CN114149469A - Ruthenium complex, rabies virus-derived peptide and liposome-modified ruthenium complex - Google Patents
Ruthenium complex, rabies virus-derived peptide and liposome-modified ruthenium complex Download PDFInfo
- Publication number
- CN114149469A CN114149469A CN202111482667.6A CN202111482667A CN114149469A CN 114149469 A CN114149469 A CN 114149469A CN 202111482667 A CN202111482667 A CN 202111482667A CN 114149469 A CN114149469 A CN 114149469A
- Authority
- CN
- China
- Prior art keywords
- ruthenium complex
- liposome
- parts
- rdp
- phospholipid
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System compounds of the platinum group
- C07F15/0046—Ruthenium compounds
- C07F15/0053—Ruthenium compounds without a metal-carbon linkage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention provides an RDP-liposome modified ruthenium complex and a preparation method and application thereof. The invention provides an application of a ruthenium complex in preparing a medicine for preventing or diagnosing cryptococcus infection. The invention also provides a ruthenium complex and an RDP-liposome modified ruthenium complex. The invention proves the treatment effect of the ruthenium complex through in vivo distribution experiments, and also proves that the RDP-liposome can be targeted into a diseased part (brain) and slowly released at the diseased part, thereby providing a new substitute medicine and a new diagnosis and treatment mode for the diagnosis and treatment of fungal diseases.
Description
Technical Field
The invention relates to a diagnosis and treatment effect of a ruthenium complex, and also relates to a ruthenium complex modified by a liposome, a ruthenium complex liposome modified by Rabies virus Derived Peptide (RDP, YTIWMPENPRPGTPCDIFTNSRGKRASNGGGG (d) RRRRRR RRR), a preparation method thereof and application of RDP in targeted diagnosis and treatment of fungal diseases (such as cryptococcal meningitis).
Background
Pathogenic bacteria (such as cryptococcus) infect various organs and tissues (such as meninges and/or brain parenchyma, etc.) of the body in an opportunistic manner when the immunity of the body is low. Because the disease symptoms are hidden and are not easy to be found, the disease cannot be effectively treated in time, and the disease incidence of fungal infection is high. In recent years, with the large-scale and wide application of broad-spectrum antibiotics, hormones and immunosuppressive drugs, the genes of pathogenic bacteria are mutated to generate wide drug resistance, so that the existing drug for treating cryptococcus neoformans is not ideal in treatment effect. The existing drugs for treating fungi comprise amphotericin B, flucytosine, fluconazole and itraconazole, the drug development is early, and the side effects of long-term use are large, so that the development of new drugs for treating fungal infection is needed.
Disclosure of Invention
In order to solve the problems of the prior art, according to a first aspect of the present invention, there is provided a polypyridyl ruthenium complex which can be used for the prevention or diagnosis of fungal (e.g., cryptococcus) infections.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the structural formula of the ruthenium complex is as follows:
the ruthenium complex disclosed by the invention is applied to preparation of a medicine for preventing or diagnosing fungal (such as cryptococcus) infection diseases.
According to one embodiment of the invention, the disease of cryptococcal infection is cryptococcal meningitis.
Liposomes are vesicles that encapsulate a drug within their lipid bilayer. Wherein, the hydrophobic end of the lipid interlayer formed by the hydrophobic parts of the two layers of phospholipid molecules can be loaded with hydrophobic drugs, and the hydrophilic inner capsule cavity can be loaded with water-soluble drugs. Because the liposome is encapsulated in the liposome, the liposome can play a certain slow release role to prolong the action time of the medicament, and the liposome has the characteristic of being fused with cell membranes, so that the stability of the medicament is greatly improved. In addition, liposomes also exhibit the advantages of improved drug solubility, improved bioavailability, etc., and thus can be combined with some drugs to form drug carriers.
In a second aspect, the present invention provides a liposome of the above ruthenium complex.
The liposome of the ruthenium complex is characterized in that: the liposome is prepared from raw materials including cholesterol, ruthenium complex, vitamin E, Tween 80 and phospholipid.
The dosage of the cholesterol, the ruthenium complex, the vitamin E, the Tween 80 and the phospholipid is 1-3 parts of cholesterol, 1 part of ruthenium complex, 2-5 parts of vitamin E, 802-5 parts of Tween and 16-30 parts of phospholipid. Preferably 1 part of cholesterol, 1 part of ruthenium complex, 2 parts of vitamin E, 802 parts of Tween and 20 parts of phospholipid.
In a third aspect, the present invention provides liposomes of an RDP modified ruthenium complex.
A RDP-modified liposome of a ruthenium complex, characterized in that: the liposome is prepared from raw materials including cholesterol, ruthenium complex, vitamin E, Tween 80, RDP and phospholipid.
The dosage of the cholesterol, the ruthenium complex, the vitamin E, the Tween 80, the RDP and the phospholipid is 1-3 parts of cholesterol, 1 part of ruthenium complex, 2-5 parts of vitamin E, 802-5 parts of Tween, 3-6 parts of RDP and 16-30 parts of phospholipid. Preferably 1 part of cholesterol, 1 part of ruthenium complex, 2 parts of vitamin E, 802 parts of Tween, 3 parts of RDP and 20 parts of phospholipid.
Precisely weighing various lipid materials according to the above formula, placing in a eggplant-shaped bottle, adding chloroform for dissolving, performing rotary evaporation at 35-37 deg.C under reduced pressure for 15-20min to uniformly form a thin film on the wall of the bottle, adding deionized water, performing hydration in a shaker at 35-37 deg.C for 45-60 min to form a suspension, performing ultrasonic treatment for 200s, passing through a 220nm film to uniformly distribute the particle size, and storing in a refrigerator at 4 deg.C.
In a fourth aspect, the invention provides an application of the ruthenium complex liposome or the RDP modified ruthenium complex liposome in preparation of a medicine for preventing or treating cryptococcal meningitis.
Has the advantages that:
the invention provides a novel ruthenium complex and application of the ruthenium complex in preparing a medicament for preventing or diagnosing cryptococcus infection. The invention also provides a ruthenium complex liposome and an RDP-liposome modified ruthenium complex. The cryptococcus and the two novel ruthenium complexes are incubated together under the condition of in vitro culture, and the antibacterial activity of the cryptococcus and the two novel ruthenium complexes is detected. The absorption of the ruthenium complex in cryptococcus was determined using a fluorescence spectrophotometer and then verified by observation under a fluorescence microscope. By utilizing the fluorescence characteristic of the complex and the target brain characteristic of RDP, the liposome treatment of meningitis model mice is carried out on the basis of in vitro experiments, and the living body imaging result is observed.
The experiment determines the optimal formula for preparing the liposome through research on the dispersity and stability of the RDP-liposome modified ruthenium complex, and provides theoretical research on the treatment aspect of cryptococcal meningitis through in vivo pharmacodynamic experiments of mice. The ruthenium complex and the RDP-liposome modified ruthenium complex are prepared, and the physical and chemical properties of the ruthenium complex and the RDP-liposome modified ruthenium complex, such as particle size distribution, Zeta potential, dispersity, encapsulation efficiency, stability and the like, are characterized, and the results show that the average particle size of the prepared ruthenium complex and the RDP-liposome modified ruthenium complex is within 100nm, the encapsulation efficiency is more than 80%, and the dispersity is good.
In the experiment, both in vivo imaging experiments and in vitro organ fluorescence observation of mice show that the prepared ruthenium complex modified by the RDP-liposome has an obvious target brain effect, and in vivo pharmacodynamic experimental results show that the RDP-liposome modified ruthenium complex treatment group can last for 20 days, the survival time is prolonged by 5 days at most compared with that of a common liposome treatment group, and the average survival time is prolonged by 2 days. Provides basis for treating invasive fungal infection.
Drawings
FIG. 1 is a graph showing the results of imaging the conventional liposome of the ruthenium complex of the present invention and the RDP-modified liposome of the ruthenium complex of the present invention on the living body of an experimental mouse, respectively; the distribution of the in-vivo ruthenium complex is mainly in organs such as lung, kidney, liver, spleen and the like (left figure 1), the ruthenium complex obviously enters the brain (right figure 1) after RDP-liposome modification, and the experimental result shows that the RDP-liposome ruthenium complex has strong target brain treatment effect.
FIG. 2 is a comparison graph of the results of two drug treatment groups of a conventional liposome and an RDP-liposome modified ruthenium complex of the present invention, wherein the control group is treated with normal saline, the blank group is a group of mice treated with the same mass of ruthenium complex without any treatment, the group of ruthenium complex without RDP-liposome is the group of ruthenium complex, and the group of ruthenium complex liposome with RDP-liposome is the group of ruthenium complex liposome.
FIG. 3 is a graph of the use of ruthenium complexes to aid in the diagnosis of pathogenic bacteria in tissue homogenates; 3a, pathogenic bacteria under a microscope, and 3b, pathogenic bacteria added with ruthenium complex; 3c is a pathogenic bacterium to which the ruthenium complex has been added.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure. Except for special description, the parts are parts by weight, and the percentages are mass percentages.
The raw materials and reagents used in the invention are all commercial products.
Novel polypyridine ruthenium complex autonomously synthesized in laboratory, peptone (Angel Yeast Co., Ltd.), yeast extract powder (Angel Yeast Co., Ltd.), glucose (Du Ke Long chemical reagent plant), agar powder (Du Ke Long chemical reagent plant), KC-39 (Jier Biochemical Shanghai Co., Ltd.), sodium chloride injection (Shijianzhuang Siyao Co., Ltd.), lecithin (Shandong Xiya chemical industry Co., Ltd.), cholesterol (Hefei Bo Mei Biotech Co., Ltd.), N, N-dimethylformamide (Hefei Bo Mei Biotech Co., Ltd.), chloroform (Du Ke Long chemical reagent plant), Tween-80 (Du Jiang Xin Jiang district Mulan industry development area), N-methylmorpholine, blood cell counting plate, microscopic microscope cover glass (Su Shi Tai laboratory appliances Co., Ltd.), india ink, 15mL eggplant-shaped bottles, and the like.
EXAMPLE 1 preparation of ruthenium complexes according to the invention
The structural formula of the ruthenium complex is as follows:
the preparation steps are as follows:
the first step is as follows: adding RuCl3(2.0mmol) and 2-phenylpyridine (4.4mmol) were combined in a 250mL round bottom flask, ethylene glycol ethyl ether (60mL) and water (20mL) were added and heated to 140 ℃ under reflux for 24h, cooled to room temperature and filtered under suction, washed with a mixed solution of acetone and ethanol during suction (60mL:60mL), dried under vacuum to give a yellow solid [ (bpy)2Ru(μ-Cl)2]20.85g (without further purification) was obtained in about 60% yield.
The second step is that: weighing [ (bpy)2Ru(μ-Cl)2]2(0.076mmol) and 1,10 phenanthroline-5, 6-dione (0.195mmol) in a sealed brown vial equipped with a septum, adding 8mL of ethylene glycol, heating to 130 deg.C, stirring for reaction for 15h, cooling to room temperature, adding the reaction solution to a centrifuge tube containing 35mL of water, mixing, adding excess ammonium hexafluorophosphate (NH)4PF6) The product was precipitated, centrifuged to remove the clear solution and washed twice with water and purified by column chromatography on silica gel using acetonitrile as eluent: water: saturated potassium nitrate solution 150:3:1, the solvent was evaporated under reduced pressure to 1mL, the product precipitated by addition of excess water and washed with water (to remove potassium nitrate in the eluent) and dried under vacuum to give 43mg of a pink solid in about 46% yield. On a nuclear magnetic resonance spectrometer Bruker Advance IIIThe 1H and 13C nuclear spectra were recorded to determine their composition. The nuclear magnetism is characterized as: δ (ppm)8.52(s,1H),8.50(s,1H),8.485(d, J ═ 2.0Hz,1H),8.475(d, J ═ 2.0Hz,1H),8.345(d, J ═ 2.7Hz,1H),8.328(d, J ═ 2.7Hz,1H),8.05(m,2H),7.86(m,2H),7.73(m,2H),7.61(d, J ═ 6.1Hz,1H),7.59(d, J ═ 6.0Hz,1H),7.48(m,1H),7.44(m,1H),7.41(m,4H),2.207(s,3H),2.198(s,3H),1.426(s,9H),1.412(s,9H), 13H, 13.13 (m,4H), 13.56 (m, 18 v ═ 50H), 18.5918 cm (ir, 18.18 cm), 18.5918 nm, 50H), 3H, (d, 18.18.18.18.5918H), 3H), (18.18.18H), (3H), (18H), (3H, 3H), (3H, 13H, 3H), (3H, C, 13H), (3H, C, 18H), C, 18H), and so forth) of NMR (m, 18H), 3H), C, 18H), 3H, C, 3H), 3H, C, 18H, C-1)2960,2872,1615,1466,1414,1244,828,762,606,555,419.CD(Δε/M-1cm-1MeCN) 279nm (-138),293.5nm (+ 315). The molecular formula of the molecular formula is C40H44F6N6PRu (M-PF6) and the molecular weight is 855.2298 through high-resolution mass spectrometry.
EXAMPLE 2 investigation of the anti-Cryptococcus Activity of ruthenium complexes
The laboratory culture method of cryptococcus comprises the following steps: 1. preparing YPD solid culture medium and YPD liquid culture medium; 2. sterilizing the prepared culture medium at high temperature in a high-pressure steam sterilization pot; 3. cooling the YPD solid culture medium, and drying the water on the surface of the container; 4. inoculating a small amount of cryptococcus in a YPD solid culture medium for culture; 5. placing the inoculated culture dish in a constant temperature incubator at 30 ℃ for culture (inverted culture); 6. the strain grown to logarithmic phase in YPD solid medium was inoculated to a small amount of YPD liquid medium and grown up (shaking at low speed).
Determination of Minimum Inhibitory Concentration (MIC) and half maximal inhibitory concentration (IC50) of polypyridyl ruthenium complex:
minimum Inhibitory Concentration (MIC) assay:
firstly, a preliminary experiment is carried out on the minimum inhibitory concentration of the drug in the experiment by a paper sheet method: the whole surface of YPD solid medium was sufficiently and uniformly coated with a suspension of Cryptococcus bacterium dipped with a sterile cotton swab at a corrected turbidity concentration of 0.5. And sticking the paper containing the ruthenium complex medicine after the moisture is completely volatilized. The drug sensitive paper pieces are pasted on the surface of the culture medium by using sterile forceps, 6 paper pieces are pasted on each flat plate, the distance between the paper pieces is not less than 24mm, and the distance between the center of each paper piece and the edge of a culture dish is not less than 15 mm. The paper sheet is attached within 15min after the bacteria inoculation. The culture dish is inverted and incubated at a constant temperature of 30 ℃ for 18-24 h.
The method for measuring the minimum inhibitory concentration of the polypyridine ruthenium complex medicament comprises the following steps: 1. adding 100 mu L of liquid culture medium into each well; 2. adding 100 μ L of medicinal liquid (2.5mg/mL) into the first hole; 3. adding 100 mu L of liquid in the first hole into the second hole, and sequentially diluting; 4. then 100. mu.L of the expanded cryptococcus strain liquid (2X 10) was added to each well4one/mL); 5. the initial concentration of the drug was 1.25mg/mL, incubated for 48h, and the wells were observed for the presence of white spots. The hole with the initial white point is recorded as the starting point for inhibiting the growth of the fungi, the last point with the white point is the minimum inhibitory concentration point, the experiment is repeated for three times, and the Minimum Inhibitory Concentration (MIC) value can be obtained according to the initial concentration of the medicament.
Determination of half inhibitory concentration (IC50) of polypyridyl ruthenium Complex drugs [11]: 1. adding 100 μ L of expanded Cryptococcus strain liquid (2 x 10) per well4One per mL), culturing for 24h until the cells grow more than 50% adherent; 2. adding 100 μ L of medicinal liquid (2.5mg/mL) into the first hole, adding 100 μ L of the first hole into the second hole, sequentially diluting, and waiting for 48 hr; 3. discarding the original culture solution and medicinal liquid, adding 100 μ L of fresh culture solution, adding 20 μ L of LMTT solution (prepared by the subject group, final concentration of 10%), and continuing culturing; 4. after 4h of incubation with MTT solution, crystals were fully formed. At the moment, the 96-well plate is slightly inverted and placed on a plurality of pieces of filter paper paved on a table top in advance, so that the supernatant can be sucked away; 5. then, 100. mu.L of dimethyl sulfoxide was added to each well, and the mixture was shaken on a low-speed shaker for about 10min to dissolve the crystals formed by the binding of MTT and DNA. Finally, the absorbance (i.e., OD) of each well was measured at 490nm using an enzyme-linked immunosorbent assay.
Observation of metabolic activity of polypyridine ruthenium complexes in cryptococcus:
the concentration of the polypyridyl ruthenium complex was determined using an F-7000 spectrofluorometer. 1 mu L of two ruthenium complex medicines with the same concentration are respectively incubated with 1mL of cryptococcus suspension cultured overnight for 2h, washed with sterile physiological saline for three times and centrifuged, and whether the polypyridine ruthenium complex is combined with the cryptococcus thalli or not is observed by using a fluorescence microscope.
Results for in vitro cryptococcus activity against polypyridyl ruthenium complexes:
general laboratory culture results for cryptococcus:
the cryptococcus has the characteristic of adherent growth, bacterial colonies on a solid culture medium are greasy, the surface is smooth and viscous, the edges of the bacterial colonies are defective, and the color of the bacterial colonies is milky; propagating in liquid culture medium in a manner of precipitation growth.
The paper method has the following preliminary experiment results on the minimum inhibitory concentration of the ruthenium complex medicament:
cryptococcus in the bacteriostatic concentration range around the paper sheet can not grow continuously, and the strains outside the bacteriostatic concentration range can grow continuously, so that a layer of transparent bacteriostatic zone is formed around the paper sheet, the bacteriostatic zones of the ruthenium complex drugs under the concentrations of 1.25mg/mL and 2.5mg/mL are both larger than those of the fluconazole drugs, and the higher the concentration is, the larger the bacteriostatic zone is, the more obvious the bacteriostatic zone is, and the reference is provided for the next determination of the minimum bacteriostatic concentration (MIC) value.
The experimental result of the Minimal Inhibitory Concentration (MIC) determination of the ruthenium complex medicament is as follows:
the MIC of the ruthenium complex drug is 1.25 x 2 obtained by three repeated experiments according to the determination method of the minimum inhibitory concentration- 3mg/mL。
Determination of half inhibitory concentration (IC50) of ruthenium complex drug:
calculating the half inhibitory concentration (IC50) value of the ruthenium complex drug by a Bliss method: 0.005655878 μmol/mL.
The result of the metabolic activity determination of the pyridine ruthenium complex in cryptococcus bacteria is as follows:
in the experiment, the polypyridine ruthenium complex has a good binding effect with cryptococcus, and the binding capacity of the ruthenium complex with cryptococcus is stronger than that of fluconazole. Under a high power microscope, the combination of the drug and the nucleus in the cryptococcus bacterium body can be observed, so that the experimental drug can penetrate through the hard shell of the cryptococcus and be combined with the cryptococcus. The research also finds that the polypyridine ruthenium complex autonomously synthesized in a laboratory has strong fluorescence, and the characteristic is utilized to provide convenience for subsequent targeted therapy.
Example 3
Preparation and characterization of ruthenium Complex liposomes
Prescription: 1mg of cholesterol, 1mg of ruthenium complex, 2mg of vitamin E, 802mg of Tween and 20mg of phospholipid.
Precisely weighing various lipid materials according to the above formula, placing in a prepared 15mL eggplant-shaped bottle, adding 4-4.5mL chloroform for dissolving, carrying out rotary evaporation at 37 ℃ under reduced pressure for 15min to uniformly form a layer of thin film on the wall of the bottle, adding 2mL deionized water, hydrating in a 37 ℃ shaking table for 45min-60min to form a suspension, carrying out intermittent ultrasonic treatment for 180s, passing through a 220nm film to uniformly distribute the particle size, and storing in a 4 ℃ refrigerator.
The inventors examined the effect of the formulation and the preparation process on the preparation of the ruthenium complex liposome, and the results are as follows.
1. The drug lipid ratio has a great influence on the encapsulation efficiency. As the amount of phospholipid is increased, more of the internal aqueous phase, and correspondingly more drug (ruthenium complex), can be encapsulated. However, the concentration of phospholipids must not be too high, otherwise stable and homogeneous liposomes cannot be formed. The results show that the highest encapsulation efficiency can be achieved when the medicine-fat ratio reaches 1: 16 (mass ratio), and the encapsulation efficiency can not be increased when the medicine-fat ratio is increased continuously.
2. Drug loading temperature when active drug loading is carried out, a certain temperature needs to be given to enhance the permeability of the liposome bilayer and enable drug molecules to cross a certain energy barrier and pass through the phospholipid bilayer. The experimental result shows that the highest encapsulation efficiency can be achieved when the temperature is about 37 ℃. When the temperature is further increased, the encapsulation efficiency is reduced to a certain degree, and the presumed reason is that the permeability of the liposome membrane is increased along with the increase of the temperature, and when the permeability is increased to a certain degree, the membrane is cracked, so that H & lt + & gt in the membrane is leaked, and the ion gradient is weakened; at the same time, it also results in an increase in the leakage rate of the drug. The influence of temperature on the stability of the liposome is comprehensively considered, and the drug loading temperature is selected to be 37 ℃.
3. When the heating time during drug loading is active, the drugs are added into the external water phase of the liposome, and enter the liposome under the driving of the ion gradient, so that the balance is quickly achieved; meanwhile, the influence of temperature on the stability of the liposome is considered, so the drug loading process is controlled in a shorter time. When the heating time is too short, the drug has not reached equilibrium of distribution yet, and thus the encapsulation efficiency is low. When the drug distribution reaches equilibrium, the encapsulation efficiency does not further increase with the increase of the heating time, and on the contrary, the drug has certain leakage at high temperature. Therefore, when the heating time is extended to 20min, the encapsulation efficiency is slightly decreased.
Example 4
Preparation of RDP-modified ruthenium complex liposome RDP-liposome-modified ruthenium complex was prepared according to the method of example 3.
Prescription: 1mg of cholesterol, 1mg of ruthenium complex, 2mg of vitamin E, 802mg of Tween, 3mg of RDP and 20mg of phospholipid.
The polypeptide RDP used in the experiment is Rabies virus derived peptide (Rabies virus derived peptide, RDP, YTIWMPENPRPGTPCTFIFSRGKRASNGGGG (d) RRRRRR RRR), a derived peptide capable of targeting the central nerve of the brain obtained by physically stirring a linker DSPE-PEG-NHS with RVG derived from Rabies virus glycoprotein (Wang Qianghua, Cheng Shang, Qin Fen, FuAiling.and Fu Chen.application of RVGpeptides to the surface of the regional viral agents, RSC Advances,2021,11(15), pp.8505-8515.).
Example 5
Characterization of liposomes prepared in examples 3-4
And (3) particle size measurement: taking 1mL of liposome solution to perform determination on a Malvern laser particle size analyzer;
potential measurement: taking 1mL of liposome solution to perform determination on a Malvern Zeta potential analyzer;
and (3) measuring the liposome encapsulation efficiency by adopting a dialysis demulsification method and combining an F-7000 fluorescence spectrophotometer: the experiment was divided into two portions (500. mu.L each), one portion was dialyzed with 200mL of absolute ethanol dialysate (1:1) for 6 hours, the liposome portion was collected, 1 drop of Tween-80 was added to break the emulsion, and the concentration C of the drug entrapped in the liposomes was measured by fluorescence spectrophotometry1(ii) a Adding 1 drop of Tween-80 into the other part, demulsifying directly, and performing fluorescence spectrophotometryDetermination of ruthenium Complex liposomes to give C2Calculated according to the following formula, the encapsulation efficiency is C1/C2X 100% calculation.
Characterization of the liposomes of physicochemical properties of the ruthenium complexes prepared in example 3:
the average particle size of the prepared ruthenium complex is about 43nm, the potential is-24.8 mV, the dispersity is good, and the encapsulation rate is about 88% by a fluorescence photometry method. The solution is stored in a refrigerator at 4 ℃, and is clear, transparent and brownish opalescent. The results show that the prepared ruthenium complex has the particle size within 100nm and good dispersity. After being placed in a refrigerator at 4 ℃ for 60 days, the appearance is still clear and transparent and respectively presents characteristic opalescence, and no obvious change is caused compared with the original preparation. The encapsulation efficiency is measured, the encapsulation efficiency is still close to 80% in 60 days, and good stability is shown.
Characterization of RDP-liposome modified ruthenium complex prepared in example 4:
the average particle size of the prepared RDP-liposome modified ruthenium complex is about 43nm, the potential is-26.5 mV, the dispersity is good, and the entrapment rate is about 87% by a fluorescence photometry method. The solution is stored in a refrigerator at 4 ℃, is clear and transparent and is light yellow opalescence, and the time can last about four weeks.
Research shows that the liposome has high penetrability to cell membranes within 100nm in particle size and can stay in inflammatory tissues for a longer time. The particle size of the RDP-liposome modified ruthenium complex prepared under the prescription condition of the experiment is within 100nm, and the dispersion degree is good. After the mixture is placed in a refrigerator at 4 ℃ for 60 days, the encapsulation efficiency is still about 80 percent, and the stability is good.
Example 6
Four groups of treatments and treatment regimens were investigated for the therapeutic effect of mice:
laboratory animal
Kunming mice with the weight of 24 +/-5 g are raised in an SPF level mouse raising room of the institute of medicine of southwest university, the temperature and the humidity are fixed, and free feeding and water feeding are carried out.
Establishment of cryptococcal meningitis model and evaluation of experimental drug diagnosis and treatment effect
The cryptococcus meningitis model establishing experiment comprises the following steps:
preparing a bacterial suspension:
1mL of sterile normal saline is taken in a sterile test tube, a proper amount of cryptococcus colonies are picked in the test tube by a sterile inoculating loop, and the test tube is slowly oscillated, so that the bacterial suspension is prepared. Covering the blood cell counting plate with a cover glass, sucking a drop of bacterial suspension, and slowly injecting the bacterial suspension from the side edge of the cover glass[7]Count under microscope (total 5 squares), calculate concentration:
repeating the above steps until the final concentration of 4.0 x 10 is prepared7pieces/mL of a physiological saline suspension of Cryptococcus, which is ready for use.
Animal treatment:
selecting experimental mice to inject cyclophosphamide injection water solution (the concentration is 10mg/mL, and the dose is 10 mu L/g body weight) into the abdominal cavity; after 24h, each mouse was anesthetized with 0.2mL of 4% chloral hydrate. Diluting the strain in logarithmic growth phase with physiological saline to obtain 4.0 x 107The concentration of each mouse is cryptococcus physiological saline suspension, 6 mu L of the bacterial suspension is injected into each mouse, the mice are normally raised after reviving, and the growth period is recorded.
Animals (cryptococcal meningitis mice) were grouped and treated:
the mice infected with cryptococcal meningitis are randomly grouped, 9 male mice with the same physiological condition and similar weight are selected for each group, 4 groups are provided, (wherein the blank group is normal mice without any reagent injection), and 1ml of 0.2mg/ml ruthenium complex liquid medicine is injected into the tail vein every day except the blank group and the control group for treatment. Once mice died, the time (unit: day) was recorded immediately and included in Excel tables for later data processing.
A. Blank groups were not treated at all
B. Control group: 1mL of physiological saline is injected
C. Ruthenium complex treatment group
D. RDP-liposome modified ruthenium complex treatment group
Live imaging results of mice treated with ruthenium complex and RDP-liposome modified ruthenium complex:
the distribution of the ruthenium complex in vivo is mainly found in organs such as lung, kidney, liver, spleen and the like by using a small animal living body imager (figure 1, left). After the RDP-liposome is modified, the ruthenium complex obviously enters the brain (right in figure 1), and the experimental result shows that the RDP-liposome ruthenium complex has a strong target brain treatment effect.
In vitro analysis of a mouse after dissecting and taking each organ, the ruthenium complex of the common liposome is mainly distributed in the organs such as liver, lung and the like, the distribution in the brain is less, the ruthenium complex modified by the RDP-liposome is distributed in a small amount in the kidney, mainly distributed in the organs such as brain, liver, lung and the like, the fluorescence intensity in the brain is obviously higher than that in other organs, and the part shows the target brain effect of the RDP more intuitively. The RDP-liposome modified ruthenium complex has good targeted treatment effect on cryptococcus meningitis.
The establishment of the cryptococcal meningitis model and the evaluation result of the treatment effect of the experimental drug are as follows:
the survival of the mice under different treatment regimes can be visualized by the statistical chart of figure 2. The control group was treated with saline. Blank groups were not treated at all. Mice were treated with the same mass of ruthenium complex, with no RDP-liposomes added being the ruthenium complex group and with RDP-liposomes added being the ruthenium complex liposome group. The experimental results found that the mice in the control group survived the shortest time, while the mice in the RDP-liposome modified ruthenium complex group survived the longest time. Wherein the survival time of the ruthenium complex group is about 2 days longer than the average survival time of the control group mice, and the survival time of the RDP-liposome modified ruthenium complex group is about 5 days longer than the average survival time of the control group disease model mice. The RDP-liposome modified ruthenium complex treatment group has the best treatment effect, and can survive for 20 days at most. The experimental results prove that the ruthenium complex has a therapeutic effect on animal disease models, and also prove that the RDP-liposome plays a certain sustained-release and targeted therapeutic effect in the process of ruthenium complex liposome treatment, so that the survival time of disease model mice is prolonged.
Example 7
The use of ruthenium complexes aids in the diagnosis of pathogenic bacteria in tissue homogenates. Tissue homogenates were extracted from euthanized mice and stained with india ink. The detection method comprises the following steps: first, a drop of 10% tissue homogenate diluted with saline is placed on a slide; 2. a small amount of india ink was also added to the tissue homogenate. 3. A small drop of ruthenium complex (final concentration of about 1%) was also mixed with the homogenate on the slide. 4. Coverslips were placed and the homogenate was observed using a fluorescence microscope (olympus, japan) and a ruthenium complex was used to aid in the detection of pathogenic bacteria in the homogenate. Under the microscope, if the homogenate emits bright circles, the infection with the pathogen is clearly diagnosed (FIG. 3).
Claims (9)
1. The ruthenium complex has the following structural formula:
2. the use of the ruthenium complex according to claim 1 for the preparation of a medicament for the prevention or diagnosis of cryptococcus infection.
3. Use according to claim 2, characterized in that: the cryptococcus infection disease is cryptococcus meningitis.
4. The liposome of a ruthenium complex of claim 1, wherein: the liposome is prepared from raw materials including cholesterol, ruthenium complex, vitamin E, Tween 80 and phospholipid, wherein the dosage of the cholesterol, the ruthenium complex, the vitamin E, the Tween 80 and the phospholipid is 1-3 parts of cholesterol, 1 part of ruthenium complex, 2-5 parts of vitamin E, 802-5 parts of Tween and 16-30 parts of phospholipid.
5. The liposome of claim 4, wherein: sterol 1 part, ruthenium complex 1 part, vitamin E2 part, Tween 802 part and phospholipid 20 part.
6. The liposome of a ruthenium complex of claim 1, wherein: the liposome is prepared from raw materials including cholesterol, ruthenium complexes, vitamin E, Tween 80, RDP and phospholipid, wherein the dosage of the cholesterol, the ruthenium complexes, the vitamin E, the Tween 80, the RDP and the phospholipid is 1-3 parts of cholesterol, 1 part of ruthenium complexes, 2-5 parts of vitamin E, 802-5 parts of Tween, 3-6 parts of RDP and 16-30 parts of phospholipid.
7. The liposome of claim 6, wherein: 1 part of cholesterol, 1 part of ruthenium complex, 2 parts of vitamin E, 802 parts of Tween, 3 parts of RDP and 20 parts of phospholipid.
8. A process for the preparation of liposomes according to any one of claims 4 to 7 wherein: precisely weighing various lipid materials, placing in a eggplant-shaped bottle, adding chloroform for dissolving, carrying out rotary evaporation at 35-37 ℃ under reduced pressure for 15-20min to uniformly form a thin film on the wall of the bottle, adding deionized water for hydrating in a shaking table at 35-37 ℃ for 45-60 min to form a suspension, carrying out ultrasonic treatment for 200s, passing through a 220nm film to uniformly distribute the particle size, and storing in a refrigerator at 4 ℃.
9. Use of a ruthenium complex according to claim 4 or 5 or a RDP-modified ruthenium complex according to claim 6 or 7 for the preparation of a medicament for the prevention or treatment of cryptococcal meningitis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111482667.6A CN114149469A (en) | 2021-12-06 | 2021-12-06 | Ruthenium complex, rabies virus-derived peptide and liposome-modified ruthenium complex |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111482667.6A CN114149469A (en) | 2021-12-06 | 2021-12-06 | Ruthenium complex, rabies virus-derived peptide and liposome-modified ruthenium complex |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114149469A true CN114149469A (en) | 2022-03-08 |
Family
ID=80452807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111482667.6A Pending CN114149469A (en) | 2021-12-06 | 2021-12-06 | Ruthenium complex, rabies virus-derived peptide and liposome-modified ruthenium complex |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114149469A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115583978A (en) * | 2022-09-28 | 2023-01-10 | 西南大学 | Phenazine ruthenium (II) complex hexafluorophosphate for inhibiting cryptococcus as well as preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4544545A (en) * | 1983-06-20 | 1985-10-01 | Trustees University Of Massachusetts | Liposomes containing modified cholesterol for organ targeting |
| US20110123451A1 (en) * | 2009-11-21 | 2011-05-26 | Max Kullberg | Method for Cell Delivery Using Targeted Liposomes Associated with a Hemolysin |
-
2021
- 2021-12-06 CN CN202111482667.6A patent/CN114149469A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4544545A (en) * | 1983-06-20 | 1985-10-01 | Trustees University Of Massachusetts | Liposomes containing modified cholesterol for organ targeting |
| US20110123451A1 (en) * | 2009-11-21 | 2011-05-26 | Max Kullberg | Method for Cell Delivery Using Targeted Liposomes Associated with a Hemolysin |
Non-Patent Citations (1)
| Title |
|---|
| QINGHUA WANG ET AL.: "Targeted Theranostic of Cryptococcal Encephalitis by a Novel Polypyridyl Ruthenium Complex", 《MOL. PHARMACEUTICS》, vol. 17, no. 1, pages 145 - 154 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115583978A (en) * | 2022-09-28 | 2023-01-10 | 西南大学 | Phenazine ruthenium (II) complex hexafluorophosphate for inhibiting cryptococcus as well as preparation method and application thereof |
| CN115583978B (en) * | 2022-09-28 | 2024-04-16 | 西南大学 | Phenazine ruthenium (II) complex hexafluorophosphate for inhibiting cryptococcus, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106137967B (en) | Target the preparation and application of the dual modified liposome drug delivery system of glioma | |
| CN108578711A (en) | A kind of acetylation sugar ester-mPEG2000-DSPE conjugate and the preparation method and application thereof | |
| CN112521388B (en) | Perylene bisimide derivative and application thereof | |
| CN107019673A (en) | A kind of Paclitaxel liposome preparation with tumor-targeting function and its preparation method and application | |
| CN103720658B (en) | Heparin modified Evacet preparation and preparation method thereof | |
| CN105477633B (en) | A kind of hypocrellin cationic liposomal formulation and the preparation method and application thereof | |
| CN114149469A (en) | Ruthenium complex, rabies virus-derived peptide and liposome-modified ruthenium complex | |
| Liu et al. | Peptide-based nano-antibiotic transformers with antibiotic adjuvant effect for multidrug resistant bacterial pneumonia therapy | |
| CN110772645A (en) | Functionalized cell-penetrating peptide modified drug delivery system | |
| CN104666247A (en) | Heparin-modified cleavable adriamycin liposome preparation and preparation method thereof | |
| CN108148193A (en) | A kind of liposome of high molecular material and its modification containing cholic acid | |
| CN112933045B (en) | Co-loaded dihydroartemisinin/chloroquine phosphate double-sensitive nano preparation and preparation method thereof | |
| CN102379850B (en) | Targeted administration liposome passing through mucus barriers of human bodies | |
| CN108143719B (en) | Polypeptide-carrying nanoliposome and preparation method and application thereof | |
| CN104208704A (en) | Preparation method for pH-sensitive carbon nano tube-targeted drug delivery system | |
| Li et al. | Preparation and characterization of baicalein-loaded nanoliposomes for antitumor therapy | |
| CN110898231B (en) | Functionalized Lalotxel liposome and preparation method and application thereof | |
| CN108619526A (en) | The sensitive preparation method for carrying chemotherapeutics and the nano-delivery system of P-gp reversal agent of drug resistance altogether of targeting reduction | |
| CN101015699A (en) | Polyethylene glycol-phosphatidyl ethanolamine polymer or medicinal acid addition salt and application thereof in pharmacy | |
| CN108653743A (en) | Double target liposomes of a kind of heart and brain and its preparation method and application | |
| CN102133191A (en) | Transferrin and albumin composite nano particle and preparation method and application thereof | |
| CN107028882B (en) | Physically-wrapped tumor-targeted nano drug delivery system, and preparation method and application thereof | |
| CN109276570A (en) | The Nano medication and preparation method thereof of biological source macrocycle molecule | |
| CN109316463A (en) | A kind of composite nano-granule and its preparation method and application | |
| CN115006351B (en) | PEG (polyethylene glycol) positively charged liposome and preparation method thereof |
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 |