodiment of the invention, typical but non-limiting mass ratios of carboxylated modified liposomes to lactoferrin are 1:1, 1.25:1, 1.5: 1. 1.75:1, 2:1, 2.25:1, 2.5:1, 2.75:1, or 3:1.
The carboxylated modified liposome is activated by a carboxyl activating agent so as to be connected with lactoferrin through chemical bonds, and in order to improve the reaction efficiency of the carboxylated modified liposome and the lactoferrin, the amount of substances of the lactoferrin is required to be excessive compared with the amount of substances of the carboxylated activated liposome, and experiments prove that when the mass ratio of the carboxylated modified liposome to the lactoferrin is 1: (1-3), the yield of the brain glioma imaging nanoprobe is higher, especially when the mass ratio of carboxylated modified liposome to lactoferrin is 1: (1.5-2) the yield of the brain glioma imaging nanoprobe is highest.
In a preferred embodiment of the present invention, in step (a), purification of the carboxylated modified liposome probes is also included, the purification including removal of organic solvent and unencapsulated contrast agent.
The organic solvent and the contrast agent in the carboxylated modified liposome probe are removed, so that the carboxylated modified liposome probe is purified, the influence of impurities on subsequent reaction with lactoferrin is avoided, and the safety and stability of the carboxylated modified liposome are improved.
In a further preferred embodiment of the invention, the organic solvent is volatilized off using an inert gas purge.
The inert gas is selected from at least one of argon, helium and nitrogen, and the organic solvent is removed by purging with the inert gas, so that the method is convenient, safe, simple and easy to implement.
In a further preferred embodiment of the invention, the non-entrapped contrast agent is removed by centrifugation, which is simple and convenient and easy to operate.
In a preferred embodiment of the present invention, in step (b), purification of the brain glioma imaging nanoprobe is also included, the purification comprising removal of the carboxyl activator and unreacted lactoferrin.
In a further preferred embodiment of the invention, the carboxy activator and unreacted lactoferrin are removed by dialysis or ultrafiltration.
In the preferred embodiment of the invention, PBS solution is used for purifying the brain glioma image nano-probe, and the carboxyl activated lactoferrin and unreacted lactoferrin are removed, so that the safety, stability and targeting of the brain glioma image nano-probe provided by the invention are improved.
In the preferred embodiment of the invention, the purification of the brain glioma image nanoprobe is more convenient and quicker by adopting ultrafiltration, and the adopted ultrafiltration membrane is of 10 ten thousandth molecular weight specification.
According to a third aspect of the invention, the invention provides an application of the brain glioma image nano probe in preparing a brain glioma diagnosis and/or treatment drug.
The technical scheme provided by the invention is further described below by combining examples and comparative examples.
Example 1
The embodiment provides a brain glioma image nano probe which is prepared according to the following steps:
(1) 2mg of 1, 2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-carboxyl-polyethylene glycol 1000 (DSPE-PEG (1000) -COOH) and 1mg of ICG are dissolved in 1mL of chloroform, after being uniformly mixed, the mixture is dropwise added into 3mL of water, the system is purged by nitrogen until the chloroform is volatilized, the mixture is centrifuged at 8000rpm/min for 10min, and free ICG is removed, so that a DSPE-PEG (2000) -COOH solution with the ICG is obtained;
(2) EDC is added into DSPE-PEG (2000) -COOH solution with ICG entrapped, carboxyl of DSPE-PEG (2000) -COOH is activated for 24h, ultrafiltration (10 ten-thousandth molecular weight specification) is carried out at 8000rpm/min for 3min for 6 times, a filter tube is filled with PBS solution each time, carboxyl activating agent is removed, finally 2ml of sample is left, 3.5mg of lactoferrin is weighed again, dissolved in 200ul of PBS solution, added into a reaction system (2 ml), stirred at room temperature (the speed is not suitable for being fast), and the reaction is carried out overnight, thus obtaining the brain glioma image nano probe solution.
Example 2
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from embodiment 1 in that in the step (a), DSPE-PEG (2000) -COOH is used instead of DSPE-PEG (1000) -COOH.
Example 3
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from embodiment 1 in that in the step (a), DSPE-PEG (3000) -COOH is used instead of DSPE-PEG (1000) -COOH.
Example 4
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from embodiment 1 in that in the step (a), DSPE-PEG (5000) -COOH is used instead of DSPE-PEG (1000) -COOH.
Example 5
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from embodiment 1 in that in the step (a), DSPE-PEG (8000) -COOH is used instead of DSPE-PEG (1000) -COOH.
Example 6
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from embodiment 1 in that in step (a), DSPE-PEG (10000) -COOH is used instead of DSPE-PEG (1000) -COOH.
Example 7
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from embodiment 1 in that in the step (a), DSPE-PEG (100) -COOH is used instead of DSPE-PEG (1000) -COOH.
Example 8
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from embodiment 1 in that in the step (a), DSPE-PEG (200) -COOH is used instead of DSPE-PEG (1000) -COOH.
The brain glioma image nano probe solutions provided in examples 1-8 are respectively injected into a brain glioma in-situ nude mouse model from tail veins for near infrared fluorescence/photoacoustic bimodal brain glioma imaging, and the results show that near infrared fluorescence and photoacoustic imaging of nude mice injected with the brain glioma image nano probes provided in examples 1-6 are clear, the boundaries of brain glioma can be accurately identified, especially the near infrared fluorescence and photoacoustic imaging of nude mice injected with the brain glioma image nano probes provided in examples 2-4 are clear, the boundaries of brain glioma can be accurately identified, help is provided for surgical excision of clean tumor tissues, and the fluorescence and photoacoustic images of nude mice injected with the brain glioma image nano probes provided in examples 7-8 can not clearly see the brain glioma boundaries, which indicates that the brain glioma image nano probes can maintain good brain glioma boundary identification functions only when the polymerization degree of polyethylene glycol is 1000-10000, especially 2000-5000.
The imaging function of the glioma image nanoprobe is described below with reference to fig. 2 and 3, and fig. 2 is a near infrared fluorescence diagram of a nude mouse injected with the glioma image nanoprobe solution provided in embodiment 2 of the present invention; fig. 3 is a photo-acoustic diagram of a nude mouse injected with the brain glioma image nanoprobe provided in embodiment 2 of the present invention; as can be seen from fig. 2 and fig. 3, the near infrared fluorescence image and the photoacoustic image of the nude mice injected with the glioma image nano probe solution provided by the embodiment 2 of the present invention are clear, and the boundary is clear, which indicates that the glioma image nano probe provided by the embodiment 2 of the present invention can pass through the blood brain barrier, actively target glioma cells, realize fluorescence and photoacoustic bimodal imaging of tumors in the microenvironment of glioma, and accurately identify the boundary of glioma from the image, thereby improving the diagnosis accuracy of glioma and providing a reference for the surgical excision of glioma.
Example 8
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (2), the mass of lactoferrin is 2mg.
Example 9
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (2), the mass of lactoferrin is 3mg.
Example 10
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (2), the mass of lactoferrin is 4mg.
Example 11
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (2), the mass of lactoferrin is 6mg.
Example 12
The present example provides a brain glioma imaging nanoprobe, the preparation method of which is different from that of example 2 in that in step (2), the mass of lactoferrin is 0.5mg.
The brain glioma image nano probe solutions provided in examples 8-12 are respectively injected into a brain glioma in-situ nude mouse model from tail veins, near infrared fluorescence/photoacoustic bimodal brain glioma imaging is carried out, and the results are compared with near infrared fluorescence images and photoacoustic images of nude mice injected with the brain glioma image nano probe solutions provided in example 2, so that the near infrared fluorescence and photoacoustic imaging of nude mice injected with the brain glioma image nano probes provided in examples 8-11 are clear, the boundaries of brain glioma can be accurately identified, especially the near infrared fluorescence and photoacoustic imaging of nude mice injected with the brain glioma image nano probes provided in examples 9-10 are clear, and the near infrared fluorescence and photoacoustic imaging definition of nude mice injected with the brain glioma image nano probes provided in example 2 is basically consistent, and the boundaries of brain glioma can be accurately identified. Whereas the fluorescence and photoacoustic images of nude mice injected with the glioma image nanoprobe provided in example 12 were less clear, and as can be seen from the fluorescence images thereof, most of the glioma image nanoprobes passing through the blood brain barrier were less, resulting in poor imaging definition of the fluorescence and photoacoustic images thereof. This demonstrates that when the mass ratio of carboxylated modified liposomes to lactoferrin is 1: in the step (1-3), the prepared brain glioma image nano probe has strong active targeting function, can penetrate through a blood brain barrier, monitor the boundary of brain glioma, and provide assistance for surgical excision of clean tumor tissues, especially when the mass ratio of carboxylated modified liposome to lactoferrin is 1: (1.5-2), the prepared brain glioma image nano probe has stronger active targeting function, can penetrate through the blood brain barrier and enter the microenvironment of the brain glioma to detect the boundary of the tumor.
Example 13
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (1), the mass of ICG is 0.2mg.
Example 14
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (1), the mass of ICG is 0.5mg.
Example 15
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (1), the mass of ICG is 1.5mg.
Example 16
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (1), the mass of ICG is 2mg.
Example 17
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (1), the mass of ICG is 0.05mg.
Example 18
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from example 2 in that in step (1), the mass of ICG is 10mg.
The brain glioma image nano probe solutions provided in examples 13-18 are respectively injected into a brain glioma in-situ nude mouse model from tail veins, near infrared fluorescence/photoacoustic bimodal brain glioma imaging is carried out, and the results are compared with near infrared fluorescence images and photoacoustic images of nude mice injected with the brain glioma image nano probe solutions provided in example 2, so that the near infrared fluorescence and photoacoustic imaging of nude mice injected with the brain glioma image nano probes provided in examples 13-16 are clear, the boundaries of brain glioma can be accurately identified, especially the near infrared fluorescence and photoacoustic imaging of nude mice injected with the brain glioma image nano probes provided in examples 15-16 are clear, and the near infrared fluorescence and photoacoustic imaging definition of nude mice injected with the brain glioma image nano probes provided in example 2 is basically consistent, and the boundaries of brain glioma can be accurately identified. Whereas the fluorescence and photoacoustic images of nude mice injected with the brain glioma imaging nanoprobes provided in examples 17-18 were less clear, this demonstrates that when the mass ratio of carboxylated modified liposomes to contrast agent was (1-10): 1, the imaging of the prepared brain glioma imaging nano probe is clear, especially when the mass ratio of carboxylated modified liposome to contrast agent is (1-2): 1, the prepared brain glioma image nano probe is clearer in imaging.
Example 19
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from that of embodiment 2 in that the adopted contrast agent is IR-780.
Example 20
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from that of embodiment 2 in that the adopted contrast agent is IR-797.
Example 21
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from that of embodiment 2 in that the adopted contrast agent is IR-808.
Example 22
The present example provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from that of example 2 in that the adopted contrast agent is IR-775.
The brain glioma image nano probe solutions provided in examples 19-22 are respectively injected into a brain glioma in-situ nude mouse model from tail veins, near infrared fluorescence/photoacoustic bimodal brain glioma imaging is carried out, and the near infrared fluorescence image and the photoacoustic image of a nude mouse injected with the brain glioma image nano probe solution provided in example 2 are compared, and the results show that the near infrared fluorescence image and the photoacoustic image of the nude mouse injected with the brain glioma image nano probe provided in examples 19-22 are clear, so that the boundary of the brain glioma can be accurately identified, and the brain glioma image nano probe provided by the invention can also pass through a blood brain barrier by adopting different types of contrast agents, actively target brain glioma cells, realize tumor imaging in the microenvironment of the brain glioma, thereby accurately identifying the boundary of the brain glioma, improving the diagnosis accuracy of the brain glioma, providing references for brain glioma surgical excision and helping to cleanly excise the brain glioma.
Example 23
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from that of embodiment 2 in that in step (1), PC-PEG (2000) -COOH is used instead of DSPE-PEG (2000) -COOH.
Example 24
The present example provides a brain glioma imaging nanoprobe, which is different from example 2 in that DPPC-PEG (2000) -COOH is used instead of DSPE-PEG (2000) -COOH in step (1). .
Example 25
The present embodiment provides a brain glioma imaging nanoprobe, and the preparation method thereof is different from that of embodiment 2 in that in step (1), PE-PEG (2000) -COOH is used instead of DSPE-PEG (2000) -COOH.
The brain glioma image nano probe solutions provided in examples 23-25 are respectively injected into a brain glioma in-situ nude mouse model from tail veins, near infrared fluorescence/photoacoustic bimodal brain glioma imaging is carried out, and the near infrared fluorescence image and the photoacoustic image of a nude mouse injected with the brain glioma image nano probe solution provided in example 2 are compared, and the results show that the near infrared fluorescence image and the photoacoustic image of the nude mouse injected with the brain glioma image nano probe provided in examples 23-25 are clear, the boundary of the brain glioma can be accurately identified, and the brain glioma image nano probe provided by the invention can also pass through a blood brain barrier by adopting different types of carboxylated polyethylene glycol modified liposome, actively target brain glioma cells, and realize tumor imaging in the microenvironment of the brain glioma, so that the boundary of the brain glioma is accurately identified, the diagnosis accuracy of the brain glioma is improved, the reference is provided for the operation excision of the brain glioma, and the brain glioma is helped to be excised cleanly.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (7)
1. The brain glioma image nano probe is characterized by comprising carboxylated modified liposome, a contrast agent and lactoferrin, wherein a hydrophobic layer of the carboxylated modified liposome encapsulates the contrast agent, a hydrophilic layer of the carboxylated modified liposome is connected with the lactoferrin through a chemical bond, and the contrast agent is a fluorescent photoacoustic bimodal contrast agent; the carboxylated modified liposome is a carboxylated polyethylene glycol modified liposome; in the carboxylated polyethylene glycol modified liposome, the molecular weight of polyethylene glycol is 1000-10000, and the carboxylated polyethylene glycol modified liposome is carboxylated polyethylene glycol modified distearoyl phosphatidylcholine;
the fluorescent photoacoustic bimodal contrast agent is selected from at least one of IR-780, IR-775, IR-797, IR-792, IR-806 or IR-808.
2. The method for preparing the brain glioma imaging nanoprobe according to claim 1, comprising the following steps:
(a) Adding the carboxylated modified liposome and the contrast agent into an organic solvent, uniformly mixing, and then adding into water, uniformly mixing, so that the contrast agent is coated in a hydrophobic layer of the carboxylated modified liposome, and obtaining a liposome probe;
(b) Uniformly mixing the liposome probe with a carboxyl activating agent, and then adding lactoferrin to uniformly mix to obtain the brain glioma image nano probe;
the organic solvent is a volatile organic solvent and is at least one of ethanol, chloroform, dichloromethane and acetone;
the carboxyl activating agent is N-hydroxysuccinimide and/or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide.
3. The method for preparing the brain glioma imaging nanoprobe according to claim 2, wherein the mass ratio of the carboxylated modified liposome to the lactoferrin is 1 (1-3).
4. The method for preparing a brain glioma imaging nanoprobe according to claim 2, wherein the mass ratio of the carboxylated modified liposome to the contrast agent is (1-10): 1.
5. The method of claim 4, wherein in step (a), the method further comprises purification of the liposome probe, wherein the purification comprises removal of the organic solvent and the non-entrapped contrast agent;
volatilizing and removing the organic solvent by adopting inert gas purging;
the non-entrapped contrast agent is removed by centrifugation.
6. The method of preparing brain glioma imaging nanoprobes according to claim 4, further comprising purification of brain glioma imaging nanoprobes in step (b), said purification comprising removal of carboxyl activator and unreacted lactoferrin;
the carboxyl activator and unreacted lactoferrin are removed by dialysis or ultrafiltration.
7. The use of a brain glioma imaging nanoprobe according to claim 1 for the preparation of a brain glioma diagnostic and/or therapeutic drug.
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