ULTRASOUND-ASSISTED DRUG DELIVERY CARRIER USING ULTRASOUND CONTRAST AGENT CONTAINING LIGAND CONJUGATED WITH DRUG THROUGH ESTER BOND

Abstract

Proposed are an ultrasound-assisted drug delivery carrier containing a ligand linked with a drug through an ester bond, a phospholipid, and a PEGylated phospholipid, a composition for drug delivery including the drug delivery carrier, and a method of preventing or treating a disease including administering the composition to an individual other than a human and releasing a drug by subjecting the site of administration of the composition to ultrasound irradiation. The ultrasound-assisted drug delivery carrier containing a ligand linked with a drug through an ester bond, a phospholipid, and a PEGylated phospholipid can be provided in the form of microbubbles or nanobubbles, and is capable of accelerating drug release due to collapse of the bubbles and promotion of hydrolysis of the ester bond during ultrasound irradiation, making it possible to deliver the drug to a desired site with high efficiency.

Claims

1. An ultrasound-assisted drug delivery carrier comprising a ligand linked with a drug through an ester bond, a phospholipid, and a PEGylated phospholipid.

2. The drug delivery carrier of claim 1, wherein the ligand is a phospholipid, a biomaterial, or an amphipathic material.

3. The drug delivery carrier of claim 2, wherein the biomaterial is a protein or a biocompatible polymer.

4. The drug delivery carrier of claim 3, wherein the protein is gelatin, collagen, fibrin, gluten, elastin, or albumin, and the biocompatible polymer is alginic acid, pectin, chitin, carrageenin, gellan gum, carboxymethyl cellulose, dextran, PCL, PLA, PGA, PVA, PAA, or hyaluronic acid.

5. The drug delivery carrier of claim 2, wherein the amphipathic material is configured such that a hydrophobic alkyl and PEG (polyethylene glycol) are linked through an ester bond.

6. The drug delivery carrier of claim 5, wherein the hydrophobic alkyl is a chain-like alkyl group having 5 to 30 carbon atoms, and the PEG is ##STR00002## (wherein x=6 to 50).

7. The drug delivery carrier of claim 1, wherein the phospholipid is at least one selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), didecanoylphosphatidylcholine (DDPC), dilauroylphosphatidylcholine (DLPC), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleyl phosphatidylethanolamine (DOPE), diarachidoyl phosphatidylethanolamine (DAPE), dilinoleyl phosphatidylethanolamine (DLPE), dipalmitoylphosphatidylglycerol (DPPG), dilauroyl phosphatidylglycerol (DL PG), distearoyl phosphatidylglycerol (DSPG), dioleoyl phosphatidylglycerol (DOPG), phosphatidylcholine (PC), and egg phosphatidylcholine (EPC).

8. The drug delivery carrier of claim 1, wherein the drug delivery carrier is provided in a form of microbubbles or nanobubbles having a diameter of 0.2 to 10 μm.

9. The drug delivery carrier of claim 1, wherein the drug delivery carrier accelerates release of the drug due to collapse of bubbles and promotion of hydrolysis of the ester bond through ultrasound irradiation.

10. The drug delivery carrier of claim 1, wherein a targeting agent (a targeting material) is additionally introduced on a surface of the ligand or the phospholipid.

11. A composition for drug delivery comprising the drug delivery carrier of claim 1.

12. A method of preventing or treating a disease, comprising: administering the composition for drug delivery of claim 11 to an individual other than a human; and releasing a drug by subjecting a site of administration of the composition to ultrasound irradiation.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 schematically shows a process of accelerating drug release by promoting hydrolysis of an ester bond between a phospholipid and a drug due to the occurrence of high temperature and high pressure when each drug delivery carrier of the present disclosure is subjected to ultrasound irradiation;

[0032] FIG. 2 shows a chemical structure in which a phospholipid and a drug (paclitaxel) or a model drug (fluorescein, cyanine 5, rhodamine B) are linked through an ester bond in the drug delivery carrier of the present disclosure;

[0033] FIG. 3 schematically shows a process of synthesizing a phospholipid linked with a drug (paclitaxel) through an ester bond in the present disclosure;

[0034] FIG. 4 schematically shows a process of synthesizing a phospholipid linked with a model drug (rhodamine B) through an ester bond in the present disclosure;

[0035] FIG. 5 schematically shows a process of synthesizing an amphipathic material (hydrophobic alkyl-PEG) linked with a drug (methotrexate) through an ester bond in the present disclosure;

[0036] FIG. 6 schematically shows a process of synthesizing an amphipathic material (hydrophobic alkyl-PEG) linked with a model drug (fluorescein) through an ester bond in the present disclosure;

[0037] FIG. 7 shows a chemical structure in which a biomaterial (hyaluronic acid, HA) and a drug (methotrexate, paclitaxel, doxorubicin) or a model drug (rhodamine B) are linked through an ester bond in the drug delivery carrier of the present disclosure;

[0038] FIG. 8 shows the three-dimensional structure of a protein (albumin) and a structure in which a drug (doxorubicin or methotrexate) is linked through an ester bond in the drug delivery carrier of the present disclosure;

[0039] FIG. 9A schematically shows a process of synthesizing a biomaterial (hyaluronic acid, HA) linked with a drug (methotrexate) through an ester bond in the present disclosure, and FIG. 9B shows NMR and UV-vis analysis data of the biomaterial before and after synthesis;

[0040] FIG. 10A schematically shows a process of synthesizing a biomaterial (hyaluronic acid, HA) linked with a model drug (rhodamine B) through an ester bond in the present disclosure, and FIG. 10B shows NMR and UV-vis analysis data of the biomaterial before and after synthesis;

[0041] FIG. 11 shows optical microscope and confocal microscope images of a drug delivery carrier (an experimental group, MB-ERPL) containing a phospholipid linked with a model drug (rhodamine B) through an ester bond, and a drug delivery carrier (a control group, MB-AFPL) containing a phospholipid linked with a model drug (fluorescein) through an amide bond;

[0042] FIG. 12 shows optical microscope and confocal microscope images of a drug delivery carrier (an experimental group, MB-EFPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a model drug (fluorescein) through an ester bond, and a drug delivery carrier (a control group, MB-ARPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a model drug (rhodamine B) through an amide bond;

[0043] FIG. 13 shows optical microscope and confocal microscope images of a drug delivery carrier (an experimental group, MB-ERHA) containing a biomaterial (hyaluronic acid) linked with a model drug (rhodamine B) through an ester bond, and drug delivery carriers (control groups) containing a biomaterial linked with a model drug (fluorescein) through an amide bond and containing a model drug (Nile red) without hyaluronic acid;

[0044] FIG. 14 shows the results of analysis of the size of the microbubble-based contrast agent using a DLS particle size analyzer, of a drug delivery carrier (an experimental group, MB-ERPL) containing a phospholipid linked with a model drug (rhodamine B) through an ester bond, a drug delivery carrier (Control Group 1, MB-AFPL) containing a phospholipid linked with a model drug (fluorescein) through an amide bond, and a drug delivery carrier (Control Group 2, MB) containing only a phospholipid and a PEGylated phospholipid;

[0045] FIG. 15 shows the results of analysis of the size of the microbubble-based contrast agent using a DLS particle size analyzer, of a drug delivery carrier (an experimental group, MB-EFPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a model drug (fluorescein) through an ester bond, a drug delivery carrier (Control Group 1, MB-ARPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a model drug (rhodamine B) through an amide bond, and a drug delivery carrier (Control Group 2, MB) containing only a phospholipid and a PEGylated phospholipid;

[0046] FIG. 16 shows the results of analysis of the size of the microbubble-based contrast agent using a DLS particle size analyzer, of a drug delivery carrier (an experimental group, MB-ERHA) containing a biomaterial (hyaluronic acid) linked with a model drug (rhodamine B) through an ester bond, a drug delivery carrier (Control Group 1, MB-AFHA) containing a biomaterial linked with a model drug (fluorescein) through an amide bond, and a drug delivery carrier (Control Group 2, MB-NR) containing a model drug (Nile red) without hyaluronic acid;

[0047] FIG. 17 shows the results of comparison of the amount of the model drug that is released through UV-vis analysis when a solution in which only a phospholipid (a control group, ERPL) linked with a model drug (rhodamine B) through an ester bond is dissolved and a drug delivery carrier (an experimental group, MB-ERPL) in the form of microbubbles containing a phospholipid linked with a model drug (rhodamine B) through an ester bond are subjected to ultrasound irradiation;

[0048] FIG. 18 shows the results of comparison of the amount of the model drug that is released through UV-vis analysis when a solution in which only an amphipathic material (a control group, EFPA) linked with a model drug (fluorescein) through an ester bond is dissolved and a drug delivery carrier (an experimental group, MB-EFPA) in the form of microbubbles containing an amphipathic material linked with a model drug (fluorescein) through an ester bond are subjected to ultrasound irradiation;

[0049] FIG. 19 shows the results of comparison of the amount of the model drug that is released through UV-vis analysis when a solution in which only a biomaterial (a control group, ERHA) linked with a model drug (rhodamine B) through an ester bond is dissolved and a drug delivery carrier (an experimental group, MB-ERHA) in the form of microbubbles containing a biomaterial linked with a model drug (rhodamine B) through an ester bond are subjected to ultrasound irradiation;

[0050] FIG. 20 schematically shows an experiment on the extent of diffusion of the drug into agarose gel during ultrasound irradiation for a drug delivery carrier (an experimental group) containing a ligand (a phospholipid, an amphipathic material, or a biomaterial) linked with a drug through an ester bond and a drug delivery carrier (a control group) containing a ligand linked with a drug through an amide bond;

[0051] FIG. 21 shows the results of confocal microscopy to determine the extent of diffusion of the drug during ultrasound irradiation for a drug delivery carrier (an experimental group, MB-EFPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a drug through an ester bond and a drug delivery carrier (a control group, MB-EMPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a drug through an amide bond;

[0052] FIG. 22 shows the results of confocal microscopy to determine the extent of diffusion of the drug during ultrasound irradiation for a drug delivery carrier (an experimental group) containing a biomaterial (hyaluronic acid) linked with a drug through an ester bond and a drug delivery carrier (a control group) containing a biomaterial linked with a drug through an amide bond;

[0053] FIG. 23 shows the results of testing on in-vitro biotoxicity of a biomaterial linked with a model drug (rhodamine B) through an ester bond and a model drug;

[0054] FIG. 24 shows the results of comparison of in-vitro cell absorption depending on whether or not ultrasound irradiation is performed on a drug delivery carrier (an experimental group, MB-ERPL) containing a phospholipid linked with a model drug (rhodamine B) through an ester bond and a drug delivery carrier (a control group, MB-AFPL) containing a phospholipid linked with a model drug (fluorescein) through an amide bond; and

[0055] FIG. 25 shows the results of comparison of the in-vitro cell absorption depending on whether or not ultrasound irradiation is performed on a drug delivery carrier (an experimental group, MB-ERHA) containing a biomaterial linked with a model drug (rhodamine B) through an ester bond and a drug delivery carrier (a control group, MB-AFHA) containing a biomaterial linked with a model drug (fluorescein) through an amide bond.

MODE FOR DISCLOSURE

[0056] A better understanding of the present disclosure may be given through the following examples. However, these examples are merely set forth to illustrate the present disclosure, and are not to be construed as limiting the scope of the present disclosure.

Example 1: Manufacture of Microbubble Drug Delivery Carrier

[0057] 1-1: Manufacture of Drug Delivery Carrier Containing Phospholipid Linked with Model Drug Through Ester Bond

[0058] A mixture of 1-10 mg of a PEG-conjugated phospholipid DSPE-PEG (2000) and 5-30 mg of a phospholipid (DSPC) at an appropriate ratio and 1-10 mg of a phospholipid linked with a drug through an ester bond were dispersed and dissolved in 2-5 mL of chloroform contained in a 30-mL vial. The vial containing the mixed solution was treated using a rotary concentrator to remove the organic solvent therefrom, thus forming a vial coated with an opaque phospholipid film. 3-5 mL of a sodium chloride (0.9%) aqueous solution and 1 mL of a liquefied gas (2H,3H-decafluoropentane) were added to the film-coated vial and ultrasound was applied thereto for 0.5-2 minutes using a tip sonicator, thereby manufacturing a microbubble drug delivery carrier. After the prepared aqueous solution was gradually cooled, the solution in the upper layer in which the residue remained was removed, and the precipitated microbubble drug delivery carrier was recovered. The microbubble solution thus prepared was confirmed through NTA, DLS, confocal microscopy, and SEM.

[0059] As a result, a drug delivery carrier (an experimental group, MB-ERPL) containing a phospholipid linked with a model drug (rhodamine B) through an ester bond was provided in the form of microbubbles having a diameter of 1-3 μm, and the surface charge thereof exhibited a negative value (−20.1 mV) due to the presence of DSPE-PEG (2000), having a negative charge.

[0060] In addition, a drug delivery carrier (a control group, MB-AFPL) containing a phospholipid linked with a model drug (fluorescein) through an amide bond was provided in the form of microbubbles having a diameter of 1-3 μm, and likewise, the surface charge thereof was about −20.4 mV due to the presence of DSPE-PEG (2000), having a negative charge.

[0061] Meanwhile, in the case of microbubbles (MB) containing only a phospholipid and a PEGylated phospholipid, microbubbles having a diameter of 1-5 μm were formed, and the surface charge thereof was relatively small, particularly about −23 mV.

[0062] 1-2: Manufacture of Drug Delivery Carrier Containing Amphipathic Material (Hydrophobic Alkyl-PEG) Linked with Model Drug Through Ester Bond

[0063] A mixture of 1-10 mg of a PEG-conjugated phospholipid DSPE-PEG (2000) and 5-30 mg of a phospholipid (DSPC) at an appropriate ratio and 1-10 mg of an amphipathic material (hydrophobic alkyl-PEG) linked with a drug through an ester bond were dispersed and dissolved in 2-5 mL of chloroform contained in a 30-mL vial. The vial containing the mixed solution was treated using a rotary concentrator to remove the organic solvent therefrom, thus forming a vial coated with an opaque phospholipid film. 3-5 mL of a sodium chloride (0.9%) aqueous solution and 1 mL of a liquefied gas (2H,3H-decafluoropentane) were added to the film-coated vial, and ultrasound was applied thereto for 0.5-2 minutes using a tip sonicator, thereby manufacturing a microbubble drug delivery carrier. After the prepared aqueous solution was gradually cooled, the solution in the upper layer in which the residue remained was removed, and the precipitated microbubble drug delivery carrier was recovered. The microbubble solution thus prepared was confirmed through NTA, DLS, confocal microscopy, and SEM.

[0064] As a result, a drug delivery carrier (an experimental group, MB-EFPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a model drug (fluorescein) through an ester bond was provided in the form of microbubbles having a diameter of 1-3 μm, and the amphipathic material (green) was selectively formed on the surface of the bubbles. The surface charge thereof exhibited a negative value (−19.8 mV) due to the presence of DSPE-PEG (2000), having a negative charge.

[0065] In addition, a drug delivery carrier (a control, MB-ARPA) containing an amphipathic material (hydrophobic alkyl-PEG) linked with a model drug (rhodamine B) through an amide bond was provided in the form of microbubbles having a diameter of 1-3 μm, and the amphipathic material (red) was selectively formed on the surface of the bubbles. Likewise, the surface charge thereof was about −19.5 mV due to the presence of DSPE-PEG (2000), having a negative charge.

[0066] Meanwhile, in the case of microbubbles (MB) containing only a phospholipid and a PEGylated phospholipid, microbubbles having a diameter of 1-5 μm were formed, and the surface charge thereof was relatively small, particularly about −23 mV.

[0067] 1-3: Manufacture of Drug Delivery Carrier Containing Biomaterial (Hyaluronic Acid) Linked with Model Drug Through Ester Bond

[0068] A mixture of 1-10 mg of a PEG-conjugated phospholipid DSPE-PEG (2000) and 5-30 mg of a phospholipid (DSPC) at an appropriate ratio was dispersed and dissolved in 2-5 mL of chloroform contained in a 30-mL vial. 1-10 μg of a biomaterial (hyaluronic acid, HA) linked with a drug through an ester bond was dispersed and dissolved in 1-3 mL of a 40% ethanol aqueous solution, after which the resulting solution was mixed with the phospholipid solution prepared above. The vial containing the mixed solution was treated using a rotary concentrator to remove the organic solvent and water therefrom, thus forming a vial coated with an opaque phospholipid film. 3-5 mL of a sodium chloride (0.9%) aqueous solution and 1 mL of a liquefied gas (2H,3H-decafluoropentane) were added to the film-coated vial and ultrasound was applied thereto for 0.5-2 minutes using a tip sonicator, thereby manufacturing a microbubble drug delivery carrier. After the prepared aqueous solution was gradually cooled, the solution in the upper layer in which the residue remained was removed, and the precipitated microbubble drug delivery carrier was recovered. The microbubble solution thus prepared was confirmed through NTA, DLS, confocal microscopy, and SEM.

[0069] As a result, a drug delivery carrier (an experimental group, MB-ERHA) containing a biomaterial (hyaluronic acid) linked with a model drug (rhodamine B) through an ester bond was provided in the form of microbubbles having a diameter of 1-3 μm, and hyaluronic acid having a negative charge (HA-RhB, red) was selectively formed on the surface of the bubbles, and the surface charge thereof showed a negative value (−28 mV) due to the presence of DSPE-PEG (2000) and hyaluronic acid, having a negative charge.

[0070] In addition, a drug delivery carrier (a control, MB-AFHA) containing a biomaterial linked with a model drug (fluorescein) through an amide bond was provided in the form of microbubbles having a diameter of 1-3 μm, and hyaluronic acid having a negative charge (HA-fluoresceine, green) was selectively formed on the surface of the bubbles, and likewise, the surface charge thereof was about −29 mV due to the presence of DSPE-PEG (2000) and hyaluronic acid, having a negative charge.

[0071] Meanwhile, in the case of microbubbles containing a fluorescent dye (Nile red) without hyaluronic acid, microbubbles having a diameter of 1-5 μm were formed, and the uniformity of the bubbles was decreased due to the presence of hydrophobic Nile red. Moreover, the bubbles in the form of a ring were mostly observed to have a spherical shape because Nile red was present only on the surface of the bubbles, but Nile red was capable of being dissolved in liquefied gas. Since DSPE-PEG was negatively charged, the surface charge of the microbubbles had a negative value, but was relatively small, particularly about −23 mV, due to the absence of hyaluronic acid.

Example 2: Manufacture of Microbubble Drug Delivery Carrier Using Contrast Agent

[0072] 1-10 mg of a phospholipid, an amphipathic material (hydrophobic alkyl-PEG) or a biomaterial (hyaluronic acid, HA) linked with a drug through an ester bond was dissolved in 5 mL of a sodium chloride (0.9%) aqueous solution. The sodium chloride solution in which the drug was dissolved was placed in a syringe, injected into a container containing a SonoVue powder via a rubber stopper, and then vigorously stirred for 20 seconds or more using a vortex mixer (SonoVue powder: sulfur hexafluoride, Macrogol 4000, distearoylphosphatidylcholine, dipalmitoylphosphatidylglycerol sodium, and palmitic acid).

Example 3: Experiment on Drug Release According to Ultrasound Irradiation

[0073] The solution of an experimental group (a drug delivery carrier containing the phospholipid, amphipathic material or biomaterial linked with the red model drug through an ester bond) dispersed in PBS and the solution of a control group (the phospholipid, amphipathic material or biomaterial dispersed without microbubbles) dispersed in PBS were placed in respective dialysis membrane bags. Each of the dialysis membrane bags containing the materials was placed in a beaker containing a PBS solution, followed by ultrasound irradiation. In the experimental group, the concentration of the released drug in the beaker was analyzed after irradiation for 10, 30 or 60 seconds, and in the control group, the concentration of the released drug was analyzed after sufficient irradiation for 60 seconds.

[0074] 3-1: Drug Delivery Carrier Containing Phospholipid Linked with Model Drug Through Ester Bond

[0075] As a result, in the control group without microbubbles, a small amount of drug was released through ultrasound irradiation, but in the experimental group, manufactured with microbubbles, it was confirmed that at least three times the amount of the drug was released compared to the control group (FIG. 17).

[0076] 3-2: Drug Delivery Carrier Containing Amphipathic Material (Hydrophobic Alkyl-PEG) Linked with Model Drug Through Ester Bond

[0077] In the control group without microbubbles, a small amount of drug was released through ultrasound irradiation, but in the experimental group, manufactured with microbubbles, it was confirmed that at least five times the amount of the drug was released compared to the control group (FIG. 18).

[0078] 3-3: Drug Delivery Carrier Containing Biomaterial (Hyaluronic Acid) Linked with Model Drug Through Ester Bond

[0079] In the control group without microbubbles, a small amount of drug was released through ultrasound irradiation, but in the experimental group, manufactured with microbubbles, it was confirmed that at least ten times the amount of the drug was released compared to the control group, and this release rate was observed to depend on the ultrasound irradiation time (FIG. 19).

Example 4: Drug Diffusion Experiment According to Ultrasound Irradiation

[0080] The solution of each of an experimental group (a drug delivery carrier containing an amphipathic material or biomaterial linked with a model drug through an ester bond) and a control group (a drug delivery carrier containing an amphipathic material or biomaterial linked with a model drug through an amide bond), dispersed in PBS, was injected into a channel formed of hydrophilic agarose gel. Thereafter, ultrasound was applied onto the channel into which the solution was injected to thereby break the microbubbles, and accordingly, the diffusion of each model drug into the hydrophilic agarose gel and the extent thereof were evaluated.

[0081] 4-1: Drug Delivery Carrier Containing Amphipathic Material Linked with Model Drug Through Ester Bond

[0082] As a result, the hydrolysis reaction of the ester bond in the drug delivery carrier was accelerated due to physical stimulation through ultrasound irradiation and the local high temperature/high pressure caused by collapse of the microbubbles, so it was confirmed with a confocal analyzer that the green model drug (fluorescein) that was linked in the experimental group was rapidly released, and the released model drug permeated and was dispersed into the hydrophilic agarose gel.

[0083] On the other hand, in the case of the red model drug (rhodamine B) conjugated through an amide bond in the control group, the amide bond was almost completely undecomposed even under high-temperature/high-pressure conditions through ultrasound irradiation, so the drug was not released but was attached to the amphipathic material (hydrophobic alkyl-PEG) and thus maintained as a macromolecule, indicating that the permeation thereof into the hydrophilic agarose gel was weak (FIG. 21).

[0084] 4-2: Drug Delivery Carrier Containing Biomaterial Linked with Model Drug Through Ester Bond

[0085] The hydrolysis reaction of the ester bond in the drug delivery carrier was accelerated due to the physical stimulation through ultrasound irradiation and the local high temperature/high pressure caused by collapse of the microbubbles, so it was confirmed with a confocal analyzer that the red model drug (rhodamine B) that was linked in the experimental group was rapidly released, and the released model drug permeated and was dispersed into the hydrophilic agarose gel.

[0086] On the other hand, in the case of the green model drug (fluorescein) conjugated through an amide bond in the control group, the amide bond was almost completely undecomposed even under high-temperature/high-pressure conditions through ultrasound irradiation, so the drug was not released but was attached to the biomaterial (hyaluronic acid) and thus maintained as a macromolecule, indicating that the permeation thereof into the hydrophilic agarose gel was weak (FIG. 22).

Example 5: In-Vitro Cell Experiment

[0087] 5-1: Cytotoxicity Test for Biomaterial Linked with Model Drug (Rhodamine B) Through Ester Bond

[0088] The biomaterial (HA-RhB) linked with the model drug (rhodamine B) through an ester bond and the model drug were added to U-251 MG cells such that the final concentration was adjusted to 1 nM, 0.01 μM, 0.1 μM, 1 μM, 10 μM and 100 μM, after which the cells were cultured for 24 hours. Thereafter, in order to confirm the in-vitro cytotoxicity of each material, cell viability was measured by adding a solution including a tetrazolium salt derivative and a cell culture medium thereto. As a result, free rhodamine B exhibited almost no toxicity at a low concentration, but toxicity thereof was observed at a concentration of 1 μM or more. On the other hand, toxicity of the biomaterial (HA-RhB) linked with the model drug through the ester bond was observed from a very high concentration, particularly 100 μM. This shows superior biocompatibility of the manufactured drug delivery carrier due to the characteristics of the hyaluronic acid platform, having superior biocompatibility (FIG. 23).

[0089] 5-2: Analysis of Intracellular Absorption According to Ultrasound Irradiation

[0090] The solution of each of the experimental group (the drug delivery carrier containing the phospholipid or biomaterial linked with the red model drug (rhodamine B) through an ester bond) and the control group (the drug delivery carrier containing the phospholipid or biomaterial linked with the green model drug (fluorescein) through an amide bond) was used to analyze the extent of in-vitro intracellular absorption depending on whether or not ultrasound irradiation was performed. The experimental group and the control group were added together to U-251 MG cells, and the cell culture medium was replaced with a fresh medium before observation in order to remove materials that were not absorbed to the cells, followed by observation with a confocal microscope.

[0091] 5-2-1: Drug Delivery Carrier Containing Phospholipid Linked with Red Model Drug Through Ester Bond

[0092] As a result, both the experimental group and the control group showed slow cell absorption before ultrasound irradiation, so almost none of red and green fluorescent colors appeared. However, it was confirmed with a confocal analyzer that the red model drug (rhodamine B) that was linked was rapidly released and absorbed by cells because of the accelerated hydrolysis reaction of the ester bond in the drug delivery carrier of the experimental group due to the physical stimulation through ultrasound irradiation and the local high temperature/high pressure caused by collapse of the microbubbles.

[0093] On the other hand, in the case of the green model drug (fluorescein) conjugated through an amide bond in the control group, it was confirmed with a confocal analyzer that the amide bond was almost completely undecomposed even under high-temperature/high-pressure conditions due to collapse of the bubbles through ultrasound irradiation, so the drug was not released but was attached to the phospholipid and thus maintained as a macromolecule, and thus cell absorption still proceeded slowly (FIG. 24).

[0094] 5-2-2: Drug Delivery Carrier Containing Biomaterial Linked with Red Model Drug Through Ester Bond

[0095] Both the experimental group and the control group showed slow cell absorption due to the high-molecular-weight hyaluronic acid microbubble platform before ultrasound irradiation, so almost none of red and green fluorescent colors appeared. However, it was confirmed with a confocal analyzer that the red model drug (rhodamine B) that was linked was rapidly released and absorbed by cells because of the accelerated hydrolysis reaction of the ester bond in the drug delivery carrier of the experimental group due to the physical stimulation through ultrasound irradiation and the local high temperature/high pressure caused by collapse of the microbubbles.

[0096] On the other hand, in the case of the green model drug (fluorescein) conjugated through an amide bond in the control group, it was confirmed with a confocal analyzer that the amide bond was almost completely undecomposed even under high-temperature/high-pressure conditions due to collapse of the bubbles through ultrasound irradiation, so the drug was not released but was attached to the biomaterial (hyaluronic acid) and thus maintained as a macromolecule, and thus cell absorption still proceeded slowly (FIG. 25).