CN114652843B - Medicament for treating ocular neovascularization, preparation method and application - Google Patents

Medicament for treating ocular neovascularization, preparation method and application Download PDF

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CN114652843B
CN114652843B CN202210072947.8A CN202210072947A CN114652843B CN 114652843 B CN114652843 B CN 114652843B CN 202210072947 A CN202210072947 A CN 202210072947A CN 114652843 B CN114652843 B CN 114652843B
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ocular neovascularization
dpa
indocyanine green
targeting
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CN114652843A (en
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楚成超
李炜
刘刚
于静雯
吴益明
代奇轩
叶锦法
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Xiamen University
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Xiamen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/54—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 compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • 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
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • 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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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
    • 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 medicament for treating ocular neovascularization and a preparation method thereof, wherein the medicament for treating ocular neovascularization comprises a targeting precursor molecule and microminiature nano particles; the targeting precursor molecules are formed by connecting two 4,4' -bipyridine-zinc (II) molecules and a new blood vessel targeting polypeptide by a benzene ring; the microminiature nanoparticle is a gold/platinum-indocyanine green nanocomposite. The targeted precursor molecules and the microminiature nano particles in the drug are self-assembled in the neovascular region to realize effective enrichment of the therapeutic drug in the neovascular region, and the microminiature nano particles in the unlabeled region are metabolized and discharged, so that the drug residue in normal tissues is reduced, and the safety is high.

Description

Medicament for treating ocular neovascularization, preparation method and application
Technical Field
The invention relates to a medicament for treating ocular neovascularization, a preparation method and application thereof, and belongs to the technical field of medicines.
Background
Neovasculature refers to the process by which a particular tissue, under internal lesions or external stimuli, sprouts from the vascular tree to form a new vascular tree, providing nutrients and oxygen to the tissue to promote metabolism. The balance of pro-angiogenic factors (VEGF, etc.) and anti-angiogenic factors in normal organisms does not lead to over-angiogenesis. However, when the eye is affected by a lesion such as dry eye, inflammation, diabetes, age-related macular degeneration, etc., is infected with bacteria, fungi, etc., or is traumatized, the balance is broken, resulting in the generation of new blood vessels in the eye. However, since the refractive tissue of the eye is a necessary condition for normal function of the eye, and the development of disease may be exacerbated by new blood vessels. For example, when the cornea is subjected to external stimuli such as trauma, infection, chemical injury, and certain diseases such as diabetes, age-related macular degeneration, etc., this physiological balance may be broken, resulting in the cornea developing new blood vessels (CNV). Corneal neovascularization has become one of the important risk factors for global blinding eye disease. In the treatment of CNV, anti-inflammatory and VEGF inhibitors are used, but they are limited by the inability to rapidly and effectively clear CNV. In addition, in recent years, the thermal radiation treatment of argon laser or Nd: YAG laser is also added to the treatment of CNV, but the traditional thermal radiation treatment may cause complications such as corneal hemorrhage, corneal thinning, iris atrophy and the like, and the clinical application safety is difficult to ensure. Aiming at the key scientific problems that the medicine enrichment is low, the clearance speed of new blood vessels is low, the effective monitoring of the treatment process can not be realized, and the like in CNV treatment, the establishment of an image-guided CNV accurate clearance medicine by combining with the design of medicine, pharmacy, materialogy, imaging and molecular biology is a problem to be solved.
Disclosure of Invention
The invention provides a medicament for treating ocular neovascularization, a preparation method and application, which can effectively solve the problems.
The invention is realized in the following way:
A drug for treating ocular neovascularization, comprising a targeting precursor molecule and ultra-small nano-particles; the targeting precursor molecules are formed by connecting two 4,4' -bipyridine-zinc (II) molecules and a new blood vessel targeting polypeptide by a benzene ring; the microminiature nanoparticle is a gold/platinum-indocyanine green nanocomposite.
As a further improvement, the neovascular targeting polypeptide is a 10 amino acid or less polypeptide.
As a further improvement, the neovascular targeting polypeptide is one of Arg-Gly-Asp(RGD)、cyclo(Arg-Gly-Asp-D-Tyr-Cys)(c(RGDyC))、Asn-Gly-Arg(NGR)、Cys-Gly-Lys-Arg-Lys、Ala-Pro-Arg-Pro-Gly.
As a further improvement, the gold/platinum-indocyanine green nanocomposite is prepared from chloroauric acid, chloroplatinic acid and indocyanine green in a one-step process.
As a further improvement, the molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green is 2:1-1:2.
As a further improvement, the size of the ultra-small nano-particles is 2 to 10nm.
The preparation method of the medicine for treating ocular neovascularization comprises the following steps:
s1, preparing the target precursor molecule;
s2, preparing the ultra-small nano particles.
As a further improvement, the step S1 specifically includes:
s11, preparing a dimer Bis (DPA) of the DPA by taking 4,4' -bipyridine (DPA) and tyramine as raw materials;
S12, adding Bis (DPA) and Sulfo-SMCC into DMSO, and vibrating until the components are uniformly dispersed for reaction for more than 2 hours to obtain a solution A; dispersing the neovascular targeting polypeptide into PBS with pH of 7.4, then adding Traut reagent, and reacting at 4 ℃ for more than 24 hours to obtain solution B;
s13, mixing the solution A and the solution B for reaction, then adding Zn (II) into the solution, stirring for reaction for more than 2 hours, and finally purifying to obtain the target precursor molecule.
As a further improvement, the molar ratio of Bis (DPA), sulfo-SMCC, neovascular targeting polypeptide and Zn (II) is 2-10:2-10:5-15.
As a further improvement, the step S2 specifically includes: and (3) adding chloroauric acid and chloroplatinic acid into water to obtain a solution C, adding indocyanine green into the water to obtain a solution D, and carrying out rapid mixing reaction on the solution C and the solution D to obtain the ultra-small nanoparticle.
As a further improvement, the molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green is 2:1-1:2.
The application of the medicament for treating ocular neovascularization in preparing medicaments for treating ocular neovascularization.
The beneficial effects of the invention are as follows:
According to the invention, the targeting precursor molecules are used for marking ocular neovascularization, and accurate and effective marking can be realized based on excellent permeability and targeting of small molecules; the ultra-small nano particles prepared by the method have excellent optical therapeutic performance and optical imaging performance, and have better tissue permeability, so that the ultra-small nano particles can effectively reach the new blood vessel; the targeted precursor molecules and the microminiature nano particles are self-assembled in the neovascular region to realize effective enrichment of the therapeutic drugs in the neovascular region, and the microminiature nano particles are metabolized and discharged in the unlabeled region so as to reduce the residues of the drugs in normal tissues.
The invention can effectively improve the safety of the treatment method based on the precise neovascular marker and the parallel eye angle irradiation form, provides a noninvasive, safe and long-acting strategy, and provides a research foundation for the design and research of clinical medicines.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic illustration of a preparation process of AuPt-ICG and an assembly process with a prodrug molecule according to an embodiment of the present invention; flow chart for the administration of a prodrug molecule (Bis (DPA-Zn) -RGD) and AuPt-ICG in the form of an eye drop.
Fig. 2 is a TEM image of AuPt-ICG, bis (DPA-Zn) -RGD and AuPt-ICG co-assembly provided by an embodiment of the present invention. (a) TEM images of AuPt-ICG; (B) Bis (DPA-Zn) -RGD and AuPt-ICG co-assembled TEM images.
Fig. 3 is a graph showing the temperature rise of each substance under laser irradiation according to the embodiment of the present invention. Temperature rise curves of water (a), ICG (b), auPt-ICG (c), bis (DPA-Zn) -RGD and AuPt-ICG assembly (d) under 808nm laser irradiation.
Fig. 4 is a plot of the slip lamp before and after optical treatment of a CNV model for a co-administered set of AuPt-ICG and DRUG-1/AuPt-ICG provided by an embodiment of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention. Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
In the description of the present invention, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.
The embodiment of the invention provides a drug for treating ocular neovascularization, which comprises a targeting precursor molecule and microminiature nano particles; the targeting precursor molecules are formed by connecting two 4,4' -bipyridine-zinc (II) molecules and a new blood vessel targeting polypeptide by a benzene ring; the microminiature nanoparticle is a gold/platinum-indocyanine green nanocomposite. The bipyridine-zinc (II) in the target precursor molecule can carry out chelation reaction with the sulfonate on the surface of the ultra-small nano particle so as to carry out assembly reaction. Large scale nanoparticles are obtained after assembly, near infrared fluorescence imaging and optical therapeutic properties are still maintained, and performance enhancement can be achieved.
The neovascular targeting polypeptide is capable of penetrating ocular biological barriers and targeting the neovascular aggregation in the focal area. The neovascular targeting polypeptide is a polypeptide with less than 10 amino acids, so that steric hindrance is not generated for precursor molecule assembly reaction, and the assembly of the AuPt-ICG nano particles can be induced. In the embodiment of the present invention, the neovascular targeting polypeptide is preferably one of Arg-Gly-Asp(RGD)、cyclo(Arg-Gly-Asp-D-Tyr-Cys)(c(RGDyC))(SEQ ID NO:1)、Asn-Gly-Arg(NGR)、Cys-Gly-Lys-Arg-Lys(SEQ ID NO:2)、Ala-Pro-Arg-Pro-Gly(SEQ ID NO:3).
The gold/platinum-indocyanine green nano-composite is prepared by a one-step method of chloroauric acid, chloroplatinic acid and indocyanine green. The molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green is 2:1-1:2. Preferably, the molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green (ICG) is 2:1, 1.5:1, 1:1, 1:1.5, 1:2. More preferably, the molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green is 1:1, and the molar ratio of chloroauric acid and chloroplatinic acid is 1:1. The scale and performance of the nano particles can be adjusted based on the change of Au, pt and ICG, and the effect of enabling the drug to pass through the biological barrier to reach the action area can be achieved.
The size of the ultra-small nano particles is 2-10 nm, the ultra-small nano particles can permeate the biological barrier of the eye to enter into the neovascular in the focus area, and the nano particles have excellent near infrared fluorescence imaging and optical treatment performances.
The embodiment of the invention also provides a preparation method of the medicament for treating ocular neovascularization, which comprises the following steps:
s1, preparing the target precursor molecule, specifically:
s11, preparing a dimer Bis (DPA) of the DPA by taking 4,4' -bipyridine (DPA) and tyramine as raw materials;
S12, adding Bis (DPA) and Sulfo-SMCC into DMSO, and vibrating until the components are uniformly dispersed for reaction for more than 2 hours to obtain a solution A; dispersing the neovascular targeting polypeptide into PBS with pH of 7.4, then adding Traut reagent, and reacting at 4 ℃ for more than 24 hours to obtain solution B;
S13, mixing the solution A and the solution B for reaction, then adding Zn (II) into the solution, stirring for reaction for more than 2 hours, and finally purifying to obtain the target precursor molecule. Zn (II) is zinc nitrate or zinc chloride.
As a further improvement, the molar ratio of Bis (DPA), sulfo-SMCC, neovascular targeting polypeptide and Zn (II) is 2-10:2-10:5-15.
S2, preparing the ultra-small nano particles, which specifically comprises the following steps: and (3) adding chloroauric acid and chloroplatinic acid into water to obtain a solution C, adding indocyanine green into the water to obtain a solution D, and carrying out rapid mixing reaction on the solution C and the solution D to obtain the ultra-small nanoparticle.
As a further improvement, the molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green is 2:1-1:2.
The embodiment of the invention also provides application of the medicament for treating ocular neovascularization in preparing medicaments for treating ocular neovascularization. The medicament for treating the ocular neovascularization can be prepared into eye drops, eye injection, ointment and other medicaments according to the needs and is used for treating diseases such as ocular neovascularization and the like.
The administration method of the drug for treating ocular neovascularization of the invention comprises the following steps: firstly, the targeting precursor molecules are administered, the targeting precursor molecules are enriched into neovasculature in a focus area under the action of the targeting peptide, and residual medicines are metabolically discharged from eyes; then, ultra-small nano particles are administrated, the nano particles can effectively permeate into a focus area neovascular based on the smaller scale of the nano particles, and the nano particles and precursor molecules are subjected to self-assembly to obtain large-scale nano particles so as to realize drug residues, and meanwhile, the residual nano particles are metabolically discharged from eyes; finally, the optical treatment is realized by irradiation with a 808nm laser. Wherein the administration route is one of eye drop and eye injection. The invention is preferably administered by eye drops, the dropping mode is as follows: dropwise adding the target precursor molecules for five times; after 6 hours, the ultra-small nano particles are dripped for five times. After the ultra-small nano particles are dripped for 12 hours, optical treatment is realized by using the parallel irradiation mode of the canthus.
Example 1
(1) First, bis (DPA) preparation: (a) Adding 4.6g (BoC) 2 O and 8.4g K 2CO3 into 2.74g tyramine, stirring and reacting for 4h to obtain a product a; (b) 3.7mL of DPA and 1.57mL of formaldehyde are taken, 1mL of 1M HCl solution is added after the mixture is uniformly mixed, 2.37g of product a is taken to be dispersed into 10mL of 1-propanol, the two solutions are mixed and stirred for 2 days, and the product b is obtained after purification; (c) 1g of the compound b was taken in 10mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and the mixture was stirred and reacted for 4 hours to obtain Bis (DPA) by purification.
(2) Followed by a coupling reaction of Bis (DPA) and RGD polypeptides: (a) 50 mu mol of Bis (DPA) and 50 mu mol of Sulfo-SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt) are added into 10mL of DMSO, and are vibrated to be uniformly dispersed and reacted for 2 hours to obtain a solution A; (b) 50 mu mol of polypeptide molecules are dispersed into 10mL of PBS (pH 7.4), 40 mu mol of Traut reagent (2-iminothiolane) is added, and the mixture is reacted for 24 hours at the temperature of 4 ℃ to obtain solution B; (c) The two preparation solutions were mixed and reacted at 4 ℃ for 24h; (d) Then, 100. Mu. Mol of zinc nitrate (Zn (II)) was added to the solution, and the reaction was stirred for 2 hours; (e) Finally, bis (DPA-Zn) -RGD is obtained by HPLC purification, and freeze-drying preservation is carried out.
(3) Firstly, 20 mu mol of chloroauric acid and 20 mu mol of chloroplatinic acid are added into 10mL of water to obtain a solution C; adding 40 mu mol of indocyanine green (ICG) into 10mL of water to obtain a solution D; the two solutions were mixed rapidly, the solution turned from green to brown rapidly, and reacted at 4℃for 6 hours, ultrafiltered using a10 kd ultrafilter tube, and the prepared AuPt-ICG-1 was stored at 4 ℃.
Example 2
(1) First, bis (DPA) preparation: (a) Adding 4.6g (BoC) 2 O and 8.4g K 2CO3 into 2.74g tyramine, stirring and reacting for 4h to obtain a product a; (b) 3.7mL of DPA and 1.57mL of formaldehyde are taken, 1mL of 1M HCl solution is added after the mixture is uniformly mixed, 2.37g of product a is taken to be dispersed into 10mL of 1-propanol, the two solutions are mixed and stirred for 2 days, and the product b is obtained after purification; (c) 1g of the compound b was taken in 10mL of methylene chloride, 4mL of trifluoroacetic acid was added thereto, and the mixture was stirred and reacted for 4 hours to obtain Bis (DPA) by purification.
(2) Followed by a coupling reaction of Bis (DPA) and NGR polypeptides: (a) 50 mu mol of Bis (DPA) and 50 mu mol of Sulfo-SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt) are added into 10mL of DMSO, and are vibrated to be uniformly dispersed and reacted for 2 hours to obtain a solution A; (b) 50 mu mol of polypeptide molecules are dispersed into 10mL of PBS (pH 7.4), 40 mu mol of Traut reagent (2-iminothiolane) is added, and the mixture is reacted for 24 hours at the temperature of 4 ℃ to obtain solution B; (c) The two preparation solutions were mixed and reacted at 4 ℃ for 24h; (d) Then, 100. Mu. Mol of zinc nitrate (Zn (II)) was added to the solution, and the reaction was stirred for 2 hours; (e) Finally, bis (DPA-Zn) -NGR was obtained by HPLC purification and freeze-dried for storage.
(3) Firstly, 20 mu mol of chloroauric acid and 10 mu mol of chloroplatinic acid are taken and added into 10mL of water to obtain solution C; adding 30 mu mol of indocyanine green (ICG) into 10mL of water to obtain a solution D; the two solutions were mixed rapidly, the solution turned from green to brown rapidly, and reacted at 4℃for 6 hours, ultrafiltered using a 10kd ultrafilter tube, and the prepared AuPt-ICG-2 was stored at 4 ℃.
Test examples
(1) The drugs for treating ocular neovascularization (including drug targeting precursor molecules and subminiature nanoparticles) prepared in example 1 were used for the treatment of CNV. Wherein Bis (DPA-Zn) -RGD (DRUG-1) was administered first followed by AuPt-ICG-1 (DRUG-2) during the course of treatment (FIG. 1). The method comprises the following specific steps: (a) Dropwise adding 10 mu L of target precursor molecules, and dropwise adding once every 30min for five times; (b) After 6h, the targeted precursor molecules are effectively enriched in the new blood vessels and excreted in the metabolism of other tissues; (c) And (3) dropwise adding 10 mu L of the ultra-small nano particles once every 30min, wherein the ultra-small nano particles effectively penetrate the cornea to reach a neovascular region, and the precursor molecules and the ultra-small nano particles are assembled in the neovascular region in a targeting way, so that the precursor molecules and the ultra-small nano particles are enriched in the neovascular region.
(2) After the ultra-small nano particles are dripped for 12h, optical treatment is realized by using a 808nm laser in the form of parallel eye angle irradiation (figure 1), and finally the treatment effect is evaluated, and the experimental results are shown in figures 2 to 4.
As shown in FIG. 2, auPt-ICG-1 was prepared as uniform nanoparticles in the present invention, and had a diameter of about 3 nm. In the presence of DRUG-1, the AuPt-ICG and DRUG-1 undergo a self-assembly reaction and form large scale nanoparticles. The smaller size of the AuPt-ICG provides a guarantee for penetrating the cornea to reach the new blood vessel.
As shown in fig. 3, the photo-thermal performance of the AuPt-ICG is significantly improved compared with that of the pure ICG, and the photo-thermal performance is further improved after the assembly with DRUG-1. Excellent photo-thermal performance can effectively remove the new blood vessel, and can reduce the dosage of the medicine and reduce the side effect of the medicine.
As shown in fig. 4, the treatment effect of CNV can be obtained by using AuPt-ICG alone, but the treatment effect of the combined administration strategy is more excellent, and the new blood vessel can be effectively removed.
In summary, the present invention proposes an optical treatment of enhanced ocular neovascularization using in vivo self-assembly strategies of the precursor molecules DRUG-1 and subminiature DRUG-2. (1) The penetration of cornea is realized by using the microminiature nano-drug, and the high-efficiency enrichment in the new blood vessel is realized under the action of the precursor molecule; (2) improved optical therapeutic performance after in vivo self-assembly; (3) The microminiature nano particles have no drug residue in normal tissues and have excellent biosafety. The invention provides a noninvasive, safe and long-acting strategy for ocular neovascularization treatment, and provides a research foundation for the design and research of clinical medicines.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Xiamen university
<120> A medicament for treating ocular neovascularization, preparation method and application
<130> 2022
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<170> PatentIn version 3.5
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Ala Pro Arg Pro Gly
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Claims (5)

1. A drug for treating ocular neovascularization, which is characterized by comprising a targeting precursor molecule and microminiature nano particles; the targeting precursor molecules are formed by connecting two 4,4' -bipyridine-zinc (II) molecules and a new blood vessel targeting polypeptide by a benzene ring; the ultra-small nano-particles are gold/platinum-indocyanine green nano-composites; the gold/platinum-indocyanine green nano-composite is prepared by chloroauric acid, chloroplatinic acid and indocyanine green by a one-step method; the molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green is 1:1, and the molar ratio of chloroauric acid to chloroplatinic acid is 1:1; the size of the ultra-small nano-particles is 3nm.
2. The drug for treating ocular neovascularization according to claim 1, characterized in that said neovascularization targeting polypeptide is a polypeptide of less than 10 amino acids.
3. A method of preparing a medicament for the treatment of ocular neovascularization as defined in any one of claims 1 to 2, comprising the steps of:
s1, preparing the target precursor molecule;
s2, preparing the ultra-small nano particles;
the step S1 specifically comprises the following steps:
s11, preparing a dimer Bis (DPA) of the DPA by taking 4,4' -bipyridine (DPA) and tyramine as raw materials;
S12, adding Bis (DPA) and Sulfo-SMCC into DMSO, and vibrating until the components are uniformly dispersed for reaction for more than 2 hours to obtain a solution A; dispersing the neovascular targeting polypeptide into PBS with pH of 7.4, then adding Traut reagent, and reacting at 4 ℃ for more than 24 hours to obtain solution B;
s13, mixing the solution A and the solution B for reaction, then adding Zn (II) into the solution, stirring for reaction for more than 2 hours, and finally purifying to obtain the target precursor molecule;
the step S2 specifically comprises the following steps: adding chloroauric acid and chloroplatinic acid into water to obtain a solution C, adding indocyanine green into the water to obtain a solution D, and carrying out rapid mixing reaction on the solution C and the solution D to obtain the ultra-small nano particles;
The molar ratio of the total amount of chloroauric acid and chloroplatinic acid to indocyanine green is 1:1, and the molar ratio of chloroauric acid and chloroplatinic acid is 1:1.
4. The method for preparing a drug for treating ocular neovascularization according to claim 3, wherein the molar ratio of Bis (DPA), sulfo-SMCC, neovascularization targeting polypeptide, zn (II) is 2-10:2-10:5-15.
5. Use of a medicament for the treatment of ocular neovascularization according to any one of claims 1 to 2 for the preparation of a medicament for the treatment of ocular neovascularization.
CN202210072947.8A 2022-01-21 2022-01-21 Medicament for treating ocular neovascularization, preparation method and application Active CN114652843B (en)

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