Low hemolytic cationic lipid and application thereof

Abstract

The invention discloses a low hemolytic cationic lipid-cholesterol monoguanidine conjugate and a preparation method of the low hemolytic cationic lipid-cholesterol monoguanidine conjugate. The cholesterol monoguanidine conjugate has the characteristic of low hemolysis rate, can be used for constructing preparations needing positive ions, such as lipid nanoparticles or lipidosome and the like, and has the potential of being used as an excellent auxiliary material.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations and relates to the preparation and application of a cholesterol monoguanidine conjugate. Background Art

[0002] Cationic lipid molecules usually contain one or more positively charged head groups, a hydrophobic tail, and a linking portion of a hydrophobic chain connecting the head group and the tail. The positively charged head group of cationic lipids can produce electrostatic effects with negatively charged biomacromolecules, which helps to encapsulate and compress biomacromolecules to form lipid complexes, thereby increasing their cellular uptake rate. In addition, cationic lipids can be used to prepare cationic liposomes or lipid nanoparticles to encapsulate active ingredients other than biomacromolecules. Compared with ordinary lipid preparations, the prepared preparations have advantages in improving tumor tissue targeting and cellular uptake rate.

[0003] However, cationic lipids are usually highly hemolytic. After intravenous administration, they are likely to damage red blood cells in the blood circulation system, causing them to rupture, thereby leading to hemolysis. In addition, cationic lipids themselves are also toxic to cells. These properties limit the application of cationic lipids in formulation development and clinical practice. It is necessary to find cationic lipids with low toxicity and low hemolysis. Summary of the invention

[0004] In order to overcome some deficiencies of the prior art, one of the purposes of the present invention is to provide an application of a cholesterol monoguanidine conjugate, wherein the cholesterol monoguanidine conjugate is used to prepare a microparticle drug delivery system. The cholesterol monoguanidine conjugate has the characteristic of low hemolysis.

[0005] A second object of the present invention is to provide a cholesterol monoguanidine conjugate.

[0006] The third object of the present invention is to provide a method for synthesizing the above-mentioned cholesterol monoguanidine conjugate.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] An application of a cholesterol monoguanidine conjugate, wherein the cholesterol monoguanidine conjugate is used for preparing a microparticle drug delivery system.

[0009] Preferably, the microparticle drug delivery system is a microparticle, a nanoparticle, a liposome, an emulsion, a polymer micelle, a gel or a mixture of the above types.

[0010] Furthermore, the cholesterol monoguanidine conjugate is used to prepare a microparticle drug delivery system by self-assembly encapsulating the drug, or the cholesterol monoguanidine conjugate is used to prepare a microparticle drug delivery system by sharing the drug with other carrier excipients.

[0011] Furthermore, the cholesterol monoguanidine conjugate is prepared into a microparticle drug delivery system by self-assembly encapsulating the drug, which is one of microparticles, nanoparticles, and gels; the cholesterol monoguanidine conjugate is prepared into a microparticle drug delivery system by sharing the drug with other carrier excipients, which is one of microparticles, microspheres, nanoparticles, liposomes, emulsions, and polymer micelles.

[0012] Furthermore, the process of preparing the cholesterol monoguanidine conjugate into a microparticle drug delivery system by self-assembly encapsulating the drug is as follows:

[0013] When the drug to be encapsulated is a fat-soluble drug, the preparation process is as follows: cholesterol monoguanidine conjugate and the drug to be encapsulated are dissolved in an organic solvent and mixed to obtain an organic mixed solution, and an aqueous solution and the organic mixed solution are mixed to obtain a suspension; then the suspension is dispersed, and the organic solvent is removed to obtain an aqueous solution of a microparticle delivery system of cholesterol monoguanidine conjugate encapsulating a fat-soluble drug;

[0014] When the drug to be encapsulated is a water-soluble drug, the preparation process can be divided into two types, one of which is: mixing the cholesterol monoguanidine conjugate with an organic solvent to obtain an organic mixed solution, mixing the aqueous solution with the organic mixed solution to obtain a suspension; then dispersing the suspension to remove the organic solvent; adding the water-soluble drug or its aqueous solution to the above suspension for incubation to obtain an aqueous solution of a microparticle drug delivery system of cholesterol monoguanidine conjugate encapsulating a water-soluble drug;

[0015] The second method is: mixing the cholesterol monoguanidine conjugate with an organic solvent to obtain an organic mixed solution, mixing a water-soluble drug or its aqueous solution or aqueous solution with the organic mixed solution to obtain a suspension; then dispersing the suspension and removing the organic solvent to obtain an aqueous solution of a microparticle drug delivery system containing a cholesterol monoguanidine conjugate and a water-soluble drug;

[0016] The aqueous solution is water or an aqueous solution containing dissolved aqueous auxiliary materials.

[0017] Furthermore, the mass ratio of the cholesterol monoguanidine conjugate to the encapsulated drug is 10000 to 0.1:1.

[0018] Preferably, the aqueous solution is water or an aqueous solution containing a water-soluble auxiliary material, wherein the water-soluble auxiliary material is a stabilizer, a surfactant, a pH regulator, an osmotic pressure regulator or an antioxidant.

[0019] Preferably, the surfactant or stabilizer is PVA, PEG, poloxamer 188, poloxamer 407, hyaluronic acid or Tween 80. Common pH regulators, osmotic pressure regulators or antioxidants can be used in the preparation.

[0020] The concentration of the conjugate affects the final form of the preparation. At low concentrations, the preparation is a nanoparticle or microparticle with good fluidity; at higher concentrations, the preparation first forms nanoparticles or microparticles, and then forms a gel at room temperature. Generally, the concentration of the conjugate is below 40 mg / mL, and the preparation has good fluidity. The self-assembled nanoparticles or microparticles or the gel formed later have drug-carrying functions. Therefore, fat-soluble drugs can be co-dissolved with the conjugate in an organic solvent and then prepared later. Water-soluble drugs, such as negatively charged water-soluble drugs, can be dissolved in an appropriate amount of aqueous solution and then incubated with the prepared nanoparticles to carry the drug.

[0021] Furthermore, the cholesterol monoguanidine conjugate is used together with other carrier excipients to encapsulate the drug to prepare a microparticle drug delivery system as follows:

[0022] When the drug to be encapsulated is a fat-soluble drug, the preparation process is as follows: dissolving the cholesterol monoguanidine conjugate, other carrier excipients and the drug to be encapsulated in an organic solvent to obtain an organic mixed solution, or dispersing the cholesterol monoguanidine conjugate and the drug to be encapsulated in other carrier excipients to obtain an organic mixed solution; mixing the aqueous solution with the organic mixed solution to obtain a suspension; then dispersing the suspension and removing the organic solvent to obtain a microparticle drug delivery system in which the cholesterol monoguanidine conjugate and the carrier excipients encapsulate the drug together;

[0023] When used in combination with other carriers, the corresponding preparation method can be flexibly adopted according to the properties of the carrier. For example, when the combined carrier excipient is a solid lipid, the lipid can be heated and melted, and then the drug and cholesterol monoguanidine conjugate can be added and mixed; when the combined excipient is a liquid lipid, such as when preparing an emulsion, the drug and cholesterol monoguanidine conjugate can be dissolved in the liquid lipid. Therefore, the organic solvent in the preparation method is not indispensable.

[0024] When the drug to be encapsulated is a water-soluble drug, its preparation process can be divided into two types, one of which is: mixing the cholesterol monoguanidine conjugate, other carrier excipients and an organic solvent to obtain an organic mixed solution, or dispersing the cholesterol monoguanidine conjugate in other carrier excipients to obtain an organic mixed solution; mixing the aqueous solution with the organic mixed solution to obtain a suspension; then dispersing the suspension to remove the organic solvent; adding the water-soluble drug or its aqueous solution to the above suspension for incubation, thereby obtaining an aqueous solution of a microparticle drug delivery system in which the cholesterol monoguanidine conjugate and the carrier excipients encapsulate the water-soluble drug;

[0025] The second method is: mixing cholesterol monoguanidine conjugate, other carrier excipients and an organic solvent to obtain an organic mixed solution, or dispersing cholesterol monoguanidine conjugate in other carrier excipients to obtain an organic mixed solution; mixing a water-soluble drug or its aqueous solution or aqueous solution with the organic mixed solution to obtain a suspension; then dispersing the suspension and removing the organic solvent to obtain an aqueous solution of a microparticle drug delivery system in which cholesterol monoguanidine conjugate and carrier excipients are used together to encapsulate the water-soluble drug;

[0026] The aqueous solution is water or an aqueous solution containing dissolved aqueous auxiliary materials.

[0027] Furthermore, the mass ratio of the total mass of the cholesterol monoguanidine conjugate and the carrier excipient to the drug is 10000 to 0.1:1; the mass ratio of the cholesterol monoguanidine conjugate to the carrier excipient is 1:0.001 to 1000.

[0028] The preparation containing cholesterol monoguanidine conjugate can adsorb negatively charged water-soluble drugs, so the encapsulation rate and drug loading of such drugs are high. At the same time, such preparations can also take advantage of other dosage forms or carriers to encapsulate water-soluble drugs of different properties. For example, if prepared into liposomes, the pH gradient method can be used to encapsulate suitable drugs, usually weak acid or weak base drugs, such as doxorubicin hydrochloride. Therefore, the protection of water-soluble drugs by the present invention is not limited to negatively charged water-soluble drugs.

[0029] Furthermore, the other carrier excipients are phospholipids, cholesterol, fatty acid monoglycerides, fatty acid diglycerides, fatty acid triglycerides, fatty acids, polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, methoxypolyethylene glycol polylactic acid-glycolic acid copolymer, methoxypolyethylene glycol polycaprolactone or methoxypolyethylene glycol polylactide, and a mixture of one or more of the products synthesized from the above carrier materials and small molecule functional groups.

[0030] Preferably, the organic solvent is selected from one or more of dichloromethane, chloroform, tetrahydrofuran, ethyl acetate, methanol, ethanol, acetone and DMSO.

[0031] Many drugs can be loaded by the cholesterol monoguanidine conjugate, such as magnolol, honokiol, paclitaxel, curcumin, cucurbitacin, SN-38, docetaxel, camptothecin, 5-fluorouracil, doxorubicin, indirubin, IR-780 iodide, 5-aminolevulinic acid, gossypol acetate, cyanine fluorescein, teniposide, methotrexate, oridonin, silybin, etc. The embodiments of the present invention are mainly anti-tumor drugs, but are not limited to this. Because the cholesterol monoguanidine conjugate referred to in the present invention is mainly used as a non-toxic excipient, its scope of use is not limited by the disease.

[0032] The second object of the present invention is achieved by adopting the following technical solution:

[0033] A cholesterol monoguanidine conjugate having a general structural formula I

[0034]

[0035] Where X is C n H 2n, n is an integer greater than or equal to 2, preferably 2-22.

[0036] The third object of the present invention is achieved by adopting the following technical solution:

[0037] The preparation method of cholesterol monoguanidine conjugate comprises the following steps:

[0038]

[0039] (1) Adding cholesterol formyl chloride solution to the diamine solution at -20°C to 10°C with constant stirring, stirring and reacting for 8 to 24 hours under inert gas protection to obtain intermediate a;

[0040] (2) The intermediate product a obtained in step (1), cyanamide and the catalyst ferric chloride are dispersed in a solvent according to the molar ratio of the intermediate product a to cyanamide and the catalyst ferric chloride of 1:1 to 9:0.1 to 1.0, and stirred for reaction at 75° C. to 120° C. in an inert gas for 15 to 24 hours to obtain cholesterol monoguanidine conjugate I.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention provides an application of a cholesterol monoguanidine conjugate, wherein the cholesterol monoguanidine conjugate is prepared into a microparticle drug delivery system by encapsulating a drug. The cholesterol monoguanidine conjugate provided by the present invention has amphiphilicity, and as a drug carrier, it can encapsulate fat-soluble drugs on the one hand, and on the other hand, due to the presence of the monoguanidine group, the compound is positively charged, and negatively charged water-soluble drugs can also be encapsulated by electrostatic adsorption. The presence of the guanidine group is also conducive to the intracellular membrane transport of the drug, so the monoguanidine cholesterol conjugate can be used as an excellent carrier material for microparticle preparations to encapsulate drugs. The monoguanidine cholesterol conjugate has good compatibility with commonly used carrier excipients, and this type of compound has low hemolysis and can be used as a dosage form excipient for intravenous administration; when it is used as a drug carrier, it helps to exert better drug efficacy. The present invention also provides a method for preparing the above-mentioned cholesterol monoguanidine conjugate, which has good reproducibility and can prepare a high-purity product. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The synthetic route of the cholesterol monoguanidine conjugate of the present invention is as follows;

[0044] Figure 2 These are digital photos of the preparations of Experimental Examples 1 to 10 of the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0046] The synthetic route of cholesterol monoguanidine conjugate is as follows Figure 1 shown.

[0047] Example 1

[0048] Cholesterol monoguanidine conjugate and preparation method thereof:

[0049] (1) 8 mL of anhydrous dichloromethane was added with 8 mL of dibutyleneamine and 0.3 mL of pyridine was added dropwise to prepare solution 1; 0.45 g of cholesterol acetyl chloride was weighed and dissolved in 4 mL of anhydrous dichloromethane to prepare solution 2; solution 2 was added dropwise to solution 1 under ice bath, and then stirred at room temperature for 20 h under nitrogen protection, centrifuged, and the supernatant was taken, 50 mL of acetonitrile was added thereto and rotary evaporated to dryness, and the dried product was washed with ultrapure water and dried to obtain an intermediate product with a yield of 97%. The structural formula of the intermediate product is as follows:

[0050]

[0051] H 1 -NMR (400MHz, DMSO-d 6 ),δ(ppm):7.04(T,1H,-N 31 -H), 5.34(T,1H,=C 11 -H),4.30

[0052] (m,1H,-OC 17 -H)2.94(dd,2H,-NC 33 -H 2 -),2.54(T,2H,NC 36 H 2 -), 0.65-2.4 (m, 49H, the remaining 49 H in the molecule).

[0053] (2) Take 236.2 mg of the intermediate product obtained in step (1), 178.41 mg of cyanamide, and anhydrous FeCl 3 61.36 mg was placed in a round-bottom flask, and 5 mL of anhydrous ethanol was added to dissolve; a condenser was then installed on the round-bottom flask, and the mixture was placed in an oil bath at 85°C for magnetic stirring, and refluxed for 21 hours under nitrogen protection; the red precipitate at the bottom was removed by centrifugation to obtain a supernatant containing AG-Chol, with a yield of about 10%. A high-purity product can be obtained by silica gel column chromatography (chloroform: methanol = 10:1) to obtain compound 1 (AG-Chol), whose structural formula is as follows:

[0054]

[0055] H 1-NMR (400 MHz, DMSO-d 6 ), δ (ppm): δ7.05 (s, 1H, N8-H), 5.91 (s, 1H, C2 = N13-H), 5.35

[0056] (s,3H,N1-H&C15=C19-H),4.30(s,1H,C11-H),3.17(d,J=5.1Hz,1H),2.93(s ,3H),2.38~2.09(m,2H),2.06~1.67(m,5H),1.62~0.74(m,38H),0.65(s,3H).

[0057] Example 2

[0058] Cholesterol monoguanidine conjugate and preparation method thereof:

[0059] (1) 8 mL of anhydrous dichloromethane was added with 8 mL of ethylenediamine and 0.3 mL of pyridine was added dropwise to prepare solution 1; 0.45 g of cholesterol acetyl chloride was weighed and dissolved in 4 mL of anhydrous dichloromethane to prepare solution 2; solution 2 was added dropwise to solution 1 under ice bath, and then stirred at room temperature for 20 h under nitrogen protection, centrifuged, and the supernatant was taken, 50 mL of acetonitrile was added thereto and rotary evaporated to dryness, and the dried product was washed with ultrapure water and dried to obtain an intermediate product with a yield of 98%. The structural formula of the intermediate product is as follows:

[0060]

[0061] H 1 -NMR (400MHz, DMSO-d 6 ),δ(ppm):7.04(T,1H,-N 31 -H), 5.34(T,1H,=C 11 -H),4.30

[0062] (m,1H,-OC 17 -H)2.94(dd,2H,-NC 33 -H 2 -),2.54(T,2H,NC 36 H 2 -), 0.65-2.4 (m, 49H, the remaining 49 H in the molecule).

[0063] (2) Take 222.7 mg of the intermediate product obtained in step (1), 178.41 mg of cyanamide, and anhydrous FeCl 361.36 mg was placed in a round-bottom flask, and 5 mL of anhydrous ethanol was added to dissolve; then a condenser was installed on the round-bottom flask, and the flask was placed in an oil bath at 85°C for magnetic stirring, and nitrogen was charged to protect the reflux reaction for 21 hours; the red precipitate at the bottom was removed by centrifugation to obtain a supernatant containing AE-Chol, with a yield of about 10%. The high-purity product can be obtained by silica gel column chromatography (chloroform: methanol = 10:1) to obtain compound 2 (AE-Chol) whose structural formula is as follows:

[0064]

[0065] H 1 -NMR (400 MHz, DMSO-d 6 ), δ (ppm): δ7.17 (s, 1H, N8-H), 5.91 (s, 1H, C2 = N13-H), 5.35

[0066] (s,3H,N1-H&C15=C19-H),4.30(s,1H,C11-H),3.27(s,2H),3.03(s,2H) ,2.38~2.09(m,2H),2.06~1.67(m,5H),1.56~0.82(m,34H),0.65(s,3H).

[0067] The digital photos of the appearance of the preparations prepared in Experimental Examples 1 to 10 are as follows: Figure 2 shown.

[0068] Experimental Example 1

[0069] Preparation and Characterization of Pure AG-Chol Nanoparticles

[0070] 5 mg of AG-Chol obtained in Example 1 was accurately weighed and dissolved in 1 mL of a mixed solution of dichloromethane and anhydrous ethanol (the volume ratio of dichloromethane to anhydrous ethanol was V:V=4:1), and then 5 mL of ultrapure water was added. After ultrasonic dispersion by a cell crusher (power 360 W, time 5 min), the organic solvent was removed by rotary evaporation, and the suspension of AG-Chol nanoparticles was obtained after filtering through a filter membrane with a pore size of 0.22 μm. The measured average particle size was 56.75 nm, the polydispersity index (PDI) was 0.133, and the zeta potential was +10.1 mV. The prepared AG-Chol nanoparticles had no significant change when placed at 4° C. for 6 months, and had good stability.

[0071] Experimental Example 2

[0072] Preparation and Characterization of Pure AG-Chol Gel

[0073] 40 mg of AG-Chol obtained in Example 1 was accurately weighed and dissolved in 1 mL of a mixed solution of dichloromethane and anhydrous ethanol (the volume ratio of dichloromethane to anhydrous ethanol was V:V=4:1), and then 1 mL of a 2% aqueous solution of poloxamer 188 was added. After ultrasonic dispersion by a cell crusher (power 360 W, time 5 min), the organic solvent was removed by rotary evaporation to obtain AG-Chol gel. The prepared pure AG-Chol gel had no significant change when placed at 4°C for 6 months, and had good stability.

[0074] Experimental Example 3

[0075] Preparation and Characterization of Paclitaxel-Loaded AE-Chol Nanoparticles

[0076] 5 mg of AE-Chol and 1 mg of paclitaxel obtained in Example 2 were accurately weighed and dissolved in 0.5 mL of a mixed solution of dichloromethane and methanol (V:V=5:1), and then 2 mL of a 2% aqueous solution of poloxamer 188 was added. After ultrasonic dispersion in a cell crusher (power 360 W, time 5 min), magnetic stirring was performed for 12 h, and the mixture was filtered through a microporous filter membrane with a pore size of 0.22 μm to obtain AE-Chol nanoparticles encapsulating paclitaxel.

[0077] The average particle size of the paclitaxel-encapsulated AE-Chol nanoparticles was measured to be 78.71 nm, the PDI was 0.150, the zeta potential was +0.223 mV, and the prepared nanoparticles had no significant change after being stored at 4° C. for 6 months, indicating good stability.

[0078] Experimental Example 4

[0079] Preparation and Characterization of AG-Chol Nanoparticles Loaded with Doxorubicin

[0080] 5 mg of AG-Chol and 1 mg of doxorubicin obtained in Example 1 were accurately weighed and dissolved in 0.5 mL of anhydrous ethanol. Then, 2 mL of a 2% aqueous solution of poloxamer 188 was added. The mixture was ultrasonically dispersed in a cell crusher (power 360 W, time 5 min), magnetically stirred for 15 h, and filtered through a 0.22 μm filter membrane to obtain nanoparticles encapsulating doxorubicin.

[0081] The average particle size of the doxorubicin-loaded nanoparticles was measured to be 66.93 nm, the PDI was 0.145, the zeta potential was +15 mV, and the prepared nanoparticles showed no significant change after being stored at 4° C. for 6 months, indicating good stability.

[0082] Experimental Example 5

[0083] Preparation and Characterization of AG-Chol Liposomes Encapsulating Doxorubicin Hydrochloride

[0084] Accurately weigh 5 mg of AG-Chol obtained in Example 1 and 15 mg of egg yolk lecithin, dissolve in 2 mL of a mixed solution of chloroform and anhydrous ethanol (V:V=6:1), evaporate the organic solvent, add 1 mL of an aqueous solution of ammonium sulfate, ultrasonically disperse with a cell crusher (power 360 W, time 5 min), dialyze for 4 h, add 2 mg of doxorubicin hydrochloride (DOX) and dissolve in 500 μL of ultrapure water to obtain a liposome suspension encapsulating doxorubicin hydrochloride.

[0085] The average particle size of the AG-Chol liposome suspension containing doxorubicin hydrochloride is 105.7 nm, the PDI is 0.110, the zeta potential is +8.04 mV, and the prepared nanoparticles have no significant changes when placed at 4° C. for 6 months, and have good stability.

[0086] Experimental Example 6

[0087] Preparation and Characterization of Paclitaxel-Loaded AG-Chol Liposomes

[0088] 5 mg of AG-Chol, 15 mg of egg yolk lecithin and 2 mg of paclitaxel obtained in Example 1 were accurately weighed and dissolved in 2 mL of a mixed solution of chloroform and anhydrous ethanol (V:V=6:1). Then 4 mL of the aqueous solution was added, and the mixture was ultrasonically dispersed using a cell crusher (power 360 W, time 5 min), magnetically stirred for 14 h, and filtered through a 0.22 μm filter membrane to obtain a liposome suspension containing paclitaxel.

[0089] The average particle size of the AG-Chol liposome suspension containing paclitaxel is 115.2 nm, the PDI is 0.213, the zeta potential is -4.99 mV, and the prepared nanoparticles have no significant changes when placed at 4° C. for 6 months, and have good stability.

[0090] Experimental Example 7

[0091] Preparation and Characterization of AG-Chol Nanoparticles Encapsulating STAT-siRNA

[0092] 50 mg of AG-Chol obtained in Example 1 was accurately weighed and dissolved in a mixed solution of 2.0 mL of dichloromethane and anhydrous ethanol (V:V=4:1), and then 10 mL of a 1.5% PVA1788 aqueous solution was added. After ultrasonic dispersion by a cell crusher (power 360 W, time 5 min), magnetic stirring was performed for 12 h to evaporate the organic solvent, and a nanoparticle suspension was obtained after filtering through a 0.45 μm filter membrane. Then 1 μg of STAT-siRNA was added, vortexed for 10 s, and allowed to stand for 1 h to obtain an AG-Chol nanoparticle suspension encapsulating siRNA.

[0093] The average particle size of the AG-Chol nanoparticle suspension containing siRNA is 84.55 nm, the PDI is 0.080, the zeta potential is +1.84 mV, and the prepared nanoparticles have no significant changes after being placed at 4° C. for 6 months, and have good stability.

[0094] Experimental Example 8

[0095] Preparation and Characterization of Nanoparticles Containing Paclitaxel, AG-Chol and PLGA

[0096] Accurately weigh 3 mg of AG-Chol, 2 mg of paclitaxel, and 2 mg of PLGA obtained in Example 1. 9k 5 mg was dissolved in 1 mL of dichloromethane: anhydrous ethanol organic phase (V:V = 4:1). After complete dissolution, 5 mL of 2% poloxamer 188 aqueous solution was added. After ultrasonic dispersion in a cell crusher, the mixture was stirred with an open magnetic stirrer in a fume hood for 12 h. The mixture was filtered through a 0.22 μm filter membrane to obtain a nanoparticle suspension containing paclitaxel.

[0097] The nanoparticle suspension containing paclitaxel has an average particle size of 122.4 nm, a PDI of 0.219, and a zeta potential of -0.838 mV. The prepared nanoparticles have no significant changes when placed at 4° C. for 6 months, and have good stability.

[0098] Experimental Example 9

[0099] Preparation and Characterization of Nanoparticles Containing Paclitaxel, AG-Chol and mPEG-PLGA

[0100] Weigh 3 mg of AG-Chol, 2 mg of paclitaxel, and 2 mg of mPEG obtained in Example 1. 3.5k -PLGA 9k 5 mg was dissolved in 1 mL of a mixed organic phase of dichloromethane and anhydrous ethanol (V:V=3:1). After complete dissolution, 5 mL of a 2 wt% poloxamer 407 aqueous solution was added. After ultrasonic dispersion in a cell crusher, the mixture was stirred magnetically for 12 h. After filtering through a 0.22 μm filter membrane, a nanoparticle suspension containing paclitaxel, AG-Chol and mPEG-PLGA was obtained.

[0101] The nanoparticle suspension containing paclitaxel, AG-Chol and mPEG-PLGA has an average particle size of 134.5 nm, a PDI of 0.180, and a zeta potential of -0.636 mV. The prepared nanoparticles have no significant changes after being placed at 4° C. for 6 months, and have good stability.

[0102] Experimental Example 10

[0103] Preparation and characterization of solid lipid nanoparticles containing paclitaxel and AG-Chol

[0104] Accurately weigh 5 mg of AG-Chol and 2 mg of paclitaxel obtained in Example 1, heat and melt 0.075 g of glyceryl monostearate, 0.175 g of precirol ATO5, and 0.125 g of poloxamer 188, add the prescribed amount of paclitaxel and AG-Chol, stir rapidly to disperse the drugs, and then put them in the freezer to solidify for 4 hours. Heat the water in the constant temperature water bath to 60°C, transfer the solidified mixture to a heat preservation sleeve connected to a constant temperature water bath, add the prescribed amount of ultrapure water to the heat preservation sleeve, disperse it in a cell crusher by ultrasound, and filter it while hot to obtain solid lipid nanoparticles containing paclitaxel.

[0105] The average particle size of the solid lipid nanoparticles loaded with paclitaxel was measured to be 238.0 nm, the PDI was 0.310, the zeta potential was -12.7 mV, and the prepared nanoparticles had no significant change after being placed at 4° C. for 6 months, and had good stability.

[0106] Experimental Example 11

[0107] Preparation of liposomes containing IR780, AEAA-PEG-PLGA, and AG-Chol

[0108] Take 15 mg of egg yolk lecithin, 5 mg of AG-Chol obtained in Example 1 and 1 mg of AEAA-PEG 3.5K -PLGA 9K (AEAA is the abbreviation of aminoethylanisamide), 1 mg IR780 was dissolved in a mixed solution of 2 mL ethanol and chloroform (V:V=1:1), and the organic solvent was evaporated under reduced pressure using a rotary evaporator in a 50°C water bath to form a uniform lipid film, 1 mL PBS solution was added, and it was hydrated by ultrasound at 40°C (5 min, working 3 s, rest 5 s, power 360 W) to form uniform milky yellow liposomes.

[0109] Experimental Example 12

[0110] Hemolytic experiment of pure AG-Chol nanoparticles

[0111] Use a clean beaker to hold fresh mouse plasma, and stir with a glass rod to remove the fibrin clots in the mouse plasma. Add 2 to 3 times the volume of 0.9% saline injection to the blood from which the fibrin clots have been removed, mix thoroughly, and then divide into 10mL centrifuge tubes and centrifuge at a speed of 1500r / min for 5min. Discard the supernatant and repeat the above operation until the supernatant is no longer red, and obtain the red blood cells (RBC) at the bottom of the centrifuge tube. Transfer the RBC and saline injection to a 100mL volumetric flask, make it into 2% RBC, and place it in a 4℃ refrigerator for standby use. Add the AG-Chol nanoparticles obtained in Experimental Example 1 to 2% RBC, prepare a series of samples of different concentrations and incubate at 37℃ for 4h. Use the blank control group that does not produce hemolysis as the negative control, and use the completely hemolyzed water as the diluent as the positive control. After the incubation is completed, centrifuge at 3000r / min for 10min, observe whether the supernatant remains clear and transparent, and judge whether hemolysis occurs. The supernatant of each group was transferred to a 96-well plate, and the absorbance at a wavelength of 545 nm was measured with an ELISA reader to calculate the hemolysis rate. Hemolysis occurred when the hemolysis rate was higher than 5%.

[0112] The experimental results showed that when the administration concentration of AG-Chol was less than 37.5 mg / kg, the hemolysis rate of the sample was less than 5%, and intravenous injection was safe, indicating that AG-Chol can be used as a dosage form excipient for intravenous administration.

[0113] Experimental Example 13

[0114] Effects of AG-Chol liposomes loaded with doxorubicin hydrochloride on the activity of mouse hepatocellular carcinoma H22 cells

[0115] The present invention adopts CCK-8 method to determine the inhibition rate of H22 cells. H22 cells in the logarithmic growth phase are collected, centrifuged, diluted, and the cells are concentrated to a cell concentration of 1×10 4 10 μL of ultra-high sensitivity CCK-8 reagent was added to the well of a 96-well plate for 24 hours, and then the well was incubated in a dark place for 24 hours. After 10 μL of ultra-high sensitivity CCK-8 reagent was added to the well in a dark place, and then the well was shaken on a micro-oscillator for 10 minutes. After culturing for 4 hours, the absorbance (OD value) of each well at 490 nm was measured with an enzyme marker. The concentration of doxorubicin hydrochloride (DOX) in the experiment was designed as follows: 0.125, 0.25, 0.5, 1, 2, 4 μg / mL; a blank control group and a zero adjustment group were set up respectively, and 5 replicate wells were set up for each concentration.

[0116] The results showed that as the concentration increased, the AG-Chol liposomes loaded with doxorubicin hydrochloride prepared in Experimental Example 5 had an increased toxicity to mouse liver cancer H22 cells, with an IC50 value of 0.137±0.003μg / mL. Compared with the IC50 of ordinary liposomes (0.198±0.002μg / mL), the liposomes prepared in Experimental Example 5 had a stronger toxicity to H22 cells.

[0117] Experimental Example 14

[0118] In vivo pharmacodynamic studies

[0119] Take out the frozen H22 liver cancer cells from the -80℃ freezer, quickly place them in a 37℃ water bath to thaw, and then inoculate them into the abdominal cavity of female Kunming mice. One week later, use a 10mL syringe to extract the ascites of the tumor-bearing mice, and take 40 mice to inoculate them under the armpits of Kunming mice at a cell concentration of 1 million / 100μL / mouse. 5-7 days after inoculation, when the tumor volume reaches 100mm 3 When the mice were about 2 hours old, 28 mice were divided into 4 groups, each with 7 mice. The drug was administered once every 3 days (DOX concentration was 5 mg / kg) by intravenous injection. The experimental groups were blank control group, DOX solution group, DOX liposome group, and experimental group 5. After 8 days of treatment, all mice were killed, their appearance was observed, and the tumor weight was measured by dissection. The calculation formula of tumor weight inhibition rate is as follows:

[0120]

[0121] The results showed that there was no significant difference in the body weight of the above groups, and the mice were in good condition, indicating that the toxicity of the preparation was low. Compared with the blank control group, the tumor weight inhibition rates of the DOX solution group, the DOX liposome group, and the experimental example 5 group were 64.28%, 81.70%, and 94.61%, respectively. The results showed that the AG-Chol liposomes prepared by the present invention encapsulated doxorubicin hydrochloride helped it to exert a higher tumor inhibition effect.

[0122] In summary, the present invention provides an application of a cholesterol monoguanidine conjugate, and the cholesterol monoguanidine conjugate is prepared into a microparticle drug delivery system by encapsulating drugs. The cholesterol monoguanidine conjugate provided by the present invention is amphiphilic, and as a drug carrier, it can encapsulate fat-soluble drugs on the one hand, and on the other hand, due to the presence of the monoguanidine group, the compound is positively charged, and negatively charged water-soluble drugs can also be encapsulated by electrostatic adsorption. The presence of the guanidine group is also conducive to the intracellular membrane transport of the drug, so the monoguanidine cholesterol conjugate can be used as an excellent carrier material for microparticle preparations to encapsulate drugs. The monoguanidine cholesterol conjugate has good compatibility with commonly used carrier excipients, and the compound has low hemolysis and can be used as a dosage form excipient for intravenous administration; when it is used as a drug carrier, it helps to exert better drug efficacy. The present invention also provides a method for preparing the above-mentioned cholesterol monoguanidine conjugate, which has good reproducibility and can prepare a high-purity product.

[0123] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A low hemolytic cationic lipid that can be used as a pharmaceutical excipient, the basic structural formula of which is: Wherein X is CnH2n, and n is an integer greater than or equal to 2.

2. The cationic lipid according to claim 1, wherein Cholesterol monoguanidine conjugates can be used to prepare low hemolytic cationic liposomes or nanoparticles.

3. The cationic lipid according to claim 1, wherein Cholesterol monoguanidine conjugates can replace cholesterol for the preparation of low hemolytic cationic liposomes.

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