A positively charged degradable double polysaccharide microsphere for glioma postoperative vasculogenic mimicry channel plugging and a preparation method thereof

Abstract

This invention discloses a positively charged, biodegradable dual-polysaccharide microsphere for occlusion of vascular mimicry channels after glioma surgery and its preparation method, belonging to the field of biomedicine. Based on a novel targeted therapy strategy rooted in the understanding of "whole-channel network" pathology, this invention provides dual-polysaccharide microspheres prepared from sodium alginate and chitosan through emulsification, cross-linking, washing, and sieving, and provides their application in interventional treatment for preventing recurrence after glioma surgery. Experimental results show that the dual-polysaccharide microspheres have a positively charged surface, possessing advantages such as strong adsorption and appropriate degradation cycle. They can block vascular mimicry channels through a dual mechanism of physical embedding and charge adsorption during the critical period of glioma recurrence, thereby cutting off the tumor's nutrient supply and inhibiting early postoperative recurrence. This invention provides a novel embolization product and treatment strategy for the prevention of postoperative recurrence of gliomas that is directly effective, cost-effective, and easy to operate.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a positively charged degradable dipolysaccharide microsphere for vascular mimicry channel occlusion after glioma surgery and its preparation method. Background Technology

[0002] Glioblastoma (GBM) is the most common and most malignant primary brain tumor of the adult central nervous system, accounting for 50.1% of all primary malignant central nervous system tumors. The annual incidence of gliomas is approximately 5 to 8 per 100,000, characterized by high recurrence and mortality rates. Although the standard treatment regimen, primarily surgical resection, radiotherapy, and temozolomide chemotherapy, has been used for many years, the median overall survival remains less than 15 months, the 5-year survival rate has long hovered below 5%, and the recurrence rate is close to 100%. Since the launch of temozolomide in 2005 and the approval of bevacizumab for recurrent patients in 2009, no groundbreaking new therapies have emerged, and the treatment of gliomas remains a serious challenge.

[0003] One of the core reasons why gliomas exhibit extremely high recurrence rates and treatment resistance lies in the presence of various abnormal channel structures in the tumor microenvironment that bypass conventional anti-angiogenic therapy targets. Vasculogenic mimicry (VM) is a non-endothelial cell-dependent microcirculatory channel formed by highly aggressive tumor cells with endothelial-like functions through self-deformation and extracellular matrix remodeling. Plasma components and erythrocytes can be detected passing through these channels. VM channels persist throughout all stages of glioma development and progression, and are closely related to tumor invasiveness, chemotherapy resistance, and poor prognosis.

[0004] In recent years, local interventional therapy has gained increasing attention due to the challenge of glioma recurrence after surgery. Selective intracerebral artery infusion chemotherapy combined with blood-brain barrier opening techniques has demonstrated feasibility and preliminary tumor control effects in several clinical studies. However, existing interventional strategies primarily focus on optimizing drug delivery pathways and have not effectively addressed the fundamental problem of high local tumor recurrence rates—namely, the nutrient supply network established by the abnormal vascular system within the tumor microenvironment. How to effectively physically block this complex vascular network remains a blind spot in current research.

[0005] Embolization microspheres, as a core material for interventional tumor therapy, have had their clinical value fully validated in the treatment of various hypervascular tumors, including liver cancer. Classic embolization materials include super-liquefied iodized oil, gelatin sponge, and polyvinyl alcohol (PVA) particles. Embolization microspheres based on PVA (such as DC Beads) are another example. ® and LC Bead ®Biocompatibles UK Ltd. Embolization microspheres are widely used clinically, offering advantages such as regular shape, controllable particle size, and drug-carrying capacity. However, existing embolization microsphere technology faces three systemic drawbacks. First, most existing products are permanent or semi-permanent materials, and long-term retention within blood vessels can induce chronic inflammatory responses and foreign body granulomas. Although some research has begun exploring biodegradable microspheres, the degradation cycle of existing biodegradable products lacks targeted design for the biological patterns of specific tumor recurrence, generally resulting in either excessively rapid degradation leading to embolization failure or excessively slow degradation causing long-term complications. Second, the surface charge of most existing embolization microspheres is neutral or negative. In the high blood flow shear stress environment of VM channels, passive embedding limits the occlusion effect, easily leading to microsphere displacement and incomplete occlusion. This directly results in early recanalization of the VM channel after surgery, creating a hidden danger for tumor recurrence. Domestic research on the design and registration review of embolization microspheres also indicates that improving the anchoring efficiency of microspheres within target vessels remains a critical technical bottleneck that urgently needs to be overcome in this field. Third, existing embolization materials mostly utilize synthetic polymers (such as PVA and PLGA) or animal-derived proteins (such as gelatin), which have inherent limitations in applications requiring extremely high biocompatibility, such as neurosurgery. Natural polysaccharide materials such as chitosan and sodium alginate have been extensively studied in the biomedical field due to their unique biocompatibility, biodegradability, and low immunogenicity. The abundant amino groups on the chitosan molecular chain endow it with cationic properties under acidic conditions, providing a structural basis for the active design of surface charges. However, existing chitosan / sodium alginate composite microspheres are mostly used as drug delivery carriers or hemostatic materials, and there are no systematic research reports on combining their unique charge-tunable properties with the needs of embolization therapy. Summary of the Invention

[0006] The purpose of this invention is to provide a positively charged degradable dual-polysaccharide microsphere for vascular mimicry channel occlusion after glioma surgery and its preparation method, in order to solve the problems existing in the prior art. This invention provides a dual-polysaccharide microsphere made from sodium alginate and chitosan through emulsification, cross-linking, washing and sieving. It can block vascular mimicry channels through a dual mechanism of physical embedding and charge adsorption during the critical period of glioma recurrence, thereby cutting off the nutrient supply to the tumor and inhibiting early recurrence after surgery.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing disaccharide microspheres, comprising the following steps: Chitosan solution and sodium alginate solution are mixed to obtain a mixed aqueous phase. The mixed aqueous phase is then added dropwise to the oil phase and stirred to emulsify, thus obtaining an emulsion. Genipin was added to the emulsion to carry out a cross-linking reaction, resulting in microspheres. These microspheres were then washed, sieved, and dried to obtain the dipolysaccharide microspheres.

[0008] Furthermore, in the chitosan solution, the chitosan has a molecular weight of 100,000 and a content of 0.2 g / L.

[0009] Furthermore, the sodium alginate solution contains 0.2 g / L of sodium alginate.

[0010] Furthermore, the volume ratio of the chitosan solution to the sodium alginate solution is 1:1.

[0011] Furthermore, the oil phase is liquid paraffin containing Span-80.

[0012] Furthermore, the volume fraction of Span-80 in the oil phase is 2%.

[0013] Furthermore, the sieving process involves collecting microspheres with a particle size of 80-150 μm through a standard sieve.

[0014] The present invention also provides a double polysaccharide microsphere obtained according to the above preparation method.

[0015] The present invention also provides the application of the above-mentioned double polysaccharide microspheres in the preparation of products that inhibit postoperative recurrence of glioma.

[0016] The present invention also provides a product for inhibiting postoperative recurrence of glioma, with the above-mentioned double polysaccharide microspheres as the active ingredient.

[0017] The present invention discloses the following technical effects: This invention, based on a novel targeted therapy strategy rooted in the understanding of "whole-channel network" pathology, provides a dual-polysaccharide microsphere made from sodium alginate and chitosan through emulsification, cross-linking, washing, and sieving. It also demonstrates its application in interventional treatment for preventing recurrence after glioma surgery. Experimental results show that the positively charged surface of these dual-polysaccharide microspheres exhibits advantages such as strong adsorption and appropriate degradation cycle. During the critical period of glioma recurrence, they can block vascular mimicry channels through a dual mechanism of physical embedding and charge adsorption, thereby cutting off the tumor's nutrient supply and inhibiting early postoperative recurrence. This invention provides a novel embolization product and treatment strategy for preventing postoperative recurrence of gliomas that is directly effective, cost-effective, and easy to operate.

[0018] The present invention has the following advantages: 1. A revolutionary treatment logic: from "drug-based treatment" to "physical intervention" Existing technologies rely on chemotherapy drugs such as temozolomide or anti-angiogenic drugs such as bevacizumab, but their effectiveness is limited and drug resistance is easily developed due to the blood-brain barrier and tumor heterogeneity. This invention, based on systematic pathological findings of the tumor's "channel network," innovatively employs a physical blocking strategy to directly block the tumor's nutrient input (VM channel). This "dimensional reduction attack" approach fundamentally changes the treatment paradigm for preventing recurrence after glioma surgery.

[0019] 2. Positive charge active anchoring solves the microsphere displacement problem. Traditional embolic microspheres have negatively or neutral surfaces, making them easily washed away under the high blood flow shear forces in VM channels, leading to incomplete occlusion. This invention utilizes the natural amino groups of chitosan to impart a positive potential of +5 to +15 mV to the microsphere surface, enabling active adsorption to the negatively charged inner wall of the VM channel via electrostatic attraction, achieving a dual locking mechanism of mechanical embedding and charge anchoring. This design significantly improves the targeted retention capability of the microspheres, solving the long-standing technical challenges of microsphere displacement and incomplete occlusion in the industry.

[0020] 3. The "7-14 day intelligent degradation window" precisely matches the recurrence window. Existing permanent embolization materials (such as PVA) are prone to causing chronic inflammation and foreign body granulomas; while rapidly degradable materials such as gelatin sponge (degradation time <3 days) cannot cover the critical period of tumor recurrence. This invention precisely controls the degradation cycle of microspheres within 7-14 days through genipin crosslinking. This cycle closely coincides with the acute recurrence window of angiogenesis after glioma surgery: physical occlusion is completed during the period when the tumor most needs blood supply, and the microspheres automatically disappear after the task is completed, avoiding the safety risks caused by long-term foreign body residue. This timely and precisely time-matched design represents the development direction of next-generation smart embolization materials.

[0021] 4. All-natural disaccharide system: the optimal solution for biocompatibility Unlike synthetic polymers (PVA, PLGA) or animal-derived proteins (gelatin), this invention utilizes two natural polysaccharides: chitosan and sodium alginate. Both exhibit excellent biocompatibility, biodegradability, and low immunogenicity. In particular, the amino structure of chitosan provides a natural basis for the design of a positive surface charge, avoiding the additional toxicity risks associated with chemical modifications. This material system is especially suitable for clinical applications such as neurosurgery, where local biocompatibility is paramount.

[0022] 5. Addressing the pain point of high recurrence rate after glioma surgery in clinical practice. The recurrence rate of glioma surgery is close to 100%, and currently there is a lack of effective local recurrence prevention methods both domestically and internationally. The product of this invention can be implanted into the surgical cavity, physically blocking the tumor's nutrient channels like "smart glue." Compared to expensive targeted drugs with significant side effects and repeated radiotherapy and chemotherapy, the treatment method based on this invention is direct in effect, cost-effective, and easy to operate, and is expected to become an essential adjunct device in standard postoperative treatment for gliomas. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Image showing in vivo IVIS imaging results of nude mice bearing an orthotopic brain tumor. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1: Preparation of positively charged disaccharide microspheres The formulation of the positively charged disaccharide microspheres of the present invention is shown in Table 1.

[0031] Table 1 Formulation of positively charged dual-polysaccharide microspheres Preparation steps: Chitosan solution preparation: Weigh 2g of chitosan, add 100mL of 1% acetic acid solution, and stir magnetically for 2 hours until completely dissolved; Preparation of sodium alginate solution: Weigh 2g of sodium alginate, add 100mL of deionized water, and stir magnetically for 1 hour until completely dissolved; Mixed aqueous phase: Mix chitosan solution and sodium alginate solution at a volume ratio of 1:1 and stir until homogeneous; Emulsification: The mixed aqueous phase was slowly dripped into liquid paraffin containing 2% Span-80 while stirring at 600 rpm to form a W / O emulsion; Cross-linking: Add genipin (0.1%, dissolved in PBS buffer) and cross-link at 45°C for 3 hours to form microspheres; Washing: The microspheres were washed sequentially with petroleum ether, isopropanol, and deionized water to remove residual oil phase; Sieving: Collect microspheres with a particle size of 80-150 μm through a standard sieve; Drying: Vacuum dry for 24 hours, then store in a sealed container.

[0032] The particle size, zeta potential, degradation cycle, and morphology of the prepared positively charged disaccharide microspheres were tested, and the results are shown in Table 2. It can be seen that all the indicators of the positively charged disaccharide microspheres obtained according to the formulation and preparation method of this embodiment meet the target values.

[0033] Table 2. Detection results of various indicators of positively charged double polysaccharide microspheres Example 2 Comparison of different chitosan molecular weights The chitosan molecular weight in Example 1 was replaced with 50,000, 100,000, 200,000 and 300,000 respectively, and the zeta potential, degradation period and mechanical strength of the prepared positively charged disaccharide microspheres were tested.

[0034] Table 3. Detection results of various indicators for positively charged disaccharide microspheres with different chitosan molecular weights. The results are shown in Table 3. Conclusion: Chitosan with a molecular weight of 100,000-200,000 can meet the requirements of 80-150 μm particle size and 7-14 days of degradation.

[0035] Example 3: Validation of therapeutic effect based on in vivo imaging 1. Construction of animal models Cell line: LN229-Luc (expressing luciferase); Animals: BALB / c Nude nude mice, 4-6 weeks old, male; Modeling: In situ brain tumor-bearing model. Mice were first anesthetized and fixed on a stereotaxic apparatus. After disinfection, the scalp was incised to expose the skull. The injection coordinates (1 mm posterior to the anterior fontanelle, 2 mm lateral to the midline, and 3-4 mm subdurally) were determined using a stereotaxic map. A bone window was then drilled using a cranial drill. Subsequently, a suspension containing tumor cells (5 μL, approximately 1 × 10⁻⁶ cells) was injected. 5 (Number of cells) were slowly injected into the brain using a microinjector. The needle was left in place for 5 minutes to allow for full cell colonization before being slowly withdrawn. The bone hole was sealed with bone wax, and the scalp was sutured. The mice were kept warm post-operatively until they recovered and were then fed as usual. Tumor formation was observed and recorded regularly. The experimental groups are shown in Table 4.

[0036] Table 4 Experimental Group Information 2. Imaging monitoring IVIS in vivo imaging was performed on postoperative days 7, 14, and 21.

[0037] Table 5 IVIS live imaging results The experimental results are shown in Table 5 and Figure 1 As shown, the conclusion is that glioma growth was significantly inhibited after implantation of positively charged microspheres, confirming that the microspheres effectively blocked the VM channels and cut off the tumor's nutrient supply. The positively charged design enhanced the retention capacity of the microspheres at the tumor site.

[0038] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing disaccharide microspheres, characterized in that, Includes the following steps: Chitosan solution and sodium alginate solution are mixed to obtain a mixed aqueous phase. The mixed aqueous phase is then added dropwise to the oil phase and stirred to emulsify, thus obtaining an emulsion. Genipin was added to the emulsion to carry out a cross-linking reaction, resulting in microspheres. These microspheres were then washed, sieved, and dried to obtain the dipolysaccharide microspheres.

2. The preparation method according to claim 1, characterized in that, The chitosan solution contains chitosan with a molecular weight of 100,000 and a content of 0.2 g / L.

3. The preparation method according to claim 1, characterized in that, The sodium alginate solution contains 0.2 g / L of sodium alginate.

4. The preparation method according to claim 1, characterized in that, The volume ratio of the chitosan solution to the sodium alginate solution is 1:

1.

5. The preparation method according to claim 1, characterized in that, The oil phase is liquid paraffin containing Span-80.

6. The preparation method according to claim 5, characterized in that, The volume fraction of Span-80 in the oil phase is 2%.

7. The preparation method according to claim 1, characterized in that, The sieving process involves collecting microspheres with a particle size of 80-150 μm through a standard sieve.

8. A type of double polysaccharide microsphere obtained by the preparation method according to any one of claims 1-7.

9. The use of the double polysaccharide microspheres of claim 8 in the preparation of a product for inhibiting postoperative recurrence of glioma.

10. A product for inhibiting postoperative recurrence of glioma, characterized in that, The active ingredient is the double polysaccharide microspheres as described in claim 8.

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