CHITOSAN POROUS STRUCTURE-BASED MAGNETICALLY ACTUATED MICROROBOT

Abstract

The present invention relates to a porous structure-based magnetically actuated microrobot and a fabricating method therefor, wherein the porous structure-based magnetically actuated microrobot is based on a natural polymer having biocompatibility and biodegradability, so that the precise targeting of the porous microrobot through the attachment of magnetic nanoparticles and the drug and cell delivery using the porous microrobot can be attained.

Claims

1. A microrobot comprising: a porous film; and magnetic nanoparticles attached to pore spaces of the porous film.

2. The microrobot of claim 1, wherein the porous film contains at least one type of natural polymer selected from the group consisting of chitosan, gelatin, alginic acid, and hyaluronic acid.

3. The microrobot of claim 1, wherein the porous film contains at least one type of element selected from the group consisting of C, O, and N, which are main ingredients of a polymer.

4. The microrobot of claim 1, wherein the diameter of the pore spaces is 35 to 130 μm.

5. The microrobot of claim 1, wherein the magnetic nanoparticles contain at least one type selected from the group consisting of Fe, Co, Mn, Ni, Gd, Mo, MM′.sub.2O.sub.4, M.sub.xO.sub.y, CoCu, CoPt, FePt, CoSm, NiFe, and NiFeCo, in which: M and M′ each are 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.

6. The microrobot of claim 5, wherein the diameter of the magnetic nanoparticles is 1 to 1,000 nm.

7. The microrobot of claim 1, wherein the magnetic nanoparticles are microporous.

8. The microrobot of claim 1, wherein cells or a drug is loaded in the microrobot.

9. The microrobot of claim 8, wherein the microrobot contains a cell adhesion-related protein.

10. A method for fabricating a microrobot, the method comprising: a porous film manufacturing step of manufacturing a porous film; a porous structure forming step of processing the porous film to form a porous structure; a microrobot preparing step of attaching magnetic nanoparticles to the porous structure to prepare a microrobot; and a material loading step of loading a material to be delivered in the microrobot.

11. The method of claim 10, wherein the porous film contains at least one type of natural polymer selected from the group consisting of chitosan, gelatin, alginic acid, and hyaluronic acid.

12. The method of claim 10, wherein in the porous film manufacturing step, the porous film is manufactured at −80 to −10° C.

13. The method of claim 10, wherein in the porous structure forming step, the porous structure is formed by laser processing of the film.

14. The method of claim 10, wherein in the microrobot preparing step, the magnetic nanoparticles are attached to the structure by electrostatic binding.

15. The method of claim 10, wherein in the material loading step, cells or a drug is loaded in the microrobot.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1A provides images showing the thickness of a chitosan porous film depending on the volume of a chitosan solution according to a preparative example of the present disclosure.

[0080] FIG. 1B is a graph showing the thickness of a chitosan porous film depending on the volume of a chitosan solution according to a preparative example of the present disclosure.

[0081] FIG. 1C provides images showing the pore space size of a chitosan porous film depending on the temperature according to a preparative example of the present disclosure.

[0082] FIG. 1D is a graph showing the pore space size of a chitosan porous film depending on the temperature according to a preparative example of the present disclosure.

[0083] FIG. 1E is an image showing a chitosan porous film manufactured according to a preparative example of the present disclosure.

[0084] FIG. 2A is an image showing a chitosan porous structure obtained by laser micro-processing according to a preparative example of the present disclosure.

[0085] FIG. 2B provides scanning electron microscope (SEM) images of porous structures processed in various shapes according to a preparative example of the present disclosure.

[0086] FIG. 3A is an image showing a magnetic particle-attached magnetically-actuated chitosan porous microrobot according to a preparative example of the present disclosure.

[0087] FIG. 3B is a scanning electron microscope image of a porous structure before the attachment of magnetic nanoparticles according to a preparative example of the present disclosure.

[0088] FIG. 3C is a scanning electron microscope image of a figure in which magnetic nanoparticles are bound to pore spaces according to a preparative example of the present disclosure.

[0089] FIG. 3D provides images showing elements of the microrobot to which magnetic particles are attached, as analyzed by energy dispersive spectrometry (EDS) according to a preparative example of the present disclosure.

[0090] FIG. 4A provides images showing the experimental results of verifying magnetic reactivity of a microrobot by using a permanent magnet according to an example of the present disclosure.

[0091] FIG. 4B provides images showing chitosan porous microrobots assembled in several shapes by external magnetic fields according to an example of the present disclosure.

[0092] FIG. 5A provides images showing the actuation figure of a microrobot delivering mesenchymal stem cells (MSC) with stained cytoplasm according to an example of the present disclosure.

[0093] FIG. 6A provides fluorescent images of fibronectin, a cell adhesion-related protein, of a microrobot according to an example of the present disclosure.

[0094] FIG. 6B is a graph showing the evaluation results of viability of adipose-derived mesenchymal stem cells loaded in a microrobot according to an example of the present disclosure.

[0095] FIG. 6C is a graph showing the evaluation results of viability of macrophages loaded in a microrobot according to an example of the present disclosure.

[0096] FIG. 7 is a diagram showing ex vivo degradation of a microrobot over time according to an example of the present disclosure.

BEST MODE FOR CARRYING OUT THE INVENTION

[0097] The present disclosure relates to a microrobot including: a porous film; and magnetic nanoparticles attached to pore spaces of the porous film.

DETAILED DESCRIPTION

[0098] Hereinafter, the present disclosure will be described in more detail with reference to examples. These examples are only for illustrating the present invention, and it would be obvious to those skilled in the art that the scope of the present invention is not construed as being limited to the examples.

Preparative Example 1: Manufacturing of Chitosan Porous Film

[0099] A chitosan porous film with multiple pore spaces existing therein was manufactured using chitosan, which is a material for fabricating a microrobot.

1-1. Observation of Volume Change of Chitosan Film Depending on Volume of Chitosan Solution

[0100] A chitosan powder was dissolved in 1% v/v acetic acid to prepare a 1.5% v/v chitosan solution. Then, residual impurities were removed using a filter with a filter pore size of 100 μm. Thereafter, 500, 625, 750, 875, and 1000 uL of solutions were poured on respective polystyrene molds with a diameter of 35 mm, and the thickness change depending on the volume of the solution was observed. There was a difference in thickness of the porous film depending on the volume of the chitosan solution, and it was verified that the thickness of the porous film increased with the increase in volume of the chitosan solution (FIGS. 1A and 1B).

[0101] The size of the microrobot increased with the increase in thickness of the porous film, which may lead to an increase in amount of cells or a drug that can be loaded, but a too large thickness of the porous film may restrict the accessible site of the microrobot within the body due to a large volume of the microrobot. Therefore, it is important to select the thickness of the porous film as appropriate.

1-2. Manufacturing of Chitosan Porous Films Having Various-Sized Pore Spaces

[0102] During the freezing of a chitosan solution, water was changed into ice crystals to undergo an emulsification step with chitosan. Only the ice as a solvent was removed by freeze-drying, and the remaining chitosan has a porous structure.

[0103] Therefore, the ice crystals formed in the emulsification step determine the size of pore spaces constituting the porous structure. The size of these ice crystals gradually decreases with the lowering in freezing temperature, and in order to investigate this, the manufactured molds were placed in a refrigerator and stored at −10, −15, −20, and −80° C. for 12 hours. The ice crystals were grown in the chitosan solution, followed by freeze drying for 12 hours in a freeze-dryer (FDCD-12003, OPERON, Korea), thereby manufacturing chitosan porous films (FIGS. 1C to 1E).

[0104] It was verified that the size of pore spaces was reduced the most at −80° C., which corresponds to the lowest temperature (FIG. 1C). It could be verified that as the lower the temperature, the smaller the size of pore spaces of the chitosan porous film, and thus the size of pore spaces of the chitosan porous film was adjusted through temperature control (FIG. 1D).

[0105] The chitosan porous film for fabrication of a microrobot was manufactured so as to have pore spaces of 35 to 130 μm at −15° C. in order to load large-sized cells as a cell therapeutic agent.

[0106] When two photons were controlled by existing photo-polymerization through two-photon lithography in order to form uniform pore spaces, photo cross-linkers and additives contained in a precursor of a microrobot potentially changed biological properties of the microrobot, causing unpredicted toxicity. Whereas, the chitosan porous film manufactured as shown in FIG. 1E was composed of a biocompatible material, so that the chitosan porous film can minimize an immune response in vivo when inserted in the living body.

Preparative Example 2: Manufacturing Method for Chitosan Porous Structure of Chitosan Porous Film Through Laser Micro-Processing

[0107] The chitosan porous film manufactured in Preparative Example 1 was cut by the Femtosecond pulse UV laser cutting machine (maximum 6 W, 343 nm wavelength) using various microrobot shapes designed by a computer-aided design; CAD) software, and subjected to laser micro-processing. After the laser micro-processing, the laser-unprocessed portion of the chitosan porous film was mechanically exfoliated from the mold by using a tweezer. In order to remove residual acetic acid introduced while the chitosan powder was dissolved in acetic acid during the manufacturing of the chitosan porous film, the laser processing-finished chitosan porous structure was immersed in an ethanol solution at 100, 80, 50, 30, and 0% for 2 hours, thereby finishing the processing of the chitosan porous structure (FIGS. 2A and 2B).

[0108] As shown in FIG. 2A, it can be verified that pore spaces were formed in the chitosan porous structure. In addition, as shown in FIG. 2B, confirmed by scanning electron microscope (SEM), the chitosan porous structure can be manufactured to have various sizes and various shapes of a circle, a donut, a square, a triangle, a hexagon, a heart, or a cross.

Preparative Example 3: Fabrication of Chitosan Porous Robot Containing Magnetic Nanoparticles

[0109] A chitosan porous microrobot containing magnetic nanoparticles was fabricated using the laser micro-processing-finished chitosan porous structure.

[0110] Referring to FIG. 3A, a chitosan porous microrobot according to the preparative example of the present disclosure is composed of a chitosan porous structure that was three-dimensionally manufactured using a chitosan porous film. The chitosan porous structure disclosed in (a) of FIG. 3A has a plurality of pore spaces. The chitosan porous microrobot disclosed in (b) of FIG. 3B was fabricated by binding magnetic nanoparticles to the plurality of pore spaces.

[0111] The magnetic nanoparticles bound to respective pore spaces can move independently by a magnetic field applied from the outside, and especially, when inserted in the living body, the magnetic nanoparticles serve to accurately move the chitosan porous microrobot to a target lesion site.

3-1. Manufacturing of Magnetic Nanoparticles

[0112] The magnetic nanoparticles, which are formed of ferumoxytol, were small-sized particles of 30 nm, approved by the U.S. Food and Drug Administration (FDA), and had a high negative charge (about −37.28 mV). This ferumoxytol, together with collagen type I, formed magnetic nanoparticles through electrostatic binding.

[0113] As a specific manufacturing method for the magnetic nanoparticles, 0.5 mg/mL collagen type I was first dissolved in 1% v/v acetic acid. Thereafter, 1 mL of ferumoxytol was poured into 20 mL of a collagen solution, followed by mixing. The mixed solution was stirred at 2500 rpm for 2 hours, and then washed six times with deionized water by centrifugation and stirring, thereby obtaining magnetic nanoparticles.

3-2. Fabrication of Chitosan Porous Microrobot

[0114] The prepared magnetic nanoparticles were attached to the surface of the laser-processed chitosan porous structure through electrostatic binding.

[0115] During the adsorption of magnetic nanoparticles, the magnetization direction of the microrobot was determined through two permanent neodymium magnets (N35 grade, 50 mm diameter and 10 mm thickness) separated by 50 mm. After magnetizing for 6 hours, the residual magnetic nanoparticles were washed three times with deionized water, and the microrobot thus fabricated was stored in deionized water at room temperature before use.

[0116] As a result of observing the chitosan porous microrobot thus fabricated through a scanning microscope, the chitosan porous structure before the attachment of the magnetic nanoparticles had smooth surface characteristics (FIG. 3B). Whereas, it was verified that the structure having magnetic nanoparticles attached to pore spaces thereof had rough surface characteristics due to the attachment of magnetic particles (FIG. 3C).

[0117] In addition, as a result of analyzing elements of the chitosan porous microrobot through energy dispersive spectrometry (EDS), C and O, which are ingredients derived from the chitosan porous structure, were confirmed. Whereas, Fe was derived from the magnetic nanoparticles, and this indicated that in the chitosan porous microrobot, the magnetic particles were bound to the respective pore spaces of the chitosan porous structure (FIG. 3D).

Example 1: Test of Magnetic Reactivity of Chitosan Porous Microrobots

[0118] In order to examine magnetic reactivity of chitosan porous microrobots, microrobots were placed in a physiological salt solution similar to the inside of the body, followed by physical shaking, and then the magnetic reactivity of the microrobots was investigated using a permanent magnet, and the results are shown in FIGS. 4A and 4B.

[0119] It was verified that the chitosan porous microrobots were concentrated toward the permanent magnet (FIG. 4A). Especially, the chitosan porous microrobots were assembled in several shapes by external magnetic fields (FIG. 4B). That is, the movement of the chitosan porous microrobots according to the direction of a magnetic field was confirmed. This indicates that when chitosan porous microrobots were injected into the body, the movement of the chitosan porous microrobots can be controlled according to the direction of the magnetic field formed outside the body.

Example 2: Test of Actuation of Chitosan Porous Microrobot

[0120] In order to investigate the actual movement of a chitosan porous microrobot, the microrobot was placed and actuated in a chamber containing a physiological salt solution. The microrobot was moved in a desired direction by controlling magnetic fields of 40 mT and 1.8 T/m generated from an electromagnetically actuated coil device.

[0121] The actuation state of the chitosan porous microrobot delivering cells with stained cytoplasm was checked using a fluorescence microscope (FIG. 5). It was verified that the chitosan porous microrobot could be actuated while containing cells, and especially, the chitosan porous microrobot could be actuated without losing magnetic reactivity thereof even after containing cells. These results indicate that the chitosan porous microrobot containing cells can be manipulatively oriented through a magnetic field, and accurately deliver a therapeutic agent (cells or a drug) to be delivered to a lesion.

Example 3: Evaluation of Viability of Adipose-Derived Stem Cells and Macrophages Contained in Chitosan Porous Microrobot

[0122] The viability of adipose-derived stem cells and macrophages contained in a cell spheroid composed of only stem cells and macrophages, in a chitosan porous structure (CPS), and in a chitosan porous microrobot (CPM) was evaluated.

[0123] In the culture of adipose-derived mesenchymal stem cells and macrophages (Raw 246.7) for cell viability evaluation, fetal bovine serum was added, so that fibronectin contained in the serum was allowed to bind to the surface and the inside of the microrobot well through an integrin receptor (FIG. 6A). The fibronectin bound to the microrobot serves to help a therapeutic agent (cells or a drug) to bind to the surface of the microrobot well.

[0124] After each type of cell spheroids, chitosan porous structures, and chitosan porous microrobots was dispensed into a round-bottom 96-well plate (Corning, USA), adipose-derived stem cells and macrophages cultured by adding fetal bovine serum were seeded with 30,000 cells/well each. At 24 hours (Day 1), 72 hours (Day 3), and 120 hours (Day 5) after cell seeding, the culture was removed from each well, and then 100 μL of the AlamarBlue cell viability reagent (Thermo Fisher Scientific Inc., USA) was added to each well together with the culture. At four hours after the addition, the fluorescent value of the supernatant was analyzed using a microplate reader (Varioskan Flash, Thermo Fisher Scientific) having excitation and emission wavelengths of 560 nm and 590 nm, respectively. The intensity detected in each sample on Day 1 was 100% as a basis, and based on this intensity, the intensities on Day 3 and Day 5 were calculated. In addition, the viability of adipose-derived stem cells and macrophages contained in the cell spheroid, chitosan porous structure, and chitosan porous microrobot were compared (FIGS. 6B and 6C).

[0125] As shown in FIGS. 6B and 6C, the spheroid composed of only adipose-derived stem cells and macrophages caused low cell viability due to a low-oxygen environment in the center portion of the cell spheroid. The chitosan porous structure lacks an amino group (e.g., fibronectin) that can serve to allow cells to adhere to the surface of chitosan, resulting in a deterioration in cell adhesion ability, causing low cell viability.

[0126] Whereas, the adipose-derived stem cells and macrophages contained in the chitosan porous microrobot showed the highest viability. The reasoning is that the surface of the chitosan porous microrobot was modified through the attachment of magnetic nanoparticles, and the fibronectin entirely bound to the inside or surface of the chitosan porous microrobot can help the adhesion of cells to effectively load the cells used as a therapeutic agent. Through such effective loading, the chitosan porous microrobot can deliver more cells to a target site.

[0127] Since the chitosan porous microrobot has a porous structure, the cells adhering to the inside or surface of the microrobot can receive sufficient oxygen and nutrients through the pore spaces constituting the porous structure.

[0128] Therefore, the fabricated microrobot does not affect cytotoxicity and can be easily degraded in vivo, and the magnetic nanoparticles attached to the surface of the chitosan porous microrobot can provide an environment suitable for cell growth and adhesion.

Example 4: Experiment of Degradation of Chitosan Porous Microrobot

[0129] Chitosan, which is a main ingredient of the chitosan porous microrobot, is known to be degraded by the enzyme lysozyme. Lysozyme is an enzyme that prevents bacterial infection by hydrolyzing mucus polysaccharides and the like contained in the cell walls of bacteria, and is an antibacterial enzyme produced by humans or animals. This lysozyme cleaves the glycosidic bonds of polysaccharide units in a polymer, and the degradation products having a small molecular weight are removed from the body. The chitosan degraded by the lysozyme enzyme in the body is present at a concentration of approximately 1 to 120 μg/mL in tissues in the body. In order to investigate the degradation of microrobot in the body, chitosan porous microrobots were cultured in 15 μg/mL and 120 μg/mL lysozyme and phosphate-buffered saline (PBS) for 31 days. In order to avoid the contamination of samples, lysozyme and phosphate buffered saline were exchanged every day. The size of the chitosan porous microrobot was observed using an optical microscope.

[0130] As a result of the experiment, the phosphate buffered saline had little effect on the degradation of the chitosan porous microrobot, but the size of the chitosan porous microrobot in the lysozyme solution gradually decreased by enzymatic degradation for 31 days. In particular, the degradation of the chitosan porous microrobot was accelerated in a high-concentration (120 μg/mL) lysozyme solution than a low-concentration (15 μg/mL) lysozyme solution, and a maximum size reduction of 47.6% was shown on Day 31. These results indicate that after the microrobots are delivered to a lesion site in the body and then release drugs and cells, the microrobots can be slowly degraded.