CN114426554A - Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof - Google Patents

Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof Download PDF

Info

Publication number
CN114426554A
CN114426554A CN202210012796.7A CN202210012796A CN114426554A CN 114426554 A CN114426554 A CN 114426554A CN 202210012796 A CN202210012796 A CN 202210012796A CN 114426554 A CN114426554 A CN 114426554A
Authority
CN
China
Prior art keywords
organic fluorescent
drug
fluorescent small
compound
small molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210012796.7A
Other languages
Chinese (zh)
Inventor
洪学传
何胜楠
崔岩
田甜
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tibet University
Original Assignee
Tibet University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tibet University filed Critical Tibet University
Priority to CN202210012796.7A priority Critical patent/CN114426554A/en
Publication of CN114426554A publication Critical patent/CN114426554A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
    • C09K2211/1051—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with sulfur
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Materials Engineering (AREA)
  • Molecular Biology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention provides an organic fluorescent small molecular compound, an organic fluorescent nano-carrier, a preparation method and application thereof, wherein the structural formula of the organic fluorescent small molecular compound is shown as a formula 1:
Figure DDA0003459630470000011
the organic fluorescent small molecular compound is modified with polypeptide, protein, polyethylene glycol, aptamer or folic acid and derivatives thereof at the adjustable and controllable sites thereof to obtain a fluorophore which can be used as an anti-tumor drug carrier; the inventionCompared with free antitumor drugs, the antitumor drug has the characteristics of tumor targeting, high sustained release rate, good treatment effect, low cardiac toxicity and the like; the invention also provides a preparation method of the anti-tumor drug, which has the advantages of simple synthetic route, high reaction efficiency, high yield and higher industrial application prospect.

Description

Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedical materials, in particular to an organic fluorescent micromolecule compound, an organic fluorescent nano-carrier, and a preparation method and application thereof.
Background
Generally, the longer the circulation time in vivo, the more time the nanocarriers have to accumulate to the lesion site, thereby better performing their diagnostic or therapeutic effects. However, conventional nanocarriers in the prior art are still insufficient for long cycling performance. In recent years, the nanocarrier has been receiving wide attention because of its high curvature and small size, and has the effects of improving the distribution of the drug in vivo and having high permeability and retention effect (EPR effect) to tumor sites. Once the nanocarrier is applied in vivo, the long-circulating property becomes an important factor for its application.
The chemotherapy drugs widely used in clinic at present are widely distributed in vivo, especially in some normal tissues, so that the bioavailability of the drugs is reduced, and obvious toxic and side effects on the normal tissues are caused. In addition, the circulation time of the drug in the blood is too short to reach the focal site. Therefore, the key to improve the cancer treatment drug efficacy is to improve the selectivity of the chemotherapy drug to cancer cells, reduce the distribution of the chemotherapy drug in non-target positions and improve the drug utilization rate. For example, doxorubicin, a member of the anthracycline class of antibiotics, has a strong antitumor activity. However, the tissue distribution is poor, the toxic and side effects are large, and the drug effect exertion is influenced by multi-drug resistance and the like easily caused by long-term taking, so that the clinical application of the traditional Chinese medicine is severely limited by the adverse factors.
In summary, there is a need for a drug carrier with long-circulating properties that improves the availability of drugs, particularly tumor drugs.
Disclosure of Invention
The polymer micelle, as one of the nano drug-carrying systems, attracts attention in recent years, and shows obvious advantages compared with other carriers: (1) the critical micelle concentration is low, and the anti-blood dilution property is strong; (2) the particle size is small, the distribution range is narrow, and the passive targeting can be achieved by utilizing the ERP effect (high permeability and retention effect of solid tumors); (3) the core-shell structure is special, the hydrophilic shell can avoid the identification of RES (reticuloendothelial system) to realize long circulation, and hydrophobic drugs enter the core to increase the drug solubility and reduce the toxic and side effects; (4) the surface is provided with a modified functional ligand to achieve the active targeting effect. Based on this, studies of prodrug micelles have also emerged.
HLA4P @ DOX consists of a small molecule near-infrared two-region fluorophore and an anthraquinone antibiotic. And NIR-II imaging of continuous real-time tracking drug delivery and treatment in vivo, compared with free DOX, the method has the characteristics of high sustained release rate, good treatment effect, low cardiotoxicity and the like.
The present invention is directed to solving at least some of the problems of the prior art, and therefore, in a first aspect of the present invention, the present invention provides a use of an organic fluorescent small molecule compound for preparing a drug carrier, wherein the structural formula of the organic fluorescent small molecule compound is shown as formula 1:
Figure BDA0003459630450000021
wherein R is1One selected from S and Se, R0、R2Are each independently selected from O, S, Se and N-R11One of (1), R11One selected from the group consisting of H, methyl and ethyl; r3、R4、R5、R6Are each independently selected from
Figure BDA0003459630450000022
Figure BDA0003459630450000023
And H, n is an integer from 0 to 18, and m is an integer from 0 to 20;
R7、R8、R9、R10are each independently selected from
Figure BDA0003459630450000024
Figure BDA0003459630450000025
Figure BDA0003459630450000026
N is an integer of 0 to 18, m is an integer of 0 to 20, and X is selected from F, Cl, Br, I and N3One kind of (1).
In one or more embodiments of the present invention, the organic fluorescent small molecule compound has the following structural formula:
Figure BDA0003459630450000031
in one or more embodiments of the present invention, the application of the above-mentioned organic fluorescent small molecule compound in preparing a drug carrier includes: modifying polypeptide, protein, polyethylene glycol, aptamer or folic acid and derivatives thereof at the adjustable and controllable sites of the organic fluorescent small molecular compound to obtain a fluorophore, and taking the fluorophore as an anti-tumor drug carrier.
Preferably, the fluorophore is used as an anthraquinone antibiotic antitumor drug carrier.
Further, the anthraquinone antibiotic antitumor drug comprises one or more of adriamycin, daunorubicin, doxorubicin, epirubicin, idarubicin and mitoxantrone.
In a second aspect of the present invention, the present invention provides an anti-tumor drug, including the organic fluorescent small molecule compound described in the first aspect of the present invention or a fluorophore obtained by modifying polypeptide, protein, polyethylene glycol, aptamer, or folic acid and derivatives thereof at a controllable site of the organic fluorescent small molecule compound.
In one or more embodiments of the invention, the antineoplastic drug targets a tumor.
In a third aspect of the present invention, there is provided a method for producing an antitumor agent described in the second aspect of the present invention, comprising:
step 1): removing acid in the active ingredient salt of the anti-tumor drug to obtain free drugs, and adding fluorophores and the free drugs, which are obtained by modifying polypeptide, protein, polyethylene glycol, aptamer or folic acid and derivatives thereof at the adjustable and controllable sites of the organic fluorescent small molecular compound, into DMSO to obtain mixed solution;
step 2): adding deionized water or PBS into a container, then dripping the mixed solution obtained in the step 1) into the bottom of the container, and carrying out ultrasonic treatment to obtain a mixture;
step 3): dialyzing the mixture obtained in step 2) to remove the free drug which is not successfully entrapped.
In one or more embodiments of the present invention, in the step 1), the mass ratio of the free drug to the fluorophore in the mixed solution is 1: 5; preferably, the antitumor drug active ingredient is selected from one or more of daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone. Preferably, the anti-tumor drug active ingredient salt is doxorubicin hydrochloride.
In one or more embodiments of the present invention, in the step 2), the dropping speed of the mixed liquid into the container is 60 to 110 drops per minute; and (3) carrying out ultrasonic treatment by using an ultrasonic cleaning machine, wherein the ultrasonic power is controlled to be 230-250W, the working frequency is controlled to be 40KHz, and the ultrasonic time is controlled to be 5-20 seconds.
In one or more embodiments of the invention, in the step 3), the cut-off molecular weight of the dialysis bag used for dialysis is 10kDa, deionized water is used as an external liquid, the dialysis time is controlled to be 24-60 hours, and the external liquid is replaced more than 6 times.
In a fourth aspect of the present invention, the present invention provides an organic fluorescent nanocarrier, wherein the organic fluorescent nanocarrier comprises the organic fluorescent small molecule compound of the first aspect of the present invention or a fluorophore obtained by modifying polypeptide, protein, polyethylene glycol, aptamer, or folic acid and derivatives thereof at a controllable site of the organic fluorescent small molecule compound.
The invention has the beneficial effects that:
1. the invention provides an application of an organic fluorescent small molecular compound in preparation of a drug carrier, wherein the organic fluorescent small molecular compound modifies polypeptide, protein, polyethylene glycol, aptamer or folic acid and derivatives thereof at an adjustable site of the organic fluorescent small molecular compound to obtain a fluorophore which can be used as an anti-tumor drug carrier;
2. compared with free antitumor drugs, the antitumor drug provided by the invention has the characteristics of tumor targeting, high sustained release rate, good treatment effect, low cardiotoxicity and the like;
3. the preparation method of the anti-tumor drug provided by the invention has the advantages of simple synthetic route, high reaction efficiency, high yield and higher industrial application prospect;
4. the invention provides a drug carrier, and the anti-tumor drug has the characteristics of tumor targeting, high slow release rate, good treatment effect, low cardiac toxicity and the like under the entrapment of the drug carrier.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of HLA 4;
FIG. 2 is a nuclear magnetic carbon spectrum of HLA 4;
fig. 3 is a graph of the absorption and emission spectra of HLA 4;
FIG. 4 shows the preparation process of HLA4 compound converted into HLA4P probe for biological imaging;
FIG. 5 is a nuclear magnetic hydrogen spectrum representation of compound HLA 4P;
FIG. 6 is a transmission electron microscope image of a compound HLA4 linked to polyethylene glycol that can self-assemble to form nanoparticles;
FIG. 7 is a schematic representation of HLA4P @ DOX self-assembly prepared in example 2;
FIG. 8 is the cellular uptake imaging of confocal microscopy of example 3;
FIG. 9 is a TEM and DLS plot of HLA4P @ DOX prepared in example 2;
FIG. 10 is the biodistribution plots of DOX and HLA4P @ DOX at different time nodes after tail vein injection in example 5;
FIG. 11 is the biodistribution of different organs at different time nodes after tail vein injection of DOX in example 5;
FIG. 12 is a biodistribution plot of different organs at different time nodes after tail vein injection of HLA4P @ DOX in example 5;
FIG. 13 is a graph of the tumor size of mice administered various treatments of example 6;
FIG. 14 is a graph of near infrared two zone images of mice administered with various treatments in example 7;
FIG. 15 is a graph of H & E staining of different organs of mice given various treatments in example 8.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The following examples, in which specific conditions are not specified, were carried out under conventional conditions or conditions recommended by the manufacturer, by using conventional methods known in the art unless otherwise specified, and by using consumables and reagents which were commercially available unless otherwise specified. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
Example 1
The structural formula of the organic fluorescent micromolecule compound is shown as formula 1:
Figure BDA0003459630450000051
wherein Y, Z are independently selected from O, S, Se and N-R9One of (1), R9One selected from the group consisting of H, methyl and ethyl; r1、R2、R3、R4Are each independently selected from
Figure BDA0003459630450000052
Figure BDA0003459630450000053
And H, n is an integer from 0 to 18, and m is an integer from 0 to 20;
R5、R6、R7、R8are each independently selected from
Figure BDA0003459630450000054
Figure BDA0003459630450000055
Figure BDA0003459630450000061
N is an integer of 0 to 18, m is an integer of 0 to 20, and X is selected from F, Cl, Br, I and N3To (3) is provided.
The preparation route of the organic fluorescent small molecule compound (the compound shown in the formula 1) is shown as follows:
Figure BDA0003459630450000062
the following experimental group 1 illustrates the preparation of an organic fluorescent small molecule compound (compound represented by formula 1) by using compound HLA4 as an example.
Experimental group 1: preparation of compound HLA4
Step 1): preparation of compound 3 a:
taking the compound 2a (2g, 5.9mmol), zinc powder (13.8g, 212.4mmol) and ammonium chloride (18.8g, 354mmol), adding the mixture into a 500mL round-bottom flask, adding 100mL of methanol-water (v/v, 9:1) and 100mL of dichloromethane under the protection of argon, introducing argon into the reaction solution, bubbling for 5min to remove oxygen in the system, and reacting at room temperature for 2 hours under the protection of argon. After completion of the reaction, the reaction mixture was cooled to room temperature, methanol was removed by rotary evaporation, and the residue was redissolved in 150mL of dichloromethane, washed with water (30 mL. times.3) three times, and washed with saturated brine (30 mL. times.3) three times. The organic phase was dried over anhydrous magnesium sulfate for 3 hours, filtered, and the filtrate was spin-dried to give an intermediate. The intermediate, N-sulfinanilide (2.47g, 17.8mmol) and trimethylchlorosilane (2.57g, 23.7mmol) were added into a 50mL round-bottom flask, 20mL pyridine was added under the protection of argon, argon was introduced into the reaction solution to bubble for 5min to remove oxygen in the system, and the reaction was carried out at room temperature for 2 hours under the protection of argon. After completion of the reaction, the reaction mixture was cooled to room temperature, pyridine was removed by rotary evaporation, and the residue was redissolved in 150mL of dichloromethane, washed with water (30 mL. times.3) three times, and washed with saturated brine (30 mL. times.3) three times. The organic phase was dried over anhydrous magnesium sulfate for 3 hours, filtered, and the filtrate was spin-dried to give 1.62g of compound 3a, yield: and 90 percent.
The data for the structural determination of compound 3a are as follows:
1H NMR(400MHz,CDCl3)δ7.43(s,1H),6.93(s,1H),2.65(t,J＝7.7Hz,2H),1.93–1.62(m,2H),1.46–1.14(m,19H),0.90(t,J＝6.8Hz,3H).13C NMR(101MHz,CDCl3)δ156.18,144.66,134.65,125.64,120.24,112.46,31.94,30.49,30.43,29.69,29.62,29.48,29.38,29.35,22.71,14.14.
step 2): preparation of compound 4 a:
the blue compound 3a (840mg,1.31mmol) and N-bromosuccinimide (NBS) (780mg, 3.93mmol) were taken and charged into a 50mL round-bottomed flask, 20mL pyridine was added under the protection of argon, argon was introduced into the reaction solution and bubbled for 5min to remove oxygen in the system, and the reaction was carried out at room temperature for 2 hours under the protection of argon. After completion of the reaction, the reaction mixture was cooled to room temperature, pyridine was removed by rotary evaporation, and the residue was redissolved in 150mL of dichloromethane, washed with water (30 mL. times.3) three times, and washed with saturated brine (30 mL. times.3) three times. The organic phase was dried over anhydrous magnesium sulfate for 3 hours, filtered and the filtrate was spin-dried to give 953mg of compound 3 a. Yield: 91 percent.
The data for the structural determination of compound 4a are as follows:
HRMS(ESI)Calcd for:C52H41N6O8S4+([M+H]+):800.8769,found:800.8743.
step 3): preparation of compound 6 a:
taking the compound 4a (720mg, 0.904mmol), fifteen percent by mass of sodium bicarbonate, the compound 5a (1.23g, 2.26mmol) and tetratriphenylphosphine palladium (10mg, 0.008mmol), adding the mixture into a 50mL round-bottom flask, adding 20mL of tetrahydrofuran under the protection of argon, introducing argon into the reaction liquid, bubbling for 5min to remove oxygen in the system, and reacting at room temperature for 2 hours under the protection of argon. After completion of the reaction, the reaction mixture was cooled to room temperature, the tetrahydrofuran was removed by rotary evaporation, and the residue was redissolved in 150mL of dichloromethane, washed three times with water (30 mL. times.3), and washed three times with saturated brine (30 mL. times.3). The organic phase was dried over anhydrous magnesium sulfate for 3 hours, filtered, and the filtrate was spin-dried to give 1.03g of 6a, yield: 80 percent.
The data for the structural determination of compound 6a are as follows:1H NMR(400MHz,CDCl3)δ7.47(s,2H),7.36(d,J＝8.5Hz,4H),7.33–7.28(m,5H),7.16(d,J＝9.7Hz,8H),7.08(dd,J＝15.1,7.9Hz,10H),4.22(dd,J＝10.8,6.1Hz,4H),2.96(t,J＝7.8Hz,4H),2.71(dd,J＝16.0,8.0Hz,4H),2.65(t,J＝7.8Hz,4H),1.71(dt,J＝15.1,7.6Hz,4H),1.38–1.22(m,36H),1.06–0.98(m,4H),0.90(t,J＝6.7Hz,6H),0.08(s,18H).13C NMR(101MHz,CDCl3)δ173.11,147.49,145.59,135.77,129.63,129.33,129.27,125.07,124.61,123.14,122.64,62.70,36.11,31.94,30.96,30.42,29.72,29.70,29.67,29.64,29.60,29.50,29.38,28.99,22.71,17.35,14.15,-1.44.
MALDI-TOF-MS Calcd for:C74H80N6O4S4([M+H]+):1474.51,found:1474.9806.
step 4): preparation of compound HLA 4:
taking the compound 6a (100mg, 0.068mmol) and trifluoroacetic acid (5mL), adding the compound into a 50mL round-bottom flask, adding 20mL dichloromethane under the protection of argon, introducing argon into the reaction liquid, bubbling for 5min to remove oxygen in the system, and reacting at room temperature for 2 hours under the protection of argon. After the reaction was completed, the reaction mixture was cooled to room temperature, and methylene chloride was removed by rotary evaporation to obtain 85mg of HLA 4. Yield: 98 percent.
The structural determination data of the compound HLA4 are as follows:
MALDI-TOF-MS Calcd for:C74H80N6O4S4([M+H]+):1272.57.,found:1272.4865.
example 1 the nuclear magnetic hydrogen spectrum characterization map of compound HLA4 prepared in experimental group 1 is shown in FIG. 1; example 1 the nuclear magnetic carbon spectrum of HLA4 of compound prepared in experimental group 1 is shown in fig. 2; example 1 the absorption and emission spectra of compound HLA4 prepared in experimental group 1 are shown in fig. 3.
The following experimental group 2 prepared a probe HLA4P for biological imaging, which was a compound HLA4 prepared in the above experimental group 1.
Experimental group 2: preparation of fluorophore HLA4P
Collecting compound HLA4(123mg, 0.226mmol), MPEG2000NH2(1.23g,0.565mmol), 100. mu.L of DIPEA, N- (2-aminoethyl) maleimide trifluoroacetate (0.7611mg, 0.030mmol) and HATU (11.410mg, 0.030mmol) were charged in a 50mL round-bottomed flask, 20mL of N, N-dimethylformamide was added under argon protection, argon was bubbled through the reaction mixture for 5min to remove oxygen in the system, and the reaction was carried out at room temperature under argon protection for 2 hours. After the reaction, the reaction mixture was cooled to room temperature, and N, N-dimethylformamide was removed by rotary evaporation to obtain 1.1g of HLA 4P. Yield: 90 percent.
The structural determination data of the compound HLA4P is characterized by a nuclear magnetic hydrogen spectrum as shown in figure 5.
FIG. 6 is a transmission electron microscope image of a compound HLA4 which is linked to polyethylene glycol and can self-assemble to form nanoparticles.
Example 2
Preparation of fluorophore-entrapped antitumor drug (HLA4P @ DOX)
First, hydrochloric acid in dox.hcl (doxorubicin hydrochloride) is removed. The fluorophore HLA4P5mg prepared in example 1 and 1mg of the hydrophobic free drug DOX (doxorubicin) were added to the DMSO solution to obtain a mixed solution. Deionized water or PBS was added to the round bottom flask and then the mixture of HLA4P and DOX was slowly dropped into the bottom. The dropping speed of the mixed liquid into the container is 60-110 drops per minute, and after the mixed liquid is added, ultrasonic treatment is carried out for 10 s. The ultrasonic power is controlled to be 240W, and the working frequency is controlled to be 40 KHz. And (3) obtaining a mixture, dialyzing the mixture to remove free DOX for about 48 hours, wherein the cut-off molecular weight of a dialysis bag used for dialysis is 10kDa, deionized water is used as external liquid, the dialysis time is controlled to be 48 hours, and the external liquid is replaced for more than 6 times in the period, so that the HLA4P @ DOX is obtained.
FIG. 6 is a schematic diagram of the self-assembly process for preparing HLA4P @ DOX in example 2. Anticancer drug doxorubicin hydrochloride (DOX) is taken as a model drug, and the drug loading capacity and the controlled release capacity of HLA4P are discussed. HLA4P @ DOX was prepared by a typical nano-encapsulation process. HLA4P @ DOX was characterized by TEM and DLS and had average sizes of 150nm and 180nm, respectively, slightly above the self-assembled size of individual HLA 4P. The encapsulation efficiency of HLA4P @ DOX was 65% as determined by UV-vis absorbance of DOX, FIG. 9 is a TEM and DLS plot of HLA4P @ DOX prepared in example 2.
Example 3
Confocal laser scanning
CT-26 cells were cultured in culture flasks. After 24 hours, 1ml of medium was added to the confocal dish, followed by HLA4P @ DOX. After 4 hours, the medium in the small dish was removed and 1mL of PBS was added. A 4% paraformaldehyde solution (1mL) was added to each small dish and held for 15 minutes for cell fixation. After 15min, the paraformaldehyde fixing solution was removed, PBS buffer was added 3 times, and 200. mu.L of DAPI staining solution was added. After 10 min incubation with DAPI staining solution, the staining solution was also removed and washed 3 times with PBS buffer. Cells were observed under a confocal microscope Leica-LCS-SP 8-STED. CT-26 cells were imaged for cellular uptake using confocal microscopy using free DOX (CDOX 60 μ M) and HLA4P @ DOX (CDOX 60 μ M) over 8 hours. FIG. 8 is the cellular uptake imaging of confocal microscopy of example 3. The results show that the drug uptake efficiency was about 50% when CT26 cells were cultured with free DOX for 6 h. In contrast, drug uptake increased to about 80% after 6h incubation with HLA4P @ DOX, suggesting that HLA4P @ DOX promotes cellular internalization and accumulation of DOX. Confocal laser scanning microscopy further demonstrated enhanced uptake of HLA4P @ DOX by CT-26 cells. The red fluorescence of HLA4P @ DOX was much stronger than free DOX, indicating a higher uptake by CT-26 cells.
Example 4
Calculation of encapsulation efficiency and drug load
Calibration curves were performed for different concentrations of DOX dissolved in DMSO. After dialysis, the HLA4P @ DOX was isolated from the blood by dissolving DOX in dimethyl sulfoxide to measure the absorbance, wherein the absorption wavelength was 480 nm. Drug Loading Efficiency (DLE) and Drug Loading Content (DLC) were analyzed by the following calculation formulas:
Figure BDA0003459630450000091
Figure BDA0003459630450000092
HLA4P can effectively encapsulate antitumor drug adriamycin (DOX, encapsulation rate is about 65%), and realize sustained release of DOX.
Example 5
Biodistribution of HLA4P @ DOX
ICR mice were randomly divided into different groups. HLA4P @ DOX solutions of free DOX were injected intravenously into ICR mice. Blood samples were collected from different time points and the whole blood samples were centrifuged at 13000rpm for 15 minutes to collect plasma. We randomly divided CT-26 tumor-bearing Balb/c mice into different groups. HLA4P @ DOX free DOX solutions were injected intravenously into CT-26 mouse models. Mice were sacrificed at various time points post injection, and heart, spleen, liver, lung, kidney and tumor were harvested and weighed. These plasma and tissue were suspended in 70% ethanol solution containing 0.3N HCl and the mixture was then homogenized well. After a further centrifugation step, the absorbance of DOX in the supernatant was measured at a wavelength of 480 nm. The DOX content in mouse blood and various tissues was determined according to a standard curve. FIG. 10 is the biodistribution plots of DOX and HLA4P @ DOX at different time nodes after tail vein injection in example 5; FIG. 11 is the biodistribution of different organs at different time nodes after tail vein injection of DOX in example 5; FIG. 12 is the biodistribution of different organs at different time nodes after tail vein injection of HLA4P @ DOX in example 5. As can be seen from the results of fig. 4, 5 and 6, even though 72H HLA4P @ DOX still has a certain concentration and is higher than free DOX, the profile reflects that HLA4P @ DOX has excellent long-circulating therapeutic potential.
Example 6
Anti-tumor effect of HLA4P @ DOX on living bodies
Mice bearing CT-26 (19-25g) were randomly divided into 4 groups (n-5) when the tumor volume of CT-26 mice was at about 70mm3OfTreatment was performed as the mean size increased (this time point was recorded as "day 1"). The group of CT-26 tumor-bearing mice was injected intravenously 1 time. Free DOX (6mg/kg), HLA4P, PBS and HLA4P @ DOX (6mg/kg) were injected intravenously into mice, respectively. Tumor volume was measured with an electronic vernier caliper. The analytical formula for CT-26 tumor volume is: e ═ V2xE/2, where E and E are the longest and shortest diameters, respectively, of CT-26 tumors. FIG. 13 is a graph of tumor size in mice given various treatments (PBS, DOX (6mg/kg), HLA4P, and HLA4P @ DOX (6 mg/kg)). CT-26 mice were weighed every two days and then tested for survival.
Tumor size and body weight were measured every 2 days. CT-26 tumors grew very rapidly after PBS and HLA4P treatment, and the tumor volumes of the two control groups increased 13-fold and 12-fold after 14 days of treatment, respectively. However, CT-26 tumors treated with free DOX grew at a slower rate within the first 6 days after injection. The mean tumor volume in the free DOX treated group increased 6-fold after 14 days of treatment.
Example 7
Imaging of HLA4P @ DOX tracking therapy
The mice of the different treatment groups of example 6 were subjected to near-infrared two-zone fluorescence imaging, and FIG. 14 is a near-infrared two-zone imaging graph of the mice of the different treatment groups (DOX (6mg/kg) and HLA4P @ DOX (6 mg/kg)).
The results show that HLA4P @ DOX not only has bright NIR-II luminescence (-1055 nm), with tumor retention lasting 14 days, but also better treatment in vitro and in vivo than free DOX with fewer side effects. HLA4P @ DOX was excited by 808nm laser, showed real-time tracking ability, and was continuously NIR-II imaged in vivo for a long period of time. Has certain therapeutic tracking effect.
The entire course of treatment is accurately monitored by NIR-II fluorescence imaging. Significant NIR-II signals were obtained at high T/TN (>23) tumor sites and fluorescence signals could persist for 14 days, indicating that HLA4P @ DOX has superior tumor targeting ability and ultra-long tumor retention.
Example 8
Histological analysis
Tumors or major organs were obtained from the different treatment groups of mice of example 6 (free DOX (6mg/kg), HLA4P, PBS, and HLA4P @ DOX (6mg/kg)) and fixed with EDTA/formalin solution. After embedding, various tissue samples were stained by H & E staining. FIG. 15 is a graph of H & E staining of different organs of mice of different treatment groups (PBS, DOX (6mg/kg) and HLA4P @ DOX (6 mg/kg)).
Although the embodiments of the present invention have been shown and described, it is understood that the above embodiments are illustrative and not restrictive, and that those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention and that they should be included in the protection scope of the present invention.

Claims (10)

1. The application of the organic fluorescent small molecular compound in preparing a drug carrier is characterized in that the structural formula of the organic fluorescent small molecular compound is shown as a formula 1:
Figure FDA0003459630440000011
wherein R is1One selected from S and Se, R0、R2Are each independently selected from O, S, Se and N-R11One of (1), R11One selected from the group consisting of H, methyl and ethyl; r3、R4、R5、R6Are each independently selected from
Figure FDA0003459630440000012
Figure FDA0003459630440000013
And H, n is an integer from 0 to 18, and m is an integer from 0 to 20;
R7、R8、R9、R10are each independently selected from
Figure FDA0003459630440000014
Figure FDA0003459630440000015
Figure FDA0003459630440000016
N is an integer of 0 to 18, m is an integer of 0 to 20, and X is selected from F, Cl, Br, I and N3One kind of (1).
2. The application of the organic fluorescent small molecule compound in the preparation of the drug carrier according to claim 1, wherein the structural formula of the organic fluorescent small molecule compound is as follows:
Figure FDA0003459630440000021
3. the use of the organic fluorescent small molecule compound of claim 1 in the preparation of a pharmaceutical carrier, comprising: modifying polypeptide, protein, polyethylene glycol, aptamer or folic acid and derivatives thereof at the adjustable and controllable sites of the organic fluorescent small molecular compound to obtain a fluorophore, and taking the fluorophore as an anti-tumor drug carrier.
4. The use of the organic fluorescent small molecule compound of claim 3 in the preparation of a drug carrier, wherein the fluorophore is used as an anthraquinone-based antibiotic anti-tumor drug carrier.
5. The use of the organic fluorescent small molecule compound in the preparation of a drug carrier according to claim 4, wherein the anthraquinone antibiotic antineoplastic drug comprises one or more of doxorubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.
6. An antitumor drug, comprising the organic fluorescent small molecule compound of claim 1 or a fluorophore obtained by modifying a polypeptide, a protein, polyethylene glycol, a nucleic acid aptamer or folic acid and derivatives thereof at a controllable site of the organic fluorescent small molecule compound.
7. The antitumor drug according to claim 6, wherein said antitumor drug is targeted to a tumor.
8. A method for preparing an antitumor agent according to claim 6, comprising:
step 1): removing acid in the active ingredient salt of the anti-tumor drug to obtain free drugs, and adding fluorophores and the free drugs, which are obtained by modifying polypeptide, protein, polyethylene glycol, aptamer or folic acid and derivatives thereof at the adjustable and controllable sites of the organic fluorescent small molecular compound, into DMSO to obtain mixed solution;
step 2): adding deionized water or PBS into a container, then dripping the mixed solution obtained in the step 1) into the bottom of the container, and carrying out ultrasonic treatment to obtain a mixture;
step 3): dialyzing the mixture obtained in step 2) to remove the free drug which is not successfully entrapped;
preferably, in the step 1), the mass ratio of the free drug to the fluorophore in the mixed solution is 1: 5; preferably, the antitumor drug active ingredient is selected from one or more of daunorubicin, doxorubicin, epirubicin, idarubicin and mitoxantrone;
preferably, in the step 2), the dropping speed of the mixed liquid into the container is 60-110 drops per minute; and (3) carrying out ultrasonic treatment by using an ultrasonic cleaning machine, wherein the ultrasonic power is controlled to be 230-250W, the working frequency is controlled to be 40KHz, and the ultrasonic time is controlled to be 5-20 seconds.
9. The method for preparing an antitumor drug as claimed in claim 8, wherein in the step 3), the cut-off molecular weight of the dialysis bag used for dialysis is 10kDa, deionized water is used as external liquid, the dialysis time is controlled to be 24-60 hours, and the external liquid is replaced more than 6 times.
10. An organic fluorescent nano-carrier, characterized in that the organic fluorescent nano-carrier comprises the organic fluorescent small molecule compound of claim 1 or a fluorophore obtained by modifying polypeptide, protein, polyethylene glycol, aptamer or folic acid and derivatives thereof at adjustable sites of the organic fluorescent small molecule compound.
CN202210012796.7A 2022-01-07 2022-01-07 Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof Pending CN114426554A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210012796.7A CN114426554A (en) 2022-01-07 2022-01-07 Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210012796.7A CN114426554A (en) 2022-01-07 2022-01-07 Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN114426554A true CN114426554A (en) 2022-05-03

Family

ID=81310670

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210012796.7A Pending CN114426554A (en) 2022-01-07 2022-01-07 Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN114426554A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106967096A (en) * 2017-05-17 2017-07-21 中节能万润股份有限公司 A kind of diazole heterocyclic compounds and its application containing thiophene-structure
CN110357873A (en) * 2019-07-30 2019-10-22 西安交通大学 For the organic fluorescence materials of super-resolution cell imaging and its preparation and application
US20200313104A1 (en) * 2019-03-26 2020-10-01 New York University Organic solar cells and methods of making the same
CN113651911A (en) * 2021-07-05 2021-11-16 南京邮电大学 Near-infrared two-region fluorescence imaging contrast agent prepared through atom transfer radical polymerization and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106967096A (en) * 2017-05-17 2017-07-21 中节能万润股份有限公司 A kind of diazole heterocyclic compounds and its application containing thiophene-structure
US20200313104A1 (en) * 2019-03-26 2020-10-01 New York University Organic solar cells and methods of making the same
CN110357873A (en) * 2019-07-30 2019-10-22 西安交通大学 For the organic fluorescence materials of super-resolution cell imaging and its preparation and application
CN113651911A (en) * 2021-07-05 2021-11-16 南京邮电大学 Near-infrared two-region fluorescence imaging contrast agent prepared through atom transfer radical polymerization and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG LI等，: ""Organic NIR-II dyes with ultralong circulation persistence for image-guided delivery and therapy"", 《JOURNAL OF CONTROLLED RELEASE》 *

Similar Documents

Publication Publication Date Title
Tang et al. Design, preparation and evaluation of different branched biotin modified liposomes for targeting breast cancer
Gao et al. Glutathione-responsive nanoparticles based on a sodium alginate derivative for selective release of doxorubicin in tumor cells
CN106806344A (en) Poly-dopamine and the mesoporous silicon dioxide nano particle of TPGS modification and preparation method and application
EP3421519B1 (en) Ovarian cancer specifically targeted biodegradable amphiphilic polymer, polymer vesicle prepared thereby and use thereof
WO2012040513A1 (en) Compositions and methods for the delivery of beta lapachone
CN103131005B (en) Amino acid block copolymer and preparation method thereof and mixture
CN112773766B (en) Liposome delivery system for tumor treatment and preparation method and application thereof
CN113952463B (en) Nanometer diagnosis and treatment agent and preparation method and application thereof
EP4230224A1 (en) Affibody-cytotoxin conjugate used for active tumor targeting therapy, and nanoparticles, preparation method, and application thereof
CN110152012A (en) The poly ion complexes and its preparation method and application that poly sialic acid and cationic compound are formed
Yi et al. Synthesis, characterization, and formulation of poly-puerarin as a biodegradable and biosafe drug delivery platform for anti-cancer therapy
Chu et al. Preparation and evaluation of long circulating erythrocyte membrane-cloaked anti-cancer drug delivery system
CN111097052B (en) Amphiphilic prodrug for active targeted therapy of tumors and preparation method and application of nanoparticles of amphiphilic prodrug
CN114748639A (en) Photosensitizer-hydroxyalkyl starch-polypeptide coupled amphiphilic macromolecular compound, nano drug delivery system and preparation method thereof
CN110448699B (en) Tumor cell nucleus targeted drug-loaded nanoparticle containing functional polypeptide modified heptamethine cyanine dyes and preparation method thereof
CN109369625B (en) Ortho ester 5-fluorouracil prodrug molecule, preparation method thereof, acid-sensitive nanoparticle thereof and application
CN112603908B (en) Nano drug-loading system based on amino acid polymer and preparation method and application thereof
CN108186571B (en) Application of reversible cross-linked asymmetric vesicle in preparation of acute leukemia treatment drug
CN107007550B (en) Redox-responsive amphiphilic copolymer and preparation method and application thereof
CN113651959A (en) Nano drug loading system based on amino acid-hydroxy acid copolymer and preparation method and application thereof
CN106620714B (en) 7-ethyl-10-hydroxycamptothecin-polymer coupling drug and preparation method of nano preparation thereof
CN101015699A (en) Polyethylene glycol-phosphatidyl ethanolamine polymer or medicinal acid addition salt and application thereof in pharmacy
CN107028882B (en) Physically-wrapped tumor-targeted nano drug delivery system, and preparation method and application thereof
Zhang et al. Surface-adaptive nanoparticles with near-infrared aggregation-induced emission for image-guided tumor resection
Jia et al. Specific modification and self-transport of porphyrins and their multi-mechanism cooperative antitumor studies

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination