KR101586866B1 - pH sensitive nano particle for drug delivery and preparation method thereof - Google Patents

Abstract

The present invention relates to a pH sensitive drug delivery nanocomposite and a method for preparing the same. More specifically, the present invention relates to a pH sensitive drug delivery system in which 3-diethylaminopropyl (DEAP) is introduced into an amine group of a polysaccharide Nanocomposites. The nanocomposite for pH-sensitive drug delivery according to the present invention is a nano-sized drug delivery vehicle that can easily and rapidly penetrate into target cells and can selectively and efficiently deliver drugs to specific disease sites having mild acidic environmental changes The therapeutic effect can be enhanced, so that there is an advantageous effect that it can be effectively used as a drug delivery vehicle for treating various diseases.

Description

[0001] The present invention relates to a pH-sensitive nanoparticle for drug delivery and a method for preparing the same,

The present invention relates to a nanocomposite for pH-sensitive drug delivery and a method for preparing the same, and more particularly, to a pH-sensitive drug delivery nanocomposite comprising a polysaccharide grafted with functional 3-diethylaminopropyl (DEAP).

Recent advances in industrialization have led to the practical use of peptides and proteins as therapeutics for clinical applications in the biotechnology field. However, in the case of drugs using these peptides or proteins, how to deliver them in vivo is a continuing part of pharmaceutical research for many years.

In particular, among the methods for drug delivery, oral delivery is the most traditional and desirable method for drug delivery, which is more so when repeated or regular administration is required (BY Kim, et al., J. Control. Release 102, 525 -38, 2005), there are fewer patients suffering compared to other methods of administration, such as injection, and less of the discomfort associated with injection (A. des Rieux et al., J. Control. Release 116, 1-27, 2006). However, when the drug is orally administered, the concentration gradient between the digestive lumen and the blood increases immediately after the drug is released from the tablet or capsule form preparation, There is a problem that a concentration gradient is drastically reduced as it moves to the digestive tract, resulting in formation of an unfavorable condition for drug absorption. Particularly, since the protein or peptide drug is hydrolyzed by digestive enzymes secreted to the digestive tract, There is a problem that the temperature is reduced.

In addition to the formulations for oral administration, various types of pharmaceuticals that have been used up to now are effective only when they are administered in an amount larger than that required to exhibit an appropriate drug effect at a diseased site. Therefore, , Many pharmaceutical companies have a need for the development of formulations that can reduce these side effects while at the same time maximizing their efficacy.

In order to solve the above problems, the drug delivery system (hereinafter referred to as DDS) has been increasingly utilized. The DDS is a method of maximizing the efficacy and effectiveness by minimizing side effects of existing medicines. It is useful for improving the selectivity of drugs which are difficult to administer. The use of special delivery systems is required if the drug being administered exhibits physico-chemical or pharmacokinetic specific properties such as high water solubility, high fat solubility, insolubility, and the like.

It is also possible to use drugs with special requirements, such as poisonous single-use injectable drugs, cytotoxic unstable drugs, high clearance drugs, easily inactivated drugs, topical application The required drugs, etc. should be considered to have the appropriate DDS.

The DDS technology has been actively researched since the 1970s by advanced countries in advanced technology. Since the introduction of the substance patent system in 1987, Korean pharmaceutical companies who have felt a sense of crisis have developed a new drug product that uses DDS, which improves the disadvantages of existing drugs rather than developing new drugs that cost 15 million years and costs more than $ 200 million. We found that the time and cost required for development was shortened to about one third and the probability of success was very high. In Korea, full-scale research has been started since 1990, and related patent applications have been steadily increasing since 1992.

In the drug delivery system, a change in pH in the human body may be used. In particular, changes in pH in the human body are observed in three places. First, as a change in the gastrointestinal tract, the difference between the low pH and the high pH of the stomach is used for drug delivery. The second is the pH change in the blood. The pH of the human body is kept at 7.4, and the change width does not generally exceed ± 0.04. However, when the pH change in the blood is higher than 0.04, alkademia is induced. When the pH is lower than 0.04, acidemia is induced. Third, the change in pH observed in the human body is observed in a local cell, which is lowered in pH than in normal cells when a disease such as cancer or hypoxia is induced.

In addition, the lesion cells are known to exhibit a certain weakly acidic pH depending on the type and site of disease. For example, in the case of cancer cells, the pH around the cancer is 0.6 And a pH of about 6.8.

As described above, there is a difference in the pH somewhat lower than the normal at the sites where various diseases such as cancer occur. Therefore, when the drug delivery system using the pH change is used, it is possible to efficiently deliver the drug specifically to the disease site However, there has not been developed a drug delivery system capable of recognizing such a pH difference yet.

Accordingly, the present inventors have found that a nanocomposite nanogel formed by introducing 3-diethylaminopropyl (DEAP) into an amine group of a polysaccharide reacts even at a slight pH change to decompose at a weak acidity, Specifically, can effectively transfer and release the drug, and the present invention has been completed.

It is an object of the present invention to provide a pH sensitive drug delivery nanocomposite comprising 3-diethylaminopropyl (DEAP) grafted polysaccharide and a process for its preparation.

It is still another object of the present invention to provide a drug delivery system in which a drug is encapsulated in the pH sensitive drug delivery nanocomposite.

Still another object of the present invention is to provide a method for preparing a pH sensitive drug delivery nanocomposite according to the present invention.

In order to achieve the above object, the present invention provides a pH sensitive drug delivery nanocomposite comprising a polysaccharide grafted with 3-diethylaminopropyl (DEAP).

According to an embodiment of the present invention, the grafting may be a combination of an amine group of the polysaccharide and an isothiocyanate group of the 3-diethylaminopropyl group.

According to one embodiment of the present invention, the polysaccharide is selected from the group consisting of pullulan, cudlan, hyaluronic acid, pectin, chitosan, dextran, heparin, ≪ / RTI > and starch.

According to one embodiment of the present invention, the polysaccharide may be chitosan.

According to one embodiment of the present invention, the chitosan may be at least one selected from water-soluble glycol chitosan, chitosan oligomer, and acetylated chitosan.

According to one embodiment of the present invention, the nanocomposite can be destabilized at pH 7.0 or lower.

According to one embodiment of the present invention, the 3-diethylaminopropyl (DEAP) may be substituted with 0.90 to 0.97 per one primary amine of the polysaccharide.

According to an embodiment of the present invention, the particle size of the nanocomposite may be 100-110 nm.

The present invention also provides a drug delivery system comprising the nanocomposite for pH-sensitive drug delivery according to the present invention and a drug capable of being encapsulated in the nanocomposite.

According to one embodiment of the present invention, the drug may be selected from the group consisting of proteins, peptides and synthetic compounds.

According to an embodiment of the present invention, the drug may be at least one drug selected from paclitaxel, docetaxel, doxorubicin, cisplatin, carboplatin, 5-FU, etoposide and camptothecin.

According to an embodiment of the present invention, the mixing ratio of the nanocomposite and the drug may be 1: 1 to 10: 1.

Further, according to the present invention,

Dissolving the polysaccharide in a solvent;

Adding 3-diethylaminopropyl to a solution in which the polysaccharide is dissolved to bind an amine group of the polysaccharide and an isothiocyanate group of the 3-diethylaminopropyl group; And

And dialyzing and lyophilizing the combined polymer. The present invention also provides a method of manufacturing a nanocomposite for pH-sensitive drug delivery.

According to one embodiment of the present invention, the polysaccharide is selected from the group consisting of pullulan, cudlan, hyaluronic acid, pectin, chitosan, dextran, heparin, And a starch.

According to one embodiment of the present invention, the chitosan may be glycol chitosan.

The pH sensitive drug delivery nanocomposite according to the present invention is based on a polysaccharide having excellent biodegradability and biocompatibility, and is a nano-sized drug prepared using 3-diethylaminopropyl (DEAP) As a drug delivery vehicle, it can easily and rapidly penetrate into target cells, and can selectively and efficiently deliver drugs to specific disease sites having environmental changes under weakly acidic conditions, thereby increasing the therapeutic effect. Therefore, Can be usefully used.

Figure 1 is a graph of the pH profile of GCS- g- DEAP nanogels by acid-base titration.
2 is a graph showing the particle size distribution of GCS- g- DEAP nanogels.
3 is a graph showing changes in permeability according to pH of GCS- g- DEAP nanogel and GCS solution.
FIG. 4 is a graph showing changes in CAC values according to pH of GCS- g- DEAP nanogels.
5 is a graph showing changes in zeta potential according to pH of GCS- g- DEAP nanogels.
Figure 6 is an FE-SEM image of GCS- g- DEAP nanogels.
Figure 7 is a graph of pH-dependent DOX release from DOX-loaded GCS- g- DEAP nanogels.
Figure 8 is a fluorescence image of A549 cells treated with DOX-loaded GCS- g- DEAP nanogel or glass DOX.

The terms used in the present invention are defined as follows.

"Sol" is the dispersion of colloidal particles in a liquid, the term "gel" refers to a pore of a submicrometer dimension and an average length, typically greater than 1 micrometer Quot; refers to a tightly interconnected network of polymer chains that exhibit a semi-solid phase that occurs spontaneously when the temperature of the polymer solution rises above the gelation temperature of the block copolymer.

"Controlled release" refers to the modulation of the rate and / or amount of a drug or therapeutic agent delivered in accordance with the drug delivery formulations of the invention. The controlled emission may be continuous or discontinuous and / or linear or nonlinear. This may be accomplished using one or more types of polymer compositions, drug loading, excipients or degradation-improving agents, or other modifying agents, alone, in combination, or sequentially to provide the desired effect.

"Drug" means an organic or inorganic compound or substance that has viability and is used or modified for therapeutic use. Proteins, oligonucleotides, DNA, and gene therapy agents are included in the broad definition of drugs.

"Therapeutic agent" refers to any compound or composition that, when administered to an organism (human or non-human animal), elicits the desired pharmacological, immunogenic and / or physiological effect by local and / or systemic action . Thus, the term encompasses compounds or chemicals traditionally considered as biomedicals including drugs, vaccines, and molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. "Therapeutic agent" includes compounds or compositions used in all major therapeutic applications including, but not limited to: anti-infectives such as antibiotics and antiviral agents; Analgesics and analgesic combinations; Topical and common anesthetics; Anorexia nervosa; Anti-arthritic agents; Anti-asthmatics; Anticonvulsants; Antidepressants; Antihistamines; Anti-inflammatory agents; Antistatic agent; Anti-migraine agent; Antineoplastic agents; Anti-itch agents; Antipsychotic agents; fever remedy; Animal festival; Cardiovascular products (including calcium channel blockers, beta-blockers, beta-agonists and antiarrhythmics); Antihypertensive agents; Chemotherapeutic agents; diuretic; Vasodilators; Central nervous system stimulants; Cough and cold products; Decongestants; Diagnostic agent; hormone; Osteogenic stimulators and bone resorption inhibitors; Immunosuppressants; Muscle relaxants; Psychostimulants; sedative; Nerve stabilizers; Proteins, peptides and fragments thereof (naturally occurring, chemically synthesized or recombinantly produced); And nucleic acid molecules (ribonucleotides (RNA) or deoxyribonucleotides (DNA), gene constructs, expression vectors, plasmids, polynucleotides, polynucleotides, polynucleotides, polynucleotides, Antisense molecules, etc.).

"Peptides ", " polypeptides "," oligopeptides ", and "proteins" can be used equally when referring to peptides or protein drugs and are not limited to specific molecular weights, peptide sequences or lengths,

"Therapeutic effect" means any improvement in a subject, human or animal disease that has been treated according to the method, and includes any prophylactic or preventive effect, or a disease that may be detected by physical examination, , And to obtain any relief in the severity of the symptoms and symptoms of the disease or disorder.

The terms "treating" or "treating ", as used herein, unless otherwise defined with respect to a particular disease or disorder, are intended to encompass all types of diseases, disorders, Preventing a disease, a disease or a disease from occurring in a person; (ii) inhibiting a disease, disorder or disease, i.e., inhibiting its progression; And / or (iii) alleviating the disease, disorder, or disease, i. E. Causing the disease, disorder and / or disease.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included. In one embodiment of the present invention, the present invention was applied to humans.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

Optionally, tissue or cell samples are obtained from primary or metastatic tumors. Tissue samples may include compounds that are not mixed with the tissue by nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. For purposes of the present invention, a "section" of a tissue sample refers to a portion or piece of tissue sample, eg, a thin slice of tissue or cell that has been cut from a tissue sample. It will be appreciated that multiple sections of a tissue sample may be taken for analysis in accordance with the present invention if the present invention is understood to include methods in which the same section of tissue sample is analyzed both at the morphological and molecular levels, It is understood that it can do.

"Cancer "," tumor ", or "malignant" refers to or represents the physiological condition of a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma.

An "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

The terms "about "," substantially ", etc. used to the extent that they are used herein are intended to be taken to mean an approximation of, or approximation to, the numerical values of manufacturing and material tolerances inherent in the meanings mentioned, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure.

Hereinafter, the present invention will be described in detail.

The present invention relates to a pH-sensitive drug delivery nanocomposite comprising a polysaccharide grafted with 3-diethylaminopropyl (DEAP), which is in the form of a "nanogel".

The pH sensitive drug delivery nanocomposite according to the present invention can be synthesized by organic binding between a polysaccharide having excellent biodegradability and biocompatibility and a pH-sensitive material.

In the nanocomposite of the present invention, the polysaccharide which is responsible for the hydrophobic block is characterized in that it has a block copolymer structure composed of a hydrophilic region having a high degree of substitution and a hydrophilic region having a low degree of substitution or no substitution in order to form a physical gel. The hydrophobic region locally stabilizes the water structure and, upon heating, destroys the water structure and enhances the hydrophobic interaction, resulting in a gelation phenomenon.

Amphiphilic polymers with both hydrophilic and hydrophobic properties form micelles or self-aggregates through interaction between hydrophobic blocks to stabilize interfacial energy in aqueous solution. Polymeric colloidal particles formed by amphiphilic polymers are known to exhibit different sizes, distributions, rheological properties, and thermodynamic stability depending on the degree of hydrophilicity and hydrophobicity

In addition, the natural polymer polysaccharide is characterized by biodegradable biodegradability and biocompatibility.

"Biodegradable" means that the block copolymer can be chemically degraded in the body to form a non-toxic compound. The rate of degradation is the same or different from the drug release rate. And "biocompatibility" refers to the ability to interact with the body without undesirable subsequent effects.

The polysaccharides used in the present invention include, but are not limited to, pullulan, cudlan, hyaluronic acid, pectin, chitosan, dextran, heparin Heparin, and starch. Among them, chitosan can be used. More preferably, water-soluble glycol chitosan, chitosan oligomer, or acetylated chitosan And still more preferably glycol chitosan, which is a chitosan derivative of the following structural formula, can be used.

Chitosan is a compound that is obtained by deacetylation of chitin, which is a natural polymer material rich in natural substances after cellulose in the natural world. The chitosan includes 2-amino-2-deoxy-β-D-glucopyranose It is a polysaccharide composed of 2-amino-2-deoxy-β-D-glucopyranose. Unlike other polysaccharides present in the natural world, chitosan contains primary amine in the main chain and thus has very unique properties. It is applied in various fields such as environment, agriculture, medicine, etc. Especially, it has excellent biocompatibility and biodegradability Gene and drug delivery system, scaffold for tissue engineering, injection type hydrogel, and the like.

The chitosan exhibits sol-gel transfer characteristics at a specific pH. The chitosan exhibits gelation at a pH of about 7.0 to 7.4 similar to that of the body. When the chitosan is solubilized below the above range, the chitosan shows clogging The gel can be formed safely in the body without occurrence.

In the present invention, 3-diethylaminopropyl (DEAP) having the following formula 1 may be used as a pH-sensitive material.

The DEAP block has a tertiary amine and may be sensitive to pH in the body.

Due to the tertiary amine group, the solubility in water varies depending on the pH, so that the gel can be formed or the sol state can be maintained according to the pH change. The tertiary amine groups are chemically non-reactive and typically exhibit reduced toxicity in normal tissues relative to secondary or primary amine groups. Also, unlike primary or secondary amine groups, tertiary amines are not converted to acyl derivatives coupled with carboxylic acids of biological proteins.

In the present invention, the organic bond of the polysaccharide and 3-diethylaminopropyl (DEAP), which is a pH-sensitive substance, reacts with the isothiocyanate group of 3-diethylaminopropyl (DEAP) Can be synthesized.

In particular, by coupling the pH-sensitive 3-diethylaminopropyl (DEAP) component to the biodegradable and completely in vitro release of the pH sensitive polymer in the human body, Gel can be produced.

In one embodiment of the present invention, pH-sensitive nanogel GCS- g- DEAP was prepared by grafting 3-diethylaminopropyl (DEAP) groups onto glycol chitosan (GCS).

The pH-sensitive DEAP group according to the present invention preferably has a degree of substitution of 0.90 to 0.97 with respect to one primary amine group of chitosan, and most preferably a degree of substitution of 0.95. The optimal nanocomposite for encapsulating the drug can be formed within the range of the degree of substitution.

The polysaccharide complex containing the pH-sensitive DEAP group of the present invention can form a spherical self-assembled nanogel in the water due to the amphiphilic property of the hydrophobic group of the pH-sensitive DEAP group and the hydrophilic group of the chitosan.

Generally, self-assembling forms spherical aggregates in which amphiphilic molecules having both hydrophilic and hydrophobic associations form in an aqueous solution. The hydrophilic group is collected inside the spherical aggregate and the hydrophobic group is collected inside. In particular, the hydrophobicity of the DEAP block, especially the DEAP block, in the GCS and hydrophobic core on the hydrophilic shell in the present invention indicates that the ionization of the DEAP block under acidic conditions (~ pK _b ) will increase the hydrophilicity of the block, It is pH dependent.

In addition, the particle size of the nanocomposite may range from 100 nm to 110 nm in diameter, and such a size is suitable for the nanocomposite to migrate to the target cell.

The nanocomposite for drug delivery according to the present invention is destabilized in a weakly acidic environment having a pH of 7.0 or less, that is, the structure of the nanocomposite is changed so that the drug having the pharmacological activity enclosed in the nanocomposite can be released to the outside have.

Therefore, it has a characteristic of being able to specifically react to a disease site showing a weak acidity at a pH of 7.0 or less, and can specifically react at a site showing a weak acidity of pH 5.0 to 7.0. Most preferably in a slightly acidic environment of pH 6.0 to 7.0.

Therefore, the nanocomposite according to the present invention can be effectively used for drug release at about pH 6.0 to 7.0, which is a weak acidic condition formed at a diseased site such as cancer. That is, the nanocomposite of the present invention is widely used as a system for efficiently transferring various anticancer drugs having hydrophobicity due to the hydrophobic nature of the core.

That is, at neutral pH, the DEAP block can be provided as a depot for hydrophobic drugs, while in an acidic pH environment the DEAP block causes degradation of the nanogel due to the protonation of the inner core , Resulting in the release of the drug out of the nanogel.

Therefore, the nanocomposite containing the pH-sensitive DEAP group of the present invention has higher selectivity to cancer tissues due to enhanced permeability and retention (EPR) than general low molecular weight anticancer agents, It has the advantage of accumulating and effectively exhibiting the anticancer effect. It forms voluntary spherical self-aggregates of several tens to several hundred nanometers in size in the aqueous solution by pH-sensitive DEAP combined with the hydrophilic polysaccharide used as the main chain, And can exhibit a high anticancer effect capable of continuous drug release, and can be usefully used for the treatment of diseases such as cancer.

In one embodiment of the present invention, the relative permeability of the nano-gel solution indicating destabilization of the nanocomposite (i.e., nanogel) of the present invention was confirmed. In particular, with respect to the permeability at pH 7.4, Relative transmittance was measured in the selected pH range. As a result, while the relative permeability of the nanogel solution increased sharply while the pH was reduced from 7.2 to 6.8, it was confirmed that the GCS- g- DEAP nanogel of the present invention had a property of being weakly acid-labile.

In addition, the zeta potential displacement of GCS- g- DEAP nanogels at different pH conditions also shows that exposure of the cation was increased due to degradation of the assembly under slightly acidic conditions.

In general, the zeta potential is used as an important factor for controlling the electrostatic dispersion as a unit for the repulsion between particles or the magnitude of attraction. The GCS- g- DEAP nanogel zeta potential of the present invention is -0.9 mV The zeta potential increased with decreasing pH and was measured as +0.95 mV at pH 6.8. From these results, it can be seen that the cations are exposed to an increased amount of cations within the nanocomposite of the present invention at a slightly acidic condition. That is, it means that the negative zeta potential generated from ethylene glycol of GCS at pH 7.4 is offset by the protonation of the DEAP block at pH 6.8.

In addition, for GCS- g- DEAP nanogels, morphological images obtained from FE-SEM also show that at pH 7.4, nearly spherical GCS- g- DEAP nanogels become unstable and partially disappear at pH 6.8.

That is, in the GCS- g- DEAP nanogel, which is an embodiment of the present invention, the structure is stably maintained at a neutral pH of 7.4, but the pH is lowered so that the nanocomposite structure is degraded under weakly acidic conditions.

Therefore, the drug delivery method using the nanocomposite polymer is a drug delivery system that sufficiently shows the selectivity to the target cell and significantly reduces the toxicity to the normal cells and continuously releases the drug for a long period of time, and treats a serious disease such as cancer It can be used in the study of a new concept of therapy.

Furthermore, the present invention provides a method for preparing the pH sensitive drug delivery nanocomposite according to the present invention.

The pH-sensitive drug delivery nanocomposite of the present invention can be prepared using methods that will be apparent to those skilled in the art, but preferably include: (i) dissolving the polysaccharide in a solvent; (ii) synthesizing a polymer by adding 3-diethylaminopropyl (DEAP) to a solution in which the polysaccharide is dissolved to bind an amine group of the polysaccharide to an isothiocyanate of 3-diethylaminopropyl (DEAP); And (iii) dialyzing and lyophilizing the synthesized polymer.

In addition, the stability of the gel to the sensitivity of nanocomposites to various pHs, such as reversible changes from liquid (sol) to gel, can be measured using standard assays or techniques to measure viscosity and volume changes at various pHs, for example using a viscometer .

In the method for preparing the pH sensitive drug delivery nanocomposite according to the present invention, the solvent capable of dissolving the polysaccharide may be any solvent that can dissolve the polysaccharide, preferably dimethylsulfoxide (DMSO) ) Can be used.

Also, the amount of 3-diethylaminopropyl (DEAP) added to the solution in which the polysaccharide is dissolved may be added in the same amount as the molar ratio of the polysaccharide added. When triethylamine or the like is added to the reaction solution, The polymer can be synthesized.

In one embodiment of the present invention, glycol chitosan was used as the polysaccharide. GCS- g- DEAP was synthesized using glycol chitosan and 3-diethylaminopropyl (DEAP). As shown below, the free amine group of glycol chitosan (GSC) reacts with an isothiocyanate group of 3-diethylaminopropyl (DEAP) to form 3-diethylaminopropyl (GCS- g -DEAP) DEAP), which can be reacted at room temperature for 5 to 7 days.

The synthesized nanocomposite can be obtained in a powder state through dialysis and freeze-drying, and when the nanocomposite is dissolved in the water phase, it has a negative charge (-).

Therefore, when a drug having a positive charge (+) is added to an aqueous solution of a polymer synthesized by the method of the present invention, the drug is encapsulated in the polymer by electrostatic attraction, that is, ionic bonding . At this time, the synthesized polymer and the pharmacologically active drug may be mixed with the synthesized polymer and drug having pharmacological activity at a mixing ratio (weight%) of 1: 1 to 10: 1.

The present invention also relates to a pH-sensitive drug delivery system in which a drug having pharmacological activity is encapsulated in the nanocomposite synthesized in the present invention. The drug delivery system also has a nanoparticle size.

Drug Delivery System is a technology to control the concentration and position of drugs introduced into the body to minimize side effects and to deliver drugs for treatment of disease to the treatment site efficiently. It can change the pharmacokinetics of existing drugs to improve efficacy and effectiveness And the drug formulation technique which is desired to be maximized. These drug delivery systems have been intensively developed in recent years in the rapid development of micro-technology or nanotechnology to deliver pharmaceutical ingredients or formulations into cells using nanoscale carriers.

 In addition, nanoparticles are dispersed particles of colloidal imbalance that have a large surface area ranging from several nanometers to several hundreds of nanometers in size, and when the nanoparticles are put into the human body, they are delivered through various methods such as injection, oral administration and skin The distribution of the drug is slightly different depending on the characteristics of the nanoparticles.

Meanwhile, in the drug delivery system according to the present invention, the nanocomposite synthesized in the present invention has a negative charge (-) in an aqueous solution, and forms a spherical drug delivery system by ionic bonding with a drug having a positive charge. The particle size of the nanocomposite nanogel of the present invention may be 100-110 nm, preferably 100-105 nm.

The particle size of the nanocomposite according to the present invention may be slightly different depending on the mixing ratio of the synthesized polymer and the drug having pharmacological activity.

Since the nanocomposite drug carrier according to the present invention has a nano-sized form as described above, it can be easily and quickly inserted into a target site, particularly a target cell.

Furthermore, the drug delivery system of the present invention is characterized in that it can have a weak action range from a weak acidity in response to a change in the pH. Specifically, the drug delivery system of the present invention has pharmacological activity Lt; / RTI > can be released.

Generally, in the body's developing process, the removal of a considerable amount of acid produced in the body must be carried out rapidly to maintain normal hydrogen ion concentration. However, when the hydrogen ion concentration is lowered below 7.35 due to a failure in the maintenance function, acidosis is induced, and when it is increased to 7.45 or higher, alkalosis is induced. In addition, the acidosis is one of the phenomena seen when a disease occurs, and it is a disease such as neuromuscular disease such as myasthenia gravis, Lou Gehrig's disease and muscular dystrophy, chronic obstructive pulmonary disease, rheumatoid arthritis, The disease site is characterized by a weak acidity, especially extracellular pH of solid cancer is 7.2 to 6.0.

Therefore, when a drug delivery system having pH-sensitive characteristics is used by using a change in pH caused upon disease occurrence, a disease-specific drug delivery system can act to diagnose or treat diseases more accurately and effectively. Recently, a drug delivery system having pH-sensitive properties has been developed, and a nanotransporter capable of introducing a polymer containing a protein drug into the cytoplasm has been developed (Y. Lee et al., J. Am. Chem Soc., 2007, 129, 5362-5363). However, the developed pH-sensitive drug delivery system has a problem in that it operates only in a low acidic region having a pH of 5.5.

However, the drug delivery system according to the present invention is characterized in that it can specifically react to a disease site showing a weak acidity at pH 7.0 or lower. Preferably at a pH ranging from 5.0 to 7.0. More preferably 6.0 to 7.0, and most preferably pH 6.8.

The drug release principle of the drug delivery nanocomposite of the present invention is that, as shown below, the pharmacologically active substance in the polymer synthesized in the present invention forms spherical nanoparticles by ionic bonding at neutral pH, that is, pH 7.4 If the pH is lowered and the pH is lowered, the ionic bond is not balanced due to the increase of the cation, and the ionic bond is dissociated and the drug present inside is released to the outside.

As described above, the drug delivery system of the present invention is based on the principle that the nanocomposite is decomposed to release the drug at about pH 7.0, which is a weakly acidic condition formed at the disease site.

Therefore, the pH-sensitive drug delivery system of the present invention can quickly release the drug present therein through the structural modification of the complex under the condition of pH of 7.0 or less, which is weakly acidic.

The drug delivery vehicle of the present invention may contain one or more drugs or therapeutic agents for a short-term therapeutic effect or treatment, wherein the drugs or therapeutic agents are administered before or after dissolving the natural and synthetic polymeric polymers in solution, May be added to the polymer used to make the polymer. Preferably, the drug or therapeutic agent is added prior to dissolution to promote a more uniform dispersion or dissolution of the drug or therapeutic agent.

A variety of techniques are known for incorporating drugs or therapeutic agents into gels (polymers), including but not limited to spray drying, solvent evaporation, phase separation, rapid freezing and solvent extraction.

The drug or therapeutic agent is included in the gel in an amount of about 0.1 to about 100% by weight, preferably about 1 to about 50% by weight, and more preferably about 10 to about 30% by weight. However, the drug or therapeutic agent may be contained in an amount of 0.01 to 95% by weight of the gel. The amount or concentration of the drug or therapeutic agent contained in the gel will depend on the rate of absorption inactivation and release of the drug or therapeutic agent as well as the delivery rate of the polymer in the gel.

The drug carrier according to the present invention prepared by the above method is a liquid at room temperature. Immediately after injection into the subject, it becomes a gel due to body temperature. The drug or therapeutic agent contained within the nanocomposite will diffuse into the extracellular matrix of the subject and will be released in a controlled manner at the target site.

In the present invention, as the drug which can be encapsulated in the synthesized polymer, any component having a positive charge and having a pharmacological activity can be used.

Preferably, proteins, peptides and synthetic compounds can be used, more preferably positively charged proteins can be used, and the positively charged proteins include, but are not limited to, lysozyme, TRAIL (tumor necrosis factor -related apoptosis-inducing ligand, pancreatopeptidase E, calcitonin, HIV Tat protein, Antennapedia homeobox protein, HSV VP22 protein herpes simplex virus VP22 protein), trastuzumab, adalimumab, cetuximab, and alemtuzumab. Rituximab, etanercept, infliximab, alefacept, basiliximab, daclizumab, erythropoietin (erythropoietin), and the like. epoetin-α, darbepoetin-α, oprelvekin, lutropin-α, interferon-β 1a, interferon-beta 1b (interferon-beta 1b), interferon-gamma 1b, glucagon, secretin, arcitumomab, ibritumomab tiuxetan, tositumomab tositumomab, tissue plasminogen activator, tenecteplase, urokinase, salmon calcitonin, palifermin (keratinicyte growth factor), and beccaplerin and becaplermin (platelet-derived growth factor). This is because, as described above, in the case of the polymer synthesized in the present invention, when the polymer is dissolved in water, it becomes negative (-), so that when a drug having a positive charge is used, ionic bonds can be formed more easily.

For example, the compounds of the present invention may be used in combination with other therapeutic agents such as proteins, polypeptides, carbohydrates, inorganic substances, antibiotics, antineoplastics, local anesthetics, antiangiogenic agents, vasoactive agents, anticoagulants, immunomodulators, cytotoxic agents, , Oligonucleotides, lipids, cells, tissues, tissues or cell aggregates, and combinations thereof.

In addition, there are cancer chemotherapeutic agents such as cytokines, chemokines, lymphokines and actually purified nucleic acids, and vaccines such as attenuated influenza virus. Indeed purified hexanes that may be incorporated include genomic nucleic acid sequences, cDNA encoding proteins, expression vectors, antisense molecules that inhibit translation or transcription in combination with complementary nucleic acid sequences, and ribozymes. Also, topical anesthetics such as ametocaine, articaine, benzocaine, bupivacaine, chlorprocaine, dibucaine, dichlonine, etidocaine, levobupivacaine, lidocaine, mephibacaine, oxetazines , Pramoxine, prilocaine, procaine, proparacaine and lopivacaine; But are not limited to, narcotic analgesics such as alfentanil, alpha prodin, buprenorphine, butorphanol, codeine, codeine phosphate, cyclosamine, dextomolamide, dezocine, diamorphine, dihydrocodeine, diphenone, , Fentanyl, hydrocodone, hydromorphone, ketobemidone, levorphanol, mettazinol, methadone, methadyl acetate, morphine, nalbuphine, norproxycin, oxycodone, oxymorphone, Pentazocine, petidine, phenazocine, pyritramide, propoxyphene, remifentanil, sufentanil, tilidine and tramadol; And non-analgesic analgesics such as salicylate or phenylpropionic acid derivatives. Examples of suitable salicylates include, but are not limited to, aminosalicylic sodium, balsalazide, choline salicylate, mesalazine, olanzalazine, para-aminosalicylic acid, salicylic acid, salicylsalicylic acid, and sulfasalazine. Examples of suitable phenylpropionic acid derivatives include ibuprofen, fenoprofen, fluvifrophen, ketoprofen, and naproxen.

In particular, since the pH-sensitive drug delivery nanocomposite of the present invention has a drug-releasing effect in a mildly acidic environment such as a cancerous disease site, the drug can be effectively used as a drug for the treatment of cancer such as paclitaxel, docetaxel, doxorubicin, cisplatin, It is preferable that the drug is at least one drug selected from anticancer agents consisting of forcido and camptothecin.

In another aspect, the present invention provides a controlled drug release pharmaceutical composition comprising a plurality of drugs or therapeutic agents in a bioerodable, biocompatible nanogel, wherein the composition drug or therapeutic agent is introduced into a patient in need thereof A method of treating a disease, disorder, or condition, including a disease or condition, and a method of producing the system of the present invention.

The drug delivery system of the present invention can be formulated into bioabsorbable, biodegradable, and biocompatible. By bioabsorbable is meant that the polymer can disappear from the initial application in the body, without decomposition or degradation of the dispersed polymer molecules. Biodegradable means that the polymer can be disrupted or degraded in the body by hydrolysis or enzymatic degradation. Biocompatibility means that all of the components are non-toxic in the body.

The Therapeutic Drug Delivery System of the Present Invention The drug delivery system of the present invention can be used to deliver a drug contained in the drug system to a person or other mammal having a therapeutically effective disease state or symptom by injection or other means By coating the tissue surface of the body, or by coating the surface of the implantable device), but it is particularly preferred that the composition be delivered parenterally. "Parenteral" means intramuscular, intraperitoneal, intravesical, subcutaneous, intravenous, and intraarterial. Therefore, the composition of the present invention can be typically formulated in injection form.

The injectable compositions of the present invention may be injected or injected into the body of a human or other mammal by any suitable method, preferably by injection through a hypodermic needle.

For example, it can be administered by intravenous, intravenous, genitourinary, subcutaneous, intramuscular, subcutaneous, intracranial, intrathecal, intrapleural, or other bodily fluids or possible space by injection or other means. Diagnosis, or surgery, through the catheter or syringe, into the joint, for example, into the joint during arthroscopy, into the urogenital tract, into the pulmonary, into the lining or into the pleura, or into any cavity or space within the body It can be introduced during the intervention procedure.

Further, according to the drug delivery system of the present invention, the drug or therapeutic agent can be released in a controlled manner to the target site of the subject.

In one embodiment, the nanocomposite is used to provide site-specific release of the drug or therapeutic agent to the subject. In another embodiment, the pH-sensitive nanogel comprises one or more drugs or therapeutic agents that can be administered to the subject, such that the drug or therapeutic agent is released by diffusion from the nanogel and / or degradation of the nanogel.

At this time, the dosage of the composition will vary depending on the type of disease and the severity of the disease or disorder being treated. It should further be understood that for any particular subject, the particular dosage regimen should be adjusted over time according to the needs of the individual and the professional judgment of the person administering the composition or administering the administration. In vivo administration can be based on in vitro release studies in cell culture or in vivo animal models

The rate of release of a drug or therapeutic agent is dependent on many factors, in particular, on the rate of degradation of the biodegradable polymer that constitutes the gel. It is also dependent on the particle size of the drug or therapeutic agent. Thus, by adjusting the factors discussed above, decomposition, diffusion, and controlled release can be varied in a wide variety of ranges. For example, emissions may be designed to occur over time, days, and months.

As described above, the pH-sensitive nanocomposite for drug delivery according to the present invention can be biodegradable and biocompatible in vivo based on a biopolymer polysaccharide, and the polysaccharide can be organically And the polymer synthesized by such an organic bond can form a nano-sized nanocomposite for drug delivery through ionic linkage with a pharmacologically active ingredient, so that it can easily and quickly penetrate into cells.

In addition, the drug delivery nanocomposite prepared according to the present invention has a disadvantage in that the structure of the nanocomposite collapses (that is, 3-diethylaminopropyl (DEAP)), which is a pH sensitive material, the nanocomposite is chemically modified by a change in pH, and finally the amine group of the glycol chitosan is exposed and the ionic bond is disassociated, so that the structure of the nanocomposite is ultimately transformed), so that the drug existing in the nanocomposite is rapidly released .

Therefore, the pH-sensitive drug delivery nanocomposite of the present invention can selectively and efficiently deliver a drug to diseases such as specific diseases or acidosis with environmental changes under weakly acidic conditions, thereby increasing the therapeutic effect. In particular, Can be used to treat diseases such as myasthenia gravis, neuromuscular diseases such as Lou Gehrig's disease and muscular atrophy, chronic obstructive pulmonary disease, rheumatoid arthritis and solid tumors.

While various aspects of the composition have been described in detail, it will be apparent to those skilled in the art that modifications, substitutions and additions may be made without departing from the scope of the invention.

[Example]

Hereinafter, the present invention will be described in detail with reference to the following embodiments and accompanying drawings. The following examples are intended to illustrate the present invention more specifically, but the protection scope of the present invention is by no means limited to the examples described in the following examples. It is apparent that the scope of the present invention covers not only the following embodiments, but also matters which can easily be deduced therefrom by a person skilled in the art.

First, in order to prepare pH sensitive nanocomposites using 3-diethylaminopropyl (DEAP), which is a chitosan derivative and a pH-sensitive material, the following substances were dissolved in a mixture of Sigma Aldrich (St. Louis, MO, USA).

Glycol chitosan (GCS, 250 kDa, 82.7% deacetylation)

3-diethylaminopropyl isothiocyanate (DEAP),

pyrene,

pyridine,

Doxorubicin HCl (DOXHCl),

Dimethyl sulfoxide (DMSO),

Triethylamine (TEA),

Na ₂ B ₄ O ₇ ,

NaOH,

HCl, and

NaCl

< Example  1>

pH  Sensitivity GCS - g - DEAP  Produce

<1-1> GCS - g - DEAP  synthesis

First, 1 g of Glycol chitosan (GCS, 250 kDa, 82.7% deacetylated) purchased from Sigma-Aldrich (USA) was dissolved in 20 ml of DMSO, and then 1 ml of triethylamine (TEA) DEAP (2.8 g) was grafted onto GCS. After the reaction, the solution was transferred to a pre-swollen dialysis membrane tube (Spectra / Por MWCO 8K) and dialyzed against deionized water to remove unreacted DEAP and the solution was lyophilized. Subsequently, the degree of substitution (DS, the number of DEAP blocks substituted per primary amine of the glycol chitosan) was calculated from delta 1.0 (-CH3, DEAP block) and delta 2.73 (-CH-, the repetitive unit of glycol chitosan) from was measured by analyzing (DMSO-d6 with TMS) peak ¹ H-NMR by the introduction ratio of the peak derived.

As a result, in the case of GCS- g- DEAP synthesized by the above method, it was confirmed that the number of 3-diethylaminopropyl blocks substituted by primary amine groups of glycol chitosan analyzed by ¹ H-NMR was 0.95.

<1-2> Acid-base titration

GCS- g- DEAP or NaCl (control) dissolved in deionized water (30 mmol / l) was adjusted to pH 10 with 1 M NaOH. The solution was titrated by a stepwise addition of 0.1 M HCl solution to obtain a pH profile. Three averages were shown.

As a result, as shown in Fig. 1, the pK _b of GCS- g- DEAP measured from the inflection point in the acid-base titration curve was about 6.74. This is similar to the pK _b of GCS at about 6.0.

These results suggest that the DEAP blocks are responsible for most of the buffering capacity within the physiological pH range; It can be seen that the pH-buffer region of the GCS- g- DEAP is between pH 6.5 and 7.0. The results are similar to those of polyHis-b-poly (ethylene glycol) because the pKb of imidazole of polyHis-b-poly (ethylene glycol) is about 7.0. However, unlike the case of imidazole, the DEAP block of the present invention has a tertiary amine and is chemically non-reactive. Since the tertiary amine group usually exhibits reduced toxicity in normal tissues as compared to secondary or primary amine groups and is not converted to an acyl derivative bonded with a carboxylic acid of a biological protein, It is expected that the effect will be different from the solution.

< Example  2>

GCS - g - DEAP Nanogel  Produce

GCS- g- DEAP (50 mg) dissolved in DMSO (5 ml) was transferred to a pre-swollen dialysis membrane tube (Spectra / Por MWCO 8K) and dialyzed against Na ₂ B ₄ O ₇ buffer Respectively. The outer phase was replaced with fresh Na ₂ B ₄ O ₇ buffer three times. The solution was filtered through a 0.8 μm syringe filter and subsequently lyophilized. After freeze-drying for 2 days, a powder was obtained.

< Example  3>

GCS - g - DEAP Nanogel  characteristic

<3-1> Particle size distribution

In order to investigate the physicochemical properties of GCS- g- DEAP nanogels, a He-Ne laser beam was first provided and at a fixed scattering angle of 632.8 nm and 90 ° using a Zetasizer 3000 (Malvern Instruments, USA) The particle size distribution of the nanogel (0.1 g / l) was measured in PBS (phosphate buffer saline) solution (pH 7.4-5.0, ionic strength = 0.15).

As a result, as shown in Fig. 2, the average particle size of the GCS- g- DEAP nanogel was about 102 nm. These GCS- g- DEAP nanogels were stable for 7 days under optimal conditions without any precipitation, and the particle size and permeability of the GCS- g- DEAP nanogels did not change even after this period.

<3-2> Nano gel  The permeability of the solution ( transmittance ) change

The light transmittance of the solution was measured using an ultraviolet / visible spectrophotometer (Labantech Co.). Before testing, the nanogel solution (0.1 g / l) at different pH (pH 7.4-5.0, ionic strength = 0.15) was stabilized at room temperature for 4 hours. With respect to the permeability at pH 7.4, the relative transmittance of the nanogel solution was measured in the selected pH range.

As a result, while the pH was reduced from 7.2 to 6.8 as shown in FIG. 3, the relative permeability of the nanogel solution, which means destabilization of the GCS- g- DEAP nanogel, increased sharply. This is in sharp contrast to the GCS solution, which does not show a change in permeability at all of the pHs tested.

<3-3> Critical aggregate  density( critical aggregation concentration , CAC ) analysis

Fluorescence measurements for CAC determination were performed using a Shimadzu RF-5301PC fluorescence spectrometer. That is, the freeze-dried nanogel powder was dispersed in HCl (or NaOH) -Na ₂ B ₄ O ₇ buffer solution (pH 7.4-5.0, ionic strength = 0.15) and pyrene mixture, finally with 6.0 × 10 ^-7 M pyrene concentration of 1 × 10 ^- to obtain the nano-gel concentration of from ³ to 5 × 10 ^-1 g / l. The CAC was then measured by plotting the intensity of the first peak (I1) of the luminescence spectral profile (at? Ex 339 nm) versus log of the GCS- g- DEAP concentration.

As a result, as shown in Fig. 4, the CAC value of the GCS- g- DEAP nanogel at pH 7.4 was relatively low (6 μg / ml). However, due to the reduced pH of the solution, the CAC value of the GCS- g- DEAP nanogel was significantly increased (24 μg / ml at pH 6.8 and 73 μg / ml at pH 6.0).

This is because acidic pH decreases the hydrophobicity of DEAP blocks and increases the CAC value.

<3-4> GCS - g - DEAP Nano-gel  Zeta potential ( Zeta potential )

The zeta potential of the GCS- g- DEAP nanogel solution (0.1 mg / ml) at different pH values (pH 7.4-5.0, ionic strength = 0.15) was measured using a Zetasizer 3000. Prior to testing, the GCS- g- DEAP nanogel solution was stabilized at room temperature for 4 hours.

As a result, as shown in FIG. 5, the zeta potential of the GCS- g- DEAP nanogel changed from -0.9 mV to +0.95 mV while the pH of the nanogel solution was decreased from pH 7.4 to 6.8. These results suggest that the negative zeta potential from ethylene glycol in GCS at pH 7.4 is offset by the protonation of the DEAP block at pH 6.8.

<3-5> GCS - g - DEAP Nano-gel  Morphological observation

GCS- g -DEAP order to observe the shape of the nano-gel and the diluted GCS- g -DEAP nano-gel solution (0.1 mg / ml) prepared by casting the sample on a slide glass at pH 7.4 or pH 6.8, and under vacuum Lt; / RTI &gt; The morphology of the sample was imaged by Field Emission Scanning Electron Microscopy (FE-SEM, Hitachi s-4800).

As a result, as shown in Fig. 6, the morphological image obtained from FE-SEM showed that almost spherical GCS- g- DEAP nanogels at pH 7.4 became unstable and partially disappear at pH 6.8.

Overall, GCS- g -DEAP the GCS- g -DEAP nanogels in the present data are rather weak acid pH determine the physical and chemical properties of the nano-gel, the hydrophilic core of the nano-gel from hydrophobic by protonation of DEAP blocks reaction Suggesting that it is destabilized because of the transition.

< Example  4>

GCS - g - DEAP Nano gel  Manufacture of Drug Delivery System

<4-1> DOX  loading

Before loading doxorubicin (DOX) in GCS- g- DEAP nanogels, the HCl salt of DOXHCl was isolated by reacting DOXHCl with 2 mole ratio of TEA in DMSO overnight. Subsequently, GCS- g -DEAP Nano gel (20 mg) and DOX (4 mg) for 24 hours, for the HCl (or NaOH) -Na ₂ B ₄ O ₇ buffer solution (10 mM) dissolved in DMSO (2 ml) Lt; / RTI &gt; The outer phase of the dialysis bag containing DOX and the polymer were changed 3 times and the non-encapsulated glass DOX was removed during this process. GCS- g -DEAP the DOX concentration in the nano-gel, at 481nm in the ultraviolet / visible spectrum analyzer DMSO / HCl (or NaOH) -Na 2 B 4 O 7 buffer mixture (95: 5 vol%.) Dissolved in Lt; / RTI &gt; was determined by measuring the UV absorbance of the DOX-loaded nanogels. The DOX loading efficiency was 78 ± 3.1 wt.%, Which was calculated by dividing the loaded DOX content by the feeding DOX content.

<4-2> DOX  Emission test

The DOX-loaded GCS- g- DEAP nanogel solution (0.5 ml) was added to the dialysis bag (Spectra / Por MWCO 15K). After sealing the dialysis bag, the pH was adjusted to pH 7.4-5.0 And immersed in vials containing fresh PBS (10 ml, ionic strength = 0.15).

DOX release tests from GCS- g- DEAP nanogels were performed at 37 DEG C under mechanical vibration (100 rev./min). The outer phase of the dialysis bag was extracted and the fresh buffer solution was replaced at defined time intervals to maintain the DOX sink. The DOX concentration was measured using a UV / visible spectrum analyzer.

As a result, as shown in Fig. 7, the DOX emission profile at pH 7.4 and pH 6.8 was significantly different. That is, 23 wt.% Drug was released at pH 7.4 at 8 wt.% At pH 7.4 and 23 wt.% At pH 6.8 within 24 hours, and 59 wt.% Drug was released at pH 7.4 at 20 wt.% And pH 6.8 within 24 hours.

And further reduction of the pH of the solution to pH 6.0 promoted the DOX release rate of the GCS- g- DEAP nanogel: 74 wt.% Drug released at pH 6.0 for 24 hours.

These results indicate that GCS- g- DEAP nanogels can be useful for targeting cancer-associated acidic pH (e.g., pH 6.8) sites and can be used to trigger drug release at endosomal pH 6.0 It suggests. Also, as a function of time, the DOX emission pattern follows near first-order kinetics and reaches the ballast within 8 hours.

< Example  5>

Drug testing

<5-1> Cell culture

Human non-small lung carcinoma A549 cells (Korean Cell Line Bank) were cultured in RPMI-1640 / PBS supplemented with 0.5 M PBS, 2 mM L-glutamine, 5% penicillin-streptomycin, using PBS medium was kept under 37 ℃ and 5% CO ₂ air incubator in a wet condition. Prior to testing, the cells growing in a single layer were obtained by trypsinization with (0.25% (w / v) trypsin / 0.03% (w / v) EDTA solution (1x10 ⁵ cells / ml). Cells attached to RPMI-1640 medium (200 [mu] l) were seeded in 96-well plates and cultured for 24 hours before in vitro cell testing.

<5-2> Fluorescence Microscopy Analysis

Different amounts of DOX accumulation in A549 cells by DOX-loaded GCS- g- DEAP nanogels were investigated according to pH signals using fluorescence microscopy (λex 488 nm and λem 510 nm, E-SCOPE 1500F). In this test, the DOX concentration in the GCS- g- DEAP nanogel solution at pH 7.4 and 6.8 was 1 μg / ml. A549 cells ( 1 x 10 ³ cells / ml) were cultured for 8 hours with a DOX-loaded GCS- g- DEAP nano-gel solution (pH 7.4 or 6.8), the cells were washed three times with PBS (pH 7.4) And fixed with 1% formaldehyde in PBS for 10 min. The cover slip was then mounted on a microscope slide with a spot of anti-fade mounting media (5% N-propyl galate, 47.5% glycerol and 47.5% TrisHCl, pH 8.4) to reduce fluorescence photobleaching.

As a result, fluorescence images of DOX-loaded GCS- g- DEAP nanogels or A549 cells treated with free DOX are shown in Fig. The DOX concentration in the cells was observed through the red fluorescence intensity of DOX. In addition, DOX accumulation levels were different in DOX-loaded GCS- g- DEAP nano-gel treated A549 cells at each pH. This behavior is comparable to that of free DOX-treated A549 cells

In addition, accelerated DOX release from GCS- g- DEAP nanogels at pH 6.8 results in improved DOX uptake in A549 cells, which provides a high fluorescence signal. These results suggest that the nanogel system may be very useful for the selective delivery of anti-cancer drugs to cancer cells.

These results DOX--loaded nano-gel and to show markedly enhanced DOX release at pH 6.8 as compared to the case in the normal pH 7.4, which arm portions of the system of the invention the acidic pH _e. (~ PH 6.8) , It is possible to increase the therapeutic activity of the drug for in vivo cancer treatment.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (15)

A nanocomposite for doxorubicin delivery comprising chitosan grafted with 3-diethylaminopropyl (DEAP). The method according to claim 1,
Wherein the grafting is carried out by binding an amine group of the chitosan to an isothiocyanate group of the 3-diethylaminopropyl group. delete delete The method according to claim 1,
Wherein the chitosan is at least one selected from water-soluble glycol chitosan, chitosan oligomer and acetylated chitosan. The method according to claim 1,
Wherein the nanocomposite is destabilized at a pH of less than 7.0. The method according to claim 1,
Wherein the 3-diethylaminopropyl (DEAP) is substituted with 0.90 to 0.97 per one primary amine of chitosan. The method according to claim 1,
Wherein the nanocomposite has a particle size of 100 to 110 nm. A doxorubicin carrier comprising the nanocomposite for delivering doxorubicin according to claim 1 and doxorubicin which can be encapsulated in the nanocomposite. delete delete 10. The method of claim 9,
Wherein the mixing ratio of the nanocomposite and doxorubicin is 1: 1 to 10: 1 by weight of the nanocomposite: doxorubicin. Dissolving chitosan in a solvent;
Adding 3-diethylaminopropyl to the solution in which the chitosan is dissolved to bind the amine group of the chitosan and the isothiocyanate of the 3-diethylaminopropyl group; And
And dialyzing and freeze-drying the combined polymer. delete 14. The method of claim 13,
Wherein the chitosan is a glycol chitosan.

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