A biologically inert kappa-carrageenan, its preparation method and use

Abstract

The application discloses a kind of biological inert kappa-carrageenan and its preparation method and application, the molecular weight range of the kappa-carrageenan is 22.5kDa-35.2kDa and the ratio of GPC peak height and half-width is greater than 2000.The kappa-carrageenan prepared in the application is more easily dissolved, and is convenient as a basic auxiliary material for preparing various biomedical materials.Compared with undegraded kappa-carrageenan, the kappa-carrageenan with specific molecular weight and H / W 1 / 2 range in the application has reduced inflammation risk, and safety has been improved better.Compared with kappa-carrageenan with lower molecular weight and / or H / W 1 / 2 Less than 2000, the kappa-carrageenan in the application does not show the improvement of antioxidant and anticoagulant activity, and shows biological inertness, which can effectively avoid the production of unexpected biological activity of kappa-carrageenan as biomedical materials, reduce toxic side effects, and is expected to solve the problems of carrageenan in the field of biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a biologically inert κ-carrageenan, its preparation method, and its application. Background Technology

[0002] Carrageenan is a hydrophilic colloid extracted and refined from red algae (such as Euphorbia milii and Carrageenan). It is primarily composed of calcium, potassium, sodium, and ammonium salts of sulfated polysaccharides consisting of galactose and dehydrated galactose. Based on the different positions and numbers of sulfated groups on its galactose residues, it can be classified into several types, including κ-carrageenan, ι-carrageenan, and λ-carrageenan. κ-carrageenan has wide applications in the biomedical field, including: 1. Microsphere carriers: Carrageenan drug-loaded microspheres can adsorb various positively charged drugs through their negatively charged structure, and targeted drug delivery can be achieved through surface modification, reducing the toxic side effects of drugs on normal tissues; 2. Gel carriers: Carrageenan can form gels for sustained release of experimental drugs, improving drug efficacy; 3. Tissue engineering scaffolds: Carrageenan can be prepared into three-dimensional porous scaffolds through freeze-drying, electrospinning, etc., providing a suitable microenvironment for cell adhesion, proliferation, and differentiation; 4. Wound dressings: Carrageenan has antibacterial, absorbent, and moisturizing properties, absorbing wound exudate and inhibiting bacterial growth on the wound surface. Meanwhile, carrageenan can also promote the migration and proliferation of fibroblasts and accelerate the wound healing process; 5. Biosensors: Carrageenan has also been studied in the field of biosensing, such as as an immobilization carrier for bioactive substances such as enzymes and antibodies and as a construction of sensing interfaces.

[0003] While κ-carrageenan shows great potential in biomedicine, it also faces some limitations and challenges. First, its high molecular weight and poor solubility can affect its feasibility as an excipient in formulations such as microspheres, gels, scaffolds, and wound dressings. For example, reports indicate that when high molecular weight carrageenan is compounded with gelatin / hyaluronic acid and prepared into microspheres via emulsification cross-linking, it is difficult to obtain uniform and spherical microspheres. Second, high molecular weight carrageenan carries potential inflammatory risks, primarily involving the activation of immune cells to release inflammatory factors, activation of the complement system, induction of intestinal inflammation, promotion of cell adhesion molecule expression, and formation of immune complex deposition. Specific degradation treatments can reduce the molecular weight of κ-carrageenan, facilitating its preparation as a drug carrier. Furthermore, reports suggest that low molecular weight carrageenan can inhibit the release of inflammatory factors (tumor necrosis factor-α, interleukin-1β, and interleukin-6) from inflammatory cells such as macrophages and neutrophils, thereby alleviating inflammatory responses. It can also exert anti-inflammatory effects by modulating intracellular inflammatory signaling pathways, such as the nuclear factor-κB signaling pathway.

[0004] However, the decrease in the molecular weight of κ-carrageenan may be accompanied by an increase in other biological activities, such as antioxidant activity (scavenging free radicals, regulating antioxidant enzyme systems) and anticoagulant activity. These increased biological activities often cause inconvenience in the treatment of some diseases, such as cancer. However, the enhanced antioxidant capacity of low molecular weight κ-carrageenan may strengthen the antioxidant defense mechanisms of tumor cells, helping them resist oxidative damage caused by treatment while clearing oxidative stress products surrounding tumor cells. For example, studies have shown that excessive antioxidants may interfere with the effectiveness of radiotherapy and chemotherapy. Furthermore, the increased anticoagulant activity may pose uncontrollable risks to coagulation-related diseases.

[0005] Therefore, it is desirable to develop a method for producing a bioinert κ-carrageenan with low inflammatory risk and no significant improvement in antioxidant and anticoagulant capabilities, which can serve as a basic excipient for various biomedical materials. Summary of the Invention

[0006] Objective of the invention: To address the current problems of κ-carrageenan in the biomedical field, this invention provides a specific molecular weight range (22.5kDa-35.2kDa) and H / W ratio... 1 / 2 The κ-carrageenan with a molecular weight greater than 2000 exhibits low inflammatory risk and no significant improvement in antioxidant and anticoagulant activities. The κ-carrageenan prepared in this invention is readily soluble, making it convenient as a basic excipient in the preparation of various biomedical materials. This κ-carrageenan has a reduced inflammatory risk and does not show any improvement in antioxidant or anticoagulant activities, exhibiting bioinertness. This effectively solves the problem that existing low molecular weight κ-carrageenan, while exhibiting reduced anti-inflammatory properties, significantly enhances antioxidant and anticoagulant activities.

[0007] The present invention also provides a method for preparing κ-carrageenan and its applications.

[0008] Technical solution: To achieve the above objective, the present invention provides a bio-inert κ-carrageenan, wherein the κ-carrageenan has a molecular weight range of 22.5 kDa-35.2 kDa and an H / W ratio of [missing information]. 1 / 2 Greater than 2000.

[0009] The method for preparing the bio-inert κ-carrageenan of the present invention includes the following steps:

[0010] (1) Add ultrapure water to κ-carrageenan powder and heat to dissolve;

[0011] (2) Add an acidic solution and heat to react; precisely control the molecular weight range and H / W of the degraded κ-carrageenan by controlling the concentration of the acidic solution, the reaction temperature, and the reaction time. 1 / 2 ;

[0012] (3) The molecular weight and H / W of κ-carrageenan after degradation1 / 2 Once the set range is reached, immediately add an alkaline solution to adjust the pH of the κ-carrageenan solution to terminate the degradation reaction; cool to room temperature.

[0013] (4) After filtering and cooling the κ-carrageenan solution, concentrate it, collect the concentrate, add ultrapure water, continue filtering, concentrate it, collect the concentrate, and repeat this step 3-5 times; reduce the volume to 250mL by rotary evaporation, and freeze dry to obtain bio-inert κ-carrageenan.

[0014] In step (1), the volume of ultrapure water is 1.5-4L; the concentration of the κ-carrageenan solution is 8.89-17.78g / L; and the acidic solution is one or more of hydrochloric acid, sulfuric acid, nitric acid, and oxalic acid.

[0015] In step (2), the volume of the acidic solution is 100-200 mL and the concentration is 0.6-1.2 M; the volume ratio of the carrageenan solution to the acidic solution is 15-20:1; the reaction temperature is 55-63℃; and the reaction time is 0.3-0.7 h.

[0016] Preferably, the acidic solution is hydrochloric acid.

[0017] In step (3), the alkaline solution is one or more of sodium hydroxide solution, potassium hydroxide solution, and calcium hydroxide solution; the concentration is 0.6-1.2M.

[0018] In step (3), the pH of the alkaline solution is adjusted to 6.5-7.0.

[0019] Preferably, the alkaline solution is a sodium hydroxide solution.

[0020] In step (4), the filtration is one or more of flat-plate ultrafiltration and ultrafiltration centrifugation; the molecular weight cutoff of the ultrafiltration membrane is 5-10 kDa; and the volume of the added ultrapure water is 1.5-2.5 L.

[0021] Preferably, the filtration method is flat-plate ultrafiltration.

[0022] The bio-inert κ-carrageenan described in this invention is a bio-inert κ-carrageenan with low inflammatory risk and no significant improvement in antioxidant and anticoagulant capabilities.

[0023] The application of the bio-inert κ-carrageenan described in this invention in the preparation of reagents or drugs for treating tumors and coagulation disorders.

[0024] The application of the bio-inert κ-carrageenan, either alone or in combination with other materials, in the preparation of microspheres for the treatment of tumors and coagulation disorders.

[0025] This invention prepares molecules with a specific molecular weight range (22.5 kDa-35.2 kDa) and H / W ratio by degradation, controlling the molecular weight range, and concentration. 1 / 2 A method for preparing bio-inert κ-carrageenan with a molecular weight greater than 2000, exhibiting low inflammatory risk and no significant improvement in antioxidant and anticoagulant capabilities. The κ-carrageenan prepared by this invention has a specific molecular weight range of 22.5 kDa-35.2 kDa. κ-carrageenan within this specific molecular weight range is readily soluble, making it convenient as a basic excipient in the preparation of various biomedical materials. Compared to undegraded κ-carrageenan (greater than 200 kDa), the κ-carrageenan described in this invention has a specific molecular weight range and H / W... 1 / 2 κ-carrageenan with a molecular weight greater than 2000 exhibits a reduced risk of inflammation and a significantly improved safety profile. Furthermore, compared to lower molecular weight and / or H / W... 1 / 2 The κ-carrageenan with a concentration less than 2000, as described in this invention, does not exhibit enhanced antioxidant and anticoagulant activities and is biologically inert. This effectively avoids the unexpected biological activities that κ-carrageenan may produce when used as a biomedical material, reduces toxic side effects, and is expected to solve the current problems of carrageenan in the biomedical field.

[0026] The microspheres prepared from κ-carrageenan within this specific molecular weight range, either alone or in combination with other commonly used materials (such as gelatin, hyaluronic acid, etc.), exhibit uniform particle size and good sphericity. In contrast, microspheres prepared from undegraded κ-carrageenan alone or in combination with other commonly used materials (such as gelatin, hyaluronic acid, etc.) show non-uniform particle size and poor sphericity. Under the same concentration conditions, the local drug delivery gel prepared from undegraded κ-carrageenan exhibits extremely high viscosity, resulting in poor spreadability and coating properties. The local drug delivery gel prepared from this κ-carrageenan, however, demonstrates excellent spreadability, better meeting drug delivery requirements. Furthermore, the peak height to full width at half maximum (H / W) ratio in the GPC spectrum is significantly higher. 1 / 2 This reflects the molecular weight distribution of κ-carrageenan after degradation. H / W 1 / 2 When the molecular weight is lower, the proportion of low molecular weight κ-carrageenan will be higher, and its antioxidant and anticoagulant activities will also be observed. Therefore, in this invention, the specific molecular weight and H / W ratio of κ-carrageenan are... 1 / 2Simultaneous control is crucial. Specifically, the κ-carrageenan of this invention within the molecular weight range (22.5kDa-35.2kDa) exhibits no significant inflammatory response or other unexpected biological activities. However, the peak height to half-width ratio reflects the molecular weight distribution. If only the molecular weight is controlled while ignoring the peak height to half-width ratio, unexpected biological activities may occur. For example, if the peak height to half-width ratio of κ-carrageenan is small within the molecular weight range (22.5kDa-35.2kDa), then the distribution of smaller molecular weights will be more pronounced, and antioxidant and anticoagulant activities will also be observed. Therefore, this invention simultaneously controls the molecular weight within the 22.5kDa-35.2kDa range and also controls the peak height to half-width ratio within a large range to ensure that the κ-carrageenan prepared by this invention has a reduced inflammatory risk, without any increase in antioxidant and anticoagulant activities, and is biologically inert. When the molecular weight of the prepared κ-carrageenan exceeds or falls below the molecular weight range of this invention, or the H / W ratio is... 1 / 2 While the inflammatory risk of κ-carrageenan decreases at molecular weights greater than 2000, its antioxidant and anticoagulant activities are significantly enhanced, and it lacks biological inertness. Furthermore, materials with κ-carrageenan molecular weights exceeding the molecular weight range of this invention have a higher inflammatory risk, and higher molecular weights also result in higher viscosity, making them less suitable for later formulation into medical materials.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] 1. The bio-inert κ-carrageenan prepared by this invention has a specific molecular weight range of 22.5kDa-35.2kDa and an H / W ratio of 1 / 2 κ-carrageenan with a molecular weight greater than 2000 is relatively easy to dissolve, making it convenient as a basic excipient in the preparation of various biomedical materials.

[0029] 2. Compared with undegraded κ-carrageenan (greater than 200 kDa), the bio-inert κ-carrageenan prepared by this invention has a reduced inflammatory risk and a significantly improved safety profile within the specific molecular weight range described in this invention.

[0030] 3. The bio-inert κ-carrageenan prepared by this invention simultaneously achieves a good H / W ratio. 1 / 2 Compared to lower molecular weight and / or H / W 1 / 2 The κ-carrageenan of this invention, with a concentration less than 2000, does not exhibit an increasing trend in antioxidant and anticoagulant activity, demonstrating biological inertness. This characteristic is significant because when κ-carrageenan is applied to the treatment of tumors and coagulation disorders, it can effectively avoid potential risks caused by abnormal antioxidant or anticoagulant activity, providing a safer option for the treatment of these diseases. Attached Figure Description

[0031] Figure 1 The molecular weight and H / W of κ-carrageenan prepared in Example 1 were 27.9 kDa. 1 / 2 Measurement.

[0032] Figure 2 Molecular weight and H / W of κ-carrageenan obtained under different HCl concentrations, reaction temperatures, and reaction times. 1 / 2 .

[0033] Figure 3 Example 1 yielded a 27.9 kDa and H / W 1 / 2 The image shows the SEM pattern of 2051.69κ-carrageenan.

[0034] Figure 4 Cytotoxicity studies of undegraded κ-carrageenan and the 27.9 kDa κ-carrageenan prepared in Example 1 were conducted. Compared with the control group, **p≤0.01, ***p≤0.001, ****p≤0.0001 (n=6).

[0035] Figure 5 For the inflammatory study of undegraded κ-carrageenan, the 27.9 kDa κ-carrageenan prepared in Example 1 and the 43.8 kDa κ-carrageenan prepared in Comparative Example 7, compared with the blank group, ***p≤0.001, ****p≤0.0001 (n=5).

[0036] Figure 6 For mouse paw edema experiments using undegraded κ-carrageenan, the 27.9 kDa κ-carrageenan prepared in Example 1, and the 43.8 kDa κ-carrageenan prepared in Comparative Example 7, (A) 1 h (B) 2 h (C) 4 h (D) 6 h, compared with the blank group, ***p≤0.001, ****p≤0.0001; compared with 27.9 kDa κ-carrageenan, # p≤0.05, ## p≤0.01, #### p≤0.0001 (n=6).

[0037] Figure 7 For the white blood cell count in the mouse paw edema test, compared with the control group, **p≤0.01, ****p≤0.0001; compared with 27.9kDaκ-carrageenan, #p≤0.05, ####p≤0.0001 (n=6).

[0038] Figure 8 Spheroidization study of undegraded κ-carrageenan and the 27.9 kDa κ-carrageenan prepared in Example 1.

[0039] Figure 9The DPPH radical scavenging experiments of the 27.9 kDa κ-carrageenan prepared in Example 1, the 21.8 kDa κ-carrageenan prepared in Comparative Example 1, the 16.7 kDa κ-carrageenan prepared in Comparative Example 2, and the 11.3 kDa κ-carrageenan prepared in Comparative Example 3 showed that, compared with the blank group, ****p≤0.0001; compared with the 27.9 kDa κ-carrageenan prepared in Example 1, #p≤0.05, ###p≤0.001, ####p≤0.0001 (n=3).

[0040] Figure 10 The H / W prepared in Example 1 1 / 2 The H / W ratio prepared from 2051.69κ-carrageenan and Comparative Example 4 is... 1 / 2 The H / W ratio prepared from 1943.77κ-carrageenan and Comparative Example 5 was... 1 / 2 The H / W ratio prepared for 1789.96κ-carrageenan and Comparative Example 6 1 / 2 For the DPPH radical scavenging experiment of 1621.12κ-carrageenan, compared with the blank group, ***p≤0.001, ****p≤0.0001; compared with H / W 1 / 2 Compared to 2051.69κ-carrageenan, # p≤0.05, ## p≤0.01, ### p≤0.001 (n=3). Detailed Implementation

[0041] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0043] The undegraded κ-carrageenan powder in this embodiment of the invention, with a molecular weight of 200-1500 kDa (CAS: 11114-20-8, item number 23092110), was purchased from Luxin (Fujian) Food Co., Ltd.

[0044] Example 1

[0045] 1. Degradation

[0046] (1) Place 40g of undegraded κ-carrageenan powder in a round-bottom flask, add ultrapure water to 3L, and heat at 60℃ until completely dissolved;

[0047] (2) Add 150 mL of 1 M HCl solution and heat in a water bath at 60 °C for 34 min;

[0048] 2. Control of molecular weight range

[0049] When the molecular weight and H / W of κ-carrageenan after degradation 1 / 2 Once the set range is reached, immediately add 1M NaOH to adjust the pH of the solution to 6.5 and terminate the reaction; cool to room temperature.

[0050] 3. Concentrated

[0051] (1) Ultrafiltration of all the solution from the previous step was performed using a flat plate ultrafiltration system (10kDa ultrafiltration membrane) at a speed of 30 rpm. The concentrate was collected and 2000 mL of purified water was added. Ultrafiltration was continued, and the concentrate was collected. This step was repeated 3 times, with each ultrafiltration time being 8 h.

[0052] (2) The volume was reduced to 250 mL by rotary evaporation and then freeze-dried to obtain the processed κ-carrageenan, and the molecular weight was determined.

[0053] Methods for determining molecular weight: (1) Sample preparation: Prepare a solution (0.5 mg / mL) of the lyophilized sample, take 250 μL and dilute it to 1 mL with 0.1 M NaNO3 solution, and filter it through a 0.22 μm filter membrane; (2) Chromatographic conditions: Agilent 1260 high performance liquid chromatograph (HPLC); chromatographic column: TSKgel GMPWXL (7.8 mm × 30 cm, 13 μm); mobile phase: 0.1 M NaNO3; flow rate: 0.3 mL / min; column temperature: 35 ℃; injection volume: 30 μL; detector is a refractive index detector (RID).

[0054] The experimental results are as follows: The molecular weight of κ-carrageenan prepared in Example 1 is as follows: Figure 1 As shown, the molecular weight is 27.9 kDa. Measured by a GPC instrument, the half-width at half-maximum (FWHM) of the GPC peak is approximately 3.24 mV, and the peak height is 6647.47 mV. Therefore, the ratio of the GPC peak height to FWHM is 2051.69.

[0055] Example 2

[0056] κ-carrageenan molecular weight range and H / W 1 / 2 control

[0057] The molecular weight range and H / W ratio of κ-carrageenan can be precisely controlled by adjusting the HCl concentration, reaction temperature, and reaction time. 1 / 2 Single-factor experiments were conducted to investigate the effects of HCl concentration, reaction temperature, and reaction time on the molecular weight and H / W ratio of κ-carrageenan. 1 / 2 The impact.

[0058] The method is as follows: 1. The reaction time was controlled at 1 h and the reaction temperature at 50 °C. The molecular weight of κ-carrageenan prepared with HCl concentrations of 0.4, 0.6, 0.8, 1.0, and 1.2 M was investigated; 2. The HCl concentration was controlled at 0.8 M and the reaction temperature at 50 °C. The molecular weight of κ-carrageenan prepared with reaction times of 0.25, 0.5, 1, 2, and 3 h was investigated; 3. The HCl concentration was controlled at 0.8 M and the reaction time at 1 h. The molecular weight of κ-carrageenan prepared with reaction temperatures of 40, 50, 60, 70, and 80 °C was investigated. Except for the change in the factors investigated in step (2) of degradation, the other steps were the same as in Example 1.

[0059] The experimental results are as follows: The molecular weight fitting results of κ-carrageenan obtained by changing the HCl concentration, reaction temperature and reaction time in Example 1 are as follows: Figure 2 As shown in the figure, the HCl concentration, reaction temperature, and reaction time all affect the final molecular weight and H / W ratio of carrageenan. 1 / 2 Yes, it has an impact. To obtain the desired molecular weight range and H / W... 1 / 2 (Right now Figure 2 The bar chart shows the ratio of GPC peak height to full width at half maximum (FWHM) for a certain molecular weight of carrageenan. For κ-carrageenan, the acidic solution, such as hydrochloric acid, should be controlled at a concentration of 0.6-1.2 M, a reaction temperature of 55-63 °C, and a reaction time of 0.3-0.7 h. The optimal control conditions are those described in Example 1.

[0060] Example 3

[0061] SEM study of κ-carrageenan prepared in Example 1

[0062] To investigate the 27.9 kDa and H / W prepared in Example 1 1 / 2 The microstructure of κ-carrageenan with a density of 2051.69 was observed using SEM in a dry state. The experimental results are as follows: the κ-carrageenan prepared in Example 1 has a density of 27.9 kDa and an H / W... 1 / 2 The microstructure of κ-carrageenan with a density of 2051.69 is as follows: Figure 3 As shown.

[0063] Example 4

[0064] The 27.9 kDa and H / W prepared in Example 1 1 / 2 Cytotoxicity study of 2051.69κ-carrageenan

[0065] The MTT assay was used to evaluate undegraded κ-carrageenan and the 27.9 kDa H / W prepared in Example 1. 1 / 2The cytotoxicity of κ-carrageenan at 2051.69 was determined. The method was as follows: HaCat cells were cultured to the logarithmic growth phase. 100 μL of medium containing 3000 cells was added to each well of a 96-well plate and incubated for 12 h. The existing medium was then replaced with medium containing a series of concentrations of 27.9 kDa κ-carrageenan and undegraded κ-carrageenan (undegraded κ-carrageenan in Example 1), resulting in final sample concentrations of 62.5, 125, 250, 500, and 1000 μg / mL in each well, with a final volume of 200 μL per well. Incubation was continued for another 12 h. A cell-only control group was used as a blank control. 50 μL of MTT solution was added to each well under dark conditions and incubated for 4 h. The supernatant was removed, and the cells were gently washed three times with PBS buffer. The reaction was then terminated by adding 150 μL of LDMSO solution to each well. Finally, the culture plate was shaken horizontally for 10 minutes, and its optical density was measured at 490 nm using an ELISA reader. Cell viability was then calculated.

[0066] The experimental results are as follows: cytotoxicity study results are as follows Figure 4 As shown. The 27.9 kDa and H / W values ​​at each concentration (62.5, 125, 250, 500, 1000 μg / mL) are... 1 / 2 κ-carrageenan at concentrations of 2051.69 showed no significant cytotoxicity to HaCat cells, with cell viability exceeding 93%. In contrast, undegraded κ-carrageenan at concentrations of 125, 250, 500, and 1000 μg / mL exhibited significant cytotoxicity, resulting in a marked decrease in HaCat cell viability.

[0067] Example 5

[0068] The 27.9 kDa and H / W prepared in Example 1 1 / 2 Inflammatory study of κ-carrageenan at 2051.69

[0069] 1. Inflammatory Risk Study: The inflammatory risk of undegraded κ-carrageenan, the 27.9 kDa κ-carrageenan prepared in Example 1, and the 43.8 kDa κ-carrageenan prepared in Comparative Example 7 was evaluated by measuring NO content. The method was as follows: RAW264.7 macrophages were used as model cells and cultured in high-glucose DMEM complete medium (containing 10% FBS and 1% antibiotic solution) at 37°C in a 5% CO2 incubator, and passaged using trypsin digestion. Three experimental concentrations were set for each of the undegraded κ-carrageenan in Example 1, the 27.9 kDa κ-carrageenan prepared in Example 1, and the 43.8 kDa κ-carrageenan prepared in Comparative Example 7: 125, 250, and 500 μg / mL. A 100 μg / mL LPS group was used as a positive control, and the cell-only group was used as a blank control. After 12 h of cell culture, the original medium was replaced with the medium containing the series of concentrations of the sample, and the cells were cultured for another 12 h. Subsequent operations were performed using a NO detection kit, where absorbance was measured at 540 nm and the NO content of each sample was calculated according to the standard curve.

[0070] The experimental results are as follows: Inflammatory risk study results are as follows... Figure 5 As shown in the figure, compared with the control group, the NO content in the 27.9 kDa κ-carrageenan treatment groups at each concentration (125, 250, and 500 μg / mL) did not increase significantly, while the NO content in the undegraded κ-carrageenan treatment groups at concentrations of 250 and 500 μg / mL increased significantly and was close to that of the positive control group. Furthermore, the NO content in the 43.8 kDa κ-carrageenan treatment groups at concentrations of 250 and 500 μg / mL also increased significantly. This indicates that the 27.9 kDa κ-carrageenan prepared in Example 1 does not pose a significant inflammatory risk.

[0071] 2. Mouse paw edema test: The inflammatory risk was evaluated by injecting undegraded κ-carrageenan, the 27.9 kDa κ-carrageenan prepared in Example 1, and the 43.8 kDa κ-carrageenan prepared in Comparative Example 7 into the mouse paws and observing the paw edema. The method was as follows: ICR male mice (18-22 g) were randomly divided into three groups of six mice each. They were acclimatized for 7 days under standard conditions. 1% (w / w) concentrations of undegraded κ-carrageenan, the 27.9 kDa κ-carrageenan prepared in Example 1, and the 43.8 kDa κ-carrageenan prepared in Comparative Example 7 were prepared using 0.9% saline and subcutaneously injected into the left paw of each mouse (50 μL / mouse). An equal volume of 0.9% saline was injected as a blank control. The thickness (T) of the left hind paw was measured with calipers at 0 h before injection and at 1, 2, 4, and 6 h after injection. The difference in paw thickness before and after injection was used to assess the edema degree.

[0072] Swelling degree = T - T0

[0073] Swelling degree refers to the degree of swelling at a certain time point after injection, T refers to the thickness of the left hind paw of the mouse at a certain time point after injection, and T0 refers to the thickness of the left hind paw of the mouse before injection.

[0074] Six hours after injection, blood was collected from the orbital region of the mice, and the number of white blood cells in the blood of each group of mice was detected using a blood biochemistry analyzer.

[0075] The experimental results are as follows: The results of the mouse paw swelling test are as follows... Figure 6 and Figure 7 As shown in the figure, compared with the control group, no significant increase in paw thickness and swelling was observed in mice at 1, 2, 4, and 6 hours after injection of 27.9 kDa κ-carrageenan, indicating no statistically significant difference compared with normal mice. This suggests that κ-carrageenan is a bioinert material with good biocompatibility. However, significant increases in paw thickness and swelling were observed at 1, 2, 4, and 6 hours after injection of undegraded κ-carrageenan and 43.8 kDa κ-carrageenan. White blood cell counts showed no significant increase in white blood cells in the 27.9 kDa κ-carrageenan treatment group compared to the control group, while the white blood cell counts were significantly increased in the undegraded κ-carrageenan and 43.8 kDa κ-carrageenan treatment groups. This indicates that undegraded κ-carrageenan and 43.8 kDa κ-carrageenan have significant inflammatory responses and poor safety, posing a significant safety risk to the body when used as pharmaceutical excipients.

[0076] Example 6

[0077] The 27.9 kDa and H / W prepared in Example 1 1 / 2 Study on the microsphere properties of 2051.69κ-carrageenan

[0078] The κ-carrageenan prepared by the degradation method of this invention has a specific molecular weight range (22.5 kDa-35.2 kDa). κ-carrageenan within this specific molecular weight range is easily soluble, making it convenient for preparing various biomedical materials. Taking common microspheres as an example, the undegraded κ-carrageenan from Example 1 and the 27.9 kDa microspheres prepared in Example 1 with a H / W ratio of [missing information] are compared. 1 / 22051.69κ-carrageenan was used as the material in a compound with gelatin to prepare the microspheres. The method is as follows: 4g of carrageenan and 7g of gelatin were weighed into 25mL of pure water, heated in a water bath at 65℃ and mechanically stirred until completely dissolved to obtain an aqueous phase system. 500mL of liquid paraffin was measured into a three-necked flask, and 0.2mL of Span 80 was added dropwise as an emulsifier. The mixture was stirred evenly to obtain an oil phase system. The above aqueous phase was added dropwise to the oil phase, and the mixture was mechanically stirred and emulsified in a water bath at 65℃ for 2h. Then, it was cured at 4℃ for 30min, and 1mL of 50% glutaraldehyde solution was added dropwise for crosslinking in the dark for 1h. After crosslinking, residual reagents were washed off, and the microspheres were washed, dried, and observed under a microscope for sphericity.

[0079] The experimental results are as follows: sphericity results are as follows Figure 8 As shown. The 27.9 kDa and H / W prepared in Example 1 1 / 2 Microspheres prepared from κ-carrageenan alone (2051.69 g) or in combination with other commonly used materials (such as gelatin) exhibit uniform particle size and good sphericity. In contrast, microspheres prepared from undegraded κ-carrageenan alone or in combination with gelatin show non-uniform particle size and poor sphericity.

[0080] Example 7

[0081] The 27.9 kDa and H / W prepared in Example 1 1 / 2 Application study of a topical drug delivery gel prepared from 2051.69κ-carrageenan

[0082] Using vitamin C as a model drug, undegraded κ-carrageenan and the 27.9 kDa H / W prepared in Example 1 were used respectively. 1 / 2 A topical gel was prepared by combining κ-carrageenan (2051.69) with gelatin. The method was as follows: 100 mg of vitamin C was weighed and dissolved in 10 mL of pure water. 8 g of undegraded κ-carrageenan and 4 g of gelatin were weighed and dissolved in 20 mL of pure water. The mixture was heated in a 60°C water bath and mechanically stirred until completely dissolved. This solution was then added dropwise to the vitamin C aqueous solution and cooled to room temperature to form gel A. Similarly, 8 g of κ-carrageenan with a pH of 27.9 kDa and H / W was weighed... 1 / 2 κ-carrageenan (2051.69 g) and 4 g of gelatin were dissolved in 20 mL of pure water, heated in a 60°C water bath with mechanical stirring until completely dissolved, and then added dropwise to a vitamin C aqueous solution. The solution was cooled to room temperature to form gel B. Spreadability was determined using the "two-glass plate method." 2.0 g of the formulation was placed on a glass plate with a pre-drawn circle of 1 cm diameter. A second glass plate (200 g) was then placed on top of the first glass plate and left for 1 minute. The increase in the diameter of the liquid circle due to the compression of the glass plates was recorded. The experiment was repeated three times.

[0083] The experimental results are as follows: Spreadability results are shown in Table 1. The increase in the diameter of the liquid ring due to pressure from the glass plate was recorded. Gel A spread from 10 mm to 11.50 ± 0.20 mm, while gel B spread from 10 mm to 14.20 ± 0.60 mm. This indicates that the topical drug delivery gel prepared from undegraded κ-carrageenan has a higher viscosity and poorer spreadability. The gel with a viscosity of 27.9 kDa and H / W... 1 / 2 The topical drug delivery gel prepared with κ-carrageenan at a strength of 2051.69 exhibited good spreadability and could better meet the drug delivery requirements.

[0084] Table 1. Spreadability results (n=3)

[0085]

[0086]

[0087] Example 8

[0088] The 27.9 kDa and H / W prepared in Example 1 1 / 2 Study on the antioxidant and anticoagulant activities of 2051.69κ-carrageenan

[0089] Compared to κ-carrageenan with a smaller molecular weight, this invention has a molecular weight of 22.5 kDa-35.2 kDa and an H / W ratio of [missing information]. 1 / 2 κ-carrageenan with a concentration greater than 2000 did not show an increasing trend in antioxidant and anticoagulant activity, exhibiting biological inertness. This characteristic is significant because when κ-carrageenan is used to treat tumors and coagulation disorders, it can effectively avoid potential risks caused by abnormal antioxidant or anticoagulant activity, providing a safer option for the treatment of these diseases. Therefore, the 27.9 kDa and H / W concentration prepared in Example 1... 1 / 2 The antioxidant and anticoagulant activities of κ-carrageenan with a concentration of 2051.69 were investigated. The H / W ratio of the degraded κ-carrageenan was controlled. 1 / 2 The relationship between molecular weight and bioactivity was investigated. Preparation methods are described in Example 1 and Comparative Examples 1, 2, and 3, and the results are shown in Table 4. By controlling the molecular weight of the degraded κ-carrageenan to be similar, the H / W ratio was investigated. 1 / 2 Regarding the relationship with bioactivity, the preparation is described in Example 1 and Comparative Examples 4, 5, and 6, and the results are shown in Table 5.

[0090] 1. Antioxidant activity:

[0091] ①H / W 1 / 2Compared with low molecular weight κ-carrageenan: the antioxidant activity was determined using a DPPH free radical scavenging assay. The method was as follows: DPPH (4.0 mg) was dissolved in 50 mL of ethanol, and the sample was added according to Table 2. The mixture was shaken well and reacted at room temperature for 5 min. The absorbance was measured at 517 nm. The absorbance of tubes 1, 2, and 3 were recorded as A1, A2, and A3, respectively. The samples tested were 27.9 kDa κ-carrageenan prepared in Example 1, 21.8 kDa κ-carrageenan prepared in Comparative Example 1, 16.7 kDa κ-carrageenan prepared in Comparative Example 2, and 11.3 kDa κ-carrageenan prepared in Comparative Example 3, respectively. Physiological saline was used as a blank control (Control), and vitamin C solution (250 μg / mL) was used as a positive control. The DPPH free radical scavenging capacity was calculated using the following formula: DPPH free radical scavenging rate (%) = (1 - (A2 - A3) / A1) × 100%.

[0092] Table 2. Methods of adding substances to each test tube in the DPPH free radical scavenging test (n=3)

[0093]

[0094] The experimental results are as follows: DPPH free radical scavenging experimental results are as follows Figure 9 As shown: (1) The free radical scavenging ability of the 27.9kDa κ-carrageenan prepared in Example 1 was only 59.25%, 48.43%, and 40.66% of that of the 21.8, 16.7, and 11.3kDa κ-carrageenan prepared in Comparative Examples 1-3, respectively, which was significantly reduced; (2) Compared with the blank control group, the 27.9kDa κ-carrageenan treatment group prepared in Example 1 did not show a significant increase in free radical scavenging ability, so it had no antioxidant activity and was biologically inert; (3) Although it was not as good as the vitamin C drug group, the free radical scavenging ability of the 21.8, 16.7, and 11.3kDa κ-carrageenan prepared in Comparative Examples 1-3 was 2.79, 3.42, and 4.07 times that of the blank control group, respectively, which showed extremely significant free radical scavenging ability and extremely significant antioxidant activity; (4) When evaluating the safety of excipients, a dose of 50-100 times is often selected. This dose allows the excipients to more fully demonstrate the potential safety issues in the experiment. Therefore, when the dosage is doubled, the free radical scavenging ability of the κ-carrageenan prepared in Comparative Examples 1-3 will be even stronger than that of the 27.9 kDa κ-carrageenan prepared in Example 1, which may pose related safety risks.

[0095] ② When the molecular weights are similar, with low H / W 1 / 2 Compared to κ-carrageenan: the test samples were prepared in Example 1 with H / W ratios of [specific values ​​not provided in the original text]. 1 / 2 κ-carrageenan with a content of 2051.69, and the H / W prepared in Comparative Example 4 1 / 2κ-carrageenan with a content of 1943.77, and H / W prepared in Comparative Example 5 1 / 2 The H / W ratio of κ-carrageenan with a value of 1789.96 and that prepared in Comparative Example 6 is... 1 / 2 The κ-carrageenan is 1621.12. The remaining steps are the same as ①.

[0096] The experimental results are as follows: DPPH free radical scavenging experimental results are as follows Figure 10 As shown. Compared with the blank group, the H / W prepared in Example 1 1 / 2 The κ-carrageenan with a saturation of 2051.69 showed no significant improvement in antioxidant activity. In contrast, the H / W prepared in Comparative Example 4... 1 / 2 κ-carrageenan with a content of 1943.77, and H / W prepared in Comparative Example 5 1 / 2 The H / W ratio of κ-carrageenan with a value of 1789.96 and that prepared in Comparative Example 6 is... 1 / 2 The antioxidant activity of κ-carrageenan was significantly enhanced at a concentration of 1621.12. The DPPH free radical scavenging experiment results are as follows... Figure 10 As shown: (1) H / W prepared in Example 1 1 / 2 The free radical scavenging ability of 2051.69κ-carrageenan was only that of the H / W prepared in Comparative Examples 4-6. 1 / 2 The free radical scavenging capacity was 58.47%, 52.93%, and 48.20% of κ-carrageenan, respectively, at 1943.77%, 1789.96%, and 1621.12%, respectively; (2) compared with the blank control group, the H / W prepared in Example 1 showed a significant decrease in free radical scavenging capacity. 1 / 2 (2) The κ-carrageenan treatment group showed no significant improvement in free radical scavenging ability, therefore it had no antioxidant activity and was biologically inert; (3) Although it was not as good as the vitamin C drug group, the free radical scavenging ability of the κ-carrageenan prepared in Comparative Examples 4-6 was 2.53, 2.79 and 3.07 times that of the blank control group, respectively, showing extremely significant free radical scavenging ability and extremely significant antioxidant activity; (4) When evaluating the safety of excipients, a dose of 50-100 times is often selected. This dose allows the excipients to more fully demonstrate the potential safety issues in the experiment. Therefore, when the dose is doubled, the free radical scavenging ability of the κ-carrageenan prepared in Comparative Examples 4-6 will be even stronger than that of the κ-carrageenan prepared in Example 1, which may lead to related safety risks.

[0097] 2. Anticoagulant activity:

[0098] The method is as follows: Sample solutions with a 27.9 kDa κ-carrageenan mass concentration of 0.25%, 0.5%, and 1% prepared in Example 1 were prepared using 0.9% physiological saline solution as the solvent. 0.5% sample solutions for Comparative Examples 1-6 were prepared using 0.9% physiological saline solution as the solvent. A 0.9% physiological saline solution was used as a blank control. The effect of H / W was investigated.1 / 2 The effect of molecular weight on the anticoagulant activity of κ-carrageenan when molecular weights are close. The H / W ratio was also investigated when molecular weights are close. 1 / 2 The effect of κ-carrageenan on anticoagulant activity was investigated. Seven plastic test tubes were taken and labeled as blank control group, A (0.25% 27.9 kDaH / W), etc. 1 / 2 κ-carrageenan (2051.69%), B (0.5% 27.9 kDa H / W) 1 / 2 κ-carrageenan (2051.69), C (1% 27.9 kDa H / W) 1 / 2 (κ-carrageenan with a concentration of 2051.69), D (0.5% 21.8 kDa H / W) 1 / 2 κ-carrageenan (2042.37), E (0.5% 16.7 kDa H / W) 1 / 2 (κ-carrageenan with a content of 2066.63), F (0.5% 11.3 kDa H / W) 1 / 2 (κ-carrageenan of 2017.45), G (0.5% 27.2 kDa H / W) 1 / 2 κ-carrageenan (1943.77), H (0.5% 27.6 kDa H / W) 1 / 2 (κ-carrageenan at 1789.96), I (0.5% 26.9 kDa H / W) 1 / 2 (κ-carrageenan, 1621.12). Coagulation time (CT) measurement: 1 mL of blood was drawn from the rabbit heart, and timing began. 0.1 mL of the sample was added to each blood sample tube, and the tubes were placed in a 37°C water bath until the blood stopped flowing when the tubes were inverted. The coagulation time was recorded. TT, PT, and APTT measurements: Thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (APTT) were measured using a kit.

[0099] The experimental results are shown in Table 3, with a focus on the CT results. Compared with the blank control group, the concentrations of 27.9 kDaH / W prepared in Example 1... 1 / 2 The κ-carrageenan treatment group with a molecular weight of 2051.69 showed no significant prolongation of CT, TT, PT, and APTT, therefore it has no anticoagulant activity and is biologically inert. In contrast, the κ-carrageenan treatment groups prepared in Comparative Examples 1-6 showed a highly significant prolongation of CT, exhibiting highly significant anticoagulant activity; compared to the κ-carrageenan prepared in Example 1, the clotting time was prolonged by up to 54.9 s. Furthermore, when the molecular weight is similar, the H / W ratio... 1 / 2 Degradation of κ-carrageenan to less than 2000 (e.g., 1943.77, 1789.96, and 1621.12 in the experiment) significantly improved CT and APTT. When H / W 1 / 2When approaching the target molecular weight, κ-carrageenan with a molecular weight less than 22.5 kDa (e.g., 21.8, 16.7, and 11.3 kDa in the experiment) showed significantly improved CT, TT, and APTT. In summary, the present invention utilizes κ-carrageenan with a molecular weight of 22.5 kDa-35.2 kDa and H / W... 1 / 2 κ-carrageenan with a concentration greater than 2000 has no anticoagulant activity.

[0100] In summary, the analysis of Table 3... Figure 9 , Figure 10 Data from Tables 4 and 5 (where coagulation time results are statistically based on 0.5% sample results) show that the 27.9 kDa H / W prepared in Example 1... 1 / 2 The κ-carrageenan with a concentration of 2051.69 had an antioxidant activity of less than 12% and a clotting time of less than 122 s, significantly lower than the κ-carrageenan prepared in Comparative Examples 1-3 (antioxidant activity greater than 17% and clotting time greater than 135 s). Furthermore, the κ-carrageenan prepared in Comparative Examples 4-6 all had antioxidant activities greater than 16%, significantly higher than the κ-carrageenan prepared in Example 1, and their clotting times were all greater than 127 s, indicating higher anticoagulant activity than the κ-carrageenan prepared in Example 1.

[0101] Table 3 Anticoagulant activity test (n=3)

[0102]

[0103]

[0104] Note: Compared with the blank control, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001; compared with group B, # p≤0.05, ### p≤0.001, #### p≤0.0001

[0105] Table 4. Relationship between molecular weight and bioactivity of degraded κ-carrageenan with similar GPC peak half-widths.

[0106]

[0107] Table 5. Relationship between GPC peak half-width and bioactivity of degraded κ-carrageenan with similar molecular weights.

[0108]

[0109] Example 9

[0110] ① Except for the degradation step (2), where the HCl solution concentration was 1.2M, the water bath temperature was 63℃, and the heating reaction time was 42min, the remaining steps were the same as in Example 1. The molecular weight and H / W ratio of the κ-carrageenan produced in this example were also investigated.1 / 2 Inflammatory, antioxidant and anticoagulant effects (methods are the same as in Examples 5 and 8).

[0111] ② Except for the degradation step (2), where the HCl solution concentration was 0.6M, the water bath temperature was 55℃, and the heating reaction time was 18min, the remaining steps were the same as in Example 1. The molecular weight and H / W ratio of the κ-carrageenan prepared in this example were also investigated. 1 / 2 Inflammatory, antioxidant and anticoagulant effects (methods are the same as in implementations 5 and 8).

[0112] The experimental results are as follows: ① The molecular weight of the κ-carrageenan obtained is 22.5, H / W 1 / 2 The value is 2091.03. The molecular weight of the κ-carrageenan obtained in step ② is 35.2, H / W. 1 / 2 The molecular weight and H / W of the κ-carrageenan prepared in Example 9 were 2039.28. 1 / 2 The inflammatory, antioxidant, and anticoagulant effects are shown in Table 6 (wherein, in the NO concentration determination experiment, the sample concentration of Examples 9①② was 500 μg / mL, and the coagulation time results were statistically analyzed based on the results of 0.5% of the samples), all of which are consistent with the results of this invention, which have low inflammatory risk and no significant improvement in antioxidant and anticoagulant capabilities.

[0113] Table 6. Molecular weight and H / W of κ-carrageenan prepared in Example 9 1 / 2 Inflammatory, antioxidant and anticoagulant effects

[0114]

[0115] Comparative Example 1

[0116] Except for the reaction time of 0.75 h in step (2) of degradation, the other steps are the same as in Example 1.

[0117] The experimental results are as follows: the molecular weight of the κ-carrageenan prepared in Comparative Example 1 is 21.8 kDa, H / W 1 / 2 It is 2042.37.

[0118] Comparative Example 2

[0119] Except for the reaction time of 0.92h in step (2) of degradation, the other steps are the same as in Example 1.

[0120] The experimental results are as follows: the molecular weight of the κ-carrageenan prepared in Comparative Example 2 is 16.7 kDa, H / W 1 / 2 It is 2066.63.

[0121] Comparative Example 3

[0122] Except for the reaction time of 1.16 h in step (2) of degradation, the other steps are the same as in Example 1.

[0123] The experimental results are as follows: the molecular weight of the κ-carrageenan prepared in Comparative Example 3 is 11.3 kDa, H / W 1 / 2 It is 2017.45.

[0124] Comparative Example 4

[0125] Except for the hydrochloric acid concentration of 1.3M and the reaction temperature of 55℃ in step (2) of degradation, the other steps are the same as in Example 1.

[0126] The experimental results are as follows: the molecular weight of the κ-carrageenan prepared in Comparative Example 4 is 27.2 kDa, H / W 1 / 2 It is 1943.77.

[0127] Comparative Example 5

[0128] Except for the hydrochloric acid concentration of 0.79M and the reaction temperature of 65℃ in step (2) of degradation, the other steps are the same as in Example 1.

[0129] The experimental results are as follows: the molecular weight of the κ-carrageenan prepared in Comparative Example 5 is 27.6 kDa, H / W 1 / 2 It is 1789.96.

[0130] Comparative Example 6

[0131] Except for the hydrochloric acid concentration of 0.79M and the reaction time of 0.8h in step (2) of degradation, the other steps are the same as in Example 1.

[0132] The experimental results are as follows: the molecular weight of the κ-carrageenan prepared in Comparative Example 6 is 26.9 kDa, H / W 1 / 2 It is 1621.12.

[0133] Comparative Example 7

[0134] Except for the reaction time of 0.26 h in step (2) of degradation, the other steps are the same as in Example 1.

[0135] The experimental results are as follows: the molecular weight of the κ-carrageenan prepared in Comparative Example 7 is 43.8 kDa, H / W 1 / 2 It is 2078.35.

Claims

1. A biologically inert κ-carrageenan, characterized in that, The κ-carrageenan has a molecular weight range of 22.5 kDa to 35.2 kDa, and the ratio of GPC peak height to full width at half maximum (H / W) is [not specified]. 1 / 2 (Greater than 2000) 2. A method for preparing the bio-inert κ-carrageenan according to claim 1, characterized in that, Includes the following steps: (1) Add ultrapure water to κ-carrageenan powder and heat to dissolve; (2) Add an acidic solution and heat to react; precisely control the molecular weight range of degraded κ-carrageenan and the ratio of GPC peak height to full width at half maximum (H / W) by controlling the concentration of the acidic solution, the reaction temperature, and the reaction time. 1 / 2 ); (3) The molecular weight and H / W of κ-carrageenan after degradation 1 / 2 Once the set range is reached, immediately add an alkaline solution to adjust the pH of the κ-carrageenan solution to terminate the degradation reaction; cool to room temperature. (4) After filtering and cooling the κ-carrageenan solution, concentrate it, add ultrapure water, continue filtering and concentrate it, and repeat this step 3-5 times; after rotary evaporation, freeze dry to obtain bio-inert κ-carrageenan.

3. The method for preparing bio-inert κ-carrageenan according to claim 2, characterized in that, The acidic solution is one or more of hydrochloric acid, sulfuric acid, nitric acid, and oxalic acid.

4. The method for preparing bio-inert κ-carrageenan according to claim 2, characterized in that, The concentration of the κ-carrageenan solution in step (1) is 8.89-17.78 g / L.

5. The method for preparing bio-inert κ-carrageenan according to claim 2, characterized in that, In step (2), the volume of the acidic solution is 100-200 mL, the volume ratio of κ-carrageenan solution to acidic solution is 15-20:1, and the concentration is 0.6-1.2 M; the reaction temperature is 55-63 °C; and the reaction time is 0.3-0.7 h.

6. The method for preparing bio-inert κ-carrageenan according to claim 2, characterized in that, Step (3) The alkaline solution is one or more of sodium hydroxide solution, potassium hydroxide solution, and calcium hydroxide solution, with a concentration of 0.6-1.2M; Step (3) The pH of the alkaline solution is adjusted to 6.5-7.

0.

7. The method for preparing bio-inert κ-carrageenan according to claim 2, characterized in that, The filtration in step (4) is one or more of flat-plate ultrafiltration and ultrafiltration centrifugation; the molecular weight cutoff of the ultrafiltration membrane is 5-10 kDa; and the volume of the added ultrapure water is 1.5-2.5 L.

8. The bio-inert κ-carrageenan of claim 1 is preferably a bio-inert κ-carrageenan with low inflammatory risk and no significant improvement in antioxidant and anticoagulant capabilities.

9. The use of the bio-inert κ-carrageenan of claim 1 in the preparation of reagents or drugs for treating tumors and coagulation disorders.

10. The application according to claim 9, characterized in that, The application of the bio-inert κ-carrageenan, either alone or in combination with other materials, in the preparation of microspheres for the treatment of tumors and coagulation disorders.

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