Preparation method and application of colon cancer targeting drug delivery system loaded with hydroxyl camptothecin

Abstract

The application discloses a preparation method and application of a colon cancer targeted drug delivery system loaded with hydroxyl camptothecin. The drug delivery system is composed of HCPT and ferritin (Fn), and the HCPT is wrapped in the Fn by a solvent evaporation method under alkaline conditions by using the characteristics that the HCPT is dissolved in an alkaline solution and hardly soluble in a weak acid environment. Under weak acid conditions, the open ring HCPT is converted into lactone type hydroxyl camptothecin, and the solubility is reduced, so that the HCPT is efficiently loaded into the Fn (HCPT@Fn). The HCPT@Fn can be combined with the transferrin receptor-1 highly expressed on the surface of colon cancer cells, promote the efficient accumulation of the HCPT in the colon cancer cells, and release the lactone type HCPT under the acidic environment of lysosomes, so as to efficiently kill the colon cancer cells.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to a novel, highly efficient targeted drug delivery system for the treatment of colon cancer. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors worldwide, ranking second in mortality among all cancers, and its incidence rate in my country has been on the rise in recent years. Current treatment methods for colorectal cancer include surgery, radiotherapy, and chemotherapy. Chemotherapy, which uses chemical drugs to kill cancer cells, plays a crucial role in the treatment of colorectal cancer.

[0003] Hydroxycamptothecine (HCPT) is an alkaloid with highly potent anti-colon cancer activity. It inhibits the activity of DNA topoisomerase I in colon cancer cells, preventing DNA double-strand unwinding and thus inhibiting DNA replication and transcription, ultimately inducing apoptosis in colon cancer cells. Clinically, it is commonly used as an adjunct therapy for various primary liver cancers, gastric cancers, colon cancers, esophageal cancers, and other malignant tumors. However, studies show that HCPT is poorly soluble in both water and lipid-soluble solutions. Therefore, in clinical practice, sodium salts of HCPT open-ring derivatives are often used. However, the open-ring derivatives of HCPT have low antitumor activity, significant adverse reactions, and a short half-life (30 min), greatly limiting their clinical application. Therefore, constructing a suitable drug delivery system to improve the solubility of HCPT is key to its effective treatment of colon cancer.

[0004] Recently, a series of biocompatible endogenous biomacromolecules have been used as drug delivery carriers. Among them, ferritin (Fn) is considered an ideal chemotherapeutic drug delivery carrier due to its unique structure and properties. Fn is heat-resistant and remains stable over a wide pH range. By changing the solution polarity or pH, the Fn subunits can undergo interconversion between denaturation / depolymerization and renaturation / assembly / aggregation, thereby loading drugs into the Fn cavity. Therefore, Fn is a novel targeted delivery carrier for chemotherapeutic drugs capable of carrying multiple drugs. Furthermore, Fn can recognize and bind to transferrin receptor 1 (TfR1) on the cell surface, and enter the cell in large quantities via TfR1 mediation. Because colon cancer cells grow and proliferate rapidly, their demand for iron ions is much greater than that of normal cells; therefore, the expression level of TfR1 on the surface of colon cancer cells is often much higher than that of normal cells. Thus, Fn can selectively accumulate in colon cancer cells.

[0005] Our study found that under alkaline conditions, the lactone-type HCPT can be transformed into an open-ring derivative of HCPT, which significantly increases its solubility, thus enabling HCPT to efficiently enter the Fn cavity structure. In a weakly acidic environment, the open-ring HCPT in the Fn cavity structure can be transformed into the lactone-type HCPT, which decreases its solubility and is thus efficiently loaded into Fn, while retaining the high antitumor activity of the lactone-type HCPT.

[0006] Therefore, we constructed a hydroxycamptothecin ferritin inclusion complex (HCPT@Fn) using a combination of alkali dissolution, acid precipitation, and solvent evaporation method. This increased the accumulation of HCPT in CT-26 colon cancer cells and released lactone-type HCPT in an acidic lysosomal environment, thus effectively killing colon cancer cells. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing and applying a colon cancer targeted drug delivery system loaded with hydroxycamptothecin. A novel method is used to load HCPT into the hollow structure of ferritin for the treatment of colon cancer. HCPT@Fn can bind to transferrin receptor-1, which is highly expressed on the surface of colon cancer cells, promoting the efficient accumulation of hydroxycamptothecin in colon cancer cells. Furthermore, it releases lactone-type HCPT in an acidic lysosomal environment, effectively killing colon cancer cells.

[0008] The technical solution of this invention is: a method for preparing a colon cancer targeted drug delivery system loaded with hydroxycamptothecin, characterized by utilizing the characteristic that HCPT is soluble in alkaline solutions but poorly soluble in weakly acidic environments. HCPT is dissolved in an ammonia solution with a pH of 10, and ferritin is dispersed in 55% acetone (acetone:water = 55:45). After mixing, the mixture is stirred in a sealed container for 30 minutes, then the acetone is evaporated and the pH is adjusted to 7.0. The ferritin structure is restored to its intact state, while the solubility of HCPT decreases, forming an inclusion complex HCPT@Fn. The HCPT@Fn has a particle size of 25.9 nm, a potential of -14.8 mV, and a drug loading of 9.79%. HCPT@Fn can bind to transferrin receptor-1, which is highly expressed on the surface of colon cancer cells, promoting the efficient accumulation of hydroxycamptothecin in colon cancer cells and releasing lactone-type HCPT in an acidic lysosomal environment, thus efficiently killing colon cancer cells.

[0009] Features of the technical solution of this invention:

[0010] ① This invention utilizes the fact that lactone-type HCPT can be transformed into an open-ring derivative of HCPT under alkaline conditions, which significantly increases its solubility, thereby enabling HCPT to efficiently enter the Fn cavity structure. In a weakly acidic environment, the open-ring HCPT in the Fn cavity structure can be transformed into lactone-type HCPT, which reduces its solubility, thus allowing it to be efficiently loaded in Fn while retaining the high antitumor activity of lactone-type HCPT.

[0011] ② This invention utilizes the ability of ferritin to bind to transferrin receptor-1, which is highly expressed on the surface of colon cancer cells, to promote the efficient accumulation of hydroxycamptothecin in colon cancer cells and release lactone-type HCPT in the acidic environment of lysosomes, thereby efficiently killing colon cancer cells.

[0012] The innovations of this invention are: ① A novel method for loading hydroxycamptothecin (HCPT) into ferritin, utilizing the characteristic that HCPT is soluble in alkaline solutions but poorly soluble in weakly acidic environments, employing a combination of alkali dissolution, acid precipitation, and solvent evaporation to efficiently load HCPT into the hollow structure of ferritin. ② The development of a drug delivery system capable of specifically accumulating in colon cancer cells and efficiently treating colon cancer. Attached Figure Description

[0013] Figure 1 The particle size distribution and zeta potential of HCPT@Fn are as follows: (A) Particle size of HCPT@Fn; (B) Zeta potential of HCPT@Fn.

[0014] Figure 2 The cytotoxicity of HCPT@Fn against HT-29 colon cancer cells. (A) MTT assay results; (B) IC50 of HCPT@Fn. 50 Statistics. n=5, * P < 0.05.

[0015] Figure 3 This study observes the toxicity of HCPT@Fn to HT-29 cells using live and dead cell staining. (A) Typical images of live and dead cell staining after HCPT@Fn treatment; (B) Statistical results of the percentage of live cells after HCPT@Fn treatment; (C) Statistical results of the percentage of dead cells after HCPT@Fn treatment. n=3, * P < 0.05, ** P < 0.01.

[0016] Figure 4 This study investigated the effect of HCPT@Fn on colony formation in HT-29 cells. (A) Typical cell colony staining pattern (20×); (B) Statistical results of colony formation. n = 3. ** P < 0.01.

[0017] Figure 5 This refers to the uptake of HCPT@Fn by HT-29 cells. (A) Fluorescence microscopy observation of HCPT@Fn uptake by HT-29 cells (200×); (B) Statistical results of uptake. n=3, * P < 0.05 ** P < 0.01.

[0018] Figure 6 These are the H&E staining results of the heart, liver, spleen, lungs, and kidneys of normal mice. Detailed Implementation

[0019] 1. Research Methods

[0020] 1.1 Preparation and characterization of HCPT@Fn

[0021] HCPT@Fn was prepared by a combination of alkali dissolution, acid precipitation, and solvent evaporation: An aqueous solution with pH 9.5 was prepared using ammonia. 27.5 mL of acetone was added, followed by 22.5 mL of deionized water, and ammonia was added to adjust the pH to 9.5, preparing a mixed solvent (acetone:water = 55:45) with a pH of 9.5. 4 mL of this mixed solvent was added, and 5 mg of ferritin was added. The mixture was stirred on a magnetic stirrer for 50 min. 12 mg of HCPT was weighed, dissolved in the pH 9.5 aqueous solution, and added to the ferritin-containing mixed solvent. After stirring for 10 min, the solution was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da. Dialysis was performed for 4 h using distilled water as the dialysate. The dialysate was replaced every 1 h, and the pH of the solution in the dialysis bag was monitored. When the pH of the solution in the dialysis bag approached neutral, the solution was filtered through a 0.8 μm filter membrane to obtain HCPT@Fn. The particle size and zeta potential of HCPT@Fn were measured using a laser particle size analyzer.

[0022] Determination of HCPT@Fn drug loading: The chromatographic column was a BEH C18 column, 1.7 μm, 2.1 mm × 100 mm; the mobile phase was methanol:water (60:40); the flow rate was 0.1 mL·min. -1 Column temperature: 35℃; injection volume: 5μL; detection wavelength: 384nm; injection time: 6min. Under the above chromatographic conditions, inject 5μL, record the peak area, calculate the concentration of HCPT based on the working curve, and calculate the drug loading of HCPT in HCPT@Fn.

[0023] 1.2 MTT Experiment

[0024] HT-29 cells were grown to the logarithmic growth phase and seeded into 96-well plates, 200 μL per well, and incubated in a CO2 incubator. After cell attachment, the cell culture medium was aspirated from the 96-well plates, and 200 μL of different concentrations of HCPT and HCPT@Fn solutions were added sequentially (HCPT concentrations were: 0.05, 0.1, 0.5, 1, and 5 μg / mL). -1 The samples were placed in a CO2 incubator and incubated for 48 hours. After incubation, 20 μL of MTT solution (5 mg / mL) was added to each well. -1The cells were incubated in a CO2 incubator for 4 hours. The supernatant was then aspirated, and 150 μL of DMSO solution was added to each well. The cells were shaken for 10 minutes. The absorbance of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 492 nm. The relative cell viability of each group was calculated. Relative cell viability (%) = (Absorbance of drug group / Absorbance of control group) × 100%.

[0025] 1.3 Live and Dead Cell Staining Assay

[0026] Logarithmic growth phase HT-29 colon cancer cells were seeded into 6-well plates, with 2 mL added to each well, and incubated in a CO2 incubator. After 24 h of culture, the culture medium was aspirated, and serum-free DMEM diluted HCPT solution (1.5, 3 μg / mL) was added. -1 ) and HCPT@Fn solutions (1.5, 3 μg·mL) -1 ), administer 2 mL to each well. After incubation for 48 h, stain the live and dead cells according to the instructions of the live / dead cell staining kit. After staining, collect the cells and incubate at 5000 rpm. -1 Centrifuge for 5 min, resuspend in 500 μL PBS buffer, add to a glass slide, air dry, and mount with glycerol. Observe the number and proportion of live and dead cells using a fluorescence microscope.

[0027] 1.4 Cloning Experiment

[0028] Logarithmic growth phase HT-29 colon cancer cells were collected and diluted to 300 cells / mL using DMEM complete culture medium. The cells were then seeded into 6-well plates, 2 mL per well, and incubated in a CO2 incubator. After 24 h of culture, the culture medium was aspirated, and serum-free DMEM diluted HCPT solution (1.5, 3 μg / mL) was added. -1 ) and HCPT@Fn solutions (1.5, 3 μg·mL) -1 ), administer 2 mL of drug to each well. After incubation for 24 h, replace with fresh DMEM complete culture medium, changing the medium every two days until visible cell clones are observed. Discard the culture medium, add 2 mL of 4% paraformaldehyde, fix for 15 min, then add crystal violet staining solution for 15 min, rinse with tap water, and take photos to record data.

[0029] 1.5HT-29 cells uptake of HCPT@Fn

[0030] Logarithmic growth phase HT-29 colon cancer cells were seeded into 24-well plates pre-placed with discs, 1 mL per well, and incubated in a CO2 incubator. After 24 h of culture, the culture medium was aspirated, and serum-free DMEM diluted HCPT solution and HCPT@Fn solution (HCPT concentration 30 μg / mL) were added. -1 ), administer 1 mL to each well. After incubation for 1, 2, and 4 hours, discard the drug solution, wash three times with 37°C PBS buffer, add 1 mL of 4% paraformaldehyde, fix for 15 min, discard the 4% paraformaldehyde, wash three times with 37°C PBS buffer, and add DAPI solution (concentration 500 μg / mL). -1 Stain at room temperature for 10 min, discard DAPI, wash 3 times with 37℃ PBS buffer, invert the disc onto a glass slide with glycerol, and observe the uptake of HCPT@Fn by colon cancer HT-29 cells using a fluorescence microscope.

[0031] 1.6 In vivo safety evaluation

[0032] Normal mice were randomly divided into 5 groups and administered physiological saline, free HCPT (4 mg·kg⁻¹), HCPT@Fn (4 mg·kg⁻¹), HCPT@Fn (8 mg·kg⁻¹), and HCPT@Fn (12 mg·kg⁻¹), respectively. The drugs were administered once every 2 days for 5 consecutive times. After administration, the mice were sacrificed, and the heart, liver, spleen, lung, and kidney of each group were obtained. The tissues were fixed in 4% paraformaldehyde for 24 h, paraffin sections were stained with H&E, and the effects of each group on the morphology of normal tissues were observed using an inverted microscope.

[0033] 2 Experimental Results

[0034] 2.1 Characterization of HCPT@Fn

[0035] The particle size and potential of HCPT@Fn were determined using a nanoparticle size and zeta potential analyzer, and the results are as follows: Figure 1 As shown, the particle size of HCPT@Fn is 25.9 nm, the zeta potential is -14.8 mV, and the HCPT drug loading is 9.79%.

[0036] 2.2 Study on the anti-colon cancer activity of HCPT@Fn

[0037] MTT assay results showed that HCPT@Fn could inhibit the proliferation of colon cancer HT-29 cells in a concentration-dependent manner, and its antitumor activity was significantly better than that of free HCPT. Figure 2 Live and dead cell staining experiments showed that HCPT@Fn could increase the proportion of dead cells in colon cancer HT-29 cells in a concentration-dependent manner. Moreover, at the same concentration, HCPT@Fn had a significantly stronger killing effect on HT-29 cells than free HCPT. Figure 3 Colony formation assays showed that HCPT@Fn inhibited HT-29 cell colony formation in a concentration-dependent manner, and the inhibitory effect was significantly better than that of free HCPT at the same concentration. Figure 4 ).

[0038] 2.3 HT-29 cells uptake HCPT@Fn

[0039] Fluorescence microscopy revealed that HCPT@Fn accumulated significantly more in HT-29 cells compared to free HCPT, and the accumulation of HCPT@Fn in HT-29 cells gradually increased with prolonged incubation time. Figure 5 ).

[0040] 2.4 In vivo safety evaluation

[0041] H&E staining results showed that no obvious abnormal morphological changes were found in the heart, liver, spleen, lungs, and kidneys of mice in each group, indicating that HCPT@Fn did not cause significant damage to the heart, liver, spleen, lungs, and kidneys of mice at the set dosage. Figure 6 ).

[0042] 3. Conclusion:

[0043] The alkaline dissolution-acid precipitation combined with solvent evaporation method can efficiently load HCPT into ferritin (Fn) nanocages. HCPT@Fn can effectively deliver HCPT to colon cancer cells and release lactone-type HCPT in the acidic environment of lysosomes, which can effectively kill colon cancer cells and has certain application prospects.

Claims

1. A method for preparing a colon cancer targeted drug delivery system loaded with hydroxycamptothecin, characterized in that: An aqueous solution with a pH of 9.5 was prepared using ammonia. Take 27.5 mL of acetone, add 22.5 mL of deionized water to it, and add ammonia to adjust the pH to 9.5 to prepare a mixed solvent with pH 9.5; Take 4 mL of the above mixed solvent, add 5 mg of ferritin Fn to it, and stir on a magnetic stirrer for 50 min; Weigh 12 mg of hydroxycamptothecin (HCPT) and dissolve it in an aqueous solution with a pH of 9.

5. Add the solution to the above-mentioned mixed solvent containing ferritin and stir for 10 min. Transfer the solution to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyze for 4 h. Use distilled water as the dialysate. Change the dialysate every 1 h and check the pH value of the solution in the dialysis bag. When the pH value of the solution in the dialysis bag is close to neutral, filter it through a 0.8 μm filter membrane to obtain HCPT@Fn.

2. The application of HCPT@Fn, prepared by the method of the colorectal cancer targeted drug delivery system loaded with hydroxycamptothecin as described in claim 1, in the preparation of drugs for treating colorectal cancer.

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