KR102366334B1 - Porous Microscaffold for tissue regeneration having increased Magnetic driving force and Preparation Method thereof - Google Patents

Abstract

The present invention relates to a microscaffold and a method for manufacturing the same, wherein the microscaffold includes a porous polymer scaffold and magnetic nanoparticles coated with a positively charged polymer, so that the magnetic driving force is increased. , it can be usefully used for tissue regeneration.

Description

Porous microscaffold for tissue regeneration having increased Magnetic driving force and Preparation Method thereof

The present invention relates to a porous self-actuating microscaffold for tissue regeneration with increased magnetic driving force and a method for manufacturing the same.

Tissue regeneration is an important paradigm considered as a next-generation medical treatment to restore the function of damaged and degenerated tissues. As possible means in tissue regeneration therapy, therapeutic molecules such as drugs, growth factors (GF) and nucleic acids, as well as tissue cells, particularly progenitor cells and/or stem cells, may be used.

In order to restore damaged tissue using cells, a scaffold, which is a skeleton that helps cells form a tissue shape, is essential. These scaffolds play a very important role in restoring the original shape and connection with existing tissues in the field of tissue regeneration.

However, the magnetically driven scaffold under development has a problem in that a high magnetic field is required for smooth operation due to a low degree of attachment of magnetic nanoparticles, and thus a large magnetic field generating device is required for application to large animals.

Accordingly, there is an urgent need to study a scaffold having an increased magnetic driving force.

Domestic Patent Publication No. 10-2011-0028019

The present inventors have tried to develop a microscaffold with increased magnetic driving force. As a result, the present invention was completed by finding that a scaffold with increased magnetic driving force can be manufactured by binding magnetic nanoparticles coated with a polymer having a positive charge to a porous polymer.

Therefore, an object of the present invention is a porous (Porous) polymer scaffold (Scaffold); and magnetic nanoparticles bonded to the surface of the porous polymer scaffold.

Another object of the present invention is to provide a method for manufacturing a microscaffold.

The present inventors have tried to develop a microscaffold with increased magnetic driving force. As a result, it was confirmed that a scaffold with increased magnetic driving force could be prepared by binding magnetic nanoparticles coated with a positively charged polymer to a porous polymer.

The present invention relates to a biodegradable porous polymer scaffold, a microscaffold comprising magnetic nanoparticles bonded to the surface of the porous polymer scaffold, and a method for manufacturing the same.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a porous (Porous) polymer scaffold (Scaffold); and magnetic nanoparticles bonded to the surface of the porous polymer scaffold.

In the present invention, "porous" refers to an arrangement of pores, channels, and cages, and the arrangement of pores, channels and cages may be irregular, regular, or periodic. Further, the pores or channels may be isolated or interconnected and may be one-dimensional, two-dimensional or three-dimensional.

In the present invention, "scaffold" refers to a structure that serves as one of the important basic elements in the field of tissue engineering and serves to provide an environment suitable for cell adhesion, differentiation, and proliferation and differentiation of cells moving from the surrounding tissue, and three-dimensional It may be a spherical structure.

Specifically, tissue engineering technology is applied to almost all organs of the human body, such as artificial skin, artificial bone, artificial cartilage, artificial cornea, artificial blood vessel, and artificial muscle. A tissue-like scaffold should be provided. The basic requirements of an ideal scaffold are largely non-toxic, mechanical properties, and porosity.

On the other hand, the scaffold must be biocompatible, and cells must be able to attach and proliferate on the scaffold to form the equivalent of a tissue or organ. Therefore, the porous polymer can be considered as a substrate for cell growth in vitro or in vivo.

The specific type of the porous polymer is not particularly limited as long as it is a particle capable of forming a porous structure, but may include a natural polymer or a synthetic polymer.

The natural polymer may be collagen, hyaluronic acid, gelatin, or chitosan, and the synthetic polymer may be poly(lactic-co-glycolic acid) (PLGA), poly(PGA) glycolic acid)), PLA (poly(lactic acid)), PEG (polyethylene glycol), polyester, teflon, PET (polyethylene terephthalate), nylon, polypropylene, or polystyrene ( polystyrene), but is not limited thereto.

As the porous polymer scaffold exhibits the porous shape as described above, growth factors can be effectively loaded into the pores, channels and cages.

The porous polymer scaffold can be biodegradable and degraded through hydrolysis and/or oxidative degradation, or enzymatically or within a suitable time under the influence of microorganisms such as bacteria, yeast, fungi and algae.

The diameter of the porous polymer scaffold may be 100-500 μm.

Specifically, the diameter of the porous polymer scaffold may be 100 μm or more to have pores of sufficient size, and may be 500 μm or less to facilitate injection into the joint cavity through a syringe needle.

In addition, the diameter of the pores of the porous polymer scaffold may be 20 μm or more.

Specifically, when the pore diameter of the porous polymer scaffold is less than 20 μm, there is a limitation in that it is difficult for cells to penetrate into the porous polymer scaffold as well as difficult to deliver nutrients to the cells.

The magnetic nanoparticles refer to nanoparticles of various materials having magnetic sensitivity including a magnetic material therein, and if the magnetic nanoparticles are particles having magnetic sensitivity, the specific type is not particularly limited, but a magnetic material or a magnetic alloy can

The magnetic material is Fe, Co, Mn, Ni, Gd, Mo, MM' ₂ O ₄ or M _x O _y (M and M' are each independently Fe, Co, Ni, Mn, Zn, Gd or Cr, x is an integer of 1 to 3, and y is an integer of 1 to 5), but is not limited thereto.

The magnetic alloy may be CoCu, CoPt, FePt, CoSm, NiFe or NiFeCo, but is not limited thereto.

Since the magnetic nanoparticles exhibit the magnetic sensitivity as described above, it is possible to precisely target the microscaffold of the present invention to a tissue requiring regeneration through magnetic field control.

In addition, since the magnetic nanoparticles are hydrophilic and have a negative ionic charge, smooth decomposition and discharge through the body circulation of the magnetic nanoparticles are possible.

The diameter of the magnetic nanoparticles may be 1-1,000 nm or 50-500 nm. When the diameter of the magnetic nanoparticles exceeds 1,000 nm, there is a fear that the applicability to the living body may be reduced, such as blocking blood vessels when injected into the living body.

The magnetic nanoparticles may be coated with a polymer having a positive charge.

The positively charged polymer may be chitosan, collagen, or polyethyleneimine, but is not limited thereto.

The positively charged polymer may be electrostatically bound to the magnetic nanoparticles.

In one embodiment of the present invention, as the magnetic nanoparticles, ferahme (Clinical application) safety verified from the FDA was used.

The ferrahem has a small size of 30 nm, a high negative ionic charge of -37.27 mV, high colloidal stability and high in vivo dose safety, while the limited surface area of the porous polymer scaffold and its A strong anionic charge may limit the amount of adsorbable perahem on the surface.

Accordingly, in order to induce aggregation according to an increase in the size of the ferraheme (magnetic nanoparticles) and a change in ionic charge, chitosan (a polymer having a positive charge) was used by coating the ferraheme.

That is, since the magnetic nanoparticles are coated with the positively charged polymer, the magnetic properties of the microscaffold can be improved by increasing the loading amount of the magnetic nanoparticles in the microscaffold of the present invention.

Specifically, in general, magnetic nanoparticles may be covalently bonded to the surface of the porous polymer scaffold. However, in this case, the magnetic nanoparticles can be stably attached to the surface of the scaffold due to the strong bonding force of the covalent bond, but a problem in that the decomposition period is prolonged due to the long time for the bond to be broken may occur.

Accordingly, since the microscaffold of the present invention includes the magnetic nanoparticles coated with the positively charged polymer, the magnetic nanoparticles are weakly attached to the surface of the porous polymer scaffold due to the binding force of the electrostatic force, which is weaker than the covalent bond. and, thus, has the advantage of being decomposed in a shorter time than covalent bonding.

The magnetic nanoparticles bound to the positively charged polymer may have a size of micrometers (μm), and may be electrostatically coupled to the surface of the porous polymer scaffold.

Specifically, when the negatively charged magnetic nanoparticles are bonded to the positively charged chitosan, they may be electrostatically bonded to the negatively charged porous polymer scaffold surface.

The diameter of the microscaffold may be 100-500 μm.

Specifically, the diameter of the microscaffold may be 100 μm or more to have a pore of sufficient size, and may be 500 μm or less to facilitate injection into the joint cavity through a syringe needle.

The microscaffold can be widely used as a material for delivery of drugs, cells, proteins and growth factors or artificial skin in the fields of tissue engineering, medicine, pharmaceuticals and materials science.

Specifically, the microscaffold can be used for tissue regeneration.

In particular, the microscaffold can be used for articular cartilage regeneration that requires a relatively short biodegradation period (1-2 months).

Accordingly, another aspect of the present invention relates to a composition for tissue regeneration comprising the microscaffold.

Accordingly, the microscaffold can be used as an active ingredient for tissue regeneration through transplantation into a defect site.

The composition may include not only the microscaffold according to the present invention, but also an aqueous solution for maintaining it in a state suitable for implantation, for example, a buffer solution or an aqueous solution for injection.

Another aspect of the present invention relates to a method for manufacturing a microscaffold comprising the following steps.

A scaffold manufacturing step of manufacturing a porous polymer scaffold;

A coating step of coating the magnetic nanoparticles with a polymer having a positive charge; and

A magnetic scaffold manufacturing step of combining the porous polymer scaffold and the coated magnetic nanoparticles.

Hereinafter, the microscaffold manufacturing method of the present invention will be described in detail.

Scaffold Manufacturing Steps

The scaffold manufacturing step may be performed by emulsification using a fluidic device.

The emulsion generally refers to a mixture of two or more immiscible liquids, and the two or more liquids may be mixed to form various types of emulsions.

Specifically, the emulsion may be, for example, an oil-in-water emulsion in which oil is a dispersed phase and water is a dispersion medium, water is a dispersed phase and oil is a dispersion medium It may also be a phosphorus water-in-oil emulsion. It may also be a water-in-oil-in-water emulsion, wherein the water-in-oil emulsion is the dispersed phase and water is the dispersion medium, and the oil-in-oil emulsion is the dispersed phase and the oil is the dispersion medium. -water-in-oil) It may be an emulsion, but is not limited thereto.

The emulsification includes all methods commonly used in (multi) emulsion production, and may be a method using mass transfer in flow channels using a fluidic device, but this It is not limited.

coating step

This step may be performed independently of the scaffold manufacturing step, and the order is irrelevant.

In this step, the positively charged polymer may be coated with the magnetic nanoparticles by electrostatically binding to the magnetic nanoparticles.

Magnetic-scaffold fabrication steps

The magnetic-scaffold manufacturing step is a method of electrostatically binding magnetic nanoparticles coated with a polymer having a positive charge in the first coating step to the surface of the porous polymer scaffold prepared in the scaffold manufacturing step. can be performed by

In the method of manufacturing the microscaffold, overlapping contents of the microscaffold are omitted in consideration of the complexity of the present specification.

The present invention relates to a microscaffold and a method for manufacturing the same, wherein the microscaffold includes a porous polymer scaffold and magnetic nanoparticles coated with a positively charged polymer, so that the magnetic driving force is increased. , it can be usefully used for tissue regeneration.

1 is a schematic diagram of a microscaffold according to an embodiment of the present invention.
2A is a scanning electron microscope (SEM) image photograph of chitosan-coated magnetic nanoparticles prepared according to an embodiment of the present invention.
2B is a result of measuring the zeta potential value of the microscaffold according to an embodiment of the present invention.
3A is a scanning electron microscope (SEM) image photograph of a microscaffold according to an embodiment of the present invention.
Figure 3b is a result of confirming the components of the microscaffold according to an embodiment of the present invention.
3C is a result of measuring the size of the microscaffold and the size of the pores according to an embodiment of the present invention.
3D is a result of confirming the chemical composition of the microscaffold according to an embodiment of the present invention.
4 is a result of confirming the magnetic reactivity of the microscaffold according to an embodiment of the present invention.
6 is a result of confirming the magnetic drivability of the microscaffold according to an embodiment of the present invention.
7 is a result confirming the stem cell culture possibility and toxicity of the microscaffold according to an embodiment of the present invention.
8 is a result of confirming the stem cell differentiation ability of the microscaffold according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

manufacturing example. Preparation of microscaffolds

go. Fabrication of fluidic devices

polymer solution storage unit; emulsifier solution storage unit; a transport gas providing unit for transporting the polymer solution and the emulsifier solution; a pressurizing unit for providing pressure to the conveying gas; an injector unit for generating and discharging a scaffold dispersion by mixing the polymer solution and the emulsifier solution; A fluidic device comprising; a scaffold generating unit for receiving the scaffold dispersion and removing the organic solvent and dispersion medium contained therein to generate a microscaffold was manufactured.

me. Preparation of porous polymer scaffolds

After adding PVA (Polyvinyl alcohol) to distilled water in a weight ratio of 1%, the mixture was stirred at 270° C. at 500 rpm for 1 hour to prepare an external solution (water phase).

Next, 0.07 g of PLGA (poly(lactic-co-glycolic acid)) was dissolved in 0.931 mL of dichloromethane, and then 15 uL of Span 80 was added. An oil phase internal solution was prepared by stirring at 3200 rpm for 7 minutes with a vortex mixer. 3 g of gelatin was added to a 1% PVA solution in a weight ratio of 6%, and heated and stirred at 41.5° C. to prepare a water phase internal solution. After adding 850 uL of the aqueous phase internal solution to the oil phase internal solution, it was mixed by stirring at 3200 rpm for 2 minutes and 30 seconds with a vortex mixer.

The external solution was flowed to the external microtubule of the injector (dual microtubule) of the fluid device manufactured in A. at an injection pressure of 10 kPa, and the mixed internal solution was flowed to the internal microtubule at an injection pressure of 20 kPa, so that A polymer scaffold was prepared.

The prepared scaffold was left for at least 3 hours in distilled water at 10° C., washed 3 times in distilled water at 35° C. to remove gelatin in the structure, and dried at room temperature and stored.

all. Preparation of chitosan-coated magnetic nanoparticles

By dissolving 10 mg of chitosan (Chitosan) in 20 mL of 1% acetic acid, 0.5 mg/mL of a chitosan solution was prepared, and 1 mL of ferahme (30 mg/mL) was added thereto and mixed. Then, the chitosan @ ferraheme particles were adhered to each other with a magnet so that the particles were not dispersed, and then centrifuged (10000 rpm, 5 minutes) to remove the supernatant except for the particles. After adding 30 mL of deionized water, each step was repeated 3 times to remove residual acetic acid.

Example. Preparation of microscaffolds of the present invention (porous polymer scaffolds bound with chitosan-coated magnetic nanoparticles)

10-30 mg/mL of the chitosan-coated magnetic nanoparticles (corresponding to the chitosan@ferrahem particle) solution prepared in C. above was put into the tube containing the porous polymer scaffold prepared in B., and reacted for 6 hours. After washing with deionized water, it was stored.

Experimental Example 1. Chitosan-Coated Magnetic Nanoparticle Characteristics Confirmation

Using a scanning electron microscope (scanning electron microscopy, SEM), the image of the chitosan-coated magnetic nanoparticles prepared in Preparation Example C. was taken.

As can be seen in Figure 2a, the prepared chitosan-coated magnetic nanoparticles were found to be magnetic aggregates generated by ionic interaction between chitosan and ferrahem.

Next, the size and zeta potential of the chitosan-coated magnetic nanoparticles prepared in Preparation Example C. were measured using a surface zeta potential and particle size analyzer (ELS-8000, Japan). .

As can be seen in FIG. 2b, the size (diameter) of the chitosan-coated magnetic nanoparticles was 1.48 μm on average, and it was found that they exhibited a weaker anionic charge (-13.17 mV) than that of uncoated ferraheme. These results mean that chitosan and magnetic nanoparticles (ferrahem) reacted through an ionic charge interaction.

Experimental Example 2. Chitosan-Coated magnetic nanoparticles bound porous polymer scaffold properties

Images of the microscaffolds of the above examples were taken using a scanning electron microscope (SEM).

As can be seen in Figure 3a, the size of the prepared microscaffold and the size of the pores regardless of the attachment of the chitosan-coated magnetic nanoparticles were almost unchanged compared to the porous polymer scaffold prepared in Preparation Example B. Could know.

The components of the above examples were confirmed using a metal component analyzer (energy dispersive X-ray spectrometry, EDX).

As can be seen in FIG. 3b , signals of carbon (C), oxygen (O), and iron (Fe) were detected in the microscaffolds of Examples. These results mean that the chitosan-coated magnetic nanoparticles are present in the microscaffolds of the examples.

Based on the SEM image of FIG. 3A, the size of the microscaffolds and pores thereof of the Example was measured using ImageJ software (National Institutes of Health, USA).

As can be seen in FIG. 3c , the size of the microscaffold and the pore size of the example were 357.55±18.57 μm and 43.85±13.39 μm, respectively.

Finally, the chemical composition was analyzed by measuring the IR absorption band of the Example using a spectrophotometer (Nicolet Nexus 670 FTIR spectrophotometer, Thermo Nicolet Corporation, USA). At this time, chitosan, ferrahem, the chitosan-coated magnetic nanoparticles of Preparation Example C., and the porous polymer scaffold of Preparation Example B. were also measured and compared.

As can be seen in FIG. 3D, the Example showed the main IR absorption band of the chitosan-coated magnetic nanoparticles of Preparation Example C., and the porous polymer scaffold of Preparation Example B. Therefore, it was found that each support mixture was included in the above examples.

In particular, peaks near 630 and 550 cm ^-1 indicate the presence of maghemite (γ-Fe2O3), which is one of iron oxide, and similar peaks also appeared in ferraheme and chitosan-coated magnetic nanoparticles. These results mean that ferrahem was composed of iron oxide core of γ-Fe2O3.

Experimental Example 3. Confirmation of magnetic reactivity

After putting the microscaffold of Example into a vial filled with physiological saline and shaking, magnetic reactivity was confirmed by applying a permanent magnet.

As can be seen in FIG. 4 , in the state of physical shaking (ii), the microscaffolds in the vial were located on the floor or spread in the liquid, but when the permanent magnet was brought close (iii), it moved rapidly toward the permanent magnet. knew what to do These results suggest that the microscaffolds respond sensitively to external magnetic fields.

Experimental Example 4. Analysis of magnetic properties

Using a vibrating sample magnetometer (VSM, Lake Shore Cryotronics 7404, USA), chitosan-coated magnetic nanoparticles of Perahem, Preparation Example C., and magnetic hysteresis curves for the microscaffolds of Examples hysteresis curve) was obtained.

As can be seen in FIG. 5, the magnetization property values of ferraheme (magnetic nanoparticles) and chitosan-coated magnetic nanoparticles are 58.94 emu/g and 53.67 emu/g, respectively, and the magnetization property values of the magnetic nanoparticles due to the chitosan coating are was found to have decreased. This result means that the binding between chitosan and ferrahem occurred, and it can be seen that about 9.11 wt% of chitosan is present in the chitosan-coated magnetic nanoparticles.

In addition, it can be seen that when the chitosan-coated magnetic nanoparticle solution was supported at different concentrations (10, 20, 30 mg/mL), the magnetic saturation of the microscaffold gradually increased to 7.78, 11.35, and 15.98 emu/g. there was. These results mean that more chitosan-coated magnetic nanoparticles can be attached to the surface of the microscaffold when a high concentration of chitosan-coated magnetic nanoparticles is used, and in particular, 30 mg/mL of chitosan-coated magnetic nanoparticles It can be seen that when the magnetic nanoparticle solution is used, about 27.15 wt% of ferrahem is present in the microscaffold.

Finally, it was found that the magnetic saturation of the chitosan-coated magnetic nanoparticles-supported microstructure was about twice as large as that of the conventional magnetic nanoparticles (PEI-MNP)-supported microstructure using a covalent bond.

Experimental Example 5. Confirmation of magnetic drivability

First, a knee phantom was manufactured from a 3D CAD model of the knee using a 3D printer. After that, a target area was formed on the inner side of the femur, and the manufactured knee phantom was placed in an acrylic chamber filled with saline. Then, the movement to the target area for about 100 microscaffolds located between the joint defect sites of the knee phantom was observed.

As can be seen in FIG. 6 , it was found that the microscaffolds can move to a desired point through the magnetic field generated by the electromagnetic field generating device. This result means that swarm motion by the magnetic field of the microscaffold is possible. Therefore, it is expected that the microscaffolds of the present invention can be usefully used in clinical applications for confirming cartilage regeneration in vivo, which requires a minimum of several hundred to several thousand microscaffolds.

Experimental Example 6. Stem cell culture and toxicity evaluation

About 5.8Х10 ³ pieces of mouse bone marrow-derived stem cells (ATCC, VA, USA) were loaded on the microscaffolds of the above Examples and the porous polymer scaffolds of Preparation Example B.

Cell viability was measured using an alamar blue solution on the 2nd, 4th and 6th days of loading, and the cytotoxicity of cells to the microscaffold compared to the porous polymer scaffold was calculated and This was evaluated.

[formula]

Cytotoxicity =

As can be seen in FIG. 7 , the cell viability of the microscaffolds compared to the porous polymer scaffolds was 90% or more, confirming that there was little toxicity to the cells.

Experimental Example 7. Confirmation of stem cell differentiation ability

About 5.8Х10 ³ of mouse bone marrow-derived stem cells (ATCC, VA, USA) were loaded on the microscaffold (3D-culture) and 24-well microplate (2D-culture) of the above example. As a control group, undifferentiated mouse bone marrow-derived stem cells were used.

TGF-β1 (Peprotech), dexamethasone (Sigma Aldrich), ascorbic acid (Sigma Aldrich), sodium pyruvate (Sigma Aldrich), proline (Sigma Aldrich), human transferrin (human transferrin) ) and selenous acid (Gibco), the microscaffold, microplate, and undifferentiated mouse bone marrow-derived stem cells were put in a differentiation medium containing 5% CO ₂ , and differentiated for 3 weeks at 37° C. culture conditions. . At this time, the differentiation medium was replaced once every 2-3 days.

After differentiation, RNA was isolated using an RNA extraction kit (Takara), and cDNA was synthesized using a cDNA synthesis kit (Takara). Then, the expression levels of Collagen II, SOX9 and agreecan, which are genetic factors related to cartilage differentiation, were measured using RT-PCR (real-time PCR).

As can be seen in FIG. 8 , the expression levels of Collagen II, SOX9 and agreecan were high in 2D-culture at the 1st week of differentiation, but at the 2nd and 3rd weeks of differentiation, the stems in the microscaffolds of Example were compared with the 2D-culture. When the cells were differentiated, the expression levels of the genes were higher.

Claims (11)

Porous (Porous) Polymer Scaffold (Scaffold); and
Including; magnetic nanoparticles bonded to the surface of the porous polymer scaffold;
The magnetic nanoparticles are coated with a polymer having a positive charge, the polymer having a positive charge is electrostatically bonded to the magnetic nanoparticles, and the magnetic nanoparticles bonded to the polymer having a positive charge are Electrostatically bonded to the surface of the porous polymer scaffold, the microscaffold (Microscaffold). According to claim 1, wherein the porous polymer is PLGA (poly(lactic-co-glycolic acid)), PGA (poly(glycolic acid)), PLA (poly(lactic acid)), PEG (polyethylene glycol), collagen (Collagen) ), hyaluronic acid (Hyaluronic acid), gelatin (Gelatin) and chitosan (Chitosan) will include any one or more selected from the group consisting of, the microscaffold. According to claim 1, wherein the magnetic nanoparticles are Fe, Co, Mn, Ni, Gd, Mo, MM' ₂ O ₄ , M _x O _y (M and M' are each independently Fe, Co, Ni, Mn, Zn, Gd, or Cr, x is an integer of 1 to 3, y is an integer of 1 to 5), CoCu, CoPt, FePt, CoSm, NiFe and NiFeCo comprising any one or more selected from the group consisting of , microscaffolds. delete The microscaffold according to claim 1, wherein the polymer having a positive charge is at least one selected from the group consisting of chitosan, collagen, and polyethyleneimine. delete A composition for tissue regeneration comprising the microscaffold of any one of claims 1 to 3 and 5. A method for preparing a microscaffold comprising the steps of:
A scaffold manufacturing step of manufacturing a porous polymer scaffold;
A coating step of electrostatically bonding a polymer having a positive charge to the magnetic nanoparticles and coating; and
A microscaffold manufacturing step of electrostatically bonding the coated magnetic nanoparticles to the surface of the porous polymer scaffold. The method according to claim 8, wherein the scaffold manufacturing step is performed by emulsification using a fluidic device. The method of claim 8, wherein the polymer having a positive charge is performed using at least one selected from the group consisting of chitosan, collagen, and polyethyleneimine. delete

Images