NL2032645B1 - Bone-level targeted-ultrasonic triggered drug delivery system, preparation method and application thereof - Google Patents

Bone-level targeted-ultrasonic triggered drug delivery system, preparation method and application thereof Download PDF

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NL2032645B1
NL2032645B1 NL2032645A NL2032645A NL2032645B1 NL 2032645 B1 NL2032645 B1 NL 2032645B1 NL 2032645 A NL2032645 A NL 2032645A NL 2032645 A NL2032645 A NL 2032645A NL 2032645 B1 NL2032645 B1 NL 2032645B1
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targeted
bone
liposome
delivery system
ultrasound
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Liu Fuwei
Lv Qianxin
Hou Yan
Chen Yicheng
Dai Taiqiang
Jin Dan
Cai Bulei
Wang Le
Xin He
Shi Qianwen
Gao Ye
Cao Feng
Kong Liang
Li Yunpeng
Zhang Kai
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Air Force Medical Univ
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    • A61K47/6911—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
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Abstract

Disclosed is a bone-level targeted-ultrasound triggered drug delivery system, which includes an external primary bone tissue targeted-ultrasound triggered liposome and an internal secondary osteoblast targeted drug-carrying liposome, wherein the internal secondary osteoblast targeted drug-carrying liposome is encapsulated in the hydrophilic core of the external primary bone tissue targeted-ultrasound triggered liposome. The drug delivery system of the invention is obtained by respectively preparing bone tissue targeted-ultrasonic triggered liposome and osteoblast targeted drug-carrying liposome, and then fusing them. The drug delivery system of the invention accurately and controllably releases the encapsulated drugs to the fracture and bone defect sites under the condition of osteoporosis (OP), improves the drug concentration and drug bioavailability of the bone injury sites, reduces the toxicity of normal tissues, and effectively promotes the healing of fracture and bone defect under OP conditions.

Description

BONE-LEVEL TARGETED-ULTRASONIC TRIGGERED DRUG DELIVERY SYSTEM,
PREPARATION METHOD AND APPLICATION THEREOF
TECHNICAL FIELD
The invention relates to the technical field of drug delivery systems, and in particular to a bone-level targeted-ultrasonic triggered drug delivery system, a preparation method and application thereof.
BACKGROUND
Osteoporosis (OP) is a systemic bone disease with abnormal bone metabolism. Abnormal metabolism leads to the decline of osteogenic ability of osteoblast (OB), bone loss, bone mass reduction, bone microstructure damage, bone trabecula reduction and bone thinning, and then leads to the increase of bone fragility and the most prone to fracture. After OP patients get fracture, due to the decrease osteogenic ability of OB at the fracture site, new bone formation and healing delay, the fracture even does not heal, and it is easy to be accompanied by bone defect.
At present, the clinical treatment methods are mainly surgical treatment and drug treatment. The surgical treatment mainly includes surgical fixation and bone defect repair with autogenous or allogeneic bone, bone repair bracket and bone cement transplantation, but all of them have certain shortcomings, such as: it is difficult to effectively fix the broken end with pressure; the interface bonding effect between bone and graft is poor; there are biological safety problems such as pathogens and immune rejection in allogeneic bone and artificial repair materials; drug treatment is mainly through the application of various drugs that promote bone formation or inhibit osteoclast, such as pro-bone formation drugs: teriparatide, raloxifene, alendronate sodium, risedronate sodium, ibandronate sodium, zoledronic acid, dinosemide, cathepsin K inhibitor, V-ATPase inhibitor, avB 3 integrin receptor antagonist, strontium ranelate and nucleic acid drugs that promote bone formation; among them, biopeptides and nucleic acid macromolecular drugs have poor physical and chemical stability, short half-life of blood circulation, and limited drug effect; these osteogenic therapeutic drugs lack specificity in the treatment of bone tissue or bone-related cells, and have potential toxicity to their normal organs and tissues, and that can easily lead to a variety of side effects. Moreover, because OP fractures and bone defects generally occur in local bone tissue, these drugs have no targeting and are distributed all over the body, and have little effect on promoting the bone healing of OP fractures and bone defects.
With the development of nanotechnology, a variety of artificial nanocarriers have been developed for drug delivery in vivo. Because human tissues contain a layered structure with nanometre scale (1-100 nm), the size of nanocarriers is close to that microstructure of human tissues. Controlling the size of nanocarriers makes it easy to penetrate the biological barrier in vivo, effectively improving the drug delivery efficiency in vivo. Compared with traditional drugs, these nanocarriers have the following advantages: increasing drug solubility, improving drug stability and blood circulation time, and changing the biological distribution of drugs, such as liposomes, polymers, polymeric peptides, calcium phosphate and metal nanoparticles.
Liposome is a kind of vesicle composed of phospholipid bilayer, and it is mainly composed of phospholipid and cholesterol. Liposome has the advantages of high entrapment efficiency, good biocompatibility, no immunogenicity and modifiability, and has been widely used for the in vivo delivery of many drugs. However, these artificial delivery systems lack targeting, and have problems of liver accumulation and clearance of reticular endothelial system (RES), resulting in low drug distribution concentration in bone tissue. By modifying PEG, bone tissue and OB targeting molecules on the surface of liposomes, bone targeting molecules include bisphosphonates, tetracycline, repeated polypeptide sequences Asp 8 and (DSS)s, and single- stranded small nucleic acid aptamer CH6 targeting OB, the drug bone tissue and OB effectiveness may be effectively improved. However, bone diseases related to fracture and bone defect caused by OP occur at the fracture and bone defect sites. The existing targeted bone tissues or OB molecules distribute the drugs in the whole body bones and OB, and that greatly reduces the bioavailability of the drugs at the injured sites, reduces the therapeutic effect, makes the drugs used in high doses, and also has potential systemic toxicity.
With the development of multi-disciplines, more and more researchers and medical workers pay attention to drug controlled release technology. Controlled drug delivery may be realized through external stimuli, such as temperature, pH, light, sound, electricity, magnetic field and other common external stimuli, and among them, ultrasound penetrates tissues and deep tissues non-invasively, and focused ultrasound accurately controls the action site. Research reports show that sonosensitizer responds to ultrasound and trigger for drug release. Low- frequency pulsed ultrasound promotes osteogenesis, and at the same time, ultrasound obtains structural images of tissues in the process of treatment, so as to diagnose the onset and treatment process, and that is an effective means to achieve accurate and integrated diagnosis and treatment. It is of great theoretical and practical significance to develop a suitable drug targeting and triggering system for OP fracture and bone defect, combined with external physical triggering means, for accurate and controllable treatment of OP bone-related diseases.
SUMMARY
The objectives of the present invention are to overcome the shortcomings of the prior art and provide a bone-level targeted-ultrasound triggered drug delivery system, a preparation method and application thereof.
First objective of the present invention is to provide a bone-level targeted-ultrasound triggered drug delivery system. which includes an external primary bone tissue targeted- ultrasound triggered liposome and an internal secondary osteoblast targeted drug-carrying liposome, wherein both the external primary bone tissue targeted-ultrasound triggered liposome and the internal secondary osteoblast targeted drug-carrying liposome are liposomes formed by amphiphilic phospholipid molecules and have a hydrophilic shell and a hydrophobic double- layer structure, and the internal secondary osteoblast targeted drug-carrying liposome is wrapped in the hydrophilic core of the external primary bone tissue targeted-ultrasound triggered liposome; the hydrophilic shells of the external primary bone tissue targeted-ultrasound triggered liposome and the internal secondary osteoblast targeted drug-carrying liposome are respectively connected with primary bone tissue targeted molecules and secondary osteoblast targeted molecules, the hydrophilic shell or hydrophobic double-layer of the external primary bone tissue targeted-ultrasonic triggered liposome is connected with sound sensitive molecules, and the hydrophilic core of the internal secondary osteoblast targeted drug-carrying liposome is loaded with osteogenic therapeutic drugs.
Optionally, the primary bone tissue targeted molecule is one of bisphosphate, tetracycline, aspartate hexarepeat (Asp)s, aspartate octarepeat (Asp)s and aspartate-serine-serine hexarepeat (DSS)s, and the secondary osteoblast targeting molecule is nucleic acid aptamer
CHS.
Optionally, the sonosensitizer molecule is one of porphyrin, rose red, chlorophyll and its derivatives, phthalocyanine and indocyanine green (ICG).
Optionally, the osteogenic therapeutic drug is one of teriparatide, raloxifene, bisphosphonates, dinosemide, cathepsin K inhibitor, V-ATPase inhibitor, avp3 integrin receptor antagonist, strontium ranelate, CKIP-1 siRNA, SOST siRNA, miRNA and overexpression plasmid.
Second objective of the present invention is to provide a preparation method of the bone- level targeted-ultrasound triggered drug delivery system, which includes the following steps: step 1: reacting primary bone tissue targeted molecule with cholesterol-polyethylene glycol- maleimide (Chol-PEG-Mal}, and freeze-drying after the reaction to obtain bone tissue targeting lipid; step 2: reacting secondary osteoblast targeted molecule with cholesterol-polyethylene glycol-maleimide (Chol-PEG-Mal), and freeze-drying after the reaction to obtain osteoblast targeted lipid; step 3: reacting cholesterol-polyethylene glycol-maleimide (Chol-PEG-Mal) with acoustic sensitive molecules at room temperature in the dark, and freeze-drying after the reaction to obtain acoustic sensitive lipid, step 4: mixing distearyl phosphatidylcholine (DSPC), cholesterol (Chol), distearyl phosphatidylacetamide-polyethylene glycol 2000 (DSPE-PEG-2000), bone tissue targeted lipid obtained in step 1 and acoustic sensitive lipid obtained in step 3 in a solvent, blow-drying with nitrogen, drying, hydrating, freezing, extruding, and freeze-drying with freeze-drying protective agent to obtain external primary bone tissue targeted-ultrasonic triggered liposome; step 5: mixing (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), dioleoyl phosphatidylcholine (DOPE), cholesterol (Chol), distearyl phosphatidylacetamide-polyethylene glycol 2000 (DSPE-
PEG-2000) and the osteoblast targeted lipid obtained in step 2 in a solvent, blow-drying with nitrogen, drying, hydrating, freezing, and extruding to obtain osteoblast targeted liposome, adding osteogenic therapeutic drugs, incubating at room temperature for 20-60 min, dialyzing to remove unencapsulated osteogenic therapeutic drugs, adding freeze-drying protective agent, and freeze-drying to obtain internal secondary osteoblast targeted drug-carrying liposome; step 6: mixing the external primary bone tissue targeted-ultrasonic triggered liposome obtained in step 4 with the internal secondary osteoblast targeted drug-carrying liposome obtained in step 5 at room temperature, adding magnesium chloride to incubate for 20-60 min, so that the internal secondary osteoblast targeted drug-carrying liposome is fused into the external primary bone tissue targeted-ultrasonic triggered liposome, and removing excess Mg?* to obtain the bone-level targeted-ultrasonic triggered drug delivery system.
Optionally, in step 1, the molar ratio of the primary bone tissue targeted molecule to cholesterol-polyethylene glycol-maleimide (Chol-PEG-Mal) is 1-3:1, the reaction solvent is N, N- dimethylformamide (DMF), and the reaction time is 24 h.
Optionally, in step 2, the molar ratio of the secondary osteoblast targeted molecule to cholesterol-polyethylene glycol-maleimide (Chol-PEG-Mal) is 1-3:1, the reaction solvent is N, N- dimethylformamide (DMF), and the reaction time is 24 h; in steps 1 and step 2, unreacted reactants are also removed.
Optionally, in step 3, the molar ratio of the acoustic sensitive molecule to cholesterol- polyethylene glycol-maleimide (Chol-PEG-Mal) is 1-3:1, the reaction solvent is N, N- dimethylformamide, and the reaction time is 24 h; in steps 3, unreacted reactants are also removed.
Optionally, in step 4, the molar ratio of distearyl phosphatidylcholine, cholesterol, distearyl phosphatidylacetamide-polyethylene glycol 2000, bone tissue targeted lipid obtained in step 1 and acoustic sensitive lipid obtained in step 3 is 40-60:30-40:5:1-5:1-5, and the solvent is chloroform; in step 5, the molar ratio of (2,3-dioleoyl-propyl)-trimethylamine, dioleoyl phosphatidylcholine, cholesterol, distearyl phosphatidylacetamide-polyethylene glycol 2000 and the osteoblast targeted lipid obtained in step 2 is 40-70:15:8-38:1-5:1-2, and the molar ratio of osteogenic therapeutic drugs to osteoblast targeted liposomes is 1:10, and the solvent is chloroform; in step 6, the molar ratio of the external primary bone tissue targeted-ultrasonic triggered liposome obtained in step 4 to the internal secondary osteoblast targeted drug- carrying liposome obtained in step 5 is 1:1, and the solvent is chloroform; in step 4 and step 5, the freeze-drying protective agent is one or at least two combinations of mannose, lactose, glucose, amino acids, sucrose and polyethylene glycol.
Third objective of the present invention is to provide an application of the above bone-level targeted-ultrasound triggered drug delivery system in the preparation of a preparation for treating bone injury.
Compared with the prior art, the invention has the following advantages: 5 (1) the bone level targeted-ultrasonic triggered type drug delivery system provided by the invention accurately targets bone in a level targeted mode, and the drug delivery system is efficiently enriched in bone tissue by using the first-level targeted bone tissue, and targets osteoblasts by the second-level, so as to effectively improve the stability of drugs and the efficiency of drugs entering osteoblasts; (2) the invention through external ultrasonic physical means, accurately controls the treatment time and treatment site, controls the trigger of acoustic sensitive molecules, and increases the permeability of external liposomes, so as to release internal liposomes. The secondary osteoblast targeted molecules on the surface of internal liposomes are used to accurately target osteoblasts, and the encapsulated drugs are effectively released to fracture and bone defect sites without affecting other normal bone tissues, thus improving the drug enrichment concentration and drug bioavailability of bone injury sites and reducing the toxicity of normal tissues.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 is a schematic structural diagram of a bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention.
Fig. 2 is a schematic diagram of the in vivo release of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention.
Fig. 3 is a particle size diagram of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention.
Fig. 4 is a Zeta potential diagram of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention.
Fig. 5 is a transmission electron microscope diagram of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention.
Fig. 6 is a diagram of the encapsulation efficiency of encapsulated drugs in the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention.
Fig. 7 is a stability diagram of encapsulated drugs in the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention.
Fig. 8 is an in vitro targeting performance diagram of a bone-level targeted-ultrasound triggered drug delivery system and a non-targeted group provided in Embodiment 1 of the present invention.
Fig. 8 (a) is an in vitro targeting performance diagram of the drug delivery system provided in Embodiment 1 of the present invention; and Fig. 8 (b} is an in vitro targeting performance of the non-targeted group.
Fig. 9 is an in vitro ultrasound response drug release performance diagram of the bone- level targeted-ultrasound triggered delivery system provided in Embodiment 1 of the present invention.
DESCRIPTION OF THE INVENTION
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein.
On the contrary, these embodiments are provided for a more thorough understanding of the present disclosure, and to fully convey the scope of the present disclosure to those skilled in the art
Embodiment 1
As shown in Fig. 1, the bone-level targeted-ultrasound triggered drug delivery system provided by the embodiment of the present invention includes an external primary bone tissue targeted-ultrasound triggered liposome and an internal secondary osteoblast targeted drug- carrying liposome, wherein both the external primary bone tissue targeted-ultrasound triggered liposome and the internal secondary osteoblast targeted drug-carrying liposome are liposomes formed by amphiphilic phospholipid molecules and have a hydrophilic shell and a hydrophobic double-layer structure, and the internal secondary osteoblast targeted drug-carrying liposome is wrapped in the hydrophilic core of the external primary bone tissue targeted-ultrasound triggered liposome; the hydrophilic shells of the external primary bone tissue targeted-ultrasound triggered liposome and the internal secondary osteoblast targeted drug-carrying liposome are respectively connected with primary bone tissue targeted molecules and secondary osteoblast targeted molecules, the hydrophilic shell or hydrophobic double-layer of the external primary bone tissue targeted-ultrasonic triggered liposome is connected with acoustic sensitive molecules, and the hydrophilic core of the internal secondary osteoblast targeted drug-carrying liposome is loaded with osteogenic therapeutic drugs.
In the embodiment of the invention, the primary bone tissue targeted molecule is aspartic acid-serine-serine 6 repeat sequence (DSS)es, the secondary osteoblast targeting molecule is nucleic acid fragment aptamer CH6, the acoustic sensitive molecule is indocyanine green (ICG), and the osteogenic therapeutic drug is CKIP-1 siRNA.
The embodiment of the invention also provides a preparation method of the bone-level targeted-ultrasonic triggered drug delivery system, which includes the following steps: step 1: dissolving 122.2 mg of Chol-PEG-Mal and 92.85 mg of (DSS)s in 10 ml of N, N- dimethylformamide (DMF), introducing argon, stirring at room temperature for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted
Chol-PEG-Mal by dialysis bag (molecular weight of dialysis bag is 3000 Da) after the complete reaction of (DSS), drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at -20°C to obtain the bone tissue targeted liposome Chol-PEG-(DSS)s; step 2: dissolving 122.2 mg of Chol-PEG-Mal and 458.75 mg of nucleic aptamer CH6 in 10 ml of N, N-dimethylformamide (DMF), introducing argon, stirring at room temperature for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted Chol-PEG-Mal by dialysis bag (molecular weight of dialysis bag is 10000
Da) after the complete reaction of nucleic aptamer CHB, drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at - 20°C to obtain the osteogenic targeted liposome Chol-PEG-CH6; step 3: dissolving 122.2 mg Chol-PEG-Mal and 38.748 mg ICG in 10 ml DMF, introducing argon, stirring at room temperature in the dark for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted Lysophospholipid -Mal by dialysis bag (molecular weight of dialysis bag is 3000 Da) after the complete reaction of ICG, drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at -20°C in the dark to obtain the acoustic sensitive liposome
Chol-PEG-ICG; step 4: dissolving 10.86 mg of DSPC, 3.48 mg of Chol, 3.51 mg of DSPE-PEG-2000, 2 mg of Chol-PEG-(DSS)s obtained in step 1 and 1.6 mg of Chol-PEG-ICG obtained in step 3 in chloroform, adding into a test tube, fixing the test tube on a nitrogen blow-drying device, turning on nitrogen until the air flow is stable and evenly blown out, blowing the nitrogen into the test tube, removing chloroform with the nitrogen blowing, and forming a uniform lipid film with no liquid at the bottom of the test tube; putting it into a vacuum drying tank, vacuum drying for 3-4 h, fully volatilizing the residual solvent, using 1 ml of 0.01M PBS with pH=7.2-7.4 to oscillate in a 50°C water bath, hydrating the lipid film in the test tube, oscillating for 2 min, and vortexing it on a vortex apparatus for 20 s (after 5 cycles); freezing the test tube with liquid nitrogen after the film on the test tube wall falls off, melting it quickly at 40°C and vortexing for 5 cycles, and squeezing the hydrated liposome liquid with an extruder, passing through 200 nm and 100 nm polycarbonate membranes in turn, cooling to room temperature after 10 cycles, adding mannose into the prepared liposome, putting it in a refrigerator at -80°C, pre-freezing overnight, and then freeze-drying it in a freeze dryer for 24 h to obtain external first-class bone tissue targeted-ultrasonic triggered liposome; step 5: dissolving 6.99 mg of DOTAP, 2.79 mg of DOPE, 3.67 mg of Chol, 3.51 mg of
DSPE-PEG-2000 and 8.17 mg of DSPE-PEG-CH6 obtained in step 2 in chloroform, adding into a test tube, fixing the test tube on a nitrogen blow-drying device, turning on nitrogen until the air flow is stable and evenly blown out, blowing the nitrogen into the test tube, removing chloroform with the nitrogen blowing, and forming a uniform lipid film with no liquid at the bottom of the test tube; putting it into a vacuum drying tank, vacuum drying for 3-4 h, fully volatilizing the residual solvent, using 1 ml of 0.01M PBS with pH=7.2-7.4 to oscillate in a 50°C water bath, hydrating the lipid film in the test tube, freezing the test tube with liquid nitrogen after the film on the test tube wall falls off, melting it quickly at 40°C and vortexing for 5 cycles, squeezing the hydrated liposome liquid with an extruder, passing through 200 nm, 100 nm and 50 nm polycarbonate membranes in turn, cooling to room temperature after 10 cycles, adding glucose into the prepared liposome, putting it in a refrigerator at -80°C, pre-freezing overnight, freeze-drying it in a freeze dryer for 24 h, adding an aqueous solution containing 25 nmol CKIP-1 siRNA after
DEPC treatment, incubating at room temperature for 20 min, dialysing and removing the excess
CKIP-1 siRNA not encapsulated in the liposome to obtain the internal secondary osteoblast targeted drug-loaded liposome; step 6: mixing the external primary bone tissue targeted-ultrasonic triggered liposome obtained in step 4 and the internal secondary osteoblast targeted drug-carrying liposome obtained in step 5 at room temperature according to the molar ratio of 1:1, adding MgCl for incubation for 20 min, so that the internal secondary osteoblast targeted drug-carrying liposome is fused into the external primary bone tissue targeted-ultrasonic triggered liposome, and dialysing and removing the excess Mg?* to obtain the bone-level targeted-ultrasonic triggered drug delivery system.
Embodiment 2
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 1 is different. Specific operation process of the step 1 of the embodiment of the present invention is as follows: step 1: dissolving 244.4 mg of Chol-PEG-Mal and 92.85 mg of (DSS)s in 10 ml of N, N- dimethylformamide (DMF), introducing argon, stirring at room temperature for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted
Chol-PEG-Mal by dialysis bag (molecular weight of dialysis bag is 3000 Da) after the complete reaction of (DSS), drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at -20°C to obtain the bone tissue targeted liposome Chol-PEG-(DSS)s.
Embodiment 3
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 1 is different. Specific operation process of the step 1 of the embodiment of the present invention is as follows: dissolving 366.6 mg of Chol-PEG-Mal and 92.85 mg of (DSS)s in 10 ml of N, N- dimethylformamide (DMF), introducing argon, stirring at room temperature for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted
Chol-PEG-Mal by dialysis bag (molecular weight of dialysis bag is 3000 Da) after the complete reaction of (DSS)s, drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at -20°C to obtain the bone tissue targeted liposome Chol-PEG-(DSS)s.
Embodiment 4
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 2 is different. Specific operation process of the step 2 of the embodiment of the present invention is as follows: dissolving 244.4 mg of Chol-PEG-Mal and 458.75 mg of nucleic aptamer CH6 in 10 ml of
N, N-dimethylformamide (DMF), introducing argon, stirring at room temperature for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted Chol-PEG-Mal by dialysis bag (molecular weight of dialysis bag is 10000
Da) after the complete reaction of nucleic aptamer CH, drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at - 20°C to obtain the osteogenic targeted liposome Chol-PEG-CHS.
Embodiment 5
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 2 is different. Specific operation process of the step 2 of the embodiment of the present invention is as follows: dissolving 366.6 mg of Chol-PEG-Mal and 458.75 mg of nucleic aptamer CH6 in 10 ml of
N, N-dimethylformamide (DMF), introducing argon, stirring at room temperature for 24 h,
monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted Chol-PEG-Mal by dialysis bag (molecular weight of dialysis bag is 10000
Da) after the complete reaction of nucleic aptamer CH, drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at - 20°C to obtain the osteogenic targeted liposome Chol-PEG-CH86.
Embodiment 6
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 3 is different. Specific operation process of the step 3 of the embodiment of the present invention is as follows: dissolving 244.4 mg Chol-PEG-Mal and 38.75 mg ICG in 10 ml DMF, introducing argon, stirring at room temperature in the dark for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted Lysophosphalipid-Mal by dialysis bag (molecular weight of dialysis bag is 3000 Da) after the complete reaction of ICG, drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at -20°C in the dark to obtain the acoustic sensitive liposome Chol-PEG-ICG.
Embodiment 7
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 3 is different. Specific operation process of the step 3 of the embodiment of the present invention is as follows: dissolving 366.6 mg Chol-PEG-Mal and 38.75 mg ICG in 10 ml DMF, introducing argon, stirring at room temperature in the dark for 24 h, monitoring reaction process by high performance liquid chromatography, dialysing and removing unreacted Lysophospholipid-Mal by dialysis bag (molecular weight of dialysis bag is 3000 Da) after the complete reaction of ICG, drying final product of the reaction by freeze dryer, detecting by nuclear magnetic resonance and infrared spectrum to further determine the synthesized product as target product, and storing sample in a refrigerator at -20°C in the dark to obtain the acoustic sensitive liposome
Chol-PEG-ICG.
Embodiment 8
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 4 is different. Specific operation process of the step 4 of the embodiment of the present invention is as follows: dissolving 10.86 mg of DSPC, 3.58 mg of Chol, 3.51 mg of DSPE-PEG-2000, 2 mg of Chol-
PEG-(DSS)s obtained in step 1 and 0.8 mg of Chol-PEG-ICG obtained in step 3 in chloroform, adding into a test tube, fixing the test tube on a nitrogen blow-drying device, turning on nitrogen until the air flow is stable and evenly blown out, blowing the nitrogen into the test tube, removing chloroform with the nitrogen blowing, and forming a uniform lipid film with no liquid at the bottom of the test tube; putting it into a vacuum drying tank, vacuum drying for 3-4 h, fully volatilizing the residual solvent, using 1 ml of 0.01M PBS with pH=7.2-7.4 to oscillate in a 50°C water bath, hydrating the lipid film in the test tube, oscillating for 2 min, and vortexing it on a vortex apparatus for 20 s (after 5 cycles); freezing the test tube with liquid nitrogen after the film on the test tube wall falls off, melting it quickly at 40°C and vortexing for 5 cycles, and squeezing the hydrated liposome liquid with an extruder, passing through 200 nm and 100 nm polycarbonate membranes in turn, cooling to room temperature after 10 cycles, adding mannose into the prepared liposome, putting it in a refrigerator at -80°C, pre-freezing overnight, and then freeze- drying it in a freeze dryer for 24 h to obtain external first-class bone tissue targeted-ultrasonic triggered liposome.
Embodiment 9
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 4 is different. Specific operation process of the step 4 of the embodiment of the present invention is as follows: dissolving 10.86 mg of DSPC, 3.29 mg of Chol, 3.51 mg of DSPE-PEG-2000, 2 mg of Chol-
PEG-(DSS)s obtained in step 1 and 3.2 mg of Chol-PEG-ICG obtained in step 3 in chloroform, adding into a test tube, fixing the test tube on a nitrogen blow-drying device, turning on nitrogen until the air flow is stable and evenly blown out, blowing the nitrogen into the test tube, removing chloroform with the nitrogen blowing, and forming a uniform lipid film with no liquid at the bottom of the test tube; putting it into a vacuum drying tank, vacuum drying for 3-4 h, fully volatilizing the residual solvent, using 1 ml of 0.01M PBS with pH=7.2-7.4 to oscillate in a 50°C water bath, hydrating the lipid film in the test tube, oscillating for 2 min, and vortexing it on a vortex apparatus for 20 s (after 5 cycles), freezing the test tube with liquid nitrogen after the film on the test tube wall falls off, melting it quickly at 40°C and vortexing for 5 cycles, and squeezing the hydrated liposome liquid with an extruder, passing through 200 nm and 100 nm polycarbonate membranes in turn, cooling to room temperature after 10 cycles, adding mannose into the prepared liposome, putting it in a refrigerator at -80°C, pre-freezing overnight, and then freeze- drying it in a freeze dryer for 24 h to obtain external first-class bone tissue targeted-ultrasonic triggered liposome.
Embodiment 10
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 5 is different. Specific operation process of the step 5 of the embodiment of the present invention is as follows: dissolving 8.74 mg of DOTAP, 2.79 mg of DOPE, 2.70 mg of Chol, 3.51 mg of DSPE-PEG- 2000 and 9.17 mg of DSPE-PEG-CH6 obtained in step 2 in chloroform, adding into a test tube, fixing the test tube on a nitrogen blow-drying device, turning on nitrogen until the air flow is stable and evenly blown out, blowing the nitrogen into the test tube, removing chloroform with the nitrogen blowing, and forming a uniform lipid film with no liquid at the bottom of the test tube; putting it into a vacuum drying tank, vacuum drying for 3-4 h, fully volatilizing the residual solvent, using 1 ml of 0.01M PBS with pH=7.2-7.4 to oscillate in a 50°C water bath, hydrating the lipid film in the test tube, freezing the test tube with liquid nitrogen after the film on the test tube wall falls off, melting it quickly at 40°C and vortexing for 5 cycles, squeezing the hydrated liposome liquid with an extruder, passing through 200 nm, 100 nm and 50 nm polycarbonate membranes in turn, cooling to room temperature after 10 cycles, adding glucose into the prepared liposome, putting it in a refrigerator at -80°C, pre-freezing overnight, freeze-drying it in a freeze dryer for 24 h, adding an aqueous solution containing CKIP-1 siRNA after DEPC treatment, incubating at room temperature for 40 min, and using dialysis method to remove the excess siRNA that is not encapsulated in the liposome to obtain the internal secondary osteoblast targeted drug-loaded liposome.
Embodiment 11
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 5 and step 6 are different. Specific operation process of the step 5 and step 6 of the embodiment of the present invention is as follows: step 5: dissolving 10.49 mg of DOTAP, 2.79 mg of DOPE, 1.74 mg of Chol, 3.51 mg of
DSPE-PEG-2000 and 9.17 mg of DSPE-PEG-CH6 obtained in step 2 in chloroform, adding into atest tube, fixing the test tube on a nitrogen blow-drying device, turning on nitrogen until the air flow is stable and evenly blown out, blowing the nitrogen into the test tube, removing chloroform with the nitrogen blowing, and forming a uniform lipid film with no liquid at the bottom of the test tube; putting it into a vacuum drying tank, vacuum drying for 3-4 h, fully volatilizing the residual solvent, using 1 ml of 0.01M PBS with pH=7.2-7.4 to oscillate in a 50°C water bath, hydrating the lipid film in the test tube, freezing the test tube with liquid nitrogen after the film on the test tube wall falls off, melting it quickly at 40°C and vortexing for 5 cycles, squeezing the hydrated liposome liquid with an extruder, passing through 200 nm, 100 nm and 50 nm polycarbonate membranes in turn, cooling to room temperature after 10 cycles, adding glucose into the prepared liposome, putting it in a refrigerator at -80°C, pre-freezing overnight, freeze-drying it in a freeze dryer for 24 h, adding an aqueous solution containing CKIP-1 siRNA after DEPC treatment, incubating at room temperature for 60 min, and using dialysis method to remove the excess siRNA that is not encapsulated in the liposome to obtain the internal secondary osteoblast targeted drug-loaded liposome; step 6: mixing the external primary bone tissue targeted-ultrasonic triggered liposome obtained in step 4 and the internal secondary osteoblast targeted drug-carrying liposome obtained in step 5 at room temperature according to the molar ratio of 1:1, adding MgCl; for incubation for 40 min, so that the internal secondary osteoblast targeted drug-carrying liposome is fused into the external primary bone tissue targeted-ultrasonic triggered liposome, and using dialysis method to remove the excess Mg?" to obtain the bone-level targeted-ultrasonic triggered drug delivery system.
Embodiment 12
The bone-level targeted-ultrasound triggered drug delivery system and its preparation method provided by the embodiment of the present invention are the same as those of the
Embodiment 1, except that the step 5 and step 6 are different. Specific operation process of the step 5 and step 6 of the embodiment of the present invention is as follows: step 5: dissolving 12.24 mg of DOTAP, 2.79 mg of DOPE, 0.77 mg of Chol, 3.51 mg of
DSPE-PEG-2000 and 9.17 mg of DSPE-PEG-CH6 obtained in step 2 in chloroform, adding into a test tube, fixing the test tube on a nitrogen blow-drying device, turning on nitrogen until the air flow is stable and evenly blown out, blowing the nitrogen into the test tube, removing chloroform with the nitrogen blowing, and forming a uniform lipid film with no liquid at the bottom of the test tube; putting it into a vacuum drying tank, vacuum drying for 3-4 h, fully volatilizing the residual solvent, using 1 ml of 0.01M PBS with pH=7.2-7.4 to oscillate in a 50°C water bath, hydrating the lipid film in the test tube, freezing the test tube with liquid nitrogen after the film on the test tube wall falls off, melting it quickly at 40°C and vortexing for 5 cycles, squeezing the hydrated liposome liquid with an extruder, passing through 200 nm, 100 nm and 50 nm polycarbonate membranes in turn, cooling to room temperature after 10 cycles, adding glucose into the prepared liposome, putting it in a refrigerator at -80°C, pre-freezing overnight, freeze-drying it in a freeze dryer for 24 h, adding an aqueous solution containing CKIP-1 siRNA after DEPC treatment, incubating at room temperature for 60 min, and using dialysis method to remove the excess siRNA that is not encapsulated in the liposome to obtain the internal secondary osteoblast targeted drug-loaded liposome; step 6: mixing the external primary bone tissue targeted-ultrasonic triggered liposome obtained in step 4 and the internal secondary osteoblast targeted drug-carrying liposome obtained in step 5 at room temperature according to the molar ratio of 1:1, adding MgCl for incubation for 80 min, so that the internal secondary osteoblast targeted drug-carrying liposome is fused into the external primary bone tissue targeted-ultrasonic triggered liposome, and using dialysis method to remove the excess Mg?* to obtain the bone-level targeted-ultrasonic triggered drug delivery system.
The performance of the bone-level targeted-ultrasound triggered drug delivery system provided by Embodiments 1-12 of the present invention is basically the same. Therefore, only the bone-level targeted-ultrasound-triggered drug delivery system provided in Embodiment 1 of the present invention is taken as an example to study its internal release principle and various properties.
Fig. 2 is a schematic diagram of the in vivo release of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention. As shown in
Fig. 2, the drug delivery system provided by Embodiment 1 of the present invention is injected in vivo by intravenous injection, and with blood circulation, the delivery system is distributed throughout the body after entering the blood. The delivery system firstly targets bone tissue with the aid of bone targeting molecules modified on external liposomes, and is enriched in the bone tissue. Local ultrasound is used to trigger the acoustic sensitive molecules on external liposomes according to the fracture site and bone defect site to be treated, so that the permeability of external liposomes is increased. Therefore, the internal liposome is released accurately and controllably, and the internal liposome is further enriched to osteoblasts by means of osteoblast targeting molecules, so that the carried osteogenic therapeutic drugs are efficiently delivered to osteoblasts, and the osteogenic ability of osteoblasts is improved. The precise control and hierarchical targeted treatment of fractures and bone injuries are realized, the drug utilization is effectively improved, the drugs are released accurately as needed, the toxic and side effects are reduced, and the treatment process is accelerated.
Fig. 3 is a particle size diagram of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention. It can be seen from Fig. 3 that the particle size distribution of the bone-level targeted-ultrasound triggered drug delivery system prepared in Embodiment 1 of the present invention is about 100 nm, and the particle size dispersion is uniform.
Fig. 4 is a Zeta potential diagram of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention. It can be seen from Fig. 4 that the Zeta potential of the bone-level targeted-ultrasound triggered drug delivery system prepared in Embodiment 1 of the present invention is positive, and its absolute value is greater than 30 mV, and that proves the prepared drug delivery system is stably dispersed in the solution and is not easy to aggregate and precipitate.
Fig. 5 is a transmission electron microscope diagram of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention. It can be seen from Fig. 5 that the prepared bone-level targeted-ultrasound triggered drug delivery system has a spherical structure, and the external primary bone tissue targeted-ultrasound triggered liposome wraps the internal secondary bone cell targeted drug-carrying liposome to form the delivery system together.
Fig. 6 is a diagram of the drug entrapment efficiency of the bone-level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention. The siRNA is incubated with internal lipid at room temperature, and the unencapsulated siRNA is removed by dialysis. The gene drug entrapment efficiency of the system is calculated. It can be seen from
Fig. 6 that the entrapment efficiency of the delivery system is as high as 75.67%.
Fig. 7 is a comparative diagram of stability of the encapsulated drug of the bone-level targeted-ultrasound triggered drug delivery system and the free siRNA provided in Embodiment 1 of the present invention. By simulating the in vivo conditions, the drug delivery system prepared in Embodiment 1 of the present invention and the free siRNA are respectively placed in PBS containing 50% foetal bovine serum, incubated at 37°C, the siRNA in the sample is extracted, and the remaining siRNA in the sample is detected by agarose gel electrophoresis. It can be seen from Fig. 7 that the stability of the drug delivery system prepared in Embodiment 1 of the present invention is significantly enhanced, and that indicates the drug delivery system prepared in Embodiment 1 of the present invention effectively protects the gene therapy drug from degradation.
Fig. 8 is an in vitro targeting performance diagram of the bone-level targeting-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention. The bone- level targeted-ultrasound triggered drug delivery system provided in Embodiment 1 of the present invention and the non-targeted group (liposome obtained by not adding targeted bone tissue or targeted osteoblast molecules in the liposome preparation process) are respectively labelled with membrane dye Dir, incubated with MC3T3 cells, then fixed overnight at 4°C with 4% formaldehyde, and photographed with laser confocal to detect osteoblast targeting performance. It can be seen from Fig. 8, compared with the non-targeted group, the bone-level targeted-ultrasound triggered drug delivery system prepared in Embodiment 1 of the present invention has enhanced fluorescence intensity in osteoblasts, and that indicates the delivery system has osteoblast targeting ability and effectively targets osteoblasts.
Fig. 9 is an in vitro ultrasound response drug release performance diagram of the bone- level targeted-ultrasound triggered delivery system provided in Embodiment 1 of the present invention. Specifically, the drug release performance test is as follow: placing the bone-level targeted-ultrasonic triggered drug delivery system provided in Embodiment 1 of the present invention under ultrasonic treatment, and selecting the treatment time for 1 min, 2 min, and 4 min, respectively; using a dialysis bag of 10000 Da for 2 h after treatment to remove the released calcein, detecting the fluorescence intensity content of the unreleased calcein in the delivery system in the dialysis bag, and calculating the drug release rate to evaluate the ultrasonic-triggered drug release performance of liposomes. It can be seen from Fig. 9, the drug release is realized by the delivery system under ultrasonic excitation, and with the extension of ultrasonic time, the drug release in the delivery system is larger, indicating that the controlled release of the drug is realized by ultrasonic.
In conclusion, in the bone-level targeted-ultrasonic triggered drug delivery system provided by the embodiment of the present invention, the bone-targeting molecules modified on the external liposomes firstly target bone tissues and enrich in the bone tissues, according to the fracture sites and bone injury sites to be treated, the local ultrasonic trigger is adopted to accurately control the release of the internal liposomes, and the internal liposomes are further enriched to osteoblasts by the osteoblast targeted molecules, so that the carried osteogenic therapeutic drugs are efficiently delivered to osteoblasts, the osteogenic ability of osteoblasts is improved, realizing the precise targeting and controllable drug release treatment of fractures and bone injuries, and it effectively improves the drug utilization, accurately releases the drugs as needed, reduces the toxic and side effects, and speeds up the treatment process.
Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims (10)

CONCLUSIESCONCLUSIONS 1. Een op botniveau gericht door ultrageluid geactiveerd geneesmiddelafgiftesysteem dat een uitwendig op primair botweefsel gericht door ultrageluid geactiveerd liposoom en een inwendig op secundaire osteoblasten gericht geneesmiddel bevattend liposoom omvat, waarbij — zowel het uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom als het inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom liposomen zijn die zijn gevormd door amfifiele fosfolipidemoleculen en die een hydrofiele omhulling en een hydrofobe dubbellaagse structuur hebben — het inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom is omhuld door de hydrofiele kern van het uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom; — de hydrofiele omhulsels van het uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom en inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom respectievelijk zijn verbonden met op primair botweefsel gerichte moleculen en op secundaire osteoblasten gerichte moleculen, — de hydrofiele omhulling of de hydrofobe dubbele laag van het uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom is verbonden met akoestisch gevoelige moleculen, en — de hydrofiele kern van inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom is geladen met osteogene therapeutische geneesmiddelen.A bone-level targeted ultrasound-activated drug delivery system comprising an external primary bone tissue-targeted ultrasound-activated liposome and an internal drug-containing liposome targeted at secondary osteoblasts, wherein — both the external primary bone tissue-targeted ultrasound-activated liposome and the internal secondary osteoblast-targeted drug-containing liposome are liposomes formed by amphiphilic phospholipid molecules and having a hydrophilic shell and a hydrophobic bilayer structure — the inner secondary osteoblast-targeted drug-containing liposome is enveloped by the hydrophilic core of the external ultrasound-targeted primary bone tissue activated liposome; — the hydrophilic shells of the outer primary bone targeting ultrasound activated liposome and inner drug-containing liposome targeting secondary osteoblasts are connected to primary bone targeting molecules and secondary osteoblast targeting molecules, respectively, — the hydrophilic shell or the hydrophobic bilayer of the external primary bone targeting ultrasonic activated liposome is associated with acoustically sensitive molecules, and the hydrophilic core of internal drug containing liposome targeting secondary osteoblasts is loaded with osteogenic therapeutic drugs. 2. Het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens conclusie 1, waarbij — het op primair botweefsel gerichte molecuul gekozen is uit de groep die bestaat uit bisfosfaat, tetracycline, een zesvoudige herhaling van aspartaat (Asp)s, een achtvoudige herhaling van aspartaat (Asp)s en een zesvoudige herhaling van aspartaat- serine-serine (DSS)s, en — het op secundaire osteoblasten gerichte molecuul het nucleinezuuraptameer CHS is.The bone-level targeted ultrasound-activated drug delivery system of claim 1, wherein - the primary bone targeting molecule is selected from the group consisting of bisphosphate, tetracycline, a six-fold repeat of aspartate (Asp)s, an eight-fold repeat of aspartate ( Asp)s and a six-fold repeat of aspartate-serine-serine (DSS)s, and - the molecule targeting secondary osteoblasts is the nucleic acid aptamer CHS. 3. Het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens conclusie 1, waarbij het akoestisch gevoelige molecuul gekozen is uit de groep die bestaat uit porfyrine, rozerood, chlorofyl en zijn derivaten, ftalocyanine en indocyaninegroen (ICG).The bone-level targeted ultrasound-activated drug delivery system of claim 1, wherein the acoustically sensitive molecule is selected from the group consisting of porphyrin, rose red, chlorophyll and its derivatives, phthalocyanine, and indocyanine green (ICG). 4. Het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens conclusie 1, waarbij het osteogene therapeutische geneesmiddel gekozen is uit de groep die bestaat uit teriparatide, raloxifeen, bisfosfonaten, dinosemide, kathepsine K- remmer, V-ATPase-remmer, avp3 integrinereceptorantagonist, strontium ranelaat, CKIP-1 siRNA, SOST siRNA, miRNA en overexpressie-plasmide.The bone-level targeted ultrasound-activated drug delivery system of claim 1, wherein the osteogenic therapeutic drug is selected from the group consisting of teriparatide, raloxifene, bisphosphonates, dinosemide, kathepsin K inhibitor, V-ATPase inhibitor, avp3 integrin receptor antagonist, strontium ranelate, CKIP-1 siRNA, SOST siRNA, miRNA and overexpression plasmid. 5. Een werkwijze voor het bereiden van het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens willekeurig welke van conclusies 1 -4, die de volgende stappen omvat: stap 1: het laten reageren van het op primair botweefsel gerichte molecuul met cholesterol- polyethyleenglycol-maleimide (Chol-PEG-Mal), en vriesdrogen na de reactie om op botweefsel gerichte lipide te verkrijgen; stap 2: het laten reageren van op secundaire osteoblasten gerichte molecuul met cholesterol-polyethyleenglycol-maleimide (Chol-PEG-Mal), en vriesdrogen na de reactie om het op osteoblast gerichte lipide te verkrijgen; stap 3: het laten reageren van cholesterol-polyethyleenglycol-maleimide (Chol-PEG-Mal) met akoestisch gevoelige moleculen bij kamertemperatuur in het donker, en vriesdrogen na de reactie om akoestisch gevoelige lipide te verkrijgen; stap 4: het in een oplosmiddel mengen van distearylfosfatidylcholine (DSPC), cholesterol (Chol), distearylfosfatidylacetamide-polyethyleenglycol 2000 (DSPE-PEG-2000), het in stap 1 verkregen op botweefsel gerichte lipide en het in stap 3 verkregen akoestisch gevoelige lipide, doorblazen met stikstof, drogen, hydrateren, invriezen, extruderen, en vriesdrogen met vriesdroogbeschermmiddel om het uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom te verkrijgen; stap 5: het in een oplosmiddel, mengen van (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), dioleoyl-fosfatidylcholine (DOPE), cholesterol (Chol), distearylfosfatidylacetamide- polyethyleenglycol 2000 (DSPE-PEG-2000) en de in stap 2 verkregen op osteoblast gerichte lipide, doorblazen met stikstof, drogen, hydrateren, invriezen, en extruderen om een op osteoblast gericht liposoom te verkrijgen, het toevoegen van osteogene therapeutische geneesmiddelen, het incuberen gedurende 20 - 60 minuten bij kamertemperatuur, dialyseren om niet-ingekapselde osteogene therapeutische geneesmiddelen te verwijderen, toevoegen van een vriesdroogbeschermingsmiddel en vriesdrogen om het inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom te verkrijgen; stap 6: het bij kamertemperatuur, mengen van het in stap 4 verkregen uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom met het in stap 5 verkregen inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom, het toevoegen van magnesiumchloride en incuberen gedurende 20 - 60 min zodat het inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom gefuseerd is met het uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom, en het verwijderen van de overmaat Mg?* om het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem te verkrijgen.A method of preparing the bone-level targeted ultrasound-activated drug delivery system according to any of claims 1-4, comprising the steps of: step 1: reacting the primary bone targeting molecule with cholesterol polyethylene glycol maleimide (Chol-PEG-Mal), and freeze-drying after the reaction to obtain bone tissue targeting lipid; step 2: reacting secondary osteoblast-targeting molecule with cholesterol-polyethylene glycol-maleimide (Chol-PEG-Mal), and freeze-drying after the reaction to obtain the osteoblast-targeting lipid; step 3: reacting cholesterol-polyethylene glycol-maleimide (Chol-PEG-Mal) with acoustically sensitive molecules at room temperature in the dark, and freeze-drying after the reaction to obtain acoustically sensitive lipid; step 4: mixing in a solvent distearyl phosphatidylcholine (DSPC), cholesterol (Chol), distearyl phosphatidylacetamide-polyethylene glycol 2000 (DSPE-PEG-2000), the bone targeting lipid obtained in step 1 and the acoustically sensitive lipid obtained in step 3, purging with nitrogen, drying, hydrating, freezing, extruding, and freeze-drying with freeze-drying protectant to obtain the external ultrasound-activated liposome targeting primary bone tissue; step 5: mixing (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), dioleoyl-phosphatidylcholine (DOPE), cholesterol (Chol), distearylphosphatidylacetamide-polyethylene glycol 2000 (DSPE-PEG-2000) and the osteoblast-targeted lipid obtained in step 2, purged with nitrogen, dried, hydrated, frozen, and extruded to obtain an osteoblast-targeted liposome, adding osteogenic therapeutic drugs, incubating for 20 - 60 minutes at room temperature, dialyzing free of charge removing encapsulated osteogenic therapeutic drugs, adding a freeze-drying protectant and freeze-drying to obtain the internal drug-containing liposome targeting secondary osteoblasts; step 6: mixing the external primary bone targeting ultrasound-activated liposome obtained in step 4 at room temperature with the internal drug-containing liposome obtained in step 5 targeting secondary osteoblasts, adding magnesium chloride and incubating for 20-60 min so that the internal secondary osteoblast-targeted drug-containing liposome is fused to the external primary bone tissue-targeted ultrasound-activated liposome, and removing the excess Mg 2 * to obtain the bone-level targeted ultrasound-activated drug delivery system. 6. De werkwijze voor het bereiden van het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens 5, waarbij in stap 1 de molaire verhouding van het op primair botweefsel gerichte molecuul ten opzichte van cholesterol-polyethyleenglycol- maleimide (Chol-PEG-Mal) 1-3: 1 is, het reactieoplosmiddel N, N-dimethylformamide (DMF) is, en de reactietijd 24 uur bedraagt.6. The method for preparing the bone-level targeted ultrasound-activated drug delivery system according to 5, wherein in step 1, the molar ratio of the primary bone tissue-targeted molecule to cholesterol-polyethylene glycol-maleimide (Chol-PEG-Mal) 1- 3:1, the reaction solvent is N,N-dimethylformamide (DMF), and the reaction time is 24 hours. 7. De werkwijze voor het bereiden van het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens 5, waarbij in stap 2 de molaire verhouding van het op secundaire osteoblasten gerichte molecuul ten opzichte van cholesterol-polyethyleenglycal- maleimide (Chol-PEG-Mal) 1-3: 1 is, het reactieoplosmiddel N, N-dimethylformamide (DMF) is, en de reactietijd 24 uur bedraagt; waarbij in stap 1 en stap 2 tevens de reactanten worden verwijderd die niet gereageerd hebben.7. The method for preparing the bone-level targeted ultrasound-activated drug delivery system according to 5, wherein in step 2, the molar ratio of the secondary osteoblast-targeting molecule to cholesterol-polyethyleneglycal-maleimide (Chol-PEG-Mal) 1- 3:1, the reaction solvent is N,N-dimethylformamide (DMF), and the reaction time is 24 hours; wherein in step 1 and step 2 also the reactants that have not reacted are removed. 8. De werkwijze voor het bereiden van het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens 5, waarbij in stap 3 de molaire verhouding van het akoestisch gevoelige molecuul ten opzichte van cholesterol-polyethyleenglycol-maleimide (Chol-PEG-Mal} 1-3: 1 is, het reactieoplosmiddel N,N-dimethylformamide is, en de reactietijd 24 uur bedraagt; waarbij in stap 3 ook de niet-gereageerde reactanten worden verwijderd.8. The method for preparing the bone level targeted ultrasound activated drug delivery system according to 5, wherein in step 3 the molar ratio of the acoustically sensitive molecule to cholesterol polyethylene glycol maleimide (Chol-PEG-Mal} 1-3 is: 1, the reaction solvent is N,N-dimethylformamide, and the reaction time is 24 hours, the unreacted reactants being also removed in step 3. 9. De werkwijze voor het bereiden van het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens 5, waarbij — in stap 4 de molaire verhouding van distearyl fosfatidylcholine, cholesterol, distearyl fosfatidylacetamide-polyethyleenglycol 2000, de in stap 1 verkregen op botweefsel gerichte lipide en de in stap 3 verkregen akoestisch gevoelige lipide 40 -60 : 30 -40:5 :1-5:1-5 bedraagt en het oplosmiddel chloroform is; — in stap 5 de molaire verhouding van (2,3-diocleoylpropyl)-trimethylamine, dioleoyl fosfatidylcholine, cholesterol, distearyl fosfatidylacetamide-polyethyleenglycol 2000 en de in stap 2 verkregen op osteoblast gerichte lipide 2:40-70:15:8-38:1-5 1-2 bedraagt en de molaire verhouding van osteogene therapeutische geneesmiddelen ten opzichte van op osteoblast gerichte liposomen 1 : 10 bedraagt en het oplosmiddel chloroform is;9. The method for preparing the bone-level targeted ultrasound-activated drug delivery system according to 5, wherein — in step 4, the molar ratio of distearyl phosphatidylcholine, cholesterol, distearyl phosphatidylacetamide-polyethylene glycol 2000, the bone tissue-targeted lipid obtained in step 1, and the acoustically sensitive lipid obtained in step 3 is 40 -60 : 30 -40:5 :1-5:1-5 and the solvent is chloroform; — in step 5, the molar ratio of (2,3-diocleoylpropyl)-trimethylamine, dioleoyl phosphatidylcholine, cholesterol, distearyl phosphatidylacetamide-polyethylene glycol 2000 and the osteoblast-targeted lipid obtained in step 2 2:40-70:15:8-38: 1-5 is 1-2 and the molar ratio of osteogenic therapeutic drugs to osteoblast-targeting liposomes is 1:10 and the solvent is chloroform; — in stap 6 de molaire verhouding van het in stap verkregen het uitwendige op primair botweefsel gerichte door ultrageluid geactiveerde liposoom ten opzichte van het in stap verkregen inwendige op secundaire osteoblasten gerichte geneesmiddel bevattende liposoom 1:1 bedraagt en het oplosmiddel is chloroform is; waarbij in stap 4 en stap 5 5 het vriesdroogbeschermingsmiddel gekozen is uit ten minste een of een combinatie van ten minste twee van mannose, lactose, glucose, aminozuren, sacharose en polyethyleenglycol is.- in step 6, the molar ratio of the external primary bone targeted drug-containing liposome obtained in step to the internal drug-containing liposome obtained in step is 1:1 and the solvent is chloroform; wherein in step 4 and step 5 the freeze-drying protectant is selected from at least one or a combination of at least two of mannose, lactose, glucose, amino acids, sucrose and polyethylene glycol. 10. Een toepassing van het op botniveau gerichte door ultrageluid geactiveerde geneesmiddelafgiftesysteem volgens willekeurig welke van conclusies 1 — 4 voor het bereiden van een preparaat voor de behandeling van botletsel.A use of the bone level targeted ultrasound activated drug delivery system according to any one of claims 1-4 for preparing a composition for the treatment of bone injury.
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