CN112870178A - Phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition - Google Patents

Phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition Download PDF

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CN112870178A
CN112870178A CN201911195675.5A CN201911195675A CN112870178A CN 112870178 A CN112870178 A CN 112870178A CN 201911195675 A CN201911195675 A CN 201911195675A CN 112870178 A CN112870178 A CN 112870178A
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alkaloid derivative
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phenanthroindolizidine alkaloid
fatty acid
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刘玉玲
王洪亮
汪仁芸
夏学军
李琳
庾石山
陈晓光
李燕
扈金萍
高丽丽
王汝冰
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Abstract

The invention belongs to the field of pharmaceutical preparations, and discloses a phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, a fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition and a preparation method thereof. The composition of the invention consists of lipid, phospholipid, emulsifier and water, and can be directly used or diluted by adding water for use. The method has simple and easy process, does not use organic solvent, is beneficial to industrial amplification, and has good popularization and application prospects.

Description

Phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, a fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition, and a preparation method and application thereof.
Background
The phenanthroindolizidine alkaloid derivative is one or more compounds with new structure and antitumor activity separated from plants of Tylophora japonica or Scopolia dichotoma of Asclepiadaceae. It can inhibit the Hedgehog pathway over-expressed in tumor cells to achieve antitumor effect[1]. However, the compounds belong to BCS IV, and not only have poor solubility in an aqueous medium, but also have low solubility in a lipid adjuvant, and have weak membrane penetration capability, so that the oral bioavailability is low.
Drugs with poor solubility in aqueous media and lipid excipients are more difficult to design for drug delivery vehicles. Meanwhile, the drug effect and the toxicity are important indexes for evaluating the clinical application possibility of the compound, and the drug absorption and the toxicity of preparation auxiliary materials are two important factors. The increased absorption of the medicine in vivo is helpful for the exertion of the medicine effect, and the low-toxicity auxiliary materials can ensure the safety. The oral absorption and the drug effect of the medicine are increased, and the pharmaceutical research of simultaneously using low-toxicity auxiliary materials is an important means for increasing the drug property of the compound.
Many studies have reported that Drug Delivery Systems (DDS) are used to increase the oral bioabsorption of drugs, such as solid lipid nanoparticles, micelles, and liquid lipid nanoparticle delivery systems[2]。
The Solid Lipid Nanoparticle (SLN) is a novel nanoparticle drug delivery system with particle size of 10-1000nm prepared by using natural or artificial lipid material as carrier and adsorbing or coating drug in lipid core. Compared with the common nano preparation, the solid lipid nanoparticle can effectively improve the oral bioavailability of the drug, is suitable for administration in various ways, has the characteristics of almost no toxicity to organisms, good biocompatibility, strong drug-loading capacity, sustained and controlled release and targeting effects, stable physical and chemical storage, low cost, contribution to large-scale production and the like, and becomes a new technology for researching oral preparations. The solid lipid nanoparticle can improve drug solubility, increase gastrointestinal mucosa permeability, and increase oral absorption of drug[3]. However, the phenanthroindolizidine alkaloid derivative has low solubility in the common medicinal solid lipid nanoparticles, cannot meet the requirements of preparations, needs to be comprehensively adjusted by a prescription to meet the preparation requirements, and meets the requirements of effective dose. At present, the reports of solid lipid nanoparticles of phenanthroindolizidine alkaloid derivatives are not found.
In order to promote the biological absorption of the BCS IV medicament phenanthroindolizidine alkaloid derivative, a medicament delivery system of the derivative needs to be deeply researched.
Disclosure of Invention
The technical problem to be solved by the invention is that the oral administration bioavailability of the phenanthroindolizidine alkaloid derivative is low, the water solubility and the fat solubility are poor, and the preparation of an administration carrier is difficult. The invention discloses two novel phenanthroindolizidine alkaloid derivative solid lipid nanoparticle compositions and a preparation method thereof for the first time, the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition has small particle size and high entrapment rate, 70-90% is released in 72 hours, and the gastrointestinal mucosa permeability of the phenanthroindolizidine alkaloid derivative can be increased and the bioavailability can be improved.
The phenanthroindolizidine alkaloid derivative is as follows: (13aS) -3-pivaloyloxy-6, 7-dimethoxy-9-phenanthro [9,10-b ]]Indolizidine (CAT3), (13aS) -3-hydroxy-6, 7-dimethoxy-9-phenanthro [9, 10-b)]-indolizidine (PF430) or dextro-deoxytylophorinine (CAT)[4，5]。
IC of CAT and CAT3 in cells50Are all 10-7-10-8mol/L. The two can be regarded as prodrugs, and the main metabolite is PF403 in vivo, so that the prodrug has higher biological activity and IC for tumor cells of various tissue sources50Is 10-10-10-12mol/L. PF403 can cross blood brain barrier and reach brain tissue to exert effect in resisting brain diseases[6]。
The invention aims to improve the water solubility of phenanthroindolizidine alkaloid derivatives through a solid lipid nanoparticle composition.
The invention also aims to prepare the solid lipid nanoparticles after forming conjugates of fatty acid and drugs, which is more favorable for encapsulating the phenanthroindolizidine alkaloid derivatives in the lipid core and increasing the encapsulation efficiency.
Another purpose of the invention is to form conjugate of fatty acid and drug, then prepare solid lipid nanoparticles, wrap phenanthroindolizidine alkaloid derivative in oil drops, convert crystalline drug into amorphous state, and contribute to increase bioavailability.
Another purpose of the invention is to prepare solid lipid nanoparticles by forming a conjugate of fatty acid and a drug, and wrap the phenanthroindolizidine alkaloid derivative in oil drops, so that the phenanthroindolizidine alkaloid derivative with electropositivity can be transferred from the surface of a lipid core to the interior of the lipid core, the electronegativity absolute value of the nanoparticles is increased, the particle same-electric repulsion force is increased, and aggregation is avoided.
The invention also aims to form the solid lipid nanoparticles, improve the surface hydrophobic property of the phenanthroindolizidine alkaloid derivative through amphiphilic molecules on the surface of a lipid core, increase the specific surface area, increase the permeation of intestinal mucosa and improve the bioavailability.
Another object of the present invention is to enhance tumor cell inhibitory ability by the nano-composition.
In order to solve the technical problems and achieve the above object, the present invention provides the following technical solutions:
the first aspect of the technical scheme of the invention provides a phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition which is characterized by containing phenanthroindolizidine alkaloid derivatives, lipid, phospholipid, an emulsifier and water;
phenanthroindolizidine-containing alkaloid derivatives: 0.1-50mg/mL,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 10 percent of the total weight of the mixture,
phospholipid: 0.05 to 10 percent of the total weight of the mixture,
emulsifier: 0.05 to 10 percent of the total weight of the mixture,
water: 70 to 99.8 percent.
Preferably, the phenanthroindolizidine-containing alkaloid derivative: 0.1-50mg/mL,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 7 percent of the total weight of the mixture,
phospholipid: 0.1 to 5 percent of the total weight of the mixture,
emulsifier: 0.1 to 5 percent of the total weight of the mixture,
water: 70 to 99.7 percent.
More preferably, the phenanthroindolizidine-containing alkaloid derivative: 0.1-50mg/mL,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 2 to 5 percent of the total weight of the mixture,
phospholipid: 0.5 to 2.5 percent of,
emulsifier: 0.5 to 2.5 percent of,
water: 90 to 97 percent.
The second aspect of the technical scheme of the invention provides a fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition, which is characterized by containing a fatty acid-phenanthroindolizidine alkaloid derivative conjugate, lipid, phospholipid, an emulsifier and water;
fatty acid-phenanthroindolizidine alkaloid derivative conjugate: 0.2-100mg/mL,
the weight ratio of the fatty acid to the phenanthroindolizidine alkaloid derivative is 0.5: 1 to 10: 1,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 10 percent of the total weight of the mixture,
phospholipid: 0.05 to 10 percent of the total weight of the mixture,
emulsifier: 0.05 to 10 percent of the total weight of the mixture,
water: 70 to 99.8 percent.
Preferably, the conjugate contains fatty acid-phenanthroindolizidine alkaloid derivatives: 0.2-100mg/mL,
the weight ratio of the fatty acid to the phenanthroindolizidine alkaloid derivative is 2: 1 to 8: 1,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 7 percent of the total weight of the mixture,
phospholipid: 0.1 to 5 percent of the total weight of the mixture,
emulsifier: 0.1 to 5 percent of the total weight of the mixture,
water: 70 to 99.7 percent.
More preferably, the conjugate containing fatty acid-phenanthroindolizidine alkaloid derivative: 0.2-100mg/mL,
the weight ratio of the fatty acid to the phenanthroindolizidine alkaloid derivative is 4: 1 to 7: 1,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 2 to 5 percent of the total weight of the mixture,
phospholipid: 0.5 to 2.5 percent of,
emulsifier: 0.5 to 2.5 percent of,
water: 90 to 97 percent.
In the first and second aspects, the phenanthroindolizidine alkaloid derivative comprises (13aS) -3-pivaloyloxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine, (13aS) -3-hydroxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine or dextrodeoxytylosin, and the structural formula is shown aS follows:
Figure BDA0002294597040000041
(13aS) -3-pivaloyloxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine
Figure BDA0002294597040000051
(13aS) -3-hydroxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine
Figure BDA0002294597040000052
Dextrorotation deoxidation tylophorinine.
The lipid is selected from one or more of fatty acid, glyceride, cholesterol and phosphorus wax lipid; wherein the fatty acid is one or more of oleic acid, stearic acid, palmitic acid, myristic acid, behenic acid and caprylic acid mixed in any proportion; the glyceride is one or more of glyceryl monostearate, glyceryl distearate, glyceryl tristearate, glyceryl monopalmitate, glyceryl dipalmitate, glyceryl tripalmitate, glyceryl monomyristate, glyceryl dimyristate, glyceryl trimyristate, glyceryl monobehenate, glyceryl dibehenate, glyceryl tribehenate, glyceryl trioleate and glyceryl trilaurate; the phosphorus wax ester is one or more of microcrystalline paraffin and whale fat wax in any proportion.
The phospholipid comprises one or more of soybean phospholipid, yolk phospholipid, lecithin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl glycerol, dipalmitoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dipalmitoyl phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine.
The emulsifier comprises one or more of poloxamer, cholate, short-chain alcohol, polysorbate, polyoxyethylene fatty alcohol ether and polyoxyethylene fatty acid ester which are mixed in any proportion; wherein the poloxamer comprises one or more of poloxamer 188, poloxamer 182, poloxamer 407 and poloxamer 908 in any proportion; the cholate comprises one or more of cholate, glycocholate and sodium taurocholate in any proportion; the short chain alcohol comprises one or more of glycerol, butanol and propylene glycol in any proportion; the polysorbate comprises one or more of tween 20 and tween 80, and is mixed at any ratio; the polyoxyethylene fatty alcohol ether comprises one or more of polyoxyethylene fatty alcohol ether Brij 78, polyoxyethylene fatty alcohol ether Brij 35 and polyoxyethylene fatty alcohol ether Brij 30 in any proportion; the polyoxyethylene fatty acid ester comprises one or more of polyoxyethylene fatty acid ester Myrj 53 and polyoxyethylene fatty acid ester Myrj 59.
In the second aspect, the fatty acid is selected from one or a mixture of more than one of oleic acid, stearic acid, palmitic acid, myristic acid, behenic acid and caprylic acid in any ratio.
The solid lipid nanoparticle composition may further comprise a lyoprotectant. The freeze-drying protective agent comprises one or two of trehalose, mannitol, glucose, mannose, sucrose and maltose.
The solid lipid nanoparticle composition can be further prepared into tablets, capsules, soft capsules, injections, enemas, nasal drops, transdermal patches or mucosal patches.
The third aspect of the technical scheme of the invention provides a preparation method of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, which is characterized in that in the first step, phenanthroindolizidine alkaloid derivative, lipid and phospholipid are mixed, heated and melted at a temperature which is 10-15 ℃ higher than the melting point of a lipid material to obtain an organic phase, and a required amount of emulsifier is dissolved in water and heated to the same temperature of the organic phase to obtain a water phase; secondly, under the high shearing action with the shearing speed of 10000 to 16000rmp and the temperature of 10 to 15 ℃ higher than the melting point of the lipid material, uniformly mixing the organic phase and the water phase to prepare primary emulsion; thirdly, carrying out ultrasonic treatment on the primary emulsion for 3-15min in a probe ultrasonic instrument under the power of 100 plus one 500W at the temperature of 10-15 ℃ higher than the melting point of the lipid material, or homogenizing for 5-15 times in a high-pressure homogenizer under the pressure of 500 plus one 1500bar to obtain liquid lipid nanoparticles; fourthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of between 0 and 30 ℃ to obtain the lipid nanoparticles.
The fourth aspect of the technical scheme of the invention also provides a preparation method of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition in the second aspect, which is characterized in that in the first step, the phenanthroindolizidine alkaloid derivative and fatty acid react for 1 to 24 hours at the temperature of 40 to 80 ℃ to form a conjugate; secondly, mixing lipid and phospholipid, heating and melting and dissolving at the temperature of 10-15 ℃ higher than the melting point of the lipid material, adding the conjugate to dissolve to obtain an organic phase, dissolving a required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; thirdly, uniformly mixing the organic phase and the water phase under the high shearing action with the shearing speed of 10000 to 16000rmp at the temperature of 10 to 15 ℃ higher than the melting point of the lipid material to prepare primary emulsion; fourthly, at the temperature of 10-15 ℃ higher than the melting point of the lipid material, carrying out ultrasonic treatment on the primary emulsion for 3-15min under the power of 100-500W in a probe ultrasonic instrument, or homogenizing for 5-15 times under the pressure of 500-1500bar in a high-pressure homogenizer to obtain liquid lipid nanoparticles; fifthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 0-30 ℃ to obtain the lipid nanoparticle.
The fifth aspect of the technical scheme of the invention provides an application of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition in the first and second aspects in preparation of a medicament for preventing and/or treating cancer and/or inflammatory diseases. The cancer is selected from glioma, bone marrow neuroblastoma, colon cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, lung cancer and pancreatic cancer.
Through the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, the following beneficial effects can be realized:
the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition can be diluted with water, artificial gastrointestinal fluid or physiological body fluid in any proportion under the action of slight stirring or esophageal peristalsis and the like.
The nanometer solid lipid nanoparticles improve the water solubility and encapsulation rate of the phenanthroindolizidine alkaloid derivative.
The nano particle size has large specific surface area, hydrophilic surface and electronegativity, can promote mutual electrostatic repulsion of lipid particles, and has better physical stability.
The phenanthroindolizidine alkaloid derivative is physically wrapped by the lipid nanoparticles, so that the crystal form order of the raw material medicines can be reduced or the raw material medicines exist in an amorphous state, and the bioavailability is increased.
By combining the characteristics, the solid lipid nanoparticle composition of the phenanthroindolizidine alkaloid derivative can improve the bioavailability of the medicine by increasing one or more characteristics of the solubility and the gastrointestinal mucosa intake and transportation of the phenanthroindolizidine alkaloid derivative. Has better action time-cytotoxic effect in tumor cells and is beneficial to the treatment effect of tumors.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition can be further prepared into various corresponding dosage forms such as tablets, capsules, soft capsules, injections, enemas, nasal drops, transdermal patches or mucosal patches.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition can be used for treating cancers and/or inflammatory diseases, and is particularly applied to cancers such as human glioma, human marrow neuroblastoma, human colon cancer, human gastric cancer, human ovarian cancer, cervical cancer, liver cancer, lung cancer, pancreatic cancer and the like.
Drawings
Figure 1 is a schematic representation of the reaction of oleic acid with phenanthroindolizidine alkaloid derivative conjugates.
Figure 2 is a graph of the infrared absorption of oleic acid and phenanthroindolizidine alkaloid derivative conjugates.
Fig. 3 is a DSC thermogram of conjugate of oleic acid and phenanthroindolizidine alkaloid derivative, a: CAT3, b: oleic acid, c: OA-CAT 3.
FIG. 4 shows the particle size distribution of the solid lipid nanoparticle composition containing phenanthroindolizidine alkaloid derivatives.
FIG. 5 is the surface charge measurement of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition.
Fig. 6 shows drug loading and encapsulation efficiency of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition.
Fig. 7 is an in vitro release curve of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition.
Fig. 8 is a PXRD pattern of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, a: CAT3, b: physical mixture of CAT3 with blank SLN, c: blank SLN, d: CAT3-SLN, e: OA-CAT 3-SLN.
Fig. 9 is a DSC chart of a phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, a: CAT3, b: physical mixture of CAT3 with blank SLN, c: blank SLN, d: CAT3-SLN, e: OA-CAT 3-SLN.
Fig. 10 is a graph of the permeability of the solid lipid nanoparticle composition in a single layer of MDCK-MDR1 cell membrane.
Fig. 11 is a graph of the results of in vitro glioma cell inhibition tests of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition.
Fig. 12 shows the effect of increasing plasma bioavailability of active metabolite (PF403) after oral administration of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition.
Examples
Example 1: solid lipid nanoparticle composition containing phenanthroindolizidine alkaloid derivatives with different lipid components
The formation of the solid lipid nanoparticle composition of the phenanthroindolizidine alkaloid derivative is examined by taking (13aS) -3-pivaloyloxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine (CAT3) aS a model drug (API) when different lipid components are used. The composition consists of the following components in percentage by weight:
TABLE 1 Phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition formula
Figure BDA0002294597040000081
Figure BDA0002294597040000091
The preparation method comprises the following steps: firstly, mixing CAT3, lipid and phospholipid, heating and melting at 80 ℃ and dissolving to obtain an organic phase, dissolving required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; secondly, uniformly mixing an organic phase and a water phase under the high shearing action with the shearing speed of 13000rmp at the temperature of 80 ℃ to prepare primary emulsion; thirdly, carrying out ultrasonic treatment on the primary emulsion in a probe ultrasonic instrument for 8min under the power of 300W at the temperature of 80 ℃ to obtain liquid lipid nanoparticles; and fourthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 0 ℃ to obtain the phenanthroindolizidine alkaloid derivative solid lipid nanoparticles.
The physical stability is an index for representing the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, and the determination method comprises the following steps: the composition is not agglomerated and does not delaminate after being placed overnight at 4 ℃, and is considered to have better stability.
The particle size and distribution of the solid lipid nanoparticle composition and the Zeta potential are important physical indexes of the dosage form, the biological properties of the solid lipid nanoparticle composition are possibly different when the particle size is different, and the electrostatic repulsion force of the solid lipid nanoparticle composition is influenced by the Zeta potential when the particle size is different from the Zeta potential, so that the shape of the solid lipid nanoparticle composition is necessarily represented. The particle size and distribution and the Zeta potential can be characterized by the following methods:
taking a proper amount of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, slowly dispersing the composition in water according to the proportion of 1:100, and observing whether the solution is transparent or not, wherein if a transparent solution can be obtained, the particle size is less than 200 nm. The particle size and distribution and the Zeta potential of the specific values can also be determined by means of laser dynamic light scattering. The results show that: the product is placed overnight at the temperature of 4 ℃ without agglomeration and delamination, and has better stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Example 2: solid lipid nanoparticle composition containing phenanthroindolizidine alkaloid derivatives with different lipid ratios
CAT3 was used as a model drug to examine the formation of solid lipid nanoparticle compositions of phenanthroindolizidine alkaloid derivatives at different oil component ratios. The composition consists of the following components in percentage by weight:
TABLE 2 Phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition formula
Figure BDA0002294597040000092
Figure BDA0002294597040000101
The preparation method comprises the following steps: firstly, mixing CAT3, lipid and phospholipid, heating and melting at 80 ℃ and dissolving to obtain an organic phase, dissolving required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; secondly, under the high shearing action with the shearing speed of 10000rmp to 16000rmp and the temperature of 80 ℃, uniformly mixing the organic phase and the water phase to prepare primary emulsion; thirdly, carrying out ultrasonic treatment on the primary emulsion in a probe ultrasonic instrument for 15min under the power of 100W at the temperature of 80 ℃ to obtain liquid lipid nanoparticles; and fourthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 0 ℃ to obtain the phenanthroindolizidine alkaloid derivative solid lipid nanoparticles. Recipe A1 was prepared in the same manner as in example 1.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition is placed overnight at 4 ℃ without agglomeration and delamination, and has good stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Example 3: phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition with different phospholipid components
CAT3 was used as a model drug to examine the formation of solid lipid nanoparticle compositions of phenanthroindolizidine alkaloid derivatives with different phospholipid compositions and dosages. The composition consists of the following components in percentage by weight:
TABLE 3 Phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition formula
Figure BDA0002294597040000102
The preparation method comprises the following steps: firstly, mixing CAT3, lipid and phospholipid, heating and melting at 80 ℃ and dissolving to obtain an organic phase, dissolving required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; secondly, under the high shearing action with the shearing speed of 10000rmp and at the temperature of 80 ℃, uniformly mixing an organic phase and a water phase to prepare primary emulsion; thirdly, carrying out ultrasonic treatment on the primary emulsion in a probe ultrasonic instrument for 3min under the power of 500W at the temperature of 80 ℃ to obtain liquid lipid nanoparticles; and fourthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 30 ℃ to obtain the phenanthroindolizidine alkaloid derivative solid lipid nanoparticles. Recipe A1 was prepared in the same manner as in example 1.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition is placed overnight at 4 ℃ without agglomeration and delamination, and has good stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Example 4: phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition with different emulsifier components
CAT3 was used as a model drug to examine the formation of solid lipid nanoparticle compositions of phenanthroindolizidine alkaloid derivatives when the compositions of different emulsifiers were combined. The composition consists of the following components in percentage by weight:
TABLE 4 Phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition formula
Figure BDA0002294597040000111
The preparation method comprises the following steps: firstly, mixing CAT3, lipid and phospholipid, heating and melting at 80 ℃ and dissolving to obtain an organic phase, dissolving required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; secondly, under the high shearing action with the shearing speed of 10000rmp and at the temperature of 80 ℃, uniformly mixing an organic phase and a water phase to prepare primary emulsion; thirdly, carrying out ultrasonic treatment on the primary emulsion in a probe ultrasonic instrument for 3min under the power of 500W at the temperature of 80 ℃ to obtain liquid lipid nanoparticles; fourthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 30 ℃ to obtain the lipid nanoparticle. Recipe A1 was prepared in the same manner as in example 1.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition is placed overnight at 4 ℃ without agglomeration and delamination, and has good stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Example 5: solid lipid nanoparticle composition containing fatty acid-phenanthroindolizidine alkaloid derivative conjugate with different lipid components
CAT3 is used as a model drug to investigate the formation of the fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition when different lipid components are used. The composition consists of the following components in percentage by weight:
solid lipid nanoparticle composition formula of compound conjugated by 5 fatty acid-phenanthroindolizidine alkaloid derivatives
Figure BDA0002294597040000121
The preparation method comprises the following steps: a first step of reacting CAT3 with a fatty acid at a temperature of 45 ℃ for 1 to 24h to form a conjugate (OA-CAT 3); secondly, mixing the lipid and the phospholipid, heating and melting at the temperature of 80 ℃, dissolving, adding OA-CAT3 to dissolve to obtain an organic phase, dissolving the required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; thirdly, uniformly mixing the organic phase and the water phase under the high shearing action with the shearing speed of 16000rmp at the temperature of 80 ℃ to prepare primary emulsion; fourthly, carrying out ultrasonic treatment on the primary emulsion in a probe ultrasonic instrument at the temperature of 80 ℃ for 8min under the power of 300W to obtain liquid lipid nanoparticles; and fifthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 0 ℃ to obtain the lipid nanoparticle.
The physical stability is an index for representing the solid lipid nanoparticle composition, and the determination method comprises the following steps: the composition is not agglomerated and does not delaminate after being placed overnight at 4 ℃, and is considered to have better stability.
The particle size and distribution of the solid lipid nanoparticle composition and the Zeta potential are important physical indexes of the dosage form, the biological properties of the solid lipid nanoparticle composition are possibly different when the particle size is different, and the electrostatic repulsion force of the solid lipid nanoparticle composition is influenced by the Zeta potential when the particle size is different from the Zeta potential, so that the shape of the solid lipid nanoparticle composition is necessarily represented. The particle size and distribution and the Zeta potential can be characterized by the following methods:
taking a proper amount of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition, slowly dispersing the composition in water according to the proportion of 1:100, and observing whether the solution is transparent or not, wherein if a transparent solution can be obtained, the particle size is less than 200 nm. The particle size and distribution and the Zeta potential of the specific values can also be determined by means of laser dynamic light scattering. The results show that: the product is placed overnight at the temperature of 4 ℃ without agglomeration and delamination, and has better stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Example 6: fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition with different lipid ratios
CAT3 is used as a model drug to examine the formation of the fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition in different oil component ratios. The composition consists of the following components in percentage by weight:
solid lipid nanoparticle composition formula of compound conjugated by 6 fatty acid-phenanthroindolizidine alkaloid derivatives
Figure BDA0002294597040000131
The preparation method comprises the following steps: firstly, reacting phenanthroindolizidine alkaloid derivative with fatty acid at the temperature of 45 ℃ for 10 hours to form OA-CAT 3; secondly, mixing the lipid and the phospholipid, heating and melting at the temperature of 80 ℃, dissolving, adding OA-CAT3 to dissolve to obtain an organic phase, dissolving the required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; thirdly, uniformly mixing the organic phase and the water phase under the high shearing action with the shearing speed of 10000rmp at the temperature of 80 ℃ to prepare primary emulsion; fourthly, carrying out ultrasonic treatment on the primary emulsion in a probe ultrasonic instrument at the temperature of 80 ℃ for 3min under the power of 500W to obtain liquid lipid nanoparticles; and fifthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 30 ℃ to obtain the lipid nanoparticle. Recipe B1 was performed as in example 5.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition is placed overnight at 4 ℃ without agglomeration and delamination, and has good stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Example 7: fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition with different phospholipid components
CAT3 is used as a model drug to examine the formation of the fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition under different phospholipid compositions and dosage. The composition consists of the following components in percentage by weight:
solid lipid nanoparticle composition formula of 7 phenanthrene fatty acid-phenanthroindolizidine alkaloid derivative conjugate
Figure BDA0002294597040000132
Figure BDA0002294597040000141
The preparation method comprises the following steps: firstly, reacting phenanthroindolizidine alkaloid derivative with fatty acid at the temperature of 45 ℃ for 10 hours to form OA-CAT 3; secondly, mixing the lipid and the phospholipid, heating and melting at the temperature of 80 ℃, dissolving, adding OA-CAT3 to dissolve to obtain an organic phase, dissolving the required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; thirdly, uniformly mixing the organic phase and the water phase under the high shearing action with the shearing speed of 16000rmp at the temperature of 80 ℃ to prepare primary emulsion; fourthly, carrying out ultrasonic treatment on the primary emulsion in a probe ultrasonic instrument for 15min under the power of 100W at the temperature of 80 ℃ to obtain liquid lipid nanoparticles; and fifthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 0 ℃ to obtain the lipid nanoparticle. Recipe B1 was performed as in example 5.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition is placed overnight at 4 ℃ without agglomeration and delamination, and has good stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Example 8: fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition with different emulsifier components
CAT3 is used as a model drug to investigate the formation of the fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition when different emulsifiers are composed. The composition consists of the following components in percentage by weight:
solid lipid nanoparticle composition formula of conjugate of epi 8 fatty acid-phenanthroindolizidine alkaloid derivative
Figure BDA0002294597040000142
The preparation method comprises the following steps: firstly, reacting phenanthroindolizidine alkaloid derivative with fatty acid at the temperature of 45 ℃ for 10 hours to form OA-CAT 3; secondly, mixing the lipid and the phospholipid, heating and melting at the temperature of 80 ℃, dissolving, adding OA-CAT3 to dissolve to obtain an organic phase, dissolving the required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; thirdly, uniformly mixing the organic phase and the water phase under the high shearing action with the shearing speed of 10000rmp at the temperature of 80 ℃ to prepare primary emulsion; fourthly, homogenizing the primary emulsion for 7 times in a high-pressure homogenizer at the temperature of 80 ℃ under the pressure of 800bar to obtain liquid lipid nanoparticles; and fifthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 0 ℃ to obtain the lipid nanoparticle. Recipe B1 was performed as in example 5.
The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition is placed overnight at 4 ℃ without agglomeration and delamination, and has good stability. Diluting with 1:100 times of water under slight stirring to form transparent emulsion with particle size less than 200 nm.
Experimental example 1: CAT3 and oleic acid conjugates (OA-CAT3 for short)
This example was characterized using the intermediate CAT3 of formula B1 and an oleic acid conjugate. The two conjugation process is schematically shown in figure 1. OA-CAT3 was taken and characterized by FTIR (FIG. 2) and DSC (FIG. 3).
The results show that: the infrared absorption spectrum shows that the OA-CAT3 spectrum is obviously different from the individual spectra of CAT3 and oleic acid. OA-CAT3 at 2793cm-1Obvious asymmetric stretching vibration peak (v) appears atas NH+) Features of tertiary amine salts are shown; meanwhile, the peak of asymmetric stretching vibration of the dissociated carboxyl group is also shown at 1560cm-1At least one of (1) and (b); and, at 1330cm of the map-1And 1172cm-1Showing a dissociated carboxyl group stretching vibration peak and a C-N stretching vibration peak in CAT 3; and, the hydroxyl group in the oleic acid molecule out-of-plane bending vibration (938 cm)-1) Is obviously reduced. The change in peak positions above confirms the conjugated interaction of CAT3 with oleic acid.
DSC shows that the endothermic peak at-11.8 ℃ of the oleic acid is respectively the endothermic peak of the transformation from the gamma crystal form to the alpha crystal form, and the endothermic peak at 13.7 ℃ is the melting peak of the alpha crystal form. CAT3 has crystalline melting peaks at 195.8 ℃ and 213.7 ℃. In the OA-CAT3 conjugate, the characteristic endothermic peaks of oleic acid and CAT3 disappeared and a conjugate melting peak at 7.5 ℃ appeared.
Experimental example 2: characterization of phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition
Particle size and distribution, surface charge:
the solid lipid nanoparticles in formulas A1 and B1 were slowly dispersed in water at a ratio of 1:100, and the particle size and surface charge were measured by laser dynamic light scattering method, as shown in Table 9 and FIGS. 4 and 5.
Drug loading and encapsulation efficiency:
the method for measuring the drug loading rate comprises the following steps: precisely measuring the solid lipid nanoparticles in the formulas A1 and B1, respectively dissolving the solid lipid nanoparticles in hot ethanol at 50 ℃, and filtering the solid lipid nanoparticles by using a PVDF (polyvinylidene fluoride) microporous filter membrane with the diameter of 0.22 mu m. Measured by UV spectrophotometry at 263 nm. The encapsulation efficiency is determined by ultrafiltration. Precisely measuring 1mL of test solution, and placing
Figure BDA0002294597040000164
In an Ultra-4100 k ultrafiltration tube, centrifugation was carried out at 15,000g centrifugal force at 4 ℃ for 30 min. The filtrate was measured by UV spectrophotometry at 263 nm. The calculation formula is as follows, and the results are shown in FIG. 6 and Table 9.
Figure BDA0002294597040000161
Figure BDA0002294597040000162
In vitro release profile:
precisely measuring 2mL of the solid lipid nanoparticles in the formulas A1 and B1 respectively, diluting the solid lipid nanoparticles in a release medium of 0.1% SDS (w/v) at 37 ℃ in 400mL, and simultaneously taking 5mL of a blank medium of 0.1% SDS (w/v) and placing the blank medium in an 8-10 kDa MWCO dialysis bag as a test solution. The determination method of the raw material medicine comprises the following steps: precisely weighing 5mg, placing in 1000mL of release medium, and operating in the same way. Slowly stirring in 37 deg.C thermostatic water bath, sampling 1mL at different time within 72h, centrifuging at 12000rpm for 5min, collecting supernatant, placing in ultraviolet spectrophotometer, measuring absorbance at 262nm, calculating CAT3 content, and drawing release curve, as shown in FIG. 7. Meanwhile, the dialysis bag was supplemented with 1mL of blank medium at 37 ℃.
PXRD and DSC:
taking formulas A1 and B1, freeze-drying in a freeze dryer for 48h, and performing PXRD and DSC measurement on the freeze-dried substance respectively. In the same formula proportion, blank solid lipid nanoparticles of formulas A1 and B1 are prepared according to the method by using the components except CAT3, and the measurement is carried out according to the method. The results are shown in FIGS. 8 and 9.
Table 9 characterization of phenanthroindolizidine alkaloid derivative solid lipid nanoparticle compositions (n ═ 3).
Figure BDA0002294597040000163
The results show that:
the formulas A1 and B1 are nano-scale particles, the particle size is about 150nm, the particle size and the distribution have no obvious difference and are in normal distribution; the drug loading of the two prescriptions is about 1mg/mL, and no statistical difference exists; the CAT3 bulk drug is hardly soluble in water, but the formulas A1 and B1 greatly improve the solubility in water.
Both the formulas A1 and B1 have higher encapsulation efficiencies of 58.48 +/-3.35 and 80.65 +/-6.79 percent respectively;
in vitro release results show that the bulk drugs in the formula A1 have better controlled release effect than the bulk drugs in the formula B1, and the controlled release effect of the formula B1 is better than that of the formula A1; the bulk drug API release conforms to the Higuchi model, the prescription a1 release conforms to the Weibull distribution model, and the prescription B1 release conforms to the 0-level linear release model with F0;
PXRD results indicated that the drug substance had diffraction peaks at d values of 10.663,5.363,5.217,4.002,7.748,3.778,6.094, and 11.487, indicating that it is a crystalline drug. The blank solid lipid nanoparticle had diffraction peaks at d values of 4.229,21.191,3.865, and 4.689, indicating that it had a crystalline lipid core. The physical mixture showed diffraction results as an overlay of both the blank nanoparticles and CAT 3. The diffraction pattern of formula A1 contains a small amount of diffraction peaks of CAT3, indicating that it contains a small amount of CAT3 crystals. The diffraction pattern of formula B1 shows no diffraction peak containing CAT3, indicating that CAT3 is amorphous.
DSC results are consistent with PXRD results.
Experimental example 3: permeability of fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition in single-layer cell membrane
In the experimental example, the intestinal mucosa permeability coefficient increasing effect is measured according to the following method, wherein the fatty acid-phenanthroindolizidine alkaloid derivative prepared from CAT3 and oleic acid is conjugated with a solid lipid nanoparticle composition formula B1(OA-CAT3-SLN, abbreviated as SLN):
a monolayer of MDCK-MDR1 cells grown on a Transwell plate, which met transport conditions and grew well, was taken and the absorption direction (AP-BL) permeability coefficient was determined. The specific determination method comprises the following steps: before the test, the cell surface was washed three times with HBSS solution at 37 ℃ to remove the cell surface attachments, and incubated at 37 ℃ for 30min to remove HBSS solution. 0.5mL of HBSS solution containing CAT3 at a concentration of 100ng/mL, prepared by diluting HBSS solution with prescription B1, was added to the AP side as a feed solution, and 1.5mL of blank HBSS solution was added to the BL side as a receiving solution. The same method is used for the operation of CAT3 bulk drug suspension (API for short).
Each sample was assayed in parallel with three wells. The Transwell plate was placed in a 37 ℃ incubator set at 50rpm, and 50. mu.L of the receiving solution was aspirated from the receiving cell at 0.5, 1, 1.5 and 2 hours, respectively, to measure the concentration of the transporter by HPLC-MS/MS method, and simultaneously, an equal amount of blank HBSS solution was added. In the same manner, SLN was measured by incubation in a constant temperature shaking chamber at 4 ℃.
The apparent permeability coefficients (Papp) of CAT3 on the AP-BL side and BL-AP side were measured and calculated from the received solutions at different time points, as shown in FIG. 10.
The results show that: the nano-scale emulsion droplets formed after the CAT3 self-emulsifying composition is spontaneously emulsified with an aqueous medium greatly increase the membrane permeability. Papp (AP-BL) increased 2.42 times compared to the drug substance.
Experimental example 4: in-vitro cytotoxic activity of fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition
In the experimental example, the in vitro cytotoxic activity of the fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition formula B1(OA-CAT3-SLN for short) prepared by CAT3 was measured according to the following method:
rat C6 brain glioma cells were in a bottle and observed to grow no less than 80% under a microscope. The medium was discarded, rinsed once with PBS, and 1.5ml of 0.1% pancreatin was added, followed by standing at 37 ℃ for 10 min. Cells should be detached from the culture flask under the observation of the lens. Complete medium neutralization was performed at3 volumes and blown to make single cell suspensions. The cells were centrifuged and 20ml of complete MEM medium was added to resuspend the cells. The cell suspension was diluted appropriately to 2X 104cells/mL. Cells were seeded in 96-well plates at 2X 10 per well4cells/mL cell suspension 100 uL. Placing CO2Incubator at 37 ℃ 5% CO2After culturing for 24h under the condition, discarding the original culture solution after the cells are completely attached to the wall, and administering the drug. OA-CAT3-SLN, blank nanoparticles and bulk drug suspension were administered at a concentration of 0.64ng/mL, and cell viability was determined by MTT method at 48 and 72h, respectively, as shown in FIG. 11.
The results show that OA-CAT3-SLN has good tumor inhibition effect in 48 and 72 hours compared with the bulk drug, and the inhibition rates are 29.77 +/-2.13 and 10.75 +/-3.12 percent respectively, while the inhibition rates of the bulk drug at the time point are only 46.20 +/-3.12 and 16.53 +/-3.95 percent respectively. The solid lipid nanoparticle composition containing the fatty acid-phenanthroindolizidine alkaloid derivative conjugate is possible to have a better in-vivo effect, and blank nanoparticles are not toxic.
Experimental example 5: relative bioavailability of fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition
The bioavailability of a metabolite PF403 in blood plasma was determined according to the following method, taking fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition formula B1(OA-CAT3-SLN for short) prepared from CAT3 in this experimental example:
SD rats were taken and fasted for 12h before administration, and were randomly divided into two groups. Separately perfusing 10mg/kg doses of CAT3-API and OA-CAT3-SLN, each group was collected at each time point of 5, 10, 15, 30min after administration and 1, 2, 4, 6, 8, 12, 24h after administration in BNPP (lipase inhibitor) -treated EP tubes, heparin was stored on ice after anticoagulation, and plasma was separated for use after centrifugation.
The amounts of CAT3 and PF403 were determined by HPLC-MS/MS. The method comprises the following steps:
HPLC：C18type (50mm gamma 2.1mm,1.8 μm) chromatography column, column temperature: 25 ℃, mobile phase: acetonitrile (0.1% FA): water 80:20, flow rate: 0.2mL/min, sample size: 5 mu L of the solution;
MS: CAT3, PF403 and ISTD (CAT) mass spectrum detection conditions are as follows: CAT 3: ESI + ion model, MRM detected ion pair 434.3 → 70.2, fragment 135, CE 25; the metabolite of CAT3 PF 403: ESI + ion model, MRM detected ion pair 350.2 → 70.2, fragment 135, CE 20; istd (cat): ESI + ion model, MRM detected ion pair 364.2 → 70.2, Fragmentor 135, CE 20.
Plasma samples were measured according to the established HPLC-MS/MS method and substituted into the running standard curve to calculate the concentration of PF 403. Unless otherwise indicated, experimental data are presented as mean ± standard deviation (n ═ 3). The main pharmacokinetic parameters are shown in table 10 and the bioavailability changes are shown in table 11. Since the antitumor activity of the active metabolite PF403 is about 1000 times that of the prodrug CAT3, the bioavailability of PF403 plays an important role in the exertion of the drug effect, and this experimental example also uses this as an index. The time-course curve of the oral administration of CAT3-API or OA-CAT3-SLN was plotted on the abscissa and the concentration of the in vivo metabolite PF403 of CAT3 in plasma on the ordinate, as shown in FIG. 12.
The results show that: OA-CAT3-SLN clearly increased the bioavailability of the active metabolite PF 403. Compared with the bulk drugs, the AUC of the PF403 is increased by 1.48 times, which shows a definite absorption promoting effect and is beneficial to the exertion of in-vivo drug effects.
TABLE 10 major pharmacokinetic parameters PF403 in plasma following oral administration of CAT3-API or OA-CAT3-SLN to rats
Figure BDA0002294597040000191
TABLE 11 relative bioavailability of PF403 in plasma following oral administration of CAT3-API or OA-CAT3-SLN to rats
Figure BDA0002294597040000192
**P<0.01vs.CAT3-API
Reference to the literature
[1]Chen J,Lv H,Hu J,et al.CAT3,a novel agent for medulloblastoma and glioblastoma treatment,inhibits tumor growth by disrupting the Hedgehog signaling pathway[J].Cancer letters,2016,381(2):391-403.
[2]Beloqui A,Solinís M
Figure BDA0002294597040000201
Rodríguez-Gascón A,et al.Nanostructured lipid carriers:Promising drug delivery systems for future clinics[J].Nanomedicine:Nanotechnology,Biology and Medicine,2016,12(1):143-161.
[3]Shen,M.-Y.,et al.,Hierarchically targetable polysaccharide-coated solid lipid nanoparticles as an oral chemo/thermotherapy delivery system for local treatment of colon cancer.Biomaterials,2019.197:p.86-100.
[4]Liu,Z.,et al.,Interaction studies of an anticancer alkaloid,(+)-(13aS)-deoxytylophorinine,with calf thymus DNA and four repeated double-helical DNAs.Chemotherapy,2011.57(4):p.310-20.
[5]Lv,H.,et al.,Synthesis,biological evaluation and mechanism studies of deoxytylophorinine and its derivatives as potential anticancer agents.PLoS One,2012.7(1):p.e30342.
[6]Ji,M.,et al.,CAT3,a prodrug of 13a(S)-3-hydroxyl-6,7-dimethoxyphenanthro[9,10-b]-indolizidine,circumvents temozolomide-resistant glioblastoma via the Hedgehog signaling pathway,independently of O6-methylguanine DNA methyltransferase expression.OncoTargets and therapy,2018.11:p.3671.

Claims (18)

1. A phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition is characterized by comprising a phenanthroindolizidine alkaloid derivative, lipid, phospholipid, an emulsifier and water;
phenanthroindolizidine-containing alkaloid derivatives: 0.1-50mg/mL,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 10 percent of the total weight of the mixture,
phospholipid: 0.05 to 10 percent of the total weight of the mixture,
emulsifier: 0.05 to 10 percent of the total weight of the mixture,
water: 70 to 99.8 percent.
2. A fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition is characterized by comprising a fatty acid-phenanthroindolizidine alkaloid derivative conjugate, lipid, phospholipid, an emulsifier and water;
fatty acid-phenanthroindolizidine alkaloid derivative conjugate: 0.2-100mg/mL,
in the fatty acid-phenanthroindolizidine alkaloid derivative conjugate, the weight ratio of fatty acid to phenanthroindolizidine alkaloid derivative is 0.5: 1 to 10: 1,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 10 percent of the total weight of the mixture,
phospholipid: 0.05 to 10 percent of the total weight of the mixture,
emulsifier: 0.05 to 10 percent of the total weight of the mixture,
water: 70 to 99.8 percent.
3. The solid lipid nanoparticle composition of phenanthroindolizidine alkaloid derivative according to claim 1 or 2, wherein the phenanthroindolizidine alkaloid derivative comprises (13aS) -3-pivaloyloxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine, (13aS) -3-hydroxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine or dextrodeoxytylosin, and the structural formula is aS follows:
Figure FDA0002294597030000011
(13aS) -3-pivaloyloxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine
Figure FDA0002294597030000021
(13aS) -3-hydroxy-6, 7-dimethoxy-9-phenanthro [9,10-b ] -indolizidine
Figure FDA0002294597030000022
Dextrorotation deoxidation tylophorinine.
4. The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition according to claim 1 or 2, wherein the lipid is one or a mixture of more than one of fatty acid, glyceride, cholesterol and phosphorus wax lipid in any proportion; wherein,
the fatty acid is one or more of stearic acid, oleic acid, palmitic acid, myristic acid, behenic acid and caprylic acid which are mixed in any proportion;
the glyceride is one or more of glyceryl monostearate, glyceryl distearate, glyceryl tristearate, glyceryl monopalmitate, glyceryl dipalmitate, glyceryl tripalmitate, glyceryl monomyristate, glyceryl dimyristate, glyceryl trimyristate, glyceryl monobehenate, glyceryl dibehenate, glyceryl tribehenate, glyceryl trioleate and glyceryl trilaurate;
the phosphorus wax ester is one or more of microcrystalline paraffin and whale fat wax in any proportion.
5. The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition according to claim 1 or 2, characterized in that the phospholipids comprise one or a mixture of more than one of soybean phospholipids, egg yolk phospholipids, lecithin, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl glycerol, dipalmitoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine in any proportion.
6. The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition according to claim 1 or 2, wherein the emulsifier comprises one or more of poloxamer, cholates, short-chain alcohols, polysorbates, polyoxyethylene fatty alcohol ethers and polyoxyethylene fatty acid esters in any proportion; wherein the poloxamer comprises one or more of poloxamer 188, poloxamer 182, poloxamer 407 and poloxamer 908 in any proportion; the cholate comprises one or more of cholate, glycocholate and sodium taurocholate in any proportion; the short chain alcohol comprises one or more of glycerol, butanol and propylene glycol in any proportion; the polysorbate comprises one or more of tween 20 and tween 80, and is mixed at any ratio; the polyoxyethylene fatty alcohol ether comprises one or more of polyoxyethylene fatty alcohol ether Brij 78, polyoxyethylene fatty alcohol ether Brij 35 and polyoxyethylene fatty alcohol ether Brij 30 in any proportion; the polyoxyethylene fatty acid ester comprises one or more of polyoxyethylene fatty acid ester Myrj 53 and polyoxyethylene fatty acid ester Myrj 59.
7. The fatty acid-phenanthroindolizidine alkaloid derivative conjugate according to claim 2, wherein the fatty acid is one or more of oleic acid, stearic acid, palmitic acid, myristic acid, behenic acid and caprylic acid in any proportion.
8. The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition according to claim 1, characterized by comprising a phenanthroindolizidine alkaloid derivative: 0.1-50mg/mL,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 7 percent of the total weight of the mixture,
phospholipid: 0.1 to 5 percent of the total weight of the mixture,
emulsifier: 0.1 to 5 percent of the total weight of the mixture,
water: 83 to 99.7 percent.
9. The phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition according to claim 8, characterized by comprising a phenanthroindolizidine alkaloid derivative: 0.1-50mg/mL,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 2 to 5 percent of the total weight of the mixture,
phospholipid: 0.5 to 2.5 percent of,
emulsifier: 0.5 to 2.5 percent of,
water: 90 to 97 percent.
10. The fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition as claimed in claim 2, wherein the fatty acid-phenanthroindolizidine alkaloid derivative conjugate comprises: 0.2-100mg/mL,
the weight ratio of the fatty acid to the phenanthroindolizidine alkaloid derivative is 2: 1 to 8: 1,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 0.1 to 7 percent of the total weight of the mixture,
phospholipid: 0.1 to 5 percent of the total weight of the mixture,
emulsifier: 0.1 to 5 percent of the total weight of the mixture,
water: 83 to 99.7 percent.
11. The fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition as claimed in claim 10, wherein the fatty acid-phenanthroindolizidine alkaloid derivative conjugate comprises: 0.2-100mg/mL,
in the fatty acid-phenanthroindolizidine alkaloid derivative conjugate, the weight ratio of fatty acid to phenanthroindolizidine alkaloid derivative is 4: 1 to 7: 1,
the weight ratio of the lipid, the phospholipid, the emulsifier and the water is as follows according to 100 percent:
lipid: 2 to 5 percent of the total weight of the mixture,
phospholipid: 0.5 to 2.5 percent of,
emulsifier: 0.5 to 2.5 percent of,
water: 90 to 97 percent.
12. The solid lipid nanoparticle composition according to claim 1 or 2, characterized in that: the composition may also contain a lyoprotectant.
13. The solid lipid nanoparticle composition according to claim 12, characterized in that: the freeze-drying protective agent comprises one or two of trehalose, mannitol, glucose, mannose, sucrose and maltose.
14. The solid lipid nanoparticle composition of phenanthroindolizidine alkaloid derivatives according to any of claims 1-11, wherein the solid lipid nanoparticle composition can be further formulated into tablets, capsules, soft capsules, injections, enemas, nasal drops, transdermal patches or mucosal patches.
15. A method for preparing the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition according to any one of claims 1, 3-9, which is characterized in that, in the first step, the phenanthroindolizidine alkaloid derivative, the lipid and the phospholipid are mixed, heated and melted at a temperature of 10-15 ℃ higher than the melting point of the lipid material to obtain an organic phase, and a required amount of emulsifier is dissolved in water and heated to obtain an aqueous phase after the same temperature of the organic phase is reached; secondly, under the high shearing action with the shearing speed of 10000 to 16000rmp and the temperature of 10 to 15 ℃ higher than the melting point of the lipid material, uniformly mixing the organic phase and the water phase to prepare primary emulsion; thirdly, carrying out ultrasonic treatment on the primary emulsion for 3-15min in a probe ultrasonic instrument under the power of 100 plus one 500W at the temperature of 10-15 ℃ higher than the melting point of the lipid material, or homogenizing for 5-15 times in a high-pressure homogenizer under the pressure of 500 plus one 1500bar to obtain liquid lipid nanoparticles; fourthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of between 0 and 30 ℃ to obtain the lipid nanoparticles.
16. A method for preparing a fatty acid-phenanthroindolizidine alkaloid derivative conjugate solid lipid nanoparticle composition as claimed in any one of claims 2 to 7 and 10 to 11, wherein in the first step, the phenanthroindolizidine alkaloid derivative and the fatty acid are reacted for 1 to 24 hours at a temperature of 40 ℃ to 80 ℃ to form a conjugate; secondly, mixing lipid and phospholipid, heating and melting and dissolving at the temperature of 10-15 ℃ higher than the melting point of the lipid material, adding the conjugate to dissolve to obtain an organic phase, dissolving a required amount of emulsifier in water, and heating to the same temperature of the organic phase to obtain a water phase; thirdly, uniformly mixing the organic phase and the water phase under the high shearing action with the shearing speed of 10000 to 16000rmp at the temperature of 10 to 15 ℃ higher than the melting point of the lipid material to prepare primary emulsion; fourthly, at the temperature of 10-15 ℃ higher than the melting point of the lipid material, carrying out ultrasonic treatment on the primary emulsion for 3-15min under the power of 100-500W in a probe ultrasonic instrument, or homogenizing for 5-15 times under the pressure of 500-1500bar in a high-pressure homogenizer to obtain liquid lipid nanoparticles; fifthly, cooling and solidifying the liquid lipid nanoparticles at the temperature of 0-30 ℃ to obtain the lipid nanoparticle.
17. Use of the phenanthroindolizidine alkaloid derivative solid lipid nanoparticle composition according to any of claims 1-10 in the preparation of a medicament for preventing and/or treating cancer and/or inflammatory diseases.
18. The use of claim 17, wherein the cancer is selected from the group consisting of glioma, medulloblastoma, colon cancer, gastric cancer, ovarian cancer, cervical cancer, liver cancer, lung cancer and pancreatic cancer.
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* Cited by examiner, † Cited by third party
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