CN112546219B - Nanotherapeutic structures and methods of making the same - Google Patents

Nanotherapeutic structures and methods of making the same Download PDF

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CN112546219B
CN112546219B CN202011417807.7A CN202011417807A CN112546219B CN 112546219 B CN112546219 B CN 112546219B CN 202011417807 A CN202011417807 A CN 202011417807A CN 112546219 B CN112546219 B CN 112546219B
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nano
scintillator
dendrimer
nanotherapeutic
photosensitizer
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CN112546219A (en
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李万万
姜朝
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Shanghai Jiaotong University
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

The invention provides a nano therapeutic structure and a preparation method thereof, wherein the structure comprises the following components: the nano-scintillator, the nano-scintillator surface modification has dendrimer, the surface grafting photosensitizer of dendrimer, just inside being provided with the cavity that is used for loading treatment drug. The nano treatment structure has the capability of loading multiple medicines, and can be used as a universal template to build a cooperative treatment platform for the X-ray photodynamic therapy and other therapies, such as: the X-ray photodynamic therapy + radiotherapy, the X-ray photodynamic therapy + chemotherapy, the X-ray photodynamic therapy + immunotherapy, the X-ray photodynamic therapy + anti-angiogenesis therapy have extremely high clinical treatment benefit, and can realize the high-efficiency removal of deep tumors under the condition of low dose.

Description

Nanotherapeutic structures and methods of making the same
Technical Field
The invention relates to the technical field of nano materials, in particular to a nano treatment structure and a preparation method thereof.
Background
The development of effective therapies to combat cancer remains one of the primary tasks of modern clinical medicine. X-ray induced photodynamic therapy uses a nano scintillator to convert X-rays into ultraviolet or visible light, which then excites the photosensitizer and initiates the production of reactive oxygen species, which can kill cancer cells. X-ray induced photodynamic therapy has great potential for the treatment of deep cancers due to the extremely high penetration depth of X-rays in biological tissues.
Through the search of the prior art, the application numbers are as follows: cn201780031000.x, disclosing prodrugs of lipid moieties linked to a drug derivative, such as an anticancer drug derivative, via a linker comprising a disulfide group, and nanoparticles coated with a lipid layer comprising the prodrug, formulations comprising the nanoparticles and the use of the nanoparticles in methods of treating diseases, such as cancer, alone or in combination with other drug compounds, targeting agents and/or immunotherapeutic agents. However, this drug has the following drawbacks: (1) medical nano scintillators playing a role in transduction are often low in efficiency, have large toxic and side effects, cannot stably exist for a long time in a physiological environment, and keep the optical performance; (2) the low energy transfer efficiency between the nano-scintillator and the photosensitizer severely restricts the generation of active oxygen and finally reduces the therapeutic effect of the nano-platform on cancer; (3) the X-ray induced photodynamic therapy consumes oxygen in the tumor microenvironment, resulting in extreme hypoxia of the tumor which is already in a hypoxic state, and further inducing a series of events with poor prognosis, such as tumor angiogenesis, invasion and metastasis; (4) due to the aforementioned limitations, the X-ray induced photodynamic therapy reported so far requires the use of a large dose of radiation to produce a sufficient therapeutic effect. However, excessive radiation exposure is always accompanied by damage to normal tissue.
Currently, much work is focused mainly on the following two aspects. On one hand, the development of a new nano scintillator is realized, but the efficiency and the biological safety of the nano scintillator are not fully paid attention; another aspect is the proof-of-concept of X-ray induced photodynamic therapy in combination with other therapies, but the energy transfer of the nano-platform and the rational building between the multiple components on the nano-platform are generally neglected. Therefore, the treatment nano platform is systematically optimized, a nano platform which is efficient, stable, good in biocompatibility, high in energy transfer efficiency, capable of regulating and controlling the tumor microenvironment (immunity, hypoxia, angiogenesis and the like), capable of providing multi-mode cooperative treatment and capable of achieving efficient cancer elimination under the condition of low dose is developed, and the nano platform has great clinical benefit.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a nano therapeutic structure and a preparation method thereof.
In a first aspect, the present invention provides a nanotherapeutic structure comprising: the nano-scintillator, the nano-scintillator surface modification has dendrimer, the surface grafting photosensitizer of dendrimer, just inside being provided with the cavity that is used for loading treatment drug.
Optionally, the active ions in the nano-scintillator include: ce3+、Nd3+、Eu3+、Tb3+、Ho3+、Er3+、Tm3+、Yb2+、Yb3+。
Optionally, the nano-scintillator comprises: CaF2:Eu2+、CaF2:Ce3+、BaF2:Ce3+、LaF3:Tb3+、CeF3:Tb3 +、ZnO、TiO2、Y2O3:Eu3+、Lu2O3:Tb3+、Tb2O3、HfO2:Tb3+、Gd2O2S:Tb3+、LuBO3:Ce3+、SrAl2O4:Eu2+、YAlO3:Ce3+、LuAlO3:Ce3+、GdAlO3:Ce3+、YGaO3:Ce3+、LuGaO3:Ce3+、GdGaO3:Ce3+、Gd3Al2Ga3O12:Ce3+、Gd3Ga5O12:Ce3+、ZnGa2O4:Cr3+、Y2SiO5:Ce3+、Lu2SiO5:Ce3+、Gd2SiO5:Ce3+、CaWO4、ZnWO4、SrHfO3:Ce3+、BaHfO3:Ce3+、Bi4Ge3O12、Gd2(WO4)3:Tb3+、ZnS:Ag+。
Optionally, the dendrimer comprises: polyamidoamine dendrimers, polypropyleneimine dendrimers, peptide dendrimers, glycodendrimers.
Optionally, the dendrimer is less than 10nm in size.
Optionally, the photosensitizer comprises: chlorin e6, rose bengal, merocyanine 540, hematoporphyrin, protoporphyrin IX, zinc phthalocyanine tetrasulfonate.
Optionally, the therapeutic agent comprises: radiotherapy sensitizer, chemotherapy medicine, immunotherapy medicine and anti-angiogenesis medicine.
In a second aspect, the present invention provides a method of fabricating a nanotherapeutic structure for fabricating a nanotherapeutic structure according to any one of the first aspects, the method comprising:
step 1: synthesizing a nano scintillator, and constructing a functional group modification layer on the surface of the nano scintillator;
step 2: constructing a framework consisting of dendritic macromolecules on the surface of the scintillator;
and step 3: grafting a photosensitizer on the surface of the dendrimer;
and 4, step 4: therapeutic drugs are loaded in the cavities of the dendrimers.
Optionally, the step 1 includes: preparing a nano scintillator by any one of a hydrothermal method, a solvothermal method, a sol-gel method and a high-temperature thermal decomposition method; the nano-scintillator includes: CaF2:Eu2+、CaF2:Ce3+、BaF2:Ce3+、LaF3:Tb3+、CeF3:Tb3+、ZnO、TiO2、Y2O3:Eu3+、Lu2O3:Tb3+、Tb2O3、HfO2:Tb3+、Gd2O2S:Tb3+、LuBO3:Ce3+、SrAl2O4:Eu2+、YAlO3:Ce3+、LuAlO3:Ce3+、GdAlO3:Ce3+、YGaO3:Ce3+、LuGaO3:Ce3+、GdGaO3:Ce3+、Gd3Al2Ga3O12:Ce3+、Gd3Ga5O12:Ce3+、ZnGa2O4:Cr3+、Y2SiO5:Ce3+、Lu2SiO5:Ce3+、Gd2SiO5:Ce3+、CaWO4、ZnWO4、SrHfO3:Ce3+、BaHfO3:Ce3+、Bi4Ge3O12、Gd2(WO4)3:Tb3+、ZnS:Ag+。
Optionally, the active ions of the nano-scintillators are used in combination as required, and the active ions include: ce3+、Nd3+、Eu3+、Tb3+、Ho3+、Er3+、Tm3+、Yb2+、Yb3+。
Compared with the prior art, the invention has the following beneficial effects:
the nano treatment structure and the preparation method thereof provided by the invention have the capability of loading multiple medicines, and provide a universal template to build a cooperative treatment platform for the X-ray photodynamic therapy and other therapies, such as: x-ray photodynamic therapy + radiotherapy, X-ray photodynamic therapy + chemotherapy, X-ray photodynamic therapy + immunotherapy, X-ray photodynamic therapy + anti-angiogenesis therapy. The nano treatment structure has extremely high clinical treatment benefit, and can realize high-efficiency removal of deep tumors under the condition of low dose.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic diagram illustrating the fabrication of a nanotherapeutic structure according to an embodiment of the present invention;
FIG. 2 shows CaF synthesized in accordance with an embodiment of the present invention2:Tb3+Transmission electron microscopy images of the nano-scintillators;
FIG. 3 is a graph showing the emission spectrum of a nano-scintillator under X-ray excitation and the absorption spectrum of rose bengal, a photosensitizer selected for use in an embodiment of the present invention;
FIG. 4 is a Fourier transform infrared spectrum of an embodiment of the present invention (used to characterize dendrimer-modified polyethylene glycol and modify dendrimers onto the surface of a nano-scintillator);
FIG. 5 is a spectrum of UV-visible absorption (used to characterize the loading effect of the photosensitizer rose bengal and the anti-angiogenic drug sunitinib) in an example of the present invention;
FIG. 6 is a schematic diagram of the effect of the nanotherapeutic structures provided in the practice of the present invention in an in vitro assay;
fig. 7 is a schematic diagram of the anti-tumor effect of the nano-therapeutic structure in the in vivo test provided by the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.
Fig. 1 is a schematic diagram illustrating a preparation principle of a nano therapeutic structure according to an embodiment of the present invention, and as shown in fig. 1, a nano scintillator is first synthesized, then a dendrimer is modified on the surface of the nano scintillator, and a photosensitizer is grafted on the surface of the dendrimer; wherein, the inside of the dendrimer is provided with a cavity for loading therapeutic drugs.
In the embodiment, the required stable and efficient scintillator type is determined, the nano scintillator is synthesized, and the surface functional group modification is carried out on the nano scintillator; constructing a dendritic macromolecular framework on the surface of the nano scintillator through chemical bonds; selecting a photosensitizer with characteristic absorption matched with the scintillation body luminescence, and grafting the photosensitizer on the surface of the dendrimer; according to the clinical characteristics of the disease, a specific combined treatment strategy is designed, therapeutic drugs are selected, and the therapeutic drugs are loaded in the internal cavities of the dendrimers. The method specifically comprises the following steps:
step a: selection, synthesis and surface modification of the nano scintillator.
Step a 1: the nano-scintillator working under physiological conditions must have extremely high chemical stability and scintillation luminescence yield. CaF2:Eu2+、CaF2:Ce3+、BaF2:Ce3+、LaF3:Tb3+、CeF3:Tb3+、ZnO、TiO2、Y2O3:Eu3+、Lu2O3:Tb3+、Tb2O3、HfO2:Tb3+、Gd2O2S:Tb3+、LuBO3:Ce3+、SrAl2O4:Eu2+、YAlO3:Ce3+、LuAlO3:Ce3+、GdAlO3:Ce3 +、YGaO3:Ce3+、LuGaO3:Ce3+、GdGaO3:Ce3+、Gd3Al2Ga3O12:Ce3+、Gd3Ga5O12:Ce3+、ZnGa2O4:Cr3+、Y2SiO5:Ce3+、Lu2SiO5:Ce3+、Gd2SiO5:Ce3+、CaWO4、ZnWO4、SrHfO3:Ce3+、BaHfO3:Ce3+、Bi4Ge3O12、Gd2(WO4)3:Tb3+、ZnS:Ag+The traditional scintillators have higher chemical stability and scintillation luminous efficiency and better biocompatibility; meanwhile, the active ions in the scintillators can be changed according to actual needs (such as Ce)3+、Nd3+、Eu3+、Tb3+、Ho3+、Er3+、Tm3+、Yb2+、Yb3+) And the radiation luminescence with different wavelengths is obtained, and the range of the selectable medical nano-scintillator is further expanded.
Step a 2: the nano-scintillator is synthesized by a proper method, such as a hydrothermal method, a solvothermal method, a sol-gel method, a high-temperature thermal decomposition method and the like.
Illustratively, the particle size of the prepared nano-scintillator is 10-200 nm.
Step a 3: the synthesized nano-scintillator is subjected to surface functional group modification (usually amino or carboxyl). The modification of the surface functional group can be realized by coating silicon dioxide on the surface of the nano particle or modifying hydrophilic organic matter with the functional group. Optional hydrophilic organic substances includeDendrimers (polyamidoamine dendrimers, polypropyleneimine dendrimers, peptide dendrimers, glycodendrimers), polyethylene glycols (PEG-COOH, PEG-NH2、PEG-NHS、PEG-SH、DSPE-PEG-COOH、DSPE-PEG-NH2) Polylysine, chitosan, and the like.
Step b: and constructing a dendrimer framework on the surface of the nano scintillator.
Illustratively, the size of the dendrimer should be less than 10nm (ensuring fluorescence resonance energy transfer efficiency).
Step b 1: the dendrimers were first grafted with polyethylene glycol to improve their hydrophilicity. Commercial dendrimers have a variety of surface groups available for selection. If the surface of the nano scintillator is carboxyl, the dendritic macromolecule with the surface group being amino is adopted, and a part of amino on the surface of the dendritic macromolecule is connected with polyethylene glycol through covalent bonds. The molecular weight of the polyethylene glycol is 500-5000D.
Illustratively, the molecular weight of the polyethylene glycol used for grafting should be less than 5 kD. The proportion of polyethylene glycol grafted on the surface of the dendrimer is 10-30%.
Step b 2: the dendrimer is connected to the nano-scintillator surface through a covalent bond.
Step c: grafting photosensitizer on the surface of the dendrimer.
Specifically, a photosensitizer with functional groups is selected, which is attached to the surface of the dendrimer by covalent bonds. Common photosensitizers with high singlet oxygen quantum yield and functional groups include chlorin e6, rose Bengal, merocyanine 540, hematoporphyrin, protoporphyrin IX, zinc phthalocyanine tetrasulfonate, and the like.
Step d: the therapeutic drug is loaded in the inner cavity of the dendrimer.
Step d 1: according to the disease characteristics and clinical experience, the needed therapeutic drugs are selected pertinently, such as radiosensitizers (nitroimidazoles, nitroaromatics, nitroheterocycles and the like); chemotherapeutic drugs (paclitaxel, cisplatin, doxorubicin, irinotecan, hydroxycamptothecin, methotrexate, gemcitabine, semustine, cyclophosphamide, lomustine, carmustine, nimustine, etc.); immunotherapeutic drugs (CTLA-4 inhibitors, PD-1/PD-L1 inhibitors, etc.); anti-angiogenesis drugs (Sunitinib, Sorafenib, Regorafenib, Pazopanib, Lenvatinib, Cabozamtibib, Axitinib and the like) construct nano platforms with different synergistic treatment functions.
Step d 2: the therapeutic drug is loaded into the cavity inside the dendrimer.
It is noted that the therapeutic agent of choice should be a small molecule compound or a small size antibody. The solvent loaded by the drug in the last step is water, physiological saline, phosphate buffer solution or other medium with good biocompatibility.
The embodiment provides a universal strategy to realize the unique nano treatment structure, and the energy resonance transmission efficiency from the nano scintillator to the photosensitizer is extremely high, so that the excellent deep photodynamic treatment curative effect can be ensured. The unique nano treatment structure has the capability of multiple loading, so that a universal template is provided to build a cooperative treatment platform for the X-ray photodynamic therapy and other therapies, such as X-ray photodynamic therapy + radiotherapy, X-ray photodynamic therapy + chemotherapy, X-ray photodynamic therapy + immunotherapy, X-ray photodynamic therapy + anti-angiogenesis therapy. The unique nano treatment structure separates the loading of multiple drugs in the implementation step and the loading space, so that the drug failure caused by the possible mutual interference among various drugs is completely not considered, the application range of the drugs is greatly expanded, and the development process of a nano treatment platform is simplified.
Example 1: x-ray photodynamic therapy and chemotherapy
Lu of the embodiment of the invention2O3:Tb3+The construction of the nano scintillator dual-core-satellite structure nano platform comprises the following steps: step S1, synthesizing surface carboxyl modified Lu2O3:Tb3+A nano scintillator; s2, grafting polyethylene glycol on the surface of the dendrimer, and then modifying the surface of the nano scintillator; step S3, connecting the photosensitizer rose bengal to the surface of the dendrimer; step S4, loading chemotherapeutic drug adriamycin to dendrimerInside.
Specifically, the step S1 includes:
step S11: 0.5 mmol of lutetium acetylacetonate and 0.05 mmol of terbium acetylacetonate were weighed into a 50 mL flask, and 15 mL of oleic acid and 5 mL of oleylamine were added thereto, followed by stirring at 90 ℃ for 10 min.
Step S12: heating to 120 deg.C, and vacuumizing for 30 min.
Step S13: introducing nitrogen, heating to 280 ℃ and reacting for 60 min.
Step S14: after the reaction was completed, the reaction mixture was cooled to 50 ℃ and 40 mL of ethanol was added to precipitate the product, which was centrifuged at 10000 RPM for 5 min.
Step S15: the washing was repeated three times with n-hexane/ethanol (1: 3) and the final product was dispersed in chloroform (30 mg/mL).
Step S16: 5 mL of chloroform solution of the nanoparticles is taken, 150 uL of tritphobic propionic acid is dropped into the chloroform solution under the condition of vigorous stirring, and the solution is stirred for 2 hours. And after the reaction is finished, adding excessive ethanol, centrifuging at 10000 RPM for 5 min, collecting precipitate, washing for three times by using absolute ethyl alcohol and deionized water, and dispersing the final product in DMSO for later use.
Specifically, the step S2 includes:
step S21: 30 mg mPEG-NHS was weighed, 1 mL DMSO was added, and the mixture was sonicated for 10 min to allow sufficient dissolution.
Step S22: adding 0.0015 mmol of G3-PAMAM dendrimer into the solution, and reacting for 24 h on a shaker (10000 rpm) under the condition of keeping out of the light to obtain dendrimer/polyethylene glycol (DP).
Step S23: mixing 40 mg Lu2O3:Tb3+Dispersing the nano scintillator in 4 mL DMSO, performing ultrasonic treatment for 15 min to fully disperse the nano scintillator, adding 20 mg EDC and 20 mg NHS, and stirring for 15 min to activate carboxyl on the surface of the nano scintillator. This step is carried out under protection from light.
Step S24: and adding the DP solution into the NP solution, and stirring and reacting for 24 hours under the condition of keeping out of the sun to obtain the NP-DP.
Specifically, the step S3 includes:
step S31: 40 mg Rose Bengal was weighed, 1 mL DMSO was added, and dissolved well by sonication.
Step S32: 20 mg EDC and 20 mg NHS were added to the above solution and reacted for 15 min on a shaker (10000 rpm) in the absence of light to activate the carboxyl group of the photosensitizer.
Step S33: and adding the activated photosensitizer into the NP-DP solution under the condition of vigorous stirring, and stirring for 24 hours in the dark.
Step S34: after the reaction is finished, the reactant needs to be fully cleaned so as to remove the free photosensitizer remained in the dendritic macromolecule. Centrifuging at 12000 rpm for 6 min to obtain product, washing with deionized water and ethanol twice, and dispersing the final product (NP-DPP) in PBS.
Specifically, the step S4 includes:
step S41: 30 mg of DOX hydrochloride is dissolved in 2 mL of methanol, 60 uL of triethylamine is added to neutralize the hydrochloric acid, and the mixture is subjected to ultrasonic treatment for 5 min to obtain an orange-red clear solution.
Step S42: and (2) under the condition of violent stirring, dropwise adding the solution into a PBS (phosphate buffer solution) of NP-DPP (dipeptidyl peptidase), stirring for 24 hours in the dark under an open condition, volatilizing methanol, loading the medicine into the dendritic macromolecule, and finally obtaining the nano platform (NP-DPPD) with the binuclear-satellite structure.
Example 2: x-ray photodynamic therapy and anti-angiogenesis therapy
CaF of the embodiment of the invention2:Tb3+The construction of the nano scintillator dual-core-satellite structure nano platform comprises the following steps: step P1: synthesis of surface carboxyl modified CaF2:Tb3+A nano scintillator; step P2: grafting the surface polyethylene glycol on the dendritic macromolecule, and then modifying the surface of the nano scintillator; step P3: attaching the photosensitizer Rose Bengal to the dendrimer surface; step P4: the anti-angiogenic drug Sunitinib was loaded inside the dendrimer.
Specifically, the step P1 includes:
step P11: 0.5 mmol of calcium chloride, 0.05 mmol of terbium chloride and 0.5 mmol of sodium citrate are weighed, dissolved in 25 mL of deionized water, and fully stirred for 30 min to ensure that the citrate and the metal cation form stable combination.
Step P12: 3.0 mmol of ammonium fluoride is weighed and dissolved in 5 mL of deionized water, and the solution is fully dissolved by ultrasonic treatment for 5 minutes to obtain a clear and transparent solution.
Step P13: under vigorous stirring, the ammonium fluoride solution was added dropwise to the solution of step (1) and stirring was continued at room temperature for 30 min.
Step P14: the clear and transparent solution finally obtained is transferred into a 40 mL polytetrafluoroethylene lining and placed in a reaction kettle for treatment for 6 h at 140 ℃.
Step P15: after the reaction is finished, the reaction kettle is naturally cooled to room temperature. The reacted liquid was taken out, and two volumes of tetrahydrofuran were added thereto, followed by centrifugation at 12000 rmp for 6 min. The resulting precipitate was collected and washing with deionized water and tetrahydrofuran was continued three times (deionized water/tetrahydrofuran =1: 2).
Step P16: the final pellet (NP) from centrifugation was dispersed in DMSO for use.
Specifically, the step P2 includes:
step P21: 30 mg mPEG-NHS was weighed, 1 mL DMSO was added, and the mixture was sonicated for 10 min to allow sufficient dissolution.
Step P22: adding 0.0015 mmol of G3-PAMAM dendrimer into the solution, and reacting for 24 h on a shaker (10000 rpm) under the condition of keeping out of the light to obtain dendrimer/polyethylene glycol (DP).
Step P23: 40 mg of CaF2:Tb3+Dispersing the nano scintillator in 4 mL DMSO, performing ultrasonic treatment for 15 min to fully disperse the nano scintillator, adding 20 mg EDC and 20 mg NHS, and stirring for 15 min to activate carboxyl on the surface of the nano scintillator. This step is carried out under protection from light.
Step P24: and adding the DP solution into the NP solution, and stirring and reacting for 24 hours under the condition of keeping out of the sun to obtain the NP-DP.
Specifically, the step P3 includes:
step P31: 40 mg Rose Bengal was weighed, 1 mL DMSO was added, and dissolved well by sonication.
Step P32: 20 mg EDC and 20 mg NHS were added to the above solution and reacted for 15 min on a shaker (10000 rpm) in the absence of light to activate the carboxyl group of the photosensitizer.
Step P33: and adding the activated photosensitizer into the NP-DP solution under the condition of vigorous stirring, and stirring for 24 hours in the dark.
Step P34: after the reaction is finished, the reactant needs to be fully cleaned so as to remove the free photosensitizer remained in the dendritic macromolecule. Centrifuging at 12000 rpm for 6 min to obtain product, washing with deionized water and ethanol twice, and dispersing the final product (NP-DPP) in PBS.
Specifically, the step P4 includes:
step P41: 20 mg of Sunitinib was dissolved in 2 mL of methanol and sonicated for 5 min to give a yellow, clear solution.
Step P42: and (3) under the condition of violent stirring, dropwise adding the solution into a PBS (phosphate buffer solution) of NP-DPP (dipeptidyl peptidase), stirring for 24 hours in the open under the dark condition, volatilizing methanol, loading the medicine into the dendritic macromolecules, and finally obtaining the nano platform (NP-DPPS) with the binuclear-satellite structure.
Further, as shown in FIGS. 2 and 3, CaF prepared in this example2The nano-scintillator has small size, uniform distribution and strong green fluorescence emission under the X-ray excitation condition. The Fourier transform infrared spectroscopy in FIG. 4 demonstrates that polyethylene glycol grafted dendrimers successfully decorated the nano-scintillator surface. The UV-VIS absorption spectrum in FIG. 5 demonstrates that both photosensitizers and drugs were successfully loaded on NP-DP, resulting in a nano-platform with dual functions of X-ray photodynamic therapy and anti-angiogenic therapy. In vitro and in vivo experiments of fig. 6 and 7 show that the constructed nano platform has excellent anti-tumor effect.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Claims (6)

1. A nanotherapeutic structure, comprising: the surface of the nano scintillator is modified with a dendrimer, the surface of the dendrimer is grafted with a photosensitizer, and a cavity for loading therapeutic drugs is arranged inside the dendrimer;
the dendrimer comprises: a polyamidoamine dendrimer;
the nano-scintillator includes: lu (Lu)2O3:Tb3+。
2. The nanotherapeutic structure of claim 1, wherein said dendrimer is less than 10nm in size.
3. The nanotherapeutic structure of claim 1, wherein said photosensitizer comprises: chlorin e6, rose bengal, merocyanine 540, hematoporphyrin, protoporphyrin IX, zinc phthalocyanine tetrasulfonate.
4. The nanotherapeutic structure of any of claims 1-3, wherein said therapeutic agent comprises: radiotherapy sensitizer, chemotherapy medicine, immunotherapy medicine and anti-angiogenesis medicine.
5. A method of preparing a nanotherapeutic structure for use in preparing a nanotherapeutic structure according to any one of claims 1-4, said method comprising:
step 1: synthesizing a nano scintillator, and constructing a functional group modification layer on the surface of the nano scintillator;
step 2: constructing a framework consisting of dendritic macromolecules on the surface of the scintillator;
and step 3: grafting a photosensitizer on the surface of the dendrimer;
and 4, step 4: therapeutic drugs are loaded in the cavities of the dendrimers.
6. The method of claim 5The method for preparing a nanotherapeutic structure of (1), wherein said step 1 comprises: preparing a nano scintillator by any one of a hydrothermal method, a solvothermal method, a sol-gel method and a high-temperature thermal decomposition method; the nano-scintillator includes: lu (Lu)2O3:Tb3+。
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