Self-assembled nanotube of camptothecin-phospholipid conjugate as well as preparation method and application of self-assembled nanotube
By using camptothecin-phospholipid conjugates to self-assemble nanotubes and their click chemical modification, the problems of easy dissociation of nanostructures and uncontrollable drug loading in existing technologies have been solved, achieving precise drug release and synergistic therapeutic effects in tumor tissues while reducing toxicity risks.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINXIANG MEDICAL UNIV
- Filing Date
- 2026-01-09
- Publication Date
- 2026-04-17
AI Technical Summary
Existing supramolecular self-assembled nanostructures are prone to dissociation during chemical modification, making it difficult to achieve site-controlled loading of various drugs. This results in inconsistent drug leakage rates, uncontrollable drug loading ratios, and systemic toxicity risks.
A self-assembled nanotube was formed by linking camptothecin-phospholipid conjugates through disulfide bonds, and functional molecules were modified into the inner cavity of the nanotubes through click chemistry to prepare a dual-drug nanotherapy agent co-loaded with camptothecin and mitomycin. The hollow cavity and azide functional groups were used to achieve precise modification.
This approach achieves the stability of nanotube structures and the site-specific loading of functional molecules, significantly improving drug accumulation and release in tumor tissues, reducing systemic toxicity, and enhancing tumor inhibition rates and synergistic therapeutic effects.
Smart Images
Figure CN121868503A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical nanotechnology, and in particular relates to self-assembled nanotubes of camptothecin-phospholipid conjugates, their preparation methods, and applications. Background Technology
[0002] Supramolecular self-assembled nanostructures rely on dynamic non-covalent bonds (such as π-π stacking and hydrophobic interactions), resulting in inherent structural stability issues. Any subsequent chemical modification can easily disrupt their delicate non-covalent force balance, leading to assembly dissociation, morphological collapse, and ultimately, loss of function.
[0003] Furthermore, another major limitation of existing technologies lies in their limited functionality. In combination chemotherapy requiring precise drug ratios (such as the treatment of highly malignant triple-negative breast cancer), traditional nanocarriers struggle to achieve precise and site-controllable loading of multiple drugs onto the carrier. Conventional physical embedding or simple mixing methods result in inconsistent drug leakage rates and uncontrollable drug loading ratios, making it difficult to achieve ideal synergistic therapeutic effects and posing significant systemic toxicity risks.
[0004] Therefore, developing a structurally stable supramolecular nanoplatform that can achieve site-specific post-modification and can be used for the precise loading of various functional molecules (such as fluorescent probes and synergistic drugs) has become a technical challenge that urgently needs to be solved in this field. Summary of the Invention
[0005] In view of this, the present invention aims to propose a self-assembled nanotube of camptothecin-phospholipid coupling, its preparation method and application, in order to solve at least one technical problem in the background art.
[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A phospholipid-camptothecin coupling compound, which links phospholipids to camptothecin via a redox-responsive linker containing a disulfide bond, has the structural formula shown in (Ⅰ). (I).
[0007] Self-assembled nanotubes of camptothecin-phospholipid conjugates are formed by the self-assembly of the above-mentioned phospholipid-camptothecin conjugates. The supramolecular nanotubes have a hollow inner cavity, and the surface of the inner cavity is distributed with azide functional groups.
[0008] Furthermore, the self-assembled nanotubes of the camptothecin-phospholipid conjugate have a tubular structure with an outer diameter of 6-7 nm, an inner cavity diameter of 2-3 nm, and a length of 500 nm to 1 μm.
[0009] A method for preparing self-assembled nanotubes of the above-mentioned camptothecin-phospholipid conjugate includes the following steps: S1: Dissolve the above phospholipid-camptothecin coupling compound in an organic solvent to form an organic phase; S2: Mix the organic phase obtained in step S1 with an aqueous buffer solution to obtain a mixed system; S3: Remove the organic solvent from the mixed system prepared in step S2, and induce the phospholipid-camptothecin coupling to self-assemble and mature, forming a self-assembled nanotube of camptothecin-phospholipid coupling with azide functional groups in the inner cavity. Preferably, the final concentration of the phospholipid-camptothecin coupling compound in the aqueous solvent in step S1 is 0.1–5 mM, more preferably about 1.0 mM; Preferably, the organic solvent in step S1 is ethanol; Preferably, the aqueous buffer solution in step S2 is deionized water; Preferably, removing the organic solvent in step S3 involves dialysis of the mixed system obtained in step S2; Preferably, the time for inducing the self-assembly and maturation of the phospholipid-camptothecin conjugate in step S3 is 5-7 h.
[0010] A self-assembled nanotube of camptothecin-phospholipid conjugate with in-cavity functionalization is obtained by a click chemical reaction between the self-assembled nanotube of camptothecin-phospholipid conjugate prepared above and an alkyne-modified functional molecule, wherein the click chemical reaction is carried out in the cavity of the nanotube.
[0011] Preferably, a click chemistry reaction is carried out in the inner cavity of the nanotube under copper catalysis to anchor the functional molecules in the inner cavity; more preferably, the copper catalyst is copper sulfate pentahydrate. Preferably, a reducing agent, sodium ascorbate, is added to the click chemical reaction carried out in the inner cavity of the nanotube under copper catalysis. Preferably, after the click chemical reaction, a purification step is also included to remove unreacted alkynyl-modified functional molecules and catalysts.
[0012] Furthermore, the alkynyl-modified functional molecule is alkynyl-modified mitomycin, thus preparing a dual-drug nanotherapy agent (dPCC9NT–MMC) co-loaded with camptothecin and mitomycin. The drug loading rates of CPT and mitomycin in the dual-drug nanotherapy agent are 23.8% and 22.8%, respectively. In the dual-drug nanotherapy agent, CPT responds to the high glutathione microenvironment of tumors, while mitomycin is gradually released through the slow hydrolysis of its ester bonds.
[0013] Furthermore, the alkyne-modified functional molecule is an alkyne-modified fluorescent molecule, and self-assembled nanotubes of fluorescently labeled camptothecin-phospholipid conjugates are prepared, preferably alkyne-modified anthocyanin 5. 1 mol% Cy5-alkynyl groups are introduced into the dPCC9NT–MMC system to study the tumor-targeting ability of the dual-drug nanotherapy agent on 4T1 tumor-bearing mice. The inhibition rate of the dual-drug nanotherapy agent on 4T1 tumor-bearing mice is significantly higher than that of single drugs and physical drug mixtures.
[0014] A pharmaceutical composition comprising a dual-drug nanotherapy agent co-loaded with camptothecin and mitomycin prepared above, and a pharmaceutically acceptable carrier. In in vivo experiments, it exhibits a tumor inhibition rate of up to 85% and a significantly prolonged circulating half-life.
[0015] The use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating triple-negative breast cancer.
[0016] The self-assembled nanotubes of the fluorescently labeled camptothecin-phospholipid conjugate prepared above were applied to fluorescence imaging in vivo to track the distribution and targeting ability of nanocarriers in tumor model animals.
[0017] Compared with existing technologies, the self-assembled nanotubes of camptothecin-phospholipid couplings described in this invention, their preparation method, and their applications have the following advantages: 1. This application adopts a strategy of self-assembly followed by modification to successfully construct supramolecular nanotubes with hollow cavities. The structure is highly controllable in terms of the diameter and length of the outer and inner cavities, and maintains morphological integrity during subsequent click chemistry reactions without collapse or dissociation. The azide groups are uniformly distributed on the inner surface of the nanotube, providing precise sites for subsequent specific modification of the cavity through click chemistry, thereby achieving site-specific loading of functional molecules.
[0018] 2. This application uses intracavitary click chemistry to covalently anchor alkyne-modified mitomycin inside nanotubes, forming a dual-drug nanotherapy agent (dPCC9NT-MMC) co-loaded with camptothecin and mitomycin, with drug loading rates of 23.8% and 22.8%, respectively, achieving precise control of drug ratio.
[0019] 3. The nanotube structure of this application significantly prolongs the time the drug remains in the bloodstream, far exceeding that of free drug, which is beneficial for drug accumulation in tumor tissue. In vivo fluorescence imaging showed that Cy5-labeled nanotubes exhibited significantly enriched fluorescence signals at the tumor site, which remained stable within 24 hours, demonstrating their excellent passive tumor targeting capability.
[0020] 4. In a triple-negative breast cancer mouse model, the tumor inhibition rate of the dual-drug nanotherapy was as high as 85%, significantly higher than that of the single-drug or physical drug combination groups (p<0.01). H&E staining and TUNEL staining results showed that the tumor tissue in the treatment group exhibited large-area necrosis and apoptosis, demonstrating that the synergistic effect of the dual drugs significantly enhanced the anti-tumor effect.
[0021] 5. Compared with free MMC, the nanotube drug delivery system significantly reduced toxicity to the liver (ALT, AST) and kidneys (BUN, CRE), with stable mouse weight and no obvious pathological damage to major organs. Targeted drug delivery and controlled release via nanocarriers significantly improved biosafety while maintaining highly effective antitumor activity.
[0022] 6. The nanotube-intraluminal click chemistry platform of this application can flexibly graft functional molecules with different alkyne modifications (such as fluorescent probes, targeting ligands, and other drugs). This strategy is particularly suitable for refractory tumors requiring precise drug ratios and synergistic administration (such as triple-negative breast cancer) and has broad clinical application potential. Attached Figure Description
[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the structure of the azide-functionalized phospholipid-camptothecin conjugate (dPCC9-N3) described in Example 1 of the present invention; Figure 2 The 1H NMR spectrum of the product dPCC9-N3 obtained in Example 1 is shown below. 1 H-NMR is used to confirm its chemical structure; Figure 3 The mass spectrum (MS) of the product dPCC9-N3 obtained in Example 1 (used to further confirm its molecular weight and chemical structure); Figure 4 An atomic force microscope (AFM) image of the supramolecular nanotubes (dPCC9NT-N3) prepared in Example 2 (showing their nanotube morphology, outer diameter, and length distribution); Figure 5 High-resolution transmission electron microscope (TEM) image of the supramolecular nanotube (dPCC9NT-N3) prepared in Example 2 (clearly showing its hollow internal cavity structure); Figure 6 This is a schematic diagram of the structure of the alkyne-modified mitomycin in Example 3; Figure 7An atomic force microscope (AFM) image of the nanotube (dPCC9NT-MMC) after intracavitary click chemical modification of MMC in Example 3 (showing that the structure remains intact); Figure 8 The image shows a transmission electron microscope (TEM) image of the nanotube (dPCC9NT-MMC) after intracavitary click chemical modification of MMC in Example 3 (showing that the structure is not destroyed); Figure 9 The image shows the purified product obtained by click chemistry reaction of dPCC9NT-N3 and MMC-acetylene, characterized by liquid chromatography-mass spectrometry (LC-MS) in Example 3. Figure 10 The release curve of camptothecin from the dual-drug nanotherapy agent (dPCC9NT-MMC) of Example 4 in PBS solution containing 10 mM GSH (simulating tumor environment); Figure 11 This is a comparison of the release behavior of mitomycin C from the dual-drug nanotherapy agent of Example 4 under different pH conditions (pH 7.4 and pH 5.4). Figure 12 The in vivo pharmacokinetic curves of the dPCC9NT-MMC dual-drug nanotherapy agent described in Example 5 and the control group Cy5 (showing its significantly prolonged blood circulation half-life); Figure 13 The tumor growth curves of the dPCC9NT-MMC dual-drug nanotherapy agent and different control groups in the 4T1 tumor-bearing mouse model described in Example 6 are shown to demonstrate its synergistic anti-tumor effect. (a) is the curve showing the change in tumor volume with treatment time, which shows the tumor volume growth of different groups such as PBS, MMC, and dPCC9NT-N3 during the treatment process (0-20 days), and intuitively reflects the inhibitory trend of each treatment group on tumor growth. (b) is a bar chart of tumor inhibition rate, which compares the tumor inhibition ratio of different groups, reflects the difference in the inhibition effect of each group, and highlights the synergistic anti-tumor effect of dPCC9NT-MMC.) Figure 14 The results of tumor tissue section staining (H&E staining and TUNEL staining (showing cell necrosis and apoptosis in the dPCC9NT-MMC group tumor tissue)); Figure 15The chart shows a comparison of serum biochemical indicators (ALT, AST, BUN, CRE) in Example 7 (illustrating that the dPCC9NT-MMC group had lower hepatotoxicity and nephrotoxicity than the free MMC group; a is a bar chart showing the relative levels of serum biochemical indicators (ALT, AST) related to liver injury: ALT and AST are key indicators reflecting liver function, and the chart shows the ALT and AST levels of different groups, demonstrating that the hepatotoxicity of the dPCC9NT-MMC group was lower than that of the free MMC group; b is a bar chart showing the relative levels of serum biochemical indicators (CRE, BUN) related to kidney injury: CRE (creatinine) and BUN (blood urea nitrogen) are core indicators reflecting kidney function, and the chart compares the CRE and BUN levels of different groups, indicating that the dPCC9NT-MMC group had weaker nephrotoxicity). Figure 16 The curve of mouse body weight change during treatment (showing that the body weight of the dPCC9NT-MMC group was stable and there were no obvious toxic side effects). Figure 17 H&E stained sections of major organs (heart, liver, spleen, lung, kidney) (showing that the organ structures of the dPCC9NT-MMC group are intact and no obvious pathological damage is observed); Figure 18 This is a schematic diagram of the structure of an alkyne-modified Cy5 fluorescent probe (Cy5-alkyne); Figure 19 The UV-Vis spectrum of the nanotube (Cy5-dPCC9NT-MMC) after intracavity modification with Cy5 (showing the characteristic absorption peak of Cy5); Figure 20 The image shows the results of in vivo fluorescence imaging (comparing the fluorescence enrichment and retention at the tumor site between the Cy5-dPCC9NT-MMC group and the free Cy5 group). Detailed Implementation
[0024] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. In this invention, the following embodiments are provided to better illustrate the invention and are not intended to limit the scope of the invention. Any modifications or alterations made by those skilled in the art to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the invention's technical solution fall within the protection scope of this invention.
[0025] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.
[0026] Example 1: Preparation of azide-functionalized phospholipid-camptothecin conjugate (dPCC9-N3) Reference Figure 1The synthetic route shown indicates that dPCC9-N3 was prepared according to the literature method (Angew. Chem. Int. Ed. 2025, e202509799) under hydroxyl-activated conditions. Specifically, mPCC9 (90.7 mg, 0.1 mmol), 5-azidopentanoic acid (57.2 mg, 0.4 mmol), N,N'-diisopropylcarbodiimide (DIC, 100.8 mg, 0.8 mmol), and 4-dimethylaminopyridine (DMAP, 24.4 mg, 0.2 mmol) were added to a 50 mL round-bottom flask and dissolved in 10 mL dichloromethane (DCM). After reacting for 6 h, the mixture was directly subjected to silica gel column chromatography (dichloromethane:methanol:water = 65:25:4) to give a pale yellow solid (51.6 mg, yield: 50%). The product was analyzed by 1H-NMR. Figure 2 ) and mass spectrometry (MS, Figure 3 Structural verification Example 2: Preparation and characterization of supramolecular nanotubes (dPCC9NT-N3) A 10 mM ethanol stock solution of dPCC9-N3 was prepared. Under vigorous stirring, the stock solution was rapidly injected into deionized water to achieve a final concentration of 1.0 mM for dPCC9-N3. Stirring continued for 5 min after injection to ensure thorough dispersion. A slightly milky appearance was observed during this process, indicating the formation of a spontaneously self-assembled structure.
[0027] Subsequently, the obtained suspension was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed three times in 500 mL of deionized water for 6 h each time to remove residual ethanol. After dialysis, the clear dispersion was allowed to stand at room temperature for 6 h to promote further maturation of the self-assembled structure. The nanotube dispersion (dPCC9NT-N3) after standing treatment was directly characterized by atomic force microscopy. AFM observation showed that it formed nanotube-like structures with an outer diameter of 6-7 nm and a length of 500 nm-10 μm. Figure 4 High-resolution transmission electron microscopy (TEM) confirmed that it has a hollow structure with an internal cavity diameter of 2-3 nm. Figure 5 ).
[0028] Example 3: Click chemistry modification of drug molecules within the nanotube lumen to obtain a dual-drug nanotherapy agent (dPCC9NT–MMC) The alkynyl-modified mitomycin C was purchased from Xi'an Ruixi Biotechnology Co., Ltd. The alkynyl-modified mitomycin C (MMC-alkynyl) was... Figure 6For example, 196 μL of the nanotube dispersion (1.0 mM dPCC9NT-N3) prepared in Example 2 was taken, and MMC-acetylene (2 μL, 100 mM), sodium ascorbate (1 μL, 50 mM), and copper sulfate pentahydrate (1 μL, 10 mM) were added sequentially. The reaction mixture was gently stirred at room temperature and in the dark for 2 h. After the reaction was completed, the nanotubes were retained using an ultrafiltration centrifuge tube and washed three times with PBS buffer to completely remove unreacted drug molecules and catalysts, thus obtaining nanotubes with intracavitary covalently modified MMC.
[0029] Compared to unmodified nanotubes, its atomic force microscopy image ( Figure 7 ) and transmission electron microscope images ( Figure 8 No significant changes were observed, indicating that the click chemistry reaction did not destroy the overall structure of the nanotubes. Furthermore, the purified product obtained by the CuAAC reaction of dPCC9NT-N3 with the drug molecule alkyne substrate MMC-alkyne in the cavity, characterized by liquid chromatography-mass spectrometry (LC-MS), was further confirmed. Figure 9 ).
[0030] The drug loading rates of CPT and mitomycin were determined to be 23.8% and 22.8%, respectively, by high performance liquid chromatography.
[0031] Example 4: Drug release monitoring of dual-drug nanotherapy agent (dPCC9NT–MMC) The dPCC9NT-MMC nanotubes prepared in Example 3 were incubated in a phosphate-buffered saline (PBS) buffer solution (pH 7.4) containing 10 mM glutathione (GSH, simulating a tumor environment). Analysis showed that camptothecin was rapidly released in the PBS solution containing 10 Mm GSH, with approximately 90% released within 48 h. Figure 10 Nanotubes were placed in PBS solutions of different pH values, and the results showed ( Figure 11 MMC is gradually released due to the hydrolysis of its carbonate bonds, and the release amount is greater in acidic solution (pH=5.4, simulating tumor environment) than in neutral solution (pH=7.4, simulating normal in vivo environment).
[0032] Example 5: Circulating half-life of a dual-drug nanotherapy agent (dPCC9NT–MMC) Pharmacokinetic study data indicate that ( Figure 12 The dual-drug nanotherapy agent (dPCC9NT-MMC) showed a significant advantage in terms of blood circulation half-life, with its blood circulation half-life (t) 1 / 2 =3.6 h) is significantly longer than free Cy5 (t 1 / 2 =0.11h), which directly supports the technical effect of "significantly extended cyclic half-life" described in the invention.
[0033] Example 6: Evaluation of the in vivo antitumor effect of the dual-drug nanotherapy agent dPCC9NT-MMC Using 4T1 tumor-bearing BALB / c mice as a model, the therapeutic effect of the dual-drug nanotherapy agent prepared in Example 4 was evaluated by monitoring the tumor inhibition rate of mice over 21 days. The experiment included a blank control group, a free mitomycin C group, a supramolecular nanotube (dPCC9NT-N3) group, a supramolecular nanotube (dPCC9NT-N3) and free mitomycin C group, and a dPCC9NT-MMC group. Each therapeutic agent was administered via tail vein injection on days 0, 2, 4, 10, 12, and 14, with CPT and MMC concentrations controlled at 5.0 mg / kg. -1 and 4.8 mg kg -1 .
[0034] The results showed that ( Figure 13 In the 4T1 tumor-bearing mouse model, the dual-drug nanotherapy agent (dPCC9NT–MMC) showed a tumor inhibition rate of up to 85% after 21 days of administration, which was significantly more synergistic than the free mitomycin group (41%), dPCC9NT-N3 group (60%), supramolecular nanotubes (dPCC9NT-N3) and free mitomycin group (68%) (p<0.01). Camptothecin and mitomycin showed a significant synergistic anti-tumor effect within the nanotubes.
[0035] Twenty-one days after drug administration, mice were sacrificed, and tumor tissue was dissected for histological analysis. Tumor tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL staining) were then used for observation. H&E staining results showed that, compared with the PBS-treated group, the free MMC-treated group, and the blank nanotube-treated group, the tumor tissue treated with dPCC9NT-MMC showed large areas of cell necrosis, while the tumor tissue in the control group had a relatively intact structure, with densely packed tumor cells, and was still predominantly composed of surviving cells. TUNEL staining results showed that the number of apoptotic cells in the tumor tissue of the dPCC9NT-MMC-treated group was significantly higher than that in the PBS group, the free MMC group, and the blank nanotube group, indicating that the dPCC9NT-MMC nanotube system can induce a higher level of apoptosis in tumor tissue. Figure 14 ).
[0036] Example 7: Safety evaluation of the dual-drug nanotherapy agent dPCC9NT–MMC To evaluate the in vivo safety of the dPCC9NT–MMC nanotube system, blood samples were collected from experimental animals in each treatment group at predetermined time points after the tumor treatment experiment, and serum biochemical indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE), were detected.
[0037] The test results showed that, compared with the free MMC treatment group, the levels of ALT, AST, BUN, and CRE in the dPCC9NT–MMC treatment group were close to the baseline control levels, while the above indicators in the free MMC treatment group were significantly increased, suggesting that free MMC has a certain impact on liver and kidney function. Figure 15 ).
[0038] Meanwhile, the changes in body weight of the animals in each group were continuously monitored during the experiment, and the results showed that ( Figure 16 The weight of animals in the dPCC9NT–MMC treatment group remained stable, with no significant downward trend. Further tissue samples from major organs, including the heart, liver, spleen, lungs, and kidneys, were collected for histological sectioning and hematoxylin-eosin (H&E) staining. The results showed that the major organ structures in the dPCC9NT–MMC treatment group remained intact, with no obvious tissue damage or pathological abnormalities observed. Figure 17 ).
[0039] The above results indicate that by covalently integrating MMC into supramolecular nanotube structures, the systemic toxicity in vivo can be effectively reduced while maintaining the antitumor effect, thereby improving the biosafety of the formulation.
[0040] Example 8: Study on the in vivo distribution of the dual-drug therapeutic agent First, the inner cavity of the nanotube was chemically modified with fluorescent molecule Cy5 to obtain Cy5–dPCC9NT–MMC. Alkyne-modified anthocyanin 5 (Cy5-acetylenic...) Figure 18 The nanotubes were purchased from Xi'an Ruixi Biotechnology Co., Ltd. Using Cy5-acetylene as the imaging agent, 196 μL of the nanotube dispersion (1.0 mM dPCC9NT-MMC) prepared in Example 3 was taken, and acetylenic-Cy5 dye (2 μL, 10 μM), sodium ascorbate (1 μL, 50 mM), and copper sulfate pentahydrate (1 μL, 10 mM) were added sequentially. The reaction mixture was gently stirred for 2 h at room temperature in the dark. After the reaction, the nanotubes were retained using an ultrafiltration centrifuge tube and washed three times with PBS buffer to completely remove unreacted dye and catalyst, obtaining nanotubes covalently modified with Cy5 and MMC in the cavity. The nanotubes were then analyzed by UV-Vis spectroscopy (UV-Vis spectroscopy). Figure 19 Characterizing the product revealed characteristic peaks of Cy5, proving that Cy5 had been successfully attached to the nanotube.
[0041] Mice were randomly divided into groups, with a control group receiving a tail vein injection of free Cy5. Results showed that Cy5-dPCC9NT-MMC gradually produced significant fluorescence signal enrichment at the tumor site, reaching a high fluorescence intensity approximately 12 hours post-administration, and the fluorescence signal was still clearly detectable in the tumor area 24 hours post-administration. As a control, an equal amount of free Cy5 was injected into tumor-bearing mice in the same manner. The results indicated that the fluorescence signal of free Cy5 decayed more rapidly over time in vivo, with significantly lower fluorescence intensity at the tumor site compared to the Cy5-dPCC9NT-MMC group, and a shorter overall retention time. Figure 20 This indicates that the nanotube system can be effectively enriched in tumor tissue through passive targeting.
[0042] Comparative Example In contrast, when alkynyl-Cy5 was reacted with unself-assembled dPCC9-N3 monomers under the same click chemistry conditions, the reaction products could not self-assemble into regular nanotube structures. Instead, they formed amorphous aggregates, and the tubular structure could not be observed by either atomic force microscopy (AFM) or transmission electron microscopy (TEM).
[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A phospholipid-camptothecin coupling compound, characterized in that: Phospholipids are linked to camptothecin via redox-responsive linkers containing disulfide bonds, as shown in (Ⅰ); （Ⅰ）。 2. Self-assembled nanotubes of camptothecin-phospholipid coupling, characterized by: The phospholipid-camptothecin coupling compound of claim 1 is self-assembled to form a camptothecin-phospholipid coupling compound self-assembled nanotube, wherein the camptothecin-phospholipid coupling compound self-assembled nanotube has a hollow inner cavity, and the surface of the inner cavity is distributed with azide functional groups.
3. The self-assembled nanotubes of camptothecin-phospholipid coupling according to claim 2, characterized in that: The self-assembled nanotubes of the camptothecin-phospholipid conjugate have a tubular structure with an outer diameter of 6-7 nm, an inner cavity diameter of 2-3 nm, and a length of 500 nm to 1 μm.
4. A method for preparing self-assembled nanotubes of camptothecin-phospholipid coupling as described in claim 2 or 3, characterized in that, Includes the following steps: S1: Dissolve the phospholipid-camptothecin coupling compound according to claim 1 in an organic solvent to form an organic phase; S2: Mix the organic phase obtained in step S1 with an aqueous buffer solution to obtain a mixed system; S3: Remove the organic solvent from the mixed system prepared in step S2, and induce the phospholipid-camptothecin coupling to self-assemble and mature, forming a self-assembled nanotube of camptothecin-phospholipid coupling with azide functional groups in the inner cavity. Preferably, the final concentration of the phospholipid-camptothecin coupling compound in the aqueous solvent in step S1 is 0.1–5 mM, more preferably about 1.0 mM; Preferably, the organic solvent in step S1 is ethanol; Preferably, the aqueous buffer solution in step S2 is deionized water or a buffer solution; Preferably, removing the organic solvent in step S3 involves dialysis of the mixed system obtained in step S2; Preferably, the time for inducing the self-assembly and maturation of the phospholipid-camptothecin conjugate in step S3 is 5-7 h.
5. A self-assembled nanotube of camptothecin-phospholipid conjugate with intracavitary functionalization, characterized in that: The self-assembled nanotubes of camptothecin-phospholipid coupling prepared according to claim 4 are subjected to click chemistry with alkyne-modified functional molecules to obtain self-assembled nanotubes of camptothecin-phospholipid coupling with in-cavity functionalization, wherein the click chemistry reaction is carried out in the cavity of the nanotube. Preferably, a click chemistry reaction is carried out in the inner cavity of the nanotube under copper catalysis to anchor the functional molecules in the inner cavity; more preferably, the copper catalyst is copper sulfate pentahydrate. Preferably, a reducing agent, sodium ascorbate, is added to the click chemical reaction carried out in the inner cavity of the nanotube under copper catalysis. Preferably, after the click chemical reaction, a purification step is also included to remove unreacted alkynyl-modified functional molecules and catalysts.
6. The self-assembled nanotube of camptothecin-phospholipid conjugate with intracavitary functionalization according to claim 5, characterized in that: The alkyne-modified functional molecule is alkyne-modified mitomycin, and a dual-drug nanotherapy agent co-loaded with camptothecin and mitomycin is prepared.
7. The self-assembled nanotube of camptothecin-phospholipid conjugate with intracavitary functionalization according to claim 5, characterized in that: The alkyne-modified functional molecule is an alkyne-modified fluorescent molecule, which is used to prepare self-assembled nanotubes of fluorescently labeled camptothecin-phospholipid conjugates, preferably alkyne-modified anthocyanin 5.
8. A pharmaceutical composition, characterized in that: This includes the dual-drug nanotherapy agent co-loaded with camptothecin and mitomycin prepared according to claim 6, and a pharmaceutically acceptable carrier.
9. Use of the pharmaceutical composition of claim 8 in the preparation of a medicament for treating triple-negative breast cancer.
10. The self-assembled nanotubes of the fluorescently labeled camptothecin-phospholipid conjugate prepared according to claim 7 are used for fluorescence imaging in vivo, characterized in that: Used to track the distribution and targeting ability of nanocarriers in tumor model animals.