A hydroxybutyl chitosan and graphene oxide-based rectal gel and a preparation method and application thereof

Abstract

This invention relates to the field of rectal drug delivery system preparation, specifically to a method for preparing a novel rectal gel based on hydroxybutyl chitosan and graphene oxide, and its application. First, HBC is prepared via homogeneous synthesis. Then, GO is prepared using a modified Hummers method, and the anticancer model drug PYM is loaded onto its surface to obtain GO@PYM. Finally, GO@PYM is crosslinked with HBC to obtain HBC / GO@PYM. This invention improves upon the shortcomings of in-situ thermosensitive gels by introducing the nanomaterial GO, increasing drug loading and mechanical strength, enhancing drug release and bioavailability, and exhibiting a significant sustained-release effect. This invention provides a new method to solve the problems of insufficient mechanical strength, low drug loading, and low bioavailability of in-situ thermosensitive gels for rectal drug delivery.

Description

Technical Field

[0001] This invention relates to the field of rectal drug delivery system preparation, specifically to a rectal gel based on hydroxybutyl chitosan and graphene oxide, its preparation method, and its application. Background Technology

[0002] Rectal drug delivery systems are drug delivery systems that allow drugs to be absorbed through the rectal mucosa into the systemic circulation. They are characterized by high safety, rapid drug absorption, and high bioavailability, and are particularly suitable for elderly patients, children, and other patients who cannot tolerate oral administration. After rectal administration, approximately 50-75% of the drug is directly absorbed through the rectal mucosa into the systemic circulation, reducing the first-pass effect in the liver. This allows for both local and systemic treatment, such as for ulcerative colitis, chronic prostatitis, chronic pelvic inflammatory disease, and tumors. Currently, rectal drug delivery systems are mainly divided into enemas, suppositories, and rectal gels. Enemas have poor patient compliance and pose a risk of leakage during administration; suppositories suffer from limited drug distribution, resulting in insufficient efficacy for most; traditional rectal gels are difficult to administer due to their high viscosity. In-situ thermosensitive gels, as a novel rectal drug delivery system, are attracting increasing attention.

[0003] In-situ thermosensitive gels are temperature-sensitive gel formulations that exist as a sol at room temperature but transform into a semi-solid gel when the temperature rises to their gelation point. They possess advantages such as good biocompatibility, strong permeability, and reduced drug toxicity, and can be widely used in various drug delivery routes, including injection, oral administration, rectal administration, and mucosal administration. Novel rectal gels based on this type of gel reduce the difficulty of administration at room temperature. When the temperature rises to the rectal temperature, exceeding the gelation point, they transform into a semi-solid gel, which facilitates fixation at the rectal lesion site and enables targeted drug delivery. Hydroxybutyl chitosan (HBC) is an excellent matrix for in-situ thermosensitive hydrogels, but its low drug loading capacity, low mechanical strength, and poor stability limit its efficacy for rectal administration.

[0004] Graphene oxide (GO) is a high-performance biomedical nanomaterial with characteristics such as large specific surface area, high mechanical strength, and good biocompatibility. It has seen continuous research progress in fields such as biosensors, drug delivery, tissue engineering, and antibacterial materials. Multiple domestic and international publications have shown that introducing graphene oxide into in-situ thermosensitive hydrogels significantly improves both its mechanical properties and drug loading capacity, thanks to its large specific surface area and high mechanical strength. Furthermore, the introduction of graphene oxide into in-situ thermosensitive hydrogels is expected to further improve its bioavailability, alter drug dissolution characteristics, and enhance antibacterial activity, demonstrating strong research and development potential. Based on this, this invention provides a method for preparing a novel rectal gel based on hydroxybutyl chitosan and graphene oxide, and its application. Summary of the Invention

[0005] The purpose of this invention is to provide a novel rectal gel based on hydroxybutyl chitosan and graphene oxide. Introducing graphene oxide into hydroxybutyl chitosan in-situ thermosensitive hydrogels can not only effectively improve the problems of low mechanical properties and low drug loading in hydroxybutyl chitosan in-situ thermosensitive hydrogels, but also improve drug bioavailability and produce a sustained-release effect, thus obtaining a novel rectal gel.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a rectal gel based on hydroxybutyl chitosan and graphene oxide, wherein HBC is prepared by homogeneous synthesis, GO is prepared by modified Hummers method, and an anticancer model drug X is loaded on the surface of GO to obtain GO@X, and finally GO@X is crosslinked with HBC to obtain HBC / GO@X.

[0007] The above-mentioned method for preparing a rectal gel based on hydroxybutyl chitosan and graphene oxide includes the following steps:

[0008] 1) In a container, KOH, urea, distilled water and CS are added in sequence, mixed evenly and refrigerated for a certain period of time and then restored to room temperature. 1,2-epoxybutane is added and the reaction is carried out under nitrogen protection. Then the pH is adjusted, dialyzed and freeze-dried to obtain HBC.

[0009] 2) In a container, add concentrated sulfuric acid, graphite powder, sodium nitrate and potassium permanganate in sequence. After reacting at a certain temperature for a period of time, stop the reaction and let it stand. Then add hydrogen peroxide solution until the reaction solution turns yellow. Centrifuge, wash and freeze dry the product to obtain GO.

[0010] 3) A certain amount of GO dispersion was prepared by ultrasonic exfoliation of the GO obtained in step 2), and drug X aqueous solution was added. The reaction was carried out in the dark. After the reaction was completed, the product was centrifuged and the lower precipitate was freeze-dried to obtain GO@X.

[0011] 4) Add an appropriate amount of HBC and Bletilla striata polysaccharide obtained in step 1) to the GO@X obtained in step 3), and stop the reaction after a period of time to obtain HBC / GO@X.

[0012] Preferably, in the above preparation method, in step 1), the refrigeration time is 2 hours and the reaction time is 48 hours.

[0013] Preferably, in the above preparation method, in step 2), the reaction time at a certain temperature is sequentially carried out at 5°C for 1 hour, at 35°C for 30 minutes, at 65°C for 30 minutes, and at 85°C for 30 minutes.

[0014] Preferably, in the preparation method described above, in step 3), the mass ratio of GO to drug X is 5:1-3.

[0015] Preferably, in the above preparation method, in step 3), the light-shielding reaction time is 10-120 min and the reaction temperature is 20-120℃.

[0016] Preferably, in the above preparation method, in step 3), drug X is an anti-tumor drug.

[0017] Preferably, in step 3) of the above preparation method, drug X is bleomycin.

[0018] Preferably, in step 4) of the above preparation method, the mass ratio of GO@X:HBC:Bletilla striata polysaccharide is 1:18-22:0.08-0.16. This relates to the application of the above-mentioned hydroxybutyl chitosan and graphene oxide-based rectal gel in rectal drug delivery carriers.

[0019] The present invention has the following beneficial effects:

[0020] 1. This invention introduces GO, which has high mechanical strength and large specific surface area, into hydroxybutyl chitosan in-situ thermosensitive hydrogel, effectively improving the problems of low mechanical properties and low drug loading of hydroxybutyl chitosan in-situ thermosensitive hydrogel.

[0021] 2. This invention introduces GO into hydroxybutyl chitosan in situ thermosensitive hydrogel. The structural modification increases the amount of drug released, improves its bioavailability, and produces a sustained-release effect.

[0022] 3. This invention uses bleomycin as a model drug. In addition, this rectal drug delivery carrier can also load other types of antitumor drugs, providing a carrier option for rectal administration. Attached Figure Description

[0023] Figure 1 These are the infrared spectra of chitosan and hydroxybutyl chitosan.

[0024] Figure 2This is the infrared spectrum of graphene oxide.

[0025] Figure 3(a) is a scanning electron microscope image of HBC.

[0026] Figure 3(b) is a scanning electron microscope image of HBC / GO.

[0027] Figure 4(a) is a mechanical loss diagram showing the changes in the storage modulus G' and loss modulus G” of HBC with temperature.

[0028] Figure 4(b) is a mechanical loss diagram showing the changes in the energy storage modulus G' and loss modulus G” of HBC / GO with temperature.

[0029] Figure 5 This is a comparison chart of the energy storage modulus G' of HBC and HBC / GO.

[0030] Figure 6 The release curves are those of HBC@PYM and HBC / GO@PYM when they were respectively loaded into the rectal mucosa of rats.

[0031] Figure 7 This is a blood concentration-time curve of HBC@PYM and HBC / GO@PYM. Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the embodiments are only for illustrating the present invention and not for limiting the scope of protection of the present invention.

[0033] Example 1

[0034] Preparation of hydroxybutyl chitosan and graphene oxide rectal gel

[0035] (I) Preparation of hydroxybutyl chitosan

[0036] Weigh 16g KOH, 8g urea, 73mL distilled water, and 3g purified lyophilized CS into a 100ml beaker, mix well, and refrigerate for 2 hours. Take 20mL of the thawed chitosan solution at room temperature, add 10mL of 1,2-epoxybutane, and react under nitrogen protection in a magnetic stirrer at 25℃ for 48 hours. Then add an appropriate amount of dilute hydrochloric acid to adjust the pH to neutral. Finally, dialyze for three days using a prepared dialysis bag (8000-14000), and lyophilize to obtain HBC.

[0037] (II) Preparation of graphene oxide

[0038] 84.4 mL of concentrated sulfuric acid was measured into a three-necked flask, and 2.5 g of graphite powder and 2 g of sodium nitrate were added. 11.25 g of potassium permanganate was added in batches. After reacting at 5 °C for 1 h, the reaction was transferred to an oil bath. The reaction was then carried out sequentially at 35 °C for 30 min, 65 °C for 30 min, and 85 °C for 30 min. The reaction was then stopped, the flask was sealed, and the mixture was allowed to stand for one week. Afterward, hot water was added to evenly disperse the reactants, and 5 mL of 30% hydrogen peroxide solution was added until the reaction solution turned yellow. The mixture was centrifuged while hot (12000 r / min, 5 min), and repeatedly washed until the lower precipitate turned brownish-black and the supernatant became colorless. The precipitate was freeze-dried for 48 h to obtain GO powder.

[0039] (III) Preparation of bleomycin-loaded graphene oxide (GO@PYM)

[0040] 10 mg of powdered GO was weighed and dissolved in 10 mL of distilled water to prepare a GO dispersion using an ultrasonic exfoliation method. 4 mg of PYM was weighed, dissolved in an appropriate amount of water, and slowly added to the above GO aqueous solution at a reaction temperature of 20 °C. The reaction was carried out under light for 60 min. After the reaction was completed, the mixture was centrifuged in a 10 mL centrifuge tube (10000 r / min, 10 min, 4 °C). The lower precipitate was freeze-dried to obtain GO@PYM.

[0041] (iv) Preparation of hydroxybutyl chitosan and graphene oxide (HBC / GO) gel

[0042] 10 mg of powdered GO was weighed and a uniformly dispersed GO dispersion was prepared by ultrasonic exfoliation. 200 mg of lyophilized HBC was added to the GO dispersion, and the mixture was stirred at low speed in a low-temperature cooling reaction bath at 4 °C for 6 h. After reaction, the mixture was placed in a refrigerator at 4 °C to obtain HBC / GO.

[0043] (V) Preparation of bleomycin-loaded hydroxybutyl chitosan (HBC@PYM) gel

[0044] 4 mg of PYM was loaded into a hydrogel formed by 200 mg of HBC, and 1.2 mg of Bletilla striata polysaccharide was added to obtain HBC@PYM, which was then stored in a refrigerator at 4°C for later use.

[0045] (VI) Preparation of a novel rectal gel containing hydroxybutyl chitosan and bleomycin-loaded graphene oxide (HBC / GO@PYM)

[0046] Take an appropriate amount of the product from Example 1 (III) into a beaker, add 200 mg HBC and 1.2 mg Bletilla striata polysaccharide, mix well and react for 4 h to obtain HBC / GO@PYM, and store in a 4℃ refrigerator for later use.

[0047] (vii) Test Results

[0048] Figure 1 These are the infrared spectra of chitosan (CS) and hydroxybutyl chitosan. In both the CS and HBC infrared spectra, a region located at 3442 cm⁻¹ can be observed. -1 The absorption peak at 2873 cm⁻¹ indicates that HBC retains the -CH and -NH stretching vibrations on the CS framework; the HBC infrared spectrum is located at 2873 cm⁻¹. -1 2925cm -1 2965cm -1 The new absorption peak at 1521 cm⁻¹ is due to the introduction of a new -CH₃ group; -1 A new absorption peak also appeared at 1158 cm⁻¹, which is due to the asymmetric extension and vibration of the -CH group, indicating that a new hydroxybutyl group was successfully introduced into the CS backbone, representing the successful synthesis of HBC. CS is located at 1158 cm⁻¹. -1 The absorption peak at 1000-1400 cm⁻¹ disappears in HBC and is located in the range of 1000-1400 cm⁻¹. -1 The peak intensities between the peaks decreased or disappeared to varying degrees. These absorption peaks are characteristic peaks at the C6-OH position, indicating that the etherification reaction in the preparation of HBC mainly occurs at the C6-OH of CS.

[0049] Figure 2 This is the infrared spectrum of graphene oxide, located at 3432 cm⁻¹. -1 The absorption peak at 1728 cm⁻¹ is the -OH group. -1 The absorption peak is at C=O, 1629 cm⁻¹. -1 The absorption peak at 1408 cm⁻¹ is C=C, indicating that a large number of oxygen-containing functional groups have been successfully introduced into graphite; -1 The absorption peak at 1225 cm⁻¹ is due to the stretching vibration of C-OH. -1 With 1056cm -1 The absorption peaks at these locations all belong to CO groups. The appearance of these oxygen-containing functional group absorption peaks indicates the successful synthesis of GO.

[0050] Figure 3(a) is a scanning electron microscope image of HBC, and Figure 3(b) is a scanning electron microscope image of HBC / GO. In Figure 3(a), it can be clearly seen that the interior of the HBC cross section has a uniform three-dimensional porous structure with a pore size of about 40-60 μm. The shape of the pores is close to that of cylinders, and the inner walls of the pores are relatively smooth. In Figure 3(b), it can be seen that the internal structure of HBC / GO also has a continuous and uniform 3D porous structure, which is similar to the internal structure of HBC. However, the pore size is significantly reduced to about 5 μm. This indicates that after the addition of GO, the smaller and denser pores increased the mechanical strength of the hydrogel and enhanced its mechanical strength.

[0051] Figure 4(a) shows the mechanical loss diagram of the storage modulus G' and loss modulus G” of HBC as a function of temperature, and Figure 4(b) shows the mechanical loss diagram of the storage modulus G' and loss modulus G” of HBC / GO as a function of temperature. From Figure 4(a), the gel point temperature of HBC is 36.5℃, while from Figure 4(b), the gel point temperature of HBC / GO is 31.7℃. This indicates that the introduction of GO lowers the gel point temperature, possibly because GO and HBC are cross-linked by a large number of hydrogen bonds, allowing the hydrogel to gel at a lower temperature. However, this gel point temperature is still within the temperature range of 30-37℃ for thermosensitive rectal gels, and it is easier to gel in vivo, indicating that the prepared HBC / GO can be used for rectal administration.

[0052] Figure 5 This is a comparison graph of the storage modulus G' of HBC and HBC / GO. It can be observed that within the temperature range of 20–40℃, the storage modulus G' of HBC is within 0–100 Pa, while that of HBC / GO is within 90–2000 Pa. Particularly at 40℃, the storage modulus G' of HBC is 24 Pa, while that of HBC / GO is 1966 Pa. Whether in the sol or gel state, the storage modulus G' is significantly improved after the introduction of GO, indicating that GO substantially enhances the mechanical properties of HBC.

[0053] Example 2

[0054] In vitro membrane release assays of HBC / GO@PYM and HBC@PYM

[0055] To evaluate the drug release effect of the HBC / GO@PYM system, an in vitro drug release experiment simulating the transrectal mucosa of rats was conducted, with the HBC@PYM group serving as a control.

[0056] Rats weighing 200g were sacrificed after being deprived of water and food for 48 hours. The rectal segment (8cm above the anus) was harvested, rinsed with physiological saline, and then HBC@PYM and HBC / GO@PYM were placed inside the rectal segment. The ends of the rectal segment were tied tightly with string and placed in a centrifuge tube containing 25mL of PBS buffer (pH=7.4). The centrifuge tube was placed in a constant temperature shaker at 37℃ and 100r / min. At 5min, 10min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, and 24h, 1mL of sample was transferred to a sample tube. After each sampling, 1mL of the same temperature PBS buffer was added to the centrifuge tube. The samples were then filtered through a 0.22μm microporous membrane, and the PYM components were analyzed using high-performance liquid chromatography (HPLC).

[0057] Figure 6Release curves of HBC@PYM and HBC / GO@PYM after being incorporated into the rectal mucosa of rats are shown. HBC@PYM exhibits a rapid drug release rate in the first hour, with a cumulative release of approximately 60%; release essentially ceases after 2 hours, ultimately reaching a cumulative release of 73.23% at 24 hours. HBC / GO@PYM, on the other hand, shows a relatively rapid release in the first 0.5 hours, with a cumulative release of approximately 20%, followed by a significant slowdown in the release rate, essentially ceasing release at 8 hours, and reaching a cumulative release of 79.34% at 24 hours. This indicates that the introduction of GO enhances the drug release from the HBC hydrogel, significantly delays the drug release time, and provides a better sustained-release effect.

[0058] Example 3

[0059] Pharmacokinetic studies of HBC / GO@PYM and HBC@PYM

[0060] HBC / GO@PYM was selected as the experimental group, and HBC@PYM was used as the control group to investigate the pharmacokinetic properties of PYM in rats. Compared with Example 2, Example 3 directly conducted drug release experiments in rats, and obtained more accurate and comprehensive drug release data (Table 1) using pharmacokinetic data processing software.

[0061] Ten healthy male SD rats weighing 200±25g were randomly divided into an HBC@PYM control group and an HBC / GO@PYM experimental group, with five rats in each group. First, the rats were given an enema with glycerin to empty their feces. Then, the rats were anesthetized with ether, and the HBC@PYM and HBC / GO@PYM were administered rectally to the comatose rats 2cm above the anus. After administration, the rats were placed upside down with their anus facing upwards for 3 minutes. Blood samples were then collected from the orbital sinus at 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h. Finally, the drug content was analyzed by high-performance liquid chromatography (HPLC).

[0062] Figure 7 The plasma concentration-time curves for HBC@PYM and HBC / GO@PYM show that at almost all time points, the plasma drug concentration in the HBC / GO@PYM group was higher than that in the HBC@PYM group. This indicates that the HBC hydrogel drug carrier with added GO has better drug dissolution and absorption, resulting in better bioavailability. Around 8 hours, the drug in the plasma of the HBC@PYM group was almost completely eliminated, while a small amount of drug remained in the HBC / GO@PYM group. This suggests that the HBC hydrogel drug carrier with added GO prolongs the drug's residence time in the body, producing a sustained-release effect and improving therapeutic efficacy.

[0063] Table 1. Basic pharmacokinetic parameters in rats after rectal administration of HBC@PYM and HBC / GO@PYM (comparison between HBC / GO@PYM group and HBC@PYM group, *P<0.05, **P<0.01)

[0064]

[0065] In Table 1, the HBC@PYM group reached its peak in about half an hour. 1 / 2 The time to peak was 2.21 hours, while the HBC / GO@PYM group with added GO reached its peak at approximately 2 hours. 1 / 2 The half-life of the HBC / GO@PYM group was 2.21 h, which was 1.44 times that of the HBC@PYM group, and the mean residence time (MRT) was 2.21 h. 0-∞ The plasma clearance rate (CL) of the HBC / GO@PYM group was 1.51 times that of the HBC@PYM group, indicating that the addition of GO prolonged the duration of drug action and further improved the drug's efficacy. The area under the curve (AUC) of the HBC / GO@PYM group was also higher than that of the HBC@PYM group. 0-∞ The bioavailability of the drug was 2.31 times that of the HBC@PYM group, indicating that the addition of GO significantly improved the bioavailability of the drug.

Claims

1. A rectal gel based on hydroxybutyl chitosan and graphene oxide, characterized in that, HBC was prepared by homogeneous synthesis, GO was prepared by modified Hummers method, and anticancer model drug X was loaded onto the surface of GO to obtain GO@X. Finally, GO@X was crosslinked with HBC to obtain HBC / GO@X. The preparation method of the rectal gel based on hydroxybutyl chitosan and graphene oxide includes the following steps: 1) In a container, KOH, urea, distilled water and CS are added in sequence, mixed evenly and refrigerated for a certain period of time and then restored to room temperature. 1,2-epoxybutane is added and the reaction is carried out under nitrogen protection. Then the pH is adjusted, dialyzed and freeze-dried to obtain HBC. 2) In a container, add concentrated sulfuric acid, graphite powder, sodium nitrate and potassium permanganate in sequence. After reacting at a certain temperature for a period of time, stop the reaction and let it stand. Then add hydrogen peroxide solution until the reaction solution turns yellow. Centrifuge, wash and freeze dry the product to obtain GO. 3) A certain amount of GO dispersion was prepared by ultrasonic exfoliation of the GO obtained in step 2), and drug X aqueous solution was added. The reaction was carried out in the dark. After the reaction was completed, the product was centrifuged and the lower precipitate was freeze-dried to obtain GO@X. 4) Add an appropriate amount of HBC and Bletilla striata polysaccharide obtained in step 1) to the GO@X obtained in step 3), and stop the reaction after a period of time to obtain HBC / GO@X; Drug X is bleomycin; By mass ratio, GO@X:HBC:Bletilla striata polysaccharide = 1:18-22:0.08-0.

16.

2. The rectal gel based on hydroxybutyl chitosan and graphene oxide according to claim 1, characterized in that: In step 1), the refrigeration time is 2 hours and the reaction time is 48 hours.

3. The rectal gel based on hydroxybutyl chitosan and graphene oxide according to claim 1, characterized in that: In step 2), the reaction time at a certain temperature is sequentially 1 hour at 5°C, 30 minutes at 35°C, 30 minutes at 65°C, and 30 minutes at 85°C.

4. The rectal gel based on hydroxybutyl chitosan and graphene oxide according to claim 1, characterized in that: In step 3), the mass ratio of GO to drug X is 5:1-3.

5. The rectal gel based on hydroxybutyl chitosan and graphene oxide according to claim 1, characterized in that: In step 3), the light-shielding reaction time is 10~120 min and the light-shielding reaction temperature is 20~120℃.

6. The application of the rectal gel based on hydroxybutyl chitosan and graphene oxide as described in claim 1 in the preparation of a rectal drug carrier.

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