CN111494316A - Preparation method and application of disulfiram-loaded nano-emulsion in-situ gel - Google Patents
Preparation method and application of disulfiram-loaded nano-emulsion in-situ gel Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0043—Nose
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention relates to the technical field of medicines, in particular to a preparation method and application of a disulfiram-loaded nano-emulsion in-situ gel, which comprises the following steps: (1) mixing the oil phase, the emulsifier and the co-emulsifier, adding the phospholipid modified by polyethylene glycol, and uniformly mixing to obtain a first mixed solution; (2) adding disulfiram into the first mixed solution, and obtaining a second mixed solution after the disulfiram is completely dissolved; (3) and adding ultrapure water containing the in-situ gel into the nano-emulsion to obtain the disulfiram-loaded nano-emulsion in-situ gel through self-assembly. The disulfiram-loaded nano-emulsion in-situ gel provided by the invention has the advantages that the solubility of disulfiram is obviously improved, an ideal slow-release effect is realized, the in-vivo stability of disulfiram is improved, the safety is higher, and the disulfiram-loaded nano-emulsion in-situ gel has the characteristic of obvious enrichment in the brain when being administrated through a nasal cavity.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method and application of a disulfiram-loaded nano-emulsion in-situ gel.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Brain glioma is the most common malignant tumor in the brain and is one of the most common tumors in the brain. The invasive growth of brain glioma brings great difficulty to surgical excision, and has poor prognosis, and relapse occurs more than 95%. Glioma stem cells can promote radiation resistance by preferentially activating the DNA damage response. The growing environment of glioma also presents a significant challenge to chemotherapy. The temozolomide which is the first-line medicament for treating brain glioma at present also has the toxic and side effects of nausea, vomiting, alopecia, bone marrow suppression and the like, and is easy to generate drug resistance. Therefore, the research on new treatment strategies to improve the treatment effect of brain glioma is not easy.
Disulfiram (DSF) has been proved to be effective on various tumors in preclinical research, has the characteristics of inhibiting tumor growth, promoting tumor apoptosis, preventing migration and invasion of tumor cells, reducing angiogenesis, reversing tumor multidrug resistance, inhibiting tumor stem cells and the like, and has low drug safety and toxicity. Disulfiram has shown a very potential anti-glioma activity in preclinical studies, and its anti-tumour activity is significantly increased by chelation with copper ions. The combined treatment of tumors with disulfiram and copper is currently in clinical research.
The earliest formulation of disulfiram is tablet, disulfiram has the characteristics of low solubility, easy degradation, short half-life, less brain drug aggregation and low oral bioavailability, and in order to further improve the drug effect, brain targeting research of nasal administration is concerned by more and more researchers at home and abroad in recent years. The medicine is administrated through nose, can bypass blood brain barrier, and directly acts on the focus part of the brain, thereby being beneficial to improving the drug effect and reducing the toxic and side effect on the whole body, and the compliance of patients is improved by a non-invasive administration mode.
Disclosure of Invention
The invention aims to improve the water solubility and stability of disulfiram, promote the absorption of disulfiram in a nasal cavity, prolong the half-life period of disulfiram in vivo and promote the high enrichment of disulfiram in brain tumors, which is one of key technologies for improving the chemotherapy effect of brain glioma. Therefore, the invention provides a preparation method and application of the disulfiram-loaded nanoemulsion in-situ gel. The disulfiram-loaded nano-emulsion in-situ gel remarkably improves the solubility of disulfiram and the in-vivo stability of disulfiram, has an ideal slow release effect, and has the characteristic of remarkable enrichment in brain when being administrated through a nasal cavity.
In order to achieve the purpose, the invention discloses the following technical scheme.
The invention discloses a preparation method of a disulfiram-loaded nano-emulsion in-situ gel, which comprises the following steps:
(1) and mixing the oil phase, the emulsifier and the co-emulsifier, adding the phospholipid modified by polyethylene glycol, and uniformly mixing to obtain a first mixed solution.
(2) And (2) adding disulfiram into the first mixed solution obtained in the step (1), and obtaining a second mixed solution after the disulfiram is completely dissolved.
(3) And adding ultrapure water containing the in-situ gelling agent into the second mixed solution, and carrying out self-assembly to obtain the disulfiram-loaded nano-emulsion in-situ gelling agent.
Further, in the step (1), the oil phase includes any one or more of oleic acid, ethyl oleate, castor oil, isopropyl myristate, soybean oil, rapeseed oil and the like.
Further, in the step (1), the emulsifier includes any one or more of tween, span, polyethylene glycol-15 hydroxystearate, caprylic/capric macrogol glyceride, polyoxyethylene hydrogenated castor oil, emulsifier OP, poloxamer, lecithin, and the like.
Further, in the step (1), the co-emulsifier comprises any one or more of ethanol, isopropanol, 1, 2-propylene glycol, PEG 400, Transartol HP, n-butanol and the like.
Preferably, in step (1), the oil phase is ethyl oleate or isopropyl myristate. The emulsifier is polyethylene glycol-15 hydroxystearate or tween 80. The coemulsifier is Transartol HP. When the oil phase, the emulsifier and the co-emulsifier are adopted, the disulfiram has better solubility in the oil phase, the emulsifier and the co-emulsifier, and the components have better compatibility.
Further, in step (1), the phospholipid modified by polyethylene glycol is preferably DSPE-PEG 2000. By adding the phospholipid modified by polyethylene glycol, the surface of the nano-emulsion is covered by PEG 2000, so that the solubility of disulfiram is improved, collision and aggregation among nano-particles are avoided, the biological stability and biocompatibility of the nano-emulsion are improved, the toxicity is reduced, and the immunogenicity is reduced.
Further, in the step (1), the oil phase accounts for 14-29% by mass, the total proportion of the emulsifier and the co-emulsifier accounts for 71-86% by mass, and the PEG-modified phospholipid accounts for 5-20% by mass of the total weight of the emulsifier and the co-emulsifier. The mass ratio of the emulsifier to the co-emulsifier is 1: 1-3: 1.
In step (2), the concentration of disulfiram in the second mixed solution is 0.1mg/m L-70 mg/m L, preferably 10-40 mg/m L, when the concentration is selected, the prepared disulfiram-loaded nanoemulsion has good stability, when the concentration of disulfiram is too high, the nanoemulsion is unstable and precipitates, and the disulfiram precipitates from the nanoemulsion, the water solubility of the disulfiram is only 4.09 μ g/m L, and the maximum concentration of the prepared disulfiram-loaded liposome in-situ gel is 1.5mg/m L.
Further, in the step (2), the disulfiram is completely dissolved by stirring or a vortex or ultrasonic process.
Further, step (2) includes a step of centrifuging the obtained second mixed solution, so as to separate excess undissolved disulfiram by centrifugation in the case of excessive disulfiram addition.
Further, in the step (3), the in-situ gelling agent contained in the ultrapure water comprises any one or more of ion-sensitive in-situ gelling agents (such as deacetylated gellan gum and sodium alginate), temperature-sensitive in-situ gelling agents (such as poloxamer and chitosan), and pH-sensitive in-situ gelling agents (such as carbomer). Preferably, the in-situ gel contained in the ultrapure water is deacetylated gellan gum, and preferably, the content of the deacetylated gellan gum in the ultrapure water is 0.1-0.5%. The disulfiram-loaded nano-emulsion in-situ gel prepared by the invention is a solution with light blue opalescence, can be quickly converted into a gel after being added with a simulated nasal solution, and is beneficial to reducing the clearing effect of nasal cavities on medicines and further improving the absorption of disulfiram in the nasal cavities.
Further, in step (3), the self-assembly process is completed by vortexing or shaking. The emulsifier, the co-emulsifier and the DSPE-PEG 2000 are self-assembled into a sphere-like structure, an oil phase containing disulfiram is wrapped inside a sphere, and the PEG 2000 is dissolved in water and is covered on the surface of the sphere to extend into the water.
The invention discloses an application of a product prepared by the preparation method of the disulfiram-loaded nano-emulsion in-situ gel in the medical field.
Compared with the prior art, the invention has the following beneficial effects:
(1) the preparation process of the disulfiram-loaded nano-emulsion in-situ gel is stable and feasible, and the obtained nano-emulsion in-situ gel is in a nano-size spheroidal shape, has a smooth surface and is uniform in particle size distribution.
(2) According to the invention, the disulfiram is prepared into the nanoemulsion, so that the solubility of the disulfiram is obviously improved, the drug is prevented from being oxidized and degraded, the disulfiram is further wrapped in a sphere formed by the emulsifier, the co-emulsifier and the phospholipid to form the nanoemulsion after being dissolved in the first mixed solution, the surface of the phospholipid is covered with polyethylene glycol, the polyethylene glycol is water-soluble, the polyethylene glycol extends into water to avoid collision and settlement among nano-emulsion particles, the solubility of the disulfiram is improved, the biological stability and biocompatibility of the nanoemulsion are improved, the half-life period of the drug is prolonged, the toxicity is reduced, and the immunogenicity is reduced.
(3) The disulfiram nanoemulsion disclosed by the invention can effectively avoid the problem of disulfiram degradation in nasal administration, because the disulfiram is easy to degrade after entering a human body through a nasal cavity, the problem can be effectively avoided by preparing the disulfiram-coated nanoemulsion, the in-vivo stability of the disulfiram is improved, and the disulfiram can be rapidly enriched to a brain focus position after being administered through the nasal cavity. In addition, the nanoemulsion can increase the fluidity of stratum corneum lipid bilayers and promote transdermal absorption.
(4) In-vitro drug release research results show that the disulfiram-loaded nano-emulsion in-situ gel prepared by the invention effectively realizes the slow release of the drug, basically realizes the complete release of the drug after 12 hours, and maintains the concentration of the drug in the body.
(5) In-vitro and in-vivo experimental research results show that the disulfiram-loaded nano-emulsion in-situ gel prepared by the invention has higher safety, better glioma cell proliferation resisting activity in vitro, and the drug can be transferred to brain tumor through nasal cavity in vivo, thereby effectively realizing the tumor growth inhibition of a rat C6 in-situ brain glioma model and prolonging the median survival period of cancer rats.
In conclusion, the disulfiram-loaded nanoemulsion in-situ gel prepared by the invention obviously increases the solubility of the medicine, improves the medicine absorption and improves the bioavailability, the medicine can bypass the blood brain barrier by nasal administration and directly acts on the focus part of the brain, the curative effect is improved, the toxic and side effect of the whole body is reduced, and the compliance of a patient is improved by a non-invasive administration mode.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a TEM image of disulfiram-loaded ion-sensitive nanoemulsion in-situ gel (DSF-INEG) prepared by a fifth embodiment of the present invention.
FIG. 2 is a diagram showing the particle size distribution of the disulfiram-loaded ion-sensitive nanoemulsion in-situ gel (DSF-INEG) prepared by the fifth embodiment of the present invention.
FIG. 3 is a graph showing the in vitro release profile of disulfiram-loaded ion-sensitive nanoemulsion in situ gel (DSF-INEG) prepared according to a fifth embodiment of the present invention.
FIG. 4 shows the in vitro 48h antitumor cell proliferation activity of the disulfiram-loaded ion-sensitive nanoemulsion in situ gel preparation prepared by the fifth embodiment of the invention in combination with copper (DSF-INEG/Cu) on human glioma cell U87 cells.
FIG. 5 shows the in vitro 48h anti-tumor proliferation activity of disulfiram-loaded ion-sensitive nanoemulsion in situ gel preparation combined with copper (DSF-INEG/Cu) on rat glioma cell C6 cells.
FIG. 6 is a graph showing the effect of disulfiram-loaded ion-sensitive nanoemulsion in situ gel formulation combined with copper (DSF-INEG/Cu) prepared according to a fifth embodiment of the present invention on the survival of SD rat C6 in situ glioma model. Control is a physiological saline nasal administration group, namely a blank group; intra administration is a DSF-INEG/Cu nasal administration group.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, the preparation of a disulfiram nasal administration preparation with stable properties, high enrichment in brain tumors and high safety is one of the key technologies for improving the chemotherapy effect of brain gliomas. Therefore, the invention provides a preparation method of the disulfiram-loaded nano-emulsion in-situ gel; the invention will now be further described with reference to the drawings and detailed description.
First embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
150mg of isopropyl myristate, 330mg of emulsifier OP and 220mg of Transartol HP are mixed (the ratio of the emulsifier to the co-emulsifier is 3:2), 28mg of DSPE-PEG 2000 is added, the mixture is subjected to ultrasonic treatment for 3min to be uniformly mixed, 20mg of disulfiram is added into the obtained mixed solution, the mixed solution is subjected to ultrasonic treatment for 3min to be fully dissolved, centrifugal separation is carried out, ultrapure water containing 0.2% of deacetylated gellan gum is added to 2m L, the mixture is subjected to vortex at the speed of 1500rpm, and the DSF-loaded ion-sensitive nanoemulsion in-situ gel preparation is obtained through self-assembly.
Second embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
mixing 98mg of ethyl oleate, 361mg of Tween 80 and 241mg of Transartol HP (the ratio of the emulsifier to the co-emulsifier is 3:2), adding 30mg of DSPE-PEG 2000, performing ultrasonic treatment for 3min to uniformly mix the mixture, adding 20mg of disulfiram to the obtained mixed solution, performing ultrasonic treatment for 3min to fully dissolve the mixture, performing centrifugal separation, adding ultrapure water containing 0.2% of deacetylated gellan gum to 2m L, performing vortex at the speed of 1500rpm, and performing self-assembly to obtain the DSF-loaded ion-sensitive nanoemulsion in-situ gel.
Third embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
mixing 200mg of ethyl oleate, 300mg of Tween 80 and 200mg of Transartol HP (the ratio of the emulsifier to the co-emulsifier is 3:2), adding 100mg of DSPE-PEG 2000, performing ultrasonic treatment for 3min to uniformly mix the mixture, adding 20mg of disulfiram into the mixed solution, performing ultrasonic treatment for 3min to fully dissolve the mixture, performing centrifugal separation, adding ultrapure water containing 0.2% of deacetylated gellan gum to 2m L, performing vortex at the speed of 1500rpm, and performing self-assembly to obtain the DSF-loaded ion-sensitive nanoemulsion in-situ gel.
Fourth embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
mixing 150mg of ethyl oleate, 275mg of tween 80 and 275mg of Transartol HP (the ratio of the emulsifier to the co-emulsifier is 1:1), adding 55mg of DSPE-PEG 2000, performing ultrasonic treatment for 3min to uniformly mix the mixture, adding 30mg of disulfiram to the mixed solution, performing ultrasonic treatment for 3min to fully dissolve the mixture, performing centrifugal separation, adding ultrapure water containing 0.1% of deacetylated gellan gum to 2m L, performing vortex at the speed of 1500rpm, and performing self-assembly to obtain the DSF-loaded ion-sensitive nanoemulsion in-situ gel.
Fifth embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
mixing 150mg of ethyl oleate, 330mg of Tween 80 and 220mg of Transartol HP (the ratio of the emulsifier to the co-emulsifier is 3:2), adding 28mg of DSPE-PEG 2000, performing ultrasonic treatment for 3min to uniformly mix the mixture, adding 20mg of disulfiram to the mixed solution, performing ultrasonic treatment for 3min to fully dissolve the mixture, performing centrifugal separation, adding ultrapure water containing 0.2% of deacetylated gellan gum to 2m L, performing vortex at the speed of 1500rpm, and performing self-assembly to obtain the DSF-loaded ion-sensitive nanoemulsion in-situ gel.
Sixth embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
mixing 150mg of ethyl oleate, 330mg of Tween 80 and 220mg of Transartol HP (the ratio of the emulsifier to the co-emulsifier is 3:2), adding 28mg of DSPE-PEG 2000, performing ultrasonic treatment for 3min to uniformly mix, adding 30mg of disulfiram into the mixed solution, performing ultrasonic treatment for 3min to fully dissolve the disulfiram, performing centrifugal separation, adding ultrapure water containing 0.5% of deacetylated gellan gum to 2m L, shaking, and performing self-assembly to obtain the DSF-loaded ion sensitive nanoemulsion in-situ gel.
Seventh embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
150mg of isopropyl myristate, 412.5mg of tween 80 and 137.5mg of Transartol HP are mixed (the ratio of the emulsifier to the co-emulsifier is 3:1), 28mg of DSPE-PEG 2000 is added, the mixture is uniformly mixed by ultrasonic treatment for 3min, 0.1mg of disulfiram is added into the mixed solution, the mixture is fully dissolved by ultrasonic treatment for 3min, centrifugal separation is carried out, ultrapure water containing 0.3% deacetylated gellan gum is added to 2m L, vortex is carried out at the speed of 1500rpm, and the DSF-loaded ion-sensitive nanoemulsion in-situ gel is obtained by self-assembly.
Eighth embodiment
A method for preparing disulfiram-loaded nano-emulsion in-situ gel comprises the following steps:
mixing 150mg of ethyl oleate, 330mg of Tween 80 and 220mg of Transartol HP (the ratio of the emulsifier to the co-emulsifier is 3:2), adding 28mg of DSPE-PEG 2000, performing ultrasonic treatment for 3min to uniformly mix, adding 40mg of disulfiram into the mixed solution, performing ultrasonic treatment for 3min to fully dissolve the disulfiram, performing centrifugal separation, adding ultrapure water containing 0.2% of deacetylated gellan gum to 2m L, shaking, and performing self-assembly to obtain the DSF-loaded ion sensitive nanoemulsion in-situ gel.
Ninth embodiment
A preparation method of DSF-loaded ion-sensitive liposome in-situ gel comprises the following steps:
dissolving 120mg of lecithin, 24mg of cholesterol and 6mg of disulfiram in 900 mu L of absolute ethyl alcohol, adding the mixture into 4m L of ultrapure water containing 0.2% of deacetylated gellan gum under the stirring and heating conditions at 40 ℃, stirring for 5min, and performing ultrasonic treatment for 3min to obtain the DSF-loaded ion sensitive liposome in-situ gel.
Performance testing
The disulfiram-loaded ion-sensitive nanoemulsion in-situ gel prepared in the fifth embodiment is used as a test object, and the test results are shown in fig. 1 to 6.
In fig. 1, the disulfiram-loaded ion-sensitive nanoemulsion in-situ gel has a spheroidal structure, a smooth surface and uniform particle size distribution. Because the PEG modified phospholipid (DSPE-PEG 2000) is added, the prepared disulfiram-loaded ion-sensitive nanoemulsion in-situ gel can be uniformly and stably dispersed.
In fig. 2, the excessively large or small particle size is not favorable for the drug to be absorbed into the brain in the nasal cavity, and the dynamic light scattering result shows that the average particle size of the disulfiram-loaded ion-sensitive nanoemulsion in-situ gel prepared by the invention is about 64nm and is favorable for the drug to be absorbed in the nasal cavity according to the optimal preparation process including the solubility, the particle size and the PDI according to three levels by screening the proportion of each component of the disulfiram-loaded ion-sensitive nanoemulsion in-situ gel.
In figure 3, both the nanoemulsion and the in-situ gel can slow down the release of disulfiram, and the in-vitro drug release research result shows that the disulfiram-loaded ion-sensitive nanoemulsion in-situ gel prepared by the invention effectively realizes the slow release of the drug, basically realizes the complete release of the drug after 12h, and maintains the concentration of the drug in the body.
In FIG. 4, the inhibition of DSF-INEG/Cu proliferation in vitro on human glioblastoma cell U87 was evaluated using the MTT assay. The experiment was divided into 4 groups of copper (CuCl) each2) The administration group, the blank ion-sensitive nanoemulsion in-situ gel (INEG) administration group, the disulfiram-loaded ion-sensitive nanoemulsion in-situ gel and copper combined (DSF-INEG/Cu) administration group and the disulfiram and copper combined (DSF/Cu) administration group (DSF/Cu administration group in which disulfiram is dissolved in<0.1% of DMSO), the results are shown in the figure, the blank ion-sensitive nanoemulsion in-situ gel has almost no influence on the cell proliferation activity, because the components of the ion-sensitive nanoemulsion in-situ gel are preferably selected from auxiliary materials approved by pharmacopoeia, the toxicity is low, the safety is high, disulfiram also has higher safety, and the prepared ion-sensitive nanoemulsion in-situ gel has better stability and biocompatibility, so the ion-sensitive nanoemulsion in-situ gel has higher safety.
In FIG. 5, MTT assay was usedTo evaluate the proliferation inhibition effect of DSF-INEG/Cu on rat glioma cell C6 cells in vitro. The experiment was divided into 4 groups of copper (CuCl) each2) The administration group, the blank ion-sensitive nanoemulsion in-situ gel (INEG) administration group, the disulfiram-loaded ion-sensitive nanoemulsion in-situ gel and copper combined (DSF-INEG/Cu) administration group and the disulfiram and copper combined (DSF/Cu) administration group (DSF/Cu administration group in which disulfiram is dissolved in<0.1% of DMSO), the results are shown in the figure, the blank ion-sensitive nanoemulsion in-situ gel has almost no influence on the cell proliferation activity, because the components of the ion-sensitive nanoemulsion in-situ gel are preferably selected from auxiliary materials approved by pharmacopoeia, the toxicity is low, the safety is high, disulfiram also has higher safety, and the prepared ion-sensitive nanoemulsion in-situ gel has better stability and biocompatibility, so the ion-sensitive nanoemulsion in-situ gel has higher safety.
In FIG. 6, Control is a saline nasal administration group, and Intra administration is a DSF-INEG/Cu nasal administration group. The mean survival time of the SD rat C6 in-situ brain glioma model can be obviously prolonged by combining the disulfiram-loaded ion-sensitive nano-emulsion in-situ gel with copper and DSF-INEG/Cu. The ion sensitive type nano-emulsion in-situ gel reduces the cleaning effect of a nasal cavity on a medicine, and on the other hand, the nano-emulsion can promote the medicine to be absorbed in the nasal cavity, the medicine can bypass a blood brain barrier to directly reach brain tumor after nasal administration, so that the large enrichment of the medicine in the brain is realized, the growth inhibition of the tumor is effectively realized, and the median survival time of a rat of an in-situ brain glioma model is obviously prolonged.
For the disulfiram-loaded ion-sensitive liposome in-situ gel prepared in the ninth embodiment, as the mass of disulfiram increases, the concentration of disulfiram of the prepared disulfiram-loaded ion-sensitive liposome in-situ gel is gradually increased, the solubility of disulfiram in water is increased to 1.5mg/m L at most, and then the amount of disulfiram, even lecithin and cholesterol is increased to obtain milky white precipitate, so that the ion-sensitive liposome in-situ gel with light blue opalescence cannot be obtained.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a disulfiram-loaded nano-emulsion in-situ gel is characterized by comprising the following steps:
(1) mixing the oil phase, the emulsifier and the co-emulsifier, adding the phospholipid modified by polyethylene glycol, and uniformly mixing to obtain a first mixed solution;
(2) adding disulfiram into the first mixed solution, and obtaining a second mixed solution after the disulfiram is completely dissolved;
(3) and adding ultrapure water containing the in-situ gel into the obtained second mixed solution, and carrying out self-assembly to obtain the disulfiram-loaded nano-emulsion in-situ gel.
2. The method for preparing the disulfiram-loaded nanoemulsion in-situ gel according to claim 1, wherein the oil phase comprises any one or more of oleic acid, ethyl oleate, castor oil, isopropyl myristate, soybean oil and rapeseed oil;
or the emulsifier comprises any one or more of tween, span, polyethylene glycol-15 hydroxystearate, caprylic/capric macrogol glyceride, polyoxyethylene hydrogenated castor oil, emulsifier OP, poloxamer, lecithin and the like;
or the auxiliary emulsifier comprises any one or more of ethanol, isopropanol, 1, 2-propylene glycol, PEG 400, TransartolHP and n-butanol.
3. The method for preparing the disulfiram-loaded nanoemulsion in-situ gel of claim 2, wherein the oil phase is ethyl oleate or isopropyl myristate; the emulsifier is polyethylene glycol-15 hydroxystearate or tween 80; the coemulsifier is TransartolHP.
4. The method for preparing the disulfiram-loaded nanoemulsion in-situ gel of claim 1, wherein the polyethylene glycol-modified phospholipid is DSPE-PEG.
5. The method for preparing the disulfiram-loaded nanoemulsion in-situ gel according to claim 1, wherein in the step (1), the oil phase accounts for 14-29% by mass, the total proportion of the emulsifier and the co-emulsifier accounts for 71-86% by mass, and the PEG-modified phospholipid accounts for 5-20% by mass of the total proportion of the emulsifier and the co-emulsifier; preferably, the mass ratio of the emulsifier to the co-emulsifier is 1: 1-3: 1.
6. The preparation method of the disulfiram-loaded nanoemulsion in-situ gel agent as claimed in claim 1, wherein in the step (2), the concentration of the disulfiram in the second mixed solution is 0.1mg/m L-70 mg/m L, preferably 10-40 mg/m L, or the disulfiram is completely dissolved by stirring, vortexing or ultrasonic process.
7. The method for preparing the disulfiram-loaded nanoemulsion in-situ gel according to claim 1, wherein the step (2) further comprises the step of centrifuging the obtained nanoemulsion.
8. The method for preparing the disulfiram-loaded nanoemulsion in-situ gel according to claim 1, wherein in the step (3), the in-situ gel contained in the ultrapure water comprises any one or more of an ion-sensitive in-situ gel, a temperature-sensitive in-situ gel and a pH-sensitive in-situ gel;
preferably, the ion-sensitive in-situ gel comprises acetylated gellan gum or sodium alginate, and the temperature-sensitive in-situ gel comprises poloxamer or chitosan; the pH sensitive in-situ gel comprises carbomer;
preferably, the in-situ gel contained in the ultrapure water is deacetylated gellan gum, and preferably, the content of the deacetylated gellan gum in the ultrapure water is 0.1-0.5%.
9. The method for preparing the disulfiram-loaded nanoemulsion in-situ gel of claim 1, wherein in step (3), the self-assembly process is completed by vortex or oscillation.
10. The use of the product obtained by the method for preparing a disulfiram-loaded nanoemulsion in-situ gel according to any one of claims 1 to 9 in the medical field.
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