Porous carbon-based dual-drug nanoscale sustained-release material and uterine support and application thereof

Abstract

This invention discloses a porous carbon-based dual-drug-loaded nanomaterial for sustained release. The material uses a thermosensitive polymer as a matrix and cellulose nanocrystals modified with cationic surfactants as a reinforcing phase, loading both hydrophilic and hydrophobic drugs. This material can be used to prepare therapeutic drugs for gynecological diseases and infertility, as well as drug delivery devices. Furthermore, this invention provides a uterine scaffold prepared using this material, which can be fabricated using 3D printing technology. Utilizing the PNIPAm hydrogel network and its temperature-responsive properties, hydrophilic drugs can be loaded at low temperatures. The high mechanical strength of CNC machining allows for the loading of hydrophobic drugs while improving the mechanical properties of the hydrogel matrix, thus solving the problems of easy detachment and bleeding in existing technologies.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a porous carbon-based dual-drug-carrying nanomaterial for sustained release and a uterine stent made from the material. Background Technology

[0002] Endometriosis is a common gynecological disease in women, caused by the growth of endometrial tissue (glands or stroma) outside the uterine cavity and uterine wall. It is also one of the causes of female infertility. The main pathological changes in endometriosis are periodic bleeding of ectopic endometrial tissue and fibrosis of surrounding tissues, forming ectopic nodules. Dysmenorrhea, chronic pelvic pain, menstrual abnormalities, and infertility are its main symptoms. The lesions can affect all pelvic tissues and organs, most commonly the ovaries, rectouterine pouch, and uterosacral ligaments, but can also occur in the abdominal cavity, thoracic cavity, and limbs.

[0003] Adenomyosis is a condition in which endometrial glands and stroma invade the myometrium and maintain cyclical proliferation, shedding, and bleeding, resulting in diffuse or localized lesions. Like endometriosis, it is a common gynecological disease, most often occurring in multiparous women aged 30 to 50. Patients mainly present with increased menstrual flow, prolonged menstrual periods, and progressively worsening dysmenorrhea.

[0004] The two common gynecological diseases mentioned above can be treated with medication or surgery. Medication treatment usually involves suppressing the cyclical endocrine stimulation of the ovaries. Pseudorabies therapy uses progestin-based drugs to cause menstruation to cease, creating a pregnancy-like response that lasts for at least six months. This causes the ectopic endometrium to stop moving and eventually atrophy, thus producing a therapeutic effect.

[0005] The optimal drug treatment option is direct, continuous intrauterine administration, which allows the medication to reach the lesion site directly and reduces systemic side effects. Intrauterine devices (IUDs) are a reliable method for achieving this, but current technology has drawbacks. The lack of hemostatic agents in the medication mix leads to prolonged bleeding after IUD insertion, and oral medication is less effective. Furthermore, for some patients with significantly enlarged uterine cavities and loose cervixes after childbirth, the lack of individualized IUD design often results in IUD expulsion, rendering the treatment ineffective. Commonly used hormonal medications are hydrophobic, while hemostatic agents are hydrophilic, making it difficult to simultaneously load both hydrophobic and hydrophilic drugs.

[0006] Therefore, finding a suitable intrauterine ring material for simultaneously loading two drugs, as well as its preparation and loading methods, is a relentless pursuit for those skilled in the art. Summary of the Invention

[0007] To address the aforementioned problems in the prior art, this invention first provides a porous carbon-based dual-drug-loaded nanomaterial for sustained release. This material uses a thermosensitive polymer as the matrix and cellulose nanocrystals (CNC) modified with cationic surfactants as the reinforcing phase, and is loaded with both hydrophilic and hydrophobic drugs.

[0008] Preferably, the temperature-sensitive polymer is poly(N-isopropylacrylamide) (PNIPAm).

[0009] Preferably, the cationic surfactant is hexadecyltrimethylammonium bromide (CTAB).

[0010] Preferably, the hydrophilic drug is hemocoagulase, and more preferably viper hemocoagulase.

[0011] Preferably, the hydrophobic drug is levonorgestrel.

[0012] Furthermore, the preparation method of the porous carbon-based dual-drug-loaded nanomaterial includes the following steps:

[0013] 1) Cellulose nanocrystals and cationic surfactants were mixed in water, heated and stirred for a period of time to allow them to fully combine, then centrifuged to collect the precipitate, and a hydrophobic drug solution was added to mix it thoroughly to obtain cationic modified cellulose nanocrystals loaded with hydrophobic drugs.

[0014] 2) Mix the cellulose nanocrystals obtained in step 1) with N-isopropylacrylamide (NIPAm), add water as a solvent, and add a crosslinking agent, initiator and accelerator to crosslink NIPAm and bind the cellulose nanocrystals into the PNIPAm crosslinked structure;

[0015] 3) Add hydrophilic drugs during the process of binding cellulose nanocrystals with PNIPAm, or load hydrophilic drugs into the cellulose nanocrystals and PNIPAm after binding is completed by gel swelling absorption.

[0016] Preferably, the heating temperature in step 1) is 100-200°C, and the stirring time is at least 6 hours, preferably 8-12 hours.

[0017] Preferably, the solvent of the hydrophobic drug solution in step 1) is an organic solvent miscible with water. When the hydrophobic drug is levonorgestrel, the solvent is preferably a formaldehyde solution.

[0018] Preferably, the cationic modified cellulose nanocrystals loaded with hydrophobic drugs prepared in step 1) have a specific surface area ≥ 0.01 m². 2 / g, with a pore size of 0.5–100 nm.

[0019] Preferably, the crosslinking agent in step 2) is N,N'-methylenebisacrylamide (BIS).

[0020] Preferably, the initiator in step 2) is ammonium persulfate (APS).

[0021] Preferably, the accelerator in step 2) is tetramethylethylenediamine (TEMED).

[0022] Furthermore, when the hydrophilic drug is viper hemocoagulase and the hydrophobic drug is levonorgestrel, the mass ratio is preferably 6:1 to 24:1, such as 6:1, 12:1, or 24:1.

[0023] Secondly, the present invention provides the application of the above-mentioned porous carbon-based dual-drug-carrying nano-sustained-release material in the preparation of drugs for the treatment of gynecological diseases and infertility, and drug-carrying tools, wherein the gynecological diseases include, but are not limited to, endometriosis and adenomyosis.

[0024] Thirdly, the present invention provides a uterine stent comprising the porous carbon-based dual-drug-carrying nanomaterial described in the present invention.

[0025] Preferably, the uterine stent is manufactured using layer-by-layer 3D printing technology with the porous carbon-based dual-drug-loaded nanomaterial, based on the size and volume of each patient's uterine cavity as analyzed by computer.

[0026] Preferably, the hydrophilic drug hemocoagulase loading in the uterine stent is 0.5–2 mg, optionally 0.5 mg, 1 mg, 2 mg, etc., and the hydrophobic drug levonorgestrel loading in the uterine stent is less than 0.1 mg.

[0027] Preferably, the uterine support includes a main shaft and two side arms, the length of which and the angle between the side arms and the main shaft are designed according to individual differences.

[0028] This invention utilizes the high mechanical strength, tunable surface chemistry, and excellent biocompatibility of CNC machining. After modification with CTAB, it is combined with levonorgestrel, enabling the loading of hydrophobic drugs while simultaneously improving the mechanical properties of the hydrogel matrix. Furthermore, by constructing a PNIPAm hydrogel network and leveraging its temperature-responsive characteristics, hydrophilic drugs can be loaded at low temperatures. This invention provides a novel method for preparing high-strength hydrogel materials that simultaneously carry both hydrophilic and hydrophobic drugs. This porous carbon-based dual-drug-loaded nanomaterial can be used to prepare drugs for gynecological diseases and infertility treatments, as well as drug-loaded devices. It can also be used to fabricate drug-loaded intrauterine stents, and through 3D printing technology, personalized stent structures can be created for patients, overcoming the shortcomings of traditional drug-loaded devices such as easy dislodgement and bleeding.

[0029] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0030] Figure 1 SEM images of hydrogels with different CNCs contents: (a, b) 6% CNCs@PNIPAm; (c, d) 10% CNCs@PNIPAm; (e, f) 20% CNCs@PNIPAm;

[0031] Figure 2 This is a schematic diagram of the uterine support structure of the present invention. Detailed Implementation

[0032] Example 1: Preparation of porous carbon-based dual-drug-carrying nanomaterials for sustained release

[0033] For CNCs, 100g of commercial cotton moistened with sodium hydroxide was first mixed with 200mL of concentrated sulfuric acid and water and heated to 100-200℃ for one day. The heated mixture was then washed, centrifuged with water, and dialyzed for 3 days to obtain a CNCs solution. The obtained CNCs solution was diluted 2-5 times with water and slowly added to 2000mL of 2% hexadecyltrimethylammonium bromide (CTAB) solution (formulation: CTAB 40g, NaCl 163.64g, 1M / L Tris-HCl 200mL, 0.5M / L EDTA 80mL; bring the volume to 2000mL). The solution was heated and stirred overnight, then centrifuged at 8000r / min for 30 minutes. The precipitate was collected, and 0.75μg of levonorgestrel (a hydrophobic drug dissolved in 35% formaldehyde solution) was added.

[0034] The obtained cationic modified CNCs were mixed with isopropylacrylamide (NIPAM) at a certain mass ratio (in this example, the CNCs accounted for 6%, 10%, and 20% of PNIPAm by weight, respectively). 4 mL of water was added as a solvent, followed by 3 mL of BIS, and the mixture was gently mixed. Then, 50 μL of 10% APS was added and the mixture was gently mixed again. Finally, 5 μL of tetramethylethylenediamine (TEMED) was added as a crosslinking agent to bind the CNCs into the crosslinked PNIPAm material, serving as a reinforcing phase for PNIPAm and enabling the loading of hydrophobic drugs. During the binding process between PNIPAm and CNCs, the hydrophilic drug viper hemocoagulase was added in proportion to achieve the co-loading of both hydrophilic and hydrophobic drugs. In this example, the mass ratio of viper hemocoagulase to levonorgestrel could be selected as 6:1, 12:1, or 24:1, or the hydrophilic drug could be loaded into the PNIPAm matrix through gel swelling absorption.

[0035] By controlling the different concentrations of CNCs used in matrix preparation, PNIPAm hydrogels with different moduli were obtained, and the modulus range was controlled to a suitable value. Figure 1 The image shows SEM images of hydrogels with different CNC contents at different magnifications.

[0036] Example 2: Preparation of Uterine Scaffold

[0037] The ultrasound or MRI images are input into a computer to analyze the morphology and dimensions of the patient's uterine cavity. Layer-by-layer 3D printing technology is then used to print porous carbon-based dual-drug-loaded nanomaterials into a form similar to... Figure 2 The uterine stent structure shown is a main shaft 1 and two side arms 2 and 3. The main shaft 1 is used to push and ultimately fix it in the middle of the uterus, while the side arms 2 and 3 are ultimately fixed at the entrance of the fallopian tubes. The main shaft has a diameter of 3-3.5 mm and a length of 18-20 mm. The lengths of the side arms 2 and 3, and the angle between the side arms and the main shaft, can be designed according to individual differences to prevent uterine enlargement after stent implantation and to avoid the problem of stent detachment. The length of the side arms is generally 15-16 mm. The bottom of the main shaft has a collar 4 for connecting the pushing device. The total axial length of the uterine stent along the main shaft is 30-35 mm. The uterine stent loaded with viper hemocoagulase and levonorgestrel solves the problem of easy bleeding in patients.

[0038] Example 3 In vitro experiment

[0039] The porous carbon-based dual-drug-carrying nanomaterial of this invention was used for direct / indirect co-culture with endometrial stromal cells (ESCs) and uterine NK cells (uNK) in vitro. The surface receptor phenotype of uNK cells, as well as genes and proteins related to embryo implantation, were detected to explore whether the material altered the uNK phenotype and function. The results showed that the adhesion of uNK cells to stromal cells remained unchanged after the addition of the new material, indicating that the material did not alter the uNK phenotype and function.

[0040] In addition, we collected NK cells (dNK) isolated from the decidua after induced abortion by flow cytometry and detected adhesion molecules after co-culturing with HTR8. We found that the expression of adhesion molecules CD54, CD62E and CD106 on the surface of dNK cells remained unchanged after the addition of new materials.

[0041] Example 4 In vivo experiment

[0042] Further in vivo experiments were conducted to construct a mouse model of endometriosis by intraperitoneal injection of allogeneic endometrial tissue. An intrauterine stent made of porous carbon-based dual-drug-carrying nanomaterial was used to treat endometriosis. The size of ectopic lesions and their adhesion to surrounding organs were compared among the experimental groups. It was found that the ectopic lesions in mice treated with the new material were significantly reduced.

[0043] This study investigated the regulatory effect and mechanism of a novel material on autophagy in the mouse endometrium. Subsequently, it analyzed how regulated mouse endometrial autophagy mediates CD56+ NK cell differentiation through downstream molecules and signaling. The expression levels of NK cell surface molecules NKp30 and NKp44, intracellular cytokines IFN-γ, Perforin, and Granzyme B, and the expression levels of surface tolerance molecules KIR2DL1 and KIR3DL1 were measured. Further analysis elucidated the role of trained CD56+ NK cells in embryo implantation. Preliminary experiments indicated that the novel material did not affect endometrial receptivity in mice.

[0044] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A uterine stent, characterized in that, This material is made of porous carbon-based dual-drug-loaded nanomaterials. The material uses the thermosensitive polymer poly(N-isopropylacrylamide) as the matrix and cellulose nanocrystals modified with the cationic surfactant cetyltrimethylammonium bromide as the reinforcing phase. It is loaded with the hydrophilic drug viper hemocoagulase and the hydrophobic drug levonorgestrel in a mass ratio of 6:1 to 24:

1. The cationically modified cellulose nanocrystals loaded with the hydrophobic drugs have a specific surface area ≥0.01 m². 2 / g, with a pore size of 0.5-100nm; the uterine support includes a main shaft and two side arms, the main shaft is used to push and finally fix it in the middle of the uterus, and the side arms are finally fixed at the entrance of the fallopian tube.

2. The uterine stent as described in claim 1, characterized in that, The spindle has a diameter of 3-3.5mm and a length of 18-20mm. The length of the side arm and the angle between the side arm and the spindle are designed according to individual differences.

3. A method for preparing a uterine stent as described in claim 1 or 2, characterized in that, Including the following steps: 1) Mix cellulose nanocrystals with the cationic surfactant cetyltrimethylammonium bromide in water, heat to 100-200°C and stir for at least 6 hours to ensure full combination, then centrifuge to collect the precipitate, add levonorgestrel formaldehyde solution and mix thoroughly to obtain cationic modified cellulose nanocrystals loaded with levonorgestrel. 2) Mix the cellulose nanocrystals obtained in step 1) with NIPAm, add water as a solvent, and add a crosslinking agent, initiator and accelerator to crosslink NIPAm and bind the cellulose nanocrystals into the PNIPAm crosslinked structure; 3) Viper hemocoagulase was added during the process of combining cellulose nanocrystals with PNIPAm to prepare the porous carbon-based dual-drug-loaded nano-sustained-release material. 4) Based on the size and volume of the uterine cavity of each patient as analyzed by computer, the uterine stent is fabricated using the porous carbon-based dual-drug-loaded nano-sustained-release material through layer-by-layer 3D printing technology.

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