KR20210095039A - Method for producing a temperature-sensitive cellulose-based hydrogel for preventing adhesion with mitomycin C release control function - Google Patents

Abstract

A method for producing a temperature-sensitive cellulose-based hydrogel for preventing adhesion with a mitomycin C release control function according to an embodiment of the present invention comprises the following steps of: preparing a mixture by mixing the modified tempo oxidized nanocellulose, methyl cellulose and mitomycin C; agitating the mixture; and gelling by adding sodium chloride to the stirred mixture.

Description

Method for producing a temperature-sensitive cellulose-based hydrogel for preventing adhesion with mitomycin C release control function

The present invention relates to a method for producing an anti-adhesion temperature-sensitive cellulose-based hydrogel having a mitomycin C release control function. it's about how

Post-operative peritoneal tissue adhesion (PPA) is one of the common health problems in which thick fibrous tissue bands form between the Bowel loop, parietal peritoneum and parenchyma.

PPA is caused by cessation of the normal healing process causing infection, intestinal obstruction, ectopic pregnancy, infertility and death of billions of patients has been proceeding with Several pharmacological agents have been used as monotherapy, but there have been problems that the condition cannot be improved due to altered pharmacokinetics or rapid drug removal in the abdominal cavity. In addition, safety issues have been raised due to side effects of drugs during clinical application.

On the other hand, the barrier-based system has the advantage that it can be treated independently by physically separating the surgical site from the surrounding organs during the severe adhesion period. However, none of these have been adopted as standards despite their positive effect on adhesion prevention.

An ideal barrier can have several characteristics: (a) be applicable for both laparotomy and laparoscopy, (b) have tunable biodegradability and cellular compatibility, (c) be suitable for covering tissue surface topography after application, and (d) up to a critical adhesion period be mechanically effective and (e) promote normal healing of the surgical tissue site;

In this regard, injectable temperature-sensitive hydrogel barriers have advantages over solid film and liquid solution-based barriers because they can be applied intraperitoneally at room temperature and converted into a solid gel at the body temperature.

The thermosensitive hydrogel barrier can inject drugs used in drug delivery systems to the target site to prevent fibrotic adhesions, and methyl cellulose can be a promising polymer when preparing thermo-gels at physiological temperatures, Mitomycin For C (MMC), it can be applied as an antibiotic with a potential role in reducing adhesions through fibrosis and vascular inhibition for several weeks.

Furthermore, intraperitoneal administration of antiproliferative MMCs has been shown to be effective in preventing primary or recurrent intraperitoneal adhesions in mice because of their ability to inhibit cross-link DNA and cell mitosis.

However, there are disadvantages in that the reactivity must be controlled in order to prevent side effects due to the rapid and explosive reactivity of MMC, which is a hydrophilic drug, and necrosis of the tissue site. In this case, TEMPO can be an effective carrier for loading and delivering drugs to the target site, and TEMPO can also improve the mechanical stability of the scaffold.

In addition, EDC is a cross-linker of the amine and carboxyl reaction that makes the NHS conjugation procedure stable, and the EDC/NHS combination is non-toxic and has the advantage of being easily removed by dialysis or rinsing as a widely used catalyst.

Republic of Korea Patent Publication No. 10-1666792

An object of the present invention is to provide a method for preparing a temperature-sensitive cellulose-based hydrogel for preventing adhesions having a mitomycin C release control function.

The method for preparing a temperature-sensitive cellulose-based hydrogel for anti-adhesion having a mitomycin C release control function according to an embodiment of the present invention is modified tempo-oxidation-mediated nanocellulose (Modified tempo oxidized nanocellulose), methyl cellulose (Methyl cellulose) ) and mitomycin C (Mitomycin C) to prepare a mixture by mixing; agitating the mixture and gelling by adding sodium chloride to the stirred mixture.

Before the step of preparing the mixture, the modified tempo-oxidation-mediated- nanocellulose may be further included.

The step of preparing the modified tempo-oxidation-mediated-nanocellulose is a process of forming a matrix by pouring the tempo-oxidation-mediated-nanocellulose suspension into a glass Petri and storing it in an incubator at 37° C. for 6 hours, N- ( 3Dimethylaminopropyl)-N-ethyl carbodide hydrochloride/N-hydroxy-succinimide (N-(3Dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride/N-hydroxy-succinimide) solution was added and at 4℃ It may include a process of culturing for 6 hours, a process of washing the cultured sheet, and a process of pulverizing the washed sheet.

The tempo-oxidized nanocellulose may be obtained from softwood bleached kraft pulp.

In the step of preparing the mixture, the modified tempo-oxidation-mediated-nanocellulose and methyl cellulose are mixed in a weight ratio of 1:1.5 to 3.5, and the mitomycin C may include 0 to 0.4 μg/100 μl.

In the stirring step, the mixture may be stirred at a temperature of 80 to 90 °C for 15 to 25 minutes.

In another embodiment of the present invention, an anti-adhesion temperature-sensitive cellulose-based hydrogel having a mitomycin C release control function is prepared according to the above-described method.

According to the present invention, it is possible to provide a temperature-sensitive cellulose-based hydrogel for anti-adhesion having a function of controlling the release of mitomycin C, which has excellent cell compatibility, mechanical stability, excellent injectability, optimal degradation, and excellent drug release ability at the surgical site. there is.

1A is an evaluation of the viability of L929 fibroblasts against various types of hydrogels for 14 days, FIG. 1B shows the effect of Mitomycin C on the viability of fibroblasts in various hydrogels, and FIG. 1C is a hydrogel raw material. (results are expressed as mean ± SD, ^*** P < 0.001 ^** P < 0.01 ^* P < 0.05).
Figure 2A shows an SEM image of a freeze-dried hydrogel and an EDXS image evaluating the efficiency of a combination of cTOCN and mitomycin C in various types of hydrogels, Figure 2B shows the viscosity of the hydrogel, Figure 2C shows the hydrogel shows the mechanical strength of, FIG. 2D shows the strength of the hydrogel as the methyl cellulose concentration increases, FIG. 2E shows the gelation time of the hydrogel observed through the 37°C vial inversion method, and FIG. 2F shows the 37 It shows the swelling behavior of the hydrogel at °C.
Figure 3A is a comparison of the weight loss behavior of the gel in vitro after immersion in SBF solution (pH 7.4, 37 °C), Figure 3B is the SBF medium (pH 7.4, 37 °C) for 14 days from other hydrogels of mitomycin C Figure 3C shows the release rate of Mitomycin C from a hydrogel prepared using TOCN (no catalyst) in SBF medium (pH 7.4, 37 °C), and Figure 4D shows the release rate of C2 through rheology at 37 °C. The gelation temperature of the hydrogel of .5T1M0.2 was measured (SBF = Simulated body fluid, analog body fluid).
4A shows the gelation time of C2.5T1M0.2 hydrogel observed through rheology at 37° C., and FIG. 4B shows injectability and body temperature of C2.5T1M0.2 hydrogel at room temperature (25° C.) (37 ℃), and Fig. 4C shows the degradation behavior in vivo after subcutaneous injection of various types of hydrogels (black dotted lines) in mice.
Figure 5A shows an image of the application of hydrogel after performing sidewall defect cecal abrasion in mice, Figure 5B shows the percentage of adhesion frequency with different adhesion scores in 4 treatment groups on day 7, and Figure 5C shows 14 The adhesion score is shown in mice treated with the C2.5T1M0 (without mitomycin c) hydrogel on the first day (it can be confirmed that the hydrogel containing mitomycin C (C2.5T1M0.2) completely prevents adhesion during the test period. The positive control group had no adhesions during the first week but showed adhesions the second week after surgery (blue circles indicate the presence of gel after 7 days).
Figure 6A is a histological evaluation of the cecum wall and peritoneum (negative control, positive control, C2.5T1M0 hydrogel-treated group, and C2.5T1M0.2 hydrogel-treated group adhesions after 14 days hematoxylin, It was confirmed by eosin (H&E) and Masson's trichrome (MT) staining; AW = abdominal wall, CM = cecal mucosa, Me = epithelial layer, SM = skeletal muscle. Vascularization is indicated by black dotted circles). Figure 6B shows that gene expression of adhesion markers (tumor necrosis factor-α (TNF-α) and fibronectin) in each sample-treated group of animals was examined by real-time real-time qPCR 14 days post-surgery (results are presented as mean ± SD). , ***P <0.001 **P <0.01 *P <0.05).
Figure 7A shows a method of regulating the genes of adhesion markers by MMC, Figure 7B is a histological examination of metabolic organs after 14 days for cytotoxicity in healthy mice and C2.5T1M0.2 hydrogel-treated mice, Figure 7C is an image taken by day SEM images of the abdominal wall of C2.5T1M0.2 hydrogel-treated mice, Figure 7D shows the abdominal wall images of healthy mice.
8A shows the crosslinking mechanism of EDC/NHC, TOCN, cTOCN, MMC and MC, and FIG. 8B shows the manufacturing process of the thermosensitive gel.

Hereinafter, the present invention will be described in more detail through Examples according to the present invention and Comparative Examples not according to the present invention, but the scope of the present invention is not limited by the Examples presented below.

The method for preparing a temperature-sensitive cellulose-based hydrogel for anti-adhesion having a mitomycin C release control function according to an embodiment of the present invention is a modified tempo-oxidation-mediated-nanocellulose (Modified tempo oxidized nanocellulose, cTOCN), methyl cellulose ( Preparing a mixture by mixing methyl cellulose, MC) and mitomycin C (Mitomycin C, MMC); agitating the mixture and gelling by adding sodium chloride to the stirred mixture.

Before the step of preparing the mixture, the modified tempo-oxidation-mediated- nanocellulose (TOCN) step may be further included.

The step of preparing the modified tempo-oxidation-mediated-nanocellulose (cTOCN) is, the tempo-oxidation-mediated-nanocellulose (TOCN) suspension is poured into a glass Petri and stored in an incubator at 37°C for 6 hours to form a matrix; N-(3Dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride/N-hydroxy-succinimide, EDC/ NHS) solution and incubation at 4° C. for 6 hours, washing the cultured sheet, and pulverizing the washed sheet may be included.

The tempo-oxidized nanocellulose (TOCN) may be obtained from softwood bleached kraft pulp.

In the step of preparing the mixture, in the step of preparing the mixture, the modified tempo-oxidation-mediated-nanocellulose and methyl cellulose are mixed in a weight ratio of 1:1.5 to 3.5, and the mitomycin C is 0 to 0.4 μg/ 100 μl.

In the stirring step, the mixture may be stirred at a temperature of 80 to 90 °C for 15 to 25 minutes.

The present invention can provide a temperature-sensitive cellulose-based hydrogel for anti-adhesion having a mitomycin C release control function according to the above-described method.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Example>

1. Preparation of materials

MMC (98% Potency), MC (88,000 Da, DS = 1.7), EDC and NHS were purchased from Sigma-Aldrich Co., USA. For in vitro analysis, EZ-CYTOX cell viability assay reagent (Dogen Seoul, Korea), Gibco ^TM fetal bovine serum (FBS), and Hyclone ^TM RPMI 1640 medium (GE Healthcare Life Sciences, USA) were used. L929 fibroblasts were performed using the American Type Culture Collection (ATCC).

The L929 fibroblast (LFC) line was purchased from the American Type Culture Collection (ATCC).

2. Preparation of TOCN

1% concentrated TOCN was prepared by TEMPO mediated oxidation from softwood bleached kraft pulp.

3. Preparation of Modified TOCN (TOCN)

First, 12ml of TOCN suspension was poured into a glass Petri dish to make a thin layer, and then stored in an incubator at 37°C for 6 hours.

Next, small holes were made in the matrix with a 22 gauge needle to increase the efficiency of the crosslinking reaction.

After that, a sufficient amount of EDC/NHS solution was added and incubated at 4° C. for 6 hours. Next, the TOCN matrix was washed with deionized water 6 times to remove unreacted EDC/NHC.

Next, the TOCN matrix was ground and filtered through a 100 μm mesh sieve.

Finally, the modified TOCN suspension (cTOCN) was stored at 4 °C.

4. Preparation of heat-sensitive hydrogels

MMC-loaded hydrogels by EDC/NHS were synthesized by a coupling reaction between the activated carboxyl group (-COOH) of TOCN and the primary amine group of MMC (-NH _{2 ) to form an amide bond (-CONH-)} do.

Each of the hydrogel samples listed in Table 1 below was dissolved in autoclaved water, transferred to a glass bottle, immersed in a water bath (85° C.), and stirred for 20 minutes.

After stirring, the mixture was cooled in an ice box (0°C), and the suspension was stirred until it became a clear solution.

Finally, the solution was finally stored at 4° C. and then gelled by adding 1.25 M sodium chloride.

Table 1 below shows the composition of the heat-sensitive hydrogel and the gelation time of the hydrogel in vitro determined by the vial inversion method.

5. Analysis of carboxylate content, crystallinity of TOCN and degree of substitution of MC

The carboxylate content of TOCN was analyzed using electrical conductivity titration.

The crystallinity (percentage) of TOCN was measured in reflection mode using X-ray diffraction (XRD, Rigaku RINT 2000 with Ni filtered CuK radiation).

The degree of substitution (DS) of MC was analyzed by performing ^{13 C NMR.} Samples were dissolved in DMSO-d6 at 80° C. (concentration of about 10 to 30 g/L) and analyzed using an Avance III 400 MHz spectrometer from Bruker (Wisburg, France).

6. In vitro cytotoxicity assessment and fibroblast invasion prevention assay

The prepared samples were completely covered to prevent contamination during storage. Later, the samples were terminally sterilized by exposure to ultraviolet C radiation (2 h), and L929 was used to evaluate the in vitro cell-material interaction and anti-adhesion capacity of the hydrogel.

7. Structural Analysis

The chemical composition of the raw polymer and the hydrogel was analyzed using total reflectance-Fourier transform spectroscopy at low temperature (4°C) (ATR-FTIR, Thermo Scientific Nicolet iS10, OMNIC 7.3 software).

8. Analysis of morphology, viscosity and mechanical strength

SEM (Scanning Electrospinning Microscopy, JSM-6701F, JEOL, Japan, equipped with Energy-dispersive X-ray spectroscopy (EDXS)) was used to confirm the presence of MMC in both the surface morphology, the hydrogel, and the lyophilized hydrogel.

After platinum coating, the freeze-dried hydrogel was evaluated. Since the viscosity of the hydrogel was applied at 25 °C, the viscosity was confirmed at room temperature (25 °C). The mechanical strength of the hydrogel was measured using a universal tester (Unitech, R&B, Korea).

9. Thermal Sensitivity Behavior and Injection Analysis

The thermal sensitivity and sol-gel transition of the hydrogel were measured by the vial inversion method based on the principle of fluidity and non-flow, and injectability was measured at 25°C.

10. In vitro degradation and swelling assay

In order to confirm the ability to absorb the surrounding fluid after application, the swelling behavior of the hydrogel was confirmed, and the pH of the ideal peritoneal fluid is 7.4 to 8.0.

The degradation behavior of the hydrogel was analyzed up to 14 days in a cell-free solution (SBF, pH 7.4), a cell-free solution similar in chemical composition to plasma at 37°C.

11. Analysis of mitomycin C (MMC) release rate from hydrogels

The release efficiency of mitomycin C from the hydrogels (C1.5T1M0.2, C2.5T1M0.2 and C3.5T1M0.2) was calculated by extracting the drug from the hydrogel.

12. In vivo hydrogel degradation assay

In vivo injectability, gel stability and biodegradability were confirmed.

Hydrogels (C1.5T1M0.2, C2.5T1M0.2 and C3.5T1M0.2) were administered through a 22-gauge needle into the back of the mice and for 2 weeks (2 mice in each group, 6 mice weekly) evaluated. At certain weeks, mice were euthanized and tissues surrounding the hydrogel were collected and histological observations were performed.

The middle portion of the tissue mold was cut to confirm degradation analysis.

13. In vivo post-surgical anti-adhesion analysis

13-1. experimental design

For in-vivo analysis, 9-week-old Sprague-Dawley rats (Rattus norvegicus)) were purchased from the Laboratory Animal Center (Dayun, Korea) and used. Each rat weighed 200 to 250 g each. All procedures were performed according to protocols and guidelines for the care and use of laboratory animals, and the study plan was approved by the Animal Ethics Committee of Soonchunhyang University, Korea.

64 male mice were divided into 4 experiments (16 mice in each group, 8 mice weekly): group 1 negative control washed with saline (no barrier), group 2 2 ml of C2.5T1M0.2 hydrogel on the scratched surface. (with drug), group 3 applied 2 ml of C2.5T1M0 hydrogel (without drug), group 4 was a positive control (covering scratched surface with 2 ml of commercial hydrogel barrier) (HyFence, CHA Bio & Diostech (Seoul) , Korea)) (Shin, Paik, & Yang, 2018).

In all mice, the surfaces of the abdominal wall (1X1 cm ² ) and cecum (1X1 cm ² ) were carefully scraped with a mass blade.

13-2. A novel anti-adhesion model after surgery

The novel anti-adhesion efficacy of hydrogels was evaluated using a mouse sidewall-defective-caecal abrasion model validated in previous studies.

Initially, a 5 cm midline incision was made using aseptic technique along the linea alba in the abdominal wall of the mouse, followed by scraping the surface of the abdominal wall and cecum with a scalpel blade. Then, 2ml of hydrogel was injected into the damaged cecal surface and abdominal wall defect and completely coagulated for 30 seconds. After closing the defect site with sutures, the mice were confined in cages so that they had free access to food and water.

13-4. In vivo toxicity and re-epithelialization assay

The general condition of the surgical site (activity, behavior, energy, hair, feces and respiration), water, food intake, mortality, and treatment methods were monitored for possible side effects caused by the hydrogel. On day 14, major organs (lung, liver, heart and kidney) were pathologically visualized, fixed and analyzed (H&E staining). After 14 days, the condition and remesothelialization of the hydrogel-treated mice were evaluated and compared with normal healthy mice through SEM.

<Statistics Analysis>

Results were expressed as mean ± standard deviation (SD), and 4 samples were considered for each group for experimental characterization (excluding animal studies). Analysis of variance (ANOVA) was performed using GraphPad Prism 5. Data are ^** P <0.05; ^*** P< was considered statistically significant.

<Experimental example>

1. Carboxylate content, crystallinity and substitution degree of TOCN

The content of carboxylate in oxidized cellulose is a major structural factor determining hydrophilicity, cytotoxicity and degradation. TOCN was prepared by oxidizing C6 of the carboxyl group glucopyranose unit of cellulose. On the other hand, the crystallinity of cellulosic materials plays an important role in the physico-mechanical and chemical properties. For example, as crystallinity increases, tensile strength and density increase, and chemical reactivity and expansion properties decrease. The measured carboxyl group content of TOCN was 1.5 mmol/g, and the crystallinity was 60.23%.

The degree of substitution (DS) controls the solubility and stability of the polymer. In general, MCs with a DS of 1.3 to 2.5 are water soluble, whereas MCs with a DS > 2.5 are soluble in organic solvents.

In the present invention, the DS value of MC was 1.7, so it can be used for the development of water-soluble thermo-gels. The DS of MC used in the example of the present invention was measured to be 1.7. Therefore, the MC can be easily dissolved in water to prepare a thermo-sensitive hydrogel.

2. Test tube ( in vitro) Intracytotoxicity assay

A prerequisite for the development of good biomaterials is good biocompatibility and should be safe for cells in direct contact. However, indirect cytotoxicity methods are insufficient to understand this phenomenon. For this reason, suitability was evaluated by direct contact of the hydrogel to fibroblasts seeded on well plates. All hydrogel groups without MMC showed distinct cell proliferation indicating non-cytotoxicity of cTOCN and MC (Fig. 1A). As can be seen in Figure 1A, C1.5T1M0.4, C2.5T1M0.4 and C3.5T1M0.4 hydrogels showed cytotoxicity after 5 days of culture. In addition, the C1.5T1M0.2, C2.5T1M0.2 and C3.5T1M0.2 hydrogel groups grew evidently by day 14, and in particular, C2.5T1M0.2 showed cell compatibility within the safety limit ( ^*** P < 0.001).

Growth patterns of L929 cells on different types of hydrogels were further evaluated by confocal image analysis (Fig. 1B). A significant difference in the number of cells growing on the hydrogel surface was found, which was consistent with the viability results determined by the WST method. Mitomycin C (MMC) is an anti-metabolite antibiotic with a potential role in reducing cell adhesion through inhibition of fibrosis. MMC is known as a DNA alkylating agent that can cross-link with guanine nucleotides of DNA helices and can inhibit the cell cycle of non-specific fibroblasts by wave and function of DNA helices (Ozerhan et al., 2016) (Urkan et al. ., 2017). For anti-adhesion applications, the hydrogel should not allow rapid, uncontrolled growth and should not be toxic. Through the above results, it was confirmed that C2.5T1M0.2 and C3.5T1M0.2 had superior cell compatibility compared to other hydrogels. Therefore, in an additional test to find suitable candidates for drug-loaded anti-adhesion hydrogels, 0.2 μg/100 μl MMC-loaded hydrogels (C1.5T1M0.2, C2.5T1M0.2, and C3.5T1M0.2) ) was used.

3. Spectral Analysis

1C shows the chemical structures of C1.5T1M0.2, C2.5T1M0.2 and C3.5T1M0.2 hydrogels confirmed through FI-IR.

Peaks at the three 3417cm ^-1 and 2883cm ^-1 of the hydrogel is available for the -OH stretching vibration of CH ₃ groups of the cellulose polymer and the COOH groups CH stretching vibration, indicating the presence of MC and cTOCN. The reason for the broadening of the -OH peak at 3417 cm ⁻¹ indicates that MMC was successfully introduced into the three hydrogels due to the presence of characteristic NH groups found in the MMC backbone. The peak at 1152-1156 cm ⁻¹ corresponds to the CH ring stretching found in all samples.

Furthermore, ^{the absorption band change at 1615 cm −1} confirmed the formation of amide I bonds between cTOCN and MMC in other hydrogels, which successfully indicated the formation of MMC-loaded hydrogels.

4. Morphological observation

The structural characteristics of the gel directly affect the tissue function near the site of application.

Referring to Figure 2A, C3.5T1M0.2 hydrogel showed a dense and rigid porous structure, whereas C1.5T1M0.2 showed a sponge-like structure with larger and relatively uniform pores. In addition, it can be confirmed that as the MC concentration increases in the lyophilized hydrogel, the pores of the hydrogel become smaller. The MMC of the hydrogel is attributable to EDXS by the presence of nitrogen peaks separate from the C and O peaks. Moreover, the gradual increase of C and O peaks revealed a higher amount of MC in the C3.5T1M0.2 hydrogel compared to other hydrogels. In general, a highly porous structure has the advantage of allowing a greater amount of liquid or medium to pass through the structure, and vice versa.

5. Viscosity evaluation

For optimal injectability, the anti-adhesion hydrogel should exhibit as good a viscosity as possible for application at room temperature and, most importantly, remain on the applied site for the period of adhesion (3-14 days).

As can be seen in Figure 2B, it can be confirmed that the viscosity of the hydrogel increases as the shear rate increases at 25 °C, of which the C3.5T1M0.2 hydrogel exhibited the highest viscosity. Incorporation of MC promotes the formation of hydrogen bonds with nanocellulose (Fig. 8). When the shear stress is increased, weak hydrogen bonds are broken, causing aggregation of the polymer, and for this reason, the viscosity of the hydrogel increases as the shear rate increases. However, there was no significant difference between C1.5T1M0.2 and C2.5T1M0.2 hydrogels, and although both hydrogels are suitable in terms of viscosity, C2.5T1M0.2 is more preferable in terms of cell compatibility. In addition, a high-viscosity gel such as C3.5T1M0.2 is undesirable because it may cause complexity because the gel must be administered by restricting the syringe during laparoscopic surgery.

Therefore, a hydrogel having a moderate viscosity even when the shear rate is increased is desirable.

6. Determination of mechanical strength

The lack of mechanical strength of heat-sensitive anti-adhesion hydrogels is one of the major limitations of hydrogels in tissue engineering applications.

2C and 2D show the mechanical strength of the three hydrogels.

As the content of the hydrogel increases, the compressive modulus of the hydrogel is improved, and it can be confirmed that the increase of the MC content contributes to the improvement of the strength of the hydrogel. In addition, hydrogen bonding is a factor that can directly affect the mechanical strength of the hydrogel. A large amount of MC in C3.5T1M0.2 promotes hydrogen bonding and ultimately exhibits superior mechanical strength compared to other hydrogels.

Mechanical stability also influences the biodegradability of the hydrogel: in humans, the abdomen operates as a hydraulic system with a normal intra-abdominal pressure of about 5-14 mmHg (667-1866 Pa). The hydrogel according to the present invention is strong enough to maintain a high compressibility and is suitable for application to the moving abdominal surface inside the body.

Figure 2D shows the stiffness of the hydrogel as a visual image, and the C2.5T1M0.2 hydrogel is not too strong like the C3.5T1M0.2 hydrogel, and is not too easily broken like the C1.5T1M0.2 hydrogel. It can be seen that the flexibility is shown.

7. Observation of gelation behavior

Gelation of injectable hydrogels is important because it can determine the processing time during surgery. After administration, gelation should occur as soon as possible so that a protective barrier is formed and conforms to the shape of the injured site, and thus, it can be confirmed that the gelation time of the hydrogel is proportional to the MC concentration determined by the vial inversion method (Table 1 and Fig. 2E). ).

At body temperature (37°C), the gelation time of C2.5T1M0.2 was 36±4 seconds, and in the case of C3.5T1M0.2 hydrogel, the fastest (27±1s) gelation time was confirmed. The gelation time of the longest time (56±6 seconds) for C1.5T1M0.2 hydrogel to be converted into a solid gel was confirmed. At this time, the three hydrogels contained 1% cTOCN.

Therefore, in the present invention, thermal sensitivity is determined by the hydrophobic interaction between MC molecules substituted with methoxy.

At low temperature (4°C), these molecules hydrate and interact between the polymer and the polymer, and as the temperature rises, the water molecules move into salt ions, and the polymer gradually loses water of hydration, resulting in a solid opaque hydrogel. A three-dimensional network formed of a single gel is formed.

8. In vitro swelling behavior

Hydrogels are three-dimensional polymer matrix components that can absorb and retain water or various biological fluids. Therefore, the swelling behavior of the hydrogel plays an important role in decomposition, blood absorption, and the pattern of irrigation and drug release at the wound site.

2F shows the expansion ratio over time, and it can be seen that the swelling phenomenon is reduced when MC is added to the hydrogel.

The swelling behavior of the hydrogel is mainly changed depending on the polymer properties, the degree of crosslinking and porosity. In general, large porous scaffolds can absorb more drug liquid than small porous scaffolds.

As can be seen from the SEM images, the C3.5T1M0.2 hydrogel has smaller pores and shows a lower swelling behavior in PBS with pH = 7.4 at 37 °C after 24 h.

In addition, increasing the content of MC increases the formation of hydrogen bonds with the amide, increasing molecular entanglement between the polysaccharide chains of cTOCN, MC, and mitomycin C. As a result, the C3.5T1M0.2 hydrogel network becomes dense, and the solvent The ability to absorb is lowered and the expansion behavior is lowered.

9. In vitro degradation assay

The development of a suitable biodegradable system for rapid removal of non-biodegradable or anti-adhesion agents is required, and in order to obtain optimal results, the barrier system must be lowered within 14 days after application to the surgical site.

3A shows the in vitro quantitative degradation percentage of various types of hydrogels.

After 14 days, no trace of C1.5T1M0.2 hydrogel was found in the Petri dish (96.54 ± 4.02%), and in the case of C2.5T1M0.2 hydrogel, it slowly degraded and almost designed time point (89.47 ± 5.24% at 14 days). ), and the C3.5T1M0.2 hydrogel was resistant to SBF washing and did not significantly change its shape after 14 days (21.25 ± 2.39%).

To further confirm the degradation phenomenon, MC gels (MC1.5, MC2.5 and MC3.5) and MC-bound TOCN hydrogels (MC1.5TOCN1, MC2.5TOCN1 and MC3.5TOCN1) were also identified. As a result, when nano-cellulose (TOCN) was added to the hydrogel, the decomposition rate was decreased compared to the MC hydrogel. On the other hand, the decomposition rate of the polymer interacting through hydrogen bonding was slowed (FIG. 8). For this reason, the C3.5T1M0.2 hydrogel maintained minimal degradation for 14 days.

In the present invention, it was confirmed that the hydrogel is hydrolyzed, and the degradability is inversely proportional to the hydrogel MC concentration.

In addition, the washing resistance of the hydrogel depends on its mechanical strength. Thus, the C3.5T1M0.2 hydrogel can provide adequate support for proliferating cells for 14 days (Figure 1A and Figure 1B).

10. MMC release rate in hydrogel

To prevent adhesions, drug release must be controllable during a critical period of adhesions (3-14 days). Referring to Figure 3B, it can be seen that the C1.5T1M0.2 hydrogel releases 24.69 ± 3.4% (0.5 μg/ml) of mitomycin during one day, and the C2.5T1M0.2 and C3.5T1M0.2 hydrogels It can be confirmed that they release 19.25±3.4% (0.39㎍/ml) and 7.45±2.01% (0.15㎍/ml), respectively. The drug release on day 5 of the C1.5T1M0.2, C2.5T1M0.2, and C3.5T1M0.2 hydrogels was approximately 61% (1.22 μg/ml), 40% (0.8 μg/ml) and 19.36 in the release medium, respectively. % (0.38 μg/ml). On days 7 and 14, it was confirmed that drug release from C2.5T1M0.2 and C3.5T1M0.2 hydrogels was much slower than that of C1.5T1M0.2 hydrogels.

The effect of MC modification and drug release was confirmed, and referring to FIG. 3C , the mitomycin C release of the hydrogel prepared using TOCN (without the use of EDC/NHC) was explosively released within 1 day, and the release pattern can be explained by the pH, degradation, drug loading and porosity of the hydrogel.

All hydrogels in acidic PBS (pH 5.4) degraded more rapidly in the intestinal physiological environment (pH 7.4), and MMC dissolves rapidly at acidic pH (pH <6).

In addition, at acidic pH, the amide cross-linking point is destroyed by protonation of the carboxyl group of the cellulose structure, and intermolecular electrostatic repulsion occurs between the polymers, causing the hydrogel to expand and finally drug release occurs explosively ( S3 ).

MMC in the hydrogel of the present invention is covalently bonded to cTOCN, and MC is attached to cTOCN and MMC by hydrogen bonding.

When the MC content is increased, the number of hydrogen bonds increases, so the breakage of intermolecular bonds is reduced and the decomposition rate of the hydrogel is lowered at pH 7.4.

For this reason, the C3.5T1M0.2 hydrogel showed a slower release profile, and the C1.5T1M0.2 hydrogel appeared faster in the pH 7.4 environment.

In the present invention, it can be confirmed that the C2.5T1M0.2 hydrogel can reduce the risk of peritoneal adhesion after surgery and can control the release of MMC. And on days 7 and 14, MMC release was lowered due to degradation of the hydrogel without breaking the amide bond between cTOCN and MMC.

11. Observation of rheological behavior (thermal-sensitivity, gelation) and injectability

Referring to FIGS. 3D and 4A and 4B, the optimized C2.5T1M0.2 hydrogel exhibits a phase transition (change in G' and G”, change in storage modulus and loss modulus) and excellent injectability according to temperature and time. can be checked

The initial G' (storage modulus) increased with temperature and approached 37 °C, and G' and G'' (loss modulus) crossed each other, confirming that the transition from the initial viscous liquid to the solid occurred. The corresponding temperature is defined as the gel temperature (T-Gel).

As can be seen in Figure 4A, G' and G" crossed at 30 seconds.

In the present invention, the gelation time of the C2.5T1M0.2 hydrogel was shown to be 30 seconds at 37 °C, which can be applied to laparoscopy by providing faster gelation. The gel was easily extruded into a syringe at room temperature (25 °C) and was stable without being detached even if it was turned over after being attached to a Petri dish at 37 °C.

Therefore, it can be confirmed that the C2.5T1M0.2 hydrogel can be an appropriate injectable barrier and can form a stable gel in the abdominal cavity.

12. In Vivo Assay

12-1 In vivo degradation of hydrogels

Figure 4C shows the time-dependent subcutaneous degradation of hydrogels by type.

All polymers were stable during injection and confirmed the conversion of non-flowing sol to gel within 1 minute. The rate of mass loss was consistent with the in vitro degradation pattern, and increased gel strength decreased the rate of hydrolysis. As a result, C3.5T1M0.2 showed lower mass loss than C2.5T1M0.2 and C1.5T1M0.2 hydrogels for 14 days.

12-2. Evaluation of anti-adhesion potential in vivo

Polishing and scraping the peritoneum with gauze, blades, brushes, and other instruments can damage the epithelial surface, triggering a series of reactions that form postoperative adhesions. The formed fibrin gel matrix converts the damaged area into thick connective tissue by accumulating fibroblasts, other substances, particularly collagen, and the like. For this reason, collagen plays an important role in adhesion formation. Typically, it takes 3 hours to initiate adhesion and 3-7 days to form an abdominal adhesion. However, since the risk of adhesion recurrence persists up to 14 days after surgery, the anti-adhesion agent must have an effective barrier effect within the application site for 14 days.

5A is a temporal image of the peritoneal state of mice in four different treatment groups.

The negative control group (no barrier) and the group treated with the hydrogel without mitomycin C (C2.5T1M0) showed strong adhesion after 7 days of application. In the case of these two groups, it can be seen that the adhesion area formed by the crosslinking of the fibrous tissues between the peritoneum and the cecum became more dense on the 14th day than on the 7th day.

On the other hand, in the commercial hydrogel treatment group, some hydrogel residues were found in the peritoneal region, but there was no adhesion on the 7th day, and strong adhesion on the 14th day was observed. This is because the remnant serves as the subsequent migration of fibroblasts (Fig. 5A).

Referring to Figure 5A, the commercial hydrogel is composed of hyaluronic acid cross-linked by 1,4-butanediol diglycidyl ether (BDDE), and is a water-insoluble and transparent viscoelastic gel that generally stays for a long time in the abdominal region, and the heat-reactive hydrogel is no. In addition, long-term retention times of hydrogels may cause stability and efficacy issues, and may indicate altered hemostasis.

5B and 5C show the percentage frequency (%), adhesion scores at 7 days and 14 days.

The adhesion frequency of SCORE 5 was higher in mice treated with C2.5T1M0 hydrogel than the percentage of negative controls after 14 days, and SCORE 5 was lower in the first week in the positive control group, and hydrogel residues in the second week. It was conspicuous because of the adhesion by All groups administered with heat-sensitive hydrogels (C2.5T1M0, C2.5T1M0.2) were converted from liquid to solid opaque gel within 30±3.45 seconds, and C2.5T1M0.2 hydrogel (mitomycin C-containing hydrogel) The anti-adhesion effect was confirmed in all animals treated with

Referring to Figure 3B, the highest release of MMC was achieved on day 5, and in the present invention, 2ml of C2.5T1M0.2 hydrogel ^{was treated in a 1x1cm 2} area of the peritoneum, and the result of confirming the release result C2.5T1M0.2 MMC released from the hydrogel was found to be 40% (0.8 μg/ml meaning ^{1.6 μg/cm 2 ) on day 5.} In addition, for patients with gastric and pancreatic cancer, ^{doses of 2 μg/cm 2} or higher may be toxic. The applied dose successfully reduced the initial invasion and proliferation phase of fibroblasts in the wound healing cascade, which usually begins on day 7 of injury, and subsequent controlled release of the drug from the hydrogel could prevent adhesions with normal tissue healing. confirmed that there is.

6A shows the area and density of collagen regenerated at the surgical site of different treatment groups by tricolor staining of tissue sections of H&E and Masson.

The C2.5T1M0 hydrogel-treated group showed sharp collagen fibers (blue stained) and muscle (red stained) between the cecum and abdominal wall surface after 14 days of study period than the control group. These results are because the C2.5T1M0 hydrogel contains cell-compatible cTOCN and MC, which can provide a robust platform for the deposition of fibrin and ECM, which in turn can form a thick network of collagen and muscle cells. can

Collagen deposition was also obtained in commercial gel-treated tissue areas, which confirmed adhesion formation in groups of animals after 2 weeks of experimentation (Fig. 5A).

In addition, collagen deposition and smooth muscle in the cecum and skeletal muscle of mice treated with the optimized hydrogel (C2.5T1M0.2) were confirmed, and adhesion was confirmed with complete decomposition inside the abdominal cavity on the 14th day.

Therefore, the C2.5T1M0.2 hydrogel can not only prevent adhesions after surgical intervention, but also maintain the in vitro degradation pattern during in vivo application.

The hydrogel of the present invention can prepare an anti-adhesion hydrogel by combining MMC (Mitomycin C), MC (methyl cellulose) and cTOCN (modified TEMPO-oxidized cellulose nanofiber). Except for MMC, it is suitable as an anti-adhesion hydrogel don't

TOCN (fiber width 2-50 nm) is attracting attention in biomedical and tissue engineering fields due to its nanostructure composition, large surface area, non-toxicity and biodegradability. It can promote cell proliferation and differentiation, and MC is a water-soluble cellulose derivative that can serve as a binder for drug delivery and a water retention agent.

Cellular compatibility of heat_sensitive methyl cellulose hydrogels was verified by identifying their potential for wound healing in a mouse full-thickness skin burn model.

Therefore, the combination of MC and TOCN may be suitable for the cells in the present invention with results obtained in in vitro cytotoxicity (C1.5T1M0, C2.5T1M0, C3.5T1M0 hydrogel, Figure 1A) and in vivo animal model (C2.5T1M0). can, and the reason for using mitomycin C in hydrogels is its anti-tumor, anti-mitotic effect.

MMC is a DNA alkylating agent capable of cross-linking with the guanine nucleotides of the DNA helix, disrupting the DNA helix and inhibiting the non-specific fibroblast cell cycle by functioning. Therefore, the C2.5T1M0.2 hydrogel according to the present invention has an anti-adhesion effect by inhibiting the proliferation of fibroblasts.

12-3: Inflammation, Fibrosis and Vascularization SCORE

6A is a Hematoxylin-eosin stained slide of peritoneal tissue treated with negative control, C2.5T1M0, C2.5T1M0.2 and positive control, showing scattered macrophages, neutrophils and blood vessels penetrating the abdominal muscular system. it has been shown

Fibrous bands and adhesions are formed by connective tissue composed of an abundant extracellular fibrous matrix containing fibroblast-like cells and capillaries.

As can be seen in Figure 6A and Table 3S, the tissue treated with C2.5T1M0.2 hydrogel did not show vascularization.

12-4. Real-time RT-qPCR

In the case of the commercial hydrogel-treated group, unexpected adhesion occurred during the experiment, so it was excluded from further in vivo characterization. During peritoneal injury, epithelial cells can rapidly convert to myofibroblasts via MMT cascades. The myofibroblasts can continuously synthesize extracellular matrix components such as fibronectin, collagen I and angiogenic factors.

The mesothelial to mesenchymal transition (MMT) is an automatically controlled physiological process in the wound healing phase. However, during abnormal and excessive wound healing, this regulatory mechanism can be altered to create tissue adhesion by replacing normal tissue with bands of fibrous tissue between surrounding organs.

TNF-α is a multifunctional pro-inflammatory cytokine that can mediate inflammation, apoptosis, immune response and organ development. It is produced mainly by macrophages (classical macrophages (M1)) but can also be released from fibroblast endothelial and epithelial cells. Low concentrations of TNF-α can act as growth inducers in fibroblasts by stimulating the proliferation of collagen and glycosaminoglycan, and vice versa. High TNF-α can trigger opposing signaling pathways involved in angiogenesis, leading to cell death or cell survival and proliferation.

Referring to FIG. 6B, it can be seen that the fibronectin gene expression was significantly reduced in the MMC group compared to the negative control group (p < 0.001) and the untreated hydrogel group (p < 0.001), see FIGS. 6B and 7A . Then, it can be confirmed that the high apoptosis induction of the TNF-α gene due to MMC application (p <0.001) is down-regulated by fibronectin after 14 days.

12-5. In vivo acute toxicity assay and re-epithelialization

MMC is an anti-tumor antibiotic that can cause tissue damage and interfere with normal wound healing, and MMC is mainly eliminated by metabolism in the liver, but metabolism can occur especially in the lungs and kidneys as well as other tissues.

The lack of cytotoxicity of the C2.5T1M0.2 hydrogel was analyzed after a 14-day study period to identify the metabolic organs of treated mice and compare them with those of normal mice.

Referring to Figure 7B, it was confirmed that there was no noticeable abnormality in the H & E staining specimen of the metabolic organs of the hydrogel-treated mice, and the ventral surface morphology was analyzed by SEM to find the healing pattern after C2.5T1M0.2 hydrogel administration. did.

SEM images showed gradual healing of the abdominal wall, which eventually heals due to remesothelialization occurring on the scratched surface.

In Figure 7C, it can be seen that the abdominal wall healed by C2.5T1M0.2 treatment after 14 days of the experiment is similar to that of normal mice (Figure 7D), and a heat-sensitive hydrogel ( The stability of the optimized C2.5T1M0.2) was confirmed.

Claims (7)

Modified tempo-oxidation-mediated nanocellulose (Modified tempo oxidized nanocellulose), methyl cellulose (Methyl cellulose) and mitomycin C (Mitomycin C) by mixing to prepare a mixture;
stirring the mixture and
Including the step of gelling by adding sodium chloride to the stirred mixture,
Method for producing a temperature-sensitive cellulose-based hydrogel for anti-adhesion with mitomycin C release control function. According to claim 1,
Before the step of preparing the mixture,
The modified tempo-oxidation-mediated-characterized in that it further comprises the step of preparing a nanocellulose,
Method for producing a temperature-sensitive cellulose-based hydrogel for anti-adhesion with mitomycin C release control function. 3. The method of claim 2,
The modified tempo-oxidation-mediated-production of nanocellulose comprises:
The process of forming a matrix by pouring the tempo-oxidation-mediated-nanocellulose suspension into a glass Petri and storing it in an incubator at 37°C for 6 hours,
A solution of N-(3-dimethylaminopropyl)-N-ethyl carbodide hydrochloride/N-hydroxy-succinimide (N-(3Dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride/N-hydroxy-succinimide) was added to the matrix. The process of input and incubation for 6 hours at 4 ℃ temperature,
The process of washing the cultured matrix and
characterized in that it comprises the process of pulverizing the washed matrix,
Method for producing a temperature-sensitive cellulose-based hydrogel for anti-adhesion with mitomycin C release control function. 4. The method of claim 3,
The tempo-oxidized nanocellulose (Tempo-oxidized nanocellulose) is characterized in that obtained from softwood bleached kraft pulp,
Method for producing a temperature-sensitive cellulose-based hydrogel for anti-adhesion with mitomycin C release control function. According to claim 1,
The step of preparing the mixture,
The modified tempo-oxidation-mediated mixing of nanocellulose and methyl cellulose in a weight ratio of 1:1.5 to 3.5,
The mitomycin C is characterized in that it contains 0-0.4 μg/100 μl,
Method for producing a temperature-sensitive cellulose-based hydrogel for anti-adhesion with mitomycin C release control function. According to claim 1,
The stirring step is,
Characterized in that the mixture is stirred for 15 to 25 minutes at a temperature of 80 to 90 ℃,
Method for producing a temperature-sensitive cellulose-based hydrogel for anti-adhesion with mitomycin C release control function. A temperature-sensitive cellulose-based hydrogel for preventing adhesions having a mitomycin C release control function prepared by the method of any one of claims 1 to 6.

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