DRUG DELIVERY CARRIER INCLUDING PLGA AND BETA-CYCLODEXTRIN CONTAINING DRUG

Abstract

Provided is a drug delivery carrier including PLGA and β-cyclodextrin containing a drug. According to the drug delivery carrier, the time during which a drug stays in the living body may be prolonged, and due to the biodegradation thereof, few side effects occur.

Claims

1. A drug delivery carrier comprising: polylactic-co-glycolic acid (PLGA) and β-cyclodextrin containing a drug, wherein PLGA and the β-cyclodextrin are linked to each other by a linker.

2. The drug delivery carrier of claim 1, wherein PLGA and the β-cyclodextrin each have a thiol group.

3. The drug delivery carrier of claim 1, wherein the linker is a disulfide bond, and the thiol group of PLGA and the thiol group of the β-cyclodextrin form a disulfide bond.

4. The drug delivery carrier of claim 1, wherein a ratio of a glycolic acid to a lactic acid in PLGA is 3:1.

5. The drug delivery carrier of claim 1, wherein the drug delivery carrier has a porosity of about 30 vol % to about 50 vol %.

6. The drug delivery carrier of claim 1, wherein the drug is a pain treatment agent or an anesthetic agent.

7. The drug delivery carrier of claim 6, wherein the pain treatment agent is selected from the group consisting of celecoxib, diclofenac, diflunisal, piroxicam, meloxicam, etodolac, mefenamic acid, meclofenamic acid, ibuprofen, indometacin, ketoprofen, ketorolac, nabumetone, naproxen, nimesulide, sulindac, tepoxalin, tolmetin, neostigmine, magnesium, atropine, dexamethasone, prednisolone, prednisone, methyl prednisolone, triamcinolone, hydrocortisone, deflazacourt, betamethasone, budenoside, ketorolac, octreotide, ziconitide, droperidol, methotrexate, and haloperidol.

8. The drug delivery carrier of claim 6, wherein the anesthetic agent is selected from the group consisting of bupivacaine, levobupivacaine, ropivacaine, prilocaine, mepivacaine, benzocaine, tetracaine, and lidocaine.

9. The drug delivery carrier of claim 1, wherein the drug is released in a sustained manner.

10. The drug delivery carrier of claim 1, wherein the drug delivery carrier is produced by linking the β-cyclodextrin containing the drug to PLGA.

11. The drug delivery carrier of claim 1, wherein PLGA is electrospun to form nanofibers after a thiol end group is formed.

12. A method of preventing or treating a pain disorder comprising: administering, to a subject in need thereof, a composition including PLGA and 3-cyclodextrin containing a pain treatment agent, wherein PLGA and the β-cyclodextrin are linked to each other by a linker.

13. The method of claim 12, wherein PLGA and the β-cyclodextrin each have a thiol group.

14. The method of claim 12, wherein the linker is a disulfide bond, and the thiol group of PLGA and the thiol group of the β-cyclodextrin form a disulfide bond.

15. The method of claim 12, wherein the β-cyclodextrin further contains an anesthetic agent.

16. The method of claim 12, wherein the pain disorder is caused by one selected from the group consisting of neuropathic pain, osteoarthritis, rheumatoid arthritis, fibromyalgia, back and musculoskeletal pain, spondylitis, intervertebral disk escape, spinal canal stenosis, juvenile rheumatoid arthritis, diabetic neuropathy, spontaneous pain, hypersensitivity pain, phantom limb pain, complex regional pain syndrome migraine, toothache, abdominal pain, ischemic pain, and post-operative pain.

17. A method of preparing a drug delivery carrier, the method comprising forming a thiol end group in polylactic-co-glycolic acid (PLGA); entrapping a drug in β-cyclodextrin having a thiol group; and linking PLGA and the β-cyclodextrin via a disulfide bond.

18. The method of claim 17, further comprising electrospinning PLGA having the thiol end group therein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0061] FIG. 1A, FIG. 1B, and FIG. 1C show diagrams illustrating a method of preparing a polylactic-co-glycolic acid (PLGA)-CD-DEX-RVC according to an embodiment of the present disclosure:

[0062] FIG. 1A shows a diagram illustrating a synthesis process and the formula of PLGA-SH; FIG. 1B shows a diagram illustrating the structure of SH-β-CD; and FIG. 1C shows a diagram illustrating the process of entrapping a drug in SH-β-CD and linking the resultant structure to electrospun PLGA-SH;

[0063] FIGS. 2A-2E show images of the surface and porosity of PLGA-CD-DEX-RVC according to an embodiment and other nanofibers:

[0064] FIG. 2A shows a scanning electron microscope (SEM) image of PLGA-SH; FIG. 2B shows a SEM image of a PLGA-CD-DEX-RVC according to an embodiment; FIG. 2C shows a SEM image of PLGA-CD-DEX-RVC prepared by entrapping a drug in PLGA-S-S-CD; FIG. 2D shows a graph in which the porosity of PLGA-CD-DEX-RVC according to an embodiment is compared with of the porosities of PLGA, PLGA-SH and PLGA-CD+DEX-RVC; and FIG. 2E shows a diagram illustrating formulae of dexamethasone and ropivacaine entrapped in PLGA-CD-DEX-RVC according to an embodiment;

[0065] FIG. 3A, FIG. 3B, and FIG. 3C show graphs in which an element contained in PLGA-CD-DEX-RVC is compared with PLGA, PLGA-SH, and PLGA-S-S-CD according to an embodiment of the present disclosure:

[0066] FIG. 3A shows a graph of measurements of elements of PLGA, PLGA-SH, PLGA-S-S-CD, and PLGA-CD-DEX-RVC in a wide range; FIG. 3B shows a graph of measurements only in a narrow range in which F 1s is detected; and FIG. 3C shows a graph of measurements only in a narrow range in which N 1s is detected;

[0067] FIG. 4A and FIG. 4B show images from which the cytotoxicity of PLGA-CD-DEX-RVC according to an embodiment was confirmed:

[0068] FIG. 4A shows an image of cells from which the cytotoxicity of PLGA and PLGA-CD-DEX-RVC (Scale bar: 200 μm) was confirmed; and FIG. 4B shows a diagram from which the cytotoxicity of PLGA and PLGA-CD-DEX-RVC according to an embodiment was confirmed;

[0069] FIG. 5A and FIG. 5B show a diagram of the drug release rate of PLGA-CD-DEX and PLGA-CD-RVC according to an embodiment:

[0070] FIG. 5A shows a graph of the drug release time of the group in which PLGA was simply loaded with dexamethasone (PLGA+DEX) and the group in which dexamethasone was entrapped in SH-β-CD and then PLGA was linked thereto (PLGA-CD-DEX); and FIG. 5B shows a graph of the drug release time of the group in which PLGA was simply loaded with ropivacaine (PLGA+RVC) and the group in which dexamethasone was entrapped in SH-β-CD and then PLGA was linked thereto (PLGA-CD-RVC);

[0071] FIG. 6A, FIG. 6B, and FIG. 6C show images from which the anti-inflammatory effect of PLGA-CD-DEX-RVC according to an embodiment was confirmed:

[0072] FIG. 6A shows images of immunohistofluorescence-stained cells from which the differentiation into M1/M2 macrophages was confirmed (Scale bar: 50 μm); FIG. 6B shows a graph of the quantified expression of the inflammatory factor iNOS; and FIG. 6C shows a graph of the quantified expression of the inflammatory factor CD206;

[0073] FIG. 7A and FIG. 7B show images from which the pain reduction effect of PLGA-CD-DEX-RVC according to an embodiment was confirmed:

[0074] FIG. 7A shows a schematic diagram showing an animal experiment using a chronic constriction injury (CCI) animal model and the effect of PLGA-CD-DEX-RVC; and FIG. 7B shows a graph showing the pain reduction effect of PLGA-CD-DEX-RVC obtained through pain evaluation (cold allodynia);

[0075] FIG. 8A and FIG. 8B show in vivo decomposition effects of PLGA-CD-DEX-RVC according to an embodiment:

[0076] FIG. 8A shows images showing the degradation of PLGA-CD-DEX-RVC in vivo over time; and FIG. 8B shows a graph showing the degradation of PLGA-CD-DEX-RVC in vivo over time;

[0077] FIGS. 9A-9E show the expression levels of TRPV1, a pain factor, when PLGA-CD-DEX-RVC according to an embodiment was used for the treatment after nerve injury, wherein the expression levels were identified by immunofluorescence staining:

[0078] FIG. 9A shows a diagram illustrating that a pain signal is transferred from sensory neurons of the dorsal root ganglia (DRG) to the dorsal horn of spinal cord; FIG. 9B shows an image showing the expression of TRPV1, a pain marker, in DRG (Scale bar: 20 μm); FIG. 9C shows a graph of the quantified expression of TRPV1, a pain marker, in DRG; FIG. 9D shows an image showing the expression of TRPV1, a pain marker, in the spinal cord of dorsal horn (Scale bar: 100 μm); and FIG. 9E shows a graph of the quantified expression of TRPV1, a pain marker, in the spinal cord of dorsal horn; and

[0079] FIGS. 10A-10D show the expression levels of Iba1, an inflammatory factor, when PLGA-CD-DEX-RVC according to an embodiment was used for treatment after nerve injury, wherein the expression levels were identified by immunofluorescence staining:

[0080] FIG. 10A shows an image showing the expression of Iba1, an inflammatory marker, in DRG (Scale bar: 20 μm); FIG. 10B shows a graph of the quantified expression of Iba1, an inflammatory marker, in DRG; FIG. 10C shows an image showing the expression of Iba1, an inflammatory marker, in the spinal cord of dorsal horn (Scale bar: 20 μm); and FIG. 10D shows a graph of the quantified expression of Iba1, an inflammatory marker, in the spinal cord of dorsal horn.

DETAILED DESCRIPTION OF THE INVENTION

[0081] Hereinafter, the present disclosure will be described in more detail through Examples. However, these examples are intended to exemplarily describe the present disclosure, and the scope of the present disclosure is not limited to these examples.

[0082] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

Example 1. Preparation of Drug-Loaded PLGA-CD-DEX-RVC

[0083] Drug-loaded PLGA-CD was prepared as follows.

[0084] Specifically, polylactic-co-glycolic acid (PLGA) having a thiol (SH) end group was synthesized as follows. PLGA having a glycolic acid and lactic acid at a ratio of 75:25 was dissolved in dichloromethanol (DCM) together with N-hydroxysuccinimide and N,N′-dicyclohexylcarbodiimide at a molar ratio of 1:10:10, and then PLGA and ethylene diamine were added thereto at a molar ratio of 1:2. After 24 hours, after filtering using a 0.45 μm filter, the filtered solution was precipitated in cold diethyl ether and vacuum dried at room temperature to form an amine (NH.sub.2) end group in PLGA. The synthesized PLGA-NH.sub.2 was dissolved in DCM again, and a 5 M 2-iminothiolane hydrochloride methanol solution was added thereto to react for 1 day. The mixture was again precipitated in cold diethyl ether and vacuum dried to synthesize PLGA-SH having a thiol (SH) end group. The powder-type PLGA-SH synthesized as described above was dissolved at a concentration of 12 wt/v % in hexafluoroisopropanol (HFIP) and then electrospun at 200 rpm.

[0085] Next, the same molar concentration of dexamethasone (DEX) or ropivacaine (RVC) was added to a 1 μg/ml of mono-(6-mercapto-6-deoxy)-β-cyclodextrin (SH-β-CD) (purchased from AARON PHARMATEC.Ltd) solution, and each of these solutions was mixed by using a stirrer for 24 hours, and then freeze-dried to prepare SH-β-CD-DEX or SH-β-CD-RVC in a powder form in which DEX or RVC was entrapped.

[0086] Then, 5 μg/ml of (SH-β-CD-DEX+SH-β-CD-RVC/distilled water) in which a ratio of SH-β-CD-DEX to SH-β-CD-RVC was 1:1, was prepared, and then the resulting mixture was attached to the PLGA-SH nanofiber by disulfide bond (—S—S—), thereby preparing the final product, PLGA-CD-DEX-RVC nanofiber.

Experimental Example 1. Identification of Surface and Porosity of PLGA-CD-DEX-RVC

[0087] The surface structure and porosity of the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 were confirmed by SEM.

[0088] FIG. 2 shows images of the surface and porosity of PLGA-CD-DEX-RVC according to an embodiment and other nanofibers:

[0089] FIG. 2A shows a SEM image of PLGA-SH; FIG. 2B shows a SEM image of a PLGA-CD-DEX-RVC according to an embodiment; FIG. 2C shows a SEM image of PLGA-CD-DEX-RVC prepared by entrapping a drug in PLGA-S-S-CD; FIG. 2D shows a graph in which the porosity of PLGA-CD-DEX-RVC according to an embodiment is compared with of the porosities of PLGA and PLGA-SH; and FIG. 2E shows a diagram illustrating formulae of dexamethasone and ropivacaine entrapped in PLGA-CD-DEX-RVC according to an embodiment.

[0090] As a result, it was confirmed that even when compared with the case where the PLGA-SH solution was electrospun and the nanofibers were not bound with the drug (FIG. 2A), the PLGA-CD-DEX-RVC prepared in Example 1 also had a significantly structural shape without broken or pierced nanofibers as shown in FIG. 2B.

[0091] On the other hand, unlike the method of Example 1, in the case of the nanofiber PLGA-CD+DEX-RVC, which was prepared by obtaining PLGA-S-S-CD in which SH-β-CD was bound to PLGA-SH nanofibers, and then adding DEX or RVC drugs thereto, followed by vortexing, as shown in FIG. 2C, there were no nanofibers having normal shapes, that is, nanofibers had holes, were broken or melted.

[0092] In addition, porosity measurements show that, as shown in FIG. 2D, the degrees of porosity of PLGA nanofibers, PLGA-SH nanofibers, and PLGA-CD-DEX-RVC nanofibers prepared in Example 1 (the third graph of FIG. 2D) were almost similar to each other. On the other hand, as shown in FIG. 2C, it was confirmed that the degree of porosity of the nanofiber PLGA-CD+DEX-RVC, which was prepared by preparing PLGA-S-S-CD and then adding DEX or RVC drugs thereto, followed by vortexing, was greatly reduced. Numerical data obtained by quantitatively measuring the degree of porosity are shown in Table 1. These results indicate that the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 are suitable for use as a scaffold.

TABLE-US-00001 TABLE 1 Group Porosity (%) PLGA 40.02% ± 0.82 PLGA-SH 44.92% ± 4.10 PLGA-CD-DEX-RVC 40.11% ± 2.05 PLGA-CD + DEX-RVC 23.18% ± 4.37

Experimental Example 2. Confirmation of Presence of Drug Bound to PLGA-CD-DEX-RVC

[0093] Whether DEX and RVC drugs were well attached onto the surface of the PLGA-CD-DEX-RVC nanofiber prepared in Example 1, was confirmed by X-ray photoelectron spectroscopy (XPS).

[0094] FIG. 3 shows a graph in which an element contained in PLGA-CD-DEX-RVC is compared with PLGA, PLGA-SH, and PLGA-S-S-CD according to an embodiment of the present disclosure:

[0095] FIG. 3A shows a graph of measurements of elements of PLGA, PLGA-SH, PLGA-S-S-CD, and PLGA-CD-DEX-RVC in a wide range; FIG. 3B shows a graph of measurements only in a narrow range in which F 1s is detected; and FIG. 3C shows a graph of measurements only in a narrow range in which N 1s is detected.

[0096] As shown in FIG. 3, it was confirmed that the F element present only in DEX and the N element present only in RVC were detected in PLGA-CD-DEX-RVC, and the F element and the N element were not detected in PLGA, PLGA-SH, and PLGA-S-S-CD to which the drugs were not attached.

[0097] These results indicate that the drugs are well attached onto the PLGA-CD-DEX-RVC nanofibers prepared in Example 1.

Experimental Example 3. Cytotoxicity Confirmation

[0098] The cytotoxicity of the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 was confirmed.

[0099] Specifically, to obtain bone marrow-derived macrophage (BMM) for confirming cytotoxicity, SD rats, an experimental animal, were sacrificed and bone marrow was extracted from femur and tibia, and BMM was separated according to the manual. The isolated BMM cells were cultured on PLGA or PLGA-CD-DEX-RVC nanofibers, and one day later, live and dead staining was performed to identify live cells and dead cells (FIG. 4A). Also, cytotoxicity was quantitatively confirmed by using a cytotoxicity assay kit (EZ-Cytox, Daeil Labservice, Korea) (FIG. 4B).

[0100] FIG. 4 shows images from which the cytotoxicity of PLGA-CD-DEX-RVC according to an embodiment was confirmed:

[0101] FIG. 4A shows an image of cells from which the cytotoxicity of PLGA and PLGA-CD-DEX-RVC (Scale bar: 200 μm) was confirmed; and FIG. 4B shows a diagram from which the cytotoxicity of PLGA and PLGA-CD-DEX-RVC according to an embodiment was confirmed.

[0102] As a result, as shown in FIGS. 4A and 4B, it was confirmed that compared to PLGA, PLGA-CD-DEX-RVC had similar cell viability and no cytotoxicity.

Experimental Example 4. Confirmation of Drug Release Time

[0103] The drug release time of the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 was confirmed.

[0104] Specifically, the drug release time of a group (PLGA-DEX or PLGA-DEX) in which the drug was simply loaded on PLGA without p-CD was compared with the drug release time of a group (PLGA-CD-DEX or PLGA-CD-RVC) in which each drug was entrapped in β-CD and then bound to PLGA. To determine the degree of release of the drug over time, each group was placed in DPBS and reacted in a shaker at 37° C. at 100 RPM. At 1, 4, 8, 12, 24 and 48 hours, the amount of drug released in the supernatant was quantified using UV-Vis spectrophotometer.

[0105] FIG. 5 shows a diagram of the drug release rate of PLGA-CD-DEX and PLGA-CD-RVC according to an embodiment:

[0106] FIG. 5A shows a graph of the drug release time of the group in which PLGA was simply loaded with dexamethasone (PLGA+DEX) and the group in which dexamethasone was entrapped in SH-β-CD and then PLGA was linked thereto (PLGA-CD-DEX); and FIG. 5B shows a graph of the drug release time of the group in which PLGA was simply loaded with ropivacaine (PLGA+RVC) and the group in which dexamethasone was entrapped in SH-β-CD and then PLGA was linked thereto (PLGA-CD-RVC).

[0107] As a result, as shown in FIGS. 5A and 5B, it was confirmed that in the case of the group in which the drug was simply loaded on PLGA, 100% of the drug was released before 24 hours, and in the case of the group in which the drug was entrapped in SH-β-CD and bound to PLGA, the drug was continuously released for more than 48 hours. These results indicate that nanofibers in which the drug was entrapped in SH-β-CD and then bound to PLGA can be used as a sustained-release drug delivery carrier that slowly releases the drug.

Experimental Example 5. Confirmation of Anti-Inflammatory Effect

[0108] The anti-inflammatory effect of the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 was confirmed in vitro by immunohistochemical staining.

[0109] Specifically, a total of five groups were used to perform cell experiments: 1) a control group not treated with LPS, 2) a group induced to differentiate into macrophages by treatment with 1 μg/ml of LPS; 3) a LPS+DEX/RVC group treated with 1 μg/ml of LPS, and 0.05 mg/mL of DEX and 0.05 mg/mL of RVC, 4) a LPS+PLGA+DEX/RVC group treated with 1 μg/ml of LPS and PLGA loaded with 0.05 mg/mL of DEX and 0.05 mg/mL of RVC, and 5) a LPS+PLGA-CD-DEX-RVC group treated with 1 μg/ml of LPS and PLGA-CD-DEX-RVC in which β-CD entrapping 0.15 mg of DEX and 0.15 mg of RVC was bound to PLGA.

[0110] For each group, BMM cells (1.2×10.sup.5/well) were inoculated into 48-well culture plate and cultured. In the case of the groups 4) and 5), nanofibers were initially laid on the floor and inoculated with BMM cells. Then, the cells were treated with 1 μg/ml of LPS to induce an inflammatory reaction, and after 24 hours, the cells were immobilized by using 4% paraformaldehyde (PFA). When inflammation was induced in macrophages, an antibody against iNOS, which is a representative M1 marker secreted from macrophages, and an antibody against CD206 (Cluster of Differentiation 206), which is an M2 marker secreted to inhibit inflammation in macrophages, were used to perform staining using immunohistofluorescence, and expression levels were confirmed in a qualitative manner (FIG. 6A) and quantitative manner (FIGS. 6B and 6C). Rabbit anti-iNOS (1:500) and mouse anti-CD206 (1:500) were used as the primary antibodies, and donkey anti rabbit 647 (1:1000) and goat anti mouse 488 (1:1000) were used as the secondary antibodies, and after staining, the cells were mounted and images thereof were obtained by confocal microscopy.

[0111] FIG. 6 shows images from which the anti-inflammatory effect of PLGA-CD-DEX-RVC according to an embodiment was confirmed:

[0112] FIG. 6A shows images of immunohistofluorescence-stained cells from which the differentiation into M1/M2 macrophages was confirmed (Scale bar: 50 μm); FIG. 6B shows a graph of the quantified expression of the inflammatory factor iNOS; and FIG. 6C shows a graph of the quantified expression of the inflammatory factor CD206.

[0113] As a result, as shown in FIG. 6, it was confirmed that the group 5) treated with PLGA-CD-DEX-RVC showed a lower level of iNOS and a higher level of CD206 than the group 3) simply treated with DEX/RVC and the group 4) simply treated with PLGA loaded with DEX/RVC. These results indicate that PLGA-CD-DEX-RVC effectively inhibits inflammation compared to the treatment with the drug or the treatment with PLGA loaded with the drug.

Experimental Example 6. Confirm the Effect of Reducing Neurogenic Pain

[0114] The pain reduction effect of the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 was confirmed in vivo.

[0115] In detail, the sciatic nerve of a 8-week-old female SD rat was bound four times by using 4-0 nylon suture at 1 mm intervals to produce a chronic constriction injury (CCI) animal model (FIG. 7A). A total of four animal model groups were used in vivo, and the groups all received CCI damage: 1) a CCI group; 2) a DEX/RVC group in which 0.25 mg/kg of DEX and 0.25 mg/kg of RVC were injected in a solution state into the injured area, 3) a PLGA+DEX/RVC group in which the injured area was treated with PLGA nanofibers, simply loaded with 0.25 mg/kg of DEX and 0.25 mg/kg of RVC, and 4) a PLGA-CD-DEX-RVC group in which PLGA-CD entrapped 0.15 mg of DEX and 0.15 mg of RVC. Pain assessments (tests using cold allodynia, acetone solution) were performed at the day intervals of 1, 2, 3, 5, 7, 10 and 14 after the completion of surgery. The higher the score, the more the animal models repeat the act of kicking or licking legs, which means the degree of pain is severe. The highest score was point 9. In the case of no response, the lowest score, 0, was applied. The act of moving and kicking legs may score point 1, and the act of continuously crouching or repeatedly kicking legs may score point 2. When the animal models repeated the act of licking and kicking their legs, the score was point 3. Each mouse was repeatedly tested three times and the sum score was measured.

[0116] FIG. 7 shows images from which the pain reduction effect of PLGA-CD-DEX-RVC according to an embodiment was confirmed:

[0117] FIG. 7A shows a schematic diagram showing an animal experiment using a chronic constriction injury (CCI) animal model and the effect of PLGA-CD-DEX-RVC; and FIG. 7B shows a graph showing the pain reduction effect of PLGA-CD-DEX-RVC obtained through pain evaluation (cold allodynia).

[0118] As a result, as shown in FIG. 7B, it was confirmed that the pain was the most reduced in the group treated with PLGA-CD-DEX-RVC. These results indicate that DEX-RVC can be effectively used for neurogenic pain.

Experimental Example 7. Confirmation of In Vivo Degradation

[0119] It was confirmed whether the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 were degraded in vivo.

[0120] Specifically, in order to measure the time during which PLGA melts in vivo, CY 5.5 fluorescent dye was attached to PLGA and PLGA-CD-DEX-RVC nanofibers and the results were implanted in the back integument of mice. Pearl Impulse small animal imaging carrier (LI-COR Biosciences, Lincoln, Nebr.) equipment was used, and the degree of degradation of nanofibers was measured by measuring the degree of reduced fluorescence expression.

[0121] FIG. 8 shows an in vivo decomposition effect of PLGA-CD-DEX-RVC according to an embodiment:

[0122] FIG. 8A shows images showing the degradation of PLGA-CD-DEX-RVC in vivo over time; and FIG. 8B shows a graph showing the degradation of PLGA-CD-DEX-RVC in vivo over time.

[0123] As a result, as shown in FIGS. 8A and 8B, like PLGA, which is a biodegradable polymer, PLGA-CD-DEX-RVC was also well degraded in vivo. These results indicate that even when PLGA-CD-DEX-RVC is injected into a living body and used, it does not remain in the living body but is degraded and is stable.

Experimental Example 8. Confirmation of Pain Marker Expression

[0124] It was confirmed whether the PLGA-CD-DEX-RVC nanofibers prepared in Example 1 reduced the expression of transient receptor potential vanilloid 1 (TRPV1) marker, which is known as a nociceptor, and the expression of Iba1 (ionized calcium binding adaptor molecule 1) marker for microglia, which is an inflammatory cell.

[0125] Neuropathic pain occurs due to a signal transduction of damaged nerve cells or an increase in inflammatory response in sensory neurons. Pain signals are transmitted from sensory neurons in the dorsal root ganglia (DRG) to the dorsal horn of the spinal cord.

[0126] Specifically, as in Experimental Example 6, the mice were perfused two weeks after the surgery to extract the spinal cord and DRG, and then immobilized with 4% PFA. After paraffin embed, the cells were cut to a size of 5 μm, and attached to a slide, followed by immunohistofluorescence staining. The cells were stained with an TRPV1 marker and an NeuN marker, which were used as antibodies for staining nuclei of neurons. Mouse anti-TRPV1 and rabbit anti-NeuN were used as primary antibodies, and Alexa 488 or Alexa 568 (Molecular Probes), and streptavidin-Alexa 594 were used as secondary antibodies. After staining, the cells were mounted and images thereof were captured by Confocal, and the expression of TRPV1, a pain marker, in neurons was quantified.

[0127] In addition, neurogenic pain is often increased depending on the inflammatory response. Accordingly, microglia, which is an inflammatory cell appearing in the central nervous carrier, was confirmed by paraffin-sectioning through Iba1 marker as described above and immunohistofluorescence staining. Goat anti-iba1 (1:500) was used as the primary antibody, and donkey anti goat 647 (1:1000, Invitrogen) was used as the secondary antibody. After staining, the cells were mounted and images thereof were captured by Confocal, and the expression of Iba1 in neurons was quantified.

[0128] FIG. 9 shows the expression levels of TRPV1, a pain factor, when PLGA-CD-DEX-RVC according to an embodiment was used for treatment after nerve injury, wherein the expression levels were identified by immunofluorescence staining:

[0129] FIG. 9A shows a diagram illustrating that a pain signal is transferred from sensory neurons of the dorsal root ganglia (DRG) to the dorsal horn of spinal cord; FIG. 9B shows an image showing the expression of TRPV1, a pain marker, in DRG (Scale bar: 20 μm); FIG. 9C shows a graph of the quantified expression of TRPV1, a pain marker, in DRG; FIG. 9D shows an image showing the expression of TRPV1, a pain marker, in the spinal cord of dorsal horn (Scale bar: 100 μm); and FIG. 9E shows a graph of the quantified expression of TRPV1, a pain marker, in the spinal cord of dorsal horn.

[0130] FIG. 10 shows the expression levels of Iba1, an inflammatory factor, when PLGA-CD-DEX-RVC according to an embodiment was used for treatment after nerve injury, wherein the expression levels were identified by immunofluorescence staining:

[0131] FIG. 10A shows an image showing the expression of Iba1, an inflammatory marker, in DRG (Scale bar: 20 μm); FIG. 10B shows a graph of the quantified expression of Iba1, an inflammatory marker, in DRG; FIG. 10C shows an image showing the expression of Iba1, an inflammatory marker, in the spinal cord of dorsal horn (Scale bar: 20 μm); and FIG. 10D shows a graph of the quantified expression of Iba1, an inflammatory marker, in the spinal cord of dorsal horn.

[0132] As a result, as shown in FIG. 9, it was confirmed that the expression of TRPV1 was the most reduced in the group treated with PLGA-CD-DEX-RVC. In addition, as shown in FIG. 10, the expression of Iba1 decreased the most in the group treated with PLGA-CD-DEX-RVC, confirming that microglia were significantly reduced compared to other groups. These results indicate that PLGA-CD-DEX-RVC is very effective in reducing pain.

[0133] When the drug delivery carrier according to an aspect is used, the time during which a drug stays in the living body may be prolonged, and due to the biodegradation thereof, few side effects occur.

[0134] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.