Electrode-based liposome adriamycin entrapment rate electrochemical detection method

Abstract

The invention belongs to the field of new materials, and discloses an electrode-based liposome adriamycin entrapment rate electrochemical detection method. According to the detection method, after a bipolar vertical ordered mesoporous silica film (bp-VMSF) is modified on an electrically activated glassy carbon electrode (p-GCE), separation, enrichment and electrochemical detection are integrated, and rapid and sensitive detection of adriamycin can be realized. The glassy carbon electrode is subjected to electrochemical activation pretreatment under the alkaline condition, bp-VMSF stably grows, the surface of the inner layer close to the p-GCE electrode is negatively charged, and the surface of the outer layer away from the electrode is positively charged. The asymmetric charge configuration enhances the adsorption capacity of positively charged adriamycin molecules on an electrode interface through electrostatic enrichment. The p-GCE adsorbs adriamycin and promotes electron transfer, and is combined with the synergistic enrichment effect of the bp-VMSF double-static nano-channel, so that the electrochemical response performance to adriamycin is jointly improved, a method is provided for rapid detection of the liposome adriamycin encapsulation efficiency, and the p-GCE has a good prospect in evaluation of liposome adriamycin preparations.

Description

Technical Field

[0001] This invention belongs to the field of new materials, specifically an electrochemical detection method for the encapsulation efficiency of liposome doxorubicin based on electrodes. Background Technology

[0002] Doxorubicin (DOX), a representative anthracycline chemotherapy drug, plays an important role in clinical treatment due to its broad-spectrum and significant anti-tumor activity against various malignant tumors such as breast cancer, lung cancer, liver cancer, and ovarian cancer. However, while exerting a powerful anti-cancer effect, this drug is often accompanied by severe dose-limiting systemic toxicities such as cardiotoxicity and myelosuppression, greatly limiting its full potential for clinical application. To overcome these limitations, liposome encapsulation technology has emerged as an advanced drug delivery strategy. This technology encapsulates doxorubicin in nanoscale liposome vesicles composed of a phospholipid bilayer, significantly improving the drug's in vivo stability and enabling effective control over drug release behavior. Leveraging its unique nanoscale size and surface modifiability, liposomes can utilize the high permeability and retention effect of solid tumor tissues to achieve passive targeting, thereby enhancing drug accumulation at the tumor site while reducing exposure to normal tissues. Therefore, liposomal doxorubicin significantly reduces systemic toxicity, especially cardiotoxicity, while enhancing anti-tumor efficacy and demonstrating the potential to overcome multidrug resistance in tumors.

[0003] In the research and quality control of liposomal doxorubicin, encapsulation efficiency (EE) is a key evaluation indicator, defined as the percentage of drug successfully encapsulated within the liposomes out of the total administered amount. This parameter not only directly reflects the maturity, stability, and reproducibility of the formulation process but is also an important basis for determining whether liposomes can achieve targeted delivery. A high encapsulation efficiency means that more drug is effectively loaded, ensuring that sufficient drug can be delivered to the tumor site, laying the foundation for efficient treatment. Conversely, unencapsulated free doxorubicin will rapidly distribute throughout the body, causing toxic side effects similar to those of traditional formulations. Therefore, accurate determination of encapsulation efficiency is of great guiding significance for assessing free drug content, predicting pharmacokinetic behavior, and clinical safety. In addition, this indicator is used throughout the entire process of formulation process optimization, production process monitoring, and formulation storage stability evaluation, and is crucial for ensuring the controllability of drug quality. Establishing a rapid, simple, accurate, and reliable method for determining encapsulation efficiency is of great value for promoting the research and practical application of liposomal doxorubicin.

[0004] CN112525971B A method for photoelectrochemical detection of chloramphenicol based on bismuth tungstate: Amine-modified mesoporous silica (PMSN) is used to embed K4Fe(CN)6 or (NH4)4[Fe(CN)6]. The aptamer of the analyte chloramphenicol (CAP) serves as the biogating agent at the PMSN pores. A Bi2WO6 / ITO working electrode is used, and a three-electrode system is employed for photocurrent measurement. Photocurrent values ​​of CAP at different known concentrations are obtained. A linear model is constructed using the differences in known CAP concentrations and corresponding photocurrents. This method indirectly detects the signal molecules released after a competitive reaction, rather than directly detecting them. The working electrode is prepared by incubating on the electrode surface via drop-coating. The adsorption amount of the signal molecules on the electrode surface fluctuates significantly, affecting accuracy. Secondary incubation introduces matrix effects, leading to substantial errors.

[0005] In summary, while the method of constructing a three-electrode system using a working electrode for liposome encapsulation detection is a technology in the field of new materials, it still suffers from problems such as complex processes, low sensitivity, complex separation processes, and low overall analytical efficiency and accuracy. Therefore, it is of great significance to develop an electrochemical detection method that is process-controllable and integrated, highly sensitive, avoids complex separation processes, and enables rapid and accurate analysis of encapsulation efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide an electrode-based electrochemical detection method for the encapsulation efficiency of liposomal doxorubicin. This detection method utilizes a working electrode integrating separation, enrichment, and detection, enabling rapid electrochemical detection of the encapsulation efficiency of liposomal doxorubicin. The working electrode integrating separation, enrichment, and detection is a bipolar VMSF film stably modified on a glassy carbon electrode. To overcome the limitation that vertically ordered mesoporous silica (VMSF) films cannot be directly and stably modified on glassy carbon electrodes, the glassy carbon electrode is electrochemically activated, and a pre-activated glassy carbon electrode (p-GCE) is used as the substrate electrode. This allows for the stable modification of the bipolar VMSF film (bp-VMSF) onto the glassy carbon electrode substrate, forming a bipolar nanochannel array electrode. This achieves integrated separation, enrichment, and detection, improves the detection sensitivity of liposomal doxorubicin encapsulation efficiency, and eliminates matrix effect interference.

[0007] The objective of this invention is achieved through the following solution: An electrode-based electrochemical method for detecting the encapsulation efficiency of doxorubicin in liposomes includes the following steps: S1, pre-activated to generate p-GCE; S2, bipolar silica nanochannel films (bp-VMSF) are grown under constant current in n-VMSF precursor solution and p-VMSF precursor solution to obtain bp-VMSF / p-GCE electrode; S3, prepare a chloroform-methanol mixed solvent to dissolve a mixture of hydrogenated soybean phosphatidylcholine, cholesterol, distearate phosphatidylethanolamine, and polyethylene glycol 2000, dry it, mix it with ammonium sulfate solution, add doxorubicin solution and enrich it at a constant temperature. S4. Prepare a chloroform-methanol mixed solvent, remove unencapsulated DOX using a dialysis membrane, add an equal volume of chloroform-methanol mixed solvent to the liposome suspension, collect the aqueous phase by vortexing and centrifugation, and determine the encapsulation efficiency of liposome doxorubicin in a blank buffer solution using electrochemical detection.

[0008] Preferably, the specific steps for activating and generating p-GCE in step S1 are as follows: GCE with a diameter of 3 mm is polished on a polishing cloth with 0.5 μm, 0.3 μm and 0.05 μm alumina powder. Then, the GCE is ultrasonically cleaned in anhydrous ethanol and ultrapure water for 3 min each, dried with N2, and anolyzed in PBS solution at a constant voltage of +1.8V for 300 s. Cathodic polarization is performed by cyclic voltammetry scans of -1.3 V to 1.25 V three times. The electrode is then removed, rinsed with deionized water and dried with N2 to obtain pre-activated GCE as p-GCE.

[0009] Polishing removes old coatings, impurities, and scratches from the electrode surface, increasing the effective specific surface area. Anodizing provides chemical bonding sites, and the generated -COOH and -OH groups can undergo condensation reactions with Si-OH generated by the hydrolysis of VMSF precursors (such as tetraethyl orthosilicate) in subsequent steps to form Si-OC covalent bonds. Cathodic polarization modifies the electrode surface and stabilizes the electrode.

[0010] Preferably, the specific steps in step S2 are as follows: placing the working electrode p-GCE in a solution of n-VMSF precursor with negatively charged Si-OH on its surface at a current density of -0.74 mA / cm². 2 A constant current growth method was used for 10 seconds, followed by rapid removal and slow washing in ultrapure water to obtain SM@n-VMSF / p-GCE. This SM@n-VMSF / p-GCE was then placed in a solution of p-VMSF precursor with positively charged amino groups at a current density of -0.74 mA / cm². 2 The electrode was grown using a constant current method for 15 seconds, then quickly removed and slowly washed in deionized water to remove residual surfactant. It was then dried with N2 and aged at 80°C for 10 hours to obtain p-GCE modified with bp-VMSF containing micelles (SM). The electrode was then placed in a micelle removal solution and stirred for 5 minutes to remove micelles, resulting in the bp-VMSF / p-GCE electrode.

[0011] When a negative constant current is applied during the growth of the n-VMSF substrate, a reduction reaction occurs at the electrode, causing a sharp increase in the local pH value at the electrode / solution interface. Under strongly alkaline conditions, the hydrolysis and condensation reactions of the VMSF precursor are accelerated, and negatively charged SiO₂... -Crosslinking is deposited on the negatively charged p-GCE surface. The local high pH and electric field force overcome electrostatic repulsion, forcing the precursor to rapidly and orderly assemble into a vertical mesoporous structure on the electrode surface. The -Si-OH on the pre-activated p-GCE surface forms a strong Si-OC covalent bond with the -COOH of GCE. When growing the p-VMSF upper layer, the substrate is a negatively charged n-VMSF and the precursor is a positively charged aminosilane. Electrostatic attraction attracts the positively charged precursor to adsorb onto the negatively charged n-VMSF surface for condensation reaction, effectively enriching DOX⁺ on the electrode surface and improving detection sensitivity. VMSF micelles self-assemble into cylindrical shapes in the silica network, and silica polymerizes around them. After removing the micelles, vertically ordered nanochannels remain. DOX reaches the electrode surface through these nanochannels, achieving electrochemical detection.

[0012] Preferably, step S3 is as follows: a mixed organic solvent is prepared by mixing chloroform and methanol in a 1:1 volume ratio; 2 mL of the mixed solvent is used to dissolve a mixture of hydrogenated soybean phosphatidylcholine, cholesterol, distearate phosphatidylethanolamine, and polyethylene glycol 2000; the mixture is dried under nitrogen and vacuum for 2 h; the mixture is hydrated in ammonium sulfate solution at 60 °C for 20-30 min to form multilayer liposomes; and doxorubicin solution is added to enrich the liposomes in a constant temperature water bath at 60 °C for 10-90 min.

[0013] Hydration forms a lipid film, resulting in multilayered liposomes of relatively uniform size and number of layers. The addition of ammonium sulfate solution establishes high concentrations of ammonium and sulfate ions within the liposomes. When liposomes containing an inner aqueous ammonium sulfate phase are placed in a doxorubicin solution, the system spontaneously tends towards ion concentration equilibrium, forming transmembrane diffusion and a concentration gradient of NH3. The doxorubicin molecule possesses both a hydrophobic anthracene ring structure and a hydrophilic aminoglycoside. Its amino group is protonated at neutral pH, giving the molecule a positive charge (DOX-NH3). + It has good water solubility; under acidic pH conditions, the amino group is fully protonated (DOX-NH3). + Protonated doxorubicin binds to a large number of sulfate ions inside, forming an insoluble gel or precipitate, which can continuously drive the reaction and achieve doxorubicin liposomes with high encapsulation efficiency and high stability.

[0014] Preferably, step S4 specifically involves the following steps: preparing a mixed solvent by mixing chloroform and methanol in a 2:1 volume ratio; removing unencapsulated residual DOX using a dialysis membrane with a molecular weight cutoff of 1000 Da; adding an equal volume of the mixed organic solvent to the liposome suspension; performing vigorous vortexing to separate the phases; collecting the aqueous phase containing DOX by centrifugation; and performing electrochemical detection in a blank buffer solution to determine the encapsulation efficiency of doxorubicin in the liposomes.

[0015] Dissolving the lipid components into the organic phase and discarding them fundamentally prevents the hydrophobic lipids from contaminating the hydrophilic electrode interface, thus ensuring the sensitivity of electrochemical detection.

[0016] Preferably, the scanning medium in step S1 is phosphate-buffered saline (PBS) with a concentration of 0.1-0.5 M and a pH of 8-9.

[0017] An alkaline environment with a pH of 8-9 promotes the formation and stability of oxygen-containing functional groups. During anodic oxidation, an oxygen evolution reaction occurs on the electrode surface. In an alkaline environment, OH-... - It is more easily oxidized than water molecules, the reaction path is more efficient, and the alkaline environment can optimize the cathodic polarization effect; if the buffer concentration is too low, the reaction is slow, and if the concentration is too high, the ions will affect the mass transfer of reactants to the electrode surface.

[0018] Preferably, the n-VMSF precursor solution in step S2 comprises the following components: 20 mL ethanol, 20 mL 0.1 M NaNO3 solution, 1.585 g CTAB, and 2.833 g TEOS; the p-VMSF precursor solution in step S2 comprises the following components: 20 mL ethanol, 20 mL 0.1 M NaNO3 solution, 1.585 g CTAB, 318 μL APTES, and 2372 mL TEOS.

[0019] n-VMSF precursor solution: CTAB is a cationic surfactant. When its concentration is higher than the critical micelle concentration, it self-assembles into cylindrical micelles in solution. Driven by electrochemical deposition, the positively charged micelles are perpendicularly oriented to the negatively charged electrode surface, serving as templates for the formation of vertically ordered mesopores. The TEOS silicon source, through hydrolysis (Si-OC2H5→Si-OH) and condensation (Si-OH+HO-Si→Si-O-Si) reactions, constructs the inorganic framework of VMSF. CTAB micelles are positively charged and exhibit electrostatic repulsion. After the addition of NaNO3, Na... + and NO3 - It compresses the electric double layer of micelles, shielding them from electrostatic repulsion, allowing the micelles to pack more tightly, thus forming smaller, more uniform pores and a more ordered structure.

[0020] p-VMSF precursor solution: APTES ensures that a sufficient amount of amino groups are introduced to achieve charge reversal on the channel surface, changing from negative to positive charge in the n-layer. CTAB, TEOS, and NaNO3 have the same effects as in the n-VMSF precursor solution.

[0021] Preferably, in step S3, the concentration of doxorubicin is 5nM-5μM, and the buffer solution for preparing the solution is PBS with a concentration of 0.01-0.1mol / L and a pH of 4-8.

[0022] The PBS buffer balances drug loading efficiency and liposome stability. The stronger the acidity of the external environment, the higher the proportion of DOX protonated, the lower the proportion of neutral molecules available for transmembrane transport, and the more actively the drug loading rate is slowed down.

[0023] Preferably, in step S3, hydrogenated soybean phosphatidylcholine, cholesterol, distearate phosphatidylethanolamine, and polyethylene glycol 2000 are mixed in a weight ratio of 3:1:1, chloroform and methanol are mixed in a volume ratio of 1:1, and the ammonium sulfate solution concentration is 250 mM.

[0024] Hydrogenated soybean phosphatidylcholine (HSPC) is a saturated phospholipid with a high phase transition temperature. When loaded with drug at 60°C, it is in a liquid crystal state with good membrane fluidity, which is conducive to the transmembrane diffusion of doxorubicin. At physiological temperature (37°C), it is in a gel state with a dense and orderly membrane structure, which can reduce drug leakage in the blood circulation. Cholesterol is embedded in the fatty acid chain of HSPC, which keeps the membrane in a fluid state at any temperature, ensuring sufficient fluidity during drug loading. At the same time, it tightly fills the gaps between the fatty acid chains of phospholipid molecules, reducing passive drug diffusion, reducing membrane permeability, and maintaining the driving force for drug loading. Distearylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000) can form a hydration protective layer on the surface of liposomes, prolonging the circulating half-life of liposomes in the blood and providing long-term circulation drive.

[0025] This invention also provides an application of an electrode-based electrochemical detection method for the encapsulation efficiency of liposomal doxorubicin. The prepared bp-VMSF / p-GCE electrode is an integrated separation-enrichment-detection electrode used for rapid and sensitive electrochemical detection of the encapsulation efficiency of liposomal doxorubicin. By comparing the changes in the content of free doxorubicin in the solution before and after liposomal encapsulation, the encapsulation efficiency of liposomal doxorubicin can be determined.

[0026] The beneficial effects of this invention are as follows: (1) Under alkaline conditions, the glassy carbon electrode is pre-electrochemically activated by cyclic voltammetry to achieve stable modification of a bipolar VMSF thin film (bp-VMSF) on the glassy carbon electrode substrate, forming a bipolar nanochannel array electrode. Compared with the constant potential activation of the glassy carbon electrode in acidic electrolytes, the electroactivity mechanism of this invention is different. In acidic media, electroactivation forms a porous micro / nano structure on the surface of the glassy carbon electrode through oxidation, which greatly increases the actual surface area of ​​the electrode and improves performance by changing the physical morphology of the glassy carbon electrode. However, this process is prone to increasing the electrode background current and reducing reproducibility. The anodic electroactivation of this invention under alkaline conditions mainly generates abundant oxygen-containing functional groups (such as hydroxyl and carboxyl groups) on the electrode surface through electrochemical oxidation, which significantly enhances its hydrophilicity and electron transfer rate, and is suitable for promoting the electrochemical reaction of the positively charged substance doxorubicin. (2) The bilayer structure of bp-VMSF has a unique charge distribution characteristic: its inner surface near the p-GCE electrode is negatively charged, while its outer surface away from the electrode is positively charged. This asymmetric charge configuration significantly enhances the adsorption of positively charged doxorubicin molecules at the electrode interface through electrostatic enrichment. The high-sensitivity interface characteristics of p-GCE itself, which adsorbs doxorubicin and promotes electron transfer, combined with the synergistic enrichment effect of the bp-VMSF dual electrostatic nanochannels, jointly improve the electrochemical response performance of this sensing system to doxorubicin. In addition, the size exclusion effect of bp-VMSF can overcome the contamination of the electrode surface by macromolecules such as proteins in complex matrices, and the negatively charged inner surface can electrostatically repel interference from negatively charged electrochemical substances, including uric acid and ascorbic acid. Therefore, the working electrode of p-GCE modified with bp-VMSF of this invention can integrate separation, enrichment, and electrochemical detection. (3) By comparing the changes in the content of free doxorubicin in the solution before and after liposome encapsulation, the encapsulation efficiency can be accurately and conveniently determined electrochemically. Compared with existing technologies, no complicated sample pretreatment is required, and the encapsulation efficiency of liposome doxorubicin can be determined rapidly and sensitively. (4) The bipolar nanochannel array electrode provided by the present invention is suitable for rapid and sensitive detection of the encapsulation efficiency of liposome doxorubicin in complex matrices such as environmental, biological, food, and clinical samples. Attached Figure Description

[0027] Figure 1 TEM (A) cross-sectional view and (B) top view of .n-VMSF; TEM (C) cross-sectional view and (D) top view of bp-VMSF; different electrodes in [Fe(CN)6] 3- (50 μM, E) and [Ru(NH3)6] 3+ CV test curves in KHP (0.05M, pH=7.4) solution (50 μM, F) at a scan rate of 50 mV / s.

[0028] Figure 2 (A) Electrochemical reaction mechanism of DOX: After enrichment in 100 nM DOX for 40 min, the CV (B) and DPV (C) curves of bp VSMF / p-GCE were measured in PBS (0.1 M, pH=6) at a scan rate of 50 mV / s. (D) CV curves of p-GCE in PBS (0.03 M, pH=6) at different scan rates (v) for DOX@bp-VMSF / p-GCE, where (E) shows the changes in anodic and cathodic peak currents relative to the scan rate.

[0029] Figure 3(A) DPV current response values ​​of DOX@bp-VMSF / p-GCE in 0.1 M PBS containing different pH values; (B) Oxidation peak current as a function of pH; (C) Anode peak potential (Epa) versus pH value. Error bars represent the standard deviation of three measurements. The DPV oxidation peak current of bp-VMSF / p-GCE enriched with different concentrations of DOX (10 nM (D), 1 μM (E)) as a function of mechanical stirring time in PBS (0.03 M, pH=6) solution, with error bars representing the standard deviation of three measurements. (F) DPV current response values ​​of DOX@bp-VMSF / p-GCE in PBS (pH=6) containing different ionic strengths.

[0030] Figure 4 (A) DPV curves of DOX@bp-VMSF / p-GCE obtained in PBS (0.03 M, pH=6) with different concentrations of DOX. (B) Calibration curves at two concentrations. (C) DPV response of DOX@bp-VMSF / p-GCE in PBS (0.03 M, pH=6) containing interfering substances, with a concentration of 1 μM for all DOX (100 nM). (D) Reproducibility obtained with five parallel electrodes.

[0031] Figure 5 (A) After enrichment in different solutions diluted 10,000 times, the DPV signal of bp-VSMF / p-GCE was measured in 0.03M PBS at pH=6. (B) The UV spectrum of the solution diluted 100 times was measured.

[0032] Figure 6 (A) UV-Vis spectra of DOX at different concentrations. Insets show magnified curves; (B) corresponding calibration curves. Detailed Implementation

[0033] All reagents used in this work were analytical grade and used directly. Aqueous solutions were prepared using ultrapure water with a resistivity of 18.2 MΩ·cm.

[0034] Example 1: This embodiment provides a method for preparing a bp-VMSF / p-GCE electrode, specifically including the following steps: S1. Preparation of pre-activated electrode p-GC: GCEs with a diameter of 3 mm were polished on a polishing cloth with 0.5 μm, 0.3 μm, and 0.05 μm alumina powders. The GCEs were then ultrasonically cleaned for 3 min each in anhydrous ethanol and ultrapure water, and dried with N2. Subsequently, the polished and cleaned GCEs underwent electrochemical pretreatment. Specifically, anodization was performed in PBS (0.1 M, pH=8) solution using CV scanning for 10 cycles at a constant voltage of 0-1.0 V. After rinsing with deionized water, the pre-activated GCE was obtained and named p-GCE.

[0035] S2, rapid preparation of bp-VMSF on the surface of a pre-activated electrode using an electrochemical-assisted self-assembly method: Preparation of n-VMSF (negatively charged Si-OH) precursor solution: 1.585 g CTAB and 2.833 g TEOS were added to 20 mL ethanol solution and 20 mL 0.1 M sodium nitrate (NaNO3) solution, and stirred at room temperature for 2.5 hours to obtain the precursor solution.

[0036] Preparation of p-VMSF (positively charged amino groups) precursor solution: 1.585 g of CTAB as a template and 318 μL of APTES were added to a mixed solution containing 20 mL of ethanol and 20 mL of NaNO3 (0.1 M pH = 2.6), and stirred until dissolved. The pH was then adjusted to 2.97 with 6 M HCl, and 2372 mL of TEOS was added as a silicon source. The mixture was then vigorously stirred at room temperature for 2.5 h.

[0037] Preparation of SM@bp-VMSF / p-GCE electrode: In an n-VMSF precursor solution, using a p-GCE electrode as the working electrode, an Ag / AgCl electrode (saturated KCl) as the reference electrode, and a platinum sheet as the counter electrode, a constant current growth method was employed (current density -0.74 mA / cm²). 2 After 10 seconds, the sample was quickly removed and slowly rinsed in ultrapure water to obtain SM@n-VMSF / p-GCE; subsequently, it was placed in a p-VMSF precursor solution and grown using the aforementioned constant current growth method (current density -0.74 mA / cm²). 2 Continue for 15 seconds, then quickly remove and slowly rinse in ultrapure water to obtain SM@bp-VMSF / p-GCE, and gently dry with N2.

[0038] Preparation of bp-VMSF / p-GCE electrode: The SM@bp-VMSF / p-GCE electrode was stirred in 0.1 M HCl-ethanol solution for 5 min to remove SM, resulting in an open-pore bp-VMSF / p-GCE electrode.

[0039] Example 2: This embodiment provides a method for preparing liposomes and DOX liposomes, specifically including the following steps: S3, Preparation of liposomes: A mixed organic solvent was prepared by mixing chloroform and methanol in a 1:1 volume ratio. Hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and distearate phosphatidylethanolamine polyethylene glycol 2000 (DSPE-MPEG2000) were used as raw materials and mixed in a 3:1:1 weight ratio. The mixture was dissolved in 2 mL of the chloroform-methanol (1:1 volume ratio) mixed organic solvent, dried under nitrogen, and then dried under vacuum for 2 hours. The lipid membrane was hydrated with 250 mM ammonium sulfate solution (pH=5.5) and mixed at 60 °C to form multilayer liposomes (MLV). The hydration process lasted 20-30 minutes to ensure sufficient hydration. Preparation of doxorubicin-liposomes: Add doxorubicin solution to the treated liposome solution at a lipid to doxorubicin concentration ratio of 8:1, and incubate in a constant temperature water bath at 60°C for 30 minutes. During this process, mix gently but avoid vigorous shaking.

[0040] Example 3: This embodiment provides the detection of DOX embedding rate, specifically including the following steps: The encapsulation efficiency (EE) was indirectly determined by testing the DOX content in the free solution before and after doxorubicin (DOX) encapsulation using bp-VSMF / p-GCE. The encapsulation efficiency was directly determined by measuring the DOX content encapsulated in liposomes using bp-VSMF / p-GCE. To obtain the DOX encapsulated in liposomes, a dialysis membrane with a molecular weight cutoff of 1000 Da was used to remove unencapsulated residual DOX. A mixed organic solvent was prepared by mixing chloroform and methanol at a 2:1 volume ratio. An equal volume of the mixed organic solvent was added to the liposome suspension, and vigorous vortexing induced phase separation, forming a two-phase system in which the lipids dissolved in the organic phase. After centrifugation, the aqueous phase containing DOX was collected, and the accuracy of bp-VSMF / p-GCE was verified using UV-Vis spectrophotometry as the standard method.

[0041] Experimental Example 1: This experimental example involves transmission electron microscopy and electrochemical probe characterization of the p-GCE, n-VMSF / p-GCE, and bp-VMSF / p-GCE electrodes from Example 1. The test and analysis results are as follows: Figure 1 As shown.

[0042] The morphology of bp-VMSF was characterized using transmission electron microscopy (JEM-2100, JEOL Ltd., Japan). To prepare the TEM sample, the bp-VMSF layer was carefully scraped from the ITO electrode using a knife and dispersed in a minimum amount of ethanol, followed by sonication for approximately 45 min. The resulting dispersion was then dropped onto a copper grid, dried under an infrared lamp, and observed by TEM. The accelerating voltage was set to 200 kV. All electrochemical tests (including cyclic voltammetry (CV) and differential pulse voltammetry (DPV)) were performed on an Autolab (PGSTAT302N) electrochemical workstation (Metrohm, Switzerland). EC measurements used a conventional three-electrode system with a platinum wire or sheet as the counter electrode, Ag / AgCl as the reference electrode, and GCE, p-GCE, n-VMSF / p-GCE, or bp-VMSF / p-GCE as the working electrode. The DPV parameters used are as follows: step potential is 5mV, pulse time is 0.05 s, pulse amplitude is 50 mV, and interval time is 0.2 s.

[0043] Figure 1 The images show TEM top views of n-VMSF (A) and bp-VMSF (C). The ordered hexagonal arrangement of the pore structure is clearly visible, with each bright spot representing a channel structure. The entire film shows no obvious defects, the channel diameters are distributed between 2 and 3 nm, and there is no difference in the surface appearance between the n-VMSF and bp-VMSF films. Figure 1 (B) and (D) are TEM cross-sectional images of n-VMSF and bp-VMSF, respectively, showing the long-range ordered nanochannels of n-VMSF with a thickness of 108 nm; and the bilayer VMSF structure of bp-VMSF with vertically ordered and uniformly distributed pores, wherein the outer p-VMSF layer has a thickness of 75 nm and the inner n-VMSF layer has a thickness of 108 nm.

[0044] Figure 1 The p-GCE, n-VMSF / p-GCE, and bp-VMSF / p-GCE electrodes were respectively applied in 0.5 mM [Fe(CN)6]. 3- (E) and [Ru(NH3)6] 3+ (F) Cyclic voltammetry (CV) curves in solution. The electrochemical support solution was 0.05 M potassium hydrogen phthalate (KHP). The scan rate was 50 mV / s. The standard electrochemical redox probe [Fe(CN)6] was determined using cyclic voltammetry (CV). 3- Or [Ru(NH3)6] 3+ The signals on different electrodes demonstrate the successful preparation of bp-VMSF / p-GCE, and its charge selectivity is investigated. Figure 1EF compared p-GCE, n-VMSF / p-GCE, and bp-VMSF / p-GCE electrodes in [Fe(CN)6]. 3- Or [Ru(NH3)6] 3+ The CV curves in the solution, as shown in Figure EF, indicate that compared to p-GCE, n-VMSF / p-GCE, [Fe(CN)6] 3- The redox peak current of [Ru(NH3)6] decreased significantly. 3+ The redox peak current increases. This is attributed to the presence of a large number of silanol groups (Si-OH) on the n-VMSF surface and in the nanochannels, resulting in a low pKa (between 2 and 3). Therefore, in the test solution, the silanol groups ionize to create a negatively charged surface, repelling anions while attracting cations through electrostatic interactions. When a positively charged amino film is further grown on the electrode, [Fe(CN)6]... 3- The increased peak current indicates that the positively charged outer amino membrane channels exhibit electrostatic attraction to the negatively charged probe. This is especially true when the electrochemical probe used is the positively charged [Ru(NH3)6]. 3+ At that time, on the bp-VMSF / p-GCE electrode, [Ru(NH3)6] 3+ The peak current decreases due to the combined effect of the electrostatic repulsion of the outer p-VMSF layer and the electrostatic attraction of the inner n-VMSF layer. These phenomena demonstrate the charge-selective permeability of bp-VMSF and the successful preparation of bp-VMSF by the continuous current constant-current EASA method.

[0045] Example 2: This example uses doxorubicin as a model to investigate the electrochemical small molecule detection performance of the bp-VMSF / p-GCE electrode in complex samples. The results are as follows: Figure 2 , Figure 3 , Figure 4 As shown.

[0046] Figure 2 (A) shows that the central anthraquinone moiety in DOX undergoes two-electron, two-proton reduction at the electrode surface to form a hydroquinone structure, which is a reversible reaction process. Figure 2Images (B) and (C) show the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) curves of 100 nM DOX on bare GCE, p-GCE, n-VMSF / p-GCE, and bp-VMSF / p-GCE, respectively. All electrodes were immersed in the test solution and stirred until adsorption equilibrium was reached before electrochemical testing. The results show that no obvious redox peaks were observed on bare GCE during the scan range of –0.8 V to –0.3 V, while p-GCE exhibited a pair of weak reversible peaks, attributed to the enhanced electrochemical activity of p-GCE. In contrast, both n-VMSF / p-GCE and bp-VMSF / p-GCE showed obvious reversible redox peaks, a result of the synergistic effect of the highly active p-GCE and the electrostatic enrichment characteristics of n-VMSF (or bp-VMSF). It is worth noting that, thanks to the "electrostatic lock-in" effect of bp-VMSF, the DOX anodic peak current of bp-VMSF / p-GCE is 6.8 times higher than that of n-VMSF / p-GCE. In the bp-VMSF structure, the inner n-VMSF layer is negatively charged, while the outer p-VMSF layer is positively charged, which enables DOX molecules to be strongly and stably confined within the n-VMSF layer.

[0047] Figure 2 (D) and (E) represent the CV test results of bp-VMSF / p-GCE in 50 nM DOX solution at different scan rates (v). The results show that, within the scan rate range of 40 mV / s to 220 mV / s, both the anode and cathode peak currents exhibit a good linear relationship with the scan rate v. The obtained fitting equations are: Anode peak current Ipa (μA) = 0.082 v (mV / s)−1.92 (R 2 = 0.999), Cathode peak current Ipc (μA) =−0.093 V (mV / s) + 2.60 (R 2 = 0.998). This indicates that the electrochemical reaction of DOX in the inner n-VMSF nanochannel is a surface-controlled process.

[0048] To maximize the sensitivity of DOX separation, enrichment, and detection using bp-VMSF / p-GCE, the detection conditions of the integrated separation, enrichment, and sensing strategy, including the pH and ionic strength of the electrolyte solution and the enrichment saturation time for different DOX concentrations, were systematically optimized. First, bp-VMSF / p-GCE was immersed in a 0.1 M PBS solution containing DOX, and mechanical stirring was used to ensure that DOX fully filled the internal nanochannels, thus preparing DOX@bp-VMSF / p-GCE. Subsequently, the modified electrode was measured using differential pulse voltammetry (DPV) in blank PBS solution.

[0049] Figure 3A shows the DPV curves of DOX@bp-VMSF / p-GCE in 0.1 M PBS at different pH values ​​(4~8). On the one hand, the anodic peak current of DOX on DOX@bp-VMSF / p-GCE reaches its maximum at pH 6.0. Figure 3 B), therefore this value was selected as the optimal pH condition for subsequent measurements. On the other hand, the peak potential of DOX shifts negatively with increasing pH ( Figure 3 B) The linear regression equation for the anodic peak potential (Epa) as a function of pH is Epa = −0.057 pH − 0.23, R 2 = 0.992, the slope can be used to determine the ratio of protons to electrons involved in an electrochemical reaction. The number of protons and electrons is calculated using the following equation: Where R is the gas constant (R = 8.314 J mol) −1 K −1 T is the absolute temperature (T = 298 K), and F is the Faraday constant (F = 96485 Cmol). −1 (where m and n are the number of protons and electrons, respectively). Calculations show that m / n = 0.97, which is approximately equal to 1, indicating that the number of protons and electrons participating in the electrochemical reaction are equal.

[0050] The concentration of the supporting electrolyte affects the ionic strength, which in turn alters the thickness of the electrical double layer within the nanochannel. As the PBS concentration decreases, the ionic strength weakens while the double layer thickness increases, causing the channel to gradually exhibit ion-selective permeation characteristics. Therefore, this study used PBS concentrations ranging from 0.01 M to 0.1 M as the supporting electrolyte and systematically investigated the current response signal of DOX@bp-VMSF / p-GCE. Figure 3 As shown in Figure D, the anodic peak current of DOX increases with increasing PBS concentration from 0.01 M to 0.03 M, but decreases in the range of 0.03 M to 0.1 M. This is a result of the combined effect of system conductivity and electrostatic effects. The concentration of the supporting electrolyte affects the ionic strength, which in turn changes the thickness of the electric double layer within the nanochannel. As the PBS concentration decreases, the ionic strength weakens while the double layer thickness increases, causing the channel to gradually exhibit ion-selective permeation characteristics. Therefore, 0.03 M PBS was determined to be the optimal concentration.

[0051] In addition, the pre-enrichment time of DOX was studied by using 10 nM DOX in the low concentration range and 1 μM DOX in the high concentration range. Figure 3(EF) shows the changes in the peak anolyte current of bp-VMSF / p-GCE enriched in PBS (0.03 M, pH = 6) with mechanical stirring for 10 nM (E) and 1 μM (F) DOX. The results indicate that the peak anolyte current gradually increases with increasing stirring time; the current value tends to stabilize when the DOX entering the nanochannels through stirring reaches saturation. The pre-enrichment time for 10 nM DOX was 50 minutes, while 1 μM DOX only required 20 minutes to reach enrichment equilibrium, which is consistent with the rule that the higher the analyte concentration, the shorter the time required to reach enrichment equilibrium.

[0052] like Figure 4 As shown in (AB), the oxidation peak current gradually increases with increasing DOX concentration. Further fitting reveals two linear regression segments: 5–100 nM and 100 nM–5 μM. Within the concentration range of 10–100 nM, the linear regression equation is I(μA) = 391.4 CDOX – 1.84, R0 2 = 0.996; In the concentration range of 100 nM to 5 μM, the linear regression equation is I(μA) = 12.0 CDOX + 36.46, R 2 = 0.997, and the calculated limit of detection (LOD) is 4.4 nM (S / N=3).

[0053] like Figure 4 As shown in Figure C, when detecting DOX in a buffer solution, various potential interfering substances (including metal ions K+) are present. + , anion Cl - The sensor exhibits negligible effects on biomolecules such as glucose, ascorbic acid, uric acid, and dopamine, demonstrating excellent selectivity. To evaluate the sensor's reproducibility, five working electrodes were fabricated in parallel for detecting 100 nM DOX. Figure 4 The results show that the relative standard deviation of the measured anode peak current is 1.9%, confirming that the constructed bp-VMSF / p-GCE sensor has good reproducibility.

[0054] Table 1 shows a performance comparison of different methods for detecting DOX.

[0055] Table 1. Performance comparison of different methods for detecting DOX Note: AgNPs: silver nanoparticles; SWV: mSiO2: mesoporous silica; MWCNTs: multi-walled carbon nanotubes; MB: methylene blue.

[0056] As shown in Table 1, compared with other electrochemical sensors, bp-VMSF, based on an integrated separation-enrichment sensing strategy, exhibits a wider detection range (5 nM ~ 5 μM) and a lower detection limit (4.4 nM). Its performance is superior to silver nanoparticle-chitosan modified glassy carbon electrodes, mesoporous silica-multi-walled carbon nanotube composites, and graphene quantum dot modified electrodes.

[0057] Experimental Example 3: This experimental example was used to determine the encapsulation efficiency of DOX in liposomes. The results are as follows: Figure 5 , Figure 6 As shown.

[0058] To determine the DOX encapsulation rate in liposomes, two methods can be used: direct and indirect. The former directly detects the encapsulated DOX content, but requires pretreatment to release DOX from the liposomes; the latter calculates the encapsulation rate by detecting the change in free DOX content in the solution before and after encapsulation. Because of the electrostatic interaction between DOX and liposome components, some DOX may adsorb onto the liposome surface and not be effectively encapsulated. Therefore, the influence of adsorbed DOX must be considered in the indirect method. This patent uses a bp-VMSF / p-GCE sensor to determine the DOX encapsulation rate in liposomes using both methods.

[0059] like Figure 5 As shown in (A), the free DOX content outside the DOX-loaded liposomes can be detected by bp-VMSF / p-GCE, and its value is significantly lower than that of the total DOX, indicating that DOX has been successfully encapsulated inside the liposomes and causes a change in the free DOX content in the solution. By comparing the detection results in the DOX-liposome mixed solution and the total DOX solution, the amount of DOX adsorbed on the liposome surface can be obtained. The experimentally measured free DOX and adsorbed DOX contents were 0.103 mg / mL and 0.126 mg / mL, respectively. Figure 5 (A) According to the data in Table 1, the DOX encapsulation efficiency measured by the indirect method using the bp-VMSF / p-GCE sensor was 88.6%. In the direct assay mode, the DOX-loaded liposomes and free DOX were first separated by membrane dialysis, and then the encapsulated DOX was released by destroying the liposome structure with an organic solvent. The released DOX was directly detected using bp-VMSF / p-GCE, and the concentration was measured to be 1.75 mg / mL. The encapsulation efficiency calculated by this method was 87.5%, which is similar to the result of the indirect method described above.

[0060] Table 2 shows the results of DOX encapsulation rate detected by dual-mode electrochemical-UV spectrophotometry.

[0061] Table 2. Electrochemical-UV spectrophotometric dual-mode detection of DOX encapsulation rate Ultraviolet-visible spectrophotometry (UV-vis) was used as the standard method to verify the accuracy of the bp-VMSF / p-GCE sensor constructed in this study in detecting the DOX encapsulation efficiency in liposomes. Figure 6 As shown in Figure A, DOX forms a strong absorption band at 470-500 nm. This characteristic absorption originates from the charge transfer transition of the quinone structure in the anthracene ring core, which allows for quantitative analysis of DOX in the concentration range of 0.1-250 μM. We also determined the encapsulation efficiency of DOX in liposomes using the UV-vis method in both direct and indirect modes, and the results are listed in Table 1. The data show that the DOX encapsulation efficiencies measured by the two methods are highly consistent, confirming the good detection accuracy of the bp-VMSF / p-GCE sensor. The bp-VMSF / p-GCE sensor proposed in this invention also has advantages such as simple preparation and operation, rapid response, and flexible detection modes. Notably, this sensor integrates separation, enrichment, and sensing functions into a single electrochemical sensor in indirect detection mode, providing a more convenient analytical method for determining the DOX encapsulation efficiency in liposomes.

[0062] Experimental Example 4: This experimental example is based on the analysis of an actual sample.

[0063] The ability of the constructed electrochemical sensor to detect complex real-world samples was evaluated using the standard addition method. The detection of DOX in human whole blood is shown in Table 3. It can be seen that the sensor exhibits good spiked recoveries (98.7-101%) and low measurement RSDs (<2.1%) for DOX detection, indicating good accuracy.

[0064] Table 3. Electrochemical sensor detection of DOX in human whole blood Note: Human whole blood was diluted 50 times with PBS (0.03 M, pH=6).

[0065] In summary, an electrochemical platform based on bp-VMSF / p-GCE integrates separation, enrichment, and detection functions, enabling indirect quantification of DOX encapsulation efficiency in liposomes. By incorporating a bilayer nanochannel structure with positively and negatively charged VMSF layers, this platform achieves both electrostatic enrichment of DOX and effective exclusion of interfering substances, thus exhibiting advantages such as high sensitivity, wide linear range, and low detection limit. The proposed method significantly reduces sample pretreatment steps, and the results are highly consistent with traditional direct electrochemical and spectrophotometric methods. Given its simplicity, high sensitivity, and reliability, this strategy holds great potential for routine quality assessment of liposome DOX formulations and broader applications in drug development and clinical evaluation.

Claims

1. An electrode-based electrochemical method for detecting the encapsulation efficiency of doxorubicin in liposomes, characterized in that, Includes the following steps: S1, pre-activated to generate p-GCE; S2, bipolar silica nanochannel films (bp-VMSF) are grown under constant current in n-VMSF precursor solution and p-VMSF precursor solution to obtain bp-VMSF / p-GCE electrode; S3, prepare a chloroform-methanol mixed solvent to dissolve a mixture of hydrogenated soybean phosphatidylcholine, cholesterol, distearate phosphatidylethanolamine, and polyethylene glycol 2000, dry it, mix it with ammonium sulfate solution, add doxorubicin solution and enrich it at a constant temperature. S4. Prepare a chloroform-methanol mixed solvent, remove unencapsulated DOX using a dialysis membrane, add an equal volume of chloroform-methanol mixed solvent to the liposome suspension, collect the aqueous phase by vortexing and centrifugation, and determine the encapsulation efficiency of liposome doxorubicin in a blank buffer solution using electrochemical detection.

2. The electrochemical detection method for liposome doxorubicin encapsulation efficiency based on electrodes according to claim 1, characterized in that, The specific steps for activating and generating p-GCE in step S1 are as follows: GCE with a diameter of 3 mm is polished on a polishing cloth with 0.5 μm, 0.3 μm and 0.05 μm alumina powder. Then, the GCE is ultrasonically cleaned in anhydrous ethanol and ultrapure water for 3 min each, dried with N2, and anolyzed in PBS solution at a constant voltage of +1.8V for 300s. Cathodic polarization is performed by cyclic voltammetry scans of -1.3 V to 1.25 V three times. The electrode is then removed, rinsed with deionized water and dried with N2 to obtain pre-activated GCE as p-GCE.

3. The electrochemical detection method for liposome doxorubicin encapsulation efficiency based on electrodes according to claim 1, characterized in that, The specific steps in step S2 are as follows: The working electrode p-GCE is placed in a solution of n-VMSF precursor with negatively charged Si-OH surfaces at a current density of -0.74 mA / cm². 2 A constant current growth method was used for 10 s, followed by rapid removal and slow washing in ultrapure water to obtain SM@n-VMSF / p-GCE. This was then placed in a solution of p-VMSF precursor with positively charged amino groups and grown at a current density of -0.74 mA / cm². 2 The electrode was grown using a constant current method for 15 seconds, then quickly removed and slowly washed in deionized water to remove residual surfactant. It was then dried with N2 and aged at 80°C for 10 hours to obtain p-GCE modified with bp-VMSF containing micelles (SM). The electrode was then placed in a micelle removal solution and stirred for 5 minutes to remove micelles, resulting in the bp-VMSF / p-GCE electrode.

4. The electrochemical detection method for liposome doxorubicin encapsulation efficiency based on electrodes according to claim 1, characterized in that, The specific steps of step S3 are as follows: Prepare a mixed organic solvent by mixing chloroform and methanol in a 1:1 volume ratio, take 2 mL of the mixed solvent to dissolve a mixture of hydrogenated soybean phosphatidylcholine, cholesterol, distearate phosphatidylethanolamine, and polyethylene glycol 2000, N2, and vacuum dry for 2 h, mix with ammonium sulfate solution and hydrate at 60℃ for 20-30 min to form multilayer liposomes, add doxorubicin solution and enrich in a constant temperature water bath at 60℃ for 10-90 min.

5. The electrode-based electrochemical detection method for doxorubicin encapsulation efficiency in liposomes according to claim 1, characterized in that, The specific steps of step S4 are as follows: Prepare a mixed organic solvent by mixing chloroform and methanol in a volume ratio of 2:1; remove unencapsulated residual DOX using a dialysis membrane with a molecular weight cutoff of 1000 Da; add an equal volume of the mixed organic solvent to the liposome suspension; perform phase separation by vigorous vortexing; collect the aqueous phase containing DOX by centrifugation; perform electrochemical detection in a blank buffer solution; and determine the encapsulation efficiency of doxorubicin in the liposomes.

6. The electrode-based electrochemical detection method for doxorubicin encapsulation efficiency in liposomes according to claim 1 or 2, characterized in that, In step S1, the scanning medium is phosphate-buffered saline (PBS) with a concentration of 0.1-0.5 M and a pH of 8-9.

7. The electrode-based electrochemical detection method for doxorubicin encapsulation efficiency in liposomes according to claim 1 or 3, characterized in that, The n-VMSF precursor solution in step S2 contains the following components: 20 mL ethanol, 20 mL 0.1 M NaNO3 solution, 1.585 g CTAB, and 2.833 g TEOS; the p-VMSF precursor solution in step S2 contains the following components: 20 mL ethanol, 20 mL 0.1 M NaNO3 solution, 1.585 g CTAB, 318 μL APTES, and 2372 mL TEOS.

8. The electrode-based electrochemical detection method for doxorubicin encapsulation efficiency in liposomes according to claim 1 or 4, characterized in that, In step S3, the concentration of doxorubicin is 5 nM-5 μM, and the buffer solution is PBS with a concentration of 0.01-0.1 mol / L and a pH of 4-8.

9. The electrode-based electrochemical detection method for doxorubicin encapsulation efficiency in liposomes according to claim 1 or 4, characterized in that, In step S3, hydrogenated soybean phosphatidylcholine, cholesterol, distearate phosphatidylethanolamine, and polyethylene glycol 2000 are mixed in a weight ratio of 3:1:1, chloroform and methanol are mixed in a volume ratio of 1:1, and the ammonium sulfate solution concentration is 250 mM.

10. The application of the electrode-based electrochemical detection method for doxorubicin encapsulation efficiency in liposomes according to any one of claims 1-9, characterized in that... The prepared bp-VMSF / p-GCE electrode is an integrated separation-enrichment-detection electrode used for rapid and sensitive electrochemical detection of liposomal doxorubicin encapsulation efficiency. By comparing the changes in the content of free doxorubicin in the solution before and after liposomal encapsulation, the encapsulation efficiency of liposomal doxorubicin can be determined.

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