CN114209677A - Brain-targeting nano preparation containing rhynchophylline and preparation method thereof - Google Patents

Brain-targeting nano preparation containing rhynchophylline and preparation method thereof Download PDF

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CN114209677A
CN114209677A CN202111510473.2A CN202111510473A CN114209677A CN 114209677 A CN114209677 A CN 114209677A CN 202111510473 A CN202111510473 A CN 202111510473A CN 114209677 A CN114209677 A CN 114209677A
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rhynchophylline
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柳文媛
韩凌飞
谢路宽
冯锋
刘富垒
曲玮
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Institute Of Innovative Medicine China Pharmaceutical University Hangzhou
China Pharmaceutical University
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Abstract

The invention discloses a brain-targeting nano preparation containing rhynchophylline, which is a nano preparation with a core-shell structure, prepared by taking 60% -gamma-PFGA as a core material of a core-shell structure nanoparticle, taking PLA-SS-PEG-A2 as a shell material of the core-shell structure nanoparticle, taking E-TPGS as a nonionic surfactant, and loading rhynchophylline or simultaneously loading rhynchophylline and Tariquard. The brain targeting nano preparation containing the rhynchophylline has small particle size and stable structure, has slow release and controlled release effects, overcomes the difficulty of low encapsulation rate of the rhynchophylline, and simultaneously improves the brain targeting property of the rhynchophylline.

Description

Brain-targeting nano preparation containing rhynchophylline and preparation method thereof
Technical Field
The invention belongs to the field of targeted nano preparations, relates to a brain targeted nano preparation containing rhynchophylline and a preparation method thereof, and particularly relates to a brain targeted nano preparation containing rhynchophylline or co-delivery of rhynchophylline and Tariquard and a preparation method thereof.
Background
Rhynchophylline (Rhynchophylline) is an alkaloid isolated from uncaria rhynchophylla of Rubiaceae. Uncariaine can be used as one of the medicines for treating Parkinson (PD), has nervous system protecting effect, and can regulate glutamate and acetylcholine, regulate inflammatory factor and related pathway, resist nerve cell apoptosis, and reduce intracellular Ca2+The medicine has the effects of improving neurodegenerative diseases and the like by overloading, and has certain development value as a medicine for treating or preventing PD. However, rhynchophylline is difficult to enter the brain and central nervous system through the Blood Brain Barrier (BBB), and thus effective therapeutic concentrations cannot be achieved in the brain, which also forms a great challenge for the application of drugs.
P-glycoprotein (P-glycoprotein, P-gp) is widely present on the BBB and is effective in removing drugs from the central nervous system. Rhynchophylline is a good substrate of P-gp and can promote the expression of P-gp, so that the rhynchophylline is difficult to accumulate in the brain.
At present, the research on rhynchophylline preparations is few, and only Chinese patent CN 110755632A discloses a brain-targeted rhynchophylline nano preparation, a preparation method and application in intravenous injection drugs. The preparation method has mild conditions, effectively solves the problem of converting rhynchophylline into isocoumarine, but lacks slow release and controlled release effects, and is difficult to improve the bioavailability of rhynchophylline on the basis of keeping the activity of rhynchophylline.
Disclosure of Invention
The invention aims to provide a brain-targeted nano preparation containing rhynchophylline, which has small particle size, stable structure and slow release and controlled release functions, overcomes the difficulty of low encapsulation rate of rhynchophylline, and simultaneously improves the brain targeting property of rhynchophylline and the slow release and controlled release functions of the preparation.
The purpose of the invention is realized by the following technical scheme:
a brain-targeted nano preparation containing rhynchophylline is a nano preparation with a core-shell structure, which is prepared by taking 60% -gamma-PFGA (poly-gamma-glutamic acid with a thiamine disulfide system grafting rate of 60%) as a core material of a nanoparticle with a core-shell structure, taking PLA-SS-PEG-A2 (polylactic acid-S-S-polyethylene glycol-Angiopep-2) as a shell material of the nanoparticle with the core-shell structure, taking E-TPGS (vitamin E polyethylene glycol succinate) as a non-ionic surfactant, and loading rhynchophylline or simultaneously loading rhynchophylline and Tariquard.
The 60% -gamma-PFGA takes gamma-PGA (poly gamma-glutamic acid, commonly called as natto gum) as a raw material, and the raw material is firstly reacted with CTABr (cetyl trimethyl ammonium bromide) to obtain gamma-PGA-CTA (formula I); reacting the gamma-PGA-CTA with 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole ring-opening products, namely p-bromomethylbenzoate and bromobenzyl under an alkaline condition at 50-60 ℃, and grafting the p-bromomethylbenzoate and the bromobenzyl on the gamma-PGA
Figure BDA0003405102410000021
(benzyl) and
Figure BDA0003405102410000022
the preparation method comprises the following steps of (1) preparing; wherein, in terms of glutamic acid unit, gamma-PGAThe molar ratio of CTA and 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole ring-opening products to bromomethylbenzoate is 1: 0.6-1: 0.66, and the molar ratio of gamma-PGA-CTA and bromobenzyl is 1: 0.4-1: 0.44; the alkaline environment is to add NaHCO into a reaction solvent NMP (N-methyl pyrrolidone)3，NaHCO3The mass-to-volume ratio of NMP to NMP is 20: 1-22: 1 mg/mL.
The preparation method of the gamma-PGA-CTA comprises the following steps: dissolving gamma-PGA in water, adding 3% CTABr (cetyl trimethyl ammonium bromide) aqueous solution under stirring until no precipitate is generated, and vacuum filtering to obtain gamma-PGA-CTA.
The structure of gamma-PGA-CTA is shown as formula I:
Figure BDA0003405102410000023
the molecular weight of the gamma-PGA is 10 ten thousand Da.
The structure of the p-bromomethylbenzoate of the ring-opening product of the 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole is shown as a formula II:
Figure BDA0003405102410000024
in the 60% -gamma-PFGA, bifunctional groups
Figure BDA0003405102410000025
The grafting ratio of (a) is 58-62%, preferably 60%;
Figure BDA0003405102410000026
the graft ratio of (A) is 38 to 42%, preferably 40%.
The 60% -gamma-PFGA can be prepared according to methods well known in the art. Reference may be made in particular to the following documents:
HanL.,et al.A stage-specific cancer chemotherapy strategy through flexible combination of reduction-activated charge-conversional core-shell nanoparticles.[J].Theranostics,2019,9(22):6532-6549.
the PLA-SS-PEG-A2 is prepared by taking PLA-SS-PEG-MAL (polylactic acid-disulfide bond-polyethylene glycol-maleimide) and Angiopep-2-Cys as raw materials, taking PBS buffer solution with the pH value of 7.0-7.5 as a reaction solvent, and reacting at room temperature under the conditions of light and oxygen exclusion; wherein, in PLA-SS-PEG-MAL, the molecular weight of polylactic acid is 2000Da, and the molecular weight of polyethylene glycol is 2000 Da; the mole ratio of PLA-SS-PEG-MAL to Angiopep-2-Cys is 1: 1-1: 1.1.
The amino acid sequence of Angiopep-2-Cys is Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys.
The invention also aims to provide a preparation method of the brain targeting nano preparation containing the rhynchophylline, which comprises the following steps:
step (1), preparation of an organic phase: dissolving 60% -gamma-PFGA in an organic solvent to prepare a nuclear material solution, and then adding rhynchophylline and Tariquidar to obtain an organic phase; wherein the concentration of 60% -gamma-PFGA in the nuclear material solution is 10-20 mg/mL; the mass ratio of the rhynchophylline to the 60% -gamma-PFGA is 5: 100-10: 100, and the mass ratio of the Tariquard to the 60% -gamma-PFGA is 0: 100-5: 100;
step (2), preparation of a water phase: weighing PLA-SS-PEG-A2 and E-TPGS, and dissolving with saturated uncaria aqueous alkali to obtain a water phase; wherein the mass ratio of the 60% -gamma-PFGA to the PLA-SS-PEG-A2 is 1: 2-2: 1, and the mass ratio of the 60% -gamma-PFGA to the E-TPGS is 1: 8-1: 2; the mass-volume ratio of the PLA-SS-PEG-A2 to the saturated uncaria aqueous alkali is 1: 1-3: 1 mg/mL;
step (3), preparing a nano preparation by a nano precipitation method: heating the water phase to 50-65 ℃ to ensure that the concentration of the E-TPGS is slightly lower than the critical micelle concentration of the E-TPGS at the temperature; dropping the organic phase into the water phase while stirring, stirring for 30-60 min after dropping, and cooling to room temperature to reduce the critical micelle concentration of E-TPGS and fully entrap the medicine;
step (4), washing: and (3) carrying out ultrafiltration treatment to remove the organic solvent and free materials to obtain the rhynchophylline-loaded or rhynchophylline and Tariquard-loaded nanometer preparation.
In the step (1), the organic solvent is dimethyl sulfoxide (DMSO).
Specifically, the concentration of 60% -gamma-PFGA in the nuclear material solution is 20 mg/mL; the mass ratio of the rhynchophylline to the 60% -gamma-PFGA is 10:100, and the mass ratio of the Tariquard to the 60% -gamma-PFGA is 0:100 or 5: 100.
In the step (2), the concentration of the rhynchophylline in the saturated rhynchophylline solution is 41.2 mug/mL.
Specifically, the mass ratio of 60% -gamma-PFGA to PLA-SS-PEG-A2 is 1:1, and the mass ratio of 60% -gamma-PFGA to E-TPGS is 1: 4.
In the step (4), the ultrafiltration treatment comprises the following steps: and centrifuging the Amicon Ultra-15 centrifugal filter for 10-20 min at the rotating speed of 1500-2000 rpm.
The invention has the beneficial effects that:
the brain targeting nano preparation only containing rhynchophylline or co-delivering rhynchophylline and Tariquard is prepared by using a nano precipitation method by using 60% -gamma-PFGA as a core material and PLA-SS-PEG-A2 as a shell material, is a core-shell structure nano preparation, has good entrapment performance and high entrapment rate, and the entrapment rates of the rhynchophylline and the Tariquard can reach about 60% and 90% respectively, so that the capacity of loading drugs is high. The rhynchophylline is attached or crosslinked with a core material, so that the problems of low solubility, low encapsulation rate and rapid metabolism of the rhynchophylline are solved.
The 60% -gamma-PFGA of the core material grafted with benzyl can encapsulate the drug by pi-pi accumulation to form the core of the core-shell structure nano preparation, thereby greatly improving the encapsulation, slow release and protection effects of the drug. Control of
Figure BDA0003405102410000041
The grafting rate is 58-62%, so that the core material 60% -gamma-PFGA has reduction sensitivity, drug release and charge reversal rates are balanced, and the core material can carry most drugs to carry out deep penetration. The 60% -gamma-PFGA is a nuclear material which has the functions of drug loading and sensitive reduction to form positive charges, and is beneficial to improving the encapsulation efficiency of drugs and the penetrability across BBB. Thiamine disulfide system in core material 60% -gamma-PFGA
Figure BDA0003405102410000042
Has brain locking function, and can enterAfter the central nervous system, the lipophilic moiety is reduced by disulfide reductase, which then closes to form thiazoles
Figure BDA0003405102410000043
Cannot cross the BBB by diffusion. Therefore, the structure is introduced into a drug-loaded core material and used for encapsulating drugs, and the drug-loaded core material is locked in the brain to have slow release and controlled release effects, thereby being beneficial to the sustained release of the drugs and the improvement of the bioavailability. Particularly, based on a combined administration strategy, the brain-targeting nano preparation co-delivered by rhynchophylline and Tariquar is further prepared, wherein the Tarquidar can inhibit the change of P-gp from closed conformation to open conformation so as to inhibit the efflux of rhynchophylline, and the Tarquidar has high specificity on P-gp and preferentially plays a role in BBB.
Angiopep-2 (A2 for short, the amino acid sequence is Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr) is a polypeptide derived from the Kunitz structural domain of aprotinin, and the capacity of A2 crossing BBB through the LRP1 mediated transport mechanism is obviously stronger than other ligands such as transferrin, lactoferrin and the like. The invention takes A2 as a brain targeting ligand, and the MAL group of PLA-SS-PEG-MAL and the thiol group of Angiopep-2-Cys (A2-C) are subjected to coupling reaction to prepare PLA-SS-PEG-A2, and the surface of the shell material of the nanoparticle with the core-shell structure is modified. According to the invention, the targeted substance Angiopep-2 targets the rhynchophylline to the brain, so that the transfer rate of the rhynchophylline through BBB is improved, the difficulty that the rhynchophylline is difficult to enter the brain is overcome, and the brain locking function is exerted by a thiamine disulfide system.
The brain-targeted nano preparation containing rhynchophylline has uniform particle size distribution, stable structure and good safety in a proper concentration range.
Drawings
FIG. 1: HPLC chart of the synthesis of the shell material PLA-SS-PEG-A2.
FIG. 2: the particle size distribution diagram of the drug-loaded nanoparticles with the core-shell structure is provided in the embodiment 3 of the invention.
FIG. 3: the invention embodiment 3 is a transmission electron microscope picture of drug-loaded nanoparticles with core-shell structures.
FIG. 4: the invention embodiment 3 is a potential diagram of drug-loaded nanoparticles with core-shell structures.
FIG. 5: example 3 in vitro drug release profile of drug-loaded nanoparticles with core-shell structure.
FIG. 6: the embodiment 3 of the invention provides a stable particle size change diagram of drug-loaded nanoparticles with core-shell structures.
FIG. 7: example 3 of the present invention is the toxic effect on mouse brain microvascular endothelial cells and human neuroblastoma cells.
FIG. 8: the invention example 3 influences the results of the mouse microvascular endothelial cell simulation in vitro BBB penetration experiment.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. The described embodiments are only some embodiments of the invention, not all embodiments. 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
The synthesis route of the core material 60% -gamma-PFGA is as follows:
Figure BDA0003405102410000051
the method comprises the following steps:
step (1), 4-methyl-5-hydroxyethyl thiazole (35.19mmol) and methyl iodide (106.02mmol) react in an autoclave at 60 ℃ for 3h, the reactant is transferred to a round-bottom flask with a proper amount of methanol, the solvent is evaporated to dryness under reduced pressure, and a proper amount of ether is used for washing to obtain tan 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole;
step (2), 1-bromopropane (49.83mmol) was dissolved in 30mL of ethanol containing 0.2mL of PEG400, and 10mL of 5H containing 0.2mL of PEG400 was added dropwise2O·Na2S2O3(45.21mmol) water solution, refluxing and reacting for 7h at 69 ℃, decompressing and evaporating the solvent, and drying in vacuum to obtain white crystalline powder propyl pentote salt;
step (3), 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole (21.1mmol) is dissolved in 16mL of water and addedNaOH (127.5mmol), reacting for 30min under the protection of argon and ice salt bath, adding 60mL of ethyl acetate and propyl pentote (52.5mmol) aqueous solution, and continuing to react for 2 h; transferring ethyl acetate layer, concentrating under reduced pressure to obtain yellow oily substance, and purifying by silica gel column chromatography (eluent is CH)2Cl2And MeOH at 50:1) to give 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole ring-opening product;
step (4), p-bromomethylbenzoic acid (32.26mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (35.75mmol) and 4-dimethylaminopyridine (16.92mmol) were dissolved in 24mL CH2Cl2And 10mL of DMF, stirring for 30min in ice bath, adding a ring-opening product (20.05mmol) of 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole, and reacting for 48h at room temperature; distilling off CH under reduced pressure2Cl2Adding the residual solution into water, extracting with ethyl acetate, taking an ethyl acetate layer, washing with water, drying with anhydrous sodium sulfate, concentrating under reduced pressure to obtain a light yellow solid, and purifying by silica gel column chromatography (the volume ratio of petroleum ether to ethyl acetate is 3:1) to obtain a 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole ring-opening product, namely p-bromomethylbenzoate;
step (5), dissolving 10g of gamma-PGA (with the molecular weight of 10 ten thousand Da) in 200mL of water, adding a CTABr aqueous solution with the mass fraction of 3% while stirring until no precipitate is generated, and performing vacuum filtration to obtain gamma-PGA-CTA;
step (6), 0.1532 g of gamma-PGA-CTA (containing 0.416mmol of glutamic acid unit) is taken to be dissolved in 8mL of NMP, and 0.1715g (0.263mmol) of p-bromomethylbenzoate, 32 mu L (0.175mmol) of bromobenzyl and 0.1689g of NaHCO are added to the ring-opening product of 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole3Reacting for 8 hours at 50 ℃; standing, cooling, filtering, pouring the filtrate into pre-cooled 60mL of methanol/water (1:1, v/v) containing 1% hydrochloric acid, stirring for 10min in an ice bath, performing vacuum filtration, and freeze-drying to obtain light yellow 60% -gamma-PFGA.
Of 60% -gamma-PFGA1H NMR(300MHz,DMSO-d6)δ：1.80(m,2H,β-CH2),1.98(m,2H,β-CH2),2.21(m,2H,α-CH2),4.28(m,1H,CH),8.35(brs,1H,NH),5.08(s,2H,CH2Ar),7.31(s,5H,Ar-H),0.86(3H,FG-SCH2CH2CH3),1.53(2H,SCH2CH2CH3),1.93&1.88(3H,4-CH3),2.59(2H,FG-CH2CH2OH),2.70(3H,FG-3-CH3),3.03(2H,FG-SCH2CH2CH3),4.33(2H,FG-CH2CH2O-),5.17(2H,FG-Ph-CH2O-),7.45(2H,FG-Ph-3,5-H),7.60&7.89(1H, FG-CHO),7.91(2H, FG-Ph-2, 6-H). By 5.17(2H, FG-Ph-CH)2O-) and 5.08(s,2H, CH)2Ar) peak area calculation of characteristic peak signals
Figure BDA0003405102410000071
The actual grafting rate of the gamma-PGA grafted by the method is 59.86%, and the error is smaller than that of the theoretical grafting rate of 60%;
Figure BDA0003405102410000072
the actual grafting yield to gamma-PGA was 40.14%.
TABLE 1.60% grafting ratio of gamma-PFGA
Figure BDA0003405102410000073
Figure BDA0003405102410000074
Wherein FG-Ph-CH2O-：
Figure BDA0003405102410000075
Bz: benzyl bromide.
Example 2
Synthesis of the Shell Material PLA-SS-PEG-A2:
Figure BDA0003405102410000076
taking 8.322mg of PLA-SS-PEG-MAL (the molecular weight of polylactic acid PLA is 2000Da, and the molecular weight of polyethylene glycol PEG is 2000Da) and 5.28mg of Angiopep-2-Cys (A2-C) (the molar ratio of the PLA-SS-PEG-MAL to the Angiopep-2-Cys is 1:1.1), and carrying out oxygen-insulated reaction in PBS buffer solution with the pH value of 7.4 at room temperature in a dark place for 3 hours; and (3) putting a dialysis bag with the molecular weight cutoff of 3500Da into pure water for dialysis for 24h, and removing free A2-C and buffer salt to prepare PLA-SS-PEG-A2.
Of PLA-SS-PEG-A21H-NMR(CDCl3) And (3) map display: the chemical shift of 6.72ppm is the characteristic peak of the Maleimide (MAL) group in the starting PLA-SS-PEG-MAL, compared to that of PLA-SS-PEG-A21The characteristic peak of MAL in H-NMR spectrum disappeared, indicating that the MAL group of PLA-SS-PEG-MAL was coupled with the thiol group of A2-C. The HPLC diagram (fig. 1) likewise shows: performing coupling reaction on an MAL group of PLA-SS-PEG-MAL and a thiol group of A2-C, measuring by taking a reaction solution when the reaction is 0h, detecting a reactant A2-C, measuring by taking the reaction solution after the reaction is performed for 1, 2 and 3h, and detecting no A2-C, so that the reaction of A2-C is proved to be performed, and the PLA-SS-PEG-A2 is obtained.
Example 3
Preparing a brain-targeting nano preparation co-delivered by rhynchophylline and Tariquard:
step (1), preparation of an organic phase: taking 10mg of 60% -gamma-PFGA (example 1), adding 0.5mL of DMSO, stirring and dissolving overnight to obtain a 60% -gamma-PFGA solution with the concentration of 20 mg/mL; adding 1mg of rhynchophylline and 0.5mg of Tariquar into a 60% -gamma-PFGA solution, stirring for 1h to dissolve the rhynchophylline and the Tarquidar to obtain a 60% -gamma-PFGA solution containing the rhynchophylline and the Tarquidar, and taking the solution as an organic phase;
step (2), preparation of a water phase: adding 1mL of saturated rhynchophylline aqueous solution into 2mg of PLA-SS-PEG-A2 (example 2) and 8mg of E-TPGS, and stirring for dissolving to obtain 1mL of aqueous phase with PLA-SS-PEG-A2 concentration of 2 mg/mL;
step (3), preparing a nano preparation by a nano precipitation method: heating 1mL of water phase to 65 ℃, dropwise adding 100 mu L of organic phase into the water phase while stirring, stirring for 1h, and slowly cooling to room temperature;
washing: centrifuging at 2000rpm for 10min by an Amicon Ultra-15 centrifugal filter (molecular weight cutoff is 100000Da), adding water, centrifuging twice to obtain brain targeting nanometer preparation co-delivered by rhynchophylline and Tariquard, and suspending in deionized water.
Comparative example 1
Preparing a brain-targeting nano preparation co-delivered by rhynchophylline and Tariquard:
step (1), preparation of an organic phase: taking 10mg of 60% -gamma-PFGA, adding 0.5mL of DMSO, stirring for overnight dissolution to obtain a 60% -gamma-PFGA solution with the concentration of 20 mg/mL; adding 1mg of rhynchophylline and 0.5mg of Tariquar into a 60% -gamma-PFGA solution, stirring for 1h to dissolve the rhynchophylline and the Tarquidar to obtain a 60% -gamma-PFGA solution containing the rhynchophylline and the Tarquidar, and taking the solution as an organic phase;
step (2), preparation of a water phase: adding 1mL of deionized water into 2mg of PLA-SS-PEG-A2 and 8mg of E-TPGS, and stirring for dissolving to obtain 1mL of aqueous phase with the concentration of PLA-SS-PEG-A2 of 2 mg/mL;
step (3), preparing a nano preparation by a nano precipitation method: heating 1mL of water phase to 65 ℃, dropwise adding 100 mu L of organic phase into the water phase while stirring, stirring for 1h, and slowly cooling to room temperature;
washing: centrifuging at 2000rpm for 10min by an Amicon Ultra-15 centrifugal filter (molecular weight cutoff is 100000Da), adding water, centrifuging twice to obtain brain targeting nanometer preparation co-delivered by rhynchophylline and Tariquard, and suspending in deionized water.
Comparative example 2
Preparing a brain-targeting nano preparation co-delivered by rhynchophylline and Tariquard:
step (1), preparation of an organic phase: taking 10mg of 60% -gamma-PFGA, adding 0.5mL of DMSO, stirring for overnight dissolution to obtain a 60% -gamma-PFGA solution with the concentration of 20 mg/mL; adding 1mg of rhynchophylline and 0.5mg of Tariquar into a 60% -gamma-PFGA solution, stirring for 1h to dissolve the rhynchophylline and the Tarquidar to obtain a 60% -gamma-PFGA solution containing the rhynchophylline and the Tarquidar, and taking the solution as an organic phase;
step (2), preparation of a water phase: adding 2mg of PLA-SS-PEG-A2 into 1mL of saturated rhynchophylline aqueous solution, and stirring for dissolving to obtain 1mL of aqueous phase with PLA-SS-PEG-A2 concentration of 2 mg/mL;
step (3), preparing a nano preparation by a nano precipitation method: heating 1mL of water phase to 65 ℃, dropwise adding 100 mu L of organic phase into the water phase while stirring, stirring for 1h, and slowly cooling to room temperature;
washing: centrifuging at 2000rpm for 10min by an Amicon Ultra-15 centrifugal filter (molecular weight cutoff is 100000Da), adding water, centrifuging twice to obtain brain targeting nanometer preparation co-delivered by rhynchophylline and Tariquard, and suspending in deionized water.
Comparative example 3
Preparing a brain-targeting nano preparation co-delivered by rhynchophylline and Tariquard:
step (1), preparation of an organic phase: taking 10mg of 60% -gamma-PFGA, adding 0.5mL of DMMF (N, N-dimethylformamide), stirring for overnight dissolution to obtain a 60% -gamma-PFGA solution with the concentration of 20 mg/mL; adding 1mg of rhynchophylline and 0.5mg of Tariquar into a 60% -gamma-PFGA solution, stirring for 1h to dissolve the rhynchophylline and the Tarquidar to obtain a 60% -gamma-PFGA solution containing the rhynchophylline and the Tarquidar, and taking the solution as an organic phase;
step (2), preparation of a water phase: adding 1mL of deionized water into 2mg of PLA-SS-PEG-A2 and 8mg of E-TPGS, and stirring for dissolving to obtain 1mL of aqueous phase with the concentration of PLA-SS-PEG-A2 of 2 mg/mL;
step (3), preparing a nano preparation by a nano precipitation method: heating 1mL of water phase to 65 ℃, dropwise adding 100 mu L of organic phase into the water phase while stirring, stirring for 1h, and slowly cooling to room temperature;
washing: centrifuging at 2000rpm for 10min by an Amicon Ultra-15 centrifugal filter (molecular weight cutoff is 100000Da), adding water, centrifuging twice to obtain brain targeting nanometer preparation co-delivered by rhynchophylline and Tariquard, and suspending in deionized water.
Example 4
Preparing a rhynchophylline brain-targeting nano preparation:
step (1), preparation of an organic phase: taking 10mg of 60% -gamma-PFGA, adding 0.5mL of DMSO, stirring for overnight dissolution to obtain a 60% -gamma-PFGA solution with the concentration of 20 mg/mL; taking 1mg of rhynchophylline, adding the rhynchophylline into the 60% -gamma-PFGA solution, and stirring for 1h to dissolve the rhynchophylline to obtain a rhynchophylline-containing 60% -gamma-PFGA solution serving as an organic phase;
step (2), preparation of a water phase: adding 2mg of PLA-SS-PEG-A2 and 8mg of E-TPGS into 1mL of saturated rhynchophylline aqueous solution, and stirring for dissolving to obtain 1mL of aqueous phase with the concentration of PLA-SS-PEG-A2 of 2 mg/mL;
step (3), preparing a nano preparation by a nano precipitation method: heating 1mL of water phase to 65 ℃, dropwise adding 100 mu L of organic phase into the water phase while stirring, stirring for 1h, and slowly cooling to room temperature;
washing: centrifuging with Amicon Ultra-15 centrifugal filter (molecular weight cutoff is 100000Da) at 2000rpm for 10min, adding water, centrifuging twice to obtain rhynchophylline brain targeting nanometer preparation, and suspending in deionized water.
Product performance was verified by the following experiments:
firstly, determining the drug loading rate and encapsulation rate of the nano preparation
And (3) respectively taking a proper amount of the nano preparation and the mother liquor which is not washed and purified in the step (3), extracting with acetonitrile with the volume of 5 times, performing vortex for 15min, performing ultracentrifugation at 12000rpm for 5min, taking supernate, measuring the drug content by HPLC, and calculating the drug loading rate and the encapsulation rate according to the following formulas. In example 3, the encapsulation efficiency of Tariquard is 90.84 + -0.71%, and the drug loading is 0.74 + -0.1%; the encapsulation rate of the rhynchophylline is 65.63 +/-1.12%, and the drug loading rate is 1.07 +/-0.2%. The nano preparation has good entrapment performance, high entrapment rate and strong drug loading capacity. Comparative example 1 when the saturated solution of rhynchophylline was replaced with deionized water, the encapsulation efficiency of Tariquidar was 84.96%, and the encapsulation efficiency of rhynchophylline was 40.61%; comparative example 2 without E-TPGS, the encapsulation efficiency of Tariquard was 63.90% and the encapsulation efficiency of rhynchophylline was 49.22%; comparative example 3 the encapsulation efficiency of Tariquidar was 81.24% and the encapsulation efficiency of rhynchophylline was 4.64% when the organic phase solvent was changed to DMF. Through the optimization, the problem of low encapsulation efficiency of the rhynchophylline is solved by adopting a method of using a rhynchophylline saturated solution, adding E-TPGS and selecting DMSO as an organic phase.
Figure BDA0003405102410000101
Figure BDA0003405102410000102
W is the amount of drug in the nano-preparation suspension, W0Is the dosage of the drug, WPAmount of nuclear material, WLThe particle size and potential of the nano preparation are measured
The brain-targeted nano-formulation co-delivered with Tariquidar of example 3 was taken, diluted with ultrapure water to a core material concentration of about 100 μ g/mL, particle size and potential were measured separately using a Malvern ZS 90 particle size potentiostat, and particle morphology was observed with a transmission electron microscope.
The particle size distribution of the nano preparation is shown in fig. 2, the average particle size of the nano particles is 60.66nm, and the nano particles have a low polydispersity index (PDI ═ 0.221), which indicates that the nano preparation has small particle size and uniform distribution.
The transmission electron microscope picture is shown in figure 3, and figure 3a is a nanometer preparation picture, and it can be seen that the nanometer preparation is spherical, has a core-shell structure and is uniform in particle size; fig. 3b and c are diagrams of shell and core materials, respectively, and it can be seen that the particle size of the nanoparticle prepared by using the shell and core materials alone becomes smaller, further proving the formation of the core-shell structure. The nano-preparation of example 3 was diluted with ultrapure water to a concentration of about 1mg/mL of core material, 900. mu.L of the nano-preparation was added to 100. mu.L of 100mM GSH (glutathione) aqueous solution, incubated for 1h, and the size and surface morphology of the nanoparticles were observed using a transmission electron microscope, as shown in FIG. 3d, indicating that the preparation underwent reduction response and the core-shell structure was cleaved.
The potential distribution diagram is shown in figure 4, the average Zeta potential of the nanoparticles is-19.8 mV, and the nanoparticles have larger negative charges, which indicates that the preparation process is stable and reliable, and ensures the stability of the nano preparation in-vivo circulation.
Third, evaluation of in vitro drug release of nano-preparation
Taking 1mL of brain-targeted nano preparation co-delivered by rhynchophylline and Tariquard (example 3, prepared by ultrapure water) with the nuclear material concentration of 0.5mg/mL, shaking and dialyzing by 25mL of PBS buffer solution with pH 7.4 and pH 7.4 containing 10mMGSH and PBS buffer solution without GSH (100rpm), replacing the dialyzate every 12h, dividing and taking 50 mu L (calibrating the volume of the nano preparation suspension before dividing and mixing the nano preparation suspension lightly) at 0, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72h, adding 250 mu L of acetonitrile for extraction, whirling for 15min, centrifuging at 12000rpm for 5min, taking the supernatant, measuring the content by HPLC, measuring the difference between the drug content and 0h at each time point to obtain the drug release amount, inspecting the drug release curves of the nano preparation in PBS and GSH, and evaluating the drug release performance.
In vitro drug release curve is shown in figure 5, the release rate of the nano preparation in PBS is slow, and the release degree of rhynchophylline and Tariquidar is less than 60% when the drug release amount is 72 h; the release rate in GSH is fast, and the rhynchophylline and the Tariquidar are fully released when the drug is released for 72 h. Shows that: the shell material with the reduction sensitive bond disulfide bond can make redox response to GSH, trigger the disintegration of a core-shell structure and accelerate the release of a drug. The nano preparation is proved to have good sustained release and controlled release effects. Disulfide bonds are stable in an oxidative extracellular environment and are easy to break in a reductive intracellular environment, so that the design of the invention can effectively avoid the early release of the drug in the systemic circulation process, thereby realizing the targeted release of the drug.
In vitro stability of the nanometer preparation
Taking the brain-targeted nano preparation co-delivered by the rhynchophylline and the Tariquard in example 3, diluting the brain-targeted nano preparation with ultrapure water to the concentration of a core material of about 100 mu g/mL, taking 900 mu L of the nano preparation, respectively adding 100 mu L of PBS buffer (pH 7.4) and fetal calf serum (Shanghai-Yuye Biotechnology Co., Ltd.), incubating at 37 ℃, respectively measuring the average particle size by adopting a particle size potentiometer for 0 hour, 2 hours, 4 hours, 6 hours, 8 hours and 12 hours, and showing that the nano preparation has good physiological stability, wherein the time-average particle size curve is shown in figure 6, and the particle size of the nano preparation is not obviously changed when the nano preparation is placed in PBS and 10% fetal calf serum for 12 hours.
Fifth, the toxic effect of the nanometer preparation on the mouse brain microvascular endothelial cells
Examination of the non-toxicity of the brain-targeted nano-formulation co-delivered by rhynchophylline and Tariquard in example 3 on mouse brain microvascular endothelial (bEnd.3) cells and human neuroblastoma (SH-SY5Y) cells was performed by in vitro cytotoxicity experiments.
In vitro cell experiments: collecting bEnd.3 cells or SH-SY5Y cells in logarithmic growth phase, preparing a cell suspension with a proper concentration by using a DMEM medium containing 10% fetal calf serum, inoculating 100 mu L of the cell suspension into a 96-well culture plate, culturing for 12h, and setting an experimental group after the cells adhere to the wall: per hole addAdding into culture medium containing nanometer preparation, wherein the final concentrations of rhynchophylline in the culture medium are 0.2, 0.4, 1, 2, and 4 μ g/mL respectively; a control group is additionally arranged: cells and blank medium (without nanoformulation) were added, blank group: adding only culture medium without adding cells; placing at 37 ℃ and 5% CO2Culturing for 24h, taking out 96-well plate, adding 10 μ LCCK-8 solution (highly sensitive CCK-8 cell proliferation assay kit, specification: 500 assoys) into each well, standing at 37 deg.C and containing 5% CO2The cell culture box is continuously incubated for 4 hours in a dark place, the absorbance of each hole is measured under 450nm by using an enzyme-labeling instrument, and the cell survival rate is calculated according to the following formula:
Figure BDA0003405102410000121
wherein A issAbsorbance of Experimental group, AbAbsorbance in blank group, AcControl absorbance.
The relationship between the cell survival rate and the administration concentration of the nano preparation in example 3 is shown in figure 7, and the nano preparation has low toxicity to the bEnd.3 cells and SH-SY5Y cells, high cell survival rate and dose dependence, which indicates that the nano preparation has good safety in a proper concentration range.
Influence of nano preparation on mouse microvascular endothelial cell simulation in-vitro BBB (BBB) penetration experiment
bEnd.3 cells were seeded on polycarbonate membrane of 24-well Transwell plate, and then 0.6mL of DMEM complete medium was added to the basal side of the Transwell plate, and the plate was incubated at 37 ℃ with 5% CO2Culturing in a cell culture box, changing the culture solution every two days, and changing the culture solution every day after culturing for one week. And continuously culturing for 14 days, and evaluating the function and the state of the model by adopting a 4h leakage test and a fluorescein sodium permeability test to determine the compactness and the integrity of the cell monolayer. The medium in the well was discarded as required, and the cells were washed 2 times with HBSS buffer (pH7.35) pre-warmed at 37 ℃. Parallel into 4 groups (n ═ 3), the first group: add 300. mu.L of medium containing the nanoformulation (example 4, final concentration of rhynchophylline is 10. mu.g/mL) to the apical chamber; second group: add 300. mu.L of sodium to the apical chamberRice formulation (example 3, final concentration of rhynchophylline 10 μ g/mL) medium; third group: add 300. mu.L of media containing 10. mu.g/mL of free rhynchophylline to the apical chamber; and a fourth group: add 300. mu.L of medium containing 10. mu.g/mL of free rhynchophylline and 6.9. mu.g/mL of free Tariquidar to the apical chamber; each group was loaded with 600. mu.g/mLHBSS at the basal side, the Transwell cell plates were then placed in an incubator at 37 ℃ and transported for 0.5, 1, 2, 3, 4.5h, 100. mu.L of each group of basal side solution was taken at each set time point and collected in 1.5mL EP tubes, and 100. mu.L of fresh HBSS buffer was added to the basal side well plates. Collecting the substrate side solution, adding 500 μ L acetonitrile, extracting, vortexing for 15min, ultracentrifuging (13000rpm, 5min), collecting supernatant, and determining drug content by liquid chromatography-tandem mass spectrometry.
The results of the in vitro BBB mouse microvascular endothelial cell simulated permeation experiments are shown in fig. 8. Compared with an uncaria rhynchophylline solution, an uncaria rhynchophylline and a Tariquidar solution, an uncaria rhynchophylline brain targeting nano preparation (example 4) and a brain targeting nano preparation co-delivered by the uncaria rhynchophylline and the Tariquidar (example 3) have the capacity of penetrating through a BBB, and the capacity of the brain targeting nano preparation co-delivered by the uncaria rhynchophylline and the Tariquidar to penetrate through the BBB is the strongest.

Claims (10)

1. A brain targeting nanometer preparation containing rhynchophylline is characterized in that: the nanometer preparation with the core-shell structure is prepared by taking 60% -gamma-PFGA as a core material of the nanometer particle with the core-shell structure, taking PLA-SS-PEG-A2 as a shell material of the nanometer particle with the core-shell structure, taking E-TPGS as a nonionic surfactant, and loading rhynchophylline or simultaneously loading rhynchophylline and Tariquard.
2. The brain-targeting nano-formulation containing rhynchophylline according to claim 1, wherein: the 60% -gamma-PFGA takes gamma-PGA as a raw material, and the gamma-PGA-CTA is obtained by reacting with CTABr; reacting the gamma-PGA-CTA with 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole ring-opening products, namely p-bromomethylbenzoate and bromobenzyl under an alkaline condition at 50-60 ℃, and grafting the p-bromomethylbenzoate and the bromobenzyl on the gamma-PGA
Figure FDA0003405102400000011
The preparation method comprises the following steps of (1) preparing; wherein the molecular weight of the gamma-PGA is 10 ten thousand Da; calculated by glutamic acid units, the molar ratio of gamma-PGA-CTA and 3, 4-dimethyl-5- (2-hydroxyethyl) iodothiazole ring-opening products to bromomethylbenzoate is 1: 0.6-1: 0.66, and the molar ratio of gamma-PGA-CTA and bromobenzyl is 1: 0.4-1: 0.44.
3. The brain-targeting nano-formulation containing rhynchophylline according to claim 1 or 2, wherein: in the 60% -gamma-PFGA, bifunctional groups
Figure FDA0003405102400000012
The graft ratio of (A) is 58 to 62%.
4. The brain-targeting nano-formulation containing rhynchophylline according to claim 1, wherein: the PLA-SS-PEG-A2 is prepared by taking PLA-SS-PEG-MAL and Angiopep-2-Cys as raw materials and PBS buffer solution with the pH value of 7.0-7.5 as a reaction solvent, and reacting at room temperature under the conditions of light protection and oxygen insulation; wherein, in PLA-SS-PEG-MAL, the molecular weight of polylactic acid is 2000Da, and the molecular weight of polyethylene glycol is 2000 Da; the molar ratio of PLA-SS-PEG-MAL to Angiopep-2-Cys is 1: 1-1: 1.1; the amino acid sequence of Angiopep-2-Cys is Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-Cys.
5. The brain-targeting nano-formulation containing rhynchophylline according to claim 1, wherein: the preparation method comprises the following steps:
step (1), preparation of an organic phase: dissolving 60% -gamma-PFGA in an organic solvent to prepare a nuclear material solution, and then adding rhynchophylline and Tariquidar to obtain an organic phase; wherein the mass ratio of the rhynchophylline to the 60% -gamma-PFGA is 5: 100-10: 100, and the mass ratio of the Tariquidar to the 60% -gamma-PFGA is 0: 100-5: 100;
step (2), preparation of a water phase: weighing PLA-SS-PEG-A2 and E-TPGS, and dissolving with saturated uncaria aqueous alkali to obtain a water phase; wherein the mass ratio of the 60% -gamma-PFGA to the PLA-SS-PEG-A2 is 1: 2-2: 1, and the mass ratio of the 60% -gamma-PFGA to the E-TPGS is 1: 8-1: 2; the mass-volume ratio of the PLA-SS-PEG-A2 to the saturated uncaria aqueous alkali is 1: 1-3: 1 mg/mL;
step (3), preparing a nano preparation by a nano precipitation method: heating the water phase to 50-65 ℃; dropping the organic phase into the aqueous phase while stirring, stirring for 30-60 min after dropping, and cooling to room temperature;
step (4), washing: and (3) carrying out ultrafiltration treatment to obtain the rhynchophylline-loaded or rhynchophylline and Tariquard-loaded nanometer preparation.
6. A method for preparing the brain-targeting nano preparation containing rhynchophylline, which is characterized by comprising the following steps: the method comprises the following steps:
step (1), preparation of an organic phase: dissolving 60% -gamma-PFGA in an organic solvent to prepare a nuclear material solution, and then adding rhynchophylline and Tariquidar to obtain an organic phase; wherein the mass ratio of the rhynchophylline to the 60% -gamma-PFGA is 5: 100-10: 100, and the mass ratio of the Tariquidar to the 60% -gamma-PFGA is 0: 100-5: 100;
step (2), preparation of a water phase: weighing PLA-SS-PEG-A2 and E-TPGS, and dissolving with saturated uncaria aqueous alkali to obtain a water phase; wherein the mass ratio of the 60% -gamma-PFGA to the PLA-SS-PEG-A2 is 1: 2-2: 1, and the mass ratio of the 60% -gamma-PFGA to the E-TPGS is 1: 8-1: 2;
step (3), preparing a nano preparation by a nano precipitation method: heating the water phase to 50-65 ℃; dropping the organic phase into the aqueous phase while stirring, stirring for 30-60 min after dropping, and cooling to room temperature;
step (4), washing: and (3) carrying out ultrafiltration treatment to obtain the rhynchophylline-loaded or rhynchophylline and Tariquard-loaded nanometer preparation.
7. The preparation method of the brain-targeted nano preparation containing rhynchophylline according to claim 6, wherein the preparation method comprises the following steps: in the step (1), the organic solvent is dimethyl sulfoxide.
8. The preparation method of the brain-targeted nano preparation containing rhynchophylline according to claim 6, wherein the preparation method comprises the following steps: the concentration of 60% -gamma-PFGA in the nuclear material solution is 10-20 mg/mL; the mass-volume ratio of the PLA-SS-PEG-A2 to the saturated uncaria aqueous alkali is 1: 1-3: 1 mg/mL; .
9. The preparation method of the brain-targeted nano preparation containing rhynchophylline according to claim 6, wherein the preparation method comprises the following steps: in the step (2), the concentration of the rhynchophylline in the saturated rhynchophylline solution is 41.2 mug/mL.
10. The preparation method of the brain-targeted nano preparation containing rhynchophylline according to claim 6, wherein the preparation method comprises the following steps: in the step (4), the ultrafiltration treatment comprises the following steps: and centrifuging the Amicon Ultra-15 centrifugal filter for 10-20 min at the rotating speed of 1500-2000 rpm.
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