CN113368078A - Mitochondrion targeted SKQ-1 nano preparation, preparation method and medicine - Google Patents
Mitochondrion targeted SKQ-1 nano preparation, preparation method and medicine Download PDFInfo
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- 239000000243 solution Substances 0.000 claims description 8
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- NJDNXYGOVLYJHP-UHFFFAOYSA-L disodium;2-(3-oxido-6-oxoxanthen-9-yl)benzoate Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=CC(=O)C=C2OC2=CC([O-])=CC=C21 NJDNXYGOVLYJHP-UHFFFAOYSA-L 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- 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
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Abstract
The invention belongs to the field of pharmaceutics, and particularly relates to a mitochondrion targeted SKQ-1 nano preparation, a preparation method and a medicament. According to the invention, SKQ-1 is prepared into PEG-PGA-coated spherical nanoparticles, compared with non-coated free SKQ-1, the SKQ-1 nano preparation provided by the invention has stronger corneal epithelial cell protection effect and corneal epithelial cell mitochondria protection effect, and has better dry eye treatment effect.
Description
Technical Field
The invention belongs to the field of pharmaceutics, and particularly relates to a mitochondrion targeted SKQ-1 nano preparation, a preparation method and a medicament.
Background
Visomitin is also named as SKQ-1 and is a novel triphenylphosphine modified antioxidant. Like Mito Q, SKQ-1 can be targeted to accumulate in mitochondria through lipophilic cation (triphenylphosphine) in the molecular structure, thereby targeting and eliminating free radicals in mitochondria. Dry eye is the most common ocular surface disease in the clinical ophthalmology. Tear hyperostosis causes the accumulation of ocular surface cell free radicals, playing a critical role in the "vicious circle" of dry eye formation and development. The treatment of exogenous antioxidant is utilized to eliminate surplus free radicals, thus achieving better treatment effect. Mitochondria are the engine of cells and are also the major organelles that produce free radicals. Like Mito Q, SkQ-1, as a mitochondrially targeted antioxidant, is expected to be a major drug for dry eye treatment. Mito Q is a derivative of CoQ 10, CoQ 10 is an important coenzyme of the mitochondrial respiratory chain, and the excessive accumulation of Mito Q in mitochondria can cause the normal respiratory chain change of the mitochondria to generate side effects. SkQ-1 is a mitochondrion-targeted oxidative cardiolipin peroxidation inhibitor, and the prior literature reports show that the SkQ-1 has better biosafety than Mito Q. SkQ-1 can effectively relieve inflammatory reaction by relieving oxidative over-stimulation reaction in eye mitochondria, and improve the problems of eye tissue degeneration, tear quality reduction and the like. Currently, SKQ-1 eye drops are subjected to clinical three-phase experiments in the United states, preliminary data show that SkQ1 rapidly improves a series of symptoms and signs clinically relevant to dry eye (such as ocular discomfort and fluorescein staining), and drug tolerance comparable to that of artificial tears, and SkQ1 is expected to become an important potential treatment scheme for dry eye patients in the world.
As a mitochondrial targeting drug, SkQ-1 faces cell membrane barriers, mitochondrial membrane barriers, intracellular metabolic barriers, and the like during mitochondrial targeting delivery, reducing efficiency. Particularly, it is used as an eye drop formulation, and has a problem of low bioavailability due to structural barriers of the ocular surface, tear erosion, and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a mitochondrion targeted SKQ-1 nano preparation, a preparation method and a medicament.
The technical scheme adopted by the invention is as follows: a preparation method of a mitochondrion targeting SKQ-1 nanometer preparation comprises the following steps: dissolving PEG-PGA, mixing with SKQ-1 solution, performing ultrasonic treatment, dropwise adding the mixture into a water system solvent system under stirring to obtain a mixed solution, and then centrifuging and dialyzing to remove the non-entrapped SKQ-1 and the solvent to obtain a mitochondrion targeted SKQ-1 nano preparation;
the PEG-PGA is a block copolymer of PEG and poly benzyl glutamate formed by amino PEG-induced benzyl glutamate-N-carbonyl cyclic internal anhydride, and the copolymer is obtained by hydrolyzing and removing benzyl at the end of the poly benzyl glutamate under alkaline conditions.
Preferably, the conditions for centrifuging the mixed solution are as follows: centrifuging at 14000 rpm and 4 ℃ for 5 min and 2 times.
Preferably, the concentration of SKQ-1 in the mixed solution is 0.1 to 1.0 mM.
Preferably, the concentration of SKQ-1 in the mixed solution is 1.0 mM.
The mitochondrion targeting SKQ-1 nanometer preparation is prepared by the preparation method of the mitochondrion targeting SKQ-1 nanometer preparation.
A medicament for the treatment of dry eye comprising a mitochondrially-targeted SKQ-1 nanoformulation as described above.
Preferably, it is in the form of eye drops.
The invention has the following beneficial effects: according to the invention, SKQ-1 is prepared into PEG-PGA-coated spherical nanoparticles, compared with non-coated free SKQ-1, the SKQ-1 nano preparation provided by the invention has stronger corneal epithelial cell protection effect and corneal epithelial cell mitochondria protection effect, and has better dry eye treatment effect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.
FIG. 1 is a schematic flow chart of an embodiment of the present invention;
FIG. 2 shows PEG-PBG prepared according to an embodiment of the present invention1An H-NMR spectrum;
FIG. 3 shows PEG-PGA prepared according to an embodiment of the present invention1An H-NMR spectrum;
FIG. 4 shows the dispersion coefficient (PDI), antioxidant particle size, potential measured for SKQ-1 (Visimitin) in different amounts of self-assembled systems;
FIG. 5 shows the measured drug loading of SKQ-1 (Visemitin) in different amounts of self-assembled systems;
FIG. 6 is a scanning electron microscope image of an SKQ-1 nano-formulation prepared according to an embodiment of the present invention;
FIG. 7 is a graph of the effect of different treatment modalities on corneal epithelial cells;
FIG. 8 is a graph of the effect of different treatment modalities on the mitochondria of corneal epithelial cells;
in fig. 9, (a) is the effect of the same treatment regimen on benzalkonium chloride induced damage to the ocular surface in an animal model of dry eye; (b) change in fluorescein staining score results;
FIG. 10 is a graph of the effect of different treatment modalities on benzalkonium chloride induced tear secretion in a dry eye animal model;
figure 11 is a graph of the effect of different treatment modalities on benzalkonium chloride induced ocular surface inflammatory responses in an animal model of dry eye.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
The invention provides a mitochondrion targeted SKQ-1 nano preparation, and the preparation method comprises the following steps: dissolving PEG-PGA, mixing with SKQ-1 solution, performing ultrasonic treatment, dropwise adding the mixture into a water system solvent system under stirring to obtain a mixed solution, and then centrifuging and dialyzing to remove the non-entrapped SKQ-1 and the solvent to obtain a mitochondrion targeted SKQ-1 nano preparation;
wherein, the PEG-PGA (product 2) is obtained by amino PEG-induced benzyl glutamate-N-carbonyl cyclic internal anhydride to form a block copolymer of PEG and poly benzyl glutamate (product 1: PEG-PBG), and the copolymer is hydrolyzed under alkaline conditions to remove benzyl at the end of the poly benzyl glutamate.
Wherein, the molecular structure of SKQ-1 is as follows:
wherein, the structural formula of the product 1 is as follows:
wherein the structural formula of the product 2 is as follows:
example 1:
a preparation process of a mitochondrion targeting SKQ-1 nanometer preparation, as shown in figure 1, the specific process is as follows:
1: preparation of PEG-PGA amphoteric dissociation copolymer: 1.2 g of glutamic acid benzyl ester-N-carbonyl cyclic anhydride is dissolved in 20 mL of dry nitrogen dimethyl formamide and mixed with amino PEG to react for 48 hours at 38 ℃ under the protection of nitrogen. Dialyzing to obtain a block copolymer of PEG and poly benzyl glutamate, and removing benzyl under the action of NaOH to form PEG-PGA block copolymer.
As shown in FIGS. 2 to 3, this example succeeded in producing a block copolymer of PEG and poly benzyl glutamate (product 1) and a PEG-PGA block copolymer (product 2).
2. Preparing the antioxidant nanoparticles: weighing a certain amount of PEG-PGA, dissolving the PEG-PGA in DMSO, mixing the dissolved PEG-PGA with Visolitin solutions (solutions obtained by dissolving DMSO in a solvent) with different concentrations, carrying out ultrasonic treatment, and then dropwise adding the mixture into a water-based solvent while stirring to obtain the Visolitin NPs solution. Centrifugation is carried out for 2 times at 14000 rpm and 4 ℃ for 5 min, and the residual unencapsulated Visolimin and solvent are removed by dialysis. The indexes of the antioxidant such as the grain diameter, the electric potential, the dispersion coefficient and the like are measured by a laser particle sizer, and the morphology of the nano particles and the like are observed by a microscope.
As shown in FIG. 4, the particle size and PDI layer tended to decrease first and then increase as the content of SKQ-1 (Visemitin) in the system increased. When the content of SKQ-1 (Visimitin) is small, the PEG-PGA cannot be effectively self-assembled through charge interaction, and only a relatively loose structure is formed, so that the particle size is large, and PDI is widely distributed. With the increase of the content of SKQ-1 (Visomitin), the two are effectively self-assembled to form stable nanoparticles. The content of SKQ-1 (Visomitin) in the system is preferably 0.1-1.0 mM.
As shown in FIG. 5, the content of SKQ-1 (Visomitin) in the system is in the range of 0.1-1.0mM, and the drug loading of the nano system can be remarkably improved by increasing the content of SKQ-1 (Visomitin) in the self-assembly system, wherein the maximum drug loading can reach about 30%.
The following is data relating to nanoparticles prepared with a SKQ-1 (Visomitin) content in the range of 1.0 mM.
As shown in FIG. 6, the nanoparticles obtained in this example were spherical in shape and had a particle diameter of about 200 nm.
As shown in FIG. 7, the nano-formulation has a stronger corneal epithelial cell protective effect (against H) than the free drug2O2Damaged).
As shown in FIG. 8, the nano-formulation has a stronger mitochondrial protection effect (against H) of corneal epithelial cells than the free drug2O2Damaged).
3. Evaluation of Dry eye treatment Effect:
180-200g, female/male, no ocular infection and inflammation, no old leucoma, and 75 healthy SD rats with a sodium fluorescein staining score of less than 4 points (provided by the university of Wenzhou medical laboratory animal center, approved by the university of Wenzhou laboratory animal ethics Committee) were kept in normal environment. The illumination in the breeding environment is irradiated from the upper ceiling to the lower ground, and the illumination intensity is 18 lux. 12/12 h day/night cycle alternating light. The experimental group mice induced dry eye by 0.2% Bac by instillation 1 time each day in the morning and evening. Mice will exhibit similar ocular surface changes as humans do when dry eyes.
The experiments were divided into 5 groups, which were: 1) a normal control group placed in a normal environment without intervention; 2) placing in normal environment 0.2% Bac dissolved in NS for applying eyes 1 time in the morning and evening; 3) placing in a mixed solution of 0.2% Bac + PEG-PGA (equivalent to Visomitin-NPs) dissolved in NS in normal environment, and applying eyes 1 time in the morning and evening each day; 4) 0.2% Bac +5mM free Visolimin dissolved in NS in normal environment was applied to eyes 1 time each day in the morning and evening; 5) 0.2% Bac +5mM Visomitin-NPs in NS were placed in normal conditions and spotted 1 time each day in the morning and evening.
As shown in fig. 9, the nano-formulation was able to better inhibit benzalkonium chloride-induced ocular surface damage relative to the free drug.
As shown in fig. 10, the nano-formulation was able to better promote benzalkonium chloride to induce tear secretion in dry eye animal models relative to free drug.
As shown in fig. 11, the nano-formulation better promoted benzalkonium chloride-induced ocular surface inflammatory responses in dry eye animal models relative to free drug.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.
Claims (7)
1. A preparation method of a mitochondrion targeting SKQ-1 nanometer preparation is characterized by comprising the following steps: dissolving PEG-PGA, mixing with SKQ-1 solution, performing ultrasonic treatment, dropwise adding the mixture into a water system solvent system under stirring to obtain a mixed solution, and then centrifuging and dialyzing to remove the non-entrapped SKQ-1 and the solvent to obtain a mitochondrion targeted SKQ-1 nano preparation;
the PEG-PGA is a block copolymer of PEG and poly benzyl glutamate formed by amino PEG-induced benzyl glutamate-N-carbonyl cyclic internal anhydride, and the copolymer is obtained by hydrolyzing and removing benzyl at the end of the poly benzyl glutamate under alkaline conditions.
2. The method for preparing mitochondrion targeted SKQ-1 nanometer preparation as claimed in claim 1, wherein the method comprises the following steps: the conditions for centrifuging the mixed solution are as follows: centrifuging at 14000 rpm and 4 ℃ for 5 min and 2 times.
3. The method for preparing mitochondrion targeted SKQ-1 nanometer preparation as claimed in claim 1, wherein the method comprises the following steps: the concentration of SKQ-1 in the mixed solution is 0.1-1.0 mM.
4. The method for preparing mitochondrion targeted SKQ-1 nanometer preparation according to claim 3, which is characterized in that: the concentration of SKQ-1 in the mixed solution was 1.0 mM.
5. The mitochondrion targeting SKQ-1 nanometer preparation prepared by the method for preparing the mitochondrion targeting SKQ-1 nanometer preparation as claimed in any one of claims 1 to 4.
6. A medicament for the treatment of dry eye, characterized by: comprising the mitochondrially targeted SKQ-1 nanoformulation of claim 5.
7. The medicament for treating dry eye according to claim 6, wherein: it is in the form of eye drop.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108210932A (en) * | 2017-12-18 | 2018-06-29 | 温州医科大学 | A kind of preparation method of charge driving self-assembly chitosan base medicine-carried nano particles |
| CN109232216A (en) * | 2018-11-11 | 2019-01-18 | 苏州怡彼得生物技术有限公司 | A kind of synthetic method of bis- quaternary phosphonium salt derivative of Mitochondrially targeted antioxidant SKQ1 |
| CN109821020A (en) * | 2019-01-28 | 2019-05-31 | 温州医科大学 | A method of preparing the application and treatment xerophthalmia of dry eye drugs |
| CN110483764A (en) * | 2018-01-02 | 2019-11-22 | 温州医科大学 | Charge drives the anti-oxidant nanoparticle of self assembly and its application as fruit and vegetable fresh-keeping agent and lichee fresh-keeping |
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2021
- 2021-05-08 CN CN202110501100.2A patent/CN113368078A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108210932A (en) * | 2017-12-18 | 2018-06-29 | 温州医科大学 | A kind of preparation method of charge driving self-assembly chitosan base medicine-carried nano particles |
| CN110483764A (en) * | 2018-01-02 | 2019-11-22 | 温州医科大学 | Charge drives the anti-oxidant nanoparticle of self assembly and its application as fruit and vegetable fresh-keeping agent and lichee fresh-keeping |
| CN109232216A (en) * | 2018-11-11 | 2019-01-18 | 苏州怡彼得生物技术有限公司 | A kind of synthetic method of bis- quaternary phosphonium salt derivative of Mitochondrially targeted antioxidant SKQ1 |
| CN109821020A (en) * | 2019-01-28 | 2019-05-31 | 温州医科大学 | A method of preparing the application and treatment xerophthalmia of dry eye drugs |
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