A self-assembled targeting nanoparticle based on a dual parent camptothecin conjugate, preparation and application

Abstract

The application discloses a kind of self-assembly tumor targeting nano microparticles based on amphiphilic camptothecin conjugate, preparation and application.The nanoparticle is obtained by self-assembly of amphiphilic camptothecin conjugate using reverse phase solvent dispersion method, wherein the amphiphilic camptothecin conjugate is obtained by introducing reduction-responsive cleavage bond on camptothecin 20'-OH, and coupling with targeting peptide.The nanoparticle is spherical, with a particle size of 60-70 nm, significantly improving the water solubility of camptothecin.It can effectively release camptothecin under tumor microenvironment, efficiently kill cancer cells, inhibit 3D tumor sphere and in vivo tumor growth, reduce toxic side effects, and has good application potential.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine, specifically to the design and synthesis of amphiphilic camptothecin conjugates, the assembly and preparation of targeted nanoparticles, and their applications. Background Technology

[0002] Cancer poses a serious threat to human health. Colorectal cancer is the third most common cancer in terms of mortality and incidence, accounting for 10% of cancer cases worldwide (KDMiller, CA-Cancer J.Clin. 2019, 69(5), 363-385). Chemotherapy is one of the main treatment methods for cancer. Camptothecin (CPT) is a broad-spectrum anticancer drug that inhibits topoisomerase activity and has a good therapeutic effect on colorectal cancer. However, it has poor solubility in water (<5μg / mL), and intravenous injection can easily block blood vessels and induce toxic side effects. Therefore, in vivo delivery of hydrophobic chemotherapeutic drugs is a major challenge in the field of cancer treatment. Delivery using nanocarriers has been a research direction in recent years, but nanocarriers constructed from excipients have a relatively low drug loading rate (generally less than 10%) (S.Fu, Acta Pharm.Sin.B 2022, 12(1), 92-106). Drug excipients can also bring unpredictable potential toxicity. Targeted modification, drug loading, etc., require multiple steps, which increases the difficulty for later approval and application.

[0003] Drug self-assembly utilizes the properties of drug molecules for direct assembly without the need for excipients, resulting in high drug loading rates (generally above 50%, even reaching 100%) (Y. Fu, J. Biomed. Nanotechnol. 2022, 18(4), 939-956), attracting widespread attention. Camptothecin and its derivatives possess a quinolone planar pentacyclic structure and have been proven to have self-assembly potential. Amphiphilic drug self-assembly utilizes the hydrophilic and hydrophobic properties of amphiphilic molecules for assembly, resulting in more stable structures and richer mechanisms of action compared to pure drug self-assembly, including π-π stacking, hydrophobic interactions, and hydrogen bonding. Furthermore, stimulus-responsive groups can be introduced to regulate the stability of nanoassemblies, enabling specific drug release at tumor sites. For example, reduction-responsive disulfide bonds (-ss-) break under tumor microenvironment conditions, releasing the drug. The flexible bond angles also balance intermolecular forces, making the nanoassemblies more compact and stable (Y. Wang, Nano Lett. 2014, 14(10), 5577-83). Therefore, it is essential to design and synthesize self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates, which have great application potential. Summary of the Invention

[0004] This invention discloses a self-assembled targeted nanoparticle based on an amphiphilic camptothecin conjugate, its preparation method, and its application. These nanoparticles improve the water solubility of camptothecin, its targeted uptake by tumor cells, and its tumor cell killing ability; enhance the growth inhibition of 3D tumor spheres and its in vivo tumor-killing effect; and reduce toxic side effects.

[0005] The technical solution adopted in this invention is as follows:

[0006] The method for preparing self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates involves using camptothecin as the hydrophobic end and the targeting peptide as the hydrophilic end. The water solubility of the targeting peptide is increased by introducing hydrophilic amino acids such as lysine and arginine. The amphiphilic camptothecin conjugates are synthesized via an amidation reaction using a solid-phase synthesis method. The coupling bond is a molecularly specific responsive cleavage bond in the tumor microenvironment, and is one of a disulfide bond, diene bond, or hydrazone bond.

[0007] The method for preparing self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates involves assembling the nanoparticles using a reverse-phase solvent dispersion method followed by dialysis.

[0008] The method for preparing self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates involves dissolving the camptothecin conjugate in one or two of dimethyl sulfoxide, N,N-dimethylformamide, and methanol to form an organic phase solution of 0.5-10 mg / mL. This organic phase solution is then added dropwise at a uniform rate to an aqueous phase of pure water or PBS solution, which is 5-10 times the volume of the organic phase. The reaction is continued with stirring for 1-5 hours.

[0009] The method for preparing self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates involves transferring the product obtained by reverse solvent dispersion into a 1000-3500 Da dialysis bag, dialyzing with ultrapure water as the dialysis solvent for 24-72 hours, and then freeze-drying to obtain self-assembled nanoparticles.

[0010] The self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates are spherical with a particle size of 70-90 nm. Compared with free camptothecin, they exhibit reduced UV-NIR absorption and fluorescence emission intensity, and significantly improved solubility in water.

[0011] The amphiphilic camptothecin conjugate self-assembled targeted nanoparticles responsively release drugs in solutions containing reducing molecules such as reduced glutathione, with a drug release rate of ~48% within 120 hours.

[0012] The aforementioned amphiphilic camptothecin conjugate self-assembled targeted nanoparticles can be targeted and taken up by tumor cells, effectively killing cancer cells.

[0013] The aforementioned amphiphilic camptothecin conjugate self-assembled targeted nanoparticles can be targeted to colon cancer tumors in vivo, inhibiting the growth of 3D tumor spheres in vitro and tumors in vivo, and reducing toxic side effects. Attached Figure Description

[0014] Figure 1 CPT-ss-RGD Synthesis Flowchart

[0015] Figure 2 ESI-MS chromatograms (AB) and HPLC chromatograms (C) of CPT-ss-RGD and CPT-cc-RGD.

[0016] Figure 3 Critical aggregation concentrations of (A) CPT-ss-RGD NPs and (B) CPT-cc-RGD NPs

[0017] Figure 4 (A) DLS particle size, (B) TEM observation, (C) solubility, (D) UV-Vis absorption spectrum and (E) fluorescence emission spectrum of CPT-ss-RGD NPs.

[0018] Figure 5 Particle size stability of CPT-ss-RGD NPs at room temperature

[0019] Figure 6 CPT-ss-RGD NPs slowly release CPT under the action of the reducing molecule glutathione.

[0020] Figure 7 (A) Targeted uptake of CPT-ss-RGD NPs by cancer cells and (B) Semi-quantitative fluorescence analysis

[0021] Figure 8 CPT-ss-RGD NPs toxicity to different cells

[0022] Figure 9 CPT-ss-RGD NPs inhibit the growth of multicellular tumor spheres.

[0023] Figure 10 CPT-ss-RGD NPs target and locate colon cancer tumors.

[0024] Figure 11 Colorectal cancer tumor growth curves, tumor growth inhibition rates, and tumor H&E and TUNEL staining after CPT-ss-RGD NPs treatment.

[0025] Figure 12 H&E staining of mouse tissues and organs after CPT-ss-RGD NPs treatment Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The present invention will be further described below with reference to the accompanying drawings and implementation examples.

[0027] Example 1: Preparation of amphiphilic camptothecin conjugates with tumor cell targeting ability

[0028] Camptothecin was purchased from Shanghai Aladdin Co., Ltd., and the resin peptide was synthesized by Shanghai ChuTai Biotechnology Co., Ltd. according to the sequence provided by me.

[0029] The specific preparation process includes the following steps; see the reaction flowchart below. Figure 1 In addition, a CPT-cc-RGD molecule was synthesized as a control.

[0030] 1) Synthesis of CPT-ss-OH: Camptothecin (174 mg, 0.5 mmol), triphosgene (55 mg, 0.17 mmol), and 4-dimethylaminopyridine (195 mg, 1.6 mmol) were weighed and dissolved, and reacted at room temperature in the dark for 3 h. 2,2-Dithiodiethanol (385 mg, 2.5 mmol) was mixed with tetrahydrofuran and added to the above solution, and the reaction was carried out overnight at room temperature. The product was diluted with dichloromethane and extracted with HCl, saturated NaCl, and ultrapure water. The organic phase was dried over anhydrous Na2SO4 and separated by filtration. Dichloromethane was removed by rotary evaporation, and the product was dried under vacuum. The crude product was eluted by silica gel column chromatography with a gradient, and dried under vacuum to give a pale yellow solid.

[0031] 2) CPT-ss-COOH was synthesized via the carboxylation of CPT-ss-OH: Succinic anhydride (200 mg, 2 mmol), CPT-ss-OH (264 mg, 0.5 mmol), and DMAP (195 mg, 1.6 mmol) were weighed and dispersed in dichloromethane and stirred overnight at room temperature. Extraction was performed with ultrapure water, 50 mM HCl aqueous solution, and saturated NaCl. The organic phase was dried over anhydrous Na₂SO₄, dichloromethane was removed by rotary evaporation, and the product was dried under vacuum. The crude product was purified by gradient elution using silica gel column chromatography, and dried under vacuum to obtain a pale yellow solid.

[0032] 3) Synthesis of CPT-ss-RGD: 200 mg of Fmoc-GR(pbf)GD(tbu)K(boc)-Resin peptide was weighed into a peptide solid-phase synthesis tube and soaked in 10 mL of DCM / DMF solution for 2 h. After removing the solvent, a small amount of peptide was taken into an EP tube for ninhydrin detection (1a). 5 mL of 20% piperidine / DMF was added and reacted 3 times to remove the Fmoc group, and a small amount was used for ninhydrin detection (1b). CPT-ss-COOH (131.9 mg, 0.21 mmol), PyBop (327.9 mg, 0.63 mmol), HoBt (85.2 mg, 0.63 mmol), and DIPEA (110 μL, 0.63 mmol) were weighed and ultrasonically dispersed in 10 mL of DCM / DMF; the resin peptide with FMOC removed was transferred to the reaction system and reacted at room temperature for 72 h. After the reaction, the product was washed with DMF and DCM, and ninhydrin was detected (2a) until the resin peptide reaction was complete. The product was washed with DMF, DCM, MeOH, ultrapure water, MeOH, DMF, and DCM, and then vacuum dried. A certain volume of cutting buffer (TFA / phenol / water, v / v / v = 90:5:5) was added, and cutting was performed for 3 hours. The cutting buffer was dispersed in 5 volumes of ice-cold diethyl ether, centrifuged, the supernatant was discarded, and the product was collected. The product was analyzed by ESI-MS and HPLC. Figure 2 The synthesis was verified to be successful.

[0033] Example 2: Preparation of CPT-ss-RGD self-assembled nanoparticles (CPT-ss-RGD NPs)

[0034] Weigh 9 mg of CPT-ss-RGD product and dissolve it in 1 mL of DMSO. Stir at room temperature for 1 h. Under magnetic stirring, add 9 mL of ultrapure water dropwise to the above solution and stir at room temperature for 1 h. After stirring, transfer the above assembly solution to a dialysis bag (1000 Dacut off) and dialyze for 72 h, changing the water every 2 h, and then freeze-dry.

[0035] Example 3: Determination of Critical Aggregation Concentration (CAC) of CPT-ss-RGD NPs

[0036] 6.7 mg of pyrene was weighed and added to 10 mL of acetone, and 20 μL was dispensed into each bottle. The acetone was evaporated to remove the acetone. A 2 mg / mL nanoparticle solution was prepared and then diluted to 0.01, 0.1, 1.0, 2.0, 5.0, 10, 20, 40, 60, 80, 100, 160, 200, and 250 μg / mL, and added to the respective bottles containing pyrene. After incubation in the dark for 24 h, the fluorescence emission spectrum at an excitation wavelength of 334 nm was measured. The CAC of the nanoparticles was determined by the intersection of the curves showing the logarithmic change in fluorescence intensity ratio (I373 / I383) with the concentration of the conjugate. The final CAC was determined to be 26.23 μg / mL (see [link to relevant documentation]). Figure 3 ).

[0037] Example 4: Changes in particle size, solubility, and optical properties of CPT-ss-RGD NPs

[0038] Lyophilized nanoparticle powder was dispersed in ultrapure water to prepare a concentration of 1 mg / mL, filtered through a 450 nm filter membrane, and used for DLS detection. One to two drops of the nanoparticle solution were added to a copper grid, dried, and then observed under a TEM microscope. The nanoparticle solution (containing 500 μg / mL CPT) and free CPT were dispersed in 1 mL of ultrapure water, and the dissolution was observed. The nanoparticle solution (containing 5 μg / mL CPT) and free CPT were dispersed in 1 mL of ultrapure water and used for UV-Vis spectroscopy and fluorescence emission spectroscopy (ex = 365 nm) detection. Figure 4 AB analysis showed that the DLS particle size of CPT-ss-RGD NPs was 183.1±28.9 nm, and TEM analysis showed that the nanoparticles were spherical with a particle size of 86.84±13.37 nm. Figure 4 The C-value shows that at a CPT concentration of 500 μg / mL, free CPT is turbid, while CPT-ss-RGD NPs are clear and transparent, indicating that the assembly of CPT-ss-RGD significantly improves the solubility of CPT. Figure 4 DE indicates that the maximum absorption peak shifted blue after assembly, the maximum absorbance value decreased, and the fluorescence emission intensity decreased, suggesting that the CPT state changed after assembly.

[0039] Example 5: CPT-ss-RGD NPs exhibit certain stability under room temperature storage.

[0040] A 1 mg / mL nanoparticle solution was left at room temperature, and the changes in DLS particle size and PDI were measured daily. Results ( Figure 5 The results show that although the nanoparticle size is increasing, it remains between 200-300 nm, indicating that the nanoparticles have a certain degree of size stability when placed at room temperature.

[0041] Example 6: Slow drug release from CPT-ss-RGD NPs under the action of reduced glutathione (GSH)

[0042] 1 mL of 1 mg / mL nanoparticle solution was transferred to a dialysis bag (MWCO = 1000 Da), which was then placed into a 15 mL centrifuge tube containing 10 mL of 0 or 10 mM GSH in PBS (pH = 7.4). The tube was then shaken at 37°C. At specific time points, 1 mL of dialysate was collected, and 1 mL of fresh 10 mM PBS solution was added. The obtained dialysate was freeze-dried, the product was dissolved in DMSO, fluorescence was detected, CPT was quantified, and drug release curves were plotted. Each group was tested in quadruplicate. Figure 6 The results showed that under 10 mM GSH, the cumulative release of CPT from CPT-ss-RGD NPs was 48.07% after 120 h, while under 0 mM GSH, the release rate was only 4.88%, indicating that GSH played a role in CPT release. In contrast, the control group CPT-cc-RGD nanoparticles showed a CPT release rate of only 5.59% after 120 h under 10 mM GSH, indicating that the -cc- group was not capable of being cleaved by GSH. The release rate of free CPT reached 99.90% after 8 h, indicating that CPT-ss-RGD NPs exhibited a certain sustained-release effect compared to free CPT.

[0043] Example 7: Targeted uptake of CPT-ss-RGD NPs by colon cancer cells

[0044] 5×10 5 SW480, CT26, MCF-7, and RAW 264.7 cells were seeded into laser confocal microscopy dishes and cultured in a cell culture incubator. After the cells reached 80% confluence, they were removed and 1 mL of complete culture medium containing CPT-ss-RGDNPs (CPT concentration of 80 μg / mL) was added. The cells were incubated for 8 h and the fluorescence was observed under a laser confocal microscope. Figure 7 The results showed that SW480 and CT26 cells treated with CPT-ss-RGD NPs exhibited stronger fluorescence compared to MCF-7 and RAW 264.7 cells, indicating that the nanoparticles can be targeted and taken up by colon cancer cells with high integrin expression. The CPT-ss-RGD NPs treatment group showed stronger fluorescence than the control group treated with CPT-cc-RGD NPs, suggesting that the -ss- bond plays an important role in CPT release.

[0045] Example 8: Toxicity of CPT-ss-RGD NPs to Different Cells

[0046] 1×10 5SW480, CT26, MCF-7, and RAW 264.7 cells were seeded in 96-well plates and cultured in a cell culture incubator until 80% confluence. Then, different concentrations of CPT (10, 20, 40, 60, 80, and 100 μg / mL) were added. -1 The nanoparticle solution and free CPT were incubated for a period of time, and then 10% CCK-8 was added. After 3 hours, the absorbance value at 450 nm was measured to calculate cell viability. Figure 8 The results showed that the cytotoxicity of CPT-ss-RGD NPs gradually increased over time, and its cytotoxicity to SW480 and CT26 cells gradually exceeded that of free CPT. The calculated IC50 values ​​of CPT-ss-RGD NPs against SW480 and CT26 cells were also observed. 50 The concentrations of CPT-ss-RGD NPs were 7.48 and 5.52 μg / mL, respectively, while those of CPT were 13.12 and 11.13 μg / mL, respectively, indicating that CPT-ss-RGD NPs had a stronger tumor cell killing ability than free CPT after 72 hours. Furthermore, RAW 264.7 cell viability analysis showed that the nanoparticles had lower toxicity than CPT, indicating that CPT-ss-RGD NPs can effectively kill colorectal cancer cells and have low toxicity to normal cells.

[0047] Example 9: CPT-ss-RGD NPs inhibit the growth of multicellular tumor spheres.

[0048] Multicellular tumor spheroids were prepared using the agarose gel electrophoresis method. When the diameter reached 300-500 μm, nanoparticle solution and free CPT (80 μg / mL) were added, and the cells were cultured in a cell culture incubator. Morphological changes of the tumor spheroids in each group were recorded at 0, 1, 3, and 5 days. The tumor spheroid volume Vi = (dmax × dmin) was calculated. 2 ) / 2(dmax and dmin: the longest and shortest sides of the tumor sphere). Results ( Figure 9 The results showed that after 5 days, CPT-ss-RGD NPs could better inhibit the growth of SW480 and CT26 tumor spheres compared to the CPT and CPT-cc-RGD NPs groups.

[0049] Example 10: CPT-ss-RGD NPs targeted localization of colon cancer tumors

[0050] For in vivo tracking, nanoparticles were loaded with the cyanine dye IR780. A tumor model was constructed using BALB / c nude mice, with each mouse inoculated with 5 × 10^6 CT26 cells until the tumor volume reached approximately 100 mm². 3 Three mice were injected via tail vein with either nanoparticles or CPT (4 mg CPT / kg). The mice were then placed under a small animal in vivo imaging system for near-infrared laser irradiation, and fluorescence changes were observed and recorded. Figure 10The results showed that after 48 hours, the fluorescence intensity of CPT-ss-RGD NPs at the tumor site was significantly stronger than that of CPT. After 72 hours, the fluorescence intensity of the tumor and internal organs of the dissected mouse was significantly higher than that of free CPT, indicating that the nanoparticles prolong the systemic circulation time and can better target and accumulate at the tumor site.

[0051] Example 11: CPT-ss-RGD NPs significantly inhibited the growth of colon cancer tumors in vivo and reduced toxic side effects.

[0052] When the tumor grows to approximately 100mm 3 Mice were injected intravenously with 100 μL of nanoparticles or CPT (4 mg CPT / kg) on ​​days 1, 3, and 5, with four mice in each group. Tumor volume changes were recorded every other day. After 11 days, mice were sacrificed, and tumors, heart, liver, spleen, lungs, and kidneys were collected. Tumor weight was measured, and tumor growth inhibition rate was calculated. Simultaneously, tumors and various tissues and organs were stained with hematoxylin-eosin (H&E) and TUNEL. Results ( Figure 11 The results showed that CPT-ss-RGD NPs inhibited tumor growth by 64%, significantly higher than CPT-cc-RGD NPs (27.7%) and free CPT (35.2%). H&E and TUNEL staining images showed that CPT-cc-RGD NPs significantly killed tumors and reduced the toxic side effects of CPT on the heart and liver.

Claims

1. A method for preparing self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates, characterized in that: An amphiphilic camptothecin conjugate, with a disulfide bond-coupled hydrophilic end containing the targeting peptide GRGDK and a hydrophobic end containing camptothecin, was synthesized and assembled using a reverse-phase solvent dispersion method, followed by dialysis to obtain nanoparticles. The coupling of the targeting peptide and the camptothecin was synthesized via an amidation reaction using a solid-phase synthesis method, as follows: (1) Synthesis of CPT-ss-OH: 174 mg of camptothecin, 55 mg of triphosgene and 195 mg of 4-dimethylaminopyridine were weighed and dissolved, and the reaction was carried out at room temperature in the dark for 3 h; 385 mg of 2,2-dithiodiethanol was mixed with tetrahydrofuran and added to the above solution, and the reaction was carried out at room temperature overnight; the product was diluted with dichloromethane and extracted with HCl, saturated NaCl and ultrapure water; the organic phase was dried with anhydrous Na2SO4 and filtered to separate; dichloromethane was removed by rotary evaporation and vacuum dried; the crude product was eluted by silica gel column chromatography gradient elution and vacuum dried to obtain a pale yellow solid; (2) Synthesis of CPT-ss-COOH: 200 mg of succinic anhydride, 264 mg of CPT-ss-OH and 195 mg of DMAP were weighed and dispersed in dichloromethane and stirred overnight at room temperature; the mixture was extracted with ultrapure water, 50 mM HCl aqueous solution and saturated NaCl; the organic phase was dried with anhydrous Na2SO4, dichloromethane was removed by rotary evaporation and dried under vacuum; the crude product was purified by gradient elution by silica gel column chromatography and dried under vacuum to obtain a pale yellow solid; (3) Synthesis of CPT-ss-RGD: Weigh 200 mg of Fmoc-GR(pbf)GD(tbu)K(boc)-Resin peptide into a peptide solid-phase synthesis tube, and soak the peptide in 10 mL of DCM / DMF solution for 2 h; after removing the solvent, take a small amount of peptide into an EP tube for ninhydrin detection; add 5 mL of 20% piperidine / DMF and react 3 times to remove the Fmoc group, and use a small amount for ninhydrin detection; weigh 131.9 mg of CPT-ss-COOH, 327.9 mg of PyBop, 85.2 mg of HoBt and 110 μL of DIPEA and sonicate them in 10 mL of DCM / DMF; transfer the resin peptide with FMOC removed to the reaction system and react at room temperature for 72 hours. h; after the reaction was completed, the product was washed with DMF and DCM respectively, and ninhydrin was detected until the resin peptide reaction was complete; then washed sequentially with DMF, DCM, MeOH, ultrapure water, MeOH, DMF, and DCM, and the product was dried under vacuum; a certain volume of cutting solution TFA / phenol / water was added, v / v / v=90:5:5, and cutting was performed for 3 h; the cutting solution was dispersed in 5 volumes of ice-cold diethyl ether, centrifuged, the supernatant was discarded, and the product was collected.

2. The method for preparing self-assembled targeted nanoparticles based on amphiphilic camptothecin conjugates according to claim 1, characterized in that: Weigh 9 mg of CPT-ss-RGD product and dissolve it in 1 mL of DMSO. Stir at room temperature for 1 h. Under magnetic stirring, add 9 mL of ultrapure water dropwise to the above solution and stir at room temperature for 1 h. After stirring, transfer the above assembly solution to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze for 72 h, changing the water every 2 h. Then freeze dry.

3. CPT-SS-RGD self-assembled targeted nanoparticles prepared according to the preparation method of any one of claims 1 to 2.

4. The application of the CPT-SS-RGD self-assembled targeted nanoparticles as described in claim 3 in the preparation of anti-colon cancer drugs.

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