A supramolecular material capable of loading and targeted release of camptothecin drugs and its preparation method and application

Abstract

The present invention discloses a supramolecular material with loading and targeted release of camptothecin drugs, and its preparation method and application. The molecular structure of the supramolecular material is that the upper and lower surfaces are respectively a porphyrin molecule with four benzylamine structures, and the benzylamine structures of the porphyrin molecules on the upper and lower surfaces are connected one by one by four groups of linear terephthaloyl chloride structures with two pyridinium salt positive charges in the middle, and the connection nodes are amide bonds. The specific structural formula is as follows: X is a methyl, ethyl, isopropyl or isobutyl group. The present invention mainly addresses the problems of insufficient water solubility, biocompatibility and poor targeting of camptothecin drugs. By introducing a supramolecular material with recognition and stable encapsulation capabilities for such drugs, it has good solubility in water without modifying the drug molecules. At the same time, the supramolecular material encapsulating the drug can keenly sense the pH changes in the surrounding environment of the lesion, complete the targeted release and enrichment of the drug, and significantly improve the efficacy.

Description

Technical Field

[0001] The invention belongs to the technical field of new material synthesis and supramolecular application, and specifically relates to a supramolecular material capable of loading and targeted releasing camptothecin drugs, and a preparation method and application thereof. Background Art

[0002] Camptothecin (CPT) is a pyrroloquinoline cytotoxic alkaloid. Studies have shown that it has significant therapeutic effects on a variety of malignant tumors such as gastric cancer, colorectal cancer and leukemia. In recent years, through the modification of sites on the aromatic ring of CPT, camptothecin anti-tumor drugs with better anti-tumor activity and lower toxic side effects such as topotecan (TPT), irinotecan (CPT-11), and hydroxycamptothecin (HCPT) have been further obtained. However, this type of drug still has the disadvantages of poor water solubility and reduced anti-tumor activity after being made into water-soluble sodium salts, which greatly limits the clinical use of this type of drug.

[0003] In order to solve the water solubility problem of this type of drugs, researchers have developed a variety of drug delivery systems, which enhance the water solubility of drugs while also playing the role of targeted delivery. The most widely used methods include liposomes, hydrogels, and micelles. However, the phospholipid bilayer structure of liposomes is not stable. During the drug delivery process, once external environmental factors such as ionic strength, acidity and alkalinity change, the bilayer is prone to hydrolysis, causing the active ingredients to leak, affecting bioavailability. For example, patent CN 112933046 B discloses a doxorubicin prodrug active drug-loaded liposome and its preparation method and application. It is found that the phospholipid bilayer of the doxorubicin dimer prodrug active drug-loaded liposome is decomposed in the release medium containing 5mM GSH, and about 60% of the drug is released after 24 hours. Hydrogel is a gel material formed by physical or chemical cross-linking. The large amount of water filled in its three-dimensional network structure helps to maintain good affinity between the hydrogel and the organism. It is often used in medicine as an ointment substrate to promote wound healing. The anti-cancer drug molecules encapsulated in it can also have therapeutic effects on diseases such as tumors. However, the current hydrogel drug carriers have disadvantages such as poor mechanical properties, low drug loading rate, unstable release rate and lack of drug delivery function, and their therapeutic effects are not ideal.

[0004] In summary, improving the water solubility of camptothecin drug molecules while ensuring the stability of drug molecules during transportation and their enrichment level in the lesion area is the prerequisite for ensuring their efficient treatment of tumor diseases. Therefore, a new type of anti-tumor drug carrier that takes into account both stability and targeting is urgently needed to solve the above technical problems. Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the object of the present invention is to provide a supramolecular material capable of loading and targeted releasing camptothecin drugs, and a preparation method and application thereof.

[0006] The microstructure of the supramolecular material of the present invention is a cubic molecule with a rigid preassembled cavity, the upper and lower surfaces of the molecule carry porphyrin groups and are connected by four groups of linear terephthalene disubstituted structures with two pyridinium salt positive charges in the middle, and the connecting nodes are amide bonds. The supramolecular material has a strong binding ability with camptothecin molecules, because: 1) driven by hydrophobic interaction, non-polar drug molecules will actively enter the cavity of the supramolecular material to form a stable complex; 2) camptothecin molecules have two types of electron-withdrawing groups, carbonyl and sp2 hybridized nitrogen atoms, which make them electron-deficient as a whole, while the porphyrin structure of the supramolecular material of the present invention is an electron-rich aromatic system with 24 centers and 26 electrons, and π-π interactions can be formed between the two; 3) camptothecin molecules can form at least three hydrogen bonds with the amide structure of the supramolecular material.

[0007] The supramolecular material of the present invention is an octavalent positive ion in water, has ideal water solubility and a rigid hydrophobic cavity, and can encapsulate water-insoluble camptothecin molecules in the cavity to enhance their solubility in water. Due to the Warburg effect, the pH values ​​of tumor tissue and normal tissue are very different. The extracellular environment of tumor cells is acidic, while that of normal tissue is neutral. When the supramolecular material encapsulating the drug approaches the tumor tissue, the hydrogen bonds are broken by the impact of hydrogen ions, allowing the drug molecules to leave the cavity and enter the lesion site, thereby achieving targeted release. Finally, the counterions of the supramolecular material are converted to recover and reuse it.

[0008] The technical solution adopted by the present invention is as follows:

[0009] A supramolecular material capable of loading and targeted release of camptothecin drugs, wherein the molecular structure of the supramolecular material is that the upper and lower surfaces are respectively a porphyrin molecule with four benzylamine structures, and the benzylamine structures of the porphyrin molecules on the upper and lower surfaces are connected one by one by four groups of linear terephthaloyl chloride structures with two pyridinium salt positive charges in the middle, and the connection nodes are amide bonds. The specific structural formula is as follows:

[0010]

[0011] In the above structural formula, X is methyl, ethyl, isopropyl or isobutyl;

[0012] -Ar- is one of the following structures:

[0013] .

[0014] The present invention also provides a method for preparing the supramolecular material capable of loading and targeted release of camptothecin drugs, which is prepared by amidation reaction of tetrabenzylamine precursor molecule TPR with a porphyrin structure and divalent diacyl chloride linear precursor molecule DDC with a pyridine structure. The specific reaction steps are as follows:

[0015] Step 1: Remove the amino protecting group and dry TPR and react with DDC in a solvent at room temperature with stirring;

[0016]

[0017] Step 2: After the reaction is completed, dilute hydrochloric acid is added to the system for acidification, and the mixture is filtered and washed to obtain the target product with chloride. Water and ammonium hexafluorophosphonate are added to the product for reaction to obtain the target product with hexafluorophosphonate.

[0018] Furthermore, in step 1, the molar ratio of TPR to DDC is 1:2-2.5.

[0019] Furthermore, the molar ratio of TPR to DDC is 1:2.1-2.2.

[0020] Furthermore, the operation steps of step 1 are: TPR is dissolved in an appropriate amount of anhydrous tetrahydrofuran to obtain a TPR solution, and the molar concentration of the TPR solution is controlled at 18-29 mmol / L; DDC is dissolved in an appropriate amount of acetonitrile to obtain a DDC solution, and the molar concentration of the DDC solution is controlled at 4.2-5.1 mmol / L; then the TPR solution is added dropwise to the DDC solution, and after the addition is completed, stirring is continued at room temperature for 6-10 hours.

[0021] Furthermore, the stirring time at room temperature is 7-8 hours.

[0022] Furthermore, the concentration of the dilute hydrochloric acid in step 2 is 0.5-2M.

[0023] The present invention also provides the use of the supramolecular material capable of loading and targeted releasing camptothecin drugs in the preparation of a drug delivery system, wherein the drug delivered is a camptothecin drug.

[0024] The supramolecular material involved in the present invention can realize the loading of camptothecin drugs, specifically including camptothecin (CPT), topotecan (TPT), irinotecan (CPT-11), hydroxycamptothecin (HCPT), exitecan (DX-8951f), 9-aminocamptothecin, rubitecan (9-nitrocamptothecin), lutetotecan (GG-211), gimatidecan (ST1481), karenitecin (BNP-1305), etc.

[0025] Camptothecin drug molecules are non-polar molecules. Since they are not water-soluble, they tend to avoid water and aggregate with each other in an aqueous environment. The supramolecular material of the present invention has a rigid hydrophobic cavity. The water molecules in the cavity are relatively orderly aggregated through hydrogen bonding. When drug molecules appear, they will actively enter the cavity driven by the hydrophobic effect and squeeze out the water molecules therein to form a stable complex.

[0026] Camptothecin drug molecules are all planar and carry carbonyl groups with strong electron-withdrawing properties and sp2-hybridized nitrogen atoms. Therefore, the drug molecules are electron-deficient as a whole, which just complements the electron-rich aromatic system porphyrin with 24 centers and 26 electrons. When the two are close to each other, they can form a "face-to-face" π-π interaction and enhance the binding ability; the carbonyl oxygen and pyridine nitrogen atoms on the cyclohexanolactone and caprolactam structures of camptothecin molecules can form at least triple hydrogen bonds with the amide hydrogen atoms on the supramolecular material. Driven by the above three effects, supramolecular materials have an ideal loading capacity for camptothecin drugs. By drawing the Job-Plot working curve, it was determined that the binding mode is [1+2], that is, one supramolecular carrier can load two drug molecules. Isothermal calorimetric titration experiments (ITC) show that the binding constant (K a ) can reach 10 4 Order of magnitude.

[0027] See the schematic diagram of the structural interaction between the supramolecular material of the present invention and the drug molecule. Figure 17A-17B The binding of this material to drug molecules is driven by the triple forces of hydrophobic interaction, hydrogen bonding and π-π interaction. This figure is an interpretation of the sites and principles of the latter two forces.

[0028] After the supramolecular material is loaded with drugs, it can be injected into the blood circulation system of the body. The extracellular pH value of normal cells is about 7.4, which is neutral, and the extracellular pH value of tumor cells is about 6.0, which is weakly acidic. The drug-loaded supramolecular material will not be released near normal cell tissues. In the vicinity of cancerous tissues, it is affected by the acidic environment, and the hydrogen bonds that serve as anchor points between drug molecules and the supramolecular material cavity are destroyed, thereby achieving targeted release of drug molecules. The drug release behavior was further investigated under different pH conditions, and the experimental results showed that supramolecular materials have sustained release properties for drug molecules.

[0029] The present invention proposes a new method for preparing a supramolecular material capable of loading and targeted release of camptothecin drugs. The supramolecular material relies on its own rigid hydrophobic cavity, and uses the triple forces of hydrophobic interaction, π-π interaction and hydrogen bonding as driving force to achieve the loading of camptothecin and its derivatives. The drug-loaded supramolecular material will not release drugs near normal cell tissues. In the acidic environment near cancerous tissues, hydrogen bonds are destroyed, thereby achieving targeted release of drug molecules. The supramolecular material proposed by the present invention has significant advantages such as simple production process, stable physical and chemical properties during storage and use, and strong binding ability to camptothecin drug molecules. It has good application prospects in the field of disease-targeted functional material development.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] 1) The present invention mainly addresses the problems of insufficient water solubility, biocompatibility and poor targeting of camptothecin drugs. By introducing a supramolecular material that has the ability to recognize and stably encapsulate such drugs, it enables them to have good solubility in water without modifying the drug molecules. At the same time, the supramolecular material encapsulating the drug can keenly sense the pH changes in the surrounding environment of the lesion, complete the targeted release and enrichment of the drug, and significantly improve the efficacy.

[0032] 2) The supramolecular material is a rigid covalent bond structure with a preassembled cavity, has strong physical and chemical stability in the biological environment, and has a simple synthesis step, which is easy to recycle and reuse. The supramolecular material and the drug molecules are non-covalently bonded, and the drug loading and release process is simple to operate.

[0033] 3) This supramolecular material has a porphyrin structure and can be used as a photosensitizer in photodynamic therapy. It can produce singlet oxygen that has strong destructive power against cancer cells under irradiation with a specific wavelength. In the future, its potential as a drug targeted delivery carrier while also taking into account the main body of photodynamic therapy can be further explored. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the H NMR spectrum of compound A1;

[0035] Figure 2 is the carbon NMR spectrum of compound A1;

[0036] Figure 3 is the H NMR spectrum of compound B1;

[0037] Figure 4 is the carbon NMR spectrum of compound B1;

[0038] Figure 5 This is the H NMR spectrum of compound PYTcage-PF6;

[0039] Figure 6 This is the carbon NMR spectrum of the compound PYTcage-PF6;

[0040] Figure 7 The compound PYTcage-PF6 1 H- 1 H COSY spectrum;

[0041] Figure 8 is the DOSY spectrum of compound PYTcage-PF6;

[0042] Fig. 9 is the HRMS-ESI spectrum of compound PYTcage-PF6;

[0043] Fig.10 It is the H NMR spectrum of the compound PYTcage-Cl, camptothecin and the complex after loading the drug;

[0044] Fig.11 This is the UV / Vis absorption spectrum of the compound PYTcage-Cl after loading the drug;

[0045] Fig.12 It is the Job-Plot working curve of camptothecin and compound PYTcage-Cl at different molar ratios;

[0046] Fig.13 is the complexation isothermal calorimetric titration curve of compound PYTcage-Cl and camptothecin;

[0047] Fig.14 It is the complexation isothermal calorimetric titration curve of compound PYTcage-Cl and hydroxycamptothecin;

[0048] Fig.15 is the complexation isothermal calorimetric titration curve of compound PYTcage-Cl and topotecan;

[0049] Fig.16 It is the in vitro release rate curve of the supramolecular material loaded with drugs;

[0050] Fig.17A It is a schematic structural diagram of the hydrogen bonding interaction between the supramolecular material of the present invention and the drug molecules;

[0051] Fig. 17B It is a schematic structural diagram of the π-π interaction between the supramolecular material of the present invention and the drug molecule;

[0052] Fig.18 It is a flow chart of the whole process simulation experiment of in vitro biology performed by PYTcage-Cl of the present invention in a laboratory environment. DETAILED DESCRIPTION

[0053] The following is a more detailed description of the present invention in conjunction with the accompanying drawings and the accompanying drawings, so that those skilled in the art can implement the invention after reading the specification. It should be understood that the specific embodiments described herein are for illustration only.

[0054] Embodiment 1, this embodiment comprises the following steps:

[0055] Step 1) Synthesis of compound S1:

[0056] 3-Bromo-4-methylbenzaldehyde (50mmol), hydroxylamine hydrochloride (60mmol) and pyridine (10mL) were dissolved in dichloromethane (120ml). Then, the mixture was heated and stirred at 313K for 6h, the reaction mixture was dissolved in water, the aqueous phase was extracted 4 times with ethyl acetate (40ml), the organic layer was dried with magnesium sulfate, filtered and the filtrate was collected. After the solvent was removed by rotary evaporation, the crude sample was purified by recrystallization, specifically, the crude product was dissolved in 100ml of ethyl acetate, shaken and dispersed evenly, and then heated in a water bath until completely dissolved, cooled to room temperature, and anhydrous ether was added dropwise until white crystals were completely precipitated, which was the purified product S1 (yield 84%) as a white solid. 1 H NMR (400 MHz, CDCl3): δ = 8.07 (s, 1H), 7.75 (s, 1H), 7.41 (d, J = 7.6Hz, 1H), 7.24 (d, J = 7.6Hz, 1H), 2.41 (s, 3H). 13 C NMR (100 MHz, CDCl3): δ = 149.0, 139.9, 131.3, 131.0, 130.7, 125.8, 125.3, 22.9. HRMS-ESI of compound S1, Calcd of C8H8BrNO: m / z = 213.9789 [M+H] + , found: 213.9722[M+H] + .

[0057]

[0058] Step 2) Synthesis of compound S3:

[0059] Activation of zinc powder: Weigh 100g of zinc powder and add it to 200ml of 5% hydrochloric acid solution, stir for 5 minutes, then filter to obtain the activated zinc powder, wash it with distilled water and anhydrous ether several times, and then dry it for use.

[0060] Compound S1 (23.5 mmol) was dissolved in 100 mL of acetic acid, and then activated zinc powder (10 g, 156 mmol) was added. The solution was stirred at room temperature for 12 hours. After the reaction was completed, the insoluble matter was removed by filtration and washed several times with ethyl acetate, and the solvent was removed by rotary evaporation. Then 200 ml of 5% sodium hydroxide aqueous solution was added, the water layer was extracted three times with ethyl acetate, the organic layer was collected and washed with brine, and magnesium sulfate was added to dry. After drying, the solvent was removed by rotary evaporation to obtain the intermediate product S2 as a colorless oily compound, and then compound S2 (20 mmol) was dissolved in methanol (100 mL), and then triethylamine (4 ml) and Boc2O (22 mmol) were added, and the reaction mixture was stirred at room temperature overnight. After that, the reaction mixture was cooled to 0 ° C, and then a dilute hydrochloric acid solution (1M, 20 mL) was slowly added. The organic layer was collected, the aqueous phase was re-extracted with dichloromethane (10 mL), and then the organic phase was washed with a saturated sodium bicarbonate aqueous solution (10 mL), and then dried with magnesium sulfate. The solvent was removed by rotary evaporation to afford compound S3 (yield 74%), which was used in the next step without further purification. 1 HNMR (400MHz, CDCl3): δ=7.43 (s, 1H), 7.16 (d, J=7.6Hz, 1H), 7.10 (d, J=7.6Hz, 1H), 4.89 (s, 1H), 4.23 (s, 2H), 2.35 (s, 3H), 1.45 (s, 9H).13C NMR (100 MHz, CDCl3): δ=155.8, 138.4, 136.7, 131.1, 130.8, 126.3, 124.9, 43.7, 28.4, 27.4. HRMS-ESI for S3, C 13 H 18 Calcd of BrNO2: m / z=300.0521[M+H] + , found: 300.0577[M+H] + .

[0061]

[0062] Step 3) Synthesis of Compound A1:

[0063] Commercially available tetraphenyl borate porphyrin (1.34 mmol), compound S3 (8.1 mmol) and tetrakis(triphenylphosphine)palladium (80 mg) were dissolved in 1.4-dioxane (70 mL). After complete dissolution, potassium carbonate aqueous solution (2M, 15 mL) was added, nitrogen was passed for 30 min to ensure that the oxygen in the solvent had been completely removed, and the mixture was vigorously stirred at 373 K for 48 hours. After the reaction was completed, the mixture was cooled to room temperature and poured into deionized water (150 mL). The aqueous layer was extracted with dichloromethane three times, and the organic phase was collected and washed with water and dried with magnesium sulfate. After removing the solvent by rotary evaporation, the crude sample was purified by flash column chromatography (volume ratio EA:PE=1:1, silica gel 200-300 mesh) to obtain compound A1 (yield is 44%) as a gray-brown solid. 1 H NMR (400 MHz, CDCl3): δ=7.74 (m, 8H), 7.67 (m, 8H), 7.33 (m, 4H), 3.32 (m, 4H), 7.29 (m, 4H), 4.91 (s, 4H), 4.36 (s, 6H), 2.07 (m, 12H), 1.42 (m, 48H). 13 C NMR (100 MHz, CDCl3): δ=155.9, 140.6, 136.8, 136.6, 136.1, 130.2, 129.6, 128.8, 128.3, 126.8, 125.3, 44.4, 28.4, 24.8, 20.0. HRMS-ESI of A1, C 96 H 98 Calcd of B4N8O8: m / z=1441.7726[M+H] + , found: 1441.7785[M+H] + .

[0064]

[0065] Step 4) Synthesis of Compound B1

[0066] Dissolve isonicotinoyl chloride (42.5mmol) and p-diiodobenzene (7mmol) in 60ml acetonitrile solution, and stir the mixture vigorously at 353K for 48 hours. After the reaction is completed, cool the mixture to room temperature, filter to obtain a brown-yellow solid, wash it several times with acetonitrile, and dry it to obtain the intermediate product S4 (3.7g, yield is 86%). Dissolve the dried S4 in 150ml water, shake it thoroughly until it is completely dissolved, then add ammonium hexafluorophosphonate (2.9g, 18mmol) to the system, filter to obtain a light yellow solid, wash it several times with water, and dry it to obtain compound B1 (3.5g, yield is 90%) as a light yellow solid. 1H NMR (400MHz, CDCl3): δ=7.23 (d, 4H), 7.22 (s, 4H), 7.21 (d, 4H). 13 C NMR (100 MHz, CDCl3): δ=141.7, 141.4, 136.4, 134.5, 130.7, 129.1, 128.4, 126.6. HRMS-ESI of B1, C 18 H 12 Cl2F 12 Calcd of N2O2P2: m / z=648.9573[M+H] + , found: 648.9511[M+H] + .

[0067]

[0068] Step 5) Synthesis of compound PYTcage:

[0069] Trifluoroacetic acid (10 mL, 131 mmol) was added to a dichloromethane (30 mL) solution containing compound A1 (1.74 mmol), and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by rotary evaporation to obtain the ammonium salt of the intermediate product S5 as a white solid. Saturated sodium bicarbonate solution was added, and then the aqueous solution was extracted with dichloromethane 3 times. The combined organic phase was washed with water and dried with magnesium sulfate. After drying, the solvent was removed by rotary evaporation to obtain pure compound S5 (yield was 92%) as a white solid without further purification.

[0070]

[0071] Weigh the dried compound S5 (0.46mmol) and dissolve it in 20ml of anhydrous tetrahydrofuran. Weigh the compound B1 (0.95mmol) and dissolve it in 200ml of acetonitrile. Then use a constant pressure dropping funnel to slowly drop the tetrahydrofuran solution of S5 into the acetonitrile solution of B1. The dropping rate is maintained at 20 drops / min. After the dropping is completed, keep stirring at room temperature for 8 hours. After the reaction is completed, slowly add a dilute hydrochloric acid solution (1M, 2mL) aqueous solution to the system for acidification. Then filter to obtain a solid and wash it with acetonitrile several times. After drying, compound PYTcage-Cl (282mg, yield is 34%) is obtained as a light yellow-brown solid. For the convenience of characterization, the counter ions of some compound PYTcage-Cl are then converted to hexafluorophosphate, i.e., PYTcage-PF6, according to the above-mentioned synthesis steps of compound B1.

[0072] Proton spectrum of PYTcage-PF6 compound 1H NMR (400 MHz, CDCl3): δ=8.44 (m, 16H), 7.84 (d, 32H), 7.77 (d, 16H), 7.59 (d, 16H), 7.53 (t, 8H), 7.51 (t, 8H), 7.45 (d, 16H), 7.43 (d, 8H), 7.41 (d, 16H), 7.22 (d, 16H). 13 C NMR (100 MHz, CDCl3): δ=155.9, 141.0, 139.9, 139.5, 129.1, 128.6, 128.5, 127.5, 126.5, 126.2, 126.0, 44.7, 28.4.

[0073] HRMS-ESI of PYTcage-PF6, C 224 H 172 F 48 N 24 Calcd of O8P8: m / z=4484.7959[M+H] + , found: 4484.7922[M+H] + . Further through 1 H- 1 The HCOSY spectrum assigned the molecular structure, and the DOSY spectrum also proved that the compound PYTcage-PF6 was a single substance.

[0074] The hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum of compound A1 are shown as follows: Figure 1 , Figure 2 As shown, the H NMR spectrum and C NMR spectrum of compound B1 are shown as Figure 3 , Figure 4 As shown, the H NMR spectrum, C NMR spectrum, 1 H- 1 The H COSY and DOSY spectra are shown in Figure 5-Figure 8 As shown, Figure 1-Figure 8 Characterization spectra (NMR hydrogen spectrum and carbon spectrum, etc.) of the intermediate products A1, B1 and the final product PYTcage, which are used to support the successful synthesis of the compound PYTcage.

[0075] Fig. 9 This is the high-resolution mass spectrometry HRMS-ESI spectrum of the final product compound PYTcage-PF6. In the spectrum, a peak of PYTcage combined with a proton 4484.7922 [M+H] + can be found, which is used to support the successful synthesis of the compound PYTcage.

[0076] Example 2: Comparison of NMR spectra of supramolecular materials before and after loading camptothecin:

[0077] The nuclear magnetic resonance hydrogen spectra of three samples, single PYTcage-Cl, single camptothecin and the mixture of the two, were compared (e.g. Fig.10 ). It was found that the characteristic absorption peaks of supramolecular materials and camptothecin shifted significantly to the high field region, which is a common phenomenon when host-guest binding occurs. It can be preliminarily judged that PYTcage-Cl and camptothecin have undergone host-guest complexation.

[0078] The preparation process of the above-mentioned mixture of PYTcage-Cl and camptothecin is as follows: a certain amount of PYTcage-Cl and camptothecin are weighed and dissolved in deuterated acetonitrile (CD3CN), and each is configured to 2.0×10 -3 mol / L solution, pipette 600µL of the prepared camptothecin solution into 500µL of the prepared PYTcage-Cl solution, and shake well. The molar ratio of supramolecular material to camptothecin is 1:1.2.

[0079] Example 3: Changes in UV / Vis absorption spectra of supramolecular materials before and after loading camptothecin:

[0080] UV / Vis absorption spectroscopy test of single PYTcage-Cl (e.g. Fig.11 ), it can be seen that the absorption wavelength is distributed in the range of 300~400nm, and then camptothecin is added in batches until the molar ratio of camptothecin to PYTcage-Cl reaches 6:1. Observation of the UV / Vis absorption spectrum shows that a new group of absorption peaks appears at λ=534nm and the intensity of the absorption peak increases with the increase of the amount of camptothecin added, indicating that electron transfer occurs between the two, which further proves that the supramolecular material achieves the inclusion of camptothecin.

[0081] The specific experimental process is to weigh a certain amount of PYTcage-Cl solid and dissolve it in acetonitrile (CH3CN) to prepare 2.0×10 -5 mol / L solution, weigh a certain amount of camptothecin solid and dissolve it in acetonitrile (CH3CN) to prepare 2.0×10 - 3 mol / L solution. Fig.11 The absorption spectrum curve at the bottom corresponds to the UV / Vis absorption spectrum of PYTcage-Cl. The curves above correspond to the UV / Vis absorption spectra after adding 10µL (0.5:1), 20µL (1:1), 30µL (1.5:1), 40µL (2:1), 60µL (3:1), 80µL (4:1), 100µL (5:1), and 120µL (6:1) of camptothecin solution to 2ml of PYTcage-Cl acetonitrile solution.

[0082] Example 4: Drawing of the Job-Plot working curve of the combination of supramolecular material and camptothecin:

[0083] In order to determine the binding ratio of PYTcage-Cl to camptothecin, the change in fluorescence intensity (Δ F ), with Δ F Draw the Job-Plot working curve (such as Fig.12 The total concentration of PYTcage-Cl and camptothecin was 2.0×10 -4 mol / L), Δ F It reflects the change of the integral value of the NMR signal peak with the change of the molar ratio. Fig.12 It can be seen that the binding ratio of PYTcage-Cl and camptothecin is 1:2.

[0084] The specific experimental process is to weigh a certain amount of PYTcage-Cl and camptothecin and dissolve them in acetonitrile to prepare 2.0×10 -3 mol / L concentration of standard solution, For example, C CPT represents the concentration of camptothecin, C PYT-cage To represent the concentration of PYTcage-Cl, 200 µL of camptothecin standard solution and 800 µL of PYTcage-Cl standard solution were mixed and then diluted with 9 mL of acetonitrile to a total concentration of 2.0 × 10 -4 mol / L mixed solution, and other concentration ratios are prepared by analogy. The mixed solution of PYTcage-Cl and camptothecin was measured by fluorescence spectrometer, and the solvent was acetonitrile.

[0085] Example 5, Measurement of the binding constants and other thermodynamic parameters of supramolecular materials and camptothecin:

[0086] The isothermal calorimetric titration (ITC) method was used to obtain the binding constant of PYTcage-Cl and camptothecin and other thermodynamic parameters (such as Fig.13 ), the solvent was acetonitrile, the two were dissolved in acetonitrile, the concentration of PYTcage-Cl solution was 0.1mM, the concentration of camptothecin solution was 2.0mM, the temperature was set to 298K, and the test was performed using the isothermal calorimetric titration (ITC) method. The measurement results showed that the binding constant K a =(1.33±0.08)×10 4 / M,Δ H =(-9.139±1)kcal / mol,Δ S =-11.8cal / mol / K.

[0087] Fig.13 In the horizontal axis, the molar ratio refers to the molar ratio of camptothecin to PYTcage-Cl. The specific operation of preparing the mixed solution is to mix the PYTcage-Cl solution and the camptothecin solution according to different volume ratios to obtain a series of mixed solutions with different molar ratios of camptothecin to PYTcage-Cl for testing.

[0088] Example 6, Measurement of the binding constant and other thermodynamic parameters of supramolecular materials and hydroxycamptothecin:

[0089] The isothermal calorimetric titration (ITC) method was used to obtain the binding constant and other thermodynamic parameters of PYTcage-Cl and hydroxycamptothecin (such as Fig.14 ), PYTcage-Cl concentration was 0.1mM, hydroxycamptothecin concentration was 2.0mM, solvent was acetonitrile, and temperature was set to 298K. The measurement results showed that the binding constant K a =(1.72±0.2)10 4 / M,Δ H =(-8.233±1.4)kcal / mol, Δ S =-8.23cal / mol / K.

[0090] Fig.14 The molar ratio on the horizontal axis refers to the molar ratio of hydroxycamptothecin to PYTcage-Cl. The specific operation of preparing the mixed solution is to mix the PYTcage-Cl solution and the hydroxycamptothecin solution according to different volume ratios to obtain a series of mixed solutions with different molar ratios of hydroxycamptothecin to PYTcage-Cl for testing.

[0091] Example 7, Measurement of the binding constants and other thermodynamic parameters of supramolecular materials and topotecan:

[0092] The isothermal calorimetric titration (ITC) method was used to obtain the binding constant and other thermodynamic parameters of PYTcage-Cl and topotecan (such as Fig.15 ), PYTcage-Cl concentration was 0.1mM, topotecan concentration was 2.0mM, solvent was acetonitrile, and temperature was set to 298K. The measurement results showed that the binding constant K a =(3.95±0.29)10 4 / M,Δ H =(-9.719±0.57)kcal / mol, Δ S =-11.6cal / mol / K.

[0093] Fig.15The molar ratio on the horizontal axis refers to the molar ratio of topotecan to PYTcage-Cl. The specific operation of preparing the mixed solution is to mix the PYTcage-Cl solution and the topotecan solution according to different volume ratios to obtain a series of mixed solutions with different molar ratios of topotecan to PYTcage-Cl for testing.

[0094] The ITC isothermal titration calorimetry curve records the relationship between the titrant volume and the reaction heat effect or enthalpy change, showing the change in heat during the host-guest binding process, and the binding constant can be measured accordingly. Figure 13-Figure 15 The upper half of the figure is the thermal curve. The peak points downward, indicating that the process is exothermic. The lower half of the figure is the data obtained by integrating the peak area of ​​the upper half of the figure, which is plotted into a line graph and thermal parameters. This figure is automatically generated by the ITC software.

[0095] Example 8: In vitro release rate experiment of supramolecular materials loaded with camptothecin at different pH values:

[0096] As a drug carrier, the interaction between PYTcage-Cl and camptothecin is greatly affected by pH. The supramolecular material loaded with camptothecin prepared in Example 2 was used for the experiment. The samples to be tested were added to the buffer system with pH values ​​of 5.5 and 6.5 and the neutral system at 298K, and the in vitro release characteristics of the samples were investigated by dialysis. The substance concentration was 0.91×10 -3 5 mL of the mol / L sample solution was placed in a dialysis bag. After the two ends of the dialysis bag were clamped, they were placed in 50 ml of acetate buffer and deionized water with pH values ​​of 6.5 and 5.5, respectively. The release rate was then investigated in a 298K constant temperature oscillator at 1200 rpm / min. 1 ml (release medium on the outside of the dialysis bag) was sampled at 10, 20, 30, 40, 50, 60, 70, and 80 min, and the corresponding volume of release medium was added. The sample was filtered through a 0.5 µm microporous membrane and the camptothecin content was determined by HPLC. The experimental results are shown in the figure. Fig.16 As shown in the figure, the supramolecular material loaded with camptothecin has pH-sensitive characteristics for the release of drug molecules. When pH=5.5, after 30 minutes, about 80% of camptothecin was released, and after 70 minutes, camptothecin was almost completely released; when pH=6.5, after 30 minutes, about 55% of camptothecin was released, and after 80 minutes, camptothecin was almost completely released; after 80 minutes in a neutral environment, camptothecin was almost not released. The more acidic the environment, the faster the hydrogen bond is destroyed, and the faster the drug is released.

[0097] The present invention also provides PYTcage-Cl to perform a full-process simulation experiment in vitro in a laboratory environment, see Fig.18The specific process is as follows: (1) Weigh supramolecular materials and camptothecin drugs (mass ratio is 1:2) and disperse them in an appropriate amount of water to obtain a suspension. The concentration of supramolecular materials is controlled at 6×10 -3 ~2.5×10 -1 mmol / L; (2) subject the suspension to ultrasonic vibration (20~40KHz) or heating and stirring (323~343K) until the suspension is completely dissolved and the aqueous solution becomes clear; (3) add dilute hydrochloric acid to adjust the pH of the system to weak acidity (pH=5.5~6.5), then add an organic solvent (ethyl acetate or dichloromethane), shake well, let stand and separate the two phases, and use a separatory funnel to separate the two phases, leaving the aqueous phase; (4) add ammonium hexafluorophosphonate (with a mass ratio of 10:1~12:1 to the supramolecular material) to the aqueous phase and stir until no precipitation occurs. Wash the filtered solid with water several times, dry it, and then dissolve it in a certain amount of acetonitrile. Add dilute hydrochloric acid solution until no precipitation occurs. Wash the filtered solid with acetonitrile several times and dry it to obtain the supramolecular material for use. This completes the whole process of drug loading-drug release-material recovery of supramolecular materials.

[0098] The main content of this invention is the loading and targeted release of drugs by supramolecular materials. By simulating the in vivo environment, the whole process of drug loading (oscillation or heating) and release (encountering the acidic environment presented by tumor cells) of the material is demonstrated, and finally the recycling of the material is achieved, proving the feasibility of material design and application, which is a good supporting experiment.

Claims

1. A supramolecular material capable of loading and targeted release of camptothecin drugs, characterized in that The molecular structure of the supramolecular material is as follows: ； In the above structural formula, X is methyl, ethyl, isopropyl or isobutyl; The -Ar- structure is one of the following structures: 。 2. The method for preparing a supramolecular material capable of loading and targeted release of camptothecin drugs as claimed in claim 1, characterized in that The product is prepared by amidation reaction of tetrabenzylamine precursor molecule TPR with porphyrin structure and divalent diacyl chloride linear precursor molecule DDC with pyridine structure. The specific reaction steps are as follows: Step 1: Remove the amino protecting group and dry TPR and react with DDC in a solvent at room temperature with stirring; ； ； Step 2: After the reaction is completed, dilute hydrochloric acid is added to the system for acidification, and the mixture is filtered and washed to obtain the target product with chloride. Water and ammonium hexafluorophosphonate are added to the product for reaction to obtain the target product with hexafluorophosphonate.

3. The method for preparing a supramolecular material capable of loading and targeted release of camptothecin drugs as claimed in claim 2, characterized in that In step 1, the molar ratio of TPR to DDC is 1:2-2.

5.

4. The method for preparing a supramolecular material capable of loading and targeted release of camptothecin drugs as claimed in claim 3, characterized in that The molar ratio of TPR to DDC is 1:2.1-2.

2.

5. The method for preparing a supramolecular material capable of loading and targeted release of camptothecin drugs as claimed in claim 3, characterized in that The operation steps of step 1 are: TPR is dissolved in an appropriate amount of anhydrous tetrahydrofuran to obtain a TPR solution, and the molar concentration of the TPR solution is controlled at 18-29 mmol / L; DDC is dissolved in an appropriate amount of acetonitrile to obtain a DDC solution, and the molar concentration of the DDC solution is controlled at 4.2-5.1 mmol / L; then the TPR solution is added dropwise to the DDC solution, and after the addition is completed, stirring is continued at room temperature for 6-10 hours.

6. The method for preparing a supramolecular material capable of loading and targeted release of camptothecin drugs as claimed in claim 5, characterized in that The stirring time at room temperature is 7-8 hours.

7. The method for preparing a supramolecular material capable of loading and targeted release of camptothecin drugs as claimed in claim 2, characterized in that The concentration of dilute hydrochloric acid in step 2 is 0.5-2M.

8. Use of the supramolecular material capable of loading and targeted releasing camptothecin drugs as claimed in claim 1 in the preparation of a drug delivery system, wherein the drug delivered is a camptothecin drug.

9. The use according to claim 8, characterized in that The camptothecin drug is camptothecin, topotecan, irinotecan, hydroxycamptothecin, exitecan, 9-aminocamptothecin, rubitecan, lortotecan, gimatecan or karenitecin.

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