KR100853309B1 - Bio-degradable nanoparticles of polyDL-lactide-co-glycolide encapsulating ciprofloxacin HCl having extended-release properties, and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a sustained release and biodegradable nanoparticles of a polylactide glycolide copolymer carrying ciprofloxacin and a method for producing the same. More specifically, the present invention is characterized by the sustained release and biodegradable porous nanoparticles of polylactide glycolide copolymer having a particle size of 100-500 nm and carrying ciprofloxacin therein, and a W / O / W emulsion solvent evaporation method. A method for producing microparticles carrying water-soluble drugs, the method comprising: (1) dissolving polylactide glycolide in dichloromethane at a concentration of 2-6% (w / v, g / ml); (2) dissolving ciprofloxacin in distilled water at a concentration of 1-2% (w / v, g / ml); (3) the dichloromethane solution in which the polylactide glycolide is dissolved and the aqueous solution of ciprofloxacin are mixed so that the weight ratio of polylactide glycolide: ciprofloxacin is 8-10: 1 and then 30-40 watts (W) using an ultrasonic mill. Processing for 1 to 3 minutes per 10 ml to output to create a W / O emulsion; And (4) the W / O emulsion was added to a 1.0% (w / v) polyvinyl alcohol solution and treated with a high-pressure homogenizer at 600-800 bar at 1 minute per 100 ml for 5-7 cycles. It provides a method for producing a slow release and biodegradable porous nanoparticles of a polylactide glycolide copolymer having a particle size of 100-500nm including the step of making a W / O / W emulsion and carrying ciprofloxacin therein.

The nanoparticles loaded with ciprofloxacin prepared according to the present invention

Since it is made of a polylactide glycolide copolymer, there are no side effects and immune rejection reactions in the human body, and the drug can be controlled to be released slowly for more than a week after administration, so the blood half-life is short, which is particularly advantageous for the administration of frequently administered drugs. After all of the drug is released, the copolymer functioning as a carrier is naturally decomposed and eliminated in the human body, and thus, there is no need to remove nanoparticles separately. In the present invention, polylactide glycolide is dissolved in dichloromethane, and ciprofloxacin is dissolved in distilled water, and then the solution is mixed. A W / O emulsion is made by an ultrasonic mill and then put into an aqueous polyvinyl alcohol solution (High-pressure homogenizer). ) Provides a novel method for producing nanoparticles between 100-500nm loaded with ciprofloxacin easily.

Ciprofloxacin, polylactide glycolide copolymers, nanoparticles

Description

Bio-degradable nanoparticles of poly (DL-lactide-co-glycolide) encapsulating ciprofloxacin HCl having extended-release properties, and manufacturing of polylactide glycolide copolymer loaded with ciprofloxacin method kind}

1 is a transmission electron micrograph of (a) nanoparticles [180-20] prepared according to Example 1 of the present invention and (b) nanoparticles [360-40] prepared according to Example 2 of the present invention, respectively. ego,

Figure 2 is a graph showing the size distribution of the nanoparticles [360-40] prepared according to Example 2 of the present invention,

Figure 3 is (a) Ciprofloxacin HCl, CIP, (b) Ciprofloxacin-free nanoparticles prepared according to Comparative Example 1 of the present invention [Empty NP], (c) according to Example 1 of the present invention Nanoparticles prepared [180-20], (d) Nanoparticles prepared according to Example 2 of the present invention [360-40], (e) Nanoparticles containing ciprofloxacin prepared according to Comparative Example 1 of the present invention XRD spectrum when simple mixing of particles and ciprofloxacin [Empty NP / CIP = 20/1],

Figure 4 is a drug of (a) nanoparticles prepared according to Example 1 of the present invention [180-20, ●], (b) nanoparticles prepared according to Example 2 of the present invention [360-40, ○] Is the result of measuring the release rate,

5 is a phosphate buffer solution [PBS, ●], ciprofloxacin [Free CIP, ○], the nanoparticles containing ciprofloxacin prepared according to Comparative Example 1 of the present invention [Empty nanoparticles, ▼], Example 2 of the present invention Nanoparticles prepared according to [360-40, CIP-nanoparticles, △], a simple mixture of ciprofloxacin and nanoparticles containing ciprofloxacin prepared according to Comparative Example 1 of the present invention [Free CIP + Empty NP, ■ ] Were treated with (a) Escherichia coli (E. coli) and (b) Salmonella typhimurium (S. typhimurium) , respectively , to compare the viability of microorganisms,

6 is treated with phosphate buffer [PBS], ciprofloxacin [Free CIP] and nanoparticles [CIP-NP] prepared according to Example 2 of the present invention to mice infected with S. typhimurium (a) 3 days and (b) evaluated antibacterial performance for 5 days, and

FIG. 7 shows antibacterial performance for 3 days by treating sterile water [DW], ciprofloxacin [Free CIP] and nanoparticles [CIP-NP] prepared according to Example 2 of the present invention to mice infected with E. coli. Will be evaluated.

<Technical field to which the invention belongs>

The present invention relates to sustained-release and biodegradable nanoparticles and preparation method of polylactide glycolide (poly- (DL-lactide-co-glycolide), PLGA) copolymer loaded with ciprofloxacin. More specifically, the present invention is characterized by the sustained release and biodegradable porous nanoparticles of polylactide glycolide copolymer having a particle size of 100-500 nm and carrying ciprofloxacin therein, and a W / O / W emulsion solvent evaporation method. A method for producing microparticles carrying water-soluble drugs, the method comprising: (1) dissolving polylactide glycolide in dichloromethane at a concentration of 2-6% (w / v, g / ml); (2) dissolving ciprofloxacin in distilled water at a concentration of 1-2% (w / v, g / ml); (3) the dichloromethane solution in which the polylactide glycolide is dissolved and the aqueous solution of ciprofloxacin are mixed so that the weight ratio of polylactide glycolide: ciprofloxacin is 8-10: 1 and then 30-40 watts (W) using an ultrasonic mill. Processing for 1 to 3 minutes per 10 ml to output to create a W / O emulsion; And (4) the W / O emulsion was added to a 1.0% (w / v) polyvinyl alcohol solution and treated with a high-pressure homogenizer at 600-800 bar at 1 minute per 100 ml for 5-7 cycles. It provides a method for producing a slow release and biodegradable porous nanoparticles of a polylactide glycolide copolymer having a particle size of 100-500nm including the step of making a W / O / W emulsion and carrying ciprofloxacin therein.

<Private Technology>

Human infection by bacteria is one of the most dangerous diseases that threaten human survival. Numerous antibiotics, including quinolone antibiotics, have been developed and used to treat these diseases. Quinolone antibiotics are known to have a wide range of antibacterial properties and include urinary tract infections, respiratory tract infections, sexually transmitted diseases, skin infections, and chronic osteomyelitis. It is known to be particularly effective in the back. Ciprofloxacin is one of the most widely used quinolone antibiotics, and is known to be effective against clinically important Gram-negative and Gram-positive pathogens, including urethritis, enteritis, infectious diseases, It is widely used for skin infections, osteomyelitis and the like. In particular, ciprofloxacin, which is known to have a therapeutic effect on urethritis, has a very short half-life in blood, and therefore, oral preparations and injections are administered two or more times a day.

In order to solve the above problems, the prior art has reported a variety of fine particles and a method for producing the same as a sustained release formulation. For example, Korean Patent No. 92-7831 describes a method for preparing a sustained release microcapsule, and Korean Patent No. 202073 describes a microcapsule sustained release formulation and a method of manufacturing the same. Since the prepared microparticles have a particle size in the micrometer range (for example, 0.5 to 400 μm), when intravascular injection such as intravenous injection is required, it cannot be used as an injection and is mainly limited to local administration. It is a disadvantage, because the particle size distribution is wide, between 5 and 100 micrometers, it is relatively difficult to control the release rate of the drug, and because the shape of the particle is not constant, it is difficult to predict the decomposition rate and drug release rate of the particles, etc. The method used in Korean Registered Patent No. 202073, or Korean Registered Patent No. 92-7831 can be used to prepare small and uniform fine particles. It is difficult and difficult to encapsulate the water-soluble drug in the particles at high concentrations, and thus has a disadvantage in that the cost of the drug can be lost in the manufacturing process due to the low sealing efficiency.

In addition, as a method of increasing the encapsulation efficiency of the drug, a method of increasing the encapsulation efficiency of the water-soluble drug by using a solvent extraction method has been developed as reported in the Patent Publication No. 162872, in which case it is difficult to completely remove the solvent, so that residual solvent remains in the fine particles There is also a possibility that the particle size is also about tens of micrometers, the particle size is large, and it is difficult to control the size distribution of the particles, it is also difficult to control and release the drug. Korean Patent Publication No. 10-481540 discloses a method for preparing nanoparticles containing a therapeutic drug. As a method of encapsulating a drug at the time of preparing the nanoparticles by emulsion polymerization, the nanoparticles have a size of 50-300 nm. Although it is possible to manufacture, since the temperature during polymerization is between 40-90 ℃, the decomposition and activity of the drug can be easily lost at such high temperatures, and the removal of the polymerization initiator and the surfactant is not easy. There is a restriction.

As described above, the W / O / W double emulsion solvent evaporation method reported in the existing patent is difficult to increase the encapsulation efficiency of ciprofloxacin, which is a water-soluble drug, and the particle size is several tens of micrometers. It is difficult to control the particle size because it is large. 3. Therefore, the particle size is large, and there are many limitations in using it as an injection. 4. There is a problem that it is not easy to control the release rate of the drug as desired. Alternatively, the rate of decomposition of the microparticles may not be constant and may remain in the body for a long time after all of the drug is released. 6. In case of nanoparticles, if the particles are not uniform, the particles may not go to the infection site and are released by the human immune system. Can move to other organs, and 7. has the disadvantage that it is difficult to maximize the efficacy of the drug in the end. In the present invention, by improving the first to dissolve the polylactide glycolide to support the continuous release of the drug in dichloromethane and dissolution of ciprofloxacin in sterile distilled water, by mixing the two solutions to prepare a primary W ₁ / O emulsion, This was dispersed in an aqueous solution in which polyvinyl alcohol was dissolved at 1.0% (w / v) to prepare a secondary W ₁ / O / W ₂ emulsion, which was then refined using a high pressure homogenizer to refine the emulsion. It was possible to manufacture and improve the disadvantages of the conventional method mentioned above was easy to use as an injection, it was possible to make nanoparticles with high sealing efficiency of water-soluble drugs.

Therefore, in the present invention, by providing a 100-500nm nano-sized sustained-release microparticles containing ciprofloxacin encapsulated using polylactide glycolide, by controlling the physicochemical and drug release properties, the ciprofloxacin is released slowly for one to two days To solve the problems of the prior art.

In addition, in the present invention, polylactide glycolide is dissolved in dichloromethane, ciprofloxacin is dissolved in distilled water, the solution is mixed, a W / O emulsion is prepared by using an ultrasonic crusher, and then put in an aqueous polyvinyl alcohol solution (High-pressure homogenizer). By providing a new method for producing nanoparticles having a size range of 100-500nm easily loaded with ciprofloxacin using the present invention provides biodegradable and sustained-release nanoparticles loaded with ciprofloxacin controlled in a certain range. To provide a new way to.

Biodegradable and sustained release nanoparticles carrying ciprofloxacin having a controlled size, such as the present invention, are easily used as an injectable agent due to their appropriately small size when applied in vivo, and between cell gaps and capillaries when injected into the human body. Not only can penetration be effectively achieved, but it also has the advantage of selectively delivering the drug to the lesion site. In addition, the small size can solve the problem of blocking the passage of the medical device (eg, syringe) for drug administration, which is one of the important problems caused by the fine particles. In addition, it is possible to release the drug at a constant rate for one week to two weeks, so that the drug can be properly released and effective for a period effective for treatment.

In particular, the present invention of the various drugs, especially the proliferation of ciprofloxacin using a biodegradable polymer polylactide glycolide (HPH, high pressure homogenizer) to a certain range of particle size controlled nanoparticles with excellent encapsulation efficiency of water-soluble drugs There is a technical significance to provide an optimized method for making particles and nanoparticles having a specific particle size produced accordingly.

For this purpose, the present invention provides a sustained release and biodegradable porous nanoparticles of a polylactide glycolide copolymer having a particle size of 100-500 nm and carrying ciprofloxacin therein. In a preferred embodiment of the present invention the size of the nanoparticles is preferably 130-353nm. In addition, the content of ciprofloxacin is 4.5-5.0 (w / w)% based on the total weight of the nanoparticles, and the ciprofloxacin supported on the nanoparticles may be continuously released from the nanoparticles for 10 days or more.

Although the particle size can be manufactured to less than 100 nm, in this case, the encapsulation rate of the drug is too low, and the content of the drug increases slightly when manufacturing the nanoparticles to 500 nm or more, but it exceeds the appropriate range as an injection. Therefore, in the present invention, the nanoparticles in the range of about 130-353 nm are sufficiently high in the encapsulation efficiency of the drug, suitable for use as an injection, and the release rate of the drug is also suitable for the effective treatment period.

In addition, in the present invention, polylactide having a particle size of 100-500 nm and ciprofloxacin supported therein in the method for preparing the water-soluble drug-supported microparticles by the W / O / W emulsion solvent evaporation method. Provided is a method for preparing slow release and biodegradable porous nanoparticles of glycolide copolymers. :

(1) dissolving polylactide glycolide in dichloromethane at a concentration of 2-6% (w / v, g / ml);

(2) dissolving ciprofloxacin in distilled water at a concentration of 1-2% (w / v, g / ml);

(3) the dichloromethane solution in which the polylactide glycolide is dissolved and the aqueous solution of ciprofloxacin are mixed so that the weight ratio of polylactide glycolide: ciprofloxacin is 8-10: 1 and then 30-40 watts (W) using an ultrasonic mill. Processing for 1 to 3 minutes per 10 ml to output to create a W / O emulsion; And

(4) The W / O emulsion was added to a 1.0% (w / v) polyvinyl alcohol aqueous solution and treated with a high-pressure homogenizer at 600-800 bar at 1 minute per 100 ml for 5-7 cycles. Steps to create a W / O / W emulsion.

Ciprofloxacin hydrochloride (C ₁₇ H ₁₈ FN ₃ O _{3 ·} HCl · H ₂ O), an active ingredient used in the present invention, is an antibiotic developed by Bayer, a German chemical and pharmaceutical company. It is called '. As a specialized medicine, it is used as an antibiotic for various infections such as respiratory infections, ear, nose, throat infections and sepsis.In particular, it has a high antibiotic effect on animal anthrax and is recognized as the only drug for treating anthrax by the US Food and Drug Administration (FDA). It has the following structure.

Polylactide glycolide (PLGA, poly- (DL-lactide-co-glycolide)) used in the present invention may be prepared according to the known art, for example DL-poly lactic acid (PLA) from DL-lactide It can be prepared by reacting the glycolide with a certain ratio and drug release using a polylactide glycolide copolymer, J. Korean Ind, Eng. Chem . Vol. 12, NO. 2, April 2001, 148-153, Na Jae-un et al.), Commercially available commercially available. In the present invention, it is preferable that the composition ratio of the copolymer is 50/50 (lactide / glycolide ratio).

In one embodiment of the present invention, the polylactide glycolide in step (1) is dissolved in dichloromethane at a concentration of 2-6% (w / v, g / ml), and in step (2) ciprofloxacin The dichloromethane solution and ciprofloxacin solution in which the polylactide glycolide is dissolved in distilled water at a concentration of 1-2% (w / v, g / ml) are dissolved in the polylactide glycolide: The weight ratio of ciprofloxacin is 8-10: 1, preferably 9: 1.

When the concentration of polylactide glycolide is lower than the above range, there is no significant difference in the size of the particles, but when the relative drug encapsulation efficiency is lowered, and when the concentration of the polylactide glycolide is higher than the above range, the particle size increases while encapsulation Since there is no big gain in efficiency, it is preferable to use polylactide glycolide in the above range.

In addition, if the concentration of ciprofloxacin is lower than the above range, the content of ciprofloxacin per unit nanoparticle weight ratio is low, making it difficult to properly adjust the concentration of the drug to an effective level when used as an injection, and the solubility of ciprofloxacin in distilled water is about 2.5%. (w / v, g / ml) Since the drug has a serious problem of precipitation in distilled water at a concentration higher than this, the drug concentration range of the drug is preferable because of the problem of lowering the sealing efficiency of the drug.

In addition, when the weight ratio of polylactide glycolide: ciprofloxacin is lower than the above range, there is a problem in that the size of the particles is smaller and the encapsulation efficiency of the drug is lowered. Since the encapsulation efficiency does not increase as compared with the increase in the average size, the weight ratio range is preferable.

In one preferred embodiment of the invention, the ultrasonic mill in step (3) has an output of 30 to 40 watts (W), preferably 40 watts, for 1 to 3 minutes per 10 ml, preferably for 1 minute. Apply.

If the output is lower than the above range, there is a problem that the size of the particle is increased to the micrometer size and the drug encapsulation efficiency is low because the W / O emulsion is not sufficiently produced in detail. There was no significant change in efficiency and the temperature in the emulsion solution is increased, which may cause a decrease in the performance of the drug, and there is a problem in that the yield of particles is lowered due to aggregation after the reaction.

In addition, when the application time of the ultrasonic grinder is longer than 1-3 minutes, the temperature of the emulsion gradually increases, causing problems such as agglomeration in the refining process, which lowers the yield of the particles. Annihilation (ie, not sufficiently emulsified) was not produced, resulting in problems such as no particle generation or aggregation (aggregation) and larger particles, and also lowered the sealing efficiency of the drug.

In a preferred embodiment of the present invention, the step (4) is a mixture of the W / O emulsion and 1.0% (w / v) polyvinyl alcohol aqueous solution 600 ~ 800 in a high-pressure homogenizer bar, preferably 600 bar, is treated for 5 to 7 cycles, preferably 5 times, at 1 minute intervals per 100 ml.

If the pressure is lower than the above range, since the W ₁ / O / W ₂ emulsion is not sufficiently fine, there is a problem in that the size of the particles is increased and the encapsulation efficiency of the drug is also lowered. In addition, if the temperature is higher than this, the temperature rises and the emulsion may occur, and the particle size may increase or may not be formed.

In addition, when treated with a high-pressure homogenizer with more cycles than the above range (that is, when it is turned for a long time), the temperature rises and the emulsion agglomerates, and the particle size does not change much, but there is a problem such as agglomeration. In the case of treatment with a high pressure homogenizer with less than a range of cycles (that is, when it is turned for a short time), the W ₁ / O / W ₂ emulsion is not sufficiently formed, so that the size of the particles is increased and the encapsulation efficiency of the drug is also lowered.

The preparation method of the present invention is particularly optimized among various drugs, in particular for making ciprofloxacin into nanoparticles whose particle size is controlled in the range of 100-500 nm, preferably 130-353 nm using polylactide glycolide, and thus There is a technical significance to provide a nanoparticle having a specific particle size produced.

In a preferred embodiment of the present invention, the preparation method of the present invention (5) by adding the W / O / W emulsion to 0.4% (w / v) aqueous solution of polyvinyl alcohol (polyvinyl alcohol, molecular weight 15,000) with a stirrer After stirring for 30 to 90 minutes at 500-1500rpm further comprises the step of recovering the nanoparticles with an ultracentrifuge and freeze-dried.

The present invention also provides a sustained release and biodegradable porous nanoparticles of a polylactide glycolide copolymer having a particle size of 100-500 nm and carrying ciprofloxacin therein prepared by the above method. Preferably the size of the nanoparticles is 130-353nm.

According to the present invention, the sustained-release and biodegradable porous nanoparticles of a polylactide glycolide copolymer having a particle size of 100-500 nm and carrying ciprofloxacin therein are made of a polylactide glycolide copolymer and thus have no effect on human body. In addition to no side effects and immune rejection, the drug can be controlled to release slowly over a week after administration, which is particularly advantageous for the administration of ciprofloxacin, a drug that should be frequently administered due to its short half-life. Since the copolymer is naturally decomposed in the human body, there is no need to remove nanoparticles separately.

In addition, in the present invention, it is difficult to increase the encapsulation efficiency of ciprofloxacin, which is a water-soluble drug, difficult to control the size of particles, and therefore, there are numerous limitations in the use of injection particles as the particles having large particle sizes, and the release rate of the drug is as desired. By improving the problems of the prior art, which was not easy to control, it is possible to predict the emission control rate and the decomposition rate of particles by making the nanoparticles have a uniform size distribution of 100 to 500 nm and a uniform shape. It can be easily used as a drug such as an injection, and also has an excellent effect of encapsulation of a water-soluble drug, thereby minimizing drug loss.

Hereinafter, the present invention will be described in detail through preferred preparations and experimental examples of the present invention, and furthermore, the antibacterial performance of the nanoparticles of the present invention against pathogenic bacteria causing urethritis and infectious diseases is described in vitro and in vivo. Evaluation was done in vivo. However, it should be noted that this is not intended to limit the scope of the present invention. In addition, it should be noted that a document including domestic and foreign patent publications cited in the present invention is incorporated in its entirety.

Example 1 Preparation Example 1 of Nanoparticles Enclosed with Ciprofloxacin

180 mg of polylactide glycolides each having a carboxyl group (Resomer RG504H, lactide / glycolide ratio = 50/50, Boeringher Ingelheim Co. Germany) was dissolved in 7 ml of dichloromethane, and 20 mg of ciprofloxacin was dissolved in 2 ml of distilled water.

After the two solutions were mixed, an ultrasonic grinder (Vibra Cell VCX-400, Sonics & Materials Inc. USA) was treated at 40 watts (W) for 1 minute per 10 ml to make a W / O emulsion.

W / O emulsion was added to 10 ml of 1% (w / v) polyvinyl alcohol (PVA) solution, and then 600 bar (kg) using a high-pressure homogenizer (R5-12.38, SMT Co. Japan). / cm ² ) to 5 cycles at 1 minute intervals to create a W / O / W emulsion.

This was again added to 40 ml of polyvinyl alcohol (PVA) 0.4% (w / v) aqueous solution at 1000 rpm with a stirrer (Top-loading stirrer, Direct Driven Digital Stirrer SS-11D, Young HANA Tech., Co. Ltd. Korea). After stirring for 1 hour, the nanoparticles were recovered by ultracentrifuge (Supra 30K, Vacuum High Speed Centrifuge, Hanil Co. Korea) and freeze-dried for 3 days to obtain nanoparticles containing ciprofloxacin.

Example 2 Preparation of Nanoparticles Containing Ciprofloxacin

Ciprofloxacin-encapsulated nanoparticles were obtained in the same manner as in Example 1 except that 360 mg of polylactide glycolide was dissolved in 7 ml of dichloromethane and 40 mg of ciprofloxacin was dissolved in 2 ml of distilled water.

Example 3 Identification of Optimal Conditions for the Preparation of Nanoparticles Packed with Ciprofloxacin

3-1) Measurement of average size / encapsulation change of nanoparticles according to PLGA concentration

The average size of the nanoparticles and the encapsulation efficiency of the drug according to the concentration (w / v%) of the polylactide glycolide used in the present invention was confirmed.

Polylactide glycolide (Resomer RG504H, lactide / glycolide ratio = 50/50, Boeringher Ingelheim Co. Germany) was dissolved in 7 ml of methylene chloride in a concentration range of 0.5-10% (w / v) (Table 1). . 20 mg of ciprofloxacin was mixed with 2 ml of dissolved distilled water and treated with an ultrasonic grinder (Vibra Cell VCX-400, Sonics & Materials Inc. USA) at 40 watts (W) for 1 minute per 10 ml to make a W / O emulsion.

The W / O emulsion was added to 10 ml of 1% (w / v) polyvinyl alcohol (PVA) aqueous solution, followed by 600 bar using a high-pressure homogenizer (R5-12.38, SMT Co. Japan). W / O / W emulsions were made by 5 cycles at 1 minute intervals in kg / cm ² ).

This was added to 40 ml of polyvinyl alcohol (PVA) 0.4% (w / v) aqueous solution and 1000 rpm with a stirrer (Top-loading stirrer, Direct Driven Digital Stirrer SS-11D, Young HANA Tech., Co. Ltd. Korea). After stirring for 1 hour to recover the nanoparticles with an ultracentrifuge (Supra 30K, Vacuum High Speed Centrifuge, Hanil Co. Korea) and freeze-dried for 3 days to obtain a nanoparticles containing ciprofloxacin.

The average particle size of the obtained nanoparticles was measured using a dynamic light scattering particle size analyzer (ELS-8000, Otsuka electronics Co. Japan), the average value is shown in Table 1.

In addition, ciprofloxacin encapsulation efficiency of the obtained nanoparticles was measured as follows and the results are shown in Table 1.

5 mg of the prepared nanoparticles were dissolved in 5 ml of dichloromethane, and 5 ml of distilled water was added thereto, stirred for 3 hours, 1 ml of the distilled water layer was collected and diluted 100-fold, and then UV spectrophotometer (UV- The absorbance at 277 nm was measured using spectrophotometer 1601, Shamdzu Co. Ltd. Japan, and the encapsulation efficiency was measured using the following equation.

Inclusion Efficiency = [Weight of Ciprofloxacin Residing in Nanoparticles / Total Ciprofloxacin Dose] X 100

Polylactide Glycolide (PLGA) Concentration (w / v%) Average particle size (nm) Encapsulation Efficiency (%, w / w) 0.5 101 20.1 One 110 32.7 2 130 42.4 6 353 47.4 8 570 48.1 10 980 50.2

As a result of Table 1, it can be seen that as the polylactide glycolide concentration increases, the average size of the nanoparticles increases and the encapsulation efficiency also increases.

However, when the polylactide glycolide concentration is 8% (w / v), the average particle size is 500 nm or more. Also, when the polylactide glycolide concentration is 10% (w / v), the average particle size increases to 980 nm. In comparison, the sealing efficiency did not increase.

In addition, when the polylactide glycolide concentration is 1% (w / v) or less, there is no significant difference in particle size, but the encapsulation efficiency of the drug is relatively decreased.

Therefore, it was confirmed that it is preferable to use a polylactide glycolide concentration of 2 to 6% (w / v) in consideration of the size and encapsulation efficiency of the final nanoparticles.

3-2) Measurement of Average Size / Encapsulation Efficiency of Nanoparticles According to Ciprofloxacin Concentration

The average size of the nanoparticles and the encapsulation efficiency of the drug according to the concentration of ciprofloxacin, the drug used in the present invention, were confirmed.

180 mg of polylactide glycolide was dissolved in 7 ml of dichloromethane, and ciprofloxacin was dissolved in 2 ml of distilled water at 0.2, 0.5, 1, 2, 3, and 4% (w / v), respectively. Nanoparticles were prepared by the method. The particle average size and the encapsulation efficiency of the nanoparticles thus obtained were measured, and the results are shown in Table 2 below.

Ciprofloxacin (CIP) Concentration (w / v%) Average particle size (nm) Encapsulation Efficiency (%, w / w) 0.2 117 37.9 0.5 121 40.1 One 130 42.4 2 148 43.7 3 360 30.1 4 575 27.6

As a result of Table 2, as the concentration of ciprofloxacin increases, the average size of the nanoparticles increases, but the encapsulation efficiency shows an excellent encapsulation rate of over 40% at 1 ~ 2% (w / v).

When the concentration of ciprofloxacin is 3% (w / v), the encapsulation efficiency is reduced to 30.1%, and at 4% (w / v), the encapsulation efficiency is reduced to 27.6%, which is about 2.5% because the solubility of ciprofloxacin in distilled water is At the above concentration, since the drug is precipitated in distilled water, the encapsulation efficiency of the drug is lowered.

In addition, when the concentration of ciprofloxacin is less than 1% (w / v), there is no significant difference in particle size, and the encapsulation efficiency is not greatly reduced, but the content of ciprofloxacin per unit nanoparticle weight is lower than that of the drug. It is difficult to control the concentration to a level effective for treatment.

Therefore, in consideration of the size of the final nanoparticles prepared, the content of ciprofloxacin and the encapsulation efficiency it was confirmed that it is preferable to use a ciprofloxacin concentration of 1 ~ 2% (w / v).

3-3) Measurement of Changes in Average Size / Encapsulation Efficiency of Nanoparticles According to Polylactide Glycolide (PLGA) / Ciprofloxacin (CIP) Weight Ratio

The average particle size of the polylactide glycolide and ciprofloxacin used in the present invention and the encapsulation efficiency of the drug were confirmed.

Dissolve 180 mg of polylactide glycolide in 7 ml of dichloromethane, and dissolve 20 mg of ciprofloxacin in 2 ml of distilled water. Nanoparticles were prepared in the same manner as in −1). The average particle size and encapsulation efficiency of the nanoparticles thus obtained were measured and shown in Table 3 below.

Polylactide Glycolide (PLGA) / Ciprofloxacin (CIP) Ratio (mg / mg) Average particle size (nm) Encapsulation Efficiency (%, w / w) 6 97 37.1 8 110 39.8 9 130 42.4 10 258 43.1 12 303 43.9 15 334 45.1

From the results in Table 3, it can be seen that as the polylactide glycolide / ciprofloxacin ratio increases, the average size of the nanoparticles increases and the encapsulation efficiency also increases.

However, when the polylactide glycolide / ciprofloxacin ratio was 12 or more, the encapsulation efficiency did not increase as compared with the increase in the amount of PLGA used and the average particle size.

In addition, when the polylactide glycolide / ciprofloxacin ratio is 8 or less, the particle size is reduced and the drug encapsulation efficiency is reduced.

Therefore, it was confirmed that it is preferable to use a polylactide glycolide / ciprofloxacin ratio of 8 to 10 in consideration of the size and encapsulation efficiency of the final nanoparticles.

3-4) Measurement of Average Particle Size / Encapsulation Efficiency of Nanoparticles According to Ultrasonic Crusher Energy Output and Application Time in Emulsification

The average particle size and the encapsulation efficiency of the drug according to the energy output range of the ultrasonic crusher used in the emulsification step to the first emulsion (W / O) of the present invention was confirmed.

180 mg of polylactide glycolide was dissolved in 7 ml of dichloromethane, and 20 mg of ciprofloxacin was dissolved in 2 ml of distilled water to prepare nanoparticles in the same manner as in 3-1). The nanoparticles were prepared by changing the energy output of the ultrasonic mill used in the emulsification step to the first emulsion (W / O) to 5, 10, 20, 30, 40, and 60 watts.

The average particle size and encapsulation efficiency of the nanoparticles thus obtained were measured and shown in Table 4 below.

Output (watt) Average particle size (nm) Encapsulation Efficiency (%, w / w) 5 Particle Formation Failure - 10 3000 nm or more - 20 375 37.8 30 230 40.1 40 130 42.4 60 128 41.9

In the results of Table 4, when the output is 20 ~ 60 watts nanoparticles of several hundred nm level was formed, and among them, the higher the output, the average size of the nanoparticles is reduced and the encapsulation efficiency is higher.

When the output was increased, there was no significant change in the encapsulation efficiency of the drug, but the rise of the temperature in the emulsion solution is feared to reduce the performance of the drug, and after the reaction agglomeration occurred in the purification process to reduce the yield of the particles.

In addition, when the output is low, the W / O emulsion is not generated finely in nano units, but increases in size in micrometer units, thereby reducing the encapsulation efficiency of drugs.

Therefore, in consideration of the size and the encapsulation efficiency of the final nanoparticles can be confirmed that the output is preferably used 30 ~ 40 watts.

In addition, in the manufacturing process was measured the sealing efficiency according to the application time when emulsifying with an ultrasonic mill.

180 mg of polylactide glycolide was dissolved in 7 ml of dichloromethane, 20 mg of ciprofloxacin dissolved in 2 ml of distilled water, and the output of the ultrasonic grinder was 40 watts. In accordance The average particle size and encapsulation efficiency of the nanoparticles were measured. The results are shown in Table 5 below.

Application time (sec) Average particle size (nm) Encapsulation Efficiency (%, w / w) 10 620 25.7 30 259 37.1 60 130 42.4 120 121 41.9 180 117 40.7 240 aggregation -

In the results of Table 5, when the application time is 10 ~ 180 seconds, the nanoparticles of several hundred nm level was formed, and among them, the longer the application time, the average size of the nanoparticles is reduced and the sealing efficiency is higher. For more than 240 seconds, agglomeration occurred and the yield of particles decreased.

When the application time is long, there is no significant change in the encapsulation efficiency of the drug, but the rise of the temperature in the emulsion solution is feared to reduce the performance of the drug and agglomeration occurs in the purification process after the reaction to reduce the yield of the particles.

In addition, when the application time is short, the W / O emulsion does not form finely in nano units but increases in size to 500 nm or more, aggregates, and decreases the encapsulation efficiency of the drug.

Therefore, in consideration of the size and the encapsulation efficiency of the final nanoparticles to be applied, it was confirmed that the application time is preferably 1 to 3 minutes.

3-5) Measurement of average particle size / encapsulation efficiency change according to the pressure and application cycle / cycle control range of the high pressure homogenizer in the emulsification step to the second emulsion (W / O / W)

The average size of the nanoparticles according to the pressure and the application cycle of the high pressure homogenizer used in the emulsification step to the second emulsion (W / O / W) of the present invention was confirmed the encapsulation efficiency of the drug.

180 mg of polylactide glycolide was dissolved in 7 ml of dichloromethane, and 20 mg of ciprofloxacin was dissolved in 2 ml of distilled water to prepare nanoparticles in the same manner as in 3-1). Nanoparticles were prepared by varying the application pressure of the high pressure homogenizer used in the emulsification step to the second emulsion (W / O / W) in the range of 50 to 1000 bar. The average particle size and encapsulation efficiency of the obtained nanoparticles were measured and shown in Table 6 below.

Pressure (bar) Average particle size (nm) Encapsulation Efficiency (%, w / w) 50 791 25.3 100 536 32.7 300 311 38.1 600 130 42.4 800 121 41.0 1000 aggeregation -

The results of Table 6 show that when the applied pressure of the high pressure homogenizer is 100 ~ 800 bar, nanoparticles of several hundred nm level were formed. Among them, as the application cycle increased, the average size of nanoparticles decreased and the encapsulation efficiency increased. Able to know. At 1000 bar or more, agglomeration occurred and the yield of particles decreased.

In the case where the application pressure of the high pressure homogenizer is high, the aggregation in the emulsion occurs due to the increase in the temperature of the emulsion solution, and also problems such as the size of the particles become large or not formed.

In addition, when the application pressure of the high pressure homogenizer is low, the W / O / W emulsion is not sufficiently formed in nano units, but the particle size is increased, and the encapsulation efficiency of the drug is reduced.

Therefore, it was confirmed that the application pressure of the high pressure homogenizer was preferably 600 to 800 bar in consideration of the size and encapsulation efficiency of the final nanoparticles.

In addition, in the manufacturing process was changed to 1, 3, 5, 7, 10, 15 cycles of the application cycle per 100 ml emulsion during the emulsification with a high pressure homogenizer to measure the encapsulation efficiency according to the number of cycles.

Applicable Cycle Average particle size (nm) Encapsulation Efficiency (%, w / w) One 386 30.1 3 170 35.9 5 130 42.4 7 128 42.1 10 131 41.7 15 aggregation -

In the results of Table 7, nanoparticles of several hundred nm level were formed when the application cycle was 1 to 10 times. Among them, as the application cycle was increased, the average size of nanoparticles decreased and the encapsulation efficiency increased, but the size was larger than 5 times. The encapsulation efficiency was similar.

In the case of more than 15 times, agglomeration occurred. However, when the number of application cycles increases, it is treated with a high-pressure homogenizer for a long time. Thus, problems such as aggregation of the emulsion occur due to an increase in temperature in the emulsion solution.

In addition, when the number of application cycles is small, the W / O / W emulsion is not sufficiently formed in nano units, but the size is increased to 500 nm or more, and the encapsulation efficiency of the drug is reduced.

Therefore, in consideration of the size and the encapsulation efficiency of the final prepared nanoparticles it was confirmed that the application cycle is preferably 5 to 7 per 100 ml of emulsion.

As described above, the optimum conditions for the preparation of nanoparticles of the present invention were found by changing various conditions. As a result, polylactide glycolide 2-6% (w / v, g / ml) and ciprofloxacin 1-2% (w / v, g / ml) solution was mixed to a polylactide glycolide: ciprofloxacin weight ratio of 8-10: 1 and then treated with an ultrasonic grinder for 1 minute per 10 ml at a power of 40 watts (W). W / O / W emulsion was prepared by making an / O emulsion, and then putting it in an aqueous 1.0% (w / v) polyvinyl alcohol solution and treating it with a high-pressure homogenizer for 5 cycles at 600 bar for 1 minute per 100 ml. It was confirmed that making the optimum manufacturing conditions.

In this case, it was possible to prepare a nanoparticle encapsulated ciprofloxacin in the range 130 ~ 353 nm.

Comparative Example 1. Preparation of Nanoparticles Without Ciprofloxacin

Polylactide glycolide then dissolved in 200mg of dichloromethane 7ml mixed with 2ml of distilled water, sonicator (Vibra Cell VCX-400, Sonics & materials Inc. USA) for 1 min per 10 ml at 40 watts (W) as W ₁ / An O emulsion (primary emulsion) was made.

W ₁ / O emulsion was added to polyvinyl alcohol (PVA) 1% (w / v) aqueous solution and treated with a high-pressure homogenizer (600 bar 5 cycle, [R5-12.38, SMT Co. Japan]). W ₁ / O / W ₂ (secondary emulsion) emulsions were made.

This was again added to 40 ml of polyvinyl alcohol (PVA) 0.4% (w / v) aqueous solution and 1000 rpm with a stirrer (Top-loading stirrer, Direct Driven Digital Stirrer SS-11D, Young HANA Tech., Co. Ltd. Korea). After stirring for 1 hour to recover the nanoparticles with an ultracentrifuge (Supra 30K, Vacuum High Speed Centrifuge, Hanil Co. Korea) and freeze-dried for 3 days to obtain nanoparticles that do not contain ciprofloxacin.

Experimental Example 1. Measurement of the content and encapsulation efficiency of ciprofloxacin in the preferred nanoparticles of the present invention

 In order to measure the content of ciprofloxacin in the nanoparticles prepared in Examples 1, 2 and Comparative Example 1, the following experiment was performed.

 5 mg of each of the nanoparticles prepared in Examples 1, 2 and Comparative Example 1 was dissolved in 5 ml of dichloromethane, and 5 ml of distilled water was added thereto, followed by stirring for 3 hours. After 3 hours, 1 ml of distilled water was taken and diluted 100-fold, and then absorbance was measured at 277 nm using a UV spectrophotometer (UV-spectrophotometer 1601, Shamdzu Co. Ltd. Japan). Measured using.

Drug content = [weight of ciprofloxacin in nanoparticles / total weight of nanoparticles] X 100. Formula (1)

Inclusion Efficiency = [Weight of Ciprofloxacin Residual in Nanoparticles / Total Ciprofloxacin Dose] X 100. Formula (2)

Polylactide Glycolide (PLGA): Ciprofloxacin (CIP) Weight Ratio (mg: mg) Drug content (%, w / w) Encapsulation Efficiency (% w / w) Particle Size (nm) Theory Found Example 1 180: 20 10 4.5 42.4 130 Example 2 360: 40 10 5.0 47.4 353

As shown in Table 8, the drug content in the nanoparticles prepared in Example 1 [180-20] and Example 2 [360-40] was 4.5 and 5.0% (w / w), respectively, and the encapsulation efficiency was 42.4, 47.4% (w / w).

That is, by adjusting the concentrations of ciprofloxacin and polylactide glycolide for each solvent at the beginning of preparation, the drug content and encapsulation efficiency could be controlled. (180mg / 7ml: 20mg / 2ml-> 360mg / 7ml: 40mg / 2ml) The drug content and encapsulation efficiency increased.

In addition, it can be seen that the particle size in the range of 130-353nm can be adjusted according to the concentration of drug and polymer for each solvent.

Experimental Example 2 Analysis of Preferred Nanoparticles of the Invention

The morphology of the nanoparticles prepared in Examples 1 and 2 was observed at 120 kV using transmission electron microscope (JEOL JEM-2000, FX-II). The particle size distribution of the nanoparticles was measured using a dynamic light scattering particle size analyzer (ELS-8000, Otsuka electronics Co. Japan) and the results are shown in FIGS. 1 and 2.

1 is a transmission electron micrograph of (a) nanoparticles [180-20] prepared according to Example 1 of the present invention and (b) nanoparticles [360-40] prepared according to Example 2 of the present invention, respectively. to be. As shown in FIG. 1, the results of observing the morphology of the ciprofloxacin-encapsulated nanoparticles with an electron microscope showed that all had a spherical morphology. (A) In the case of the nanoparticles prepared in Example 1 [180-20] It was found to have a size distribution of approximately 100-200 nm, and (b) the nanoparticles [360-40] prepared in Example 2 showed a size distribution of approximately 200-400 nm.

In addition, Figure 2 is a graph showing the size distribution of the nanoparticles [360-40] prepared according to Example 2 of the present invention. As shown in FIG. 2, the particle size distribution [360-40] of the nanoparticles prepared in Example 2 showed a size distribution of about 100-500 nm, and the average size of the nanoparticles is shown in Table 8 above. It was. From the results, it can be seen that almost identical to the transmission electron micrograph shown in FIG.

Experimental Example 3. X-ray diffraction pattern analysis of preferred nanoparticles of the present invention

The crystal properties of ciprofloxacin and nanoparticles were measured in the range of 10 to 80 ^o using an X-ray powder diffractometer (Rigaku D / MAX 1200 automated powder diffractometer) and the results are shown in FIG. 3.

Figure 3 is (a) Ciprofloxacin HCl, (b) Ciprofloxacin-free nanoparticles prepared according to Comparative Example 1 of the present invention [Empty NP], (c) prepared according to Example 1 of the present invention Nanoparticles [180-20], (d) nanoparticles prepared according to Example 2 of the present invention [360-40], (e) nanoparticles containing no ciprofloxacin prepared according to Comparative Example 1 of the present invention; XRD (X-ray diffraction) spectrum when simple mixing of ciprofloxacin [Empty NP / CIP = 20/1].

As shown in FIG. 3, (a) ciprofloxacin showed a strong peak at about 20 ^° , and (b) nanoparticles not containing the drug prepared in Comparative Example 1 exhibited soft peak characteristics to around 10 to 60. (c)-(d) Ciprofloxacin-encapsulated [180-20, 360-40] nanoparticles all showed smooth peaks as did nanoparticles containing no drug, but (e) nanoparticles containing no drug. Simple mixing of and ciprofloxacin showed strong peaks of ciprofloxacin (20 ^o ) and smooth peaks of unencapsulated nanoparticles, and the results showed that the drug was well encapsulated within the nanoparticles.

Experimental Example 4 Drug Release Experiment of Preferred Nanoparticles of the Present Invention

10 mg of the nanoparticles prepared in Examples 1 and 2 were dispersed in 5 ml of phosphate buffered saline (0.1M, pH 7.4) and placed in a dialysis membrane (MWCO: 12,000 g / mol), which was then 200 In a ml-sized vial was added with 95 ml of phosphate buffer solution. Release experiment was carried out in a stirrer at 37 ℃, 100 rpm conditions. In order to maintain a constant condition, phosphate buffer solution was exchanged daily, and the concentration of drug released at regular time intervals was measured at 277 nm using a UV spectrophotometer (UV-1601, Shamdzu Co. Ltd. Japan). All experiments were repeated three times.

4 shows (a) nanoparticles [180-20] prepared according to Example 1 of the present invention [●], and (b) nanoparticles [360-40, ○] prepared according to Example 2 of the present invention. It is a graph showing the result of measuring drug release rate.

As shown, ciprofloxacin was slowly released from the nanoparticles prepared in accordance with an embodiment of the present invention for 10 days. In particular, the nanoparticles prepared in Example 2 [360-40] showed a relatively slow drug release properties, in both cases it can be seen that the drug was released within about 15 days.

These results suggest that ciprofloxacin is a water-soluble drug, so the release rate of drug is relatively higher than that of hydrophobic drug.

Experimental Example 5. Evaluation of antibacterial performance of nanoparticles of the present invention

(1) Confirmation of in vitro antibacterial effect

The in vitro antibacterial effect of the nanoparticles encapsulated with ciprofloxacin of the present invention was confirmed.

The microorganisms were Escherichia coli (E. coli) and Salmonella typhimurium (ATCC 14028) ( S. typhimurium ) and were purchased from Korea Centers for Disease Control and Prevention.

Stock solutions were prepared and diluted according to the instructions of the CLSI (Clinical Laboratory Standards Institute, formerly NCCLS) M7-A6 method (15 National Committee for Clinical Laboratory Standards 2003).

After subculturing the cryopreserved microbial solution, ciprofloxacin or the nanoparticles of Example 2 were added to the microbial solution at various concentrations (0.0005-1 μg / ml) to confirm the antibacterial effect. All experiments were repeated three times. The growth of microorganisms was evaluated by reading the optical density (OD) at 600 nm with a spectrophotometer.

5 is a phosphate buffer solution [PBS, ●], ciprofloxacin [Free CIP, ○], nanoparticles containing ciprofloxacin prepared according to Comparative Example 1 [Empty nanoparticles, ▼], nanoparticles prepared according to Example 2 [360-40, CIP-nanoparticles, △], a simple mixture of ciprofloxacin-free nanoparticles prepared according to Comparative Example 1 and ciprofloxacin [Free CIP + Empty NP, ■], respectively (a) Escherichia coli ( E. coli) and (b) Salmonella typhimurium (S. typhimurium) are shown to evaluate the antibacterial performance by comparing the viability of microorganisms.

As shown, it can be seen that the buffer and the nanoparticles of Comparative Example 1 do not significantly affect the growth of microorganisms. On the other hand, ciprofloxacin had a concentration-dependent effect of inhibiting microbial growth up to 0.05 mg / ml for E. coli (a), and showed similar tendency even with simple mixing of ciprofloxacin with unencapsulated nanoparticles. Could. In addition, the nanoparticles prepared according to Example 2 of the present invention showed a lower microbial growth inhibitory effect than ciprofloxacin at the same drug concentration as ciprofloxacin, which is thought to be due to the slow release of ciprofloxacin from the nanoparticles.

As shown in Figure 5 (b) it was shown that the nanoparticles prepared according to Example 2 of the present invention also showed antibacterial performance similar to E. coli against S. typhimurium . In this case, it was also found that the nanoparticles not containing the drug had no effect on the growth of the microorganisms, indicating that the polylactide glycolide constituting the nanoparticles had no effect on the growth of the microorganisms.

In addition, S. As for typhimurium , the nanoparticles prepared according to Example 2 of the present invention showed antibacterial efficacy similarly to ciprofloxacin.

(2) confirm the antibacterial effect in vivo 1

The antibacterial effect of the nanoparticles encapsulated with ciprofloxacin of the present invention was confirmed in vivo.

For animal experiments, S. typhimurium was incubated overnight in Luria-Bertani (LB) medium, and the pellets obtained by centrifugation were washed with phosphate buffer solution and diluted to 100 CFU / ml.

The S. typhimurium was injected in the abdominal cavity of BALB / c mice (female, 6 weeks old) to 20 CFU per horse. After 24 hours, mice were intraperitoneally injected with ciprofloxacin or the nanoparticles of Example 2 of the present invention at a dose of 25 mg / kg, and the mice were killed on the 3rd and 5th days to remove the spleen to remove 0.05% (w). / v) Homogenized with sterile buffer containing Tween 20 and S. typhimurium was plated on LB agar to assess the number of microorganisms.

6 is a phosphate buffer solution [PBS], ciprofloxacin [Free CIP] and Example 2 of the present invention after the infection of S. typhimurium in BALb / C mice for one day to evaluate the antibacterial performance of nanoparticles and ciprofloxacin. The resulting nanoparticles [CIP-NP] were treated with the same dosage, respectively, to evaluate antibacterial performance for (a) 3 days and (b) 5 days.

As shown in FIG. 6 (a), the number of microorganisms was evaluated on the third day after the treatment, and ciprofloxacin and nanoparticles recorded much lower microbial numbers compared to the case of the buffer solution and treated with ciprofloxacin. It can be seen that the difference between and the nanoparticles is not large. On the other hand, ciprofloxacin after 5 days (FIG. 6 (b)) can be seen that the number of microorganisms increased sharply, and the number of microorganisms was much smaller than that of ciprofloxacin in the nanoparticles of the present invention.

(3) In vivo  Check the antibacterial effect 2

For animal model experiments with urethritis, E. coli was incubated overnight at 37 ° C. in Luria-Bertani (LB) medium and 1 × 10 ⁶ cells / ml of cells were placed on a dialysis membrane (MWCO 10000 Da, Spectra, USA). It was inserted into the abdominal cavity of ICR mice anesthetized with ketamine (ketamine, (100 mg / Kg)). Four hours after surgery, sterile distilled water (DW), ciprofloxacin (CIP) and nanoparticles (CIP-NP) were injected subcutaneously (dose: 25 mg / kg), respectively. Three days later, the mice were killed and the number of microorganisms in the dialysis membrane was plated on HI agar for evaluation.

Figure 7 shows the growth of bacteria after 3 days of treatment with sterile water [DW], ciprofloxacin [Free CIP] and nanoparticles [CIP-NP] prepared according to Example 2 of the present invention in E. Coli -infected mice, respectively . The result is.

As shown in Figure 7, it can be seen that the growth of microorganisms is much suppressed in the case of the nanoparticles of the present invention compared to the case of treatment with ciprofloxacin.

As a result of the animal experiment, it was found that the nanoparticles prepared by the new method according to the present invention can inhibit the in vivo proliferation of pathogenic microorganisms for a longer time, and thus the nanoparticles encapsulated with ciprofloxacin of the present invention It can be effectively used for urethritis and infectious diseases.

According to the present invention, it was possible to prepare a sustained release formulation by encapsulating polylactide glycolide nanoparticles in a simple process without significant denaturation of the intrinsic antibacterial performance of ciprofloxacin widely used in pathogenic microorganisms causing urethritis. In addition, it improved the problems of ciprofloxacin that should be administered daily, and it was found that the nanoparticles had anti-bacterial performance equivalent to that of ciprofloxacin in in vitro experiments in antibacterial performance. It has been shown that it can inhibit the proliferation of in vivo and can be effectively used for urethritis and infectious diseases.

Claims (12)

delete delete delete delete In the method for producing microparticles carrying a water-soluble drug by W / O / W emulsion solvent evaporation, (1) dissolving polylactide glycolide having a carboxyl group as a terminal in dichloromethane at a concentration of 2-6% (w / v, g / ml); (2) dissolving ciprofloxacin in distilled water at a concentration of 1-2% (w / v, g / ml); (3) the dichloromethane solution in which the polylactide glycolide is dissolved and the aqueous solution of ciprofloxacin are mixed so that the weight ratio of polylactide glycolide: ciprofloxacin is 8-10: 1 and then 30-40 watts (W) using an ultrasonic mill. Processing for 1 to 3 minutes per 10 ml to output to create a W / O emulsion; And (4) 5 to 7 cycles (cycles) in a 1-minute cycle per 100 ml at 600-800 bar using a high-pressure homogenizer in the W / O emulsion in a 1.0% (w / v) polyvinyl alcohol aqueous solution Sustained release of the polylactide glycolide copolymer having a particle size of 100-500 nm including the step of making a W / O / W emulsion, wherein the supported ciprofloxacin is continuously released for 10 days or more, and Biodegradable Pore Nanoparticles Manufacturing Method (5) The W / O / W emulsion is polyvinyl alcohol 0.4. % (w / v) Siprofloxacin has a particle size of 100-500nm, and further comprising the step of recovering the nanoparticles by ultra-centrifuge after stirring for 30-90 minutes at 500-1500rpm in an aqueous solution and freeze-dried. Method for preparing slow release and biodegradable porous nanoparticles of supported polylactide glycolide copolymer [Claim 6] The ciprofloxacin has a particle size of 100-500 nm, characterized in that the W / O emulsion is formed by treating the ultrasonic mill for 1 minute per 10 ml at an output of 40 watts (W) in step 3. Method for preparing slow release and biodegradable porous nanoparticles of supported polylactide glycolide copolymer [6] The particle size of 100-500nm according to claim 5, wherein the high pressure homogenizer is processed at 600 bar for 5 (cycles) for 1 minute per 100ml in order to make a W / O / W emulsion. Method for preparing slow release and biodegradable porous nanoparticles of polylactide glycolide copolymer loaded with ciprofloxacin delete delete delete delete

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