CN111012918B - Cholesterol biguanide conjugate with anti-tumor and carrier effects and application of salt thereof in microparticle administration preparation - Google Patents
Cholesterol biguanide conjugate with anti-tumor and carrier effects and application of salt thereof in microparticle administration preparation Download PDFInfo
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Abstract
The invention provides a cholesterol biguanide conjugate and a salt thereof, a preparation method of a particle type drug delivery preparation and application thereof, aiming at the technical problems of more byproducts, low yield, difficult purification, narrow application range and the like in the preparation of the cholesterol biguanide conjugate in the prior art when the cholesterol biguanide conjugate is used for coating drugs, and the cholesterol biguanide conjugate and the salt thereof can be self-assembled into nano particles or micro particles, or the cholesterol biguanide conjugate and the salt thereof are used as carriers or used for coating drugs together with other pharmaceutic adjuvants and are prepared into the particle type drug delivery preparation. The invention can obtain the nano preparation with high encapsulation rate, good stability and more uniform grain diameter by an emulsion solvent volatilization method, and has great development potential.
Description
Technical Field
The invention belongs to the technical field of antitumor medicinal preparations, and particularly relates to an amphiphilic cholesterol biguanide conjugate and a salt thereof, and a preparation method and application thereof.
Background
At present, many commercially available antitumor drugs are fat-soluble drugs, the water solubility is poor, the oral absorption rate is not high, and even the drugs with better water solubility have the problem of poor targeting property to tumor cells after administration. The medicine is prepared into a particle type administration preparation, particularly a nano preparation, so that the requirements of various administration routes on the particle size of the preparation can be met, and the aims of improving the long circulation property, the targeting property and the membrane permeability of the preparation, reducing the toxic and side effects of the medicine and the like can be fulfilled.
The nanoparticle preparation of common antitumor drugs mostly adopts in vivo biodegradable lipoid or high molecular polymer as a carrier, and the drugs are wrapped in the nanoparticles or adsorbed on the surfaces of the nanoparticles. However, when such a carrier is used, since the nanoparticles may cause toxic and side effects in vivo as foreign substances, the smaller the amount of the carrier used, the better the safety, on the premise that the drug loading amount is satisfied. This requirement can be met by using a carrier which itself has anti-tumor activity.
Cholesterol is a substance with very good physiological compatibility, has higher affinity to tumor cells, but has strong lipid solubility, and is generally used as an auxiliary component for preparing liposome preparations. The nano-particles can be self-assembled by connecting the nano-particles with certain groups with large polarity. The biguanide group is more polar and positively charged due to the presence of the guanidino group in the structure. Some biguanide drugs are discovered to have anti-tumor activity in succession, for example, metformin is a clinical drug widely used for treating type II diabetes, and researches show that metformin is used for treating diabetes, so that the incidence rate of cancer is obviously reduced, and thus metformin is considered as a potential anti-cancer drug. However, the anti-tumor activity of the metformin is not high, and the metformin has strong water solubility, so that the metformin has a plurality of problems in preparing a nano preparation. At present, many scholars try to link a biguanide group with a lipophilic group to prepare lipid prodrugs, pharmacosomes and the like [ Meixing, etc. ], lipid prodrugs containing guanidino drugs and pharmacosomes thereof, grant No. CN 101723857B ] to obtain good effects of resisting tumors, reducing blood sugar, reducing toxic and side effects and the like. Further researchers have linked biguanides to cholesterol formyl chlorides via ethylenediamine reactions to form conjugates, but only for the entrapment of Nanoparticles formed from an EGFR siRNA drug, prepared mainly by membrane dispersion-extrusion, the conjugates being present in lipid bilayers in a formulation that is practically liposomes [ Shi K, et al.functional lipid sites to synthetic Oncogene doping and Tumor Growth inhibition ] (2017), mol.ther.25,1567-1579 ]; in addition, the specific synthesis method in this document has the following drawbacks: the first step of the synthesis method needs to drop ethylenediamine into a cholesterol formyl chloride solution, and in the former dropping process, the cholesterol formyl chloride in the solution is excessive, so that the amine groups at two ends of the firstly dropped ethylenediamine are very easy to react with the cholesterol formyl chloride to generate more byproducts, and if the ethylenediamine is inversely dropped, the expected purity is higher; in the second step, the ratio of the intermediate product to the dihydroamine and the ferric trichloride is 1:2:3, after practice, the proportion of dicyandiamide in the reaction is too small, the yield is not high, and the proportion of ferric trichloride is too large, so that the later removal is not facilitated. In addition, the reaction temperature is only 70 ℃, the reaction time is short, the yield is not high, and the forward progress of the reaction is facilitated by increasing the temperature and prolonging the reaction time. In the purification of the final product, the addition of aqueous hydrochloric acid is used in this document to remove excess dicyandiamide and ferric chloride. Practice shows that the product prepared in the way is a viscous lump in water, impurities are extremely difficult to be thoroughly washed out, part of the conjugate is washed away in each washing, the actual yield is low, and the obtained product is brown.
If the ethylenediamine in the reaction is replaced by different diamines, such as propane diamine, butane diamine and the like, the ethylenediamine also has the double functions of a carrier and an anti-tumor function, and the coating of different drugs is favorably expanded. Related studies have not been reported.
Disclosure of Invention
The invention provides a cholesterol biguanide conjugate, a salt thereof, a preparation method of a particle type administration preparation of the cholesterol biguanide conjugate, and an application of the cholesterol biguanide conjugate, aiming at the technical problems that in the prior art, the preparation of the cholesterol biguanide conjugate has a plurality of byproducts, low yield and difficult purification, and the application range of the cholesterol biguanide conjugate is narrow when the cholesterol biguanide conjugate is used for encapsulating drugs.
The invention adopts the following technical scheme:
the application of the cholesterol biguanide conjugate and the salt thereof, the cholesterol biguanide conjugate and the salt thereof are self-assembled into nano-particles or micro-particles, or the cholesterol biguanide conjugate and the salt thereof are used as carriers or used together with other pharmaceutic adjuvants to carry medicines and are prepared into micro-particle type administration preparations; the particle type administration preparation is a preparation with the particle size of micron level and nanometer level in the pharmacy category, and mainly comprises particles, nanoparticles, liposome, emulsion and polymer micelle; the medicine comprises fat-soluble medicine and negatively charged water-soluble medicine, when X (described later) is C 2 H 4 Without including epidermal growthFactor small interfering RNA (EGFR siRNA).
The application of the cholesterol biguanide conjugate and the salt thereof in the self-assembly nanoparticle preparation is carried out according to the following method and proportion: weighing 0.01-300mg of the cholesterol biguanide conjugate or the salt thereof, dissolving in 0.1-5mL of organic solvent, then adding 5mL of aqueous solution, performing ultrasonic or stirring dispersion uniformly, and evaporating to remove the organic solvent to obtain the self-assembly nanoparticle suspension of the cholesterol biguanide conjugate or the salt thereof.
The application of the cholesterol biguanide conjugate and the salt thereof in fat-soluble medicine encapsulating fat-soluble medicine nanoparticle preparation, particle, liposome, emulsion or polymer micelle is carried out according to the following method and proportion to prepare the fat-soluble medicine encapsulating fat-soluble medicine particle or nanoparticle preparation: weighing 1-300mg of the cholesterol biguanide conjugate or salt thereof and a fat-soluble drug with the mass being 0.0001-50% of the weight of the weighed cholesterol biguanide conjugate or salt thereof, dissolving the fat-soluble drug in 0.1-5mL of an organic solvent, then adding 5mL of an aqueous solution, uniformly dispersing, and evaporating to remove the organic solvent to obtain a particle or nanoparticle suspension of the cholesterol biguanide conjugate or salt thereof coated with the fat-soluble drug; or, the preparation of the fat-soluble medicine nanoparticle preparation nanoparticle, microparticle, liposome, emulsion or polymer micelle by encapsulating the fat-soluble medicine is carried out according to the following method: weighing the cholesterol biguanide conjugate or salt thereof and a fat-soluble auxiliary material a shared with the cholesterol biguanide conjugate or salt thereof, wherein the sum of the mass of the cholesterol biguanide conjugate or salt thereof and the fat-soluble auxiliary material a is 1-300mg, the cholesterol biguanide conjugate or salt thereof and the fat-soluble auxiliary material a are shared according to any mass ratio, meanwhile, weighing a fat-soluble medicament with the mass of 0.0001-50% of the mass of the weighed cholesterol biguanide conjugate or salt thereof, dissolving the fat-soluble medicament and the fat-soluble auxiliary material a into 0.1-2mL of organic solvent, then adding 5mL of aqueous solution, uniformly dispersing, and evaporating to remove the organic solvent to obtain the nanoparticle, microparticle, liposome, emulsion or polymer micelle nanoparticle suspension of the cholesterol biguanide conjugate or salt thereof encapsulating the fat-soluble medicament.
The application of the cholesterol biguanide conjugate and the salt thereof in a water-soluble drug-encapsulated water-soluble drug nanoparticle, microparticle, liposome, emulsion or polymer micelle preparation is carried out according to the following method: weighing 1-300mg of the cholesterol biguanide conjugate or the salt thereof, dissolving in 0.1-5mL of organic solvent, then adding 5mL of aqueous solution, uniformly dispersing, evaporating to remove the organic solvent, adding a negatively charged water-soluble drug with the mass of 0.0000001-10% of the weight of the weighed cholesterol biguanide conjugate or the salt thereof, uniformly dispersing again, and evaporating to remove the organic solvent to obtain a water-soluble drug-encapsulated cholesterol biguanide conjugate or salt nanoparticle suspension; or, the preparation of the water-soluble medicine-encapsulated water-soluble medicine nano-particle, liposome, emulsion or polymer micelle preparation is carried out according to the following method: weighing the cholesterol biguanide conjugate or salt thereof and a fat-soluble auxiliary material a shared with the cholesterol biguanide conjugate or salt thereof, wherein the total mass of the cholesterol biguanide conjugate or salt thereof and the fat-soluble auxiliary material a is 1-300mg, the cholesterol biguanide conjugate or salt thereof and the fat-soluble auxiliary material a are shared according to any mass ratio, the cholesterol biguanide conjugate or salt thereof and the fat-soluble auxiliary material a are jointly dissolved in 0.5-2mL of organic solvent, then 5mL of aqueous solution is added, after uniform dispersion, the organic solvent is removed by evaporation, then negatively charged water-soluble drugs with the mass of 0.000001-10% of the weight of the weighed cholesterol biguanide conjugate or salt thereof are added, after uniform dispersion again, the organic solvent is removed by evaporation, and then the water-soluble drug-encapsulated cholesterol biguanide conjugate or salt nanoparticle, microparticle, liposome, emulsion or polymer micelle suspension is obtained.
Furthermore, the fat-soluble auxiliary material a is a mixture of one or more of fatty acid monoglyceride, fatty acid diglyceride, fatty acid triglyceride, fatty acid, phospholipid, PLA, PLGA, PCL, mPEG-PLGA, mPEG-PCL, mPEG-PLA and a product synthesized by taking the auxiliary material as a main material and small molecular functional groups.
Further, the aqueous solution is water or an aqueous solution in which a water-soluble auxiliary material is dissolved.
Further, the water-soluble auxiliary material comprises PVA, PEG, poloxamer 188, poloxamer 407, hyaluronic acid or Tween 80.
Further, the chemical structure of the cholesterol biguanide conjugate is:
the chemical structure of the cholesterol biguanide conjugate salt is:
wherein X is C n H 2n 、C n H 2n -O-C m H 2m 、C n H 2n -S-C m H 2m Or C n H 2n -S-S-C m H 2m N is 2 to 22, m is 1 to 22;
y is a hydrochloride, acetate, sulfate, phosphate, carbonate or nitrate.
Further, the synthesis method of the cholesterol biguanide conjugate and the salt thereof is as follows: the first step is as follows: dropwise adding the cholesterol formyl chloride solution into the diamine solution under the condition of continuously stirring at the temperature of-20-10 ℃, stirring at room temperature under the protection of nitrogen for reaction for 12-24h, washing the residue after rotary evaporation by using a saturated sodium chloride solution or deionized water, and drying to obtain an intermediate product;
wherein the molar ratio of cholesterol formyl chloride to diamine is more than 1:10;
secondly, dispersing the intermediate product, dicyandiamide and ferric trichloride in a solvent, reacting for 15-24 hours at 75-120 ℃ under the protection of nitrogen gas, centrifuging to remove red precipitate, reserving supernatant, and purifying the supernatant to obtain the cholesterol biguanide conjugate;
wherein the molar ratio of the intermediate product to the diamine is 1:2.5-10; the molar ratio of the intermediate product to ferric trichloride is 1:0.3-0.9;
thirdly, introducing acid gas or solution into the obtained supernatant to obtain the cholesterol biguanide conjugate salt; the cholesterol biguanide conjugate can also be obtained, for example, by adding an equimolar amount of base to the salt of the cholesterol biguanide conjugate.
The invention provides a cholesterol biguanide conjugate particle type drug delivery system with drug loading function, which can be prepared by an emulsion solvent volatilization method, and in summary, the basic preparation steps are as follows: 1) Dissolving the conjugate in an organic solvent, and dissolving the conjugate in the organic solvent together with a fat-soluble medicine or a fat-soluble auxiliary material to be encapsulated; 2) Dissolving water-soluble auxiliary materials in an aqueous solvent; 3) Mixing the liquids obtained in the two steps to obtain turbid liquid; 4) Dispersing the turbid solution to proper particle size by methods such as ultrasound, stirring or high-pressure homogenization; 5) Stirring for 6-36h in a fume hood, or performing rotary evaporation under reduced pressure to remove organic solvent. The single micro-particle type drug delivery system of the cholesterol biguanide conjugate, a system carrying fat-soluble drugs and a micro-particle type drug delivery system carrying drugs together with other fat-soluble carriers can be obtained through the steps; 5) And (4) if the water-soluble medicine with negative electricity needs to be wrapped, adding the water-soluble medicine into the liquid prepared in the step (5), and stirring and incubating for a period of time to obtain the water-soluble medicine-carried microparticle preparation system.
Wherein the organic solvent is one or a mixed solvent of more than two of dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, methanol, ethanol, acetone and DMSO; preferably a mixed solvent of dichloromethane and ethanol.
The aqueous solvent is water or an aqueous solution containing a stabilizer, a pH adjuster, and the like, which are commonly used in liquid preparations.
The fat-soluble auxiliary material is common particle preparation carrier auxiliary material or auxiliary material such as fat-soluble antioxidant and the like, such as one or mixture of PLGA, mPEG-PLGA, AEAA-PEG-PLGA, fatty acid monoglyceride, fatty acid diglyceride, fatty acid triglyceride, fatty acid, phospholipid, PEG and/or the auxiliary material modified by targeting action groups.
The water-soluble auxiliary materials comprise PVP 1788, PEG, poloxamer 188, poloxamer 407, tween 80, SDS and other auxiliary materials meeting the requirement of administration route or auxiliary materials for increasing physical stability or chemical stability.
Many drugs can be entrapped by the cholesteryl biguanide conjugates and salts thereof, such as magnolol, honokiol, paclitaxel, curcumin, cucurbitacin, SN-38, docetaxel, camptothecin, 5-fluorouracil, doxorubicin, indirubin, IR-780 iodide, 5-aminolevulinic acid, gossypol acetate, anthocyanidin, teniposide, methotrexate, oridonin, heme, silybin, siRNA, and the like.
In the invention, the biguanide and cholesterol are coupled by an improved method, so that the generation of byproducts is reduced, the yield of a target product is improved, and the purification process of the target product is simplified. On one hand, the compound can entrap drugs with medium to strong lipid solubility; on the other hand, due to the existence of the biguanide group, the compound is positively charged, can also carry negatively charged water-soluble drugs in an electrostatic adsorption mode and is beneficial to the intracellular transport of the drugs, so that the biguanide cholesterol conjugate can be used as an excellent carrier for carrying the drugs. In addition, the pharmacological effects of biguanide derivatives are reported in the literature to be similar to that of metformin in many respects, for example, inhibition of mTOR activity by AMPK, and the like. Therefore, the biguanide cholesterol conjugate is not limited to the preparation of drugs for treating tumors, and can also be used as a carrier for preparing particle preparations for other indication diseases because the biguanide group also has the broad effects of reducing blood sugar and the like.
The biguanide cholesterol conjugate can encapsulate a medicament, can be matched with other carriers to encapsulate the medicament, and is usually matched with nano preparation carriers with stronger fat solubility, such as solid lipids, liquid lipids, PLGA (polylactic-co-glycolic acid) and the like. Or in combination with phospholipids. In addition, adjuvants with long circulation effect, such as mPEG-PLGA with polyethylene glycol (PEG) at one end and PLGA at the other end, can also be added into the carrier. Similarly, adjuvants with tumor targeting effect can also be added into the carrier, such as aminoethyl benzamide group (AEAA) capable of targeting sigma receptor and PEG-modified PLGA, i.e. AEAA-PEG-PLGA. The cholesterol biguanide conjugate or the salt thereof is the nanoparticle of the carrier, and the nanoparticle preparation with high encapsulation efficiency, good stability and uniform particle size can be obtained by an emulsion solvent volatilization method, and the two preparations have no obvious difference.
From the aspect of formulation, if nanoparticles can be successfully prepared, the preparation parameters, such as reduction of dispersion power, reduction of the dosage of organic solvents and the like, are simply changed, and then the dosage forms such as particles, microspheres and the like with larger particle sizes can be easily prepared. The liposome, the emulsion and the micelle can also be prepared by an emulsion solvent evaporation method, and in addition, the prepared liquid preparations such as nano-particles or micro-particles and the like can also be subjected to post-treatment to prepare dosage forms such as gels, oral solid preparations and the like. Therefore, on the basis of the preparation technology of the particles, the application range of the cholesterol biguanide conjugate and the particles taking the conjugate as a carrier can be further widened by combining the conventional preparation technology, and the development potential is great.
Drawings
FIG. 1 is a diagram showing the appearance of the formulations of application examples 1 to 13;
FIG. 2 is a graph showing the results of in vivo imaging of tumor-bearing mice.
Detailed Description
In order to make the technical purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention are further described below with reference to the accompanying drawings and specific embodiments.
Example 1: synthesis of cholesteryl biguanide conjugate Big-Chol-1 and its hydrochloride
1. Synthesis of intermediates
(1) Adding 6mL of anhydrous dichloromethane into 6mL of ethylenediamine, and dropwise adding 0.3mL of pyridine to prepare a solution 1;
(2) Weighing 0.45g of cholesterol formyl chloride, adding 4mL of anhydrous dichloromethane for dissolving, and preparing a solution 2;
(3) Dropwise adding the solution 2 into the solution 1 under ice bath, stirring at room temperature for 20 hours under the protection of nitrogen, centrifuging to absorb the lower-layer liquid, adding 40mL of acetonitrile, performing rotary evaporation to dryness, washing the dried substance with a saturated sodium chloride solution, and drying to obtain an intermediate product, wherein the yield is 98%, and the structural formula is as follows:
hydrogen spectra results (400MHz, DMSO-d) 6 )：7.14(T，1H，-N 31 -H)，5.34(T，1H,＝C 11 -H)，4.32(m，1H，-O-C 17 -H)3.12(dd，2H，-N-C 32 H 2 -)，2.74(T，2H，N-C 33 H 2 -), 0.65-2.4 (m, 45H, the remaining 45H).
2. Synthesis of Big-Chol-1 and its hydrochloride
(1) 236.2mg of intermediate product, 126.1mg of dicyandiamide and anhydrous FeCl are taken 3 40.55mg, weighing, placing in a round-bottom flask, and adding 5mL of absolute ethyl alcohol for dissolving;
(2) Assembling a condenser pipe on the round-bottom flask, placing the round-bottom flask in a 90 ℃ oil bath kettle for magnetic stirring, and filling nitrogen to protect reflux reaction for 20 hours to finish the reaction;
(3) Centrifuging to remove the red precipitate at the bottom, and obtaining the supernatant containing the Big-Chol-1. Introducing hydrogen chloride gas into the supernatant to form a fine precipitate of a hydrochloride of the product, and drying to obtain a final product of Big-Chol-1 hydrochloride, wherein the yield is about 92%, and the high-purity product can be obtained by silica gel column chromatography (chloroform: methanol = 10.
The structural formula is as follows:
hydrogen spectrum results (400MHz, DMSO-d) 6 )：7.02(s，2H，＝N 40 -H,＝N 39 -H,)，6.02(s,3H,-N 34 H-,-N 38 H 2 ),5.33(s，1H,＝C 11 -H)，4.32(m,1H,-O-C 17 -H)2.99(m,4H,-C 32 H 2 -，-C 33 H 2 -), 0.65-2.5 (m, 45H, the remaining 45H)
Carbon spectrum results: (100MHz, CDCl3): 160.07, 157.03, 139.77, 122.51, 74.66, 56.71, 56.26, 50.00, 42.33, 41.29, 40.48, 39.77, 39.52, 38.59, 37.02, 36.57, 36.23, 35.8, 31.87, 28.25, 28.17, 24.3, 23.94, 22.82, 22.57, 21.07, 19.36, 18.74, 11.88.
Example 2 Synthesis of Cholesterol biguanide conjugate Big-Chol-2 and its hydrochloride
1. Synthesis of intermediates
(1) Adding 8mL of anhydrous dichloromethane into 8mL of butanediamine and dropwise adding 0.3mL of pyridine to prepare a solution 1;
(2) Weighing 0.45g of cholesterol formyl chloride, adding 4mL of anhydrous dichloromethane for dissolving, and preparing a solution 2;
(3) Dropwise adding the solution 2 into the solution 1 under ice bath, then stirring at room temperature for 20 hours under the protection of nitrogen, centrifuging, taking supernate, adding 50mL of acetonitrile, carrying out rotary evaporation to dryness, washing the dried substance with saturated sodium chloride aqueous solution, and drying to obtain an intermediate product, wherein the yield is 97%, and the structural formula is as follows:
hydrogen spectrum results (400MHz, DMSO-d) 6 ),δ(ppm)：7.04(T，1H，-N 31 -H)，5.34(T，1H,＝C 11 -H)，4.30(m，1H，-O-C 17 -H)2.94(dd，2H，-N-C 33 -H 2 -)，2.54(T，2H，N-C 36 H 2 -), 0.65-2.4 (m, 49H, the remaining 49H in the molecule).
2. Synthesis of the end product
(1) 236.2mg of intermediate product, 168.1mg of dihydroamine and anhydrous FeCl are taken 3 40.55mg, weighing, placing in a round-bottom flask, and adding 5mL of absolute ethyl alcohol for dissolving;
(2) Assembling a condenser pipe on the round bottom flask, placing the round bottom flask in an oil bath kettle at the temperature of 85 ℃, magnetically stirring, and filling nitrogen to protect reflux reaction for 21 hours to finish;
(3) Centrifuging to remove the red precipitate at the bottom, and obtaining the supernatant containing Big-Chol-1. Introducing hydrogen chloride gas into the supernatant, centrifuging to obtain a precipitate, and drying to obtain a final product, namely Big-Chol-2 salt, wherein the yield is 90%, and the high-purity product can be obtained by silica gel column chromatography (chloroform: methanol = 10):
hydrogen spectrum results (400MHz, DMSO-d) 6 ),δ(ppm)：7.85(s，2H，＝N 42 -H，＝N 41 -H)，7.09(s，1H，N 31 -H)，5.74(s,2H,-N 40 H 2 ),5.32(s，1H,＝C 11 -H)，4.30(m,1H,-O-C 17 -H),2.96(dd,2H,-C 32 H 2 -)，2.75(t,2H,,-C 36 H 2 -), 0.65-2.4 (m, 49H, remaining 49H).
Application example 1: preparation and characterization of pure Big-Chol-1 salt nanoparticle
Precisely weighing 5mg of Big-Chol-1 salt, dissolving the Big-Chol-1 salt in 1mL of mixed solution of dichloromethane and absolute ethyl alcohol (the volume ratio of dichloromethane to absolute ethyl alcohol is V: V =4: 1), then adding 5mL of ultrapure water, performing ultrasonic dispersion by a cell crusher (200W, 3min, the conditions are shown in the specification), performing rotary evaporation to remove an organic solvent, and filtering the mixture by a filter membrane with the aperture of 0.22 mu m to obtain Big-Chol-1 salt nanoparticle suspension, wherein the measured Z average particle size is 111.2nm, the polydispersity index (PDI) is 0.21, the zeta potential +20mV, and the prepared Big-Chol-1 salt nanoparticles have no significant change after being placed at 4 ℃ for 6 months and have good stability.
Application example 2: preparation and characterization of Big-Chol-1 salt nanoparticle coated with magnolol
10mg of Big-Chol-1 salt and 5mg of magnolol are precisely weighed, dissolved in 2mL of mixed solution of dichloromethane and methanol (V: V = 5.
Application example 3: preparation and characterization of Big-Chol-2 salt nanoparticle coated with gossypol acetate
5mg of Big-Chol-2 salt and 1mg of gossypol acetate are precisely weighed, dissolved in 0.8mL of mixed solution of dichloromethane and absolute ethyl alcohol (V: V =3: 1), then 5mL of PVA1788 aqueous solution with the mass fraction of 1.5% is added, after ultrasonic dispersion by a cell crusher, magnetic stirring is carried out for 15h, the mixture is filtered through a 0.45-micrometer filter membrane to obtain gossypol acetate-encapsulated nanoparticles, the measured Z-average particle size is 203.5nm, the PDI is 0.18, and the zeta potential is +2.09mV.
Application example 4: preparation and characterization of Honokiol-encapsulated Big-grain of Big-grain Chol-2 salt
15mg of Big-Chol-1 salt, 7.5mg of honokiol and 5mg of glyceryl monostearate are precisely weighed, dissolved in 2mL of a mixed solution of dichloromethane and absolute ethyl alcohol (V: V =4 1), then 15mL of poloxamer 407 water solution with the mass fraction of 1.0% is added, the mixture is subjected to ultrasonic dispersion by a cell crusher, organic solvent is removed by rotary evaporation, and the mixture is filtered by a 0.22 mu m filter membrane to obtain the honokiol-coated nanoparticles, wherein the Z average particle size is 138.7nm, the PDI is 0.30, and the zeta potential is +27.9mV.
Application example 5: preparation and characterization of Big-Chol-2 salt lipid particles
Precisely weighing 500mg of Big-Chol-2 salt and 250mg of palmitic acid fatty glyceride, dissolving in 1mL of a mixed solution of dichloromethane and absolute ethyl alcohol (V: V =3: 1), adding into 5mL of ultrapure water, stirring and dispersing, and performing rotary evaporation to remove an organic solvent to obtain Big-Chol-2 salt lipid particles, wherein the Z-average particle size is 2217.6nm, and the PDI is 0.35.
Application example 6: preparation and characterization of doxorubicin-entrapped Big-Chol-1 salt liposome
7.5mg of Big-Chol-1 salt, 1.5mg of adriamycin, 15mg of soybean phospholipid and 2mg of cholesterol are precisely weighed and dissolved in 3mL of mixed solution of chloroform and absolute ethyl alcohol (V: V = 6.
Application example 7: preparation and characterization of Big-Chol-1 salt nanoparticle encapsulating STAT-siRNA
Accurately weighing 50mg of Big-Chol-1 salt, dissolving the Big-Chol-1 salt in 2.0mL of mixed solution of dichloromethane and absolute ethyl alcohol (V: V =4: 1), then adding 10mL of PVA1788 aqueous solution with the mass fraction of 1.5%, after ultrasonic dispersion by a cell crusher, magnetically stirring for 12h, volatilizing the organic solvent, filtering by a filter membrane of 0.45 mu m to obtain nanoparticle suspension, then adding 1 microgram of STAT-siRNA, vortex mixing for 10s, and standing for 1h to obtain the Big-Chol-1 salt nanoparticle suspension carrying the siRNA. The Z-average particle diameter was 126.3nm, PDI was 0.23, zeta potential +25.3mV.
Application example 8: preparation and characterization of SN-38-entrapped Big-pill-2 salt nanoparticle
Big-Chol-2 salt of 5mg, SN-38 (7-ethyl-10-hydroxycamptothecin) of 1mg are precisely weighed and dissolved in 1mL of mixed solution of dichloromethane and absolute ethyl alcohol (V: V =4: 1), then 5mL of PVA1788 aqueous solution with the mass fraction of 1.5% is added, after ultrasonic dispersion by a cell crusher, stirring is carried out for 12h by magnetic force, filtering is carried out by a filter membrane of 0.45 mu m, and the Big-Chol-2 salt nanoparticle suspension encapsulating SN-38 is obtained, wherein the Z average particle diameter is 130.7nm, the PDI is 0.28, the zeta potential +24.0mV.
Application example 9: preparation and characterization of docetaxel-entrapped Big-Chol-1 salt PEG-PLA micelle
2mg of Big-Chol-1 salt, 1mg of docetaxel and 1mg of PEG are precisely weighed 3.5K -PLA 2K 10mg of the docetaxel is dissolved in 1mL of a mixed solution of chloroform and methanol (V: V =4: 1), then 5mL of PVA1788 aqueous solution with the mass fraction of 1.5% is added, the mixture is stirred uniformly and is subjected to ultrasonic dispersion by a cell disruptor, the mixture is stirred magnetically for 12 hours and is filtered through a 0.45-micron filter membrane, and a docetaxel-loaded micelle suspension is obtained. The Z-average particle diameter was 102.9nm, the PDI was 0.21, and the zeta potential +32.5mV.
Application example 10: preparation and characterization of nanoparticles containing magnolol, big-Chol-1 salt and PLGA
Precisely weighing 3mg of Big-Chol-1 salt and 2mg of magnolol 9k 5mg, dissolved in 1mL dichloromethane: in an organic phase of absolute ethyl alcohol (V: V = 4).
Application example 11: preparation and characterization of nanoparticles containing magnolol, big-Chol-1 salt and mPEG-PLGA
Weighing Big-Chol-1 salt 3mg, magnolol 2mg, mPEG 3.5k -PLGA 9k 5mg, dissolved in 1mL dichloromethane: is composed ofAnd (2) adding 5ml of poloxamer 407 aqueous solution with the mass percent of 2% after completely dissolving in water and ethanol (V: V = 3) in an organic phase, performing ultrasonic dispersion by using a cell crusher, performing magnetic stirring for 12h, and filtering the solution through a 0.22 mu m filter membrane to obtain a nanoparticle suspension containing magnolol, big-Chol-1 salt and mPEG-PLGA, wherein the Z average particle size is 115nm, the PDI is 0.18, and the zeta potential is +18.5mV.
Application example 12: preparation and characterization of nanoparticles containing magnolol, big-Chol-1 salt and AEAA-PEG-PLGA
Weighing Big-Chol-1 salt 3mg, magnolol 2mg, mPEG 3.5K -PLGA 9k 1.0mg，AEAA-PEG 3.5K -PLGA 9k 4.0mg, dissolved in 1mL of a mixed solution of dichloromethane and absolute ethanol (V: V =4 1), 5mL of a 1.5% by mass aqueous solution of PVA1788 was added, and the mixture was subjected to ultrasonic dispersion by a cell disruptor under ice-bath, magnetic stirring for 14 hours, and filtration through a 0.22 μm filter to obtain a final preparation. The Z-average particle diameter was 120nm, PDI was 0.22, and zeta potential +14.5mV.
Wherein AEAA-PEG 3.5K -PLGA 9K The synthesis method comprises the following steps: 0.5g of p-methoxybenzoyl chloride and 0.66g of 2-bromoethylamine hydrobromide were dissolved in 5mL of acetonitrile, mixed, added with 1.74mL of N, N-diisopropylethylamine, and reacted under magnetic stirring at room temperature in the dark for 6 hours. 400mg of BOC-PEG 3.5K -NH 2 Adding the mixture into the system, and reacting for 12 hours under magnetic stirring. Dropwise adding the reaction liquid into 40mL of glacial ethyl ether, centrifuging, drying the precipitate, adding a small amount of water to dissolve the precipitate, carrying out ultrafiltration by using an ultrafiltration tube with 3KD, freeze-drying the residual liquid, adding 4.5mL of a dichloromethane solution of 33% trifluoroacetic acid, reacting for 2 hours under magnetic stirring, carrying out rotary evaporation, adding 5mL of dichloromethane into the residue to dissolve the residue, settling by using cold ethyl ether, centrifuging, drying, and weighing. Adding 5mL of dichloromethane into the dried product in the previous step for dissolving, and weighing PLGA with the same molar mass as the product 9K -COOH was dissolved in 5mL of DCM, and the two solutions were mixed, and 20 times the molar mass of DIPEA and N, N' -diisopropylcarbodiimide were added, respectively, and the mixture was stirred at room temperature for 48 hours. Dropping in cold ether for settling, centrifuging, and drying to obtain final product AEAA-PEG 3.5K -PLGA 9K 。
Application example 13: preparation and characterization of Big-Chol-2 salt emulsion
5mg of Big-Chol-2 salt, 10mg of soybean oil and 2mg of soybean phospholipid are precisely weighed, dissolved in 0.8mL of mixed solution of dichloromethane and absolute ethyl alcohol (V: V = 5.
MTT method for determining influence of pure Big-Chol-1 salt nanoparticles prepared in application example 1 on activity of human breast cancer MCF-7 cells
The MCF-7 cells grown in the logarithmic phase were trypsinized and plated onto 96-well plates at 150mL/well at 8X 10 3 And (4) cells. After the cells were cultured for 24h, the old culture medium was removed and the drug was added. A blank control group and a zero-adjusting group without cells are respectively arranged at 200 mu L per well, 6 concentrations are respectively arranged in each group, 5, 10, 15, 20, 25, 30 and 35 mu g/mL, 6 multiple wells are arranged at each concentration, and the cell inhibition rate of the drug preparation and the cells in co-culture for 24h is determined by adopting an MTT method. The results show that the cytotoxicity of the pure Big-Chol-1 salt nano-particle prepared by the application example 1 on MCF-7 cells is increased along with the increase of the concentration, and the IC50 value is about 25.7 mu g/mL.
MTT method for determining influence of Big-Chol-2 salt emulsion prepared in application example 13 on activity of ECA-109 cells of human esophageal cancer
The logarithmic phase-grown ECA-109 cells were trypsinized and plated onto 96-well plates at 150mL/well at 8X 10 3 And (4) cells. After the cells were cultured for 24 hours, the old culture medium was removed and the drug was added. 200 mu L of each well are respectively provided with a blank control group and a zero-adjusting group without cells, and an experimental group of Big-Chol-2, each group is provided with 6 concentrations which are respectively 1.56,3.12,6.25, 12.5, 25, 50, 100 and 105 mu g/mL, each concentration is provided with 6 multiple wells, and the cell inhibition rate of the drug preparation and the cells in co-culture for 24h is determined by adopting an MTT method. The results show that the Big-Chol-2 salt emulsion prepared in application example 13 has increased cytotoxicity to ECA-109 cells with an IC50 value of about 98.7. Mu.g/mL as the concentration increases.
In vivo pharmacodynamic study of formulations
The pharmacodynamic advantages of the preparation will be illustrated here by taking application examples 1, 10, 11 and 12 as examples. The frozen S180 cells are taken out from a refrigerator at the temperature of-80 ℃, quickly placed in a water bath at the temperature of 37 ℃ for unfreezing, and then inoculated in the abdominal cavity of a male Kunming mouse. One week later, ascites of tumor-bearing mice was extracted with a 10mL syringe, and each mouse was inoculated subcutaneously into the right axilla of a male Kunming mouse with 0.2mL of ascites. Mice with similar body weight and tumor volume were divided into 6 groups of 6 mice each. The specific experimental groups are as follows: (1) a control group of 5% glucose solution; (2) application example 1; (3) magnolol solution group; (4) application example 10; (5) application example 11; (6) Application example 12, the drug was administered by tail vein injection, every other day, and the total dose of each group was 5mg/kg (the formulation contained BIG-Chol-1 salt and magnolol at the same time, the ratio of the two was 3). Body weight was measured daily, general conditions were observed, and the following day after the last administration, mice were sacrificed and tumor tissues were removed. Tumor weight inhibition rate = (1-mean tumor weight of administration group/mean tumor weight of control group) was calculated.
The results show that the body weights of the groups have no significant difference, the mouse state is better, and the toxicity of the preparation is lower. Compared with a control group 1, tumor weight inhibition rates of 2-6 groups are respectively 68%, 25%,57%,87% and 93.12%, which indicates that the Big-Chol-1 salt nanoparticles have an inhibition effect on S180 tumors, and also indicates that the Big-Chol-1 salt prepared by the invention is used as a carrier to encapsulate magnolol to prepare long-circulating or sigma receptor targeting nanoparticles, which are all beneficial to improving the treatment effect of the magnolol.
Preparation of nanoparticle coated with IR780 iodide and in-vivo imaging experiment
Weighing 6mg of Big-Chol-1 salt, 0.2mg/mL of IR780 solution, dissolving in 0.5mL of a solution with the volume ratio of 4:1, adding 2ml of PVA solution with the mass fraction of 1.5 percent as an emulsifier into an organic phase of dichloromethane and absolute ethyl alcohol, carrying out ice bath detection and ultrasonic detection, carrying out magnetic stirring, and filtering through a 0.45um filter membrane to obtain the final preparation IR780-NP group.
Weighing PLGA 9k 4mg of big-Chol-1 salt 2mg of IR780 solution 0.2mg/mL, dissolved in 0.5mL of a volume ratio of 4:1, adding 2ml of PVA aqueous solution with the mass fraction of 1.5 percent into an organic phase of dichloromethane and absolute ethyl alcohol, detecting the ultra-high temperature, opening the magnetic stirring for 12 hours, and filtering the mixture through a 0.45um filter membraneTo obtain the final preparation PLGA-IR780-NP group.
Weighing mPEG 3.5k -PLGA 9k 4mg of big-Chol-1 salt 2mg of IR780 solution 0.2mg/mL, dissolved in 0.5mL of a volume ratio of 4:1 dichloromethane and absolute ethyl alcohol, adding 2ml PVA water solution with mass fraction of 1.5%, detecting, opening, magnetically stirring for 12h, and filtering with 0.45um filter membrane to obtain final preparation MPEG-PLGA-IR780-NP group.
Weighing mPEG 3.5 -PLGA 9k 0.8mg，AEAA-PEG 3.5k -PLGA 9k 3.2mg, big-Chol-1 salt 2mg, IR780 solution 0.2mg/mL, dissolved in 0.5mL volume ratio 4:1 dichloromethane and absolute ethyl alcohol, adding 2ml PVA water solution with mass fraction of 1.5%, detecting, opening, magnetically stirring for 12h, and filtering with 0.45um filter membrane to obtain final preparation AEAA-MPEG-PLGA-IR780-NP group.
BALC mice, breast inoculated with 4T1 tumors, were grouped according to body weight and tumor volume, 2 per group. The images were taken at 1h, 4h, 8h, 12h, and 24h after the administration, respectively. The excitation wavelength was set at 770nm, the emission wavelength at 830nm, and the exposure time at 20s. The specific experimental groups are as follows: (A) IR780-NP group; (B) PLGA-IR780-NP group; (C) mPEG-PLGA-IR780-NP group, (D) AEAA-PEG-PLGA-IR780-NP group. The injection concentration of IR-780 iodide is 5mg/kg, the injection is administered in tail vein, and the mice are anesthetized by intraperitoneal injection with 10% (M/V) chloral hydrate before being photographed by a small animal imager. The results are shown in FIG. 2. From the in vivo imaging results, it can be seen that several groups of the preparations examined can target tumor sites, and even after 24h of administration, the preparations still have strong fluorescence.
The experiments show that the cholesterol biguanide conjugate is used as a carrier, and can wrap various medicines by combining with the preparation technology provided by the invention to form particle preparations such as nanoparticle preparations, the preparation method is simple, the applicable medicine range is wide, the stability is good, the targeting effect of the wrapped medicines is improved, and the preparation method is a great progress in the field of preparations.
Finally, it should be noted that: the above embodiments are merely illustrative and not restrictive of the technical solutions of the present invention, and any equivalent substitutions and modifications or partial substitutions made without departing from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.
Claims (6)
1. A preparation method of a self-assembly nanoparticle suspension of a cholesterol biguanide conjugate or a salt thereof is characterized by comprising the following steps: weighing 0.01-300mg of the cholesterol biguanide conjugate or the salt thereof, dissolving in 0.1-5mL of organic solvent, then adding 5mL of aqueous solution, performing ultrasonic or stirring dispersion uniformly, and evaporating to remove the organic solvent to obtain a suspension of the self-assembled nanoparticles of the cholesterol biguanide conjugate or the salt thereof;
the chemical structure general formula of the cholesterol biguanide conjugate is as follows:
the chemical structure general formula of the cholesterol biguanide conjugate salt is as follows:
wherein X is C n H 2n 、C n H 2n -O-C m H 2m 、C n H 2n -S-C m H 2m Or C n H 2n -S-S-C m H 2m N is 2 to 22, m is 1 to 22; y is a hydrochloride, acetate, sulfate, phosphate, carbonate or nitrate.
2. A preparation method of a suspension of nanoparticles or microparticles of a cholesterol biguanide conjugate or a salt thereof coated with a fat-soluble drug is characterized by comprising the following steps: weighing 1-300mg of the cholesterol biguanide conjugate or the salt thereof and a fat-soluble drug with the mass of 0.0001-50% of the weight of the weighed cholesterol biguanide conjugate or the salt thereof, dissolving the fat-soluble drug in 0.1-5mL of an organic solvent, then adding 5mL of an aqueous solution, uniformly dispersing, and evaporating to remove the organic solvent to obtain a suspension of nanoparticles or particles of the cholesterol biguanide conjugate or the salt thereof loaded with the fat-soluble drug;
the chemical structure general formula of the cholesterol biguanide conjugate is as follows:
the chemical structural general formula of the cholesterol biguanide conjugate salt is as follows:
wherein X is C n H 2n 、C n H 2n -O-C m H 2m 、C n H 2n -S-C m H 2m Or C n H 2n -S-S-C m H 2m N is 2 to 22, m is 1 to 22; y is a hydrochloride, acetate, sulfate, phosphate, carbonate or nitrate.
3. A preparation method of a suspension of nanoparticles of a cholesterol biguanide conjugate or a salt thereof entrapping a water-soluble drug is characterized by comprising the following steps: the preparation method comprises the following steps: weighing 1-300mg of the cholesterol biguanide conjugate or salt thereof, dissolving in 0.1-5mL of organic solvent, then adding 5mL of aqueous solution, uniformly dispersing, evaporating to remove the organic solvent, adding a negatively charged water-soluble drug which accounts for 0.0000001-10% of the weight of the weighed cholesterol biguanide conjugate or salt thereof, and uniformly dispersing again to obtain a water-soluble drug-encapsulated cholesterol biguanide conjugate or salt nanoparticle suspension;
the chemical structure general formula of the cholesterol biguanide conjugate is as follows:
the chemical structure general formula of the cholesterol biguanide conjugate salt is as follows:
wherein X is C n H 2n 、C n H 2n -O-C m H 2m 、C n H 2n -S-C m H 2m Or C n H 2n -S-S-C m H 2m N is 2 to 22, m is 1 to 22; y is a hydrochloride, acetate, sulfate, phosphate, carbonate or nitrate.
4. The method for producing a suspension according to any one of claims 1 to 3, wherein the aqueous solution is water or an aqueous solution in which a water-soluble auxiliary is dissolved.
5. The method of claim 4, wherein the water soluble excipient comprises PVA, PEG, poloxamer 188, poloxamer 407, hyaluronic acid, or Tween 80.
6. A process for the preparation of a suspension according to any of claims 1 to 3, wherein the method for the synthesis of the cholesteryl biguanide conjugate and its salts is as follows:
the first step is as follows: dropwise adding the cholesterol formyl chloride solution into the diamine solution under the conditions of-20-10 ℃ and continuous stirring, stirring at room temperature under the protection of nitrogen for reaction for 12-24 hours, washing the residue after rotary evaporation by adopting a saturated sodium chloride solution or deionized water, and drying to obtain an intermediate product;
wherein the molar ratio of cholesterol formyl chloride to diamine is more than 1:10;
secondly, dispersing the intermediate product, dicyandiamide and ferric trichloride in a solvent, reacting for 15-24 hours at 75-120 ℃ under nitrogen protection and stirring, centrifuging to remove red precipitate, reserving supernatant, and purifying the supernatant to obtain the cholesterol biguanide conjugate;
wherein the molar ratio of the intermediate product to the diamine is 1:2.5-10; the molar ratio of the intermediate product to ferric trichloride is 1:0.3-0.9;
and thirdly, introducing acid gas or solution into the obtained supernatant to obtain the cholesterol biguanide conjugate salt.
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