CN111358948A - Camptothecin-berberine/indocyanine green nano-drug, preparation method and application - Google Patents
Camptothecin-berberine/indocyanine green nano-drug, preparation method and application Download PDFInfo
- Publication number
- CN111358948A CN111358948A CN202010243013.7A CN202010243013A CN111358948A CN 111358948 A CN111358948 A CN 111358948A CN 202010243013 A CN202010243013 A CN 202010243013A CN 111358948 A CN111358948 A CN 111358948A
- Authority
- CN
- China
- Prior art keywords
- berberine
- camptothecin
- drug
- indocyanine green
- nano
- 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
Links
Images
Classifications
-
- 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
- A61K47/55—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 the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- 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
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
- Pulmonology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
Abstract
The invention discloses a method for co-assembling camptothecin-berberine/indocyanine green with a mitochondrially targeted microenvironment-responsive anticancer nano-drug, which is characterized in that the co-assembly of the camptothecin-berberine coupling drug molecule containing a disulfide bond and a photosensitizer indocyanine green is realized by designing a camptothecin-berberine coupling drug molecule containing a disulfide bond, so that the anticancer nano-drug is obtained; the camptothecin-berberine/indocyanine green co-assembled nano-drug provided by the invention has sensitive tumor microenvironment response, can realize cancer cell mitochondria targeted drug delivery, and improves the treatment effect of the drug on lung cancer through the synergistic effect of chemotherapy and photothermal treatment.
Description
Technical Field
The invention relates to the technical field of nano-drug synthesis, in particular to a mitochondrion targeted camptothecin-berberine/indocyanine green nano-drug with stimulus response, a preparation method and application thereof.
Background
Cancer is seriously threatening the life and health of human beings, and in china, the number of people dying from cancer annually exceeds millions. Therefore, the treatment of cancer is a topic of intense research in the medical field. Due to the advantages of high delivery efficiency, specific targeting function and the like, the nano-drug has great potential in the aspect of treating cancers. Different nano-carriers such as liposome, polymeric micelle, inorganic nano-particle and the like are widely applied to preparing nano-drugs so as to improve the safety and the treatment effect of chemotherapeutic drugs. Although many drug carriers are reported in the literature, only a few of the nano-drug carriers have been approved by the FDA for clinical use to date due to problems with low drug loading and short-and long-term toxicity. Therefore, it is important to explore a simple and efficient method for preparing the carrier-free nano-drug.
The nano particles formed by supermolecule self-assembly play an important role in the fields of optimizing imaging signals, controlling drug release, improving the therapeutic effect of drugs and the like. Much work has been devoted to the development of organic molecules that self-assemble based on weak interactions. The organic molecules have structural diversity, high biodegradability and adjustable versatility, and the structure provides wide application in the field of nano-drugs. In addition, these organic nanoparticles can improve the passive targeting function of the nanoparticles through enhanced osmotic retention effect, thereby improving the diagnosis and treatment effect of diseases.
Mitochondria play an important role in the metabolic process of cells, and therefore, mitochondrially targeted drug delivery is a very potential cancer treatment. Among the numerous mitochondrial targeting approaches, non-localized lipophilic cations are the most common class of structures, which are often coupled to other small molecules or directly modified on the surface of nanocarriers. The mitochondrial membrane of cancer cells is more negatively charged than normal cells, resulting in a tendency for the cationic targeting group to accumulate in the mitochondria of cancer cells. Berberine is an isoquinoline alkaloid and possesses many biological activities, such as anticancer, antimicrobial, anti-inflammatory, etc. Notably, berberine, due to its specific delocalized positive charge structure, is also able to selectively accumulate in the mitochondria of cancer cells. Moreover, berberine can also induce apoptosis by various means such as lowering mitochondrial membrane potential, increasing activity oxygen, etc.
Camptothecin and its analogues inhibit cancer cell DNA replication by binding to topoisomerase I, thereby inhibiting cancer cell proliferation. To date, many camptothecin analogs have been used clinically and have been shown to act on lung cancer, ovarian cancer, cervical cancer, and the like. However, drug resistance has been developed with the long-term clinical use. In addition, chemotherapy, although the main treatment modality in clinical practice, has drawbacks such as severe side effects, high recurrence, and the like. Therefore, new therapeutic approaches have been developed to solve these problems. As a representative and non-invasive method for tumor treatment, photothermal therapy (PTT) integrating a photothermal conversion agent can convert absorbed light into thermal energy to destroy local cancer cells while reducing side effects on normal cells. Moreover, combination therapy combining chemotherapy and photothermal therapy has great advantages in clinical applications because it can take advantage of the advantages of both treatment modalities while overcoming the disadvantages of each treatment modality. Glutathione (GSH) is a strong bioreductive tripeptide that can break disulfide bonds at certain concentrations. The higher level of GSH in cancer cells compared to normal tissues is sufficient to reduce disulfide bonds.
Disclosure of Invention
The purpose of the invention is as follows: in view of the above problems of the existing cancer chemotherapy and nano-drug carrier, and the advantages of the combination therapy and the mitochondria targeting drug delivery, one of the objectives of the present invention is to provide a camptothecin-berberine/indocyanine green nano-drug; the second purpose of the invention is to provide a preparation method of the camptothecin-berberine/indocyanine green nano-drug; the invention also aims to provide the application of the camptothecin-berberine/indocyanine green nano-drug.
The camptothecin is coupled with the berberine through the disulfide bond, and then the camptothecin and the berberine are assembled together with the photosensitizer indocyanine green in water to form the carrier-free nano-drug with mitochondrion targeting, so that mitochondrion targeting drug delivery is achieved, the anti-lung cancer effect is realized, and meanwhile, the safety problem caused by a carrier is also solved.
The technical scheme is as follows: the synthesis method of the camptothecin-berberine/indocyanine green nano-drug comprises the following steps:
(1) placing a proper amount of berberine hydrochloride in a single-opening reaction container, and reacting under a high-temperature vacuum condition to obtain demethylberberine;
(2) carrying out reflux reaction on demethyl berberine and bromoethanol for 24 hours under the conditions of acetonitrile serving as a solvent and potassium carbonate serving as alkali to obtain hydroxyethyl berberine;
(3) dissolving the hydroxyethyl berberine obtained in the step (2) in absolute methanol, stirring in ice bath, then adding a small amount of methanol solution of sodium borohydride for many times, and reacting for 24 hours to obtain reduced hydroxyethyl berberine;
(4) refluxing dithiodipropionic acid in acetyl chloride to obtain dithiodipropionic anhydride, and then reacting with camptothecin to obtain dithiodipropionic camptothecin;
(5) carrying out a reaction on camptothecin dithiodipropionate and reduced hydroxyethyl berberine under the conditions that pyridine is used as a solvent, N, N' -Dicyclohexylcarbodiimide (DCC) is used as a dehydrating agent, and 4-Dimethylaminopyridine (DMAP) is used as a catalyst to obtain a camptothecin-berberine coupling drug precursor;
(6) reacting the camptothecin-berberine conjugate prodrug with N-bromosuccinimide (NBS) to obtain the camptothecin-berberine conjugate drug;
(7) dissolving the camptothecin-berberine coupling drug and the photosensitizer indocyanine green in an organic solvent, dripping the solution into deionized water, performing ultrasonic treatment for a period of time, and dialyzing the solution in ultrapure water to obtain the camptothecin-berberine/indocyanine green co-assembled nano-drug.
Furthermore, the ratio of the demethylberberine to the bromoethanol, the acetonitrile and the potassium carbonate in the step (2) is 1: 1-2: 60-80: 1-2.
Further, the mass ratio of the hydroxyethyl berberine to the methanol to the sodium borohydride in the step (3) is 1: 120-150: 1-3;
further, the reaction temperature of the step (1) is 180-190 ℃.
Further, the ratio of the dithiodipropionic acid, acetyl chloride and camptothecin in the step (4) is 5-10: 120-150: 1.
Further, in the step (5), the dithiodipropionic acid camptothecin, the DCC and the DMAP are sequentially dissolved in pyridine, then the mixture is stirred for 30-60 min under the protection of nitrogen to obtain a first mixed solution, and then the reduced hydroxyethyl berberine dichloromethane solution is slowly added into the first mixed solution to react for 24-48 h to obtain the camptothecin-berberine coupling prodrug.
Further, in the step (5), the ratio of the amount of the reduced hydroxyethyl berberine, the dithiodipropionic acid camptothecin, the N, N' -dicyclohexylcarbodiimide, the 4-dimethylaminopyridine and the pyridine is 1: 1.2-2: 1.5-2.5: 0.1-0.3: 55.
Further, in the step (6), the camptothecin-berberine conjugate prodrug is dissolved in a chloroform solution, stirred for 30min, then slowly added into the chloroform solution of NBS, and continuously reacted for 24 h.
Further, in the step (6), the ratio of the camptothecin-berberine coupled prodrug to the N-bromosuccinimide is 1: 1-1.2.
Further, in the step (6), the reaction temperature is 30-50 ℃.
Further, in the step (7), the amount ratio of the camptothecin-berberine to indocyanine green is 1: 0.5-1.5.
Further, in the step (7), the organic solvent is any one or more of ethanol, propanol, pyridine, isopropanol, dimethyl sulfoxide and methanol.
Further, in the step (7), the ultrasonic power is 300W, and the ultrasonic time is 20-60 min.
Further, in the step (7), the dialysis time is 12-24 h.
The application of the camptothecin-berberine/indocyanine green nano-drug prepared according to the method in preparing the anti-cancer drug preparation is also within the protection scope of the invention. Wherein the tumor is lung cancer.
The synthetic route of the camptothecin-berberine conjugate drug is as follows:
has the advantages that: the invention utilizes a supermolecule co-assembly technology to prepare the camptothecin-berberine/indocyanine green nano-drug, and realizes the combined drug use of the nano-drug, the stimulation response release, the mitochondrion targeted drug delivery, the chemotherapy and the photothermal therapy. The new nano-drug designed by the invention has determined result, is easy to repeat and characterize, and has better treatment effect.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a hydrogen spectrum of a camptothecin-berberine conjugate prodrug;
FIG. 2 is a high resolution mass spectrum of a camptothecin-berberine conjugate prodrug;
FIG. 3 is a hydrogen spectrum of a camptothecin-berberine conjugate drug;
FIG. 4 is a high resolution mass spectrum of a camptothecin-berberine conjugate drug;
FIG. 5 is a Transmission Electron Microscope (TEM) morphology of camptothecin-berberine/indocyanine green nano-drug;
fig. 6 is a distribution diagram of the particle size of the camptothecin-berberine/indocyanine green nano-drug at time 0 and 20 days after standing. The abscissa in the figure is the particle size distribution and the ordinate is the intensity;
fig. 7 is a graph of the particle size change of camptothecin-berberine/indocyanine green nano-drug at different times within 20 days. In the figure, the abscissa is time and the ordinate is particle size;
FIG. 8 shows UV absorption spectra of camptothecin-berberine conjugate drugs (CPT-ss-BBR), camptothecin-berberine/indocyanine green nanopharmaceuticals (CPT-ss-BBR NPs) and indocyanine green (ICG). In the graph, the abscissa is wavelength and the ordinate is absorbance;
FIG. 9 is a graph of the cumulative release of Camptothecin (CPT) from camptothecin-berberine/indocyanine green nano-drugs at 37 ℃ under different pH conditions with/without 20mM GSH and with/without light, wherein the abscissa is time and the ordinate is the cumulative amount of drug released;
fig. 10 is a graph of in vitro cytotoxicity (MTT) of 72h camptothecin-berberine/indocyanine green nano-drug (with/without illumination), indocyanine green (with/without illumination), camptothecin-berberine conjugate drug, camptothecin, etc. against a549, where the abscissa is drug concentration and the ordinate is cancer cell survival rate;
fig. 11 is a mitochondrial targeting confocal image of camptothecin-berberine/indocyanine green nano-drug after 45min and 90min of culture, in which the first column is confocal picture of berberine fragment, the second column is confocal picture of mitochondrial fluorescence probe, the third column is combined picture of the first and second columns, and the fourth column is mitochondrial co-localization correlation.
Detailed Description
In order to clearly understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below with reference to the accompanying drawings and examples. The preferred embodiments of the present invention are described in detail below, and the embodiments included in the present invention are not limited to these embodiments and may have other embodiments in addition to the embodiments described in detail. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1: synthesis of demethylberberine
Adding 5g berberine hydrochloride into a 100mL round bottom flask, heating for 2h under vacuum at 190 deg.C, and gradually changing yellow solid powder into dark red powder to obtain crude demethylberberine product. Column chromatography (elution machine: dichloromethane/methanol 15/1) to obtain dark red demethylberberine powder 3.68g with yield of 85%.
Example 2: synthesis of hydroxyethyl berberine
4.22g (13.14mmol) of desmethylberberine prepared in example 1, 30mL of acetonitrile, 1.82g (13.14mmol) of potassium carbonate were added to a 100mL round bottom flask, stirred at 60 ℃ for 20min, then 2.46g (19.71mmol) of bromoethanol was added, the temperature was raised to reflux for 24h, and column chromatography (elution machine: dichloromethane/methanol 15/1) was performed to obtain 5.03g of yellow hydroxyethylberberine powder with a yield of 86%.
Example 3: synthesis of reduced hydroxyethylberberine
4.48g (10.07mmol) of the hydroxyethylberberine prepared in example 2 and 35mL of methanol were added to a 100mL round bottom flask, stirred in ice bath, and then added with a methanol solution of sodium borohydride (0.76g,20.14mmol) to react for 12h to obtain a crude reduced hydroxyethylberberine product, which was separated by column chromatography (elution machine: dichloromethane/methanol 15/1) to obtain 3.06g of white reduced hydroxyethylberberine powder with a yield of 82%.
Example 4: synthesis of camptothecin dithiodipropionate
Dithiodipropionic acid (2g,9.51mmol) was dissolved in acetyl chloride (15mL) and refluxed for 5 hours. Then removing the solvent, precipitating filter residue in excessive ethyl glacial ether, and drying to obtain the crude product of the dithiodipropionic anhydride. Into a 100mL round bottom flask was added the prepared dithiodipropionic anhydride (1.24g,6.45mmol), camptothecin (0.45g,1.29mmol), then dissolved with pyridine, stirred under nitrogen, and a solution of DMAP (0.16g,1.29mmol) in pyridine was added to the system. Then the mixed solution is heated to 70 ℃, and the reaction is continued for 48 hours in an ice bath. The reaction solution was extracted with chloroform and dilute hydrochloric acid, and dried under vacuum to obtain 0.51g of camptothecin dithiodipropionate with a yield of 73%.
Example 5: synthesis of camptothecin-berberine coupled prodrug
1.57g (2.81mmol) of the camptothecin dithiodipropionate synthesized in example 4, a catalytic amount of 28.09mg (0.23mmol) of DMAP (dimethyl formamide), 0.73g (3.51mmol) of DCC (DCC) and 10mL of pyridine are added into a 100mL round-bottom flask, the mixture is stirred for 30min under ice bath, a dichloromethane solution of the reduced hydroxyethyl berberine synthesized in example 3 (2.0g and 2.34mmol) is added into a reaction system, the reaction is continued for 48h under ice bath to obtain a crude camptothecin-berberine coupling drug precursor, the solvent pyridine is removed by reduced pressure distillation, and then column chromatography (washing and dewatering machine: dichloromethane/methanol ═ 30/1) is carried out for separation to obtain 2.58g of the camptothecin-berberine coupling drug precursor with the yield of 79%.
1H NMR(600MHz,CDCl3)δ8.38(s,1H),8.22(d,J＝5.7Hz,1H),7.93(d,J＝5.5Hz,1H),7.82(t,J＝5.5Hz,1H),7.66(t,J＝5.0Hz,1H),7.26(s,1H),6.86(d,J＝5.6Hz,1H),6.77(d,J＝5.6Hz,1H),6.70(s,1H),6.57(s,1H),5.91(s,2H),5.67(d,J＝11.4Hz,1H),5.40(d,J＝11.4Hz,1H),5.29–5.27(m,2H),4.36(dd,J＝7.1,3.3Hz,2H),4.24(dd,J＝5.7,2.4Hz,2H),4.17(dd,J＝5.2,2.3Hz,1H),3.81(s,3H),3.48(s,2H),3.21(d,J＝7.2Hz,3H),3.02–2.88(m,7H),2.78(t,J＝4.8Hz,2H),2.70–2.56(m,2H),2.28(dd,J＝9.7,4.6Hz,1H),2.16(dd,J＝9.4,5.0Hz,1H),0.98(t,J＝5.0Hz,3H).；ESI-MS m/z[M+H]+＝892.25649。
Fig. 1 and fig. 2 are a hydrogen spectrum and a high-resolution mass spectrum of the camptothecin-berberine conjugate prodrug, respectively, which prove the success of the compound preparation.
Example 6: synthesis of camptothecin-berberine coupled prodrug
1.57g (2.81mmol) of the camptothecin dithiodipropionate synthesized in example 4, a catalytic amount of DMAP 56.18mg (0.46mmol), DCC 0.73g (3.51mmol) and pyridine 10mL are added into a 100mL round-bottom flask, the mixture is stirred for 30min under ice bath under the protection of nitrogen, a dichloromethane solution of the reduced hydroxyethyl berberine synthesized in example 3 (2.0g,2.34mmol) is added into a reaction system, the reaction is continued for 48h under ice bath to obtain a crude camptothecin-berberine coupling drug precursor, the solvent pyridine is removed by reduced pressure distillation, and then column chromatography (elution machine: dichloromethane/methanol ═ 30/1) is carried out for separation to obtain 2.78g of the camptothecin-berberine coupling drug precursor with the yield of 85%.
Example 7: synthesis of camptothecin-berberine conjugate drug
450mg (0.32mmol) of the camptothecin-berberine conjugate drug precursor synthesized in example 5 and 20mL of chloroform are added into a 100mL round-bottom flask, stirred at room temperature for 30min, then a chloroform solution of NBS (86.49mg,0.48mmol) is added into the reaction system, and the reaction is continued at 50 ℃ for 24h to obtain a crude camptothecin-berberine conjugate drug, and the crude camptothecin-berberine conjugate drug is separated by column chromatography (elution machine: dichloromethane/methanol ═ 10/1) to obtain 133.57mg of the camptothecin-berberine conjugate drug with the yield of 45%.
1H NMR(600MHz,DMSO-d6,ppm):δ9.70(s,1H),8.83(s,1H),8.65(s,1H),8.16(d,J＝6.1Hz,1H),8.05(dd,J＝12.5,5.5Hz,2H),7.94(d,J＝6.1Hz,1H),7.77(t,J＝5.1Hz,1H),7.70(s,1H),7.64(t,J＝5.0Hz,1H),7.13(s,1H),6.96(s,1H),6.18(s,2H),5.50–5.41(m,2H),5.23(d,J＝2.8Hz,2H),4.89–4.85(m,2H),4.48(dd,J＝7.0,3.7Hz,2H),4.43(dd,J＝6.5,4.3Hz,2H),4.03(s,3H),3.15–3.11(m,2H),3.00–2.90(m,6H),2.77(t,J＝4.5Hz,2H),2.12(dt,J＝7.1,4.7Hz,2H),0.91(t,J＝4.9Hz,3H).；ESI-MS m/z[M-Br]+＝888.22594。
Fig. 3 and 4 are a hydrogen spectrum and a high-resolution mass spectrum of the camptothecin-berberine conjugate drug, respectively, which prove the success of the compound preparation.
Example 8: synthesis of camptothecin-berberine conjugate drug
450mg (0.32mmol) of the camptothecin-berberine conjugate drug precursor synthesized in example 5 and 20mL of chloroform are added into a 100mL round-bottom flask, stirred at room temperature for 30min, then a chloroform solution of NBS (57.66mg,0.32mmol) is added into the reaction system, and the reaction is continued at 50 ℃ for 24h to obtain a crude camptothecin-berberine conjugate drug, and the crude camptothecin-berberine conjugate drug is separated by column chromatography (elution machine: dichloromethane/methanol ═ 10/1) to obtain 103.88mg of the camptothecin-berberine conjugate drug with the yield of 35%.
Example 9: synthesis of camptothecin-berberine conjugate drug
450mg (0.32mmol) of the camptothecin-berberine conjugate drug precursor synthesized in the example 5 and 20mL of chloroform are added into a 100mL round-bottom flask, stirred at room temperature for 30min, then a chloroform solution of NBS (86.49mg,0.48mmol) is added into the reaction system, and the reaction is continued at room temperature for 24h to obtain a crude camptothecin-berberine conjugate drug, and the crude camptothecin-berberine conjugate drug is separated by column chromatography (elution machine: dichloromethane/methanol 10/1) to obtain 118.72mg of the camptothecin-berberine conjugate drug with the yield of 40%.
Example 10: preparation of camptothecin-berberine/indocyanine green nano-drug
10mg (0.01mmol) and 8mg (0.01mmol) of the camptothecin-berberine conjugate drug synthesized in example 7 are dissolved in 0.4mL of dimethyl sulfoxide solvent, ultrasonic treatment is carried out at the power of 300W for 20min at room temperature, the solution is gradually injected into 10mL of deionized water, and the mixed solution system is stirred for 2 h. Then dialyzing in a dialysis bag with molecular weight cutoff of 3500Da for 12h to obtain the camptothecin-berberine/indocyanine green nano-drug.
Example 11: preparation of camptothecin-berberine/indocyanine green nano-drug
10mg (0.01mmol) and 8mg (0.01mmol) of the camptothecin-berberine conjugate drug synthesized in example 7 are dissolved in 0.4mL of dimethyl sulfoxide solvent, ultrasonic treatment is carried out at the power of 300W for 60min at room temperature, the solution is gradually injected into 10mL of deionized water, and the mixed solution system is stirred for 2 h. Then dialyzing in a dialysis bag with molecular weight cutoff of 3500Da for 12h to obtain the camptothecin-berberine/indocyanine green nano-drug.
Example 12: preparation of camptothecin-berberine/indocyanine green nano-drug
5mg (0.005mmol) and 8mg (0.01mmol) of the camptothecin-berberine coupling drug synthesized in the example 7 are dissolved in 0.4mL of dimethyl sulfoxide solvent, ultrasonic treatment is carried out at the power of 300W for 20min at room temperature, the solution is gradually injected into 10mL of deionized water, and the mixed solution system is stirred for 2 h. Then dialyzing in a dialysis bag with molecular weight cutoff of 3500Da for 12h to obtain the camptothecin-berberine/indocyanine green nano-drug.
Example 13: preparation of camptothecin-berberine/indocyanine green nano-drug
10mg (0.01mmol) and 8mg (0.01mmol) of the camptothecin-berberine conjugate drug synthesized in example 7 are dissolved in 0.4mL of methanol solvent, ultrasonic treatment is carried out at the power of 300W for 20min at room temperature, the solution is gradually injected into 10mL of deionized water, and the mixed solution system is stirred for 2 h. Then dialyzing in a dialysis bag with molecular weight cutoff of 3500Da for 12h to obtain the camptothecin-berberine/indocyanine green nano-drug.
Example 14: related characterization of camptothecin-berberine/indocyanine green nano-drug
Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) were used to observe and characterize the morphology and particle size distribution of camptothecin-berberine/indocyanine green nano-drugs. The TEM picture of FIG. 5 shows that the camptothecin-berberine/indocyanine green nano-drug is in a regular spherical shape, and the particle size is about 150 nm. The DLS analysis of fig. 6 found that the average particle size of the camptothecin-berberine/indocyanine green nano-drug is 165nm, and the distribution is narrow (PDI ═ 0.086). And after 20 days, the particle size of the camptothecin-berberine/indocyanine green nano-drug still does not change greatly (figure 7). An ultraviolet-visible spectrophotometer was used to detect the monomer interaction in camptothecin-berberine/indocyanine green nano-drugs, and as a result, it was found that there was an interaction between camptothecin-berberine and indocyanine green because the ultraviolet absorption peaks of both monomers had a distinct red shift (fig. 8).
Example 15: drug release experiment of camptothecin-berberine/indocyanine green nano-drug
The cumulative release of camptothecin from camptothecin-berberine/indocyanine green nano-drugs was measured by dialysis. 1mL of the camptothecin-berberine/indocyanine green nano-drug of example 10 was put in a dialysis bag with a molecular weight cut-off of 3500Da, and then the nano-drug was soaked in 30mL of PBS (pH 7.4 or 5.6, with/without 20Mm GSH, with/without 808nm1W/cm2Light irradiation) and continued shaking at 37 ℃ at 150 r/min. A2 mL sample was taken for UV detection at a predetermined time and supplemented with 2mL of fresh PBS.
As a result, it was found (as shown in fig. 9) that the camptothecin-berberine/indocyanine green nano-drug exhibits a significant GSH response characteristic, and the drug release process can be accelerated by low pH and light. It is noted that camptothecin released in excess of 80% under conditions of pH 5.6, light, GSH 20 mM.
Example 16: MTT method cancer cell viability experiment
Inoculating A549 human lung cancer cell in 96-well culture plate with 5% CO2After culturing in an incubator at 37 ℃ for 24 hours, different concentrations of the camptothecin-berberine/indocyanine green nano-drug of example 10 (with/without 808nm 1W/cm) were added to each well2Illumination), camptothecin, berberine, indocyanine green (with/without 808nm 1W/cm)2Light irradiation), camptothecin-berberine conjugate drug solutions each 100 μ L to final drug concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40 μ M, respectively, and culturing for 72 h; separately 50. mu.L of MTT was added, followed by 5% CO2And continuously culturing for 4h in an incubator at 37 ℃, removing the culture medium, adding 150 mu L of DMSO, shaking uniformly on a plate shaker, reading the plate at 495nm by using an enzyme-labeling instrument, and calculating the cell inhibition rate according to the measured absorbance value. Data are presented as mean ± SD (n ═ 3).
As a result, it was found (as shown in FIG. 10) that the culture was 24 hoursThen, camptothecin-berberine/indocyanine green nano-drug (808nm 1W/cm)2Illumination) (IC50The inhibitory effect of camptothecin (IC) on a549 was attributed to 0.21 μ M50＝0.43μM)。
Example 17: mitochondrial co-localization experiments
Laser confocal microscopy was used to observe the localization of camptothecin-berberine nano-drug mitochondria. A549 human Lung cancer cells were seeded in glass dishes at 5% CO2And after culturing for 12h in an incubator at 37 ℃, adding 5 mu M of camptothecin-berberine nano-drugs into each dish, and continuously culturing for 45min or 90 min. Then, glass dishes containing A549 cells were washed three times with PBS buffer solution, and Mitotracker Red (1. mu.M) was added as a mitochondrial localization reagent and incubated for an additional 25 min. After the incubation, the glass dishes were washed three times with PBS buffer solution and the cells were observed with a confocal laser microscope.
As a result, the camptothecin-berberine/indocyanine green nano-drug is found to have good mitochondrial targeting function and the co-localization coefficient exceeds 0.9 no matter the camptothecin-berberine/indocyanine green nano-drug is cultured for 45min or 90min (as shown in figure 11).
Claims (10)
1. A synthetic method of a mitochondrion-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug is characterized by comprising the following steps:
(1) reacting berberine hydrochloride under high temperature and vacuum conditions to obtain demethylberberine;
(2) carrying out reflux reaction on demethyl berberine, bromoethanol, a solvent and alkali to obtain hydroxyethyl berberine;
(3) dissolving the hydroxyethyl berberine obtained in the step (2) in methanol, stirring in ice bath, and adding sodium borohydride methanol solution for reaction to obtain reduced hydroxyethyl berberine;
(4) carrying out reflux reaction on dithiodipropionic acid and acetyl chloride to obtain dithiodipropionic anhydride, and then reacting with camptothecin to obtain dithiodipropionic camptothecin;
(5) mixing the camptothecin dithiodipropionate with a solvent, a dehydrating agent and a catalyst, and adding reduced hydroxyethyl berberine to react to obtain a camptothecin-berberine coupled prodrug;
(6) reacting the camptothecin-berberine coupling drug precursor with N-bromosuccinimide to obtain the camptothecin-berberine coupling drug;
(7) dissolving the camptothecin-berberine coupling drug and the photosensitizer in an organic solvent, and carrying out ultrasonic treatment and dialysis to obtain the camptothecin-berberine/indocyanine green co-assembled nano-drug.
2. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: in the step (2), the solvent is acetonitrile, and the base is potassium carbonate; the ratio of the demethylberberine to the bromoethanol, the acetonitrile and the potassium carbonate is 1: 1-2: 60-80: 1-2.
3. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: the mass ratio of the hydroxyethyl berberine to the methanol to the sodium borohydride in the step (3) is 1: 120-150: 1-3; in the step (4), the mass ratio of dithiodipropionic acid, acetyl chloride and camptothecin is 5-10: 120-150: 1.
4. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: in the step (5), the solvent is pyridine, the dehydrating agent is N, N' -dicyclohexylcarbodiimide, and the catalyst is 4-dimethylaminopyridine; the weight ratio of the reduced hydroxyethyl berberine to the dithiodipropionic acid camptothecin to the N, N' -dicyclohexylcarbodiimide to the 4-dimethylaminopyridine to the pyridine is 1: 1.2-2: 1.5-2.5: 0.1-0.3: 55.
5. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: in the step (6), the mass ratio of the camptothecin-berberine coupled prodrug to the N-bromosuccinimide is 1: 1-2.
6. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: the reaction temperature in the step (1) is 180-190 ℃; the reaction temperature in the step (6) is 30-50 ℃.
7. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: the photosensitizer in the step (7) is indocyanine green; the amount ratio of the camptothecin-berberine coupling drug to the indocyanine green is 1: 0.5-1.5.
8. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: in the step (7), the organic solvent is any one or more of ethanol, propanol, pyridine, isopropanol, dimethyl sulfoxide and methanol.
9. The method for synthesizing the mitochondrially-targeted camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug according to claim 1, wherein the method comprises the following steps: in the step (7), the ultrasonic power is 300W, and the ultrasonic time is 20-60 min; the dialysis time is 12-24 h.
10. The application of the camptothecin-berberine/indocyanine green co-assembled stimulus-responsive anticancer nano-drug obtained by the preparation method of any one of claims 1 to 9 in the preparation of anticancer drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010243013.7A CN111358948A (en) | 2020-03-31 | 2020-03-31 | Camptothecin-berberine/indocyanine green nano-drug, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010243013.7A CN111358948A (en) | 2020-03-31 | 2020-03-31 | Camptothecin-berberine/indocyanine green nano-drug, preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111358948A true CN111358948A (en) | 2020-07-03 |
Family
ID=71199321
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010243013.7A Pending CN111358948A (en) | 2020-03-31 | 2020-03-31 | Camptothecin-berberine/indocyanine green nano-drug, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111358948A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111840574A (en) * | 2020-08-21 | 2020-10-30 | 西南大学 | Preparation and application of 5-aminolevulinic acid-camptothecin small-molecule prodrug |
| CN113278092A (en) * | 2021-05-24 | 2021-08-20 | 山东大学齐鲁医院 | Polymer carrier material, preparation and application thereof |
| CN115607672A (en) * | 2022-10-28 | 2023-01-17 | 宁波大学 | Novel indocyanine green/berberine hydrochloride self-assembly near-infrared photo-thermal nano reagent and preparation thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105193831A (en) * | 2015-09-14 | 2015-12-30 | 郑州大学 | Preparation method and application of indocyanine green loaded self-assembled multifunctional nano targeting system |
| WO2017042634A2 (en) * | 2015-09-10 | 2017-03-16 | Del Mar Pharmaceuticals | Use of dianhydrogalactitol or derivatives and analogs thereof for treatment of non-small-cell lung carcinoma, glioblastoma, and ovarian carcinoma by induction of dna damage and stalling of cell cycle |
| CN108409756A (en) * | 2018-03-08 | 2018-08-17 | 莎穆(上海)生物科技有限公司 | Multi-functional prodrug of a kind of heterodimer based on camptothecin and its preparation method and application |
| CN108478794A (en) * | 2018-03-29 | 2018-09-04 | 沈阳药科大学 | The structure of photosensitizer-chemotherapeutic " photochemical one " small molecule prodrugs and its self-assembled nanometer grain |
| CN108586551A (en) * | 2018-04-28 | 2018-09-28 | 山东大学 | The preparation and application of IR780-LA/CPT-ss-CPT nanoparticles |
| CN109350748A (en) * | 2018-10-24 | 2019-02-19 | 沈阳药科大学 | Redox sensitive key bridging small molecule prodrugs and its self-assembled nanometer grain |
| CN110251685A (en) * | 2019-06-27 | 2019-09-20 | 东南大学 | Taxol-berberine Nano medication synthetic method and application |
-
2020
- 2020-03-31 CN CN202010243013.7A patent/CN111358948A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017042634A2 (en) * | 2015-09-10 | 2017-03-16 | Del Mar Pharmaceuticals | Use of dianhydrogalactitol or derivatives and analogs thereof for treatment of non-small-cell lung carcinoma, glioblastoma, and ovarian carcinoma by induction of dna damage and stalling of cell cycle |
| CN105193831A (en) * | 2015-09-14 | 2015-12-30 | 郑州大学 | Preparation method and application of indocyanine green loaded self-assembled multifunctional nano targeting system |
| CN108409756A (en) * | 2018-03-08 | 2018-08-17 | 莎穆(上海)生物科技有限公司 | Multi-functional prodrug of a kind of heterodimer based on camptothecin and its preparation method and application |
| CN108478794A (en) * | 2018-03-29 | 2018-09-04 | 沈阳药科大学 | The structure of photosensitizer-chemotherapeutic " photochemical one " small molecule prodrugs and its self-assembled nanometer grain |
| CN108586551A (en) * | 2018-04-28 | 2018-09-28 | 山东大学 | The preparation and application of IR780-LA/CPT-ss-CPT nanoparticles |
| CN109350748A (en) * | 2018-10-24 | 2019-02-19 | 沈阳药科大学 | Redox sensitive key bridging small molecule prodrugs and its self-assembled nanometer grain |
| CN110251685A (en) * | 2019-06-27 | 2019-09-20 | 东南大学 | Taxol-berberine Nano medication synthetic method and application |
Non-Patent Citations (4)
| Title |
|---|
| YU CHENG 等: ""Mitochondria-targeting nanomedicine self-assembled from GSH-responsive paclitaxel-ss-berberine conjugate for synergetic cancer treatment with enhanced cytotoxicity"", 《JOURNAL OF CONTROLLED RELEASE》 * |
| YU CHENG 等: ""Triple stimuli-responsive supramolecular nanoassembly with mitochondrial targetability for chemophotothermal therapy"", 《JOURNAL OF CONTROLLED RELEASE》 * |
| YUANYUAN ZHANG 等: ""Polymeric nanocarriers incorporating near-infrared absorbing agents for potent photothermal therapy of cancer"", 《POLYMER JOURNAL》 * |
| 王用超: ""基于二硫键自组装多功能靶向纳米给药系统研究"", 《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111840574A (en) * | 2020-08-21 | 2020-10-30 | 西南大学 | Preparation and application of 5-aminolevulinic acid-camptothecin small-molecule prodrug |
| CN113278092A (en) * | 2021-05-24 | 2021-08-20 | 山东大学齐鲁医院 | Polymer carrier material, preparation and application thereof |
| CN115607672A (en) * | 2022-10-28 | 2023-01-17 | 宁波大学 | Novel indocyanine green/berberine hydrochloride self-assembly near-infrared photo-thermal nano reagent and preparation thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103705940B (en) | The preparation of a kind of natural activity medicine-polysaccharide targeting complex and antineoplastic application thereof | |
| CN111358948A (en) | Camptothecin-berberine/indocyanine green nano-drug, preparation method and application | |
| CN109021026B (en) | Cisplatin prodrug, preparation method and application | |
| CN103705939A (en) | Preparation of amphiphilic ursolic acid-polysaccharide coupled substance and application thereof in treating tumors | |
| Liao et al. | A chitosan/mesoporous silica nanoparticle-based anticancer drug delivery system with a “tumor-triggered targeting” property | |
| CN112494458B (en) | Construction of triglyceride-like prodrug intravenous injection self-assembly nanoparticles | |
| CN112089845B (en) | Taxane drug-adriamycin prodrug self-assembly nanoparticles and application thereof | |
| CN107158410B (en) | Folic acid-chitosan-Cy 7 polymer with tumor targeting property and preparation method thereof | |
| CN103059005B (en) | Chrysin amide derivative and medical application thereof | |
| CN110251685B (en) | Synthesis method and application of taxol-berberine nano-drug | |
| Zhang et al. | Biotin-functionalized targeting anti-tumor complex based on β-cyclodextrin and methotrexate | |
| CN108853512A (en) | The preparation and antitumor application thereof of double Types of Medicine cis-platinum/adriamycin-poly-dopamine prodrug nanoparticles | |
| CN114668840B (en) | Application of NIR-II photothermal material in preparation of cancer cell multidrug resistance reversing medicine | |
| Wang et al. | Tumor microenvironment-responsive polymer with chlorin e6 to interface hollow mesoporous silica nanoparticles-loaded oxygen supply factor for boosted photodynamic therapy | |
| CN108586551B (en) | Preparation and application of IR 780-L A/CPT-ss-CPT nanoparticles | |
| CN108524529B (en) | Acid-sensitive adriamycin prodrug based on zwitterion and folic acid targeting as well as preparation method and application thereof | |
| CN113845551A (en) | Pt (II) complex with photodynamic anti-triple negative breast cancer activity and preparation method and application thereof | |
| Fan et al. | Self-assembly of the active lactone form of a camptothecin–phospholipid complex for sustained nuclear drug delivery | |
| CN106236733A (en) | A kind of supermolecule nano particle of targeted delivery camptothecine and preparation method thereof | |
| CN113461754B (en) | Base-modified adriamycin prodrug and preparation method and application thereof | |
| CN112641760B (en) | Ferrocene-berberine/indometacin @ glucose oxidase @ hyaluronic acid nano-drug, preparation method and application | |
| CN113797350B (en) | Glycosyl polymer and preparation method and application thereof | |
| Zhang et al. | Cyclodextrin single isomer-based vesicle for chlorin e6 delivery and enhanced efficiency of photodynamic therapy for cancer treatment | |
| CN105879040B (en) | Preparation and application of polyaspartic-RGDF-antitumor drug compound | |
| CN110368500B (en) | Amphiphilic copolymer prodrug, preparation method and calcipotriol-entrapped nanoparticles |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200703 |
|
| RJ01 | Rejection of invention patent application after publication |