KR101938339B1 - A composition for delivering bioactive material comprising Alginate-Microencapsule - Google Patents

Abstract

The present invention relates to a method for producing micro beads by a vibration nozzle technique by mixing an osteogenic protein with an alginate solution in order to prepare an injectable alginate microencapsulated osteogenic protein and an alginate microcapsule , The microencapsulation of the present invention is injectable and can be used as a method to increase osteogenic differentiation promoting ability.

Description

TECHNICAL FIELD The present invention relates to a composition for delivering a physiologically active substance containing an alginate microcapsule as an active ingredient,

The present invention relates to a composition for transporting a physiologically active substance containing an alginate microcapsule as an active ingredient and a method for producing the same.

In the field of regenerative medicine, there are many new medical methods that promote tissue regeneration through localizing agents. Examples of medical methods include regeneration of myocardium after infarction, induction of skeletal growth in spinal fusion, treatment of diabetic foot ulcers, and limitation or restoration of damage due to stroke. Topical agents such as growth factors can be obtained using a scaffold.

The scaffold provides the appropriate mechanical environment, architecture, and surface chemistry for angiogenesis and tissue formation. The use of a scaffold as a drug or cell delivery system has a great advantage, but it does not provide adequate response rates for the release of reagents such as cells or proteins acting as growth factors, while at the same time reducing the porosity, strength and collapse of scaffolds degradation, and reaction rate.

A more complex problem in the use of scaffolds as delivery systems for recovery and / or regeneration in living beings is the route of administration. In many treatments, areas where tissue repair is necessary are difficult to access (eg, cerebral palsy in stroke therapy or myocardial infarction in post-infarction treatment). Thus, there is a need for an improved injectable scaffold that can be administered via minimal invasive procedures.

In general, the scaffold is a pre-formed water-insoluble substrate or hydrogel with generally large interconnected pores. These scaffolds are implanted in patients for in vivo tissue repair and / or regeneration.

 When implanted, the previously formed water-insoluble substrate must be shaped to fill the space in the body, know the space to be filled, and the shape of the space that can be filled is limited. In addition, invasive procedures are needed to deliver the scaffold.

Conversely, several hydrogel materials were designed to be delivered directly into the body through a syringe. The gel is formed in the body in response to trigger signals, such as temperature changes or ultraviolet light exposure. These systems have the advantage that they can fill any shape space without prior knowledge of the spatial dimension. However, these hydrogels lack interconnected porous networks, limiting the release of reagents due to their low diffusion characteristics.

In addition, the weak mechanical strength of the hydrogel means that it can not withstand the compressive force applied at the time of use, which means that the reagents in the gel are actually compressed from the hydrogel and exhibit undesirable transmission characteristics.

An injectable drug delivery system is disclosed in WO2010 / 100506, and such a system comprises a composition comprising: (i) an injectable scaffold material comprising discrete particles; And (ii) a reagent required for delivery. Individual particles can interact to form a scaffold.

The use of a scaffold for delivering a formulation is in the treatment of bone, particularly in spinal fusion, in the treatment of multiple fractures and dental bones. The statin drug indirectly enhances endogenous bone morphogenetic protein-2 (BMP-2; bone conduction growth factor) activity and stimulates vascular endothelial growth factor production (VEGF; expression of osteoblast differentiation and angiogenesis promotion) Which is known to promote the formation of < RTI ID = 0.0 > Statins have long been used as oral drugs for the treatment of hyperlipidemia. The use of statins for local delivery in vivo is orthopedic.

The alginate comprises a heterobifunctional group of linear bifunctional copolymers composed of 1-4-linked beta -D-mannuronic acid and its C-5 epimer alpha-L-glucuronic acid. Alginate has long been studied as a biomaterial in a wide range of physiological and therapeutic applications. Its potential as a biocompatible graft material was first investigated in 1964 in the surgical role of artificially expanding plasma volume (Murphy et al., Surgery. 56: 1099-108, 1964). Over the last two decades, there have been remarkable advances in the alginate cell microencapsulation for the treatment of diseases such as diabetes in particular.

Several patents and patent applications attempted to perfect the materials and methods of encapsulation:

International Patent Application Publication No. WO 91/09119 discloses a method of encapsulating a biological material, more specifically an islet cell with a bead with an alginate gel, followed by a second layer, preferably comprising a poly-L-lysine, and an alginate Lt; RTI ID = 0.0 > 3-layer < / RTI >

U.S. Patent No. 5,084,350 provides a method of encapsulating a biologically active material into a large matrix, followed by liquefaction of the microcapsules.

U.S. Patent No. 4,663,286 discloses a process for preparing microcapsules by gelation of microcapsules followed by hydration to expand the microcapsules to control the permeability of the microcapsules.

It is an object of the present invention to provide a novel effective delivery system of bone morphogenetic protein-2 (BMP-2).

In order to accomplish the above object, the present invention provides a method for preparing a microbead by mixing an osteogenic protein with an alginate solution to produce an injectable alginate microencapsulated osteogenic protein,

And a vibration frequency (Hz) in the range of 300 to 2500 Hz.

In one embodiment of the present invention, the vibration frequency (Hz) is preferably 1000 Hz, but is not limited thereto.

In another embodiment of the present invention, the flow rate of the solution is preferably in the range of 15 to 28 mL / min, and the flow rate of the solution is more preferably performed at 23 mL / min, but is not limited thereto.

In another embodiment of the present invention, the voltage of the method is preferably in the range of 500 to 1000 V, but is not limited thereto.

In a preferred embodiment of the present invention, the vibration frequency (Hz) is 1000 Hz and the flow rate of the solution is preferably 23 mL / min.

In the present invention, the diameter of the microbeads is preferably from 250 to 300 μm, but is not limited thereto.

In one embodiment of the present invention, the bone-forming protein is preferably bone-forming protein 2 (BMP-2), but is not limited thereto.

The present invention also provides injectable alginate microcapsules comprising an osteogenic protein having a diameter of from 250 to 300 μm.

In one embodiment of the present invention, the microcapsules are prepared by mixing a bone-forming protein and an alginate solution and subjecting the solution to a vibration frequency (Hz) in the range of 300 to 2,500 Hz and a solution flow rate of 15 to 28 mL / , But is not limited thereto.

In a preferred embodiment of the present invention, the microcapsules are prepared by mixing a bone-forming protein with an alginate solution and producing the solution at a vibration frequency (Hz) of 1000 Hz and a solution flow rate of 23 mL / min using a vibration nozzle technique No.

In another embodiment of the present invention, the microcapsules are preferably for promoting osteogenesis differentiation, but are not limited thereto.

Hereinafter, the present invention will be described.

The encapsulation method using alginate has been used for a long time. However, in order to be clinically useful for cell therapy, a uniform bead shape and a size suitable for the purpose of use are required. When stem cells are applied to localized areas for arthritis treatment, encapsulation may be useful for maximizing therapeutic effect due to prolonged survival of cells. However, in order to apply capsule stem cells to patients non-invasively rather than surgically, a microbead smaller than the inner diameter of a clinically usable needle and of uniform size is required.

In general, the inner diameter size of injection needles used in clinical use in humans and animals is 23 gauge, and the inner diameter is 337 μm. Therefore, it is necessary to use micro beads of 250-300 um in order to easily contain the cells and to easily pass through the needles. In the present invention, a method of making a microbead having a uniform shape with a size of 250-300 μm using various parameters such as frequency, flow rate, and electrode is established, and after the stem cell is encapsulated, its shape and size Respectively.

In addition, microbid and encapsulated cells were implanted subcutaneously in the mouse using a 23 gauge needle rather than an operation to assess and compare bio - toxicity and bioavailability.

The alginate micro beads were made by the vibration method, and the production of micro beads for injection was carried out using standard parameters and using various parameters. Micro-bead toxicity was tested in animal models. The encapsulated cASCs were evaluated for viability and proliferation in vitro. Microencapsulated or uninoculated cells were injected into the subcutaneous tissue of the mice and traced using in vivo bioluminescent imaging to confirm survival and maintenance of the transplanted cells.

The optimized injected microbead had a single size and a diameter of about 250 um. The alginate bead had no significant evidence of in vivo toxicity. The cells retained encapsulation survival, evidence of in vitro proliferation in microcapsules, and in vivo bioluminescent imaging showed that alginate encapsulation improved retention of transplanted cells.

Hereinafter, the present invention will be described in detail.

Microbead  Dimension and Shape  characteristic

The effects of modified frequency (Hz), flow rate (mL / min), and voltage (V) on bead diameter and shape were evaluated using a standardized procedure. The aim of the process was to determine the optimal parameters for producing ideal microbeads for injection into 23-gauge hypodermic needles.

The microcapsules produced using optimum conditions, especially 1000 Hz frequency, 23 mL / min flow rate, and 1000 V voltage were about 250 um diameter (FIG. 1). The diameter of the microcapsules tends to decrease with higher frequencies, higher flow rates and higher voltages. The result, however, suggests that an exceeding frequency of 2500 Hz produces microbeads with increased diameters. Also, an excess flow rate of 28 mL / min produced smaller diameter microbeads; However, the severe purging effect of the sodium alginate solution caused severe loss of solution.

Of alginate microbeads  Toxicity and in vivo bioavailability

Toxicity tests were performed to determine toxicity and biocompatibility of alginate microbeads in in vivo. Toxicity was assessed in rats by subcutaneous injection of beads and subcutaneous tissue blood and tissue evaluation was performed.

The blood chemistry evaluation of the control and alginate microcapsule-injected groups showed no significant toxicity (Fig. 2).

Histological evaluation of the microcapsule injection site showed that the beads were detected after 2 weeks of implantation and that the degradation of the bead material and the defective cytosyntis occurred without inflammation (FIG. 3).

As can be seen from the present invention, the microencapsulation of the present invention is injectable and can be used in a way that enhances osteogenic differentiation promoting ability.

Fig. 1 shows the standardization of optimum bead size for injection. (A) Microbead diameter (μm) as a function of increasing frequency (Hz). (B) Microbead diameter (μm) as a function of flow rate (mL / min). (C) microbead diameter (um) as a function of increasing voltage (V). (D) a light source microscope image of a microbead; bar = 500 [mu] m.
Figure 2 is an in vitro non-toxic evaluation of alginate microbeads. There was no significant difference between body weight and hematological evaluation of PBS injection group and alginate microbead injection group, 0 day, 1 day and 2 weeks after injection. (A) Weight. (B) White blood cell count. (C) Blood urea nitrogen. (D) Creatinine. (E) Glutamic-pyruvic transaminase. (F) Transaminase to alanine.
Figure 3 is a micrograph of H & E stained sections of subcutaneous tissue injected with alginate microcapsules at 2 weeks. Decomposition of bead material and defective cytosity. (A) Alginate microcapsules were detected after 2 weeks of transplantation; bar = 500 [mu] m. (B) illustration - high magnification image of the box area; bar = 250 [mu] m. (C) Notation - a larger enlarged image of the box area; bar = 125 [mu] m.
4 shows BMP-2 Release Kinetics.
Figure 5 shows results of an Alkaline Phosphatase (ALP) assay.
FIG. 6 is a photograph of a MicroCT photograph taken after subcutaneous injection in an in-vivo experiment. FIG.
FIG. 7 is a photograph of tissue analysis after subcutaneous injection in an in-vivo experiment.
8 is a photograph showing an experimental result using a Calvarial Defect model in an in-vivo experiment.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

All animal experiments of the present invention were in accordance with the guidelines of the Experimental Animals Committee of Kangwon National University. The rats and mice used in the experiments were kept free of water and feed at 21 ° C, 55% humidity and 12 h light-dark cycle. The animals were euthanized using carbon dioxide suffocation.

Example 1: Microencapsulation  Standardization for

Alginate microbeads were purchased from Buchi Encapsulator B-395 pro. ^e using a vibrating nozzle technology. Several parameters such as nozzle size, vibrational frequency (Hz), flow rate (mL / min), and electrostatic charge (V) were set up and injected with a diameter smaller than the average medial diameter of the 23 gauge hypodermic needle (337 um) Optimized for microbeads. The bead shape was analyzed by a light source microscope.

The polymer was 1.2% sodium alginate solution and the gelation solution was 100 mM calcium chloride solution. The sterile filtered 1.8% sodium alginate solution ^f was diluted to 1.2% with saline. The gelation solution was prepared by mixing 11 g of anhydrous calcium chloride powder with 1000 mL of distilled water. The nozzle size was set at 120 μm for the standardization experiment. Other stabilization parameters include a 5 mL syringe pump size, and an 80% agitation rate.

The frequency (Hz) was tested at 0, 40, 300, 1000, and 2500 Hz as factors affecting the bead diameter. The flow rate was set at 15 mL / min. The voltage is turned off for this experiment. 100 beads were counted after bead formation and measured through a light source microscope and the average diameter was determined based on various parameters tested.

Flow rate (mL / min) as a factor affecting the bead diameter was tested at rates of 15, 18, 23, and 28 mL / min. The frequency was set at 1000 Hz, which was determined to be optimized in this experiment. This experiment also turned off the voltage. After bead production, similar coefficients and diameter measurements and averaging were used to evaluate the various parameters tested.

Voltage (V) was tested at 0, 300, 500, 800, and 1000 V as a function of bead diameter. The flow rate and frequency were set at 23 mL / min and 1000 Hz, respectively, optimized for the above experiments. A similar evaluation procedure was used to evaluate the various parameters tested.

Example 2: Alginate microbead  In-vitro test

Sprague Dawley male rats to ^g (7-8 weeks old, weighing 170-200 g) were used in this experiment. The toxicity was tested in rats of the microbeads. Control group (n = 6) treated subcutaneous injection with PBS only in the dorsal area, whereas test group (n = 6) performed similar injection in which PBS of the present invention was suspended in PBS. Toxicity was assessed by measuring the hematological parameters of the injection site for each group.

Standardized parameters were used for bead manufacture and blood tests were performed immediately, 1 week, and 2 weeks after injection. The rats were euthanized with carbon dioxide and their tissues were collected at injection sites. The collected tissues were rinsed with 1x PBS and fixed in 10% formalin buffer for 24 hours. The tissue was filled in paraffin and sectioned to a thickness of 4 [mu] m. Slides were stained with H & E solution and cell shape was evaluated.

Example 3: BMP - 2  Containing Alginate Microbeads  How to make

Mix 1.8% Na-Alginate and sterile saline to make 1.2% Na-Alginate. Mix 0.25 mg of BMP-2 powder and 1 ml of sterile physiological saline to make 1 ml of BMP-2 solution.

Next, 1.2% Na-Alginate and BMP-2 solution are mixed at a ratio of 3: 1 and placed in a syringe. Prepare 100 mM Calcium Chloride solution with gelling solution. A Buchi Encapsulator B-395 pro (Buuchi Labortechinik AG, Dottikon, Switzerland) was set up with a nozzle size of 120 μm, a frequency of 1,000 Hz, a flow rate of 23 mL / min, a rate of 8% And the gelling solution is placed at the injection position, and then the production is started. Alginate microbeads should be placed in the gelling solution for more than 20 minutes.

Example 4: In  vitro experiment

The cells used in the experiment were canine adipose tissue derived mesenchymal stem cells collected from adipose cells of 2 - year - old male Beagle. The medium used for the culture of these cells was supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Darmstadt, ) At 48-hour intervals. After incubation at 37 ℃ in 5% CO2 incubator,

BMP-2 Release Kinetics Study

BMP-2-containing alginate microbeads and BMP-2-containing collagen sponge. 6-well plates were used. Alginate microbeads containing BMP-2 were placed in 40 well cell strainer in 3 wells. Collagen sponge fragments containing BMP-2 were added to each of the remaining 3 wells, Sterile physiological saline was added and stored at 37 ° C in an incubator.

When samples were taken, 1 ml each of 2 ml sterile physiological saline solution containing the released BMP-2 was collected using two 1.5 ml eppendorf tubes and stored at -70 ° C until BMP-2 ELISA. The BMP-2 immunoassay kit (R & D Systems, Inc., MN, USA) was performed according to the guideline given after 1, 2, 4, 7, 10, The amount was confirmed.

As shown in FIG. 4, it was confirmed that large amounts of BMP-2 were released from the alginate microbeads and the collagen sponge until the fourth day. BMP-2 was released in a similar manner in both delivery systems until day 7. Since then, Alginate microbeads retained a very small amount of BMP-2 release, but the Collagen sponge continuously released more BMP-2 than Alginate microbeads.

Alkaline Phosphatase (ALP) assay study

This experiment was conducted to quantify the bone morphogenesis of BMP-2 in alginate microbeads and collagen sponge delivery system. Alginate microbeads (B +) containing BMP-2, Alginate Microbeads (B-) without BMP-2, Collagen Sponge containing BMP-2 C +) and collagen sponge group (C-) without BMP-2, and cultured in an incubator at 37 ° C and 5% CO2. Alkaline Phosphatase Assay Kit (abcam) guideline. After completion of the plate, the experiment was carried out at Week 1 and 2.

As can be seen in FIG. 5, ALP activity on day 7 showed no significant difference between Alginate microbeads with BMP-2 and Alginate microbeads without BMP-2. However, ALP activity on day 14 was found to be significantly higher than that of Alginate microbeads with BMP-2.

Example 5: In vivo  Experiment

The animals used in this experiment were 6 male ICR mice at 8 weeks and 14 male Sprague Dawley rats at 10 weeks of age. Water and feed were randomly distributed and all animal experiments were conducted according to Guideline of Animal Experiment Ethics Committee of Kangwon National University.

Subcutaneous injection

Alginate microbeads containing BMP-2 were injected subcutaneously under the mouse to test whether the bone formation was successful at the injection site and whether there were other side effects. Alginate microbeads containing BMP-2 were subcutaneously injected subcutaneously in the left side of the body of the mouse. Alginate microbeads, which did not contain BMP-2, were injected subcutaneously into the right subcutis of the trunk and then fed for 4 weeks. After 4 weeks of incubation, Micro-Computed Tomography (MicroCT) and histological examination were performed.

As can be seen from FIGS. 6 and 7, the Micro CT test showed that a tissue having a density similar to that of a hollow bone was created at the injection site of the left subcutaneous body of the body, but nothing was formed at the subcutaneous injection site of the right subcutaneous body Respectively. Histological examination revealed that the resulting tissue was osteocyte with lamellar structure and bone marrow with bone marrow. Alginate microbeads were not completely decomposed on the inside of the generated bone tissue.

Calvarial  Experiment with Defect Model

This experiment was carried out to determine the effectiveness of alginate microbeads containing BMP-2 for healing of bone defects. A Calvarial defect model with a diameter of 5 mm was prepared using an 11-week-old male Sprague Dawley rats. The defect area was filled with Alginate microbeads containing BMP-2, Alginate microbeads without BMP-2, , And those with no collagen sponge. After 8 weeks, micro-computed tomography (MicroCT) and histological examination were performed.

As can be seen from FIG. 8, it was confirmed that the defects were recovered from the defect site by the injection of Alginate microbeads with BMP-2 and collagen sponge with BMP-2. However, Alginate microbeads without BMP-2 And the untreated group did not recover the defect site. In the recovered area, there was no significant difference between the groups treated with Alginate microbeads with BMP-2 and Collagen sponge with BMP-2 at the defect site. And bone tissues were significantly larger than the group injected with collagen sponge with BMP-2.

Claims (12)

In order to prepare an injectable alginate microencapsulated osteogenic protein, a method for preparing a microbead by mixing a bone forming protein with an alginate solution and using a vibration nozzle technique,
Na-alginate and osteogenic protein solution were mixed at a ratio of 3: 1, and the mixture was placed in a syringe. A calcium chloride solution was prepared as a gelling solution. A vibration frequency of 1,000 Hz and a flow rate of 23 mL / min , A stirrer speed of 8% and a voltage of 1,000 V, and a syringe containing a mixture of Na-alginate and osteogenic protein solution was placed to position the gelation solution at the injection position. Gelling solution for 20 minutes or more to solidify the microbeads. delete delete delete delete delete delete delete delete delete delete delete

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