A glutamate-responsive nanoliposome, its preparation method, and its application in antiepileptic therapy.
By designing glutamate-responsive nanoliposomes and utilizing glutamate oxidase to catalyze the generation of hydrogen peroxide in a high-glutamate environment, precise drug release during epileptic seizures was achieved, solving the problem of insufficient drug responsiveness in existing technologies, improving therapeutic efficacy and reducing toxic side effects.
Patent Information
- Application Number
- CN202610080113.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-21
- Publication Date
- 2026-06-02
Smart Images
Figure CN122123981A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical materials, specifically to a glutamate-responsive nanoliposome, its preparation method, and its application in antiepileptic therapy. Background Technology
[0002] Epilepsy is a common chronic neurological disorder, with clinical manifestations including behavioral, cognitive, sensory, and memory impairments. In epilepsy patients, abnormally excited neurons in the epileptogenic focus release large amounts of excitatory neurotransmitters (glutamate), promoting the generation and propagation of hypersynchronous discharges in the neuronal network, ultimately leading to seizures. For example, in temporal lobe epilepsy, the glutamate concentration in the hippocampus during a seizure is 6-50 times higher than normal, reaching up to 150 μM or more. Currently, drug therapy is the main clinical means of controlling epileptic seizures. Patients maintain stable blood drug concentrations by taking antiepileptic drugs regularly over a long period. First-line antiepileptic drugs include carbamazepine (CBZ), lamotrigine, and sodium valproate. These drugs target the hypersynchronous discharge of nerve cells, controlling seizures through mechanisms such as regulating the function of ion channels in nerve cell membranes, increasing the release of inhibitory neurotransmitters, and inhibiting ionotropic glutamate receptors. However, long-term use of high doses of antiepileptic drugs can lead to toxic side effects, including central nervous system toxicity, ataxia, and liver and kidney damage, and may even result in drug-resistant epilepsy.
[0003] In recent years, the emergence of nanomedicine delivery systems has provided researchers with new design ideas. These systems help increase the concentration of antiepileptic drugs in brain tissue while reducing dosage and peripheral toxicity. However, existing nanomedicine delivery systems cannot achieve precise treatment or address the problem of central nervous system toxicity. For example, while traditional liposomes can increase the intracranial concentration of antiepileptic drugs, their drug release responsiveness in the epileptogenic focus is insufficient, failing to achieve precise release and effectively address the significantly elevated glutamate levels during seizures. Furthermore, after release, neurons throughout the brain remain in a high-concentration drug environment for an extended period, resulting in central nervous system toxicity. Therefore, there is an urgent need to develop novel liposomes capable of responsive drug release under high glutamate conditions during seizures, enabling rapid and timely drug release during seizures and non-release during non-seizure periods, thereby improving the efficacy of antiepileptic drugs and reducing their toxic side effects. Summary of the Invention
[0004] The purpose of this invention is to address the aforementioned problems by providing a glutamate-responsive drug-releasing nanoliposome, its preparation method, and its application in antiepileptic drugs. The nanoliposomes provided by this invention have suitable particle size and potential, excellent stability, and can efficiently load antiepileptic drugs. They also possess advantages such as a suitable response limit to high glutamate levels in epileptogenic foci, short response time, and high release rate, providing a solid foundation for subsequent pharmacodynamic evaluation and solving the problems in the prior art.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] In a first aspect, the present invention provides a glutamate-responsive nanoliposome, comprising a liposome, glutamate oxidase, distearate phosphatidylethanolamine-ketothiol-methoxy polyethylene glycol (DSPE-TK-mPEG), and an antiepileptic drug; wherein the glutamate oxidase is encapsulated in the hydrophilic region of the center of the liposome, the antiepileptic drug is loaded in the hydrophobic region of the liposome bilayer, and DSPE-TK-mPEG is modified on the liposome bilayer membrane.
[0007] The liposomes described in this invention acquire long-term in vivo circulation capacity through mPEG modification, thereby effectively accumulating within the epileptogenic focus. Simultaneously, the glutamate oxidase loaded at the center of the liposome rapidly converts excess glutamate during epileptic seizures into hydrogen peroxide, causing DSPE-TK-mPEG fragmentation on the liposome membrane and a decrease in bilayer structural stability, leading to liposome rupture and disintegration, releasing the antiepileptic drug. This achieves the goal of inhibiting neuronal hypersynchronous discharge and suppressing epileptic seizures. Ultimately, the liposomes of this invention can achieve the goal of rapidly releasing antiepileptic drugs in the pre-seizure phase and not releasing them when the seizure is not occurring, thus improving the efficacy of antiepileptic drugs and reducing their toxic side effects.
[0008] Secondly, the present invention provides a method for preparing the above-mentioned glutamate-responsive nanoliposomes, comprising the following steps:
[0009] Neutral phospholipids, cholesterol, and DSPE-TK-mPEG are mixed and dissolved in an organic solvent to obtain a homogeneous lipid solution. The neutral phospholipids are selected from distearyl lecithin (DSPC), dipalmitoyl lecithin (DPPC), dioleoyl lecithin (DOPC), or lecithin (SPC). The molecular weight of DSPE-TK-mPEG is 2000, 3000, or 5000. The molar ratio of neutral phospholipids, cholesterol, and DSPE-TK-mPEG is 6:4:(0.5-7).
[0010] Add the antiepileptic drug solution to the lipid solution and mix well to obtain a lipid solution containing the drug with a total lipid (neutral phospholipids, cholesterol and DSPE-TK-mPEG) to antiepileptic drug mass ratio of (5-30):1.
[0011] Organic solvents in the lipid solution containing the drug are removed by rotary evaporation to obtain a uniform film on the flask wall. Glutamate oxidase phosphate buffer is added to the solvent-removed lipid film, and the film is hydrated by uniform stirring on a magnetic stirrer to obtain a uniformly dispersed lipid aqueous solution. The mass ratio of glutamate oxidase to DSPE-TK-mPEG is (1-5):3.9.
[0012] The lipid aqueous solution was repeatedly extruded using a polycarbonate membrane and a liposome extruder to obtain drug-loaded nanoliposomes with small and uniform particle size. The nanoliposomes were then purified by dialysis to remove free and unbound drug, thus obtaining the glutamate-responsive nanoliposomes.
[0013] Preferably, the organic solvent is dichloromethane or trichloromethane.
[0014] Preferably, the hydration conditions are: stirring at a constant speed of 300-1000 rpm for 0.5-2 h at a temperature of 30-50 ℃.
[0015] Preferably, the pore sizes of the polycarbonate membrane are 200 nm and 100 nm.
[0016] Preferably, the antiepileptic drug is carbamazepine (CBZ), lamotrigine, phenytoin sodium, or topiramate.
[0017] Thirdly, the present invention provides the application of the above-mentioned glutamate-responsive nanoliposomes in the preparation of epilepsy treatment agents.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] This invention provides a glutamate-responsive drug-releasing nanoliposome, which can load an antiepileptic drug within the hydrophobic region of a lipid bilayer membrane and simultaneously load glutamate oxidase within the hydrophilic region of the liposome cavity, achieving simultaneous loading of hydrophobic and hydrophilic substances. The particle size is controlled between 100-200 nm using a polycarbonate membrane, resulting in small, stable, and uniform particle size. In a high-level glutamate environment, the glutamate oxidase within the liposome reacts glutamate to generate hydrogen peroxide. The product reacts with TK, causing DSPE-TK-mPEG to break in the middle, thereby reducing the stability of the liposome bilayer structure, leading to rapid disintegration of the liposome and release of the antiepileptic drug. It exhibits a certain response threshold, with minimal liposome release at physiological glutamate levels.
[0020] The glutamate-responsive drug-releasing nanoliposomes of this invention significantly improve the stability and bioavailability of antiepileptic drugs, overcoming the shortcomings of traditional antiepileptic drugs in the treatment process. Optimized liposome preparation provides an excellent drug delivery platform for antiepileptic drugs. Simultaneously, utilizing the synergistic effect of glutamate oxidase and DSPE-TK-mPEG on the liposomes, the drug responds to the sudden increase in glutamate during epileptic seizures, achieving specific release of the antiepileptic drug, improving therapeutic efficacy and reducing toxic side effects, and showing promising clinical application prospects. Attached Figure Description
[0021] Figure 1 This is a transmission electron microscope image of the glutamate-responsive nanoliposomes prepared in Example 1;
[0022] Figure 2 The particle size distribution of the glutamate-responsive nanoliposomes prepared in Example 1 in phosphate buffer;
[0023] Figure 3 The specific protein bands of the glutamate-responsive nanoliposomes prepared in Example 1;
[0024] Figure 4 The particle size change of the glutamate-responsive nanoliposomes prepared in Example 1 after incubation in a 200 μM glutamate solution;
[0025] Figure 5 This describes the drug release of the glutamate-responsive nanoliposomes prepared in Example 1 after incubation in a 200 μM glutamate solution over 2 hours.
[0026] Figure 6 This is an evaluation of the long-term in vivo circulation capacity of the glutamate-responsive nanoliposomes prepared in Example 1 in animals;
[0027] Figure 7 This is an evaluation of the efficacy of the glutamate-responsive drug-releasing nanoliposomes prepared in Example 1 on a pilocarpine-induced acute epilepsy model. Detailed Implementation
[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0029] Example 1
[0030] Preparation of nanoliposomes for glutamate-responsive drug release:
[0031] 5.7 mg DPPC, 2 mg cholesterol, and 7.8 mg DSPE-TK-mPEG were placed in a round-bottom flask. 1 mg carbamazepine (CBZ) was added, and the mixture was completely dissolved in 5 mL dichloromethane. A uniform thin film was then prepared using a rotary evaporator and completely dried in a fume hood. 1 mL of phosphate-buffered saline containing glutamate oxidase (2 mg / mL) was added, and the mixture was hydrated at 600 rpm and 45 °C for 1 h. The liposomes were then passed through 200 nm and 100 nm polycarbonate membranes 20 times each. Finally, after dialyzing with a 300 kDa dialysis bag for 24 h, the liposomes were stored at 4 °C and designated Lipo@CBZ.
[0032] Meanwhile, without adding CBZ, DPPC, cholesterol, DSPE-TK-mPEG and glutamate oxidase were mixed in the above proportions, and the liposome carrier, denoted as Lipo, was prepared according to the above method.
[0033] Example 2
[0034] Characterization of glutamate-responsive drug-releasing nanoliposomes Lipo@CBZ:
[0035] The nanoliposomes Lipo@CBZ prepared in Example 1 were characterized by observing the structural features such as liposome size and surface morphology using transmission electron microscopy, determining the hydrated particle size of the liposomes using dynamic laser scattering particle size analyzer, and verifying the loading of glutamate oxidase on the liposomes using Western blotting.
[0036] Among them, the results of transmission electron microscopy are as follows Figure 1 As shown, the nanoliposome structure has a clear shape. The results of dynamic laser scattering particle size analysis are as follows... Figure 2 As shown, the nanoliposomes have uniform particle size and are evenly dispersed. The results of Western blotting are as follows: Figure 3 As shown, the nanoliposomes exhibit specific protein bands associated with glutamate oxidase.
[0037] Example 3
[0038] kinetics of glutamate oxidase in liposomes:
[0039] Take 1 mL of liposome Lipo@CBZ solution, add 200 μM glutamic acid solution at 37℃, mix well and let stand for a period of time, and determine the change in hydration particle size of liposomes using a dynamic laser scattering particle size analyzer.
[0040] The results of the dynamic laser scattering particle size analyzer are as follows: Figure 4 As shown, a 200 μM glutamate solution can alter the particle size of nanoliposomes.
[0041] Example 4
[0042] Glutamate-responsive drug release kinetics studies of liposomes:
[0043] Lipo@FITC was prepared using fluorescein isothiocyanate (FITC) as a model drug. The glutamate-responsive release performance of the liposomes was evaluated using the dialysis bag method. One mL of Lipo@FITC liposome solution was added to a final concentration of 200 μM glutamate and placed in a 3500 Da dialysis bag. The dialysis bag was then immersed in 10 mL of PBS with a glutamate concentration of 200 μM. One mL of the extracorporeal solution was collected at 5, 15, 30, 60, 90, and 120 min, and an equal volume of glutamate-containing PBS was added at the same temperature. The control group did not add glutamate and used glutamate-free PBS. Finally, the fluorescence intensity of the collected extracorporeal solution was measured and statistically analyzed using a fluorometer to determine the release characteristics of the liposomes and evaluate their glutamate-responsive drug release capacity.
[0044] The results of the fluorescence spectrophotometer test are as follows Figure 5 As shown, a 200 μM glutamate solution enables nanoliposomes to release drugs.
[0045] Example 5
[0046] Liposome long-circulation capacity assessment:
[0047] Healthy male C57BL / 6J mice, approximately 8 weeks old, were selected, and a chronic epilepsy model was induced by injecting 250 ng of kainic acid solution into the CA1 region of the hippocampus. The chronic epilepsy model mice were divided into two groups: a Lipo@Cy5 experimental group (using Lipo-loaded fluorescent dye Cy5) and a Cy5 control group. Fluorescence imaging of both groups was performed using a small animal in vivo imaging system 1 hour and 72 hours after intravenous injection. Results are as follows: Figure 6 As shown, Lipo@Cy5 reached a high concentration in the mouse brain 1 hour after injection and could remain there for a long time, with strong fluorescence still observed after 72 hours; however, the control group with the same concentration of Cy5 showed weak fluorescence 1 hour after injection and the fluorescence basically disappeared after 72 hours.
[0048] Example 6
[0049] Evaluation of the efficacy of liposomes in treating epilepsy:
[0050] Healthy male C57BL / 6J mice were randomly divided into 7 groups: 1. Control group (saline), 2. 1 mg / kg CBZ, 3. 3 mg / kg CBZ, 4. 10 mg / kg CBZ, 5. Lipo@CBZ (equivalent to 1 mg / kg CBZ), 6. Lipo@CBZ (equivalent to 3 mg / kg CBZ), and 7. Lipo@CBZ (equivalent to 10 mg / kg CBZ), with 5 mice in each group. Mice in each group were intravenously injected with the treatment drugs, followed by an intraperitoneal injection of 1 mg / kg scopolamine half an hour later, and an injection of 250 mg / kg pilocarpine one hour later to induce acute epileptic seizures. The latency of grand mal seizures (GS) in the following hour was observed and recorded.
[0051] Observation results as follows Figure 7 As shown, group 7 significantly prolonged the latency of GS compared to groups 4 and 1, and the efficacy of Lipo@CBZ was positively correlated with concentration.
[0052] The contents described in this specification are merely an enumeration of the implementation forms of the inventive concept, and the scope of protection of this invention should not be regarded as limited to the specific forms described in the embodiments.
Claims
1. A glutamate-responsive nanoliposome, characterized in that, It is composed of liposomes, glutamate oxidase, distearate phosphatidylethanolamine-ketothiol-methoxy polyethylene glycol (DSPE-TK-mPEG), and an antiepileptic drug; wherein, glutamate oxidase is encapsulated in the hydrophilic region of the center of the liposome, the antiepileptic drug is loaded in the hydrophobic region of the liposome bilayer, and DSPE-TK-mPEG is modified on the liposome bilayer membrane.
2. A method for preparing glutamate-responsive nanoliposomes as described in claim 1, characterized in that, The preparation method includes the following steps: Neutral phospholipids, cholesterol, and DSPE-TK-mPEG were mixed in a molar ratio of 6:4:(0.5-7) and dissolved in an organic solvent to obtain a homogeneous lipid solution. An antiepileptic drug solution was added to a lipid solution and mixed thoroughly to obtain a lipid solution containing the drug with a mass ratio of lipid to antiepileptic drug of (5-30):
1. After removing the organic solvent, a lipid membrane was obtained. A phosphate buffer solution of glutamate oxidase was added to the lipid membrane, and the mixture was hydrated to obtain an aqueous lipid solution; wherein the mass ratio of glutamate oxidase to DSPE-TK-mPEG was (1-5):3.
9. Drug-loaded nanoliposomes were obtained by repeatedly extruding a lipid aqueous solution using a polycarbonate membrane and a liposome extruder. The nanoliposomes were then purified by dialysis to remove free unbound drug, thus obtaining the glutamate-responsive nanoliposomes.
3. The preparation method according to claim 2, characterized in that, The neutral phospholipid is selected from distearyl lecithin, dipalmitoyl lecithin, dioleoyl lecithin, or lecithin.
4. The preparation method according to claim 2, characterized in that, The molecular weight of the DSPE-TK-mPEG is 2000, 3000 or 5000.
5. The preparation method according to claim 2, characterized in that, The organic solvent is dichloromethane or trichloromethane.
6. The preparation method according to claim 2, characterized in that, The hydration conditions are: stirring at a constant speed of 300-1000 rpm for 0.5-2 h at a temperature of 30-50 ℃.
7. The preparation method according to claim 2, characterized in that, The polycarbonate membranes have pore sizes of 200 nm and 100 nm, respectively.
8. The preparation method according to claim 2, characterized in that, The antiepileptic drugs mentioned are carbamazepine, lamotrigine, phenytoin sodium, or topiramate.
9. The use of the glutamate-responsive nanoliposomes as described in claim 1 in the preparation of epilepsy treatment agents.