CN111671728B - Sodium alginate double-layer drug-loaded microsphere and preparation method thereof - Google Patents
Sodium alginate double-layer drug-loaded microsphere and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a sodium alginate double-layer drug-loaded microsphere and a preparation method thereof, wherein ENBREL + Bevacizumab is loaded on the outer layer of the microsphere, IGF-1 is loaded on the inner layer of the microsphere, and the microsphere is mainly prepared by a flow focusing method and a microfluidic preparation method. When the release agent is applied to the process of preventing osteogenic differentiation and bone bridge formation, the release of an anti-bone bridge formation factor (Enbrel + Bevacizumab) and a chondrogenic differentiation factor (IGF-1) in a damaged local sequence of an epiphyseal plate can be effectively controlled, the osteogenic differentiation and the bone bridge formation can be prevented by the outer-layer anti-bone bridge formation factor (Enbrel + Bevacizumab) released at the early stage, and the chondrogenic differentiation and the epiphyseal plate cartilage regeneration can be promoted by the released IGF-1 at the later stage, so that the release agent has important significance for repairing the damaged epiphyseal plate.
Description
Technical Field
The invention relates to a drug-loaded microsphere, in particular to a sodium alginate double-layer drug-loaded microsphere and a preparation method thereof.
Background
Epiphyseal plates, also known as growth plates, are areas of cartilage where the terminal immature long bone of a child controls the longitudinal growth of bone. Fracture, infection, tumor or iatrogenic injury can cause epiphyseal injury, and about 30% of children are fractured with epiphyseal injury and unbalanced bone growth. Once the epiphyseal plate is damaged, the epiphyseal cartilage tissue is gradually replaced by bone tissue, a bone bridge is formed, growth retardation, angulation or rotation deformity is caused, the joint function is even further influenced, and the physical and mental health of the children is greatly influenced. The currently adopted clinical treatment method mainly comprises the steps of bone bridge resection combined with corresponding material filling, such as adipose tissues, high polymer materials and the like, so as to prevent the bone bridge from relapse and allow peripheral undamaged epiphyseal plate tissues to recover the longitudinal growth capacity of bones, but the success rate of the treatment method is very low, and operations such as osteotomy correction or limb lengthening and the like are mostly required in the later stage.
After the joint efforts of related personnel in the professional field, the construction of the epiphyseal cartilage with bioactivity by adopting the cartilage tissue engineering technology becomes a new direction for treating the epiphyseal injury of children, and the technology mainly utilizes chondrocytes and/or Bone Marrow Mesenchymal Stem Cells (BMSCs) to realize cartilage regeneration by combining with a scaffold made of a specific material. Although a great deal of research is done previously, different seed cells are utilized, different types of bioactive materials are screened, and regulation and control methods are adopted, the problems that the bracket chondrogenesis efficiency is low, and the fusion of regenerated cartilage and surrounding normal tissues is poor are still faced. More importantly, the regenerated cartilage does not have a layered structure similar to a natural epiphyseal plate and cannot control the longitudinal growth of long bones. The key reasons for this are mainly the following two points: 1. the micro-composition aspect of the scaffold is as follows: at present, researches on the micro-composition of the scaffold mainly focus on the direction of promoting chondrogenic differentiation and regeneration of cartilage tissues, and neglects that local osteogenic differentiation and bone bridge formation after epiphyseal plate injury is prevented from the source in the early stage of injury; 2. in the aspect of the microstructure of the bracket: at present, the microstructure of the tissue engineering scaffold does not accord with the physiological structure characteristics of an epiphysis plate, so that the regenerated chondrocytes are in a disordered accumulation state and cannot play the biological function of controlling the longitudinal growth of osteogenesis.
With the intensive research on various animal models, the local major pathology after epiphyseal injury changes to: osteogenic changes, i.e. the formation of bone bridges, occur at the site where cartilage should otherwise form, and the process is divided into 4 stages: inflammatory response phase, fibroblastic phase, osteogenic phase and remodeling phase. The first stage (inflammatory phase) after epiphyseal injury is mainly manifested by inflammatory cell infiltrationSite of injury, high expression of inflammatory cytokines and mediators, the most important of which is Tumor Necrosis Factor alpha (TNF-alpha), followed by the TNF-alpha antagonist Enbrel in a rat epiphyseal injury model®(Etanercept, ETN) significantly reduced the proliferation of inflammatory cells. More importantly, studies report that TNF- α not only regulates the inflammatory response phase itself, but also plays an important role in regulating downstream responses following epiphyseal injury. Subsequently, the stromal cells infiltrating at the site of the epiphyseal injury, after forming "fibrous tissue", enter the third phase of bone bridge formation (ossification), which is most notably the formation of new blood vessels. Vascular Endothelial Growth Factor (VEGF) is a key mediator of angiogenesis, and invasion of new blood vessels is a prerequisite for the ossification of mesenchymal cells to form bone bridges. In addition, studies have found that VEGF is detectable at days 1-3 post epiphyseal injury in rats, suggesting that VEGF may begin to function during the inflammatory phase following epiphyseal injury until the bridge is formed. Similarly, after the epiphyseal injury of the rat, the rat is treated by using an anti-vascular endothelial growth factor antibody (anti-VEGF) Bevacizumab (Bevacizumab), and the result shows that the number of vascular-like structures at the epiphyseal injury part is obviously reduced, the expression of osteogenesis related genes is obviously reduced, and bone tissues are reduced.
Therefore, inhibition of early inflammatory responses and angiogenesis after epiphyseal injury is critical for prevention of bone bridge formation, and we can use the corresponding inhibitors (Enbrel) due to the important role of TNF-a and VEGF in this phase®And Bevacizumab) to interrupt the subsequent reaction of bone bridge formation. Of course, after controlling osteogenic differentiation to inhibit bone bridge formation, we still need to induce chondrogenic differentiation of exogenous or endogenous mesenchymal stem cells using Growth factors such as Insulin Like Growth factor 1 (IGF-1) to eventually form epiphyseal cartilage tissue at the site of injury. How to effectively control the anti-osteopontic factor (Enbrel)®+ Bevacizumab) and the chondrogenic differentiation factor (IGF-1) become a critical factor in the local sequence release of epiphyseal plate lesions.
Disclosure of Invention
In order to solve the problems, the invention provides a sodium alginate double-layer drug-loaded microsphere and a preparation method thereof, the double-layer microsphere can achieve a better controlled release effect, can release Enbrel + Bevacizumab on the outer layer in the early stage to prevent osteogenic differentiation and osteopontin formation, and then release IGF-1 on the inner layer to promote chondrogenic differentiation and epiphyseal cartilage regeneration, and the sequence release characteristic has an obvious effect on preventing osteopontin formation.
In order to achieve the technical purpose and achieve the technical effect, the invention adopts the following technical scheme: a preparation method of sodium alginate double-layer drug-loaded microspheres mainly adopts a flow focusing method and a microflow control method, and specifically comprises the following steps:
1) placing the microfluidic chip on an objective table of an optical microscope, and respectively connecting six hoses to extend out of six channel ports;
2) adding a 1% sodium alginate solution added with IGF-1 into a single-channel injection pump through an injector, exhausting air, and connecting with a No. 2 channel port through a hose;
3) sucking 1% sodium alginate solution into a two-tube injector, placing the two-tube injector in a two-channel injection pump, and respectively connecting the two 1 # channel ports through flexible tubes;
4) sucking CaCl-containing solution with two-tube syringe2The paraffin oil is arranged in a double-channel injection pump and is respectively connected with two No. 3 channel ports;
5) a No. 4 channel port is connected into an empty centrifuge tube through a hose;
6) opening the injection pump, washing the microfluidic chip and the connecting hose, and collecting waste liquid at the tail end by using a centrifugal tube;
7) the flow rate of a 1% sodium alginate solution mixed with IGF-1 was adjusted to 50. mu.L/h by a syringe pump, and the mixture was mixed with CaCl-containing solution2The flow rate of the paraffin oil injection pump is adjusted to 400 mu L/h, and the flow rate of the 1% sodium alginate solution injection pump is adjusted to 80 mu L/h; sequentially starting the three injection pumps, and observing the balling condition in the microfluidic chip through a microscope;
8) transferring the hose connected with the No. 4 channel port into a centrifugal tube added with a surfactant, and collecting microspheres;
9) sucking the upper oil layer in the centrifugal tube in a super clean bench, filtering for 2-3 times by using a filter membrane with the pore diameter of 50 mu m, and cleaning microspheres;
10) immersing the microspheres into a solution containing ENBREL and Bevacizumab, adsorbing for 1 hour, and freeze-drying to obtain the sodium alginate double-layer microspheres loaded with ENBREL + Bevacizumab and IGF-1 in layers.
Further, in step 4), CaCl dissolved in paraffin oil2The concentration of (2) is 1%.
Further, in the step 7), the synthesis of the microfluidic chip is observed under an optical microscope, and the microspheres can be judged to be formed stably by observing the uniform flow of the spheres.
The sodium alginate double-layer drug-loaded microsphere prepared by the method has the advantages that ENBREL + Bevacizumab is loaded on the outer layer of the microsphere, and IGF-1 is loaded on the inner layer.
The invention has the beneficial effects that:
the sodium alginate double-layer drug-loaded microsphere prepared by the method disclosed by the invention is applied to the process of preventing osteogenic differentiation and bone bridge formation, the release of an anti-osteopontic factor (Enbrel + Bevacizumab) and a chondrogenic differentiation factor (IGF-1) in a local sequence of epiphyseal plate injury can be effectively controlled, the osteogenic differentiation and bone bridge formation can be prevented by the outer-layer anti-osteopontic factor (Enbrel + Bevacizumab) released at the early stage, the chondrogenic differentiation and epiphyseal plate cartilage regeneration can be promoted by the inner-layer IGF-1 released subsequently, the effect on the aspect of preventing osteogenic differentiation and bone bridge formation is remarkable, and the method has important significance on the repair after epiphyseal plate injury; and the whole preparation process is simple, the operability is strong, the market application potential is large, and the popularization and the use are facilitated.
Drawings
FIG. 1 is a schematic structural diagram of a sodium alginate double-layer drug-loaded microsphere;
fig. 2 is a schematic diagram of a micro-fluidic device for preparing sodium alginate double-layer drug-loaded microspheres.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying fig. 1-2, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Example 1: preparation of sodium alginate double-layer drug-loaded microspheres
In the embodiment, the preparation of the sodium alginate double-layer drug-loaded microsphere is mainly carried out by using a flow focusing method and a microfluidic preparation method, and the preparation method mainly comprises the following steps:
1. as shown in fig. 2, the microfluidic chip is placed on the stage of the optical microscope, and six hoses are connected to the stage to extend out of six channel ports;
2. adding a 1% sodium alginate solution added with IGF-1 into a single-channel injection pump through an injector, exhausting air, and connecting with a No. 2 channel port through a hose;
3. sucking 1% sodium alginate solution into a two-tube injector, placing the two-tube injector in a two-channel injection pump, and connecting the two-tube injector with two No. 1 channel ports through a hose;
4. sucking 1% CaCl with a two-tube syringe2The paraffin oil is arranged in a double-channel injection pump and is respectively connected with two No. 3 channel ports;
a No. 5.4 channel port is connected into an empty centrifugal tube through a hose;
6. opening the injection pump, washing the microfluidic chip and the connecting hose, and collecting waste liquid at the tail end by using a centrifugal tube;
7. the flow rate of a 1% sodium alginate solution mixed with IGF-1 was adjusted to 50. mu.L/h, and the mixture was mixed with 1% CaCl2The flow rate of the paraffin oil injection pump is adjusted to 400 mu L/h, and the flow rate of the 1% sodium alginate solution injection pump is adjusted to 80 mu L/h; sequentially starting the three injection pumps, and observing the balling condition in the microfluidic chip through a microscope;
8. the hose connected with the No. 4 port is moved into a centrifuge tube added with a surfactant, microspheres are collected, the surfactant which has no biological toxicity and does not react with components in the microspheres can be used, and generally, the aseptic treatment is not needed;
9. sucking the upper oil layer in the centrifugal tube in a super clean bench, filtering for 2-3 times by using a filter membrane with the pore diameter of 50 mu m, and cleaning microspheres;
10. immersing the microspheres into an ENBREL + Bevacizumab solution, wherein the concentrations of ENBREL and Bevacizumab can be flexibly adjusted according to the specific conditions of an implanted receptor (animal or human), the effect can be screened by observing the effect under different concentrations through specific experiments, and the sodium alginate double-layer microspheres loaded with ENBREL + Bevacizumab and IGF-1 in a layered manner are obtained by freeze-drying after being adsorbed for 1 hour; sodium alginate microspheres containing different concentrations of IGF-1 can be obtained by adjusting the concentration of IGF-1.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (4)
1. A preparation method of sodium alginate double-layer drug-loaded microspheres is characterized by comprising the following steps:
1) placing the microfluidic chip on an objective table of an optical microscope, and respectively connecting six hoses to extend out of six channel ports;
2) adding a 1% sodium alginate solution added with IGF-1 into a single-channel injection pump through an injector, exhausting air, and connecting with a No. 2 channel port through a hose;
3) sucking 1% sodium alginate solution into a two-tube injector, placing the two-tube injector in a two-channel injection pump, and respectively connecting the two 1 # channel ports through flexible tubes;
4) sucking CaCl-containing solution with two-tube syringe2The paraffin oil is arranged in a double-channel injection pump and is respectively connected with two No. 3 channel ports;
5) a No. 4 channel port is connected into an empty centrifuge tube through a hose;
6) opening the injection pump, washing the microfluidic chip and the connecting hose, and collecting waste liquid at the tail end by using a centrifugal tube;
7) the flow rate of a 1% sodium alginate solution mixed with IGF-1 was adjusted to 50. mu.L/h by a syringe pump, and the mixture was mixed with CaCl-containing solution2The flow rate of the paraffin oil injection pump is adjusted to 400 mu L/h, and the flow rate of the 1% sodium alginate solution injection pump is adjusted to 80 mu L/h; sequentially starting the three injection pumps, and observing the balling condition in the microfluidic chip through a microscope;
8) transferring the hose connected with the No. 4 channel port into a centrifugal tube added with a surfactant, and collecting microspheres;
9) sucking the upper oil layer in the centrifugal tube in a super clean bench, filtering for 2-3 times by using a filter membrane with the pore diameter of 50 mu m, and cleaning microspheres;
10) immersing the microspheres into a solution containing ENBREL and Bevacizumab, adsorbing for 1 hour, and freeze-drying to obtain the sodium alginate double-layer microspheres loaded with ENBREL + Bevacizumab and IGF-1 in layers.
2. The method for preparing sodium alginate double-layer drug-loaded microspheres as claimed in claim 1, wherein in step 4), CaCl dissolved in paraffin oil is added2The concentration of (2) is 1%.
3. The preparation method of the sodium alginate double-layer drug-loaded microsphere as claimed in claim 1, wherein in the step 7), the synthesis of the microfluidic chip is observed under an optical microscope, and the microsphere can be judged to start to be stably formed by observing the uniform flow of the spheres.
4. The sodium alginate double-layer drug-loaded microsphere prepared by the preparation method of the sodium alginate double-layer drug-loaded microsphere as claimed in any one of claims 1 to 3, wherein ENBREL + Bevacizumab is loaded on the outer layer of the microsphere, and IGF-1 is loaded on the inner layer.
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| WO2015088299A1 (en) * | 2013-12-09 | 2015-06-18 | Vilnius University | Method for production of biopolymer-based droplets and particles in a microfluidic system |
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