CN112138166B - Nano medicine, its preparation method and application - Google Patents
Nano medicine, its preparation method and application Download PDFInfo
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- CN112138166B CN112138166B CN202011033917.3A CN202011033917A CN112138166B CN 112138166 B CN112138166 B CN 112138166B CN 202011033917 A CN202011033917 A CN 202011033917A CN 112138166 B CN112138166 B CN 112138166B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- 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
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4425—Pyridinium derivatives, e.g. pralidoxime, pyridostigmine
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention belongs to the field of medicines, and particularly relates to a nano-drug which takes a spherical phosphatidylserine material as a carrier, wherein the carrier contains an acetylcholinesterase heavy activator. The invention also relates to a preparation method and application of the nano-medicament. The nano-drug can effectively penetrate through a blood brain barrier to quickly release the drug to a central nervous system, has high reactivation rate on acetylcholinesterase, good drug formation property, stable performance, safety and simple and convenient preparation, and can be used for quickly treating organophosphorus compound poisoning of the central nervous system.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a nano-drug, and a preparation method and application thereof.
Background
In the Central Nervous System (CNS), the blood-brain barrier (BBB) is the most important physiological barrier, consisting of vascular Endothelial Cells (ECs), the Tight Junctions (TJs) formed between the astral nerve endings and the basement membrane. Due to the presence of BBB, nearly 100% of large molecules cannot enter the brain, and only a portion of small molecules can reach the brain through the BBB. The BBB prevents most of the heterogeneous substances from entering the brain, protects the brain from the external environment and maintains the stability of the internal environment of the brain. In order to make drugs effectively penetrate blood brain barrier and be administered to the center, previous researches mostly focus on the design and structure modification of novel drug molecules so as to change the carried charges, improve the lipophilicity and the like. However, the steps for changing the molecular structure are complicated, and may affect the binding of the drug molecule to the target, resulting in failure to exert the drug effect. Therefore, the study scheme of molecular structure modification still cannot effectively realize central drug delivery.
One of the most important measures for the cure of neurotoxic agent intoxication is reactivation, i.e. the reactivation of the acetylcholine hydrolyzing function of acetylcholinesterase bound to the neurotoxic agent molecule. The medicine capable of restoring the activity and the function of acetylcholinesterase is called as a heavy activator for short. Taking Soman as an example, soman (Soman, chemical name: ethylhexyl methylphosphonate) is a nerve poison and is the most difficult nerve poison to prevent and treat. Currently, the reactivation agents generally applied clinically, such as pralidoxime chloride, double-phosphorus and the like, have no good reactivation effect on acetylcholinesterase poisoned by soman; while the double quaternary amine monooxime reactivation agent represented by HI-6 has good reactivation effect on the acetylcholinesterase poisoned in the periphery of soman, the molecule of the double quaternary amine monooxime reactivation agent has strong hydrophilicity and positive charge, so that the double quaternary amine monooxime reactivation agent is difficult to penetrate through a Blood Brain Barrier (BBB) to enter the center, and the reactivation on the acetylcholinesterase poisoned in the center cannot be effectively realized.
At present, the existing nano-drugs for treating central nervous system diseases can effectively carry drug molecules to penetrate through a blood brain barrier and enter the center, but most of the existing nano-drugs are slow-release nano-structures aiming at treating brain tumors and the like. However, the treatment of the poisoning caused by the nerve agent is different from the above, and the nerve agent needs to rapidly release the medicine in the center so as to rapidly take effect, because the nerve agent has a very strong inhibitory effect on the activity of acetylcholinesterase, so that acetylcholine is rapidly accumulated in the body in an excessive way, serious functional disorders of peripheral and central cholinergic nervous systems are caused, and finally convulsion, brain injury and even death of the poisoned person are caused. Therefore, there is a need for a therapeutic drug that can be rapidly released in the central nervous system to exert therapeutic effects, but there is a lack of a nano-drug that can penetrate the blood brain barrier and rapidly release the drug.
Researchers prepare the human serum albumin into nanoparticles, and then use electrostatic adsorption to load HI-6 drugs on the particle surfaces to simulate an in vitro blood brain barrier model for biological efficacy evaluation. From the aspect of pharmacodynamic evaluation, the capability of the drug to penetrate the blood brain barrier is improved to a certain extent, but the efficiency is low, the structure is unstable, and the requirement of treating the poisoning of nerve toxicants (such as soman) still cannot be met.
In addition, the existing central targeting nano-drug is basically prepared by modifying corresponding targeting components on nano-particles, and not only more additives, coupling agents and the like are needed, but also harsh synthesis conditions are needed, so that the preparation of the drug becomes complicated and tedious, for example, nano-porous silicon spheres carrying the drug need to react at high temperature under strong alkali conditions, the synthesis steps are complicated, and the time consumption is long. For the above reasons, most of the existing nano-drugs have poor drug properties, which are not favorable for pilot test and amplification of the drugs.
Therefore, in the face of the urgent practical requirement that the neurotoxic agent has no drug cure, the development of a re-activating agent which can rapidly release the drug, has high drug loading, good drug effect and simple preparation and is used for treating the organophosphorus poisoning of the central system is urgently needed.
Disclosure of Invention
One of the purposes of the invention is to provide a nano-drug, which adopts spherical phosphatidylserine materials (such as POPS) as carriers to load an acetylcholinesterase heavy activator, has high drug loading rate, can effectively penetrate blood brain barriers and quickly release the drug in the center, has high reactivation rate on acetylcholinesterase, has good drug forming property, stable performance, safety and reliability, and is simple and convenient to prepare. The invention also aims to provide a preparation method and application of the nano-drug.
In order to achieve the above object, the present invention relates to a nano-drug, which uses spherical phosphatidylserine material as a carrier, wherein the carrier contains acetylcholinesterase reactivation agent.
In some embodiments of the first aspect of the present invention, "spherical" refers to spherical or spheroidal.
In some embodiments of the first aspect of the present invention, the phosphatidylserine-based material is selected from the group consisting of 1-palmitoyl-2-oleoylphosphatidylserine (POPS) and Dimyristoylphosphatidylserine (DMPS), preferably 1-palmitoyl-2-oleoylphosphatidylserine (POPS).
In certain embodiments of the first aspect of the present invention, "comprising" means that the support is packed or coated with the acetylcholinesterase reactivation agent.
In certain embodiments of the first aspect of the present invention, the acetylcholinesterase reactivation agent is an organophosphorus compound (e.g., soman) poisoned acetylcholinesterase reactivation agent.
In some embodiments of the first aspect of the present invention, the acetylcholinesterase reactivation agent is a bis-quaternary amine monoxime reactivation agent.
In some embodiments of the first aspect of the present invention, the acetylcholinesterase reactivation agent is selected from the group consisting of amidophospine, pralidoxime chloride, and bisphosphate, preferably amidophospine (HI-6).
In some embodiments of the first aspect of the present invention, the average particle size of the nano-drug is 80 to 200nm, preferably 100 to 200nm, such as 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm.
In some embodiments of the first aspect of the present invention, the encapsulation efficiency of the nano-drug is 70% to 85%, preferably 75% to 85%, such as 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%.
In some embodiments of the first aspect of the present invention, the drug loading rate of the nano-drug is 12% to 23%, preferably 14% to 20%, such as 15%, 16%, 17%, 18%, 19%.
In certain embodiments of the first aspect of the present invention, the spherical support is formed by a bilayer, wherein the inner molecular layer is hydrophilic and the outer molecular layer is hydrophobic.
In certain embodiments of the first aspect of the present invention, the polymer dispersion coefficient (PDI) of the nano-drug is 0.1 to 0.2, such as 0.13 ± 0.02.
In some embodiments of the first aspect of the present invention, the average potential of the nanomedicine is between-40 and-28 mv, such as-32.67 ± 2.51mv.
The second aspect of the present invention relates to a method for preparing a nano-drug, comprising the steps of:
(1) Rotationally evaporating the organic solvent in the solution of the phosphatidylserine material to obtain a membrane;
(2) Mixing a solution of an acetylcholinesterase reactivation agent with a membrane at a ratio of 1 (3-16) ml/mg (e.g., 1;
(3) Repeatedly extruding the mixture through a liposome extruder for 5-40 times (preferably 10-30 times, such as 11 times, 13 times, 15 times, 16 times, 17 times, 18 times, 20 times, 22 times, 25 times, 28 times and 35 times) at a temperature not lower than the phase transition temperature of the phosphatidylserine material to obtain the nano-drug; wherein the pore size of the polycarbonate membrane in the liposome extruder is 80-200 nm (preferably 100-200 nm, such as 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180 nm).
In some embodiments of the second aspect of the present invention, in step (2), the mixing is under ultrasonic conditions.
In some embodiments of the second aspect of the present invention, in step (3), the reciprocal extrusion is performed through a liposome extruder at the phase transition temperature of the phosphatidylserine-based material.
In some embodiments of the second aspect of the present invention, the method has one or more of the following features a to J:
A. in the step (1), the phosphatidylserine material is selected from 1-palmitoyl-2-oleoyl phosphatidylserine and dimyristoyl phosphatidylserine, and preferably 1-palmitoyl-2-oleoyl phosphatidylserine;
B. in the step (1), the organic solvent is at least one selected from chloroform, acetone and ethanol, preferably chloroform;
C. in the step (1), the ratio of the phosphatidylserine material to the organic solvent in the solution is 0.01-18 mg/ml, such as 5mg/ml, 8mg/ml, 10mg/ml and 15mg/ml;
D. in the step (1), the temperature of rotary evaporation is room temperature;
E. in the step (2), the acetylcholinesterase reactivation agent is selected from pralidoxime chloride, diphosphine and amidopralidoxime, preferably amidopralidoxime;
F. in the step (2), the solvent of the solution is PBS and/or sterilized normal saline;
G. in the step (2), the concentration of the solution is 1-5 mg/ml, such as 2mg/ml, 2.2mg/ml, 3mg/ml and 4mg/ml;
H. in step (2), the mixing time is 0.5 to 20 minutes, for example, 1,2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 minutes;
I. in the step (3), the phase transition temperature of the phosphatidylserine material is 0-60 ℃, such as 6 ℃,10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 41 ℃,45 ℃, 50 ℃ and 55 ℃;
J. the nano-drug is the nano-drug according to the first aspect of the invention.
In some embodiments of the second aspect of the invention, room temperature is generally understood to be from 10 ℃ to 30 ℃.
The third aspect of the invention relates to the application of the nano-medicament in the first aspect of the invention in preparing a medicament for treating organophosphorus compound poisoning in the central nervous system.
In some embodiments of the third aspect of the present invention, the organophosphorus compound is selected from soman, sarin and novigook, preferably soman.
In some embodiments of the third aspect of the present invention, the poisoning is selected from the group consisting of mild poisoning, moderate poisoning, and severe poisoning.
In the present invention, unless otherwise specified:
the term "BBB" refers to the blood brain barrier.
The term "FLU" refers to sodium fluorescein.
The term "PBS" refers to phosphate buffered saline.
The term "BMECs" refers to murine brain microvascular endothelial cells.
The term "PDI" refers to the polymer dispersion coefficient.
The term "POPS" refers to 1-palmitoyl-2-oleoyl phosphatidylserine.
The term "DMPS" refers to dimyristoyl phosphatidylserine.
The term "DPPC" refers to phosphatidylcholine dipalmitate.
The term "MPPC" refers to 1-myristoyl-2-palmitoyl lecithin.
The term "DMPC" refers to dimyristoylphosphatidylcholine.
The term "SOPC" refers to 1-stearoyl-2-oleoyl lecithin.
The term "TJ" refers to tight junctions.
The term "CNS" refers to the central nervous system.
The term "AChE" refers to acetylcholinesterase.
The term "HI-6" refers to the heavy activator amidophospine.
The term "ATCH" refers to iodothioacetylcholine.
The term "DTNB" refers to 5,5' -dithiobis (2-nitrobenzoic acid).
The term "OD value" refers to the absorbance value measured by a microplate reader.
The term "KM mouse" refers to the outcrossing Kunming mouse with the largest production and use amount in China, and is derived from swiss mice.
The term "Ellman method" refers to the method that the substrate iodothio-Acetylcholine (ATCH) is decomposed under the action of acetylcholinesterase (AChE) to generate thiocholine, the thiocholine is rapidly acted with a color reagent 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB) to generate yellow substances with light absorption at 415nm, and the OD value of the yellow substances can reflect the activity of AChE.
The term "cholinesterase extract" refers to an agent used to extract acetylcholinesterase from the brain of a mouse.
The invention has the following beneficial effects:
1. the nano-drug has high drug loading rate.
2. The nano-drug can effectively penetrate through a Blood Brain Barrier (BBB) to rapidly release the drug to a central nervous system (such as the brain), and is suitable for rapidly curing organophosphorus compound (such as soman) poisoning of the central nervous system.
3. The nano-drug has high reactivation rate to acetylcholinesterase, and the reactivation rate to the acetylcholinesterase with soman poisoning reaches more than 40%, thus realizing the leap of the working medium for curing organophosphorus poisoning.
4. The nano-drug has good pharmacy, stable performance and simple preparation, and the phosphatidylserine carrier material can be degraded in vivo and is safe and reliable.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which
FIG. 1 is a flow chart of the production process in example 1;
FIG. 2 is a TEM photograph of example 2;
FIG. 3 is a particle size distribution diagram in example 2;
FIG. 4 is the in vitro cumulative release rate profile of HI-6 and the nano-drug of example 2;
FIG. 5 is a graph of the fluorescence intensity of the BMECs in example 4 at different time uptake of the same concentration of FLU and FLU-LPs;
FIG. 6 is a graph of the fluorescence intensity of BMECs ingesting different concentrations of FLU and FLU-LPs at the same time in example 4;
FIG. 7 shows the reactivation ratio of HI-6 and nano-drug of example 5 on whole blood AChE of soman moderately infected mice;
FIG. 8 shows the reactivation ratio of HI-6 and the nano-drug of example 5 on the whole brain AChE of the soman moderately infected mouse;
FIG. 9 shows the reactivation ratio of HI-6 and the nano-drug of example 6 on whole blood AChE of soman heavily infected mice;
FIG. 10 shows the reactivation ratio of HI-6 and the nano-drug of example 6 on the whole brain AChE of the soman heavily infected mouse;
FIG. 11 shows the reactivation ratio of HI-6 and the nano-drug of example 8 on whole blood AChE of soman heavily infected mice;
FIG. 12 is the reactivation ratio of HI-6 and the nano-drug of example 8 on the whole brain AChE of soman heavily infected mice;
FIG. 13 shows the reactivation ratio of HI-6 and various groups of nano-drugs in example 10 on whole blood AChE of soman moderately infected mice;
FIG. 14 shows the reactivation ratio of HI-6 and various groups of nano-drugs in example 10 on whole brain AChE of soman moderately infected mice.
Detailed Description
The embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying examples, in which some, but not all embodiments of the invention are shown. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1: preparation of HI-6 loaded POPS nano-drugs (HI-6-POPS-LPs)
The target drug used in this example is HI-6 (1- [ [ (4-carbamoyl-pyridine) methoxy ] methyl ] -2- (hydroxyimino-methyl) dichloride) synthesized by the institute of toxicological medicine of the institute of military medicine, purity 95% or more; POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine) is available from Guangzhou Guangdong Gaiha Biotech Co., ltd., and has a purity of 99% or more.
The preparation process is shown in figure 1.
(1) Accurately weighing 160mg of POPS into a 100ml round-bottom flask, adding 20ml of chloroform, oscillating and uniformly mixing to obtain a POPS chloroform solution, and evaporating the chloroform by a rotary evaporator at room temperature to obtain a uniform phospholipid film;
(2) Precisely weighing 44mg of HI-6, adding 20ml of PBS for dissolving to obtain 2.2mg/ml of HI-6 solution; adding 20ml of HI-6 solution into the phospholipid membrane prepared in the step (1), wherein the ratio of the HI-6 solution to the membrane is 1 ml/mg, and carrying out ultrasonic treatment in an ultrasonic instrument for 1-2min to obtain nano-drug suspension;
(3) And (3) under the ice bath condition, carrying out reciprocating extrusion on the nano-drug suspension obtained in the step (2) by a liposome extruder with the aperture of a polycarbonate membrane being 100nm for 11 times to obtain the HI-6-loaded POPS nano-drug with uniform particle size distribution.
Example 2: characterization of HI-6 loaded POPS Nanoparticulates (HI-6-POPS-LPs)
(1) And (3) morphology observation: taking 50 mu l of the nano-drug prepared in the example 1, diluting the nano-drug by PBS to a proper ratio, dripping the nano-drug on a copper net for fixing for about 10min, sucking off excessive liquid, sucking 1% uranyl acetate on the copper net, standing for 2min, sucking off the excessive liquid, and naturally drying. The sample was examined under a Transmission Electron Microscope (TEM) and the photograph is shown in FIG. 2.
As can be observed from FIG. 2, the diameter of the nano-drug is about 100nm, and the nano-drug is obviously of a double-layer structure.
(2) Particle size and potential measurements: 500. Mu.l of the nano-drug prepared in example 1 was injected into the absorption cell of the particle size analyzer, and the particle size distribution chart is shown in FIG. 3.
As can be seen from FIG. 3, the nano-drug has a concentrated particle size distribution, the average particle size is 139.38 + -1.82 nm, the polymer dispersion coefficient (PDI) is 0.13 + -0.02, the average potential of the nano-drug is-32.67 + -2.51 mv, the higher potential reduces the chance of agglomeration among particles, and improves the stability of the nano-drug.
(3) Determination of in vitro cumulative release rate curves:
high performance liquid chromatography conditions: the chromatographic column is a senescon C18 with the specification of 250 multiplied by 4.6mm; the mobile phase is acetonitrile, aqueous solution containing 5mM sodium n-heptane sulfonate and 1.2 per mill (w/w) trifluoroacetic acid; the elution procedure is characterized in that the elution is carried out at the volume ratio of acetonitrile to aqueous solution of 15; the detection wavelength is 300nm; the peak emergence time is 7.4min; the flow rate is 2mL/min; the column temperature is 30 ℃; the amount of sample was 20. Mu.L.
Establishment of a standard curve: precisely weighing 10mg of HI-6, preparing an HI-6 solution with the concentration of 1mg/mL by using ultrapure water, sequentially diluting the HI-6 solution to 200 mu g/mL, 100 mu g/mL, 50 mu g/mL, 25 mu g/mL, 10 mu g/mL and 5 mu g/mL by using the ultrapure water, detecting by using high performance liquid chromatography according to the conditions, and performing linear regression by using a peak area (A) as an ordinate and a concentration (c, mu g/mL) as an abscissa and drawing a standard curve. The results show that: the quasi-curve equation is: a =12.74c +5.387 2 =0.9997; HI-6 has a good linear relation between the concentration and the peak area within the concentration range of 1-200 mug/ml.
The nano-drug prepared in the example 1 is uniformly oscillated, PBS is used as a release medium, 1mL of nano-drug and 1mL of HI-6 are respectively absorbed and put into two dialysis bags, the dialysis bags are respectively placed into 800mL of PBS at 37 ℃, 3mL of PBS is taken out of 3mL of dialysis bags at each time at a preset time point, 3mL of PBS is simultaneously supplemented to ensure that the volume of the solution outside the dialysis bags is not changed, the samples are detected by adopting high performance liquid chromatography according to the conditions, the measured peak areas are substituted into a standard curve to calculate the HI-6 concentration, further the HI-6 in-vitro cumulative release rate is calculated, and the prepared in-vitro cumulative release rate curve of the drug is drawn as shown in figure 4.
As can be seen from FIG. 4, the in vitro cumulative release rate curves of HI-6 and nano-drug show that HI-6 is released at a faster rate, and is substantially completely released at 45min, while nano-drug is released only about 45% within the same time.
(4) Determination of encapsulation and drug loading:
taking 500 mul of the nano-drug prepared in example 1, placing the nano-drug in an ultrafiltration centrifugal tube (10K), centrifuging the nano-drug at the rotation speed of 4000g for 30min at 4 ℃, recording the volume of liquid in a lower chamber of the ultrafiltration tube, diluting the nano-drug by 100 times, injecting a sample into a high performance liquid chromatograph, detecting the sample according to the conditions in the item (3), substituting the measured peak area into a standard curve in the item (3) to calculate the HI-6 concentration, and then calculating the drug encapsulation efficiency (EE%) and the drug loading efficiency (DE%) according to the following formulas:
drug encapsulation efficiency (EE%) = drug loading amount of nanocarrier/total amount of drug input × 100%
Drug loading rate (DE%) = drug loading amount of nano carrier/(drug loading amount of nano carrier + amount of nano carrier) × 100%
And calculating to obtain: the drug encapsulation efficiency of the POPS nano-drug loaded with HI-6 is 77.29 +/-2.54% (w/w), and the drug loading rate is 16.54 +/-0.03% (w/w), which indicates that the nano-drug has high drug loading rate on HI-6.
Example 3: preparation of POPS nano-drugs (FLU-LPs) loaded with fluorescein sodium (FLU)
In the embodiment, a water-soluble fluorescent dye fluorescein sodium (FLU) is adopted to mark POPS nano-drugs, the FLU is used for replacing the water-soluble drug HI-6 loaded in the embodiment 1, and then the fluorescence intensity of the nano-drugs is utilized to detect whether the nano-drugs can transport the drugs to the brain and the residual situation of the drugs in the brain.
Preparation of POPS nano-drugs (FLU-LPs) loaded with water-soluble dye fluorescein sodium (FLU):
5mg of FLU was weighed accurately and dissolved in 50ml PBS to obtain a solution of 0.1mg/ml FLU. Accurately weighing 40mg POPS, adding 5ml chloroform to obtain a POPS chloroform solution, transferring to a round bottom flask, evaporating the solvent at room temperature by a rotary evaporator, adding 5ml FLU solution, performing ultrasonic treatment for 1min, performing reciprocating extrusion on the obtained nano-drug suspension by a liposome extruder with a polycarbonate membrane pore diameter of 100nm for 11 times, and storing the obtained nano-drug at 4 ℃.
Example 4: central targeting of POPS nano-drug in vitro evaluation
Quantitative assessment of intake of FLU-LPs by BMECs:
BMECs at 5X 10 3 (ii) inoculation into 96-well plates at individual/ml density, incubation for 24h, dilution of FLU (sodium fluorescein) and FLU-LPs (liposomes, prepared as in example 3) to the indicated concentrations (312.5 ng/ml, 625ng/ml, 1250ng/ml, 2500ng/ml, 3750ng/ml, 5000ng/ml, 6250ng/ml, 7500ng/ml, 8750ng/ml, 10000 ng/ml) with PBS, addition of the diluted solutions to the individual plates, CO addition into the individual plates, CO addition 2 Incubating for 1h in incubator, removing liquid after incubation, washing with PBS for 2 times, and adding 4% polymethyl methacrylateAldehyde was fixed for 15min, liquid was aspirated again, washed 2 times with PBS, stained with DAPI for 5min, liquid was aspirated a third time, and washed 2 times with PBS.
Cells were incubated with 5000ng/ml of FLU and FLU-LPs for 0.5h, 2h, 3h, 4h, respectively, all as above.
The fluorescence intensity taken up by the cells was measured by the high content imaging analysis system for each sample prepared, and the results are shown in FIGS. 5 to 6.
As can be seen from FIGS. 5-6, the intracellular fluorescence intensity of the FLU-LPs group was significantly higher than that of the FLU group when the cells were incubated with 5000ng/ml of FLU and FLU-LPs, respectively, for different periods of time. The results of 1 hour incubation period showed that the trend of uptake of FLU by BMECs was gradual with increasing drug concentration, and that the cell uptake was 1.44, 1.33, 1.17, 1.32 and 1.27 times that of the FLU group at concentrations 3750, 5000, 6250, 7500 and 8750ng/ml in the FLU-LPs group, respectively. The above results indicate that POPS can efficiently carry FLU into BMECs.
Example 5: reactivation effect of HI-6 loaded POPS nano-drug on AChE of soman moderately infected mouse
(1) Grouping experiments: kunming mice, males, randomly divided by body weight into 4 groups (15 per group) included: a normal group (no drug and no drug), an infected group (only drug and no drug), a control group (drug and 2.2mg/ml HI-6 solution), a nano-drug group (drug and HI-6-POPS-LPs nano-drug prepared in example 1, the content of the loaded HI-6 is 2.2 mg/ml).
(2) The experimental method comprises the following steps:
(1) and (4) administration. Injecting soman subcutaneously into mice of the infected group, the control group and the nano-drug group at a dose of 100 mu g/kg, and then immediately injecting corresponding drugs into tail veins of the mice of the control group and the nano-drug group, wherein each mouse is administered at a dose of 10 mu l/g;
(2) blood and brain were collected. Taking blood and whole brain of each group of mice after administration for 10min for measuring the activity of acetylcholinesterase;
(3) enzyme activity assay (Ellman method).
a. Whole blood of each group of mice was diluted with distilled water to (1;
weighing whole brains of each group of mice, adding 1.3ml of acetylcholinesterase extracting solution, homogenizing, centrifuging (4 ℃,10000rpm, 10min), taking supernate, and diluting the supernate into (1;
b. respectively sucking 20 mul of diluted whole blood and brain supernatant of each group of mice, adding the whole blood and the brain supernatant into an enzyme label plate, adding 30 mul of ATCH (3 mM) and 50 mul of PBS into a test hole, adding 80 mul of PBS into a blank control hole, arranging 3 compound holes in the blank control hole and the test hole, and finally adding the PBS into each hole to 100 mul;
c, reacting for 30min in a constant temperature box at 37 ℃;
d. add 20. Mu.l DTNB (0.03%) to each well;
e. measuring the absorbance X (OD) at a wavelength of 415 nm;
f. calculating the reactivation ratio of each group according to the measured light absorption value, and calculating a formula:
q reactivation ratio ＝(X Administration set -X Contamination group )/(X Normal group -X Toxicant exposure group )
X Administration set Subtracting the average value of the light absorption values of the blank control holes from the average value of the light absorption values of the experimental holes of the control group or the nano-drug group;
X normal group Subtracting the average value of the light absorption value of the blank control hole from the average value of the light absorption value of the test hole of the normal group;
X toxicant exposure group The average of the absorbance values of the blank control wells was subtracted from the average of the absorbance values of the experimental wells of the infected group.
(3) The experimental results are as follows:
FIG. 7 shows the reactivation ratio of HI-6 and nano-drug on whole blood AChE of soman moderately infected mice;
FIG. 8 shows the reactivation ratio of HI-6 and nano-drug on whole brain AChE of soman moderately infected mice.
As can be seen from FIG. 7, both HI-6 and the nano-drug can ensure that the reactivation rate of acetylcholinesterase of whole blood of the soman moderately infected mouse reaches about 50%, and the nano-carrier is proved to be capable of effectively releasing the loaded HI-6 and achieving the treatment effect similar to that of the free drug with the same dose.
As can be seen from FIG. 8, the HI-6 alone can not penetrate the blood brain barrier, so that the reactivation rate of acetylcholinesterase on the whole brain of the soman moderately infected mouse can only reach 17%; the nano-drug can effectively penetrate blood brain barrier and carry the drug into the center, so that the reactivation of the toxic enzyme in the center is effectively realized, the reactivation rate of acetylcholinesterase on the whole brain of the somatotoxic mouse reaches 45%, and the nano-drug is the highest value in all reports at present. According to the records of the existing documents, the mouse brain acetylcholinesterase reactivation rate reaches 10 percent, and the antidote can be effectively used; the invention realizes that the reactivation rate of acetylcholinesterase of the soman central poisoning exceeds 25 percent for the first time, and realizes the leap of the quality of the soman poisoning curing work.
Example 6: reactivation effect of POPS nano-drug loaded with HI-6 on AChE of soman heavily-infected mouse
(1) Experimental grouping: the specific procedure is the same as in example 5.
(2) The experimental method comprises the following steps: and (4) administration. Injecting the infected mice, the control mice and the nano-drug mice with 120 mu g/kg of fusman subcutaneously, and then immediately injecting the corresponding drugs into tail veins of the control mice and the nano-drug mice, wherein each mouse is administered with 10 mu l/g of the corresponding drugs;
the rest is the same as in example 5;
(3) The experimental results are as follows:
FIG. 9 shows the reactivation ratio of HI-6 and nano-drugs on whole blood AChE of soman heavily infected mice;
FIG. 10 shows the reactivation ratio of HI-6 and nano-drugs on AChE in whole brain of soman heavily infected mice.
As can be seen from FIG. 9, both HI-6 and the nano-drug can ensure that the reactivation rate of acetylcholinesterase of whole blood of the mice with severe soman infection reaches about 20%, and the reactivation rate of acetylcholinesterase of whole blood is reduced by 30% compared with that of example 5 due to severe soman infection.
As can be seen from FIG. 10, the reactivation rate of the acetylcholinesterase of HI-6 to the whole brain of the soman heavily-infected mouse can only reach 1%; the nano-drug can effectively penetrate blood brain barrier and carry the drug to enter the central nervous system, and can effectively realize the reactivation of brain acetylcholinesterase even under the severe soman contamination condition, and the reactivation rate reaches 40 percent.
Example 7: preparation of HI-6 loaded DMPS nano-drugs (HI-6-DMPS-LPs)
The target drug used in this example is HI-6 (1- [ [ (4-carbaryl-pyridine) methoxy ] methyl ] -2- (hydroxyimino-methyl) dichloride monohydrate) synthesized by institute of toxicology medicine of military medical institute, purity is above 95%; DMPS was purchased from beijing, cheng-minded to the science and technology limited.
(1) Accurately weighing 140mg of DMPS in a 100ml round-bottom flask, adding 20ml of chloroform, oscillating and uniformly mixing to obtain a DMPS chloroform solution, and evaporating chloroform by a rotary evaporator at room temperature to obtain a uniform phospholipid film;
(2) Precisely weighing 44mg of HI-6, adding 20ml of PBS for dissolving to obtain 2.2mg/ml of HI-6 solution; adding 20ml of the HI-6 solution into the phospholipid membrane prepared in the step (1), wherein the ratio of the HI-6 solution to the phospholipid membrane is 1;
(3) And (3) carrying out reciprocating extrusion on the nano-drug suspension obtained in the step (2) by a liposome extruder with a polycarbonate membrane pore size of 100nm at the temperature of 35 ℃, wherein the reciprocating extrusion times are 11 times, so as to obtain the HI-6-loaded DMPS nano-drug with uniform particle size distribution.
Example 8: reactivation effect of HI-6-loaded DMPS nano-drug on AChE of soman heavily-infected mouse
(1) Grouping experiments: kunming mice, males, randomly divided into 4 groups by body weight (15 per group) included: a normal group (no drug exposure and no drug exposure), an infected group (only drug exposure and no drug exposure), a control group (drug exposure and 2.2mg/ml HI-6 solution), and a nano-drug group (drug exposure and 2.2mg/ml HI-6-DMPS-LPs nano-drug prepared in example 7, with the content of the loaded HI-6 being 2.2 mg/ml).
(2) The experimental method comprises the following steps:
(1) and (4) administration. Injecting the infected group mice, the control group mice and the nano-drug group mice with soman subcutaneously at the dose of 120 mu g/kg, and then immediately injecting corresponding drugs into tail veins of the control group mice and the nano-drug group mice, wherein each mouse is administrated at the dose of 10 mu l/g;
(2) blood and brain were collected. Taking blood and whole brain of each group of mice after administration for 10min for acetylcholinesterase activity determination;
(3) and (4) measuring the enzyme activity. The concrete method is the same as the embodiment 5;
(3) The experimental results are as follows:
FIG. 11 shows the reactivation ratio of HI-6 and HI-6-DMPS-LPs nano-drug on whole blood AChE of soman heavily infected mice;
FIG. 12 shows the reactivation ratio of HI-6 and HI-6-DMPS-LPs nano-drug on whole brain AChE of soman heavily infected mice.
As can be seen from FIG. 11, both HI-6 and the nano-drug can ensure that the reactivation rate of acetylcholinesterase of whole blood of the soman heavily-infected mouse reaches about 23%.
As can be seen from FIG. 12, the reactivation ratio of acetylcholinesterase of HI-6 to the whole brain of the mice heavily infected with soma can only reach 7%, the reactivation ratio of acetylcholinesterase of HI-6-DMPS-LPs to the whole brain of the mice heavily infected with soma can only reach 14%, and no statistical difference is seen, which indicates that HI-6-DMPS-LPs have no central targeting property.
Example 9: preparation of HI-6 loaded Phosphatidylcholine (PC) nano-drug
The target drug used in this example was HI-6 (1- [ [ (4-carbaryl-pyridine) methoxy ] methyl ] -2- (hydroxyimino-methyl) dichlorine monohydrate synthesized by institute of toxicology medicine of military medical institute, purity of 95% or more, and each of DPPC (1, 2-dihydroxyimino-sn-3-phosphocholine), MPPC (1-morpholino-2-dihydroxyimino-sn-glycero-3-phosphocholine), DMPC (1, 2-dihydroxyimino-sn-glycero-3-phosphocholine) and SOholpc (1-dihydroxyphenyl-2-dihydroxyphenyl-sn-glycero-3-phosphocholine) and SOhol (1-phosphono-3-phosphocholine) was commercially available in the trade of 99% or more.
(1) Respectively and precisely weighing 147mg of DPPC, 141mg of MPPC, 136mg of DMPC and 158mg of SOPC in a 100ml round-bottom flask, respectively adding 20ml of chloroform, oscillating and uniformly mixing to obtain chloroform solutions of the drug carriers, and evaporating the chloroform by a rotary evaporator at room temperature to obtain uniform phospholipid films;
(2) Precisely weighing 44mg of HI-6, and adding 20ml of PBS to obtain 2.2mg/ml of HI-6 solution; adding 20ml of HI-6 solution into each phospholipid membrane prepared in the step (1), wherein the ratio of HI-6 to the membrane is 1 ml/mg, and carrying out ultrasonic treatment in an ultrasonic instrument for 1-2min to obtain nano-drug suspension;
(3) And (3) respectively carrying out reciprocating extrusion on the nano-drug suspension obtained in the step (2) through a liposome extruder with a polycarbonate membrane pore size of 100nm at the corresponding drug carrier phase transition temperature (DPPC 41 ℃, MPPC35 ℃, DMPC23 ℃ and SOPC6 ℃), wherein the reciprocating extrusion times are 11 times, and obtaining the nano-drug with uniform particle size distribution.
Example 10: reactivation of AChE (cytotoxic peptide-mediated isothermal amplification) of soman moderately infected mouse by HI-6-loaded phosphatidylcholine nano-drug
Effect
(1) Grouping experiments: kunming mice, males, randomly divided by body weight into 7 groups (15 per group) included: a normal group (no drug exposure and no drug exposure), an infected group (only drug exposure and no drug administration), a control group (HI-6 solution exposure and 2.2mg/ml administration), a nano-drug 1 group (HI-6-loaded DPPC nano-drug prepared in example 9 with a loading of HI-6 of 2.2 mg/ml), a nano-drug 2 group (HI-6-loaded MPPC nano-drug prepared in example 9 with a loading of HI-6 of 2.2 mg/ml), a nano-drug 3 group (HI-6-loaded DMPC nano-drug prepared in example 9 with a loading of HI-6 of 2.2 mg/ml), and a nano-drug 4 group (HI-6-loaded SOPC nano-drug prepared in example 9 with a loading of 2.2 mg/ml).
(2) The experimental method comprises the following steps:
(1) and (4) administration. Injecting soman subcutaneously into mice of the infected group, the control group and the nano-drug 1-4 groups at a dose of 100 mu g/kg, and then immediately injecting corresponding drugs into tail veins of the mice of the control group and the nano-drug 1-4 groups, wherein each mouse is administered at a dose of 10 mu l/g;
(2) blood and brain were collected. Taking blood and whole brain of each group of mice after administration for 10min for measuring the activity of acetylcholinesterase;
(3) and (4) measuring the enzyme activity. The concrete method is the same as the example 5;
(3) The experimental results are as follows:
FIG. 13 shows the reactivation ratio of HI-6 and various groups of nano-drugs on whole blood AChE of soman moderately infected mice;
FIG. 14 shows the reactivation ratio of HI-6 and various groups of nano-drugs on whole brain AChE of soman moderately infected mice.
As can be seen from FIG. 13, there was no statistical difference in the acetylcholinesterase reactivation rates of HI-6 and various groups of nano-drugs on whole blood of soman moderately infected mice.
As can be seen from FIG. 14, the reactivation rates of acetylcholinesterase of HI-6 and various groups of nano-drugs on the whole brain of the soman moderately infected mouse are not statistically different, which indicates that the nano-drugs have no central targeting.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (17)
1. A nanometer medicinal preparation comprises spherical phosphatidylserine as carrier, wherein the carrier contains acetylcholinesterase reactivator.
2. The nano-drug of claim 1, wherein the phosphatidylserine material is selected from the group consisting of 1-palmitoyl-2-oleoyl phosphatidylserine and dimyristoyl phosphatidylserine.
3. The nano-drug according to claim 1, wherein the phosphatidylserine material is 1-palmitoyl-2-oleoyl phosphatidylserine.
4. The nano-drug of claim 1, wherein the acetylcholinesterase re-activator is a bis-quaternary amine monoxime re-activator.
5. The nano-drug of claim 1, wherein the acetylcholinesterase re-activator is selected from amidophosdine, pralidoxime chloride, and bisphosphate.
6. The nano-drug of claim 1, wherein the acetylcholinesterase re-activator is amidophosphidine.
7. The nano-drug according to claim 1, wherein the nano-drug has an average particle size of 80 to 200nm.
8. The nano-drug according to any one of claims 1 to 7, which has a drug loading rate of 12% to 23%.
9. The nano-drug according to any one of claims 1 to 7, having a drug encapsulation efficiency of 70% to 85%.
10. A method for preparing a nano-drug, comprising the steps of:
(1) Rotationally evaporating the organic solvent in the solution of the phosphatidylserine material to obtain a membrane;
(2) Mixing the solution of the acetylcholinesterase heavy activator with the membrane according to the proportion of 1 (3-16) ml/mg to obtain a mixture;
(3) Repeatedly extruding the mixture for 5-40 times through a liposome extruder at a temperature not lower than the phase transition temperature of the phosphatidylserine material to obtain the nano-drug; wherein the aperture of the polycarbonate membrane in the liposome extruder is 80-200 nm.
11. The method of claim 10, wherein in step (2), the mixing is under ultrasonic conditions.
12. The method according to claim 10 or 11, characterized by one or more of the following a to J:
A. in the step (1), the phosphatidylserine material is selected from 1-palmitoyl-2-oleoyl phosphatidylserine and dimyristoyl phosphatidylserine;
B. in the step (1), the organic solvent is at least one selected from chloroform, acetone and ethanol;
C. in the step (1), the ratio of the phosphatidylserine material to the organic solvent in the solution is 0.01-18 mg/ml;
D. in the step (1), the temperature of rotary evaporation is room temperature;
E. in the step (2), the acetylcholinesterase reactivation agent is selected from pralidoxime chloride, double-compound phosphate and amidophospine;
F. in the step (2), the solvent of the solution is PBS and/or sterilized normal saline;
G. in the step (2), the concentration of the solution is 1-5 mg/ml;
H. in the step (2), the mixing time is 0.5-20 minutes;
I. in the step (3), the phase transition temperature of the phosphatidylserine material is 0-60 ℃;
J. the nano-drug is the nano-drug according to any one of claims 1 to 9.
13. The method according to claim 12, wherein in item a, the phosphatidylserine material is 1-palmitoyl-2-oleoyl phosphatidylserine in step (1).
14. The method of claim 12, wherein in item E, in step (2), the acetylcholinesterase reactivation agent is amidophosphidine.
15. Use of a nano-drug as claimed in any one of claims 1 to 9 for the manufacture of a medicament for the treatment of organophosphorous compound poisoning of the central nervous system.
16. Use according to claim 15, wherein the organophosphorus compound is selected from soman, sarin and novigook.
17. Use according to claim 15, wherein the organophosphorus compound is soman.
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| CN104840448A (en) * | 2014-02-17 | 2015-08-19 | 中国人民解放军军事医学科学院毒物药物研究所 | Efficient fast blood brain barrier penetrating nano drug |
| WO2019083365A1 (en) * | 2017-10-25 | 2019-05-02 | Universiteit Leiden | Delivery vectors |
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| US5080904A (en) * | 1986-10-28 | 1992-01-14 | Takeda Chemical Industries, Ltd. | Liposome composition and its production |
| CN104840448A (en) * | 2014-02-17 | 2015-08-19 | 中国人民解放军军事医学科学院毒物药物研究所 | Efficient fast blood brain barrier penetrating nano drug |
| WO2019083365A1 (en) * | 2017-10-25 | 2019-05-02 | Universiteit Leiden | Delivery vectors |
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