CN110013471B - Nano-structure lipid carrier for synergistic treatment of brain glioma and preparation method and application thereof - Google Patents
Nano-structure lipid carrier for synergistic treatment of brain glioma and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano-structure lipid carrier for synergistic treatment of brain glioma, and a preparation method and application thereof, and belongs to the technical field of nano-medicine. The nano-structure lipid carrier for the synergistic treatment of brain glioma is prepared from glyceryl monostearate, triglyceride, temozolomide, curcumin, poloxamer 188 and absolute ethyl alcohol by a microemulsion method. The nano-structure lipid carrier prepared by the invention has the advantages of particle size less than 100nm, uniform particle size distribution, Zeta potential of-8.54 +/-0.51 mV and high encapsulation efficiency, wherein the temozolomide encapsulation efficiency is 91.53 +/-0.07%, and the curcumin encapsulation efficiency is 88.64 +/-0.99%. The preparation method provided by the invention is simple to operate and high in repeatability. The nano-structured lipid carrier prepared by the invention has the sequential release performance of two drugs and has the synergistic treatment effect on glioma.
Description
Technical Field
The invention belongs to the technical field of nano-medicine, and particularly relates to a nano-structure lipid carrier for synergistic treatment of brain glioma, and a preparation method and application thereof.
Background
Glioblastoma (GBM) is a malignant brain tumor that is one of the leading causes of death in adults under 40 years of age, children and adolescents. The incidence of glioblastoma has increased over the past three decades. Common treatment methods are surgical resection, chemotherapy, and radiation therapy. However, in order not to aggravate brain dysfunction, the surgical removal of glioma tissue can only be partial, and thus the surgical treatment cannot cure glioblastoma. In addition, glioma is very easy to recur due to the proliferative capacity of glioma stem cells. Glioma cells are invasive and therefore the survival of glioma patients is only 12-15 months. The blood brain barrier is present, so that the traditional chemotherapeutic drugs such as temozolomide are difficult to enter brain tissues, and the treatment effect is poor. The blood brain barrier refers to the barrier between the plasma formed by the walls of the brain capillaries and the glial cells and the barrier between the plasma formed by the choroid plexus and the cerebrospinal fluid. It is mainly composed of brain capillary endothelial cells, astrocytes and pericytes. Studies have shown that 98% of drugs do not cross the blood-brain barrier. The drug carrier passing through the blood brain barrier needs to have the characteristics of being less than 200nm in size, non-toxic, biodegradable, free of immune reaction and the like.
The nano-structured lipid carrier (NLC) is composed of solid lipid and liquid lipid as drug carriers, and has a particle size of about 100-200 nm. It is in solid form at room temperature and body temperature, and partially forms a crystalline lipid matrix, with good biocompatibility and biodegradability. Muller states that NLCs may exist in three structures: defective NLCs, amorphous NLCs, and complex NLCs. The advantages of the compound as a drug carrier are that the solubility of the insoluble drug is improved, the irritation of the drug is reduced, and the action time of the drug in vivo is prolonged. Compared with Solid Lipid (SLN) nanoparticles, NLC has higher drug loading and encapsulation efficiency. The particle size of the compound is generally less than 100nm, and the compound can penetrate through a blood brain barrier and is a good medicine carrier for treating central nervous system diseases. CN107375316A discloses a method for preparing a structured lipid carrier and a structured lipid carrier, in which a nanostructured lipid carrier is prepared by a high-pressure homogenization method, but the method has high requirements for instruments. The microemulsion is prepared by a microemulsion method, and has simple operation and low instrument requirement.
Temozolomide (TMZ) is a commonly used chemotherapeutic drug for clinical treatment of glioblastoma. CN108014097A discloses a TMZ sustained-release capsule and a preparation method thereof, which has the function of drug sustained release. Compared with the invention, the NLC for the synergistic treatment of the brain tumor has the advantages of small particle size and higher bioavailability. Curcumin (CUR) is a chemical component extracted from rhizome of plants of Zingiberaceae and Araceae, and has antibacterial, antitumor and antioxidant effects. Researchers load the CUR by using the nanostructured lipid carrier, and find that the tumor inhibition effect of the CUR is obviously enhanced, and the inhibition efficiency is improved from 19.5% to 82.3%. Meanwhile, animal experiments show that the CUR-NLC has certain targeting property on the brain. Therefore, in order to overcome the defects that the traditional chemotherapeutic drugs are difficult to cross blood brain barriers and have low bioavailability, the invention prepares the nano-structured lipid carrier simultaneously carrying TMZ and CUR.
Disclosure of Invention
In order to overcome the defects of low solubility and low bioavailability of the traditional chemotherapeutic drugs, the invention mainly aims to provide a nano-structured lipid carrier for synergistically treating brain glioma. The lipid carrier loaded with temozolomide and curcumin nano-structure mainly comprises glyceryl monostearate, triglyceride and poloxamer 188, and is prepared by a microemulsion method.
The invention also aims to provide a preparation method of the nano-structured lipid carrier for the synergistic treatment of the brain glioma.
Still another object of the present invention is to provide the use of the above nanostructured lipid carrier for the synergistic treatment of brain gliomas.
The purpose of the invention is realized by the following technical scheme: a nanostructured lipid carrier for synergistic treatment of brain glioma is prepared from glyceryl monostearate, triglyceride, temozolomide, curcumin, poloxamer 188, and anhydrous alcohol by microemulsion method.
The nano-structure lipid carrier for the synergistic treatment of the brain glioma specifically comprises the following components in parts by weight:
the particle size of the nano-structure lipid carrier for the synergistic treatment of the brain glioma is less than 100 nm.
The preparation method of the nano-structure lipid carrier for the synergistic treatment of the brain glioma specifically comprises the following steps:
respectively weighing 6-30mg of glyceryl monostearate, 9-64mg of triglyceride, 10-30mg of temozolomide and 10-30mg of curcumin, and stirring at constant temperature of 75-85 ℃ until the components are completely dissolved to be used as an oil phase; meanwhile, slowly dripping the mixed solution of 32-125mg of poloxamer 188, 10-45mg of absolute ethyl alcohol and 50-80ml of deionized water into a beaker filled with an oil phase, and stirring for 15-25min to obtain microemulsion; then, the microemulsion was slowly added dropwise to 500ml of deionized water at 4 ℃, stirred for 4 hours, and freeze-dried.
In one embodiment, the rotation speed of the stirring is 200-500 rpm.
In one embodiment, the freeze-drying time is 48-72 hours.
The nano-structure lipid carrier for synergistic treatment of glioma, which is obtained by the preparation method, has the characteristics of particle size less than 100nm, uniform particle size distribution, high encapsulation rate, sequential release of double drugs and the like, and has the function of synergistically inhibiting glioma cell proliferation.
The nano-structure lipid carrier for the synergistic treatment of the brain glioma is applied to the preparation of the medicine for treating the brain glioma.
Compared with the prior art, the invention has the following advantages and effects:
the method for preparing the nano-structure lipid carrier for the synergistic treatment of the brain glioma is simple to operate and high in repeatability, and does not introduce an organic solvent. The prepared nano-structure lipid carrier for the synergistic treatment of the brain glioma has the advantages of small particle size, uniform particle size distribution, high encapsulation efficiency, sequential release of double drugs and the like.
The invention takes temozolomide and curcumin as medicines and glycerin monostearate and triglyceride as main materials, and prepares a nano-structure lipid carrier loaded with temozolomide and curcumin by a microemulsion method. Cell experiments prove that temozolomide and curcumin can produce a synergistic anti-glioma cell effect. Specifically, curcumin is firstly dissolved out from a nano-structure lipid carrier to enhance the sensitivity of cancer cells, and then temozolomide is dissolved out, so that a better effect of inhibiting the proliferation of glioma cells is shown. The invention relates to a nano-structure lipid carrier for synergistic treatment of brain glioma.
The curcumin adopted in the invention has the anti-tumor effect and can generate the synergistic effect with the temozolomide, and the curcumin has the effect of being used as a sensitizer to synergistically increase the inhibition effect of the temozolomide on glioma cells.
Drawings
Fig. 1 is a transmission electron microscope image of the nano-structured lipid carrier co-loaded with temozolomide and curcumin prepared in example 1.
Fig. 2 is a particle size diagram of the nanostructured lipid carrier co-loaded with temozolomide and curcumin prepared in example 2.
Fig. 3 is the surface potential of the nanostructured lipid carrier co-loaded with temozolomide and curcumin prepared in example 3.
Fig. 4 shows the encapsulation efficiency of the nanostructured lipid carrier co-loaded with temozolomide and curcumin, prepared in example 4.
Fig. 5 is the in vitro drug release of the nanostructured lipid carrier co-loaded with temozolomide and curcumin prepared in example 5.
Fig. 6 shows the inhibitory effect of the nanostructured lipid carrier co-loaded with temozolomide and curcumin, prepared in example 6, on murine glioma cells C6.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Example 1
The embodiment provides a preparation method of a nano-structure lipid carrier for synergistic treatment of brain glioma, which specifically comprises the following steps:
precisely weighing 28mg of glyceryl monostearate, 12mg of triglyceride, 10mg of temozolomide and 20mg of curcumin, placing the mixture into a beaker, adding a magnetic stirring rotor with proper size, placing the beaker into a constant-temperature water bath magnetic stirrer with the temperature of 75-85 ℃, and rotating at 400rpm until the mixture is completely dissolved to form an oil phase; meanwhile, 80mg of poloxamer 188, 80mg of absolute ethyl alcohol and 60ml of deionized water are taken and placed in a beaker, a preservative film is sealed, and then a magnetic stirrer with a constant-temperature water bath at 75-85 ℃ is placed to keep the temperature constant; slowly dripping the mixed solution of poloxamer 188 and absolute ethyl alcohol into a beaker filled with an oil phase, and stirring for 15-25min by a magnetic stirrer; then, the microemulsion in the beaker is quickly absorbed by a rubber dropper, and then is slowly dripped into 500ml of deionized water at 4 ℃, kept at 4 ℃, stirred by a magnetic stirrer for 4 hours, and then frozen and dried. The prepared nanostructured lipid carrier has a particle size of less than 100nm and uniform particle size distribution (as shown in FIG. 1).
Example 2
The embodiment provides a preparation method of a nano-structure lipid carrier for synergistic treatment of brain glioma, which specifically comprises the following steps:
precisely weighing 27mg of glyceryl monostearate, 14mg of triglyceride, 15mg of temozolomide and 15mg of curcumin, placing the mixture into a beaker, adding a magnetic stirring rotor with proper size, placing the beaker into a constant-temperature water bath magnetic stirrer with the temperature of 75-85 ℃, and setting the rotating speed at 400rpm until the mixture is completely dissolved to form an oil phase; meanwhile, 34mg of poloxamer 188, 18mg of absolute ethyl alcohol and 50ml of deionized water are placed in a beaker, a preservative film is sealed, and then a magnetic stirrer with a constant-temperature water bath at 75-85 ℃ is placed to keep the temperature constant; slowly dripping the mixed solution of poloxamer 188 and absolute ethyl alcohol into a beaker filled with an oil phase, and stirring for 15-25min by a magnetic stirrer; then, the microemulsion in the beaker is quickly absorbed by a rubber dropper, and then is slowly dripped into 500ml of deionized water at 4 ℃, kept at 4 ℃, stirred by a magnetic stirrer for 4 hours, and then frozen and dried. The particle size was measured to be 92nm (as shown in FIG. 2).
Example 3
The embodiment provides a preparation method of a nano-structure lipid carrier for synergistic treatment of brain glioma, which specifically comprises the following steps:
precisely weighing 25mg of glyceryl monostearate, 15mg of triglyceride, 16mg of temozolomide and 14mg of curcumin, placing the mixture into a beaker, adding a magnetic stirring rotor with proper size, placing the beaker into a constant-temperature water bath magnetic stirrer with the temperature of 75-85 ℃, and setting the rotating speed at 400rpm until the mixture is completely dissolved to form an oil phase; meanwhile, 45mg of poloxamer 188, 35mg of absolute ethyl alcohol and 80ml of deionized water are placed in a beaker, a preservative film is sealed, and then a magnetic stirrer with a constant-temperature water bath at 75-85 ℃ is placed to keep the temperature constant; slowly dripping the mixed solution of poloxamer 188 and absolute ethyl alcohol into a beaker filled with an oil phase, and stirring for 15-25min by a magnetic stirrer; then, the microemulsion in the beaker is quickly absorbed by a rubber dropper, and then is slowly dripped into 500ml of deionized water at 4 ℃, kept at 4 ℃, stirred by a magnetic stirrer for 4 hours, and then frozen and dried. The Zeta potential of the compound is-8.54 +/-0.51 mV (shown in figure 3).
Example 4
The embodiment provides a preparation method of a nano-structure lipid carrier for synergistic treatment of brain glioma, which specifically comprises the following steps:
precisely weighing 29mg of glyceryl monostearate, 12mg of triglyceride, 20mg of temozolomide and 10mg of curcumin, placing the mixture into a beaker, adding a magnetic stirring rotor with proper size, placing the beaker into a constant-temperature water bath magnetic stirrer with the temperature of 75-85 ℃, and setting the rotating speed at 400rpm until the mixture is completely dissolved to form an oil phase; meanwhile, 80mg of poloxamer 188, 80mg of absolute ethyl alcohol and 75ml of deionized water are taken and placed in a beaker, a preservative film is sealed, and then a magnetic stirrer with a constant-temperature water bath at 75-85 ℃ is placed to keep the temperature constant; slowly dripping the mixed solution of poloxamer 188 and absolute ethyl alcohol into a beaker filled with an oil phase, and stirring for 15-25min by a magnetic stirrer; then, a rubber head dropper is used for quickly sucking up the microemulsion in the beaker, and then the microemulsion is slowly dripped into 500ml of deionized water at 4 ℃, the temperature is kept at 4 ℃, a magnetic stirrer is used for stirring for 4 hours, and freeze drying is carried out; the particle size of the obtained nano-structured lipid carrier is less than 100nm, and the nano-particles are uniformly distributed. Wherein the temozolomide encapsulation rate is 91.53 +/-0.07%, and the curcumin encapsulation rate is 88.64 +/-0.99% (as shown in figure 4).
Example 5
The embodiment provides a preparation method of a nano-structure lipid carrier for synergistic treatment of brain glioma, which specifically comprises the following steps:
precisely weighing 25mg of glyceryl monostearate, 16mg of triglyceride, 10mg of temozolomide and 20mg of curcumin, placing the mixture into a beaker, adding a magnetic stirring rotor with proper size, placing the beaker into a constant-temperature water bath magnetic stirrer with the temperature of 75-85 ℃, and setting the rotating speed at 400rpm until the mixture is completely dissolved to form an oil phase; meanwhile, 47mg of poloxamer 188, 29mg of absolute ethyl alcohol and 80ml of deionized water are placed in a beaker, a preservative film is sealed, and then a magnetic stirrer with a constant-temperature water bath at 75-85 ℃ is placed to keep the temperature constant; slowly dripping the mixed solution of poloxamer 188 and absolute ethyl alcohol into a beaker filled with an oil phase, and stirring for 15-25min by a magnetic stirrer; then, the microemulsion in the beaker is quickly absorbed by a rubber dropper, and then is slowly dripped into 500ml of deionized water at 4 ℃, kept at 4 ℃, stirred by a magnetic stirrer for 4 hours, and then frozen and dried.
The particle size of the obtained nano-structured lipid carrier is less than 100nm, and the nano-particles are uniformly distributed. As shown in fig. 5, it can be seen that the nanostructured lipid carrier prepared by the present invention has the characteristics of dual-drug sequential release, i.e., the curcumin release rate is faster, and the temozolomide release rate is slower.
Example 6
The embodiment provides a preparation method of a nano-structure lipid carrier for synergistic treatment of brain glioma, which specifically comprises the following steps:
precisely weighing 26mg of glyceryl monostearate, 15mg of triglyceride, 18mg of temozolomide and 12mg of curcumin, placing the mixture into a beaker, adding a magnetic stirring rotor with proper size, placing the beaker into a constant-temperature water bath magnetic stirrer with the temperature of 75-85 ℃, and rotating at 400rpm until the mixture is completely dissolved to form an oil phase; meanwhile, 80mg of poloxamer 188, 80mg of absolute ethyl alcohol and 80ml of deionized water are placed in a beaker, a preservative film is sealed, and then a thermostatic water bath magnetic stirrer at 75-85 ℃ is placed to keep the temperature constant. Slowly dripping the mixed solution of poloxamer 188 and absolute ethyl alcohol into a beaker filled with an oil phase, and stirring for 15-25min by a magnetic stirrer; then, the microemulsion in the beaker is quickly absorbed by a rubber dropper, and then is slowly dripped into 500ml of deionized water at 4 ℃, kept at 4 ℃, stirred by a magnetic stirrer for 4 hours, and then frozen and dried.
The particle size of the obtained nano-structured lipid carrier is less than 100nm, and the nano-particles are uniformly distributed. The MTT is adopted to detect the cell viability, and the result shows that the nano-structured lipid carrier prepared by the invention has obvious cytotoxicity on the murine glioma C6 cell line, which indicates that the nano-structured lipid carrier has a synergistic inhibition effect on the proliferation of glioma cells (as shown in figure 6).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (6)
1. A preparation method of a nano-structure lipid carrier for synergistic treatment of brain glioma is characterized in that: the method specifically comprises the following steps:
respectively weighing 6-30mg of glyceryl monostearate, 9-64mg of triglyceride, 10-30mg of temozolomide and 10-30mg of curcumin, and stirring at constant temperature of 75-85 ℃ until the components are completely dissolved to be used as an oil phase; meanwhile, slowly dripping the mixed solution of 32-125mg of poloxamer 188, 10-45mg of absolute ethyl alcohol and 50-80ml of deionized water into a beaker filled with an oil phase, and stirring for 15-25min to obtain microemulsion; then, the microemulsion was slowly dropped into 500ml of deionized water at 4 ℃, stirred for 4 hours, and freeze-dried.
2. The method for preparing the nanostructured lipid carrier for the synergistic treatment of brain glioma according to claim 1, characterized in that: the rotation speed of the stirring is 200-500 rpm.
3. The method for preparing the nanostructured lipid carrier for the synergistic treatment of brain glioma according to claim 1, characterized in that: the freeze drying time is 48-72 h.
4. A nanostructured lipid carrier for the synergistic treatment of brain glioma, prepared by the preparation method according to any one of claims 1 to 3.
5. The nanostructured lipid carrier for the synergistic treatment of brain gliomas according to claim 4, characterized in that: the particle size of the nano-structured lipid carrier is less than 100 nm.
6. Use of the nanostructured lipid carrier according to claim 4 or 5 for the synergistic treatment of brain gliomas in the preparation of a medicament for the treatment of brain gliomas.
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| CN101396334A (en) * | 2008-10-16 | 2009-04-01 | 南京医科大学 | Curcumin lipid carrier and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101396334A (en) * | 2008-10-16 | 2009-04-01 | 南京医科大学 | Curcumin lipid carrier and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Enhanced accumulation of curcumin and temozolomide loaded magnetic nanoparticles;Fahima Dilnawaz et al;《European Journal of Pharmaceutics and Biopharmaceutics》;20131231;第1-36页 * |
| Nanostructured lipid carriers, solid lipid nanoparticles, and polymeric nanoparticles:which kind of drug delivery system is better for glioblastoma chemotherapy?;Jie Qu et al;《Drug Delivery》;20160608;第23卷(第9期);第3408-3416页 * |
| Vincristine and temozolomide combined chemotherapy for the treatment of glioma: a comparison of solid lipid nanoparticles;Maria J. Ramalho et al;《Drug Delivery》;20151231;第1-6页 * |
| 替莫唑胺联合姜黄素对C6 胶质瘤细胞凋亡的作用;王亚华等;《实用医学杂志》;20161231;第32卷(第10期);第1564-1567页 * |
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