Microfluidic chip, drug-loaded microsphere, drug-loaded microneedle and drug-loaded microneedle patch

Abstract

The invention discloses a micro-fluidic chip, a drug-loaded microsphere, a drug-loaded microneedle and a drug-loaded microneedle patch, belongs to the technical field of drug delivery systems, particularly relates to the field of drug-loaded microneedle patches based on a micro-fluidic technology, and is particularly used for realizing collaborative treatment of acupoint stimulation and drug sustained release. The problems that in the prior art, an existing medicine carrying microsphere preparation method is uneven in particle size, high in medicine burst release rate and low in encapsulation efficiency, traditional acupuncture treatment operation dependence is high, and acupuncture point stimulation and medicine slow release cannot be coordinated are solved. The drug-loaded microneedle patch comprises a medical stainless steel main needle group, a hydrogel drug-loaded auxiliary needle group and an adhesive tape. The micro-fluidic chip, the drug-loaded microsphere, the drug-loaded microneedle and the drug-loaded microneedle patch are suitable for realizing synergistic interaction of drug sustained release and acupoint stimulation.

Description

Technical Field

[0001] This invention relates to the field of drug delivery system technology, and more particularly to the field of drug-loaded microneedle patches based on microfluidic technology, specifically for achieving synergistic treatment of acupoint stimulation and sustained drug release. Background Art

[0002] Depression is a mental illness with a high relapse rate and a high disability rate. Current treatments for depression mainly include medication and physical therapy.

[0003] in: Drug therapy primarily utilizes drug-loaded microspheres. However, existing methods for preparing drug-loaded microspheres suffer from problems such as uneven particle size, high drug burst release rates, and low encapsulation efficiency, leading to significant side effects and easy drug resistance in current drug therapies.

[0004] Physical therapy mainly includes traditional acupuncture (using traditional press needles). Traditional acupuncture has a high effectiveness rate in treating depression (53.3%~85.1%), with a low recurrence rate and few adverse reactions. However, it has the problem of high operator dependence: the efficacy is limited by the operator's experience and individual differences. Professionals are needed to accurately locate acupoints, making it unsuitable for ordinary patients to perform on their own, and thus difficult to popularize.

[0005] In addition, there is a lack of synergy between existing drug therapy and physical therapy techniques. Traditional press needles only contain a single metal needle and can only physically stimulate acupoints, while traditional drug-loaded microspheres can only provide sustained drug release. In short, existing technologies cannot achieve synergy between acupoint stimulation and sustained drug release, which limits further improvement in treatment efficacy. Summary of the Invention

[0006] This invention proposes microfluidic chips, drug-loaded microspheres, drug-loaded microneedles, and drug-loaded microneedle patches, which solve the problems of uneven particle size, high drug burst release rate, and low encapsulation rate in existing drug-loaded microsphere preparation methods, as well as the high operational dependence of traditional acupuncture treatment and the inability of acupoint stimulation and drug sustained release to work synergistically.

[0007] The microfluidic chip of the present invention includes a main channel, a first channel, and a second channel; Main pipeline: Its diameter gradually increases from upstream to downstream, and it is used to inject the internal water phase; First pipe: intersects with the main pipe at an angle, used to inject the oil phase to cut the inner aqueous phase and form the W / O emulsion core; The second pipe is located downstream of the first pipe and intersects with the main pipe at a cross shape. Its diameter differs from that of the first pipe by ≤5%. It is used to inject the external aqueous phase to encapsulate the W / O emulsion core and form W / O / W microspheres.

[0008] This invention also proposes a drug-loaded microsphere preparation system, the system comprising: a microfluidic pressure pump, a piezoelectric microdroplet generator, and a microfluidic chip; the microfluidic chip is the microfluidic chip described above. The piezoelectric droplet generator is integrated into the main channel inlet of the microfluidic chip and is used to cut the internal aqueous phase into monodisperse droplets; A microfluidic pressure pump is used to inject the internal aqueous phase, oil phase, and external aqueous phase into the microfluidic chip at a given flow rate ratio: Main pipeline: Injects internal water phase; First conduit: Injecting oil phase to cut the internal aqueous phase droplets; Second channel: Inject external aqueous phase to encapsulate oil phase, forming W / O / W microspheres.

[0009] This invention also proposes a method for preparing drug-loaded microspheres, the method comprising the following steps: Drug extraction steps: The effective components of Bupleurum chinense and Paeonia lactiflora were extracted using the ethanol reflux method to obtain the extract. Preparation steps for PLGA solution: Under constant temperature of 40℃, 180 mg of amino-terminated PLGA was added to a mixed solvent of benzyl benzoate and benzyl alcohol, and the mixture was stirred magnetically until completely dissolved. The resulting PLGA solution was used as the oil phase. Preparation steps of drug solution: The extract was prepared into liposomes, and the lipophilic components were encapsulated in the liposomes and then used as the inner aqueous phase to improve the encapsulation efficiency and achieve dual controlled release. 5% trehalose stabilizer was added to the inner aqueous phase to protect the activity of the drug during emulsification, solidification and freeze-drying processes and reduce burst release. Steps for forming a water-in-oil-in-water double emulsion: Polyvinyl alcohol solution was used as the external aqueous phase; Using the drug-loaded microsphere preparation system described above, W / O / W microspheres are prepared based on an internal aqueous phase, an oil phase, and an external aqueous phase. Microsphere solidification and purification steps: Photocuring of W / O / W microspheres in the presence of a photoinitiator enhances the mechanical strength and stability of the microspheres and reduces burst release. The photocured microspheres are then irradiated with 365 nm blue light for 120 seconds to achieve cross-linking and curing. The cross-linked and cured microspheres were washed and purified to remove residual solvents and surfactants, and purified microspheres were obtained. Microsphere post-processing steps: The purified microspheres were mixed with deionized water at a mass ratio of 2:1 and incubated in a constant temperature shaker for 1 hour. The incubated microspheres were treated with 50 kHz ultrasound for 5 minutes to promote uniform dispersion of the microspheres and obtain an ultrasound-treated suspension. Mannitol was added to the sonicated suspension as a freeze-drying protectant to improve resolubility and reduce microsphere aggregation and structural collapse during freeze-drying. The suspension containing the lyophilization protectant was transferred to a lyophilizer and freeze-dried at -60°C for 72 hours to obtain PLGA sustained-release microsphere powder as drug-loaded microspheres.

[0010] The present invention also proposes drug-loaded microspheres, which are prepared by the drug-loaded microsphere preparation method described above.

[0011] This invention also proposes a method for preparing drug-loaded microneedles, the method comprising the following steps: Vacuum casting loading steps: The drug-loaded microspheres described above are mixed with a polyvinyl alcohol-sucrose matrix solution at a mass ratio of 1:9 to form a drug-loaded suspension. The drug-loaded suspension was injected into the cavity of the microneedle mold, and a vacuum was applied for 20 minutes under a vacuum of -0.1 MPa to allow the drug-loaded microspheres to fully penetrate into the gap at the tip of the microneedle mold. Centrifugal filling and shaping steps: The vacuum-cast microneedle mold was centrifuged at 4200 rpm for 5 minutes. This process was repeated 3 times to remove excess matrix solution, so that the drug-loaded microspheres were evenly distributed at the tip of the microneedle mold to form drug-loaded microneedles.

[0012] This invention also proposes drug-loaded microneedles, which are prepared using the drug-loaded microneedle preparation method described above.

[0013] The present invention also proposes a drug-loaded microneedle patch, which includes: a medical stainless steel main needle group, a hydrogel drug-loaded auxiliary needle group, and adhesive tape. The adhesive tape is thicker in the middle and thinner around the edges; the medical stainless steel main needle group and the hydrogel drug-loaded auxiliary needle group are fixed in the middle area of ​​the adhesive tape. The medical stainless steel main needle group includes 4 medical stainless steel main needles; the 4 medical stainless steel main needles are symmetrically distributed around the center of the adhesive tape; The hydrogel drug-loaded needle group is made of hydrogel material; the hydrogel drug-loaded needle group includes multiple drug-loaded needles, and the drug-loaded needles are the drug-loaded microneedles described above. The hydrogel drug-loaded auxiliary needle group is divided into 4 regions, each region is arranged in an octagonal array; the 4 regions are distributed around the periphery of the medical stainless steel main needle group, forming the shape of 4 petals of a lilac flower; the needle height of the medical stainless steel main needle group is greater than the needle height of the hydrogel drug-loaded auxiliary needle group.

[0014] Furthermore, a preferred embodiment is provided, wherein the diameter of the medical stainless steel main needle is 0.16mm-0.30mm, its needle body length is 0.8mm-3mm, and its needle body is made of austenitic stainless steel wire; The drug-loaded accessory needle is conical in shape, with a needle length of 0.8 mm and a bottom diameter of 0.3 mm.

[0015] Furthermore, a preferred embodiment is provided in which the needle tip spacing between the drug-loaded needles in each region of the hydrogel drug-loaded needle group is 0.65 mm, the number of drug-loaded needles is 10 × 10, and the distribution area of ​​the drug-loaded needles is 6.5 mm × 6.5 mm.

[0016] Furthermore, a preferred embodiment is provided, wherein the adhesive tape includes a waterproof layer, a limiting and fixing layer, and an adhesive; The waterproof layer is a PU film, located on the outermost layer; The limiting layer is made of non-woven fabric and is located below the waterproof layer; The portion of the adhesive tape that comes into contact with the skin is an adhesive; the adhesive is polysilicon.

[0017] The present invention has the following beneficial effects: 1. The microfluidic chip of the present invention, through the integrated 3D printed gradient diameter main channel and cross channel design, combined with the piezoelectric microdroplet generator to actively cut the internal aqueous phase, can realize the preparation of monodisperse W / O / W microspheres, significantly improving the particle size uniformity.

[0018] 2. The drug-loaded microspheres of the present invention, through a liposome-PLGA microsphere dual encapsulation structure, enable lipophilic drugs to be rapidly released via liposomes and then sustained-released via PLGA degradation, thereby reducing the burst release rate and improving the encapsulation efficiency.

[0019] 3. The drug-loaded microneedle patch of the present invention, through the clove-shaped layout of the medical stainless steel main needle group and the hydrogel drug-loaded auxiliary needle group, utilizes the design that the height of the main needle is greater than that of the auxiliary needle and the enrichment of drug-loaded microspheres at the tips of the auxiliary needles to simultaneously achieve precise acupoint stimulation (main needle) and sustained release of drugs in the dermis (auxiliary needle).

[0020] The microfluidic chip, drug-loaded microspheres, drug-loaded microneedles, and drug-loaded microneedle patches described in this invention are suitable for transdermal acupoint sustained-release therapy and acupoint stimulation therapy of active ingredients of traditional Chinese medicine (such as Bupleurum-Paeonia lactiflora extract), especially for long-term intervention of chronic diseases (such as depression) at Neiguan acupoint. The combination of microneedle puncture and main needle achieves synergistic effect of drug sustained release and acupoint stimulation. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the structure of a microfluidic chip in one embodiment of the present invention; Figure 2 This is a schematic diagram of the distribution structure of the stainless steel main needle group and the hydrogel drug-loaded auxiliary needle group in a drug-loaded microneedle patch according to one embodiment of the present invention. Figure 3 This is a side view of the structure of a drug-loaded microneedle patch in one embodiment of the present invention; Figure 4 This is a schematic diagram of a drug-loaded microneedle patch according to one embodiment of the present invention; Figure 5 This is a schematic diagram of a conventional snap needle (excluding adhesive tape) in one embodiment of the present invention. Figure 6 This is a schematic diagram of a conventional snap needle (including adhesive tape) in one embodiment of the present invention.

[0023] Figures 7 to 9 In one embodiment of the present invention, a high-performance liquid chromatogram is provided; wherein, Figure 7 This is a high-performance liquid chromatogram of a pharmaceutical standard. Figure 8 The high-performance liquid chromatogram of the blank microspheres is shown. Figure 9 This is a high-performance liquid chromatogram of drug-loaded microspheres; Figure 10 This is a schematic diagram of rat acupoint location in one embodiment of the present invention; Figure reference numerals: a, main channel; b, first channel; c, second channel; d, drug-loaded microspheres; 1, medical stainless steel main needle group; 2, hydrogel drug-loaded auxiliary needle group; 3, limiting and fixing layer. Detailed Implementation

[0024] To make the technical solutions and advantages of the present invention clearer, the specific embodiments of the present invention will be described in further detail and completely below with reference to the accompanying drawings. The various embodiments described below are only some preferred embodiments of the present invention, and not all of them; the various embodiments described below are intended to explain the present invention and should not be construed as limiting the present invention; reasonable combinations of the technical features defined in the various embodiments of the present invention, as well as all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort, are all within the scope of protection of the present invention.

[0025] Implementation method 1: Microfluidic chip, the microfluidic chip includes a main channel, a first channel and a second channel; Main pipeline: Its diameter gradually increases from upstream to downstream, and it is used to inject the internal water phase; First pipe: intersects with the main pipe at an angle, used to inject the oil phase to cut the inner aqueous phase and form the W / O emulsion core; The second pipe is located downstream of the first pipe and intersects with the main pipe at a cross shape. Its diameter differs from that of the first pipe by ≤5%. It is used to inject the external aqueous phase to encapsulate the W / O emulsion core and form W / O / W microspheres.

[0026] In this embodiment, the microfluidic chip is designed using PR drawing software and fabricated using integrated 3D printing technology.

[0027] In this embodiment, the microfluidic chip further includes a substrate: It measures 4cm x 5cm, is 12mm thick, and is made of PDMS. The substrate is used to encapsulate microchannels (i.e., a network of microfluidic channels fabricated on the substrate, including a main channel, a first channel, and a second channel).

[0028] The substrate serves as a three-dimensional carrier, and the microchannel structure is encapsulated inside it using integrated 3D printing technology to form a complete flow path system.

[0029] The core functions of the substrate: (1) Physical support and packaging: Carrying channels: The designed cross (intersecting) channel structure is materialized inside the PDMS substrate through photolithography or 3D printing process; Leak-proof sealing: The elastic PDMS material automatically seals the flow channels after bonding, preventing fluid leakage.

[0030] (2) Fluid control assistance: Wettability regulation: The hydrophobicity of PDMS surface promotes oil / water phase separation and supports laminar flow at low Reynolds number; Optical transparency: Facilitates observation of the droplet formation process (such as the oil phase cutting through the internal aqueous phase).

[0031] (3) System Integration Fundamentals: The substrate serves as a mechanical interface, supporting the docking of the microfluidic chip with external devices (such as piezoelectric droplet generators and pressure pumps).

[0032] In this embodiment, the diameters of the first pipe and the second pipe are equal or similar (difference ≤ 5%).

[0033] In this embodiment, under laminar flow conditions with low Reynolds number (e.g., Re < 100), the interface between the inner aqueous phase and the oil phase remains stable within the cross-shaped channel; the shear force generated by the vertical injection of the oil phase cuts the inner aqueous phase into monodisperse water-in-oil (W / O) emulsion cores.

[0034] In this embodiment, the microfluidic chip is used to integrate with a piezoelectric droplet generator to cut the internal aqueous phase into monodisperse droplets through piezoelectric vibration; at the same time, the diameter of the main channel gradually increases from upstream to downstream to suppress droplet aggregation, and finally obtains uniform microspheres with a small coefficient of variation (CV) (e.g., <5%).

[0035] Implementation Method 2: A drug-loaded microsphere preparation system, the system comprising: a microfluidic pressure pump, a piezoelectric microdroplet generator, and a microfluidic chip; the microfluidic chip is the microfluidic chip described above; The piezoelectric droplet generator is integrated into the main channel inlet of the microfluidic chip and is used to cut the internal aqueous phase into monodisperse droplets; The microfluidic pressure pump is used to inject internal aqueous phase, oil phase, and external aqueous phase into the microfluidic chip at a given flow ratio: Main pipeline: Injects internal water phase; First conduit: Injecting oil phase to cut the internal aqueous phase droplets; Second channel: Inject external aqueous phase to encapsulate oil phase, forming W / O / W microspheres.

[0036] In this embodiment, the piezoelectric microdroplet generator has a built-in piezoelectric ceramic and is connected to the excitation power supply signal; the excitation power supply sends a pulse voltage, and the piezoelectric ceramic generates high-frequency vibration (>1 kHz) under the pulse voltage, cutting the continuous internal water phase flow into uniform droplets.

[0037] In this embodiment, W / O / W: water-in-oil-in-water.

[0038] W / O / W microspheres: water-in-oil-in-water (drug) microspheres, water-in-oil-in-water double emulsion microspheres.

[0039] In this embodiment, the drug-loaded microsphere preparation system: Piezoelectric microdroplet generator: Enables on-demand generation of monodisperse droplets, significantly improving particle size uniformity and controllability.

[0040] Microfluidic chip cross (intersecting) channel: orderly three-phase assembly to ensure the integrity of W / O / W structure.

[0041] Precise flow control of microfluidic pressure pumps: Optimized oil / water ratio for high encapsulation efficiency.

[0042] Particle size controllability: The active cutting of the piezoelectric module (piezoelectric microdroplet generator) significantly reduces the standard deviation of microsphere size compared to traditional methods.

[0043] Drug release stability: Uniform particle size and double encapsulation structure significantly reduce burst release effect.

[0044] Implementation Method 3: A method for preparing drug-loaded microspheres, the method comprising the following steps: Drug extraction steps: The effective components of Bupleurum chinense and Paeonia lactiflora were extracted using the ethanol reflux method to obtain the extract. Preparation steps for PLGA solution: Under constant temperature of 40℃, 180 mg of amino-terminated PLGA was added to a mixed solvent of benzyl benzoate and benzyl alcohol, and the mixture was stirred magnetically until completely dissolved. The resulting PLGA solution was used as the oil phase. Preparation steps of drug solution: The extract was prepared into liposomes, and the lipophilic components were encapsulated in the liposomes and then used as the inner aqueous phase to improve the encapsulation efficiency and achieve dual controlled release. 5% trehalose stabilizer was added to the inner aqueous phase to protect the activity of the drug during emulsification, solidification and freeze-drying processes and reduce burst release. Steps for forming a water-in-oil-in-water double emulsion: Polyvinyl alcohol solution was used as the external aqueous phase; Using the drug-loaded microsphere preparation system described above, W / O / W microspheres are prepared based on an internal aqueous phase, an oil phase, and an external aqueous phase. Microsphere solidification and purification steps: Photocuring of W / O / W microspheres in the presence of a photoinitiator enhances the mechanical strength and stability of the microspheres and reduces burst release. The photocured microspheres are then irradiated with 365 nm blue light for 120 seconds to achieve cross-linking and curing. The cross-linked and cured microspheres were washed and purified to remove residual solvents and surfactants, and purified microspheres were obtained. Microsphere post-processing steps: The purified microspheres were mixed with deionized water at a mass ratio of 2:1 and incubated in a constant temperature shaker for 1 hour. The incubated microspheres were treated with 50 kHz ultrasound for 5 minutes to promote uniform dispersion of the microspheres and obtain an ultrasound-treated suspension. Mannitol was added to the sonicated suspension as a freeze-drying protectant to improve resolubility and reduce microsphere aggregation and structural collapse during freeze-drying. The suspension containing the lyophilization protectant was transferred to a lyophilizer and freeze-dried at -60°C for 72 hours to obtain PLGA sustained-release microsphere powder as drug-loaded microspheres.

[0045] In this embodiment, the ethanol reflux method is also known as the alcohol extraction method, and the effective components of Bupleurum-White Peony are the active components in the Bupleurum-White Peony raw materials.

[0046] In this embodiment, the steps of the ethanol reflux method are as follows: Raw material processing: Bupleurum and white peony coarse powder (1:1, total mass 9g) were heated and refluxed three times (2h / time) with ethanol (material-liquid ratio 1:10). Concentration process: Combine the extracts and concentrate them to a semi-fluid state (approximately 5 mL volume) using a rotary evaporator (55℃ water bath, 60 Pa, 30 r / min) to obtain the extract.

[0047] In this embodiment, during the preparation step of the PLGA solution: 180 mg amino-terminated PLGA:PLA-PGA, monomer ratio 25:75, molecular weight range 5k-30k; Benzyl benzoate and benzyl alcohol mixed solvent: 1 mL, ratio 1:1; Magnetic stirring: 300 rpm; Oil phase (PLGA solution): PLGA concentration is 10%-20% (w / v).

[0048] In this embodiment, the drug solution preparation step utilizes a dual controlled release system (dual drug delivery system) consisting of liposomes and PLGA microspheres: (1) Core components:

[0049] (2) Dual controlled release mechanism: Liposome (liposome layer) release: The phospholipid bilayer breaks down to achieve initial rapid release, i.e., the rapid release phase, during which the lipophilic components diffuse into the cell; PLGA (PLGA microsphere layer) degradation: The polymer is slowly hydrolyzed to achieve long-lasting sustained release, i.e., the sustained release stage, which controls the release rate of liposomes; Liposomes + PLGA microspheres: synergistically maintain blood drug concentration.

[0050] (3) Special design of the internal water phase: Key preparation steps: "Preparing extracts into liposomes, encapsulating lipophilic components within the liposomes, and then using this as the internal aqueous phase"—this process breaks through the traditional drug delivery model. Traditional method: Lipophilic components are directly dispersed in the oil phase of PLGA, resulting in low encapsulation efficiency and high burst release rate; This technology: Liposomes: Lipophilic components are encapsulated into nanoliposomes using phospholipids (such as lecithin); Internal aqueous phase reconstruction: The liposome suspension is injected into the main channel as the microfluidic internal aqueous phase (instead of being directly dissolved in the organic phase).

[0051] Dual structure formation process: Liposome suspension → internal aqueous phase; PLGA oil phase → encapsulates liposomes → forms W / O emulsion; PVA external aqueous phase → encapsulates W / O emulsion → forms W / O / W microspheres.

[0052] The final result was a triple structure consisting of liposomes (core), PLGA (intermediate layer), and PVA (shell).

[0053] (4) Technological advantages: Dual controlled release synergistic mechanism: Improve encapsulation rate: The liposome barrier prevents lipophilic components from leaking into the oil phase during emulsification.

[0054] Reduce burst release effect: Liposomes and PLGA microspheres form a dual carrier: The PLGA layer prevents the drug from directly contacting the external environment; the liposomes must first overcome the PLGA barrier to release the drug; together they reduce the burst release rate.

[0055] Co-regulation release kinetics: Immediate-release phase: After microneedle puncture, the liposomes rapidly release the drug, resulting in a rapid onset of action (relieving acute symptoms). Sustained-release phase: PLGA controls the sustained release of liposomes, maintaining long-term blood drug concentration (reducing relapse rate).

[0056] In this embodiment, polyvinyl alcohol (PVA) is used.

[0057] In this embodiment, a drug-loaded microsphere preparation system is used to prepare W / O / W microspheres based on an internal aqueous phase, an oil phase, and an external aqueous phase, including the following steps: Three-phase fluid drive and flow control steps: The microfluidic pressure pump delivers the (drug) liquid phase to the microfluidic chip according to the following flow parameters: Main pipeline: Injection of an internal aqueous phase solution containing drug liposomes, with a flow rate range of 5–10 μL / min; First pipeline: Inject oil phase solution (a mixed solvent of benzyl benzoate / benzyl alcohol containing PLGA, or simply PLGA solution), with a flow rate range of 3–5 μL / min; Second pipeline: Inject external aqueous phase solution (polyvinyl alcohol, PVA) at a flow rate of 3–5 μL / min; Steps for piezoelectric on-demand droplet generation: When the internal aqueous phase flows through the piezoelectric microdroplet generator integrated into the main pipeline inlet, monodisperse droplet control is achieved through the following mechanism: Active cutting: Piezoelectric ceramics generate high-frequency vibrations (>1 kHz) under pulsed voltage, cutting the continuous internal water phase flow into uniform droplets (diameter deviation <±0.5μm). On-demand start / stop: Instantly starts and stops droplet generation in response to electronic control signals to avoid wasting ineffective fluid; Microfluidic channel emulsion assembly steps: After a monodisperse aqueous droplet enters the cross channel (i.e., the channel formed by the intersection of the first and second pipes with the main pipe), it sequentially undergoes the following: Oil phase cutting: The oil phase in the first channel encapsulates the water phase droplets inside, forming a water-in-oil (W / O) primary emulsion; External aqueous phase encapsulation: The external aqueous phase (PVA) of the second conduit (c) further encapsulates the W / O droplets, forming water-in-oil-in-water (W / O / W) double emulsion microspheres.

[0058] The generated W / O / W emulsion is exported through the downstream channel (of the main pipeline) and its structure is fixed by photocuring (irradiation with 365nm blue light for 120 seconds), ultimately obtaining drug-loaded (sustained-release) microspheres with a particle size of 10–20μm and an encapsulation efficiency of "≥77.8%".

[0059] In this embodiment, the cross-linked and cured microspheres are washed and purified to remove residual solvents and surfactants: The cross-linked and cured microspheres were transferred to centrifuge tubes and washed three times each with acetone and 75% ethanol (v / v) (10 mL each time, centrifugation conditions: 3000 rpm, 10 min) to remove residual solvent and surfactant.

[0060] In this embodiment, the purified microspheres were mixed with deionized water at a mass ratio of 2:1 and incubated in a constant temperature shaker for 1 hour at an incubation temperature of 25±2℃. Purpose: To fully hydrate the microspheres and restore them to their hydrated state before freeze-drying; Eliminate the damage to the microsphere structure caused by residual organic solvents (acetone / ethanol) during the purification process.

[0061] In this embodiment, the incubated microspheres are treated with 50 kHz ultrasound for 5 minutes: power density 0.5 W / cm², pulse mode: 2 seconds on and 1 second off.

[0062] Purpose: Break up microsphere aggregates to ensure monodispersity (reduce particle size distribution width); To avoid structural inhomogeneity caused by aggregation during subsequent freeze-drying.

[0063] In this embodiment, mannitol is added to the ultrasonically treated suspension as a freeze-drying protectant: 10% (w / v) mannitol, and the mixture is magnetically stirred (300 rpm) until completely dissolved.

[0064] Purpose: Improved solubility: Mannitol forms a glassy structure, reducing microsphere clumping after freeze-drying; Preventing structural collapse: A crystalline scaffold is formed during freezing to maintain the porous morphology of the microspheres.

[0065] In this embodiment, PLGA sustained-release microsphere powder: The particle size is 10-20 μm, the encapsulation efficiency is ≥77.8%, and the drug loading is ≥4.28%.

[0066] Implementation Method 4: Drug-loaded microspheres, which are prepared using the drug-loaded microsphere preparation method described above.

[0067] In this embodiment, the drug-loaded microspheres are W / O / W microspheres, comprising a liposome core, a PLGA intermediate layer, and a PVA shell; The liposome core encapsulates a lipophilic drug component; The PLGA intermediate layer encapsulates the liposome core; The PVA shell encapsulates the PLGA intermediate layer to form a W / O / W structure.

[0068] In this embodiment, the liposome core comprises Bupleurum-White Peony Extract and 5% Trehalose Stabilizer; The PLGA intermediate layer is formed by dissolving amino-terminated PLGA in a mixed solvent of benzyl benzoate and benzyl alcohol. The PVA shell is formed by curing a polyvinyl alcohol-sucrose matrix solution.

[0069] In this embodiment, the liposome core is an inner aqueous phase; the PLGA intermediate layer is an oil phase; and the PVA shell is an outer aqueous phase.

[0070] Implementation Method 5: Method for preparing drug-loaded microneedles, the method comprising the following steps: Vacuum casting loading steps: The drug-loaded microspheres described above are mixed with a polyvinyl alcohol-sucrose matrix solution at a mass ratio of 1:9 to form a drug-loaded suspension. The drug-loaded suspension was injected into the cavity of the microneedle mold, and a vacuum was applied for 20 minutes under a vacuum of -0.1 MPa to allow the drug-loaded microspheres to fully penetrate into the gap at the tip of the microneedle mold. Centrifugal filling and shaping steps: The vacuum-cast microneedle mold was centrifuged at 4200 rpm for 5 minutes. This process was repeated 3 times to remove excess matrix solution, so that the drug-loaded microspheres were evenly distributed at the tip of the microneedle mold to form drug-loaded microneedles.

[0071] In this embodiment, a polyvinyl alcohol (PVA)-sucrose matrix solution is used to form water-soluble microneedle tips: (1) Dissolution mechanism: PVA contains a large number of hydroxyl groups (-OH), while sucrose contains multiple hydroxyl groups and forms hydrogen bonds when it comes into contact with water. Upon contact with skin tissue fluid, the hydrogen bond network breaks down, leading to rapid dissolution.

[0072] (2) The functional positioning of "cutting-edge": Core area: 500–800 μm segment at the tip of the microneedle; Drug-loaded focusing: Through vacuum casting and centrifugal filling, drug-loaded microspheres are enriched at the tip (accounting for ≥80%), and the substrate is mainly blank PVA-sucrose matrix.

[0073] (3) Process implementation of water-soluble tips: Material selection: Polyvinyl alcohol: Function: Film-forming framework, providing mechanical strength; Contribution to solubility: Water-soluble polymers; sucrose: Functions: Pore-forming agent and plasticizer, improving the dissolution rate after puncture; Solubility contribution: small molecule sugars.

[0074] Tip dissolution kinetics: Puncture of the stratum corneum → infiltration of tissue fluid → hydrogen bond breakage → matrix dissolution → microsphere release.

[0075] Time threshold: swelling begins within 5 seconds → complete dissolution within 60 seconds → release of drug-loaded microspheres into the dermis.

[0076] Synergistic effect: Sucrose accelerates water penetration, while PVA controls the dissolution rate to prevent premature burst release of microspheres.

[0077] (4) Technical advantages compared to traditional water-insoluble microneedles:

[0078] The core problem to be solved: Precise acupoint delivery: After dissolution, the microspheres are positioned in the dermal layer of the Neiguan acupoint (depth ≈ 1 mm) to activate nerve endings; Avoid drug waste: The tip dissolves and releases the drug locally, reducing systemic circulation losses and increasing drug concentration at the target site.

[0079] In this embodiment, the microneedle mold corresponds to the microneedle (mechanical) structure, the needle shape is conical, the needle length is 0.8mm, the bottom diameter of the needle body is 0.3mm, and the needle body is made of hydrogel material.

[0080] Implementation method 6: Drug-loaded microneedles, wherein the drug-loaded microneedles are prepared using the drug-loaded microneedle preparation method described above.

[0081] Implementation method 7: Drug-loaded microneedle patch, the drug-loaded microneedle patch comprising: a group of medical stainless steel main needles, a group of hydrogel drug-loaded auxiliary needles, and adhesive tape; The adhesive tape is thicker in the middle and thinner around the edges; the medical stainless steel main needle group and the hydrogel drug-loaded auxiliary needle group are fixed in the middle area of ​​the adhesive tape. The medical stainless steel main needle group includes 4 medical stainless steel main needles; the 4 medical stainless steel main needles are symmetrically distributed around the center of the adhesive tape; The hydrogel drug-loaded needle group is made of hydrogel material; the hydrogel drug-loaded needle group includes multiple drug-loaded needles, and the drug-loaded needles are the drug-loaded microneedles described above. The hydrogel drug-loaded auxiliary needle group is divided into 4 regions, each region is arranged in an octagonal array; the 4 regions are distributed around the periphery of the medical stainless steel main needle group, forming the shape of 4 petals of a lilac flower; the needle height of the medical stainless steel main needle group is greater than the needle height of the hydrogel drug-loaded auxiliary needle group.

[0082] In this embodiment, the needle body of the medical stainless steel main needle is made of austenitic stainless steel wire, specifically: The needle body of the medical stainless steel main needle is made of 06Cr19Ni10 (SUS304) austenitic stainless steel wire as specified in GB / T4240-2019 "Stainless Steel Wire".

[0083] In this embodiment, the medical stainless steel main needle is conductive to provide electrical stimulation to acupoints.

[0084] In this embodiment, the four regions of the hydrogel drug-loaded auxiliary needle group are closely distributed around the periphery of the medical stainless steel main needle group.

[0085] In this embodiment, the height of the medical stainless steel main needle group is greater than the height of the hydrogel drug-loaded auxiliary needle group, that is, the medical stainless steel main needle is slightly longer, which can effectively stimulate the target acupoint.

[0086] In this embodiment, the hydrogel-loaded drug-eluting needle group occupies approximately 2 / 3 of the area on the adhesive tape, resulting in a wide drug delivery range.

[0087] In this embodiment, the patch uses a main needle group plus a secondary needle group in the center. The secondary needles in each area are located behind the main needles and are distributed in an octagon. The peripheral secondary needles can work together with the main needles to provide auxiliary stimulation to specific acupoints, thereby enhancing the therapeutic effect. The secondary needle area is adjacent to the main needle area, and there is a certain height difference between the main needle group and the secondary needle group. The metal needle group (i.e., the main needle) is slightly longer to effectively stimulate the target acupoints.

[0088] In this embodiment, because the metal main needle group and the hydrogel secondary needle tip have different heights, the height difference can ensure that the depth of the metal main needle group piercing the skin is not affected by the dense secondary needle group. At the same time, even if the patch is slightly deviated from its position, the four main needles can still effectively stimulate specific acupoints, thereby improving the fault tolerance rate of the patch and reducing the difficulty of use.

[0089] In this embodiment, the secondary needles in the four areas form a lilac shape around the central main needle area, enhancing the visual appeal.

[0090] Implementation method 8: The diameter of the medical stainless steel main needle is 0.16mm-0.30mm, the needle body length is 0.8mm-3mm, and the needle body is made of austenitic stainless steel wire; The drug-loaded accessory needle is conical in shape, with a needle length of 0.8 mm and a bottom diameter of 0.3 mm.

[0091] In this embodiment, the dense hydrogel needle cluster is 0.8 mm long and can penetrate the epidermal layer for sustained drug release.

[0092] Implementation Method 9: In each region of the hydrogel drug-loaded needle group, the needle tip spacing between the drug-loaded needles is 0.65 mm, the number of drug-loaded needles is 10 × 10 (i.e., 100), and the distribution area of ​​the drug-loaded needles is 6.5 mm × 6.5 mm.

[0093] Implementation method 10: The adhesive tape includes a waterproof layer, a limiting and fixing layer, and an adhesive; The waterproof layer is a PU film, located on the outermost layer; The limiting layer is made of non-woven fabric and is located below the waterproof layer; The portion of the adhesive tape that comes into contact with the skin is an adhesive; the adhesive is polysilicon.

[0094] In this embodiment, the waterproof layer is made of medical PU film, which is extremely thin and has the characteristics of high waterproofness, low allergy, and breathability.

[0095] In this embodiment, the material of the fixing layer is non-woven fabric, which is moisture-proof, breathable, flexible, lightweight, and inexpensive.

[0096] In this embodiment, the adhesive material is: The adhesive that comes into contact with the skin is polysiloxane, also known as silicone gel. It is a polymer of organic compounds containing siloxanes. In this polymer, it is directly bonded to Si atoms through Si-O bonds and Si-C bonds. It usually exists in a gel-like form. Polyurethane dressings containing silicone gel can quickly adhere to the skin, have a soft texture, and their multi-layered structure can relieve pressure injuries. They are also easy to separate from the skin when removed.

[0097] In this embodiment, the size of the adhesive tape is 8.9mm × 8.9mm.

[0098] In this embodiment, the adhesive used to fix the needle body and the limiting fixation layer is a hydrogel.

[0099] In this embodiment, the limiting and fixing layer and the waterproof layer are fixedly connected by hydrocolloid.

[0100] In this embodiment, the medical stainless steel main needle is fixedly connected to the adhesive tape via a hydrocolloid: The needle tip of the medical stainless steel main needle passes through the limiting and fixing layer; The bottom of the medical stainless steel main needle is wrapped between the limiting and fixing layer and the waterproof layer; The bottom surface of the medical stainless steel main needle is fixedly connected to the upper surface of the waterproof layer using a hydrocolloid.

[0101] A limiting and fixing layer is used to limit and fix the bottom of the medical stainless steel main needle, which strengthens the fixation of the main needle and enhances its stability.

[0102] In this embodiment, the hydrogel drug-loaded accessory needle group is fixedly connected to the upper surface of the limiting and fixing layer via hydrocolloid.

[0103] In this embodiment, the drug-loaded microneedle patch is also known as a hydrogel press needle.

[0104] In this embodiment, the drug-loaded microneedle patch is applied to the Neiguan acupoint, and the drug release and acupoint stimulation are synergistically achieved through microneedle puncture.

[0105] It should be noted that the structure and composition of a traditional snap button are as follows: (1) Structure: It is made of medical stainless steel wire.

[0106] (2) Composition: The needle body of the press needle is made of 06Cr19Ni10 (sus304) austenitic stainless steel wire as specified in GB / T4240-2019 "Stainless Steel Wire".

[0107] (3) Number of needles: 1.

[0108] (4) Specifications: needle diameter 0.16mm-0.30mm, needle length 0.6mm-30mm.

[0109] (5) Adhesive tape: Skin-colored hypoallergenic adhesive tape.

[0110] In this embodiment, compared with traditional press needles, the hydrogel press needle (drug-loaded microneedle patch) has the following advantages: (1) Increase the number of main needles and the number of auxiliary needles: The patch adopts a main needle group plus an auxiliary needle group in the center. When using this acupoint patch, the main needle is aligned with the acupoint to achieve targeted stimulation of the specific acupoint. By setting auxiliary needles around the main needle, the auxiliary needles can work together with the main needle to achieve auxiliary stimulation of the specific acupoint, thereby enhancing the therapeutic effect. In addition, considering that users of this patch (medicated microneedle patch) may not have very professional acupoint identification ability, this technical solution, through the layout of the main needle and the auxiliary needles, can still achieve effective stimulation of the specific acupoint even if the patch is slightly deviated from its position, thereby improving the error tolerance of the patch and reducing the difficulty of use.

[0111] (2) The auxiliary needle is a drug-loaded sustained-release needle: The auxiliary needle is a minimally invasive device that can overcome the stratum corneum barrier to achieve transdermal drug delivery. It uses a single-emulsion method to create sustained-release microspheres that encapsulate the drug, allowing it to enter the bloodstream through the skin to produce local or systemic therapeutic effects. This effectively solves the problem of large molecule drugs being unable to penetrate the stratum corneum barrier and can improve the transdermal penetration rate of the drug. This product avoids drug degradation by enzymes in the stomach and metabolism in the liver, thus reducing stomach irritation, and also avoids the pain caused by injection.

[0112] (3) Improved adhesive material: The back of the patch is made of PU film combined with silicone gel, which is more waterproof than traditional medical tape and has low allergenicity, making it suitable for long-term use.

[0113] (4) Improved backing structure: The backing structure is optimized to be thick in the middle and thin around the edges to ensure that the backing gel adheres tightly to the skin. This not only allows water to flow quickly through the patch to effectively prevent water penetration, but also allows the patch to adapt to different user environments. Patients can move freely without being restricted by the treatment method, thus improving their sense of well-being during the treatment.

[0114] It should be noted that the drug-loaded microspheres are pH-responsive microspheres, and their drug release rate can be controlled by changes in environmental pH.

[0115] In this embodiment, the drug-loaded auxiliary needle is equipped with microelectrodes, which form a weak electric field on the surface of human skin through electrolysis, thereby changing the pH value and ion concentration of the skin surface and stimulating skin cells to produce electrochemical reactions; by changing the pH value, the drug release rate of the auxiliary needle is controlled.

[0116] Implementation Method 11: A specific embodiment is provided for the determination of drug content in drug-loaded microspheres: (1) Liquid chromatography conditions: Column: Aglient C18 (4.6 × 250 mm, 5 µm); Mobile phase: 0-5 min: 10% acetonitrile; 5-20 min: 10%→35%; 20-25 min: 35%→50%; Column temperature: Low polar phase: 35℃ → High polar phase: 45℃; Flow rate: 1.0 mL⋅min⁻¹; Injection volume: 10.00; Injection volume: 20 μL; Detection wavelength: 190-400 nm; (2) Microsphere determination method: Dissolve an appropriate amount of alcohol-extracted drug in ultrapure water to prepare a 10% standard solution as a reference solution; dissolve drug microspheres (i.e., drug-loaded microspheres) in acetonitrile, perform high-pressure microfluidic jet for 15 min (to completely destroy the microsphere structure and eliminate drug encapsulation residue), add PBS to rinse the solution, centrifuge and collect the supernatant as the sample solution; PLGA blank microspheres were processed in the same way and used as blank solutions.

[0117] The determination was performed according to the chromatographic method, and the chromatographic peaks were recorded.

[0118] The formula for calculating drug component concentration is as follows: (Peak area in Ax sample; peak area in As standard; concentration of Cs standard) Data analysis system, constructing a triple validation model: ① Convolutional Neural Network - Peak Recognition: Automatically separates overlapping peaks with an AUC recognition rate of >99%.

[0119] ②PLS chemometrics eliminates matrix background interference, with a recovery rate of 98-102%.

[0120] ③ Blockchain data storage ensures that experimental data is uploaded to the blockchain in real time, guaranteeing that the original data cannot be tampered with.

[0121] By combining advanced separation technology, smart materials, artificial intelligence and blockchain, the first traditional Chinese medicine microsphere analysis platform that conforms to the FDAALCOA+ principle has been established, providing a new paradigm for quality research of complex formulations.

[0122] (3) Results: ① Liquid chromatography results: The extract, paeoniflorin, and paeoniflorin lactone were prepared into a 10% standard solution using ultrapure water. The peak areas were determined by liquid chromatography, and a standard curve was plotted with concentration and peak area as variables.

[0123] As shown in the high-performance liquid chromatogram, the retention time of paeoniflorin in the extract was 12 min. Therefore, under these chromatographic conditions, the determination of drug concentration was not affected by the excipients used in the preparation of the microspheres or the reagents used in the analysis, indicating that this chromatographic method has good specificity and can be used for the determination of drug content.

[0124] ② Results of microsphere encapsulation efficiency measurement: The concentration of the standard drug was 0.1 mg / ml. Accurately weigh 10 mg of drug-loaded microspheres into a 5 ml volumetric flask, add an appropriate amount of acetonitrile, dissolve the microspheres using a high-pressure microfluidic jet, dilute with PBS buffer, and bring to volume. After sonication to mix thoroughly, centrifuge 1 ml (3000 rpm, 15 min), and determine the drug content in the supernatant using HPLC.

[0125] The formulas for calculating drug loading and encapsulation efficiency are as follows: Drug loading (%) = Drug mass in microspheres / Total mass of microspheres × 100%.

[0126] Encapsulation efficiency (%) = Actual drug loading of microspheres / Theoretical drug loading of microspheres × 100%.

[0127]

[0128] Implementation method 12: A specific embodiment is provided for pharmacodynamic experimental research.

[0129] (1) Laboratory animals: Sixty SD rats, weighing approximately 180-220 g, were selected based on the ease of operation, high success rate, good repeatability, and low feeding cost. Half were male and half female. The rats had free access to water and food. They were kept at a temperature of 22-24 ℃ and humidity of 45%-55% for one week using a day-night cycle. Fine hairs on the rats' forelimbs and parietal region were removed with a depilatory cream, and the rats were allowed one day to acclimatize.

[0130] (2) Experimental method: ①Grouping and Dosing: Before the experiment, 60 rats were acclimatized for 7 days. To eliminate individual differences, after measuring body weight and conducting open field tests, 60 rats with similar indicators were selected and divided into 6 groups (n=10), with half males and half females. These were: blank group, model group, positive drug fluoxetine hydrochloride hydrogel microneedle group, Bupleurum-Paeonia lactiflora drug-loaded microneedle group (including only the auxiliary needle), single press needle group, and Bupleurum-Paeonia lactiflora hydrogel press needle group (i.e., drug-loaded microneedle patch, including the main needle and auxiliary needle). Drug administration was administered while the model was being established. The control group received no stimulation, while the other groups received continuous transdermal drug administration combined with electrical stimulation for 56 days. The model group received blank hydrogel press needles combined with electrical stimulation to establish the model.

[0131] During the experiment, the inner side of the rat's forelimb was routinely disinfected with 75% ethanol at the Neiguan acupoint (located approximately 3 mm from the wrist joint between the radius and ulna) and the Baihui acupoint (located at the midline of the parietal bone). The product was then applied to the skin corresponding to the acupoints using tweezers, and an electrical stimulation device (20Hz, ≤5mA) was placed on top.

[0132] ②CUMS Depression Model: After 7 days of acclimatization, rats were individually housed in all groups except the control group (which was kept together). The model group, the positive control group (fluoxetine hydrochloride hydrogel microneedle group), the Bupleurum-Paeonia lactiflora drug-loaded microneedle group, the single-needle press group, and the Bupleurum-Paeonia lactiflora drug-loaded hydrogel press group were all subjected to 56 days of chronic unpredictable mild stress (CUMS). Nine stimulation methods were used: fasting, water deprivation, ice water swimming, day-night reversal, tail clamping, damp bedding, restraint, noise stimulation, and electric shock to the paws. One to two stimulation methods were used daily without repetition, ensuring the rats could not predict the next stimulation method. Each 7-day cycle lasted for 8 weeks (56 days). The rats' condition was observed daily, and their weight was measured weekly.

[0133] Fasting: Deprive the animal of feed for 24 hours.

[0134] Water restriction: Deprivation of drinking water for 24 hours.

[0135] Ice water swimming: Rats were placed in a large bucket with a diameter of 20 cm, a height of 50 cm, and a water depth of 30 cm, and swam in ice water at 4 ℃ for 5 minutes.

[0136] Day and night reversed: 12 hours of darkness during the day, 12 hours of light at night.

[0137] Tail clamping: Clamp the rat's tail 1 cm from the base with tweezers for 2 minutes.

[0138] Moist bedding: Mice were placed in bedding that had been moistened with water for 24 hours.

[0139] Restraint: Rats were placed inside mineral water bottles to restrict their free movement for 3 hours.

[0140] Noise stimulation: Create noise for 3 hours (40 MHz) using an ultrasonic cleaner.

[0141] Electric shock to the soles of the feet: 36 V voltage, one shock every 10 seconds, each shock lasting 2 seconds, for a total of 10 shocks.

[0142] The specific stimulus schedule for the CUMS model is shown in the table below.

[0143] Stimulation schedule of the CUMS model

[0144] ③ Behavioral tests: (a) Body weight test; (b) Food intake test; (c) Open field test (OFT); (d) Sugar water preference test (SPT); (e) Forced swimming test (FST).

[0145] ④ Statistical processing: Statistical analysis and graphing were performed using GraphPad Prism 8.0 software (GraphPad Software, USA). All data are expressed as mean ± standard deviation (±s). Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA with LSD test was used for pairwise comparisons among multiple groups.

[0146] (3) Experimental results: ①Weight: In week 1, there were no statistically significant differences among the groups of rats (P>0.05), and their body weight remained relatively consistent, as shown in the table below. After 8 weeks of modeling, the body weight of the model group rats was significantly lower than that of the control group (P<0.01). Compared with the depressed rats in the model group, there were significant differences in the Bupleurum-Paeonia lactiflora drug-loaded microneedle group, the single press needle group, and the Bupleurum-Paeonia lactiflora drug-loaded hydrogel press needle group (P<0.05).

[0147] Table of weight changes of rats in each group (g) ± s , n =10)

[0148]

[0149] ②Food intake: Compared with the control group, the food intake of the CUMS-induced model rats was significantly lower than that of the control group, and the difference was statistically significant (P<0.01). Compared with the model group, the food intake of rats in the Bupleurum-White Peony hydrogel press needle group, the Bupleurum-White Peony drug-loaded microneedle group, and the single press needle group was significantly increased (P<0.05), as shown in the table below. This indicates that the combined treatment of Bupleurum-White Peony extract and press needles can, to some extent, improve the decrease in food intake induced by CUMS in rats.

[0150] Food intake table of rats in each group at week 8 ( ± s , n =10)

[0151]

[0152] ③Open field experiment: The open field test is mainly used to assess the rats' ability to move autonomously and explore in unfamiliar environments, thereby determining their depressive status. As shown in the table below, the number of times the model group rats stood upright was significantly less than that of the control group rats, with a statistically significant difference (P<0.01), indicating that the autonomous exploration ability of depressed rats was reduced. Compared with the model group, the number of times the rats stood upright was significantly increased in the Bupleurum-White Peony hydrogel press needle group, the Bupleurum-White Peony drug-loaded microneedle group, and the single press needle group (P<0.05), indicating that the combined treatment of Bupleurum-White Peony extract and press needles can significantly improve the reduced activity level in rats caused by CUMS.

[0153] Table of the number of times rats stood upright in open field experiment for each group ( ± s , n =10)

[0154]

[0155] ④ Sugar water preference experiment: The sucrose preference test is mainly used to assess anhedonia in depressed rats. Anhedonia leads to a decreased preference for sucrose in rats. Before modeling, there was no significant difference in sucrose preference rates among the different groups. After 8 weeks of CUMS modeling, compared with the control group, the sucrose preference rate in the model group was significantly lower (P<0.01), indicating anhedonia in the rats. Compared with the model group, the sucrose preference rate was higher in the Bupleurum-Paeonia lactiflora hydrogel press needle group, the Bupleurum-Paeonia lactiflora drug-loaded microneedle group, and the single press needle group (P<0.05), indicating that Bupleurum-Paeonia lactiflora extract combined with press needle therapy has a certain ameliorative effect on anhedonia induced by CUMS in depressed rats, and the combined treatment of Bupleurum-Paeonia lactiflora extract with press needle therapy is more effective than the single press needle group and the single drug administration group. (See table below.) Table of sucrose preference rates in each group of rats ( ± s , n =10)

[0156]

[0157] ⑤ Forced swimming: The forced swimming test was used to represent the despair behavior of depressed rats, with the immobility time reflecting the rats' despair state. As shown in the table below: Compared with the control group, the immobility time of the model group rats was significantly longer (P<0.01), indicating that the model group rats clearly exhibited despair behavior. Compared with the model group, the immobility time of rats in the Bupleurum-Paeonia lactiflora hydrogel press needle group, the Bupleurum-Paeonia lactiflora drug-loaded microneedle group, and the single press needle group was significantly shorter (P<0.05), indicating a stronger will to live.

[0158] The timetable for forced swimming immobility in each group of rats ( ± s , n =10)

[0159]

[0160] (4) Summary: Using CUMS-depressed rats as the research subjects, and taking rat body weight, food intake, open field test, sucrose preference test, and forced swimming test as behavioral indicators, this study observed and compared the effects of the Bupleurum-White Peony hydrogel press-fit group (drug-loaded microneedle patch, including main needle and auxiliary needle), the Bupleurum-White Peony drug-loaded microneedle group (only auxiliary needle), and the single press-fit group on improving the behavioral characteristics of CUMS-depressed rats. The results showed that, compared with the control group, the model rats had smaller body weight, lower food intake, fewer open field standing times, lower sucrose preference rate, and longer immobile time during forced swimming. This indicated that the model group rats experienced decreased appetite, loss of novelty in free exploration, a gradually decreasing preference for sucrose, and poor mental state, demonstrating the successful establishment of the CUMS-depressed rat model. Compared with the model group, the Bupleurum-White Peony hydrogel press needle group, the Bupleurum-White Peony drug-loaded microneedle group, and the single press needle group showed increased uprighting frequency, sugar water preference rate, and food intake, and shortened forced swimming immobility time. This indicates that the Bupleurum-White Peony combined with press needle and the positive drug group can significantly improve the rats' autonomous exploration activity, deepen their preference for sugar water, and improve their mental state, thus exhibiting a good antidepressant effect.

[0161] The above further describes the technical solution provided by the present invention in detail through several specific embodiments in order to highlight the advantages and benefits of the technical solution provided by the present invention. However, the several specific embodiments described above are not intended to limit the present invention. Any reasonable changes and improvements to the present invention, reasonable combinations of implementation methods and equivalent replacements based on the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A microfluidic chip, characterized in that, The microfluidic chip includes a main channel, a first channel, and a second channel; Main pipeline: Its diameter gradually increases from upstream to downstream, and it is used to inject the internal water phase; First pipe: intersects with the main pipe at an angle, used to inject the oil phase to cut the inner aqueous phase and form the W / O emulsion core; The second pipe is located downstream of the first pipe and intersects with the main pipe at a cross shape. Its diameter differs from that of the first pipe by ≤5%. It is used to inject the external aqueous phase to encapsulate the W / O emulsion core and form W / O / W microspheres.

2. A drug-loaded microsphere preparation system, characterized in that: The system includes: a microfluidic pressure pump, a piezoelectric droplet generator, and a microfluidic chip; the microfluidic chip is the microfluidic chip according to claim 1; The piezoelectric droplet generator is integrated into the main channel inlet of the microfluidic chip and is used to cut the internal aqueous phase into monodisperse droplets; The microfluidic pressure pump is used to inject internal aqueous phase, oil phase, and external aqueous phase into the microfluidic chip at a given flow ratio: Main pipeline: Injects internal water phase; First conduit: Injecting oil phase to cut the internal aqueous phase droplets; Second channel: Inject external aqueous phase to encapsulate oil phase, forming W / O / W microspheres.

3. A method for preparing drug-loaded microspheres, characterized in that, The method includes the following steps: Drug extraction steps: The effective components of Bupleurum chinense and Paeonia lactiflora were extracted using the ethanol reflux method to obtain the extract. Preparation steps for PLGA solution: Under constant temperature of 40℃, 180 mg of amino-terminated PLGA was added to a mixed solvent of benzyl benzoate and benzyl alcohol, and the mixture was stirred magnetically until completely dissolved. The resulting PLGA solution was used as the oil phase. Preparation steps of drug solution: The extract was prepared into liposomes, and the lipophilic components were encapsulated in the liposomes and then used as the inner aqueous phase to improve the encapsulation efficiency and achieve dual controlled release. 5% trehalose stabilizer was added to the inner aqueous phase to protect the activity of the drug during emulsification, solidification and freeze-drying processes and reduce burst release. Steps for forming a water-in-oil-in-water double emulsion: Polyvinyl alcohol solution was used as the external aqueous phase; The drug-loaded microsphere preparation system described in claim 2 is used to prepare W / O / W microspheres based on an internal aqueous phase, an oil phase, and an external aqueous phase. Microsphere solidification and purification steps: Photocuring of W / O / W microspheres in the presence of a photoinitiator enhances the mechanical strength and stability of the microspheres and reduces burst release. The photocured microspheres are then irradiated with 365 nm blue light for 120 seconds to achieve cross-linking and curing. The cross-linked and cured microspheres were washed and purified to remove residual solvents and surfactants, and purified microspheres were obtained. Microsphere post-processing steps: The purified microspheres were mixed with deionized water at a mass ratio of 2:1 and incubated in a constant temperature shaker for 1 hour. The incubated microspheres were treated with 50 kHz ultrasound for 5 minutes to promote uniform dispersion of the microspheres and obtain an ultrasound-treated suspension. Mannitol was added to the sonicated suspension as a freeze-drying protectant to improve resolubility and reduce microsphere aggregation and structural collapse during freeze-drying. The suspension containing the lyophilization protectant was transferred to a lyophilizer and freeze-dried at -60°C for 72 hours to obtain PLGA sustained-release microsphere powder as drug-loaded microspheres.

4. Drug-loaded microspheres, characterized in that, The drug-loaded microspheres were prepared using the drug-loaded microsphere preparation method described in claim 3.

5. A method for preparing drug-loaded microneedles, characterized in that, The method includes the following steps: Vacuum casting loading steps: The drug-loaded microspheres according to claim 4 are mixed with a polyvinyl alcohol-sucrose matrix solution at a mass ratio of 1:9 to form a drug-loaded suspension. The drug-loaded suspension was injected into the cavity of the microneedle mold, and a vacuum was applied for 20 minutes under a vacuum of -0.1 MPa to allow the drug-loaded microspheres to fully penetrate into the gap at the tip of the microneedle mold. Centrifugal filling and shaping steps: The vacuum-cast microneedle mold was centrifuged at 4200 rpm for 5 minutes. This process was repeated 3 times to remove excess matrix solution, so that the drug-loaded microspheres were evenly distributed at the tip of the microneedle mold to form drug-loaded microneedles.

6. A drug-loaded microneedle, characterized in that, The drug-loaded microneedles are prepared using the drug-loaded microneedle preparation method described in claim 5.

7. A drug-loaded microneedle patch, characterized in that, The drug-loaded microneedle patch includes: a group of medical stainless steel main needles, a group of hydrogel drug-loaded auxiliary needles, and adhesive tape. The adhesive tape is thicker in the middle and thinner around the edges; the medical stainless steel main needle group and the hydrogel drug-loaded auxiliary needle group are fixed in the middle area of ​​the adhesive tape. The medical stainless steel main needle group includes 4 medical stainless steel main needles; the 4 medical stainless steel main needles are symmetrically distributed around the center of the adhesive tape; The hydrogel drug-loaded accessory needle group is made of hydrogel material; the hydrogel drug-loaded accessory needle group includes multiple drug-loaded accessory needles, and the drug-loaded accessory needles are the drug-loaded microneedles as described in claim 6; The hydrogel drug-loaded auxiliary needle group is divided into 4 regions, each region is arranged in an octagonal array; the 4 regions are distributed around the periphery of the medical stainless steel main needle group, forming the shape of 4 petals of a lilac flower; the needle height of the medical stainless steel main needle group is greater than the needle height of the hydrogel drug-loaded auxiliary needle group.

8. The drug-loaded microneedle patch according to claim 7, characterized in that, The diameter of the medical stainless steel main needle is 0.16mm-0.30mm, its needle body length is 0.8mm-3mm, and its needle body is made of austenitic stainless steel wire. The drug-loaded accessory needle is conical in shape, with a needle length of 0.8 mm and a bottom diameter of 0.3 mm.

9. The drug-loaded microneedle patch according to claim 8, characterized in that, In each region of the hydrogel drug-loaded needle cluster, the needle tip spacing between the drug-loaded needles is 0.65 mm, the number of drug-loaded needles is 10 × 10, and the distribution area of ​​the drug-loaded needles is 6.5 mm × 6.5 mm.

10. The drug-loaded microneedle patch according to claim 7, characterized in that, The adhesive tape includes a waterproof layer, a limiting and fixing layer, and an adhesive. The waterproof layer is a PU film, located on the outermost layer; The limiting layer is made of non-woven fabric and is located below the waterproof layer; The portion of the adhesive tape that comes into contact with the skin is an adhesive; the adhesive is polysilicon.

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