KR20180062657A - Cellulosenanofiber carrier using bioprinting technology and method for specific drug release using the same - Google Patents

Abstract

The present invention relates to a method for immobilizing target materials, preferably probiotics, proteins, and drugs, in a nanocellulose carrier by using bioprinting technology, and to a method for selectively releasing a target material in vivo using the carrier manufactured by the method. According to the present invention, a nanocellulose as a carrier can be used for food by having excellent biocompatibility as an organic synthetic fiber, and can be easily transferred by having excellent acid resistance, bile resistance, and mechanical properties. As a strategy for making a three-dimensional structure elaborately, it is easy to control sustained-release release because the target material (for example, probiotics, proteins, drugs, and the like) is immobilized (supported or encapsulated, bonded, and the like) to the nanocellulose carrier by using the 3D bioprinting technique. Thus, it is possible to easily control sustained release. In addition, it is possible to protect a target material from gastric acid and bile acid according to the selection of a cross-linked material. Also, it is possible to rapidly and selectively release a target material by an enzyme in the intestinal organs (the small intestine or the large intestine). A delivery system may be usefully applied to transfer health functional food firstly, and proteins, drugs, microorganisms, and the like secondly.

Description

[0001] The present invention relates to a cellulosic nano fiber delivery system using bioprinting technology and a selective release method using the same.

The present invention relates to a method for immobilizing a target substance, preferably a probiotic, a protein, a drug, etc., on a cellulose nano fiber transporter using a bioprinting technique and a method for selectively releasing a target substance in vivo using a carrier prepared by the method .

Probiotics are health-promoting microorganisms at the proper dose, collectively referred to as useful microorganisms, including lactobacilli, such as the well-known Lactobacillus or Bifidobacterium. In addition to beneficial effects for healthy individuals, probiotics are also effective for a variety of symptoms and diseases such as lactose intolerance, irritable bowel syndrome, diarrhea caused by antibiotics, obesity, and diabetes. Known mechanisms of probiotics include 1) inhibiting the growth of harmful bacteria by acting nutrients and binding sites competitively against harmful bacteria, 2) strengthening the intestinal barrier by promoting mucin secretion and epitherial cell proliferation, and 3) stabilizing the immune system in the body have.

The beneficial effect of probiotics is assumed to be caused by live bacteria reaching the intestine (small intestine or colon). However, the number of live bacteria reaching the actual intestine by gastric acid and bile acid is less than one millionth. To overcome this problem, a method of consuming probiotics in excess of 10 times was used, but there was a limitation in that it was not a fundamental solution as well as using expensive probiotic raw materials. Therefore, an encapsulation technique that enhances acid resistance is considered, but the number of viable bacteria that retention of the probiotics in the actual field is small. Therefore, the ideal probiotic delivery vehicle must have the properties of acid-fast, bile-resistant, and selectively releasable to protect the live bacteria. Because probiotics can be inactivated by pH, temperature, enzymes, osmotic pressure, exposure to oxygen, solvents, etc., it is difficult to produce transporters containing these properties in the traditional manufacturing process. There is also a limit to material selection because the material used must be biocompatible and should not adversely affect the probiotics. Natural polysaccharides such as alginate, carrageenan, agar, and ganoderma, or gelatin or whey powder have been studied but failed to meet the required properties. Especially, the characteristic of retention of probiotics in the intestine without being discharged from the body is difficult to develop due to the arrangement of immobilization technology for acid resistance and bile resistance.

As a result, the present inventors have made efforts to develop a novel carrier, and as a result, a cellulose nanofiber carrier having immobilized a target substance as well as probiotics was prepared using a bioprinting technique. The cellulose nano fiber transporter according to the present invention is a material produced based on organic compound fiber which is most abundant in nature and has no biocompatibility, especially for edible use. It does not affect the activity of a target substance, especially probiotics, It is possible to immobilize a large amount of probiotics as compared to the previously developed probiotics encapsulation technique of a size of several hundreds of millimeters to a millimeter size and to enable the immobilization of a large amount of probiotics. (retention) is expected to be easy. In addition, the inkjet bio-3D printing technology, which is introduced for immobilizing a target substance, in particular, probiotics, is a technique capable of ejecting an ink in a very small droplet of several to several tens of picoliters. The ejection speed is as high as several to several hundred kH, It is easy to produce and uses less Drop-On-Demand (DOD) method which can print only the desired amount in desired position. It also has a high resolution because it ejects very small droplets. The present inventors have completed the present invention by confirming that they can be used for immobilizing various target substances such as drugs, proteins, bacteria, cells and the like by utilizing the advantage of uniformly discharging very small droplets.

Lee, BK, Yun YH, Choi JS, Choi YC, Kim JH and Kim YH. J. Pharm. 427 305-10 Katra W E, Palazzolo R D, Rowe C W, Giritlioglu B, Teung P and Cima M J 2000 Oral dosage forms fabricated by J. Dimensional Printing J. Control. Release 66 1-9 Iwanaga S, Saito N, Sanae H and Nakamura M 2013 Facile fabrication of uniform size-controlled microparticles and potentiality for tandem drug delivery system of micro / nanoparticles Colloids Surfaces B Biointerfaces 109 301-6

An object of the present invention is to provide a method for immobilizing a target substance on a cellulosenanofiber (CNF) carrier using 3D bio-printing technology.

Another object of the present invention is to provide a cellulosic nanofiber carrier prepared by the above method and a selective release method of a target substance in vivo using the same.

In order to achieve the above object, the present invention provides a method for immobilizing a target substance on a cellulosenanofiber (CNF) carrier using 3D bio-printing technology.

The present invention also provides a cellulose nanofiber carrier prepared by the above method.

The present invention also provides a method for selectively releasing a target substance in vivo using the carrier.

According to the present invention, the cellulose nanofiber as a carrier is an organic synthetic fiber, which has excellent biocompatibility and can be used for edible use. The cellulose nanofiber is excellent in acid resistance, bile resistance and mechanical properties, thus facilitating intestinal transmission. As a strategy for elaborating a three-dimensional structure, immobilization (carrying, encapsulating, bonding, etc.) of a target substance (for example, probiotics, proteins, drugs, etc.) to a cellulose nano fiber transporter using 3D bio- Since it produces uniform microparticles, it is easy to control the sustained release. In addition to having the property of protecting the target substance from stomach acid and bile acid according to the selection of the cross-linking substance, the delivery system can rapidly and selectively release the target substance by the enzyme in the intestine (small intestine or large intestine) Can be usefully applied to health functional foods, and secondarily to various proteins, drugs and microorganisms.

1 is a schematic view showing a method of fabricating cellulose nanofiber microparticles in which probiotics are immobilized (supported, encapsulated, bonded, etc.) using an inkjet bio-3D printer,
FIG. 2 is a schematic view showing an oxidized cellulose subjected to a TEMPO-mediated oxidation step,
FIG. 3 shows the survival of the probiotics encapsulated in CNF gel formed by using protamine, PLL, and CaCl2 as a control group after culturing for 24 hours, and lyophilized cells were plated on MRS agar plate medium and cultured for 48 hours at 37 ° C. The colonies of probiotics formed by the above-
Figure 4 shows the membrane integrity of the probiotics encapsulated in a cellulose nanofiber gel using CaCl2 as a crosslinking agent,
Figure 5 shows cell viability of probiotics in conditions corresponding to the stomach environment,
Figure 6 shows the bead stability of alginate, CNF, CNF-PL and CNF-CH beads at pH 7.4 corresponding to the intestine pH,
FIG. 7 shows changes in tryptic digestion of CNF particles in phosphate buffer under the absence of a cofactor,
FIG. 8 shows changes in tryptic digestion of CNF particles in phosphate buffer under the condition of CaCl 2 as a cofactor,
Figure 9 shows changes in tryptic digestion of CNF particles in the artificial intestinal fluid with CaCl2 as a cofactor,
Figure 10 shows the disintegration of CNF / polylysine beads by trypsin activity,
11 shows the shape of CNF particles according to the concentrations of CaCl2, protamine and PLL,
Figure 12 shows the shape of CNF particles according to the concentration of CNF and substrate (CaCl2, PLL, protamine)
13 shows the shape of a CNF particle according to a 3D printing pattern,
14 shows the shape of CNF particles according to the droplets velocity,
15 shows the shape of CNF particles according to the concentration of CaCl2, PLL, and protamine in circular jetting,
16 is a photograph showing a probiotic immobilized on 3D printing cellulose nano fiber microparticles.

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

The present invention provides a method for immobilizing a target substance on a cellulose nano fiber (CNF) carrier using a 3D bio-printing technique.

The immobilization is not limited to the concept including both carrying, encapsulating, or bonding a target substance in the cellulose nanofibers.

A system for immobilizing a target substance on a cellulosenanofiber (CNF) carrier using 3D bio-printing technology is shown in FIG.

As shown in FIG. 1, an inkjet printer head module included in a target substance-bearing cellulosic nanofiber carrier production system according to the present invention comprises: a cartridge for storing ink; A nozzle package having a micro-sized diameter for discharging the target material-bearing cellulose nano-fiber ink; And a cross-linking agent aqueous solution tank for gelating the discharged ink into microparticles.

The ink may be a cellulose-derived hydrogel precursor for protecting a target material.

The target substance collectively refers to a substance that can be contained in a carrier, and includes, without limitation, microorganisms, proteins, low-molecular or high-molecular drugs, cells, oil / inorganic substances and the like including probiotics.

The probiotics include, but are not limited to, strains belonging to the genus Lactobacillus or strains belonging to the genus Bifidobacterium.

The crosslinked material aqueous solution means a positively charged substance that gels the negative electrode cellulose-derived hydrogel precursor, and may be an ionic aqueous solution, an aqueous protein solution, or an aqueous carbohydrate solution, but is not limited thereto.

The ionic aqueous solution includes a calcium ion-containing aqueous solution, a strontium ion aqueous solution, and the like, but is not limited thereto.

The protein aqueous solution includes, but is not limited to, an aqueous protein solution that contains positively charged amino acid substances such as asparagine, glutamine, lysine, arginine, and histidine such as protamine and poly-L-lysine.

The aqueous polymer solution includes, but is not limited to, aqueous chitosan solution.

The nozzle package may be detachable, detachable, or integral with the cartridge. The nozzle package may have one or more nozzles, and the diameter of the nozzles may have a size between 20 um and 120 um, but is not limited thereto.

The brine aqueous solution water tank includes, but is not limited to, a petri dish, a chalet, a beaker, a vial, and the like, which can contain a liquid.

The crosslinked material aqueous solution tank preferably includes a stirring function through a magnetic stirrer, a stirrer bar, an ultrasonic wave or a mechanical stirrer to prevent aggregation and binding of the fine particles formed.

The present invention also provides a cellulose nanofiber carrier prepared by the above method.

The delivery may be in the form of nanogels or nanoparticles, and the average particle size may be from a few nanometers to a few tens of microsamples, but is not limited thereto.

The present invention also provides a composition for intracorporeal delivery of a target substance, which comprises a Cellulosenanofiber (CNF) carrier having a target substance immobilized using a 3D bio-printing technique.

The target substance collectively refers to a substance that can be contained in a carrier, and includes, without limitation, microorganisms, proteins, low-molecular or high-molecular drugs, cells, oil / inorganic substances and the like including probiotics.

The composition may be used for the intestinal delivery of a target substance, preferably a probiotic.

The present invention also provides a composition for organoselective delivery of a target substance, which comprises a Cellulosenanofiber (CNF) carrier having a target substance immobilized using 3D bio-printing technology.

The composition may be used for organoselective delivery of a target substance and may be selectively delivered to a target organ suitable for the target substance such as, for example, stomach, small intestine, large intestine or diseased tissue (e.g. cancer cells) . ≪ / RTI >

The present invention also provides a composition for controlling emission of a target substance, which comprises a Cellulosenanofiber (CNF) carrier in which a target substance is immobilized using a 3D bio-printing technique.

In one embodiment, the composition may be made into a sustained-release preparation and used for delayed release (delivery) of the target material. In another aspect, the composition may be used for stimulation-sensitive release (delivery) of a target substance to be released according to external stimuli such as temperature, pH, electric field, magnetic field, and metabolism, but is not limited thereto.

The present invention also provides a method for selectively releasing a target substance in vivo using the carrier.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example  One. Probiotic  pharmacy

Lactobacillus rhamnosus GG, Lactobacillus plantarum, and Lactobacillus acidophilus were inoculated into the MRS liquid medium and cultured at 37 ° C for 24 hours. The culture was centrifuged at 4800 rcf for 10 minutes, the supernatant was removed, and the cells were recovered and washed twice with 0.85% NaCl. Thereafter, an appropriate amount of 0.85% NaCl was added to obtain a 30-fold concentrated probiotic broth. To the probiotic broth, a solution containing 10% of water and Trehalose was added in an equal amount to obtain a concentrated broth of probiotics 15 times. The above-mentioned bacterial solution was subjected to freeze-drying treatment (freeze drying apparatus FD-1000, temperature: -40 degrees or less, degree of vacuum: 10 Pa or less) for 24 hours to obtain lyophilized probiotic bacteria.

Example  2. Inkjet  Using 3D printing Probiotics Bearing

In this Example, a cellulose nanofiber carrier containing probiotics as a target substance was prepared. More specifically, a polystyrene petri dish having a diameter of 35 mm was coated with a 2% (w / v) aqueous solution of calcium chloride; 2% (w / v) poly-L-lysine aqueous solution or 2% (w / v) protamine crosslinking agent solution. Lactobacillus rhamnosus GG or Lactobacillus plantarum or Lactobacillus acidophilus was added to 0.5% (w / v) nano-cellulose aqueous solution filtered with a 0.45 um syringe filter. And mixed with the strain to prepare 4 mg / ml. The prepared ink was mounted on a cartridge of a bio-inkjet 3D printing system, and then the ink was ejected at an input voltage of 80 V and a speed of 100 Hz. The discharged ink is received in the aqueous crosslinking solution and gelated. To prevent aggregation and binding of the formed fine particles and ensure uniformity, a magnetic stirrer was placed in the aqueous solution of the crosslinked material aqueous solution, followed by stirring at 200 rpm. The above experimental procedure is shown schematically in FIG.

Example  3. Cell survival analysis

Survival of the probiotics encapsulated in the cellulose nanofiber (CNF) gel formed using the protamine, PLL and CaCl2 prepared in Example 2 was confirmed by using the cells recovered after culturing for 24 hours and the cells lyophilized as a control group. For this purpose, 100 uL of 0.85% NaCl containing each substance was plated on MRS agar plate culture medium and cultured at 37 ° C for 48 hours to confirm colonies of probiotic bacteria formed. The results are shown in Fig.

Next, the probiotics encapsulated in the cellulose nanofiber gel using the CaCl 2 prepared in Example 2 as a crosslinking agent were confirmed using a Live / Dead Bacterial Viability Kit (Molecular Probes). First, 3 uL of SYTO 9 (3.34 mM) and Propidium iodide (20 mM) were mixed in 0.5 mL of distilled water to prepare a 2X solution. The cellulosic nanofiber gel prepared in Example 2 contained in 100 uL of 0.85% NaCl was added to the 2X solution in an equal amount. After that, it was left in a dark place for 15 minutes and photographed with Confocal microscopy (FV10i, Olympus). The results are shown in Fig. It can be confirmed that the probiotics are enclosed in the 3D-printed CNF particles.

Next, the survival rate of the probiotics encapsulated in the cellulose nanofiber transporter under the conditions corresponding to the stomach environment was confirmed because the probiotics had to endure the gastric acid to be administered after ingestion in order to be effective in the intestinal tract. To this end, the protective effect of the cellulose nanofiber gel prepared in Example 2 on the probiotics was evaluated by adding pH 2.3, 0.2% NaCl for 1.5 hours. The addition of 0.2% NaCl at pH 2.3 to the unencapsulated probiotics was followed by the addition of 0.68% KH2PO4 in the negative control, pH 7.4 as the stationary control. Probiotic protection against the low pH of the cellulose nanofibrous gel prepared in Example 2 The effects were evaluated. To determine the survival rate, 100uL of serial dilution samples were plated on MRS agar plate medium to confirm the number of colonies. The results are shown in Fig.

Example  4. Nano-cellulose Bead  Character analysis

The characteristics of nanocellulose beads immobilized on probiotics prepared by bio - 3D printing were measured by various methods and experimentally confirmed the release of nanocellulose beads in the intestines.

Figure 6 shows the results of evaluating the bead stability of alginate, CNF, CNF-PL and CNF-CH beads at pH 7.4 corresponding to intestine pH. In the case of alginate beads, deformation occurred under water and pH 7.4 conditions. In the case of CHF and the like, it remained stable in water and pH 7.4.

FIG. 7 shows changes in tryptic digestion of CNF beads in phosphate buffer under the absence of a cofactor. FIG. 8 shows changes in tryptic digestion of CNF beads in phosphate buffer under the condition of CaCl 2 as a cofactor. FIG. 9 shows changes in tryptic digestion of CNF beads in an artificial intestinal fluid with CaCl 2 as a cofactor. As a result, the deformation of nanocellulose beads can be confirmed after trypsin digestion. Figure 10 shows the disintegration of CNF / polylysine beads by trypsin activity, which confirms the deformation of beads after trypsin treatment.

11 shows the bead shape of CNF formed by bio-3D printing according to the concentration of CaCl2, protamine and PLL, and it can be confirmed that they have different sizes and shapes according to their concentrations. FIG. 12 shows CNF beads according to concentrations of CNFs and substrates (CaCl 2, Protein, and PLL) at different concentrations, and shows the shape of CNF beads according to various conditions of 3D printing. The formation of CNF beads of different shapes according to the printing pattern is shown in Figs. 13, 14, and 15. Fig. 3D printing patterns, droplets velocity, and circular jetting, CNF beads were prepared according to the concentration of CaCl1, protamine, and Pll, and the size and shape of each CNF bead can be controlled. 16 is a photograph showing a probiotic immobilized on 3D printed microparticles.

Claims (8)

A method of immobilizing a target substance on a cellulosenanofiber (CNF) carrier using 3D bio-printing technology.
The method of claim 1, wherein the target material is selected from the group consisting of microorganisms, proteins, drugs, cells, organic materials, and inorganic materials.
3. The method of claim 2, wherein the microorganism is a probiotic.
4. The method of claim 3, wherein the probiotics comprise a strain of the genus Lactobacillus or a strain of the genus Bifidobacterium.
The method of claim 1, wherein the immobilization is carried, encapsulated, or bonded.
(Cellulosenanofiber; CNF) carrier in which a target substance is immobilized using 3D bio-printing technology.
A composition for organoselective delivery of a target substance, comprising a Cellulosenanofiber (CNF) carrier in which a target substance is immobilized using 3D bio-printing technology.
A cellulosenanofiber (CNF) carrier comprising a target substance immobilized using 3D bio-printing technology.

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