A cannabidiol targeted sustained-release preparation for preventing and treating bilirubin encephalopathy, and a preparation method and application thereof

Abstract

The present application relates to the technical fields of biological medicine preparation, and discloses a cannabidiol targeted sustained-release preparation for preventing and treating bilirubin encephalopathy, a preparation method and application thereof, which comprises the following steps: dissolving polylactic acid-glycolic acid copolymer, cannabidiol and silymarin in an oil phase, forming an emulsion through emulsification, controlling solvent volatilization to a specific semi-cured critical interval to obtain a swollen semi-rheological state carrier, utilizing residual solvent diffusion dynamics to induce reverse diffusion of pH-sensitive hydrogel molecular chains and embed the hydrogel molecular chains into a swollen surface layer of the carrier, removing residual solvents under negative pressure to make the carrier shrink and crystallize and physically lock the embedded hydrogel molecular chains, and finally coupling specific antibodies, and the present application establishes a solvent residue-induced interfacial interpenetrating network physical locking mechanism to solve the hidden danger of peeling caused by weak interfacial bonding force between a hydrophilic shell and a hydrophobic core.

Description

Technical Field

[0001] This invention relates to a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy, its preparation method and application, belonging to the field of biopharmaceutical manufacturing technology. Background Technology

[0002] Bilirubin encephalopathy is caused by unconjugated bilirubin crossing the blood-brain barrier and depositing in the basal ganglia, leading to neurological damage. Cannabidiol (CBD) possesses neuroprotective and anti-inflammatory activities and is a potential drug for alleviating neurological damage. However, its poor water solubility and unstable physiological environment limit its clinical efficacy. Existing technologies utilize polylactic acid-glycolic acid copolymers to construct nanocarriers, loading hydrophobic drugs and modifying them with hydrophilic functional layers to enhance long-term circulation or targeting capabilities. Existing technologies often simply pursue the pharmacological superposition of active ingredients while neglecting the structural construction of delivery systems. For example, Chinese invention patent application CN116688099A discloses a combination drug and application for the prevention and / or treatment of thrombotic diseases. Although it attempts to use CBD in combination with fibroblast growth factor, it essentially only involves a simple physical mixing or stepwise administration of the two active ingredients without constructing a unified carrier system with a spatial structure. This non-integrated delivery mode exposes hydrophobic CBD directly to complex physiological fluids, failing to shield it from enzymatic degradation in the blood environment, resulting in uncontrollable pharmacokinetic behavior.

[0003] When constructing multilayer targeted formulations, conventional preparation processes suffer from interface stability bottlenecks. Conventional stepwise methods first completely solidify and desolvate the hydrophobic core, and then modify the hydrophilic hydrogel or targeting ligand by physical adsorption or surface chemical reaction. However, due to the incompatibility of interfacial tension and thermodynamics between the hydrophobic polyester core and the hydrophilic functional shell, the two are only connected by weak van der Waals forces. When the formulation enters the bloodstream, it is subjected to blood flow shear and plasma protein competitive adsorption and replacement. The functional shell with weak binding force is easily disintegrated and peeled off. Interface failure leads to premature loss of the targeting group, and the exposure of the hydrophobic core to the physiological environment triggers drug burst release, exacerbating nonspecific toxicity.

[0004] Therefore, how to develop a system that enhances the mechanical stability of the core-shell interface, maintains high biocompatibility and precise pH response characteristics, and resists structural damage and drug burst release during in vivo circulation has become the technical problem to be solved by this invention. Summary of the Invention

[0005] To address the problems mentioned in the background art, the technical solution of the present invention is as follows: A method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy, the method comprising the following steps:

[0006] Step S1: Establish a semi-cured swelling interface. Dissolve polylactic acid-glycolic acid copolymer, cannabidiol, and silymarin in dichloromethane oil phase, and use polyvinyl alcohol aqueous solution as aqueous phase. Homogenize and emulsify to form an oil-in-water emulsion. Perform controlled solvent evaporation on the emulsion and monitor the system status in real time. Stop evaporation when the residual amount of dichloromethane inside the polylactic acid-glycolic acid copolymer particles reaches the semi-cured critical range of 5.0wt% to 8.0wt%, thereby obtaining a carrier suspension with a surface in a swollen semi-rheological state.

[0007] Step S2: Construct an interpenetrating network. Add a pH-sensitive hydrogel precursor solution to the carrier suspension in a swollen semi-rheological state. Perform stirring and incubation at 25 degrees Celsius. Utilize the mass transfer force generated by the outward diffusion of residual dichloromethane inside the particles to induce the hydrogel molecular chains in the pH-sensitive hydrogel precursor solution to diffuse backward and embed into the swollen surface of the polylactic acid-glycolic acid copolymer particles.

[0008] Step S3, solidify the locking pins. Place the system obtained in step S2 in a negative pressure environment with a vacuum degree of -0.08MPa to -0.09MPa to remove the remaining dichloromethane, causing the polylactic acid-glycolic acid copolymer particle chain segments to shrink and crystallize, and physically locking the embedded hydrogel molecular chains onto the core surface. After centrifugation and washing, a core-shell precursor with an interpenetrating network interface is obtained.

[0009] Step S4, targeted modification, involves conjugating a specific antibody to the pH-sensitive layer surface of the core-shell precursor.

[0010] Preferably, in step S2, the pH-sensitive hydrogel precursor solution contains carboxymethyl chitosan or sodium alginate at a concentration of 2.0 mg / mL to 5.0 mg / mL; the stirring and incubation operation is controlled at 100 rpm to 200 rpm and the incubation time is controlled at 15 minutes to 30 minutes. The stirring speed and time parameters are used to enable the hydrogel molecular chains to penetrate to a depth of 10 nanometers to 30 nanometers in the swollen surface layer.

[0011] Preferably, the process of removing the remaining dichloromethane in step S3 follows a specific solvent removal rate, wherein the solvent removal rate is... The following relationship must be satisfied: ,in, This indicates the mass removal rate of dichloromethane. This represents the absolute value of the vacuum level in a negative pressure environment. This represents the effective evaporation surface area of ​​the system. Indicates the dynamic viscosity of the pH-sensitive hydrogel precursor solution. This is a preset constant related to the crystallinity of polylactic acid-glycolic acid copolymer; the relationship is used to synchronize the shrinkage rate of polylactic acid-glycolic acid copolymer with the curing rate of hydrogel molecular chains by controlling the matching relationship between vacuum degree and viscosity.

[0012] Preferably, in step S1, the mass ratio of cannabidiol to silymarin is 3:1 to 5:1; the total concentration of cannabidiol and silymarin in the dichloromethane oil phase is 20 mg / mL to 50 mg / mL; the molecular weight of the polylactic acid-glycolic acid copolymer is 10,000 Da to 50,000 Da, and the molar ratio of lactic acid to glycolic acid is 50:50 to 75:25.

[0013] Preferably, the homogenization speed in step S1 is 3000 rpm to 5000 rpm, and the time is 5 minutes to 10 minutes; the controlled solvent evaporation operation is carried out under a vacuum of -0.04 MPa to -0.06 MPa until the refractive index of the system changes to a preset threshold corresponding to a residual amount of 5.0 wt% to 8.0 wt%.

[0014] Preferably, the specific antibody in step S4 is an anti-neuron-specific enolase antibody or an anti-glial fibrillary acidic protein antibody; the coupling is achieved by activating the carboxyl group on the surface of the nucleoshell precursor and forming an amide bond with the amino group of the specific antibody.

[0015] Preferably, the preparation method further includes a freeze-drying step of the product obtained in step S4. The freeze-drying includes pre-freezing at -80 degrees Celsius for 4 hours and sublimation drying at -50 degrees Celsius and 10 Pa vacuum for 24 hours to obtain a dry powder formulation with an average particle size of 100 nanometers to 200 nanometers. The pH value of the pH-sensitive hydrogel precursor solution in step S2 is 7.4, and the hydrogel has the material property of undergoing volume phase change or increasing solubility in the pH range of 7.2 to 7.6. The material property is used to trigger the release of cannabidiol and silymarin in the microenvironment of bilirubin encephalopathy lesions.

[0016] Preferably, the formulation has a core-shell structure, wherein the core-shell structure has an interpenetrating network physical anchoring layer with a thickness of 10 nm to 30 nm formed in steps S2 and S3 between the polylactic acid-glycolic acid copolymer core and the pH-sensitive hydrogel shell; the cumulative drug release of the formulation is less than 20% in phosphate buffer at pH 7.4 within 48 hours, while the cumulative drug release is greater than 80% in a simulated pathological environment at pH 7.2 to 7.6 within 24 hours.

[0017] Preferably, the particle size change rate of the formulation after centrifugation at 5000 rpm for 10 minutes is less than 5%, and the retention rate of specific antibodies on the pH-sensitive hydrogel shell surface is greater than 90%.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. In the prevention and treatment of bilirubin encephalopathy, the preparation method controls the semi-curing window of the PLGA carrier after solvent removal. It utilizes the diffusion kinetics of residual solvent to induce the hydrophilic pH-sensitive hydrogel molecular chains to permeate and swell the surface of the hydrophobic core. With the rapid removal of the solvent, the infiltrated molecular chains are physically pinned to the shrinking and dense polymer lattice, and a micron-scale molecular interpenetrating network is constructed at the core-shell interface. This physical structure enables the functional shell layer and the drug core to form a high-strength mechanical interlock. The binding stability does not depend on weak van der Waals forces, ensuring that the shell structure remains intact and the target ligand is retained when the formulation is subjected to high-speed fluid shear forces in blood circulation and competitive displacement by plasma proteins. This prevents shell peeling from causing early drug leakage or failure of the targeting function.

[0020] 2. By utilizing the solvent evaporation-induced formation of a dense interfacial layer as a physical barrier for drug diffusion, and combining it with a surface-modified pH-sensitive hydrogel layer, a precise gated release mechanism targeting specific pathological parameters of bilirubin encephalopathy is established. The dense interpenetrating structure of the interface seals the outward diffusion channels of hydrophobic drug molecules, allowing the formulation to maintain drug sequestration in a non-pathological physiological pH body fluid environment, reducing burst release effects. When the formulation is enriched in a specific pH microenvironment of abnormal bilirubin accumulation, the volume phase change or conformational change of the outer hydrogel immediately triggers the opening of the interfacial channels. This dual regulatory mechanism ensures the steady-state distribution of drug molecules during systemic delivery, achieving high-concentration targeted release in the brain lesion area, increasing the drug efficacy at the lesion site while reducing non-specific effects on normal nerve tissue.

[0021] 3. Utilizing the strong hydrophobic properties of the PLGA core matrix, high-density co-loading and spatiotemporally synchronized delivery of cannabidiol and the auxiliary ingredient silymarin are achieved. The microstructure fixes the mass ratio of the two active ingredients within a single nanoparticle, ensuring that they cross the blood-brain barrier synchronously at a predetermined ratio to reach the target nerve tissue. This co-delivery mode avoids the problem of inconsistent distribution and metabolic rates of the two drugs in vivo caused by physical mixing or separate administration. This allows silymarin to exert a membrane-stabilizing effect at the lesion site in a timely manner, assisting cannabidiol in blocking the nerve damage pathway. Through the synchronization of pharmacokinetic behavior, the overall defense and repair efficacy of a unit dose of the drug against bilirubin-induced neurotoxicity is improved. Attached Figure Description

[0022] Figure 1 This is a process flow and control logic diagram of the solvent residue induction and interface physical pinning mechanism of the present invention;

[0023] Figure 2 The figure shows the hydrodynamic radius and light scattering response characteristics of the pH-sensitive hydrogel of the present invention under critical acidic conditions.

[0024] Figure 3 This is a block diagram of the cascaded preparation system that integrates vacuum solvent control and negative pressure crystallization locking functions according to the present invention. Detailed Implementation

[0025] The technical solution 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. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] This invention provides a method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy. The method involves preparing a semi-cured precursor for a substrate carrier, constructing an interpenetrating network, performing curing anchors, and targeting modification steps to build a core-shell structured targeted sustained-release formulation. A molecular weight of [insert molecular weight here] is selected. to The molar ratio of lactic acid to glycolic acid is: to Polylactic acid-glycolic acid copolymer (PLA-GAC) was used as the backbone material. PLA-GAC, cannabidiol, and silymarin were dissolved in dichloromethane to form a homogeneous oil phase. The mass ratio of cannabidiol to silymarin was set at [value missing]. to The total concentration of both in the oil phase was set as follows: to Experimental verification shows that when the proportion of cannabidiol is lower than... At the lower limit, its repair efficacy against nerve damage decreases; while above the lower limit... At the upper limit, the relative content of silymarin is insufficient to synergistically block the transmembrane transport of bilirubin; adding the above oil phase to a polyvinyl alcohol aqueous solution, in to Continuous homogenization and emulsification at high speed minutes to Minutes were required to form an oil-in-water emulsion; the emulsion was then placed in a rotary evaporator and... to Solvent evaporation was carried out under controlled vacuum, and the refractive index change of the system was monitored in real time during this process. When the residual amount of dichloromethane inside the polylactic acid-glycolic acid copolymer particles reached a certain level... to When the semi-solidification critical range is reached, the evaporation operation is stopped, resulting in a carrier suspension with a swollen, semi-rheological state on the surface. This residual range is used to maintain the high-energy swelling state of the particle surface. If it is below... The movement of the chain segment is then hindered, preventing subsequent physical embedding; if it is higher than... The particle structure collapses and cannot maintain its nano-morphology.

[0027] After obtaining the carrier suspension in a swollen, semi-rheological state, a pre-prepared pH-sensitive hydrogel precursor solution was added, containing a concentration of [missing information]. to The carboxymethyl chitosan or sodium alginate, with its pH value adjusted to Place the mixture under magnetic stirring and set the stirring speed to [value missing]. to And at normal pressure, Stirring and incubation in an environment minutes to Minutes; In this step, the dichloromethane remaining inside the polylactic acid-glycolic acid copolymer particles diffuses outward, generating mass transfer motive force, inducing the hydrogel molecular chains in the external pH-sensitive hydrogel precursor solution to diffuse backward and embed into the swollen surface layer of the particles. The above-mentioned rotation speed and time parameters ensure that the penetration depth of the hydrogel molecular chains reaches [a certain value]. to This creates a gradient interpenetrating network between the hydrophobic core and the hydrophilic shell; the incubated system is then connected to a vacuum system, and the vacuum level is adjusted to... to A negative pressure environment is used to remove residual dichloromethane from the particles, and the solvent removal rate is controlled during this process. The following relationship must be satisfied: ,in, The mass removal rate of dichloromethane. This represents the absolute value of the vacuum level in a negative pressure environment. The effective evaporation surface area of ​​the system. The dynamic viscosity of the pH-sensitive hydrogel precursor solution. This is a preset constant related to the crystallinity of polylactic acid-glycolic acid copolymer.

[0028] Based on this relationship, the matching relationship between vacuum degree and system viscosity is adjusted so that the shrinkage and crystallization rate of polylactic acid-glycolic acid copolymer particle segments is synchronized with the dehydration and solidification rate of hydrogel layer. With rapid solvent removal, the polylactic acid-glycolic acid copolymer segments shrink and rearrange crystals, physically locking the hydrogel molecular chains previously embedded in the surface layer to the core surface. After centrifugation and washing, a layer with a thickness of [missing information] is obtained. to The core-shell precursor of the interpenetrating network physical pinning layer, which is in Centrifugal force The particle size change rate after minutes is less than Furthermore, the retention rate of specific antibodies on the surface of the pH-sensitive hydrogel shell is greater than that of... The specific antibody is coupled to the pH-sensitive layer surface of the nucleoshell precursor. The specific antibody is selected from either an anti-neuron-specific enolase antibody or an anti-glial fibrillary acidic protein antibody. This activates the carboxyl groups on the surface of the nucleoshell precursor, causing them to form amide bonds with the amino groups of the specific antibody. After the reaction, unreacted free antibody is removed. The resulting product is then freeze-dried. The freeze-drying step includes... Pre-freezing Hours, at , Sublimation drying under vacuum Hours, an average particle size of was obtained to A dry powder formulation, which is available at pH In phosphate buffer, The cumulative drug release per hour is less than ; in the pH specific to bilirubin encephalopathy lesions to In a simulated pathological environment, a volume phase transition or increased solubility of the pH-sensitive layer triggers drug release. The cumulative drug release per hour is greater than .

[0029] Example 1: This example details a method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy. By controlling the amount of residual solvent, a core-shell nano-formulation with an interfacial interpenetrating network physical nail structure is constructed. A semi-cured swelling interface is established using a lactic acid to glycolic acid molar ratio of [missing information]. Molecular weight is Polylactic acid-glycolic acid copolymer (PLGA) was used as the backbone material, and a weight was taken... Polylactic acid-glycolic acid copolymer, Cannabidiol and Silymarin, dissolving all three together in In dichloromethane, a homogeneous and transparent oil phase is formed, wherein the mass ratio of cannabidiol to silymarin is [missing information]. Slowly inject the above oil phase concentration In an aqueous solution of polyvinyl alcohol, a high-shear homogenizer was used. Continuous homogenization and emulsification at high speed Within minutes, a water-in-oil emulsion with uniform particle size is formed. The emulsion is then transferred to a rotary evaporator, and the water bath temperature is set to [temperature value missing]. And adjust the vacuum level to Solvent evaporation was performed, and the refractive index-solvent residue calibration procedure was followed: standard samples of polylactic acid-glycolic acid copolymer / polyvinyl alcohol emulsion with dichloromethane mass fractions of 4.0 wt%, 6.0 wt%, 8.0 wt%, and 10.0 wt% were prepared in advance, and the refractive index of each component was measured under constant temperature conditions of 30℃. The transformation equation was obtained by fitting the data using the least squares method. (correlation coefficient) The coefficients of this equation are then entered into the online monitoring and control system.

[0030] During this process, the solvent content in the system is monitored in real time using an online refractive index meter. When the residual amount of dichloromethane inside the polylactic acid-glycolic acid copolymer particles decreases to... (in the state) to When the semi-solidification critical range is reached, the rotary evaporation operation is stopped. The product obtained at this point is a carrier suspension with a swollen, semi-rheological state on the surface. The particle skeleton is not yet fully crystallized and densified. An interpenetrating network is then constructed by adding [the following to the carrier suspension in the swollen, semi-rheological state]. A pre-prepared aqueous solution of carboxymethyl chitosan was used as a pH-sensitive hydrogel precursor, and the concentration of this precursor solution was [missing information]. pH value pre-adjusted to Place the mixture on a magnetic stirrer and set the speed to [speed value missing]. ,exist Perform stirring incubation under constant temperature conditions for a duration of [duration missing]. Minutes later, during this stage, the mass transfer force generated by the outward diffusion of residual dichloromethane inside the particles induces the carboxymethyl chitosan molecular chains in the external aqueous phase to diffuse in the opposite direction and physically embed into the relaxed, swollen surface layer of the particles, forming a gradient interpenetrating region. After the solidification of the locking pins and the core-shell forming incubation are completed, the system is connected to a vacuum pump, and the process is carried out according to the process parameters. Inverse calculation of target vacuum degree: Determination of dynamic viscosity of this batch of hydrogel precursors. and the effective evaporation surface area of ​​the system Call the preset crystallinity constant ( Value determination: The solvent removal mass rate per unit time was measured using the same batch of raw materials under a reference vacuum of -0.05 MPa. According to the formula (Originally obtained from calibration), substituted into the relational formula to calculate the matching vacuum degree. The vacuum level was rapidly increased to .

[0031] Under negative pressure, residual dichloromethane within the particles is removed. As the solvent is rapidly removed, the polylactic acid-glycolic acid copolymer segments shrink and rearrange to crystallize, physically locking the previously embedded carboxymethyl chitosan molecular chains within the dense polymer lattice, forming a mechanically interlocked interface. After the solvent is completely removed, the resulting suspension is... centrifuge After minutes, the supernatant was discarded to remove unstented free polymers. The precipitate was resuspended in deionized water and washed three times to obtain a core-shell precursor with an interpenetrating network physically stented layer. Targeted modification and formulation drying were then performed to disperse the core-shell precursor at pH [missing information - likely a pH value]. In the MES buffer, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added to activate the carboxyl groups on the shell surface. Minutes later, add anti-neuron-specific enolase (NSE) antibody. Stirred reaction For hours, the antibody is coupled to the surface of the formulation via amide bonds. After the reaction is complete, unreacted free antibody is removed by dialysis, and finally the product is freeze-dried. Pre-freezing Hours, at , Sublimation drying under vacuum Hours, an average particle size of was obtained Cannabidiol targeted sustained-release dry powder formulation.

[0032] Example 2: This experiment constructed a multi-dimensional in vitro simulation validation platform with a gradient control system. The experiment focused on the mechanical stability and drug sequestration ability of the formulation under simulated physiological conditions, and selected pH... Phosphate buffer simulates the normal body fluid environment and introduces... Fetal bovine serum was used to mimic the competitive adsorption effect of blood proteins, while simultaneously applying... The isothermal oscillation was used to simulate blood flow shear forces. Under these standard simulated physiological conditions, three parallel experimental groups were established: the present invention sample group (the formulation prepared using the complete process of Example 1), the physical adsorption control group (hydrogel modified onto the surface of fully cured polylactic acid-glycolic acid copolymer particles using the traditional physical adsorption method), and the shell-free control group (naked drug-loaded particles without modified hydrogel). High-performance liquid chromatography was used to analyze the drug-loaded particles. The concentration of cannabidiol in the supernatant of each group was measured at regular intervals within hours to calculate the cumulative release rate; and a pH-responsive release gradient verification system was constructed to address the unique pathological microenvironment of bilirubin encephalopathy, with pH values ​​of [missing values]. (Normal Physiology) (Pathological borderline) (Severe acidosis) and A series of buffer media for (extreme environments) were prepared, all containing the same concentration of fetal bovine serum. The sample group of this invention was placed in the above-mentioned media at different pH values. The release rate test is then performed. This step aims to quantify the response threshold and sensitivity of the formulation to small pH changes, and to verify whether it can achieve a precise switching effect within a specific weakly acidic offset range of the lesion, rather than being released indiscriminately under all acidic conditions.

[0033] To further explore the synergistic mechanism between cannabidiol and silymarin, an in vitro bilirubin injury model based on SH-SY5Y human neuroblastoma cells was established. The cells were divided into five groups: a blank control group (no treatment), a model group (treated with only silymarin), and a bilirubin-induced ... The study included bilirubin-induced damage, single-drug group A (bilirubin + free cannabidiol), single-drug group B (bilirubin + free silymarin), and the formulation group of this invention (bilirubin + formulation of Example 1). In each group, the final concentration of cannabidiol remained consistent, while the concentration of silymarin was adjusted according to the formulation ratio. During incubation... Hours later, cell viability was determined using the CCK-8 assay, and intracellular reactive oxygen species levels and mitochondrial membrane potential changes were detected using flow cytometry. This part of the experiment aimed to provide a direct comparison between single drugs and combination drugs, and between free drugs and nano-formulations.

[0034] Table 1: Comparison of stability data of different structural formulations under simulated physiological conditions

[0035]

[0036] Referring to Table 1, under simulated blood circulation physiological conditions, the drug leakage in the sample group of this invention was lower than that in the two control groups, while the physical adsorption control group showed lower leakage. Release rate within hours is as high as This indicates that traditional physical adsorption layers cannot effectively resist the dual effects of serum protein competition and shear forces, leading to shell peeling and premature drug ejaculation. In contrast, the sample group of this invention... The cumulative release rate within hours is still controlled at Within.

[0037] Table 2: Drug release response data of the formulation of the present invention under different pH conditions

[0038]

[0039] Referring to Table 2, the formulation exhibits high sensitivity and selectivity to environmental pH, at various pH levels. to Only within the tiny window A change of one pH unit, Hourly cumulative release rate from surge to It exhibits a switching effect.

[0040] Table 3: Comparison of the protective effects of different drug treatments on bilirubin-damaged nerve cells

[0041]

[0042] Referring to Table 3, the cell survival rate of the formulation group of the present invention was higher than that of any single drug group and approached the level of normal cells. More importantly, the formulation group showed a non-additive synergistic enhancement effect in terms of reactive oxygen species scavenging and mitochondrial protection. The reactive oxygen species level was lower than that of the single-drug A group. and Group B (single drug) ).

[0043] Example 3: This example combines Figures 1 to 3 This document describes a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy, its preparation method, and application instructions. Figure 1 As shown, in the raw material preparation and oil phase construction stage, polylactic acid-glycolic acid copolymer, cannabidiol, and silymarin are dissolved in dichloromethane to form a homogeneous oil phase. After emulsification, an O / W emulsion is formed and enters the controlled solvent evaporation stage. The refractive index is monitored by a real-time status monitoring module to ensure that evaporation stops when the residual solvent reaches the critical range of 5.0wt%-8.0wt%. At the same time, the process parameter calibration module dynamically adjusts the negative pressure vacuum degree according to the viscosity of the hydrogel precursor to ensure synchronous curing rate. The system sequentially goes through the interpenetrating network construction stage, which uses the diffusion dynamics of residual solvent to induce the reverse embedding of hydrogel molecular chains into the surface layer; the negative pressure curing and nailing stage, which removes the remaining solvent, causes the core to shrink and crystallize, and physically nails the embedded chain segments; and finally, after targeted modification and conjugation of specific antibodies to the surface of the core-shell precursor and the finished product formulation stage, the dry powder formulation with a core-shell structure is obtained by freeze drying.

[0044] like Figure 2 As shown, the horizontal axis represents the pH range of 6.0 to 7.4, and the vertical axis represents the relative percentage change. The solid line in the graph represents the change in hydrodynamic radius, and the dashed line represents the change in scattered light intensity. Both curves show that when the pH is between 7.25 and 7.4, the relative rate of change remains at a low level, while when the pH is below 7.2, the relative rate of change shows a steep upward trend and remains high in the pH range of 6.0 to 6.6. Figure 3As shown, the preparation system consists of a series of cascaded functional modules, including a raw material dissolution workstation for preparing a dichloromethane oil phase mixture containing polylactic acid-glycolic acid copolymer, the core drug cannabidiol, and the excipient drug silymarin; a high-shear homogenization workstation that operates at 3000 to 5000 rpm for 5 to 10 minutes in a polyvinyl alcohol aqueous solution environment; and a vacuum solvent control system equipped with refractive index monitoring to maintain the residual solvent of the semi-cured swollen carrier within the critical range of 5% to 8% under a vacuum of -0.04 to 0.06 MPa. The system includes: a reaction vessel for constructing an interfacial network with a penetration depth of 10 to 30 nanometers induced by solvent reverse diffusion at 25 degrees Celsius; a negative pressure crystallization nailing system for removing residual solvent at a vacuum of -0.08 to 0.09 MPa to achieve a physical nailing interpenetrating layer; a biochemical modification reaction unit for coupling specific antibodies through surface carboxyl group activation and amide bond; and a deep freeze-drying workstation for delivering finished products with a particle size of 100 to 200 nanometers after pre-freezing at -80 degrees Celsius and sublimation drying at -50 degrees Celsius and 10 MPa vacuum.

[0045] Example 4: This example constructs a standardized parameter calibration procedure based on the coupling of solvent evaporation kinetics and interfacial rheology. Through quantitative experiments, it establishes the functional relationship between the residual dichloromethane content inside polylactic acid-glycolic acid copolymer particles and the mass of interfacial interpenetrating network formation, thereby scientifically determining the relationship. to During this half-curing window, in the initiation phase of the process, a series of carrier precursors with different solvent residue gradients are prepared. By controlling the rotary evaporation time, the residual dichloromethane content is controlled to achieve specific values. , , , and Five experimental groups were prepared. Each group was given a pH-sensitive hydrogel precursor solution labeled with fluorescein isothiocyanate. The stirring and incubation operations were performed according to the speed and time parameters in Example 1. After incubation, the washed particles were scanned by laser confocal microscopy to quantify the radial penetration depth of the fluorescently labeled hydrogel molecules on the particle surface. At the same time, the particle size stability of the particles during the operation was monitored by dynamic light scattering instrument.

[0046] In-depth analysis of experimental data reveals the decisive influence of solvent residue on the evolution of interfacial structure. When the residue is... At this time, the chain segments are in a glassy, ​​frozen state, and the fluorescence signal only remains on the outermost layer of the particle, resulting in insufficient penetration depth. This indicates that the interface is too dense to form an effective interpenetrating network, and as the residual amount increases to Enhanced interfacial rheology and a leap in fluorescence penetration depth This marks the opening of the molecular chain diffusion channel, in With the residual amount, the penetration depth reached Furthermore, the particle morphology maintains the baseline sphericity, exhibiting optimal swelling-diffusion balance; however, when the residual amount reaches... At that time, although the penetration depth further increased to However, the polydispersity index of the particles began to show an upward trend, indicating that the structural stability was approaching a critical point. Based on the above gradient experiment results, this invention establishes the following judgment logic: an effective interpenetrating network construction simultaneously satisfies a penetration depth greater than [missing information]. Given the constraints of no granular structure collapse, the data shows that when the residual amount of dichloromethane is within the range of... to In the range, the degree of swelling of the surface layer is sufficient to induce the physical embedding of hydrogel molecular chains and to maintain the mechanical support of the particle skeleton.

[0047] Example 5: This example constructs a standardized offline calibration procedure based on the coupling of interfacial rheology and molecular dynamics. Through quantitative experiments, a multidimensional functional relationship is established between the residual dichloromethane content inside polylactic acid-glycolic acid copolymer particles, the viscosity of the hydrogel precursor solution, the interfacial interpenetration depth, and the stability of the particle structure. This scientifically identifies the core process window. During the execution phase of the calibration procedure, a series of carrier precursors with different dichloromethane residual gradients are prepared. By real-time monitoring of the system refractive index during rotary evaporation, samples with solvent residual amounts of [missing values] are selected. , , , and Five experimental groups were prepared, and each group was given a pH-sensitive hydrogel precursor solution labeled with fluorescein isothiocyanate, according to Example 1. to Stirring and incubation were performed within a specified rotation speed range. During this process, tomographic scanning of the washed particles was conducted using a laser confocal microscope to quantify the radial penetration depth of the fluorescently labeled hydrogel molecules on the particle surface. Simultaneously, dynamic light scattering was used to monitor changes in the polydispersity index of the particles during operation to assess structural stability. Data analysis showed that when the residual dichloromethane content was within a certain range... to Within the specified range, the degree of interfacial swelling is sufficient to induce the hydrogel molecular chains to penetrate to a depth that is sufficient to reach the specified depth. to The effective interpenetration threshold is sufficient to prevent the particle skeleton from collapsing.

[0048] In addition, to address potential fluctuations in the physical properties of raw materials from different batches, this invention further establishes a pre-calibration procedure for on-site deployment. Before large-scale production of each batch of formulation, the dynamic viscosity of the pH-sensitive hydrogel precursor solution used is measured. Based on the measured values, according to the formula Dynamically adjust the vacuum level during the negative pressure curing stage. If batch of raw materials leads to If it's too high, then increase it accordingly. The absolute value of the solvent is used to compensate for the mass transfer resistance caused by high viscosity and ensure the solvent removal rate. It is always maintained within a preset range that allows the polymer shrinkage rate to synchronize with the hydrogel curing rate.

[0049] Example 6: To eliminate potential technical problems regarding the setting of the target pH response threshold and the consistency of responses between different batches of hydrogels, this example constructs a standardized engineering procedure based on the fine calibration of the critical phase transition point and batch-to-batch consistency verification. This procedure is applied as a preliminary step before any new batch of hydrogel raw material is put into formulation production to ensure that the pH response characteristics of each batch of product fall within the designed therapeutic window. The procedure establishes a critical phase transition point calibration system based on the abrupt change in light scattering intensity. The pH-sensitive hydrogel precursor from the batch to be tested is taken and formulated into... The standard solution was placed in the sample cell of a dynamic light scattering instrument equipped with precise temperature control and pH titration function, and subjected to constant temperature and pH titration. At physiological temperature, The step size is one unit, and the solution is added dropwise using a micro-titration pump. Hydrochloric acid solution, precisely adjust the pH value of the system from Gradually down to During this process, the intensity of scattered light and the hydrodynamic radius of the solution are monitored in real time. The data acquisition system automatically captures the inflection point where the hydrodynamic radius value increases dramatically, based on a preset algorithm. The pH value corresponding to this inflection point is defined as the actual critical phase transition point of this batch of materials. When the measured critical phase transition point value falls within the pH range... Only when the material is within the narrow window is the batch deemed qualified.

[0050] Furthermore, for qualified raw materials, a release kinetics verification step can be performed under simulated pathological conditions to prepare model nanoparticles loaded with fluorescent probes, which are then placed in pH... With pH In a constant-temperature release medium, the release curve was monitored in real time using a fluorescence spectrophotometer, and the ratio of the release rate constants under two pH conditions was calculated. This procedure sets clear quantitative standards for compliance: the ratio is greater than... That is, the release rate under pathological conditions is at least as high as that under normal conditions. To ensure the integrity of the switching effect, any batch that fails to meet this standard must be corrected by fine-tuning the proportion of ionized groups on the hydrogel macromolecular chain according to the preset feedback adjustment mechanism, until it passes the above dual verification process again.

[0051] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy, characterized in that, The method includes the following steps: Step S1: Establish a semi-cured swelling interface. Dissolve polylactic acid-glycolic acid copolymer, cannabidiol, and silymarin in dichloromethane oil phase, and use polyvinyl alcohol aqueous solution as aqueous phase. Homogenize and emulsify to form an oil-in-water emulsion. Perform controlled solvent evaporation on the emulsion and monitor the system status in real time. Stop evaporation when the residual amount of dichloromethane inside the polylactic acid-glycolic acid copolymer particles reaches the semi-cured critical range of 5.0wt% to 8.0wt%, thereby obtaining a carrier suspension with a surface in a swollen semi-rheological state. Step S2: Construct an interpenetrating network. Add a pH-sensitive hydrogel precursor solution to the carrier suspension in a swollen semi-rheological state. Perform stirring and incubation at 25 degrees Celsius. Utilize the mass transfer force generated by the outward diffusion of residual dichloromethane inside the particles to induce the hydrogel molecular chains in the pH-sensitive hydrogel precursor solution to diffuse backward and embed into the swollen surface of the polylactic acid-glycolic acid copolymer particles. Step S3, solidify the locking pins. Place the system obtained in step S2 in a negative pressure environment with a vacuum degree of -0.08MPa to -0.09MPa to remove the remaining dichloromethane, causing the polylactic acid-glycolic acid copolymer particle chain segments to shrink and crystallize, and physically locking the embedded hydrogel molecular chains onto the core surface. After centrifugation and washing, a core-shell precursor with an interpenetrating network interface is obtained. Step S4, targeted modification, involves conjugating a specific antibody to the pH-sensitive layer surface of the core-shell precursor.

2. The method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy according to claim 1, characterized in that, In step S2, the pH-sensitive hydrogel precursor solution contains carboxymethyl chitosan or sodium alginate at a concentration of 2.0 mg / mL to 5.0 mg / mL; the stirring and incubation operation is controlled at a speed of 100 rpm to 200 rpm and an incubation time of 15 minutes to 30 minutes. The speed and time parameters are used to enable the hydrogel molecular chains to penetrate to a depth of 10 nanometers to 30 nanometers in the swollen surface layer.

3. The method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy according to claim 1, characterized in that, The process of removing the remaining dichloromethane in step S3 follows a specific solvent removal rate, the solvent removal rate is... The following relationship must be satisfied: ,in, This indicates the mass removal rate of dichloromethane. This represents the absolute value of the vacuum level in a negative pressure environment. This represents the effective evaporation surface area of ​​the system. Indicates the dynamic viscosity of the pH-sensitive hydrogel precursor solution. This is a preset constant related to the crystallinity of polylactic acid-glycolic acid copolymer; the relationship is used to synchronize the shrinkage rate of polylactic acid-glycolic acid copolymer with the curing rate of hydrogel molecular chains by controlling the matching relationship between vacuum degree and viscosity.

4. The method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy according to claim 1, characterized in that, In step S1, the mass ratio of cannabidiol to silymarin is 3:1 to 5:1; the total concentration of cannabidiol and silymarin in the dichloromethane oil phase is 20 mg / mL to 50 mg / mL; the molecular weight of the polylactic acid-glycolic acid copolymer is 10,000 Da to 50,000 Da, and the molar ratio of lactic acid to glycolic acid is 50:50 to 75:

25.

5. A method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy according to claim 1, characterized in that, In step S1, the homogenization emulsification speed is 3000 rpm to 5000 rpm and the time is 5 minutes to 10 minutes; the controlled solvent evaporation operation is carried out under a vacuum of -0.04 MPa to -0.06 MPa until the refractive index of the system changes to the preset threshold corresponding to a residual amount of 5.0 wt% to 8.0 wt%.

6. The method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy according to claim 1, characterized in that, In step S4, the specific antibody is either an anti-neuron-specific enolase antibody or an anti-glial fibrillary acidic protein antibody; the coupling is achieved by activating the carboxyl group on the surface of the nucleoshell precursor and forming an amide bond with the amino group of the specific antibody.

7. The method for preparing a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy according to claim 1, characterized in that, The preparation method also includes a freeze-drying step of the product obtained in step S4. The freeze-drying includes pre-freezing at -80 degrees Celsius for 4 hours and sublimation drying at -50 degrees Celsius and 10 Pa vacuum for 24 hours to obtain a dry powder formulation with an average particle size of 100 nanometers to 200 nanometers. The pH value of the pH-sensitive hydrogel precursor solution in step S2 is 7.4, and the hydrogel has the material property of undergoing volume phase change or increasing solubility in the pH range of 7.2 to 7.

6. The material property is used to trigger the release of cannabidiol and silymarin in the microenvironment of bilirubin encephalopathy lesions.

8. The cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy prepared by the method according to claim 1, characterized in that, The formulation has a core-shell structure, with an interpenetrating network physical anchoring layer of 10 to 30 nanometers thickness formed by steps S2 and S3 between the polylactic acid-glycolic acid copolymer core and the pH-sensitive hydrogel shell. The cumulative drug release of the formulation is less than 20% in phosphate buffer at pH 7.4 within 48 hours, while the cumulative drug release is greater than 80% in a simulated pathological environment at pH 7.2 to 7.6 within 24 hours.

9. A cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy according to claim 8, characterized in that, The particle size change rate of the formulation after centrifugation at 5000 rpm for 10 minutes is less than 5%, and the retention rate of specific antibodies on the pH-sensitive hydrogel shell surface is greater than 90%.

10. The application of a cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy, characterized in that, The use of the cannabidiol targeted sustained-release formulation for the prevention and treatment of bilirubin encephalopathy prepared by the preparation method described in claim 1 in the preparation of drugs for the prevention and treatment of bilirubin encephalopathy.

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