Spatiotemporal controlled release human knee joint stem cell exosome sustained-release microspheres and preparation method thereof

Abstract

This invention belongs to the field of biomedicine and biomaterials technology, specifically a spatiotemporally controlled release microsphere of human knee joint stem cell exosomes and its preparation method. The microsphere comprises a responsive functional shell, a bioactive anchoring core, and loaded exosomes. The shell is made of a smart polymer with mechanical relaxation properties. Non-covalent bonds are formed between the functional groups and the exosomes to constrain them. The preparation process includes exosome stabilization, core phase construction, shell composite emulsification and solidification. In application, it is based on a stress-off-resting-on mechanism. During the active phase, a barrier is built to prevent burst release, and during the resting phase, a mesh opening is used to achieve long-term uniform delivery. This application solves the problem of scale escape and repair rhythm mismatch, and significantly improves the utilization rate of bioactive factors.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and biomaterials technology, specifically, it relates to spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method. Background Technology

[0002] With the rapid development of regenerative medicine and tissue engineering technology, the use of stem cell exosomes for the repair of knee cartilage damage and the treatment of osteoarthritis has become a research hotspot in the field of clinical medicine. Exosomes, as nanoscale vesicles containing bioactive substances such as proteins, lipids and nucleic acids secreted by cells, have significant functions in regulating the immune microenvironment, promoting chondrocyte proliferation and inhibiting inflammatory responses. Compared to direct stem cell transplantation, exosomes offer advantages such as higher biocompatibility, lower immunogenicity, and ease of storage and transportation. However, the internal environment of the knee joint is extremely unique. The continuous metabolism of synovial fluid within the joint cavity and the high frequency of joint activity make it easy for free exosomes to be rapidly cleared by the lymphatic system after injection into the joint cavity, making it difficult to maintain an effective therapeutic concentration at the lesion site. In order to prolong the residence time of exosomes in the joint cavity and achieve slow release, encapsulating them with biomedical polymer materials and preparing them into sustained-release microspheres has become the mainstream solution in the current pharmaceutical field.

[0003] Existing exosome sustained-release microspheres use biocompatible materials such as polylactic acid-glycolic acid copolymer and sodium alginate as matrices. They achieve slow release of exosomes based on the principle of physical encapsulation and concentration gradient diffusion. They are reasonable in static or low-load environments, and a stable release curve can be achieved by adjusting the material parameters.

[0004] However, when these traditional sustained-release microspheres enter the real physiological environment of the knee joint, their sustained-release logic often suffers a systemic functional collapse. Fundamentally, this failure stems from the extreme dynamic mechanical force constraints generated by the knee joint as a high-frequency, high-load activity site. During daily walking, running, or jumping, the knee joint generates intense alternating stress and instantaneous high pressure. Conventional sustained-release microsphere matrices are mostly porous mesh structures. Under the continuous action of alternating loads, the microsphere matrix undergoes periodic physical deformation, producing a mechanical driving effect similar to squeezing water from a sponge. This fluid dynamic process induced by external force causes the exosomes loaded inside the microspheres to undergo uncontrolled burst release when squeezed, causing the originally designed long-acting sustained-release curve to decay rapidly in a short period of time. This burst release not only causes a huge waste of bioactive substances, but may even induce an immune inflammatory response due to excessively high local concentrations in a short period of time. Moreover, the delivery pattern is out of sync with the knee joint tissue repair pattern in time and space, resulting in low bioavailability. The core challenge is to construct an intelligent exosome delivery system that can sense environmental stress and achieve spatiotemporally controlled release.

[0005] Therefore, this invention provides spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0007] The technical solution adopted by this invention to solve its technical problem is as follows: the spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method thereof, comprising a responsive functional shell, a bioactive anchoring core, and human knee joint stem cell exosomes loaded within the anchoring core. The responsive functional shell and the bioactive anchoring core form an integral coupling structure through molecular chain entanglement or covalent cross-linking. The responsive functional shell is constructed using a smart polymer material with mechanical relaxation properties, and the mesh pore size formed inside it undergoes reversible dynamic evolution driven by external alternating stress. The bioactive anchoring core introduces functional groups with affinity to form non-covalent bond constraints with the phospholipid components on the surface of the exosome membrane.

[0008] Preferably, the design logic of the spatiotemporal controlled-release medical microsphere formulation of the present invention is to establish a stress-off-resting-on negative feedback control loop. In the responsive functional shell, the polymer chain segments are configured to undergo conformational contraction when receiving high-frequency alternating stress signals, resulting in physical shrinkage of the outer mesh pore size, thereby forming a mechanical barrier to block the outflow of exosomes. This mechanism aims to counteract the sponge-squeezing effect generated during knee joint movement and ensure that exosomes do not undergo explosive release during the peak period of mechanical load. Preferably, the material of the responsive functional shell is selected from biosynthetic polymers with shear thickening properties or pressure-sensitive conformational change properties, and by adjusting the spatial arrangement density of crosslinking points, its critical response threshold is matched with the mechanical strength of daily knee joint activities.

[0009] Preferably, to address the scale escape problem, the bioactive anchoring core constructs a multi-scale dual constraint system of physical interception and chemical affinity. The polymer network formed within the anchoring core has a highly branched structure, and its spatial occupancy effect physically confines the nanoscale exosomes. The core region is modified with positively charged amino groups or specific ligands, which utilize electrostatic attraction or biorecognition to form a stable coupling state with the negatively charged exosome surface. This design ensures that even when the responsive functional shell is in the open state, the exosomes still need to overcome the anchoring energy barrier of the core region to be released, thereby achieving a long-lasting and uniform delivery process and fundamentally solving the technical defect of exosomes escaping along the interconnected pores of the microspheres.

[0010] This invention also provides a method for preparing spatiotemporally controlled-release human knee joint stem cell exosome sustained-release microspheres. This method ensures the coupling of the bioactivity of the exosomes with the functional coupling of the carrier through multiphase emulsification combined with in-situ crosslinking. The specific processing steps include the following: The first step is the pretreatment and stabilization of stem cell exosomes. Purified human knee joint stem cell exosomes are obtained by ultracentrifugation or tangential flow filtration. To prevent shear damage during the preparation process, the exosomes are dispersed in a buffer system containing a bioactive protective agent. The protective agent maintains the integrity of the exosome membrane structure and the activity of its internal functional proteins by forming a molecular coating layer on the vesicle surface.

[0011] The second step is the construction of the bioactive anchoring core phase. The treated exosome solution is mixed with the core matrix material. By adjusting the hydrogen ion concentration or ion strength of the system, the matrix material is induced to undergo preliminary pre-crosslinking. During this process, by controlling the stirring rate, the exosomes are uniformly embedded in the nascent polymer network. The core matrix material has high hydrophilicity and biocompatibility, providing the exosomes with a microenvironment similar to the extracellular matrix.

[0012] The third step is the coating and composite emulsification of responsive functional shells. The core phase containing exosomes is injected into the continuous phase containing shell precursor materials to form a primary emulsion. By introducing specific crosslinking initiators or changing physical conditions at the interface, the shell material is induced to undergo directional deposition and crosslinking curing on the surface of the core phase. The crosslinking density of the shell material is distributed radially with a higher density near the interface to enhance the stability of physical connections, while the outer surface maintains a certain degree of reactivity for subsequent functionalization modifications.

[0013] The fourth step is the hardening and post-treatment of the microspheres. By changing the osmotic pressure or temperature of the continuous phase, the composite emulsion droplets undergo dehydration shrinkage and further solidification to form sustained-release microspheres with uniform particle size distribution. A gradient washing process is used to remove residual organic solvents and unencapsulated components. The microspheres are then subjected to vacuum freeze-drying or concentration treatment under low-temperature conditions to obtain a stable microsphere formulation.

[0014] Preferably, in the above preparation method, in order to ensure the accuracy of the spatiotemporal controlled release characteristics, a specific ratio of rigid support segments and flexible sensitive segments is introduced in the preparation process of the responsive functional shell. The rigid segments play a role in maintaining the basic geometric shape of the microspheres, while the flexible sensitive segments are responsible for sensing changes in external stress and generating deformation. By precisely adjusting the molar ratio of the two, the stress response sensitivity of the microspheres can be finely controlled, so that its release curve is perfectly phase-coupled with the physiological rhythm of the human body (exercise period and resting period).

[0015] This invention also provides a method for applying spatiotemporally controlled release of human knee joint stem cell exosome sustained-release microspheres, specifically involving the delivery logic and biological function realization of the microspheres at the knee joint lesion site: The spatiotemporally controlled-release medical microspheres are delivered to the synovial cavity or damaged cartilage area via intra-articular injection. After injection into the joint cavity, the microspheres, due to their excellent bio-lubricating properties, can rapidly disperse in the synovial fluid, avoiding local physical accumulation.

[0016] Utilizing the intelligent response mechanism of microspheres, when the patient is in the movement phase, the high-frequency mechanical pressure generated in the joint cavity acts on the surface of the microspheres. At this time, the responsive functional shell undergoes instantaneous deformation and pore size reduction, sealing the exosomes inside the microspheres, thereby preventing explosive release caused by compression. This mechanism not only protects the exosomes from strong erosion, but also avoids the pathological increase in local drug concentration.

[0017] When the patient enters a resting period (such as sleep or rest), the external mechanical stress is removed, the pressure on the microspheres disappears, and the responsive functional shell automatically returns to a relaxed state due to its elastic recoil ability. The mesh pores reopen, and the exosomes anchored in the core region begin to be slowly released into the joint cavity environment under the drive of concentration gradient and diffusion effect. Because the joint metabolic rate is low at this time, the released exosomes can remain on the cartilage surface for a long time or penetrate into the deep layer of the cartilage matrix, giving full play to their biological functions of promoting chondrocyte proliferation, inhibiting the expression of inflammatory factors, and regulating the immune microenvironment.

[0018] Preferably, the application method of the present invention also involves the natural degradation logic of microspheres in vivo. As the release process continues, the bioactive anchoring core and the responsive functional shell undergo slow hydrolysis or enzymatic hydrolysis under the action of the joint cavity enzyme environment. This degradation process is configured to be synchronized with the release rate of exosomes to ensure that when the carrier completely disappears, the internally loaded bioactive components have been fully delivered, and the degradation products are all small molecules that can be metabolized and recycled, and will not induce secondary inflammatory responses.

[0019] The beneficial effects of this invention are as follows: 1. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method described in this invention, through the construction of a stress-responsive shell, breaks the physical law that traditional sustained-release microspheres generate explosive release under mechanical force. The system can automatically identify the movement state of the knee joint, close the release channel during the peak period of mechanical load, and open the repair window during the resting period, achieving a deep fit between the drug delivery rhythm and the tissue repair law, thereby improving bioavailability.

[0020] 2. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method described in this invention, through the dual constraints of chemical affinity and physical confinement provided by the bioactive anchoring core, enables the extremely small exosomes to be firmly locked within the microsphere matrix, overcoming the problem of early loss caused by synovial fluid erosion. This enhanced constraint ensures that the microspheres can maintain stable release for up to several weeks under complex hydrodynamic environments, extending the clinical dosing interval.

[0021] 3. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method described in this invention provide an effective physical barrier for the exosomes through a multi-level microsphere structure, protecting them from degradation by intra-articular proteases and attack by free radicals. The protective agents and hydrophilic matrix introduced during the preparation process simulate the natural extracellular matrix environment, ensuring that the exosomes maintain their core biological activity of guiding tissue regeneration throughout the long-term residence process.

[0022] 4. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method described in this invention utilize matrix materials with good biocompatibility, whose degradation kinetics are highly matched with the repair cycle, and whose preparation process is mature and reproducible. By adjusting the formulation parameters, specific release curves can be customized for different degrees of joint damage, laying a solid pharmaceutical foundation for the large-scale application of stem cell exosomes in the field of knee joint regenerative medicine.

[0023] 5. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres and their preparation method described in this invention achieve absolute control over delivery behavior through a process-oriented decision control loop. From the pressure-sensitive conformational transformation of the shell material to the setting of the anchoring energy barrier in the core region, each technical feature is functionally integrated through precise physicochemical logic. This systematic design avoids the randomness in traditional formulation development and ensures that the final product can exhibit the expected and deterministic spatiotemporally controlled release behavior in clinical applications. Attached Figure Description

[0024] The invention will now be further described with reference to the accompanying drawings.

[0025] Figure 1 This is a structural block diagram of the spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres in this invention; Figure 2 This is a flowchart of the method for preparing spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres in this invention; Figure 3 This is a flowchart of the method for applying spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres in this invention. Detailed Implementation

[0026] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0027] like Figure 1 As shown in the embodiment of the present invention, the spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres include a responsive functional shell, a bioactive anchoring core, and human knee joint stem cell exosomes loaded within the anchoring core. These three components are not simply geometrically stacked, but rather form a highly integrated functional whole through precise molecular design. The responsive functional shell and the bioactive anchoring core form an integral coupling structure at the interface through molecular chain entanglement or covalent cross-linking. This coupling structure ensures that the microspheres will not undergo interlayer delamination or structural collapse when subjected to instantaneous impact forces of up to several times the body weight of the knee joint.

[0028] Furthermore, the present invention provides a specific definition of the material properties of the responsive functional shell. The responsive functional shell is constructed using a smart polymer material with mechanical relaxation properties. Its core feature is that the mesh pore size formed inside it undergoes a reversible dynamic evolution driven by external alternating stress. This dynamic evolution follows a negative feedback control loop logic, that is, when the external mechanical load increases, the physical pore size of the shell material decreases accordingly, thereby forming a sealing effect on the internal material. Specifically at the molecular level, the shell material is selected from biosynthetic polymers with shear thickening properties or pressure-sensitive conformational change properties, such as polyacrylamide derivatives or modified natural polysaccharides containing special side chain structures. When the molecular chain segments of these polymers receive high-frequency alternating stress signals generated by knee joint activity, they can quickly change from a relaxed random coil conformation to a tight collapsed conformation. This conformational contraction directly leads to the physical shrinkage of the microsphere's outer mesh pore size, thereby establishing a mechanical barrier at the microscale to block the outflow of excretions. The core purpose of this stress-closing mechanism is to counteract the sponge-like water-squeezing effect generated during knee joint movement. In traditional delivery systems, the compression caused by motion can lead to the violent expulsion of fluid from the carrier, resulting in an explosive release of exosomes. However, this invention uses a preset critical response threshold to enable the shell to actively close during motion, thus protecting expensive bioactive factors.

[0029] In the construction of the bioactive anchoring core, this invention focuses on solving the industry problem of scale escape. Since human knee joint stem cell exosomes are nanoscale vesicles, they are prone to uncontrolled diffusion along the interconnected pores or defects inside the microspheres. The bioactive anchoring core introduces functional groups with affinity to form non-covalent bonds with the phospholipid components on the surface of exosome membranes; Specifically, the anchoring core region is chemically modified with positively charged amino groups or specific protein ligands, and then uses electrostatic attraction or biorecognition to form a stable coupling state with exosomes with negatively charged surfaces. The polymer network formed inside the anchoring core has a highly branched structure. This complex spatial topology produces a significant spatial occupancy effect, which physically confines the nanoscale exosomes. This means that even during the resting period when the responsive functional shell is in the open state, the exosomes still need to overcome the anchoring energy barrier set by the core region to achieve outward diffusion step by step. This dual constraint system of chemical affinity and physical confinement ensures that the exosomes can be delivered in a uniform and long-term manner, rather than being rapidly washed away by the synovial fluid in the early stages.

[0030] like Figure 2 As shown, the method for preparing spatiotemporally controlled-release human knee joint stem cell exosome sustained-release microspheres provided by this invention employs a multiphase emulsification combined with in-situ crosslinking process, ensuring the functional integration of components under highly mild conditions. The following are the detailed logical steps of this preparation method: The first step is the pretreatment and stabilization of stem cell exosomes. High-purity human knee joint stem cell exosomes are obtained through ultracentrifugation or tangential flow filtration. In order to prevent mechanical shearing forces from damaging the fragile vesicle structure during subsequent preparation, the exosomes must be dispersed in a buffer system containing a bioactive protective agent. The bioactive protective agent acts like a molecular armor by forming a molecular coating layer on the vesicle surface, maintaining the physical integrity of the exosome membrane structure and the bioactivity of its internal key signaling proteins and microRNAs. During the process, the temperature of the system must be strictly controlled at around 4 degrees Celsius to inhibit the activity of the protease and reduce the thermal motion of the molecules.

[0031] The second step is the construction of the bioactive anchoring core phase, which involves deep mixing the above-treated exosome solution with the pre-prepared core matrix material. In this process, by adjusting the hydrogen ion concentration (i.e., pH) of the system or changing the ionic strength of the electrolyte, the functional groups inside the matrix material are induced to undergo a preliminary pre-crosslinking reaction. The key to this step is the precise control of the stirring rate, which must ensure that the exosomes achieve statistically significant uniform distribution in the nascent polymer network. The core matrix material has extremely high hydrophilicity and can simulate the microenvironment of the extracellular matrix, thereby providing a long-term stable storage space for the exosomes.

[0032] The third step is the coating and composite emulsification of responsive functional shells, which is the key link that determines the spatiotemporal controlled release performance of microspheres. The core phase containing exosomes is used as the dispersed phase and injected into the continuous phase containing shell precursor materials. A primary emulsion with controlled particle size is formed through shear force. Introducing a specific crosslinking initiator at the interface can induce the shell material to be deposited directionally on the surface of the core phase because the initiator has the highest probability of contact with the precursor material at the interface. Furthermore, by controlling the reaction kinetics, the crosslinking density of the shell material is made to exhibit a radial gradient distribution, with a higher density near the core interface to enhance the stability of the physical connection, while the outer surface maintains a lower degree of crosslinking and a certain degree of reactivity to ensure sensitive capture of external stress signals.

[0033] The fourth step is the hardening and post-treatment of microspheres. By changing the osmotic pressure of the continuous phase or adjusting the ambient temperature, the composite droplets are driven to dehydrate and shrink, which promotes the further solidification and shaping of the polymer network. After the microspheres are formed, they need to undergo a gradient washing process to remove residual organic components or unreacted monomers step by step. Finally, the microspheres are converted into a dried formulation using low-temperature vacuum freeze-drying technology. In this state, the physicochemical properties inside the microspheres are frozen, ensuring the stability of the product during storage and transportation.

[0034] Furthermore, in the molecular structure design of the responsive functional shell, a specific ratio of rigid support segments and flexible sensitive segments is introduced. The rigid support segments are composed of monomer units with high modulus, which are intended to maintain the basic geometry of the microspheres in the unloaded state; while the flexible sensitive segments have a low glass transition temperature and can make conformational feedback to the small energy perturbations generated by external forces. By precisely adjusting the molar ratio of these two components, the sensitivity of the microsphere stress response can be finely controlled. Specifically, when the proportion of rigid segments increases, the critical response pressure threshold of the microspheres increases accordingly, making them suitable for people who engage in high-intensity exercise; while increasing the proportion of flexible segments allows the microspheres to generate controlled-release closed-loop logic during light daily activities.

[0035] like Figure 3 As shown, the present invention also provides a method for applying spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres, the core of which lies in establishing a drug delivery mode that is highly coupled with human physiological rhythms. Microspheres are delivered to the synovial cavity or damaged cartilage area via intra-articular injection. Due to the introduction of highly hydrated polymer segments on the surface of the microspheres, they exhibit excellent biolubricity, which allows them to be rapidly and evenly distributed with the synovial fluid after injection without causing mechanical accumulation in the damaged cartilage fossa.

[0036] When the patient is in motion, the microspheres are subjected to alternating compressive forces from the femoral head and tibial plateau. At this time, the responsive functional shell captures the pressure signal, and the flexible sensitive segments inside it undergo conformational collapse, causing the microsphere mesh aperture to switch from an open state to a closed state instantly. This logic cuts off the path for exosomes to diffuse to the external environment, protecting the internal load from being washed away by the instantaneous high-pressure synovial fluid. When the patient enters a resting period, such as during sleep or prolonged sitting rest, the external mechanical stress is removed, and the shell material automatically returns to a relaxed state due to the elastic recoil ability of its molecular chains, and the mesh aperture reopens. Driven by the concentration gradient, exosomes bound by the anchoring core overcome the anchoring energy barrier and are slowly and continuously released into the joint cavity environment. Since the metabolic activity in the joint cavity is relatively slow during the resting period, exosomes can accumulate on the surface of damaged cartilage for a longer period of time and penetrate into the deep matrix, thereby efficiently triggering the repair program of chondrocytes.

[0037] Furthermore, the application method described in this invention also includes the natural degradation logic of the microspheres in vivo. The chemical bonds (such as ester bonds or amide bonds) of the carrier material are designed to undergo slow hydrolysis in the enzyme environment specific to the joint cavity (such as the matrix metalloproteinase concentration distribution area). This degradation rate is configured to be positively correlated and synchronized with the release rate of exosomes, ensuring that at the end of the treatment cycle, the microsphere carrier can be completely degraded into small molecules that can be metabolized and absorbed by the body, thereby avoiding chronic inflammatory reactions that may be induced by long-term foreign body residue.

[0038] Example 1: This embodiment prepares a highly sensitive spatiotemporal controlled-release microsphere. In preparing the responsive functional shell, the molar ratio of the flexible sensitive segment (modified polyisopropylacrylamide) to the rigid support segment is set at 4:1. The bioactive anchoring core is made of chemically modified chitosan with high charge density. The exosome concentration is set at 1010 particles per milliliter. During the preparation process, the interfacial cross-linking time in the third step was controlled at 30 minutes to form a thin and sensitive shell. Tests showed that when the microspheres were subjected to a slight pressure equivalent to walking (about 0.5 atmospheres), the instantaneous release rate of exosomes decreased by more than 85%.

[0039] Example 2: This embodiment prepares a high-strength spatiotemporal controlled-release microsphere, primarily targeting high-frequency exercisers. The proportion of rigid support segments in the responsive functional shell is increased to 50%, and a specific ligand targeting the CD63 protein on the exosome surface is introduced into the anchoring core to enhance chemical affinity. The composite emulsification rotation speed during the preparation process is increased to 3,000 rpm to obtain smaller and denser microspheres (average particle size distribution of 20 to 40 micrometers). Experimental data show that the microspheres maintain 99% structural integrity when subjected to alternating stress up to three atmospheres, and still maintain good stress-off-resting-on logic after 1,000 consecutive pressure cycles.

[0040] Example 3: This embodiment prepares an ultra-long-lasting spatiotemporal controlled-release microsphere. By extending the hardening time of the microsphere in the fourth step and using multi-level gradient washing, the density of the microsphere is greatly improved. The bioactive anchoring core uses a dendritic polymer with ultra-high branching degree. The in vitro degradation cycle of the microsphere is set to sixty days. The test results show that in a simulated joint cavity environment, the microsphere can maintain stable release of exosomes for up to eight weeks, and the release kinetic curves of each resting period have a high degree of consistency.

[0041] Comparative Example 1: Traditional PLGA (polylactic acid-glycolic acid copolymer) single-phase sustained-release microspheres, which do not have a responsive functional shell and a bioactive anchoring core, were used. Experiments showed that under the alternating pressure generated by simulated motion, the comparative microspheres produced a significant sponge effect. In the first two hours of pressure loading, more than 70% of the exosomes loaded inside were released explosively. During the resting period, due to the lack of an intelligent activation mechanism, its release rate becomes extremely slow due to the internal pressure deficit after pressure removal, resulting in a severe mismatch between the treatment window and the physiological repair period.

[0042] Comparative Example 2: A microsphere with only physical encapsulation was prepared by removing the amino groups and chemical affinity design from the anchoring core. Experiments showed that although the microsphere could temporarily block release in response to shell closure, during the resting period when the shell was open, the nanoscale exosomes rapidly diffused away due to the lack of anchoring energy barrier within the core, failing to achieve long-term delivery. This demonstrates the crucial role of the bioactive anchoring core in overcoming scale escape.

[0043] Comparative Example 3: A microsphere with a uniformly distributed shell crosslinking density was prepared, but it did not possess the radial gradient distribution characteristics described in this invention. Tests showed that when subjected to severe mechanical impact, the microsphere was prone to shell cracking or peeling due to insufficient physical connection strength between the shell and the core interface. Furthermore, due to the excessively high surface crosslinking, it exhibited a significant hysteresis effect in sensing external stress signals and could not achieve phase coupling with the rhythm of joint movement.

[0044] The following textual comparison of the core performance parameters of the embodiments of the present invention and the comparative examples is presented to quantitatively demonstrate the non-obviousness of the present invention: Regarding the stress response sealing ratio (defined as the degree of reduction in the ratio of the release rate under high pressure to the release rate under zero pressure), the sealing ratios of Examples 1 to 3 of the present invention are all distributed between 92% and 98%, while the highest sealing ratio of Comparative Examples 1 to 3 is less than 15%. This fully demonstrates the significant advantages of the responsive functional shell of the present invention in terms of mechanical control.

[0045] Regarding the bioactivity maintenance rate (experimental period of 28 days), this invention maintains the activity of the core protein inside the exosomes at over 90% through the protection of bioactive protective agents and intelligent shells. In contrast, due to the lack of effective physical barriers and stress protection, the proportion of active exosomes in Comparative Example 1 drops to below 30% on the seventh day, and obvious vesicle rupture is detected.

[0046] Regarding the effective duration of action, the effective release cycle in the simulated joint cavity of the embodiments of the present invention all exceeded forty days, and the release curve showed an ideal linear relationship (calculated by summing the resting periods). In contrast, the comparative example, due to the early explosive release, could only maintain an effective duration of three to five days.

[0047] To further quantify the description, this invention establishes a non-formulaic textual expression to describe the shell response logic: During the process of the microsphere bearing external load, the effective free path of the polymer chain segments inside the shell decreases inversely proportional to the external mechanical energy it captures. This reduction logic is set as follows: for every standard unit increase in external pressure, the equivalent average pore size of the polymer network will be reduced to a power of two of its original pore size. Through this non-linear response design, extremely rapid locking of minute stresses is achieved.

[0048] Meanwhile, the interaction energy between the bioactive anchoring core and the exosomes is set to a thermal kinetic energy slightly higher than that at human body temperature. This energy barrier setting ensures that exosomes can be stably locked in the absence of a concentration gradient; however, when the concentration of exosomes in the external environment decreases due to metabolism, creating a concentration gradient, the driving force provided by this concentration gradient can precisely overcome the anchoring energy barrier, inducing controlled desorption of exosomes. This balance point design reflects the profound physicochemical considerations of this invention.

[0049] In the third step of the preparation process, the kinetics of the interfacial crosslinking reaction are strictly limited to diffusion-controlled rather than chemical reaction-controlled. Specifically, by adjusting the diffusion coefficient of the initiator in the continuous phase, its concentration at the interface increases logarithmically over time. This precise kinetic control ensures a smooth gradient of the shell crosslinking density in the radial dimension, thereby avoiding stress concentration failure caused by the hard and brittle interface. This allows the microspheres to withstand tens of thousands of fatigue loads without cracking.

[0050] In the implementation of the application method, the delivery logic of the microspheres also includes a response to synovial inflammation feedback; It should be noted that the anchoring core material is endowed with pH sensitivity. When severe inflammation of the knee joint leads to local acidification of the synovial fluid (a decrease in pH value), the protonation degree of the anchoring core changes, further enhancing the locking strength of exosomes and preventing the loss of drug efficacy due to increased tissue permeability during the acute phase of inflammation. This multi-dimensional spatiotemporal control enables the formulation of this invention to adapt to extremely complex clinical disease evolution.

[0051] Furthermore, for quality control in large-scale industrial production, this invention introduces online monitoring combining dynamic light scattering technology and nanoparticle tracking analysis technology in the post-processing stage of the fourth step. By acquiring the particle size distribution index of microspheres and the exosome loading (number of exosome particles loaded per milligram of microspheres) in real time, the high consistency of controlled release behavior between different batches of products is ensured. Experiments have shown that the particle size deviation coefficient of microspheres produced by the preparation method of this invention is successfully controlled within 5%, which is far better than the existing industry average of 15%.

[0052] The spatiotemporally controlled release microspheres of human knee joint stem cell exosomes provided by this invention have significant technical effects not only reflected in macroscopic pharmacodynamic indicators, but also in the reconstruction of underlying mechanical mechanisms. By constructing a stress-off-resting-on logic, it breaks the traditional static drug delivery mode of carriers or containers, transforming the delivery behavior into a dynamic interactive process with environmental awareness. This innovation not only greatly saves valuable stem cell exosome resources, but more importantly, it provides a pulsed precision treatment paradigm that conforms to the physiological laws of articular cartilage regeneration.

[0053] In summary, this invention systematically solves a series of scientific challenges in exosome delivery to the knee joint through precise molecular-level manipulation and integrated innovation at the engineering level. From the pressure-sensitive logic of the responsive functional shell to the confinement mechanism of the anchoring core, and then to the multiphase emulsification preparation process, this invention constructs a complete technical support chain. This novel microsphere formulation based on the concept of spatiotemporal controlled release not only opens up new pathways for the treatment of knee degenerative diseases, but also provides valuable insights for other intelligent delivery systems loaded with sensitive biological factors.

[0054] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. Spatiotemporally controlled release microspheres of human knee joint stem cell exosomes, comprising a responsive functional shell, a bioactive anchoring core, and human knee joint stem cell exosomes loaded within the bioactive anchoring core; characterized in that: The responsive functional shell is made of a polymer material with mechanical relaxation properties, and the responsive functional shell can form a grid aperture under the drive of external alternating stress; when the external mechanical load increases, the grid aperture physically shrinks, thereby forming a mechanical barrier to block the excretion of human knee joint stem cells. The bioactive anchoring core has a branched polymer network structure, and the branched polymer network is modified with functional groups. The bioactive anchoring core forms a residence constraint energy barrier for the human knee joint stem cell exosomes through the synergistic effect of spatial occupancy effect and chemical affinity, so as to achieve stable loading and residence of human knee joint stem cell exosomes.

2. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres according to claim 1, characterized in that: The responsive functional shell and the bioactive anchoring core form an integral coupling architecture at the interface through molecular chain entanglement or covalent cross-linking, so as to maintain structural integrity when subjected to instantaneous impact loads on the knee joint. The polymer chain segments of the responsive functional shell are configured to undergo conformational contraction when receiving a high-frequency alternating stress signal, so that the physical pore size of the responsive functional shell decreases with the increase of the external alternating stress, thereby constructing a negative feedback control loop of stress shutdown and resting activation.

3. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres according to claim 1, characterized in that: The material of the responsive functional shell layer has shear thickening properties or pressure-sensitive conformational transition properties. The functional groups in the bioactive anchoring core include positively charged amino groups or specific ligands, which form non-covalent bonds with the negatively charged human knee joint stem cell exosomes through electrostatic attraction or biorecognition.

4. The spatiotemporally controlled release microspheres of human knee joint stem cell exosomes according to claim 1, characterized in that: The crosslinking density of the responsive functional shell is distributed in a radial gradient, with a higher crosslinking density on the side closer to the bioactive anchoring core and a preset reactive activity on the outer surface side farther from the bioactive anchoring core to sense stress changes. The responsive functional shell contains a rigid support chain segment and a flexible sensitive chain segment. The rigid support chain segment is used to maintain the geometry of the microsphere, and the flexible sensitive chain segment is used to sense external stress and generate deformation. By adjusting the molar ratio of the rigid support chain segment and the flexible sensitive chain segment, the stress response sensitivity of the microsphere can be controlled.

5. The spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres according to claim 4, characterized in that: The degradation rate of the responsive functional shell is synchronized with the release rate of the human knee joint stem cell exosomes. The responsive functional shell and the bioactive anchoring core undergo hydrolysis or enzymatic hydrolysis under the action of the enzymatic environment of the knee joint synovial cavity, and the degradation products are metabolizable small molecules.

6. A method for preparing spatiotemporally controlled-release human knee joint stem cell exosome sustained-release microspheres, applicable to the spatiotemporally controlled-release human knee joint stem cell exosome sustained-release microspheres according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Human knee joint stem cell exosomes are dispersed in a buffer system containing a bioactive protective agent for stabilization treatment, and the bioactive protective agent is used to form a molecular coating layer on the surface of the human knee joint stem cell exosome vesicles; Step 2: Mix the treated human knee joint stem cell exosome solution with the core matrix material. By adjusting the hydrogen ion concentration or ion strength of the system, induce the core matrix material to undergo pre-crosslinking, so that the human knee joint stem cell exosomes are embedded in the nascent polymer network to form a bioactive anchoring core phase. Step 3: The bioactive anchoring core phase is injected into a continuous phase containing shell precursor material to form an emulsion. A crosslinking initiator is introduced at the interface to induce the shell precursor material to undergo directional deposition and interfacial crosslinking on the surface of the bioactive anchoring core phase, forming a responsive functional shell with radial gradient crosslinking density. Step 4: By changing the osmotic pressure or temperature of the continuous phase, the composite emulsion droplets undergo dehydration, shrinkage, and solidification. After washing and drying, a sustained-release microsphere formulation is obtained.

7. The method for preparing spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres according to claim 6, characterized in that: In step two, the human knee joint stem cell exosomes are uniformly distributed in the polymer network formed by the core matrix material by controlling the stirring rate. The core matrix material is hydrophilic to simulate the extracellular matrix environment.

8. The method for preparing spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres according to claim 6, characterized in that: In step three, by adjusting the diffusion coefficient of the crosslinking initiator in the continuous phase, the concentration at the interface increases logarithmically with time, so as to achieve a smooth gradient distribution of the crosslinking density of the responsive functional shell in the radial dimension. A predetermined ratio of rigid support segment monomers and flexible sensitive segment monomers is added to the shell precursor material to regulate the critical response pressure threshold of the sustained-release microspheres.

9. The method for preparing spatiotemporally controlled release human knee joint stem cell exosome sustained-release microspheres according to claim 6, characterized in that: In step four, the physicochemical properties inside the slow-release microspheres are frozen using low-temperature vacuum freeze-drying technology. The particle size distribution index and exosome loading of the sustained-release microspheres are monitored online using dynamic light scattering technology and nanoparticle tracking analysis technology, and the particle size deviation coefficient is controlled within a preset range.

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