KR102632923B1 - Hydrogel and method for preparing the same - Google Patents

Abstract

The present invention provides a hydrogel with excellent drug delivery ability, pH-dependent, biocompatible, biodegradable, and antibacterial properties, and a method for producing the same. In detail, the present invention provides a hydrogel with excellent drug delivery ability, pH-dependent, biocompatible, biodegradable, and antibacterial properties, and a method for manufacturing the same. A hydrogel comprising a hydrophilic synthetic polymer and (3-mercaptopropyl)trimethoxysilane (MPTMS) and a method for producing the same are provided.

Description

Hydrogel and method for preparing the same {Hydrogel and method for preparing the same}

The present invention relates to a hydrogel with excellent drug delivery ability and a method for manufacturing the same.

The drug delivery system is a dosage form designed to efficiently deliver the required amount of drug by minimizing the side effects of existing drugs and maximizing the efficacy and effect of the drug. In these drug delivery systems, biocompatible and/or biodegradable hydrogels that can be injected into the body are being developed for efficient drug delivery of macromolecules such as proteins and genes. These hydrogels are formed through chemical and/or physical cross-linking of polymers, and some cross-linking agents are used for chemical bonding.

In addition, hydrogel is one of the materials that is attracting attention because it has a high water content and can be applied to various fields by controlling its chemical and/or physical properties. In particular, by adjusting the biocompatibility of hydrogel, it can be used for human bone, cartilage, and skin regeneration, drug delivery, and wound treatment, and the demand for tissue regeneration and application as a cell therapy agent is increasing day by day.

For example, hydrogels with wettability are being studied, such as in Korean Patent No. 1985368, and the demand for technology development for hydrogels with excellent wettability and biocompatibility continues to increase.

The present invention provides a hydrogel with excellent drug delivery ability, pH-dependent, biocompatible, and biodegradable properties as well as antibacterial properties, and a method for producing the same.

In one aspect of the present invention, the present invention includes irradiated chitosan, natural gelling polymer, hydrophilic synthetic polymer and (3-mercaptopropyl)trimethoxysilane (MPTMS), Hydrogel is provided.

In another aspect of the present invention, the present invention includes the steps of irradiating chitosan with radiation; mixing irradiated chitosan, natural gelling polymer, and hydrophilic synthetic polymer with the solvent; and adding (3-mercaptopropyl)trimethoxysilane (MPTMS) to the solvent.

The present invention can provide a hydrogel with excellent drug delivery ability, swelling properties, thermal stability, biodegradability, biocompatibility and antibacterial properties. Therefore, the hydrogel of the present invention contains radioactive peptide and/or radioactive It can also be usefully applied when radioactive drugs such as proteins must maintain stability in vivo.

Figure 1 schematically shows a method for producing a hydrogel according to an embodiment of the present invention.
Figure 2 shows the results of FTIR analysis of a hydrogel according to an example of the present invention.
Figure 3 shows the results of measuring the swelling properties of the hydrogel in distilled water according to an example of the present invention.
Figure 4 shows the results of measuring the swelling properties of hydrogels according to an example of the present invention according to pH.
Figure 5 shows the results of measuring the swelling properties of the ionic solution of the hydrogel according to an example of the present invention (NaCl in Figure 5(a), CaCl ₂ in Figure 5(b)).
Figure 6 shows the results of measuring the biodegradability of the hydrogel according to an example of the present invention.
Figure 7 shows the analysis of the antibacterial properties of the hydrogel according to an example of the present invention using the well and disk diffusion method, and shows an image of the inhibited area around the well and disk visually observed.
Figure 8 shows the minimum inhibitory concentration (MIC) of the hydrogel according to an embodiment of the present invention against Salmonella typhimurium (Gram-negative) and Listeria monocytogenes (Gram-positive) represents.
Figure 9 shows the results of measuring the drug release ability of the hydrogel according to an example of the present invention.

Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

The present invention provides a hydrogel that has excellent drug delivery ability, swelling properties, thermal stability, biodegradability, biocompatibility, and antibacterial properties.

Specifically, the present invention provides a hydrogel comprising irradiated chitosan, a natural gelling polymer, a hydrophilic synthetic polymer, and (3-mercaptopropyl)trimethoxysilane (MPTMS). .

In general, chitosan has a very large molecular weight (about 300 to 1000 kDa), so its solubility in solvents such as water is very low and its viscosity is high. On the other hand, when it has low viscosity and high solubility, it is necessary to lower the molecular weight of chitosan because better physicochemical advantages, antibacterial, antitumor, and antifungal properties can be obtained.

Methods for lowering the molecular weight of chitosan include oxidative degradation, acid hydrolysis, and enzymatic degradation known in the art. However, according to the present invention, the molecular weight of chitosan can be lowered by irradiating radiation. This method is cost-effective, safe, less toxic, and environmentally friendly. More specifically, radiation such as X-rays, gamma rays, or electron beams may be used as the radiation.

The backbone of chitosan has 1-4 glycosidic bonds, and irradiation can break the C-H and C-OH bonds of chitosan, lowering the molecular weight of chitosan. Therefore, irradiation cleaves the bonds in the chemical structure of chitosan, thereby reducing the molecular weight of the polymer.

Therefore, the weight average molecular weight of general chitosan is about 31.00×10 ⁴ to 37.5×10 ⁴ , but the weight average molecular weight of irradiated chitosan of the present invention may be 19.00×10 ⁴ to 21.50×10 ⁴ , preferably It may be 19.50×10 ⁴ to 21.00×10 ⁴ . If the weight average molecular weight of irradiated chitosan is less than 19.50×10 ^4, problems related to mechanical properties, physicochemical characteristics, crystallinity, and contact angle may occur. , if it exceeds 21.50×10 ⁴ , the viscosity increases and solubility is low, which may cause a problem in which the drug delivery ability (drug release ability) is significantly reduced.

Furthermore, the present invention can use natural gelling polymers as polymers capable of producing hydrogels. However, there are limitations in producing hydrogels using only natural gelling polymers due to the undesirable weak mechanical strength of natural gelling polymers. Therefore, the present invention can be used not only with natural gelling polymers but also with synthetic polymers.

For example, the present invention provides natural gelling polymers such as galactomannan, glucomannan, guar gum, locust bean gum, pluronic, agar, algin, carrageenan gum, xanthan gum, tamarind gum, tara gum, karaya gum, and gellan gum. A natural gelling polymer containing at least one member selected from the group consisting of may be used, but is not limited thereto.

Furthermore, the present invention can use hydrophilic synthetic polymers as synthetic polymers. For example, the hydrophilic synthetic polymer may be a synthetic polymer containing at least one selected from the group consisting of polyvinyl alcohol, polyacrylate, polyvinylpyrrolidone, and polyethylene glycol, but is not limited thereto.

On the other hand, the hydrogel for drug delivery of the present invention, for example, based on the total weight of the hydrogel, 40 to 80% by weight of the irradiated chitosan, 5 to 15% by weight of the natural gelling polymer, and the hydrophilic synthetic polymer. It may contain 3 to 10% by weight and 10 to 45% by weight of (3-mercaptopropyl)trimethoxysilane, preferably 45 to 75% by weight of irradiated chitosan, and 8 to 13 of the natural gelling polymer. By weight, it may include 4 to 8 wt% of the hydrophilic synthetic polymer and 10 to 45 wt% of (3-mercaptopropyl)trimethoxysilane.

In the present specification, the hydrogel of the present invention may be abbreviated. For example, the hydrogel containing irradiated chitosan, guar gum, and PVP of the present invention is referred to as “RCGP.” It may be referred to as “.

In detail, based on the total weight of the hydrogel, the content of the irradiated chitosan may be 40 to 80% by weight, for example 45 to 70% by weight, preferably 45 to 65% by weight, and the radiation If the irradiated chitosan content is less than 40% by weight, problems may arise with regard to mechanical properties, physicochemical characteristics, crystallinity and contact angle, and if the content of irradiated chitosan is less than 40% by weight, problems may arise with regard to mechanical properties, physicochemical characteristics, crystallinity and contact angle. If it is exceeded, the drug delivery ability (drug release ability) may be significantly reduced due to high viscosity and low solubility.

In addition, based on the total weight of the hydrogel, the content of the natural gelling polymer may be 5 to 15% by weight, preferably 8 to 13% by weight, and when the content of the natural gelling polymer is less than 5% by weight, the viscosity is high and Due to low solubility, the drug delivery ability (drug release ability) may be significantly reduced, and if it exceeds 15% by weight, the properties of the hydrogel may be weakened.

Furthermore, based on the total weight of the hydrogel, the content of the hydrophilic synthetic polymer may be 3 to 10% by weight, preferably 4 to 6% by weight, and if the content of the hydrophilic synthetic polymer is less than 3% by weight, the physicochemical properties (physiochemical characteristics) may deteriorate, and if it exceeds 10% by weight, the toughness of the hydrogel may deteriorate.

Meanwhile, the present invention includes (3-mercaptopropyl)trimethoxysilane (MPTMS), which can act as a crosslinking agent, and the use of such a component reduces the swelling of the hydrogel. It can be improved to obtain physical properties suitable for drug delivery.

The MPTMS is a non-toxic organosilane that can improve the crosslinking and chemical modification capabilities of chitosan, natural gelling polymers, and hydrophilic synthetic polymers.

The present invention may include 10 to 45 wt% of the MPTMS, and preferably 20 to 40 wt% of the MPTMS, based on the total weight of the hydrogel. If the MPTMS is added in less than 10% by weight, there is a problem that the swelling property of the hydrogel disappears or is lowered, and if it exceeds 45% by weight, a hydrogel with very strong networking and a significantly low water function is produced. Problems may arise.

Meanwhile, the hydrogel of the present invention may be in the form of a dried hydrogel film, or may be additionally impregnated with a solution such as a drug or cosmetic composition.

The use of the hydrogel is not particularly limited and may be used for cosmetics, drug delivery, etc., and is preferably used for drug delivery.

Meanwhile, according to the present invention, a method for producing the hydrogel of the present invention is provided.

In detail, the present invention includes the steps of irradiating chitosan with radiation; mixing irradiated chitosan, natural gelling polymer, and hydrophilic synthetic polymer with a solvent; and adding (3-mercaptopropyl)trimethoxysilane (MPTMS) to the solvent.

A more detailed description of the irradiated chitosan, natural gelling polymer, hydrophilic synthetic polymer, and (3-mercaptopropyl)trimethoxysilane (MPTMS) has already been described in the above-mentioned drug delivery hydrogel. Since it has been explained, it will be omitted below.

Meanwhile, the step of mixing irradiated chitosan, natural gelling polymer, and hydrophilic synthetic polymer with a solvent can be performed as a single step or sequentially mixed with a solvent, and the mixing order and method are particularly limited. It doesn't work.

Meanwhile, the radiation may use X-rays, gamma rays, or electron beams, but is not limited thereto. Preferably, gamma rays may be used to lower the molecular weight of chitosan.

Additionally, the radiation may be irradiated at a total dose of 10 to 30 kGy, preferably 15 to 25 kGy. If the total dose of radiation irradiated is less than 10kGy, the molecular weight of chitosan is not sufficiently low, which may cause problems in the manufacture of hydrogel due to problems such as high viscosity. If the total dose of radiation is irradiated exceeding 30kGy, the chitosan may be damaged. As the molecular weight is significantly lowered, problems may arise that make it difficult to demonstrate the physicochemical advantages (viscosity, solubility, etc.) of chitosan.

Meanwhile, water may be used as the solvent, and further, an acid may be added to the solvent to more smoothly dissolve chitosan, for example, acetic acid may be added, preferably 0.5 to 2 M acetic acid. An aqueous solution can be used. When using a solvent in the above range, chitosan can be easily decomposed into the molecular weight range of the present invention.

Furthermore, when the hydrogel of the present invention is used for drug delivery, it may further include the step of adding at least one selected from the group consisting of drugs and radioisotopes to the solvent, for example, adding MPTMS. It may further include the step of adding the drug to be delivered to the solvent. At this time, the type of the drug is not particularly limited, but may be, for example, at least one selected from the group consisting of ampicillin and kanamycin monosulfate monohydrate, preferably ampicillin, The radioactive isotope may be, for example, radioactive iodine, such as I-131, I-125, Tc-99m, Zr-89, In-111, Sr-90, etc.

Meanwhile, alternatively, when the hydrogel of the present invention is used for drug delivery, at least one selected from the group consisting of a drug and a radioisotope is not added to the solvent, but the hydrogel is first manufactured and then the drug and radioisotope are added to the solvent. It can also be prepared by adding at least one member selected from the group consisting of.

The hydrogel production method of the present invention includes, after all components are mixed, for example, subsequent to the step of adding MPTMS, and/or in the step of adding at least one member selected from the group consisting of a drug and a radioisotope. Subsequently, the step of drying the solution after uniformly mixing may be further included, and at this time, drying may be performed at 40 to 50° C. for 2 to 4 hours. The drying method is not particularly limited, but may be performed, for example, by hot air drying, oven drying, freeze drying, etc.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

To prepare the hydrogel for drug delivery of the present invention, the following materials were prepared.

Chitosan (Mw: 31.00-37.5×10 ⁴ g/mol, 800-2000 cP, >75% deacetylated), natural gelling polymer, guar gum (food grade extra refined, 500 cP), hydrophilic synthesis Polyvinyl pyrrolidone (PVP, Mw: 40,000 g/mol) was used as the polymer, and all were purchased from Sigma Aldrich (Yongin, Seoul). (3-mercaptopropyl)trimethoxysilane (MPTMS) was used with 95% purity and purchased from Sigma Aldrich-USA. KCl, NaCl, CaCl ₂ , monopotassium phosphate (KH ₂ PO ₄ ), sodium acetate, and NaOH were all purchased from Merck-Germany. Gram-positive Staphylococcus aureus (S. aureus), yeast extract, and tryptophan were used for antimicrobial analysis. Ampicillin sodium was supplied by Duchefa Biochemie-Netherlands. Acetic acid (≥99.7), methanol, hydrochloric acid, and boric acid were used from Daejung Chemical & Metals Co. Ltd. (Siheung, South Korea).

Examples 1 to 3

A total dose of 20 kGy was administered to the prepared chitosan at a dose of 1.02 kGy/h in an air atmosphere using a ⁶⁰ Co gamma irradiation device (150 TBq capacity, ACEL, MDS Nordion, Canada) at the Korea Atomic Energy Research Institute (Jeongeup, Korea). Gamma rays were irradiated to ensure sufficient dose.

After gamma irradiation, the irradiated chitosan was mixed with a 0.5M acetic acid aqueous solution at a concentration of 2w/v% and mixed on a hot plate at 15 - 30°C for 12 hours. And impurities were removed using a filter (Mira-cloth, Calbiochem Novabiochem Corp., San Diego, CA) to prepare an aqueous chitosan solution.

Guar gum was added to distilled water at 60°C to prepare a 1 w/v% guar gum aqueous solution, and PVP was added to distilled water at 45°C to prepare a 1 w/v% PVP aqueous solution.

The chitosan aqueous solution, guar gum aqueous solution, and PVP aqueous solution were mixed at a volume ratio of 3:1:0.5 to make a total of 45 mL. At this time, the chitosan solution and the guar gum aqueous solution were first mixed at 50°C for 2 hours, then PVP was added and mixed again at 50°C for 2 hours.

Next, 200 μl (Example 1), 400 μl (Example 2), and 500 μl (Example 3) of MPTMS as a cross-linking agent were added and mixed.

A solution containing irradiated chitosan, guar gum, PVP, and MPTMS was filled into a Petri dish and dried using desiccators at room temperature (25°C) and 20% relative humidity. As a result, a hydrogel was produced.

The above manufacturing method is briefly shown in Figure 1.

Comparative Example 1

The hydrogel was prepared in the same manner as in Example 1, except that the hydrogel was prepared from a mixed solution of irradiated chitosan, guar gum, and PVP without additional mixing of MPTMS. .

The components and contents of the hydrogels prepared in Examples 1 to 3 and Comparative Example 1 are summarized in Table 1.

number Chitosan g (% by weight) Guar gum g (% by weight) PVPg (% by weight) MPTMSg (% by weight) Comparative Example 1 0.6(80) 0.1(13.3) 0.05(6.7) - Example 1 0.6(63.2) 0.1(10.5) 0.05(5.3) 0.2(21) Example 2 0.6(52.2) 0.1(8.7) 0.05(4.3) 0.4(34.8) Example 3 0.6(48) 0.1(8) 0.05(4) 0.5(40)

Experiment example

(1) FTIR (Fourier Transform Infrared Spectroscopy) analysis

To confirm whether MPTMS was mixed in the hydrogels of Examples 1 to 3, FTIR analysis was performed.

FTIR analysis was performed using an iS10 (Thermo Fisher Scientific) spectrometer, with each spectrum having a scanning range of 4000-750 cm ^-1 and a resolution of 4 cm ^-1 Measured at resolution. The results are shown in Figure 2.

As a result, as shown in Figure 2, stretching vibrations due to internal and external hydroxyl groups present in the hydrogel could be observed at 3540-3140 cm ^-1 , saccharin structure at 1152 cm ^-1 and 890 cm ^-1 The presence of chitosan and guar gum was confirmed by the pyranose ring. Additionally, characteristic bands of chitosan were observed at 1300 cm ^-1 by cis-amide III and at 1575 and 1640 cm ^-1 by the combination of amide I and amide II. The stretching bands at 1250 and 1110 cm ^-1 are due to cyclic and acyclic -COC bonds of chitosan, guar gum and PVP. Sharp stretching bands at 1120 and 1010 cm ^-1 were observed, which were due to the -Si-O-Si- and -Si-OC- bonds of the hydrogel formed by crosslinking with a crosslinker. Examples 1 to 3 Increasing the concentration of the MPTMS cross-linking agent in the hydrogel showed an increase in band intensity, from which cross-linking between chitosan, guar gum, and PVP could be confirmed.

(2) Swelling test

[Swelling test on distilled water]

The swelling properties of Examples 1 to 3 and Comparative Example 1 were tested in distilled water. Distilled water was poured so that the hydrogel (which was first weighed) in the Petri dish was submerged in distilled water. Afterwards, distilled water was removed at 10-minute intervals, and the Petri dish was dried with tissue paper. The weighed Petri dish with the swollen hydrogel was then placed on an analytical balance and the mg value to the nearest value was recorded. The swelling value was calculated by the following equation.

Swelling (g/g)=(W _s -W _d )/(W _d ) (where W _s represents the swollen hydrogel and W _d represents the dried hydrogel)

This experiment was continued until the weight of the swollen hydrogel decreased, and just before the weight of the swollen hydrogel began to decrease, the maximum swelling limit of the prepared hydrogel was reached.

As a result, as shown in Figure 3, it was confirmed that the hydrogel of Comparative Example 1 had a swelling property of at least 19.33 g/g and that of Example 1 had a swelling property of a maximum 57.4 g/g. In addition, it was confirmed that Examples 1, 2, and 3 had excellent swelling properties compared to Comparative Example 1.

On the other hand, since the swelling of the hydrogel occurs due to diffusion of water, this diffusion can be calculated by the following equation.

F=kt ⁿ (where n represents the swelling index, k is the swelling rate constant, and F is the ratio of Wt (swelling ratio at instant t) and W _eq (representing the equilibrium time of swelling) Determined partial swelling)

A graph of ln(F) versus In(t) of F is shown in FIG. 2b, and the diffusion parameter values are summarized in Table 2 below.

parameter Comparative Example 1 Example 1 Example 2 Example 3 n 0.57 0.55 0.68 0.69 Y-intercept -2.016 -2.347 -2.864 -2.961 K 0.133 0.096 0.057 0.052 Regression analysis (Regression, %) 86 99 98 98

At this time, if the swelling index n is less than 0.5, the diffusion pattern is Fickian, and if the swelling index n is greater than 0.5, the diffusion pattern is non-Fickian, and if n is 1, it represents the release mechanism of Case II. . As shown in Table 1, it was confirmed that the hydrogels of Examples 1 to 3 showed a higher n value than the hydrogel of Comparative Example 1, which means that the hydrogels of Examples 1 to 3 had a higher water absorption ability. indicates that it is

[Swelling test by pH]

The swelling properties of the hydrogels obtained in Examples 1 to 3 and Comparative Example 1 were measured according to pH. The swelling properties of the hydrogels of the above examples and comparative examples were measured in buffer solutions with pH of 2, 4, 6, 7, 8, and 10.

The results are shown in Figure 4, and as shown in Figure 4, it was confirmed that the hydrogel showed minimal swelling at neutral and basic pH, while maximum swelling occurred at acidic pH. The hydrogel of Comparative Example 1 showed less swelling at acidic pH (2 to 6) compared to the hydrogel of Examples 1 to 3 at the same pH. The hydrogel of Example 2 at pH 4 showed maximum swelling (55.73 g/g). Hydrogels based on cationic polymers such as chitosan can exhibit maximum swelling at acidic pH due to protonation of amino (-NH ²⁺ ) groups and repulsion of protonated elements. Deprotonation of amino (-NH ²⁺ ) groups at higher pH levels may be responsible for the reduced swelling of the prepared hydrogels. Therefore, less swelling is observed at neutral and basic pH due to deswelling at higher pH.

Protonation occurs when the pKb in each experiment is greater than the pH of the surrounding medium, enhancing the amount of cations on the polymer chains. Therefore, swelling occurs due to the hydrophilicity and chain repulsion generated by irradiation of chitosan.

[Swelling test by ions]

Since swelling properties may vary depending on salt concentration, the swelling properties of the hydrogels of Examples 1 to 3 and Comparative Example 1 were measured in solutions containing NaCl and CaCl ₂ salts. The results are shown in Figure 5.

As shown in Figure 5, the swelling property of the hydrogel decreased as the concentration of NaCl and CaCl ₂ increased in the ionic solution. A large amount of electrolyte reduced the osmotic pressure between the hydrogel and the external solvent, resulting in a charge screening effect. Because of this, diffusion decreases and swelling properties decrease. As shown in Figure 5(a), the maximum expansion value was measured at 22.18 g/g for 0.2M NaCl (Figure 5(a)) and 17.18 g/g for 0.2M CaCl ₂ (Figure 5(b)). .

Meanwhile, for the CaCl ₂ electrolyte ion solution, greater swelling occurred compared to the same concentration of NaCl electrolyte ion solution. Ca ²⁺ cations form complexes with the polymer, which appears to be due to an increase in pore size at low concentrations of CaCl ₂ resulting in larger swelling values.

(3) Biodegradation measurement

To measure biodegradability, 35 mg of the hydrogels of Examples 1 to 3 and Comparative Example 1 were each immersed in PBS, and after 1, 3, 5, and 7 days, each hydrogel was extracted and accurately weighed. The weight reduction rate (%) was calculated by subtracting the initial weight from the final weight, and the results are shown in Figure 6.

As shown in Figure 6, after 7 days, it was confirmed that the hydrogel gradually decomposed over time. Specifically, it was confirmed that 91.15% of the hydrogel of Comparative Example 1 was decomposed, 79.84% of the hydrogel of Example 1, 83.45% of the hydrogel of Example 2, and 86.34% of the hydrogel of Example 3 were decomposed. I was able to.

Meanwhile, chitosan, which is composed of a glycosidic bond as a monomer, imparts biodegradable properties to the hydrogel, but it was confirmed that the mixed use of MPTMS, chitosan, guar gum, and PVP reduced weight loss due to the interaction of natural and synthetic polymers. , especially when using MPTMS at the same content as in Example 1, biodegradability was the lowest.

(4) Antibacterial analysis

The antibacterial activity of the hydrogels of Examples 1 to 3 and Comparative Example 1 was analyzed by well and disk diffusion methods.

100 μl (10 ⁸ cells/ml) log phase cultures of Salmonella typhimurium (Gram-negative) and Listeria monocytogenes (Gram-positive) were cultured at Luria Bertani (LB). Agar plates were inoculated aseptically using spread plate technology. Afterwards, wells (6-8 mm) were prepared on an LB agar plate using a sterilized cork borer, and 100 μl of the prepared hydrogels of Examples 1 to 3 and Comparative Example 1 were aseptically inoculated into the wells. .

Meanwhile, for disk diffusion analysis, a sterilized filter disk (6 mm) was placed on the surface of the LB agar plate, and 100 μl of the prepared hydrogels of Examples 1 to 3 and Comparative Example 1 were aseptically inoculated on the surface of the paper disk. did. This is shown in Figure 7.

The plates were incubated overnight at 28°C for 24 hours and the inhibition zones around the wells and discs were observed, and the results are shown in Table 3.

Agar well diffusion assay strain Sample (100㎕) Comparative Example 1 Example 1 Example 2 Example 3 Salmonella Typhimorium (Gram negative) ++ ++ ++ +++ Listeria monocytogenes (Gram positive) +++ ++ +++ ++ Paper disc diffusion assay strain Sample (100㎕) Comparative Example 1 Example 1 Example 2 Example 3 Salmonella Typhimorium (Gram negative) ++ ++ + + Listeria monocytogenes (Gram positive) ++ ++ + ± * Diameter of inhibition zone: ±: 0-3mm; +:3-6mm; ++: 6-9mm; +++: greater than 12mm

All hydrogels showed some activity against both Gram positive and Gram negative bacterial strains as shown in Figure 7 and Table 3 above. Specifically, a clear inhibition zone was observed in the agar well diffusion method. The hydrogel of Comparative Example 1 and the hydrogel of Example 2 showed an area exceeding 12 mm for Listeria monocytogenes. Additionally, an inhibition zone exceeding 12 mm was observed for Salmonella Typhimurium with the hydrogel of Example 3. Meanwhile, inhibition zones were observed in all hydrogels in the agar disk diffusion method, but none of the hydrogels exceeded 9 mm.

Meanwhile, the minimum inhibitory concentration (MIC) of the hydrogels of Examples 1 to 3 and Comparative Example 1 was determined by broth dilution. 20 μl of the bacterial culture was aseptically inoculated into LB medium (2 mL) supplemented with different concentrations of the hydrogels of Examples 1 to 3 and Comparative Example 1. The tubes were incubated with an orbital shaker (180 rpm) for 24 hours at 28°C. Furthermore, media not inoculated with hydrogel was used as a control.

After incubation, 0.1 mL of samples were serially diluted, plated on LB agar plates by spread plate technique, incubated at 28°C for 2 hours, and observed for colony forming units. The results are shown in Table 4.

dilution ratio Salmonella Typhimorium, Gram negative (CFU/mL) Comparative Example 1 Example 1 Example 2 Example 3 1/256 4.5±0.1×10 ⁶ 4.0±0.1×10 ⁶ 3.1±0.6×10 ⁶ 3.5±0.0×10 ⁶ 1/128 9.3±0.1×10 ⁵ 6.3±0.4×10 ⁵ 6.8±0.3×10 ⁵ 4.9±0.6×10 ⁵ 1/64 4.3±0.0×10 ⁵ 2.8±0.0×10 ⁵ 3.7±0.2×10 ⁵ 4.1±0.2×10 ⁵ 1/32 6.8±0.1×10 ⁴ 4.1±0.1×10 ⁴ 2.3±0.1×10 ⁴ 2.0±0.3×10 ⁴ 1/16 1.7±0.0×10 ² 0.5±0.0×10 ² 0.2±0.0×10 ² 0.3±0.0×10 ² 1/8 3.0±0.0 2.0±1.0 N.D. N.D. 1/4 N.D. N.D. N.D. N.D. 1/2 N.D. N.D. N.D. N.D. One N.D. N.D. N.D. N.D. control 9.7±0.4×10 ⁶ dilution ratio Listeria monocytogenes, Gram positive (CFU/mL) Comparative Example 1 Example 1 Example 2 Example 3 1/256 6.5±0.1×10 ⁵ 5.8±0.4×10 ⁵ 7.5±0.6×10 ⁵ 4.7±0.3×10 ⁵ 1/128 3.8±0.3×10 ⁵ 4.1±0.1×10 ⁵ 3.5±0.0×10 ⁵ 2.7±0.4×10 ⁵ 1/64 1.9±0.0×10 ⁴ 1.3±0.1×10 ⁴ 1.3±0.0×10 ⁴ 1.0±0.0×10 ⁴ 1/32 8.6±0.1×10 ³ 5.0±0.1×10 ³ 4.3±0.1×10 ³ 3.3±0.1×10 ³ 1/16 N.D. N.D. N.D. N.D. 1/8 N.D. N.D. N.D. N.D. 1/4 N.D. N.D. N.D. N.D. 1/2 N.D. N.D. N.D. N.D. One N.D. N.D. N.D. N.D. control 1.0±0.0×10 ⁶

*Minimum Bacterial Concentration (MBC) is the dilution ratio that represents the N.D. (No Data) at which colony forming units begin to be observed. The viability of the hydrogel was observed by counting CFU in diluted hydrogel samples. At a dilution of 1/16, the bacterial count appeared to be 0, and then the bacterial count increased in bacterial strains and in all hydrogels, as shown in Table 4 above.

The MICs of the hydrogels were different for the two bacterial strains. Both the hydrogels of Examples 1 and 2 were observed at 1/8, while the hydrogels of Examples 2 and 3 were both observed at 1/36 for Salmonella Typhimurium. In Listeria monocytogenes, the MICs of all hydrogels were observed at 1/32 of the original dilution as shown in Figure 8. The hydrogel of the present invention showed improved antibacterial properties due to the low weight average molecular weight of chitosan, and the hydrogel entered bacterial cells and terminated the conversion of DNA to RNA, thereby delaying growth.

The mechanism of inhibition of bacterial growth is due to the binding of microbial DNA to chitosan, which results in protein synthesis and impedance of mRNA by penetration of chitosan into the nucleus of the microorganism.

(5) Drug release ability analysis

[Preparation of SIF, SGF, and PBS]

To prepare artificial intestinal fluid digestive enzyme (SIF) at pH 6.8, 118 mL of 0.1 M sodium hydroxide (NaOH) was added to 250 mL of 0.2 M potassium phosphate (monopotassium phosphate, KH ₂ PO ₄ ). Furthermore, to prepare artificial gastric fluid digestive enzyme (SGF) at pH 1.2, 1 g of NaCl was mixed with 3.5 mL of hydrochloric acid (HCl) and diluted with distilled water until the volume reached 500 mL. Additionally, PBS at pH 7.4 was prepared.

[Manufacture of drug-loaded hydrogel]

As a drug, ampicillin sodium was used. A chitosan solution was prepared by dissolving 0.5 g of irradiated chitosan in 25 mL of 0.5 M acetic acid aqueous solution and adding 25 mL of distilled water. In addition, a guar gum aqueous solution was prepared by adding 0.2 g of guar gum to 25 mL of distilled water, and a PVP aqueous solution was prepared by adding 0.5 g of PVP to 25 mL of distilled water. Chitosan solution, guar gum solution, and PVP solution were mixed at a volume ratio of 3:1:0.5 at 45°C for 2 hours. Afterwards, the drug solution prepared by adding 35 mg of the drug to 5 mL of distilled water was added to the mixed solution of the chitosan solution, guar gum solution, and PVP solution.

Afterwards, an MPTMS solution containing 200 μl of MPTMS mixed in 5 mL of methanol was added dropwise to the solution containing the drug for dissolution. It was then mixed at 45°C for 4 hours, transferred to a Petri dish, and dried overnight in a 40°C drying oven (VS-1202D3, VISION Scientific Co., Ltd, Korea).

After drying, the prepared hydrogel was immersed in a beaker at 37°C for 3 hours in PBS, SIF, and SGF, respectively. 5 mL of these solutions were extracted from the beaker every 30 minutes, and equal amounts of solutions (PBS, SIF, and SGF) were added back to the beaker to maintain equal volumes of solutions. As a reference sample, the above-mentioned contents of drug containing PBS and SIF without hydrogel were used. UV-Vis spectroscopy was used to measure drug release amount. The results are shown in Figure 9.

As shown in Figure 9, SIF and PBS showed controlled drug release, with 81.30% drug release in SIF and 87.40% drug release in PBS over 180 minutes. This meets the US Pharmacopoeia standard of releasing more than 80% of the drug over 3 hours.

It was confirmed that the hydrogel of the present invention is suitable for administration of injectable (intravenous, IV) drugs.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. This will be self-evident to those with ordinary knowledge in the field.

Claims (15)

Contains irradiated chitosan, natural gelling polymer, hydrophilic synthetic polymer, and (3-mercaptopropyl)trimethoxysilane (MPTMS);
A hydrogel, wherein the natural gelling polymer is guar gum, and the hydrophilic synthetic polymer is polyvinylpyrrolidone (PVP).
The hydrogel according to claim 1, wherein the weight average molecular weight (Mw) of the irradiated chitosan is 19.00×10 ⁴ to 21.50×10 ⁴ .
The method of claim 1, wherein the hydrogel is comprised of 40 to 80% by weight of the irradiated chitosan, 5 to 15% by weight of the natural gelling polymer, 3 to 10% by weight of the hydrophilic synthetic polymer, and A hydrogel comprising 10 to 45% by weight of (3-mercaptopropyl)trimethoxysilane.
The hydrogel of claim 1, wherein the hydrogel contains 20 to 40% by weight of MPTMS, based on the total weight of the hydrogel.
delete delete The hydrogel according to claim 1, wherein the hydrogel is in the form of a hydrogel film.
The hydrogel of claim 1, wherein the hydrogel is for drug delivery.
Irradiating chitosan with radiation;
mixing irradiated chitosan, natural gelling polymer, and hydrophilic synthetic polymer with a solvent; and
It includes adding (3-mercaptopropyl)trimethoxysilane (MPTMS) to the solvent,
A method for producing a hydrogel, wherein the natural gelling polymer is guar gum, and the hydrophilic synthetic polymer is polyvinylpyrrolidone (PVP).
The method of claim 9, wherein the radiation is an X-ray, a gamma ray, or an electron beam.
The method of claim 9, wherein the radiation is irradiated at a total dose of 10 to 30 kGy.
The method of claim 9, wherein the solvent is water.
The method of claim 9, wherein the solvent is a 0.5 to 2 M aqueous acetic acid solution.
The method of claim 9, further comprising adding at least one selected from the group consisting of drugs and radioisotopes to the solvent.
The method of claim 14, wherein the drug includes at least one selected from the group consisting of ampicillin and kanamycin monosulfate monohydrate.