CN115806583A - A kind of amphiphilic polypeptide derivative and its application - Google Patents

Abstract

The invention discloses an amphiphilic polypeptide derivative with general formula I, its sequence is R ₁ ‑Phe‑Arg‑Phe‑Lys‑R ₂ ‑R ₃ , the compound consists of a hydrophobic drug-polypeptide conjugate and Composed of hydrophilic PEG, it has self-assembly properties, so photosensitizers (PpIX) can be entrapped in its hydrophobic core to form drug-loaded micelles. After the micelles enter the body, they are cleaved under the action of glutathione (GSH) or esterase (CES) overexpressed in cancer cells. Specific release; on the other hand, hydrophobic drugs and photosensitizers can be used in tumor chemotherapy and photodynamic therapy, respectively, so as to realize the combination of the two methods. The micelle preparation has good stability or good tumor inhibition effect and can be used for tumor treatment.

Description

A kind of amphiphilic polypeptide derivative and its application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to an amphiphilic polypeptide derivative and its application.

Background technique

Polymer micelles are nano drug carriers formed by self-assembly of amphiphilic block copolymers in aqueous solution. The size of the carrier is generally only tens to hundreds of nanometers, so it can be administered through intravenous injection. Hydrophobic anticancer drugs can be entrapped in the hydrophobic core of micelles through covalent bonds, electrostatic interactions, hydrophobic interactions, etc., so as to be delivered to tumor tissues and tumor cells. By introducing stimulus-responsive sequences into micelles, such as pH-responsive, redox-responsive, enzyme-responsive, etc., tumor microenvironment-responsive micelles can be prepared to achieve controlled release of drugs. Glutathione (GSH) and esterase (CES) are overexpressed in tumor tissues, and disulfide bonds or ester bonds are introduced into the amphiphilic blocks of micelles to prepare micelles with redox responsiveness or esterase responsiveness. The ability of micelles to achieve specific drug release in tumor cells.

Photodynamic therapy (PDT) is an effective cancer therapy. Under the irradiation of visible light, the photosensitizer used in PDT can transfer energy to molecular oxygen, resulting in the generation of singlet oxygen (ROS), leading to apoptosis of tumor cells. Protoporphyrin IX (PpIX) is the most commonly used photosensitizer, which belongs to the porphyrin class of photosensitizers, can effectively absorb light energy and generate enough singlet oxygen, and is a photosensitizer with excellent performance. However, the application of PpIX in photodynamic therapy is often limited due to its water insolubility and low selectivity to target tissues. The nano-drug loading systems developed today, such as micelles, microspheres, nanoparticles, etc., can effectively solve the problems of their water insolubility and limited specificity. Therefore, encapsulating PpIX in the hydrophobic core of micelles with tumor-specific targeting function can not only effectively improve the solubility of PpIX in vivo, but also allow it to specifically accumulate in tumor tissues, thereby exerting its anticancer efficacy. .

The existing tumor-targeting reduction-responsive carrier materials, such as amphiphilic block copolymers, have complicated synthesis steps, low product yields, and high costs, which largely limit their applications. Therefore, it is of great significance to develop an amphiphilic polypeptide derivative with glutathione (GSH) or esterase (CES) responsiveness.

Contents of the invention

Object of the invention: One of the objects of the present invention is to provide an amphiphilic polypeptide derivative of general formula I, whose sequence is R ₁ -Phe-Arg-Phe-Lys-R ₂ -R ₃ ,

in,

R₂选自

R₃选自PEG440(即PEG₈)、PEG1000或PEG3400。R ₁ is selected from

_R2 is selected from

R ₃ is selected from PEG440 (ie PEG ₈ ), PEG1000 or PEG3400.

The compounds of general formula I of the present invention are preferably the following compounds:

Another object of the present invention is to provide a preparation method for a compound of general formula I, comprising the following steps: after the dichloro resin is activated by dichloromethane, adding an Fmoc-protected amino acid to connect the first amino acid; after the reaction, remove Amino acid, clean the resin, and seal the resin with blocking solution; after the sealing is completed, wash the resin with DMF, and use 20% piperidine/DMF solution for deprotection to remove the Fmoc protecting group; after deprotection, wash with DMF resin, and add the next Fmoc-protected amino acid to connect the next amino acid; repeat DMF cleaning, deprotection, DMF cleaning, and addition of Fmoc amino acid to extend the peptide chain; after all the amino acids are connected, add to the resin cutting with trifluoroacetic acid; after the cutting, the cutting solution is collected, and the crude product is obtained by rotary evaporation, dissolved in water, and freeze-dried; the freeze-dried product is separated and purified to obtain the amphiphilic polypeptide derivative.

The amphiphilic polypeptide derivative has glutathione (GSH) specific cleavage sequence -S-S- or esterase (CES) specific cleavage sequence -COO-.

Preferably, the blocking solution is methanol:N,N-diisopropylethylamine:DCM=15:5:80, V/V/V.

The compounds of the general formula I of the present invention can be prepared by the above-mentioned or similar preparation methods, and the corresponding starting materials can be selected according to the difference of the substituent and the position of the substituent. Those skilled in the art should realize that the above-mentioned route is helpful for understanding the present invention, but does not limit the content of the present invention, unless otherwise specified, the variables are defined as mentioned in the general formula I.

Another object of the present invention is to provide the application of the above-mentioned amphiphilic polypeptide derivatives in the preparation of cancer therapeutic drugs. Specifically, the cancers are lung cancer and melanoma.

Another object of the present invention is to provide a drug-loaded micelle, comprising the above-mentioned amphiphilic polypeptide derivative and photosensitizer protoporphyrin IX.

Another object of the present invention is to provide the preparation method of the above-mentioned drug-loaded micelles. The amphiphilic polypeptide derivative and the photosensitizer protoporphyrin IX are respectively configured into a DMSO mother solution, and the two DMSO mother solutions are mixed and dispersed in a PBS solution. After mixing evenly, the solution is subjected to ultrasound or cell disruption to obtain the drug-loaded micelles.

Another object of the present invention is to provide the application of the above-mentioned drug-loaded micelles in cancer treatment.

The amphiphilic polypeptide derivatives provided by the present invention are composed of hydrophobic drug-polypeptide conjugates and hydrophilic PEG. The compound can self-assemble and simultaneously pack photosensitizer (PpIX) in its hydrophobic core to form a drug-loaded micelles. The micelles are cleaved under the action of glutathione (GSH) or esterase (CES) overexpressed in cancer cells, on the one hand, the specific release of hydrophobic drugs and photosensitizers in cancer cells is achieved; on the other hand, On the one hand, hydrophobic drugs and photosensitizers can be used in tumor chemotherapy and photodynamic therapy, respectively, so as to realize the combined use of the two methods. The micelle preparation has good stability or good tumor suppressing effect, and can be used for cancer treatment.

Specifically, the cancers are lung cancer and melanoma.

Beneficial effect:

(1) The amphiphilic polypeptide derivative provided by the present invention has glutathione (GSH) or esterase (CES) responsiveness, and can destroy micelles under the action of GSH or CES. Since GSH and CES are overexpressed in tumor cells, the specific release of hydrophobic drugs and photosensitizers in tumor cells can be achieved.

(2) The present invention designs a series of drug-loaded micelles as drug carriers, which not only have good encapsulation efficiency and drug-loading capacity for photosensitizers, but also realize efficient delivery of photosensitizers; at the same time, they can also maintain a relatively high concentration under protein conditions. The long-term stability provides the possibility for its stable transportation in the blood.

(3) The amphiphilic polypeptide derivatives of the present invention and photosensitizers are co-assembled to form drug-loaded micelles, which can release photosensitizers and small molecule hydrophobic drugs after entering cancer cells. Photosensitizers can realize photodynamic therapy of tumors, and small molecule hydrophobic drugs can realize chemotherapy of tumors, so as to realize the combination of photodynamic therapy and chemotherapy, thereby improving the antitumor activity of drugs.

Description of drawings

Figure 1 is the mass spectrum of compound 1.

Figure 2 is the mass spectrum of compound 3.

Figure 3 is a micelle size distribution diagram; Figure 3A is a micelle size distribution diagram of compound 1, Figure 3B is a micelle size distribution diagram of compound 2, and Figure 3C is a micelle size distribution diagram of compound 3.

Figure 4 is a graph of the micelles critical micelle concentration (CMC), Figure 4A is the micelles critical micelle concentration of compound 1, Figure 4B is the micelles critical micelle concentration of compound 2, Figure 4C is the micelles critical micelle concentration of compound 3 Micelle concentration, Figure 4D is the micelle critical micelle concentration of compound 4, and Figure 4E is the micelle critical micelle concentration of compound 5.

Figure 5 is a graph of the particle size change of the micelles under different conditions within 7 days, Figure 5A is a graph of the particle size change of the compound 2 micelles, and Figure 5B is a graph of the particle size change of the compound 3 micelles.

Figure 6 is a disulfide bond response liquid chromatogram and mass spectrum of compound 3, Figure 6A is a liquid chromatogram without TCEP, Figure 6B is a liquid chromatogram with TCEP, and Figure 6C is a mass spectrum of the product corresponding to Figure 6B picture.

Figure 7 is the release curve of micellar drugs, Figure 7A is the release curve of compound 1 under different GSH concentration conditions, and Figure 7B is the release curve of compound 3 under different GSH concentration conditions.

Figure 8 is a diagram of the generation of micellar singlet oxygen (ROS), Figure 8A is a diagram of the generation of singlet oxygen (ROS) in compound 1 drug-loaded micelles, Figure 8B is the generation of compound 3 drug-loaded micelles singlet oxygen (ROS) Situation diagram.

Fig. 9 is a graph showing the survival rate of A549 cells co-incubated with PpIX and PpIX+compound 1 for 48 hours.

Figure 10 is a graph of the survival rate of A375 cells co-incubated with PpIX and PpIX+compound 3 for 48 hours.

Detailed ways

The technical solutions of the present invention will be described in detail below through specific examples, but the protection scope of the present invention is not limited to the examples.

The starting materials and reaction reagents used in the specific examples of the present invention are all commercially available. The experimental methods not indicating specific conditions in the examples of the present invention are generally in accordance with conventional conditions, or in accordance with the conditions suggested by raw material or commodity manufacturers. Reagents without specific sources indicated are conventional reagents purchased in the market.

In the following examples, the amphiphilic polypeptide derivative R ₁ -Phe-Arg-Phe-Lys-R ₂ -R ₃ (abbreviated as R ₁ -FRFK-R ₂ -R ₃ ),

时，简称为VES(维生素E琥珀酸酯)；Wherein, R ₁ is selected from

When, referred to as VES (vitamin E succinate);

时，化学名为：LND-Lys(NBD)-Phe-(氯尼达明-赖氨酸(4-硝基苯基-2-氧杂-1,3-二唑)-苯丙氨酸，简写为LND-K(NBD)F-)。R ₁ is selected from

At that time, the chemical name is: LND-Lys(NBD)-Phe-(lonidamine-lysine (4-nitrophenyl-2-oxa-1,3-oxadiazole)-phenylalanine, Abbreviated as LND-K(NBD)F-).

The Fmoc-solid phase synthesis of embodiment 1VES-Phe-Arg-Phe-Lys-S-S-PEG1000 (compound 1)

(1) Swelling: Weigh 0.2 mmol of 2-chlorotrityl chloride resin (2-ChlorotritylChloride Resin) with a degree of substitution of 1.0 mmol/g in a solid-phase synthesis tube, add 10 mL of dichloromethane (DCM), and place in Shake on a shaker to make it swell for 30 minutes, then remove it by suction filtration; add 10 mL of DCM, shake it on a shaker for 2 minutes, remove it by suction filtration, repeat 5 times;

(2) Connection of the first amino acid: Weigh 0.3 mmol of Fmoc-protected PEG1000 (Fmoc-PEG1000-COOH) (purchased from Shanghai Pengshuo Biotechnology Co., Ltd.), absorb N,N-diisopropylethylamine (DIPEA ) 0.3mmol was added, dissolved in 10mL DCM, added to the solid-phase synthesis tube, and placed on a swing bed to react for 3 hours; To fully remove unreacted amino acids;

(3) Blocking of resin: Prepare 20 mL of blocking solution (methanol: DIPEA: DCM = 15: 5: 80, v/v/v), add 10 mL to the solid-phase synthesis tube in two times, and add 10 mL each time. Shake on the shaker for 10 minutes;

(4) Deprotection: remove the blocking solution by suction filtration, add 10mL N,N-dimethylformamide (DMF) to the solid-phase synthesis tube, shake on the shaker for 2min, remove by suction filtration, repeat 5 times to fully wash Remove the blocking solution; add 10mL deprotection solution (20% piperidine/DMF, v/v) to remove the Fmoc protection group, add the deprotection solution for the first time and then shake for 5 minutes, then remove it by suction filtration, and then shake for 25 minutes for the second time , removed by suction filtration; after deprotection, add 10mL DMF to wash 5 times (each time 2min), to fully remove the deprotection solution;

(5) The connection of the second amino acid: add Fmoc-protected disulfide bond (Fmoc-S-S-COOH), TBTU, HOBt (TBTU, HOBt purchased from Shanghai Biide Pharmaceutical Technology Co., Ltd.) with a molar mass of 0.6mmol and DIPEA (purchased from Aladdin Reagent (Shanghai) Co., Ltd.), add 10mL of DMF solvent to the solid-phase synthesis tube, and place it on a swing bed for 3h;

Preparation method of Fmoc-SS-COOH: Weigh cysteamine hydrochloride (1mmol, 0.225g) (purchased from Shanghai Beide Pharmaceutical Technology Co., Ltd.) and NaHCO ₃ (3mmol, 0.252g) and dissolve in 1mL water. Then add 10 mL of dioxane and stir. Cool in an ice bath to 0° C. and react for 10 min, and add succinic anhydride (1 mmol, 0.1 g) (purchased from Shanghai Beide Pharmaceutical Technology Co., Ltd.) to the above mixture. The resulting reaction mixture was stirred overnight. The next day, 0.33mL DIPEA and 1mL DMF solution containing Fmoc-OSu (1mmol, 0.337g) (purchased from Shanghai Pide Pharmaceutical Technology Co., Ltd.) were added successively in an ice bath at 0°C, and then the reaction mixture was heated at 0°C. The reaction was continued for 2 hours in an ice bath; after the reaction was completed, suction filtration was performed to collect the filtrate. Rotary evaporation filtrate, then 20mL of water was added to the viscous liquid produced after rotary evaporation, a white precipitate was precipitated, suction filtered, and a small amount of H ₂ O was used to wash the white precipitate, and the white precipitate was purified by column chromatography (dichloromethane:methanol =15:1), and then lyophilized to obtain the target product Fmoc-SS-COOH.

(6) Extension of the peptide chain: repeat steps (4), (5), add successively the lysine (Fmoc-Lys(Boc)-OH) and phenylalanine (Fmoc) that are all 0.6mmol of Fmoc protection in molar weight -Phe-OH), arginine (Fmoc-Arg(Pbf)-OH), phenylalanine (Fmoc-Phe-OH) (the above amino acids were purchased from Jill Biochemical (Shanghai) Co., Ltd.) and vitamin E succinic acid Ester (VES) (purchased from Aladdin Reagents (Shanghai) Co., Ltd.), TBTU, HOBt and DIPEA 0.6mmol, add 10mL of DMF solvent in the solid-phase synthesis tube, and place it on a swing bed for 3h;

(7) Cutting: After the reaction, first wash with 10mL DMF 5 times (2min each time), then wash 5 times with 10mL DCM (2min each time), after filtering off all the reaction solution, transfer the resin to a round bottom flask , add 10mL trifluoroacetic acid (TFA) and stir for 6h;

(8) Post-processing: after cutting, collect the filtrate by suction filtration, remove TFA by vacuum rotary evaporation in a water bath at 30°C; add 10 mL of water lysate, freeze-dry in a lyophilizer, and obtain crude polypeptide;

(9) Product purification: the crude product is dissolved with acetonitrile and water (volume ratio 1:1), and then separated and purified by preparative liquid chromatography (Shimadzu, Japan) to obtain the final product; the product is analyzed by HPLC and MS for analysis. HPLC results showed that the purity of compound 1 was 92.435%.

Result analysis: The theoretical exact molecular weight is 2325.78, and ESI-MS m/z: Calcd.[M+2H] ²⁺ 1163.9, which is consistent with the target molecular weight, proving that compound 1 was synthesized correctly (see Figure 1).

The synthesis of embodiment 2VES-Phe-Arg-Phe-Lys-S-S-PEG3400 (compound 2)

Refer to the synthetic method of compound 1 in Example 1. The difference is that the molecular weight of the PEG used is different. In this example, Fmoc-PEG3400-COOH (purchased from Shanghai Pengshuo Biotechnology Co., Ltd.) is used, and the others are the same as in Example 1.

Synthesis of Example 3LND-Lys(NBD)-Phe-Phe-Arg-Phe-Lys-S-S-PEG1000 (Compound 3)

Refer to the synthetic method of compound 1 in Example 1. The difference is: after connecting Fmoc-PEG1000-COOH, Fmoc-S-S-COOH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, need Then connect Fmoc-Phe-OH, Fmoc-K(NBD)-COOH in sequence, the dosage is 0.6mmol, and finally connect 0.6mmol of lonidamine (LND), other operations are the same.

Preparation method of Fmoc-K(NBD)-COOH: Weigh anhydrous Na ₂ CO ₃ (3.0mmol, 0.3183g) into a round bottom flask, add 15mL H ₂ O to dissolve, then add Fmoc-K to the round bottom flask (3.0mmol, 1.1052g) (purchased from Aladdin Reagent (Shanghai) Co., Ltd.), add 20mL CH ₃ OH to dissolve; fill N ₂ into the round bottom flask; weigh NBD-Cl (2.4mmol, 0.479g) ( (purchased from Shanghai McLean Biochemical Technology Co., Ltd.) was placed in a beaker, dissolved with 30mL CH ₃ OH to obtain NBD-Cl methanol solution; put the round bottom flask on a magnetic stirrer, turn on the stirring, and then use a syringe to slowly round Add NBD-Cl methanol solution dropwise in the bottom flask; : 1) extract multiple times, collect the aqueous phase, and adjust the solution to pH 3-4 with 1M HCl; add the aqueous phase to a separatory funnel, extract several times with ether, and collect the organic phase; rotary evaporation removes the organic phase solvent, and then freezes Dry to obtain Fmoc-K(NBD)-COOH.

HPLC results showed that the purity of compound 3 was 94.1%.

Result analysis: The theoretical precise molecular weight is 2524.79, ESI-MS m/z: Calcd.[M+4H] ⁴⁺ 632.4, consistent with the target molecular weight, proving that compound 3 was synthesized correctly (see Figure 2).

Synthesis of Example 4LND-Lys(NBD)-Phe-Phe-Arg-Phe-Lys-S-S-PEG440 (Compound 4)

Refer to the synthetic method of compound 1 in Example 1. The difference is that the molecular weight of the PEG used is different. In this example, Fmoc-PEG440-COOH (purchased from Shanghai Macklin Biochemical Technology Co., Ltd.); After Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-K(NBD)-COOH need to be connected in turn, dosage Both were 0.6 mmol, and finally 0.6 mmol of lonidamine (LND) was added, and other operations were the same.

Synthesis of Example 5LND-Lys(NBD)-Phe-Phe-Arg-Phe-Lys-COO-PEG1000 (Compound 5)

Refer to the synthetic method of compound 1 in Example 1. The difference is: replace Fmoc-S-S-COOH with Fmoc-COO-COOH, the molar amount used is the same; After -OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, then connect Fmoc-Phe-OH, Fmoc-K(NBD)-COOH in sequence, the dosage is 0.6mmol, and finally connect 0.6mmol The lonidamine (LND), in addition to other operations are the same.

Fmoc-COO-COOH preparation method: Weigh 9-fluorenylmethoxycarbonyl-glycinol (1mmol, 0.2830g) (purchased from Gil Biochemical (Shanghai) Co., Ltd.), succinic anhydride (1.1mmol, 0.110g) (purchased 4-Dimethylaminopyridine (0.16mmol, 0.020g) (purchased from Shanghai Pide Pharmaceutical Technology Co., Ltd.) and 4-dimethylaminopyridine (purchased from Shanghai Pide Pharmaceutical Technology Co., Ltd.) were dissolved in 10mL DCM, and then added to the reaction bottle Add 260μL DIPEA to the solution, and react at room temperature for 5h; after the reaction, add 20mL DCM to the reaction solution; extract with 1M HCl solution (40mL) and deionized water (30mL×3), keep the organic phase; then The organic phase was washed with NaCl/H ₂ O solution (10 mL×3), and then dried with anhydrous Na ₂ SO ₄ ; the solution was subjected to rotary evaporation and purified by column chromatography (dichloromethane:methanol=10: 1), and then lyophilized to obtain the target product Fmoc-COO-COOH.

The preparation of embodiment 6 drug-loaded micelles

Weigh an appropriate amount of amphiphilic polypeptide derivative powder prepared in Examples 1-5, add DMSO to dissolve, and prepare a 50 mg/mL mother solution; take another appropriate amount of PpIX powder, add DMSO solution, and prepare a 2 mg/mL mother solution. Take 10 μL of DMSO mother solution of amphiphilic polypeptide derivatives and 50 μL of PpIX DMSO mother solution in EP tubes, then add 940 μL of PBS to it, vortex and mix well, and then ultrasonicate the solution or disrupt cells to obtain drug-loaded micelles.

The particle diameter of embodiment 7 drug-loaded micelles and the mensuration of electric potential

The micelles prepared in Example 6 were filtered with a 0.22 μm filter to remove impurities and large aggregates. The particle size and polydispersity index (PdI) of the micelles were determined by a Malvern particle size analyzer (Malven Nano ZS90, purchased from Malvern, UK). The test temperature was 25°C, the measurement was repeated three times, and the average value was taken. The results are shown in Table 1.

Table 1 Particle and PdI results of drug-loaded micelles

Figure 3 is a micelle size distribution diagram; Figure 3A is a micelle size distribution diagram of compound 1, Figure 3B is a micelle size distribution diagram of compound 2, and Figure 3C is a micelle size distribution diagram of compound 3.

As shown in Table 1 and Figure 3, most of the PdI are less than 0.3, indicating that the micelles of the above compounds are well formed, the particle size distribution is uniform, and they have good dispersibility.

The mensuration of embodiment 8 critical micelle concentration (CMC)

The concentration of the amphiphilic polypeptide derivatives was prepared to be 2mg/mL, and the micellar solution with a PpIX concentration of 0.4mg/mL was prepared respectively, and then gradient dilution was carried out to obtain the concentrations of the amphiphilic polypeptide derivatives as 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 0.0625mg/mL, 0.03125mg/mL, 0.015625mg/mL, 0.0078125mg/mL, PpIX concentration is 0.2mg/mL, 0.1mg/mL, 0.05mg/mL, 0.025mg /mL, 0.0125mg/mL, 0.00625mg/mL, 0.003125mg/mL, 0.0015625mg/mL micellar solution. Use a Malvern particle size analyzer (Malven Nano ZS90, purchased from Malvern, UK) at room temperature (25°C), fix 632.8nm as the excitation light source, measure the light scattering intensity (Kcps) at each concentration, take the concentration as the abscissa, and the light The scattering intensity (Kcps) is plotted on the ordinate, and the concentration corresponding to the intersection point of the two straight lines is the critical micelle concentration (CMC) of the amphiphilic polypeptide derivative. The results are shown in Table 2.

Table 2 CMC results of drug-loaded micelles

Figure 4 is a micelle critical micelle concentration (CMC) result graph; Figure 4A is the micelle critical micelle concentration of compound 1, Figure 4B is the micelle critical micelle concentration of compound 2, and Figure 4C is the micelle critical micelle concentration of compound 3 Micelle concentration, Figure 4D is the micelle critical micelle concentration of compound 4, and Figure 4E is the micelle critical micelle concentration of compound 5.

As shown in Figure 4, the critical micelle concentrations of compounds 1, 2, 3, 4, and 5 were 11.51, 1.63, 17.25, 4.39, and 11.8 μM, respectively, indicating that the above compounds have good self-assembly ability. The above-mentioned critical micelle concentration is very small, which can ensure that the drug-loaded micelles can still maintain the form of micelles after entering the body and being diluted by blood, so as to prevent the drugs from being released in advance.

The determination of embodiment 9 micelle encapsulation efficiency and drug loading

Drawing of PpIX standard curve: Accurately weigh 1 mg of PpIX, add 1 mL of DMSO and 1 mL of acetonitrile to dissolve, and obtain 0.5 mg/mL of PpIX mother solution. Aspirate 10 μL of the mother solution, and dilute to 10 mL with the same volume of DMSO and acetonitrile to obtain a 500 ng/mL solution. The solution was serially diluted with the same volume of DMSO and acetonitrile to obtain 250, 125, 62.5, 31.25, 15.625, 7.3125 ng/mL solutions. A fluorescence spectrophotometer was used for detection, the excitation wavelength was set to 405 nm, and the emission wavelength was set to 600-700 nm to measure the fluorescence intensity of PpIX. A standard curve was established with the concentration as the abscissa and the fluorescence intensity at a wavelength of 633nm as the ordinate.

Determination of the drug loading and encapsulation efficiency of micelles: Three micellar solutions with amphiphilic peptide derivative concentration of 1 mg/mL and PpIX concentration of 0.2 mg/mL were prepared in parallel. Use a high-speed centrifuge to centrifuge at 6000 rpm for 3 minutes to remove PpIX that is not packed into the micelles. The supernatant is freeze-dried with a freeze-dryer, and after freeze-drying, weigh the quality of the freeze-dried powder, which is m _total ; dissolve the freeze-dried powder with equal volumes of DMSO and acetonitrile, and then use a fluorescence spectrophotometer to Determination, calculate the content of PpIX in the micelle by Beverage, which is m ₁ .

Encapsulation rate = m ₁ /m ₂ × 100%

Drug loading = m ₁ /m _total × 100%

(m ₁ is the mass of PpIX in self-assembled micelles; m ₂ is the feeding amount of PpIX; m is the _total mass of drug-loaded micelles)

The results of micelle encapsulation efficiency and drug loading are shown in Table 3:

Table 3 The results of micelle encapsulation efficiency and drug loading

As shown in Table 3, compounds 1, 2, and 3 all have good encapsulation efficiency and drug loading, especially compound 2, the encapsulation efficiency can reach 89.43±5.50%, and the drug loading can reach 16.99±2.56%.

The test of embodiment 10 micelle stability

Two micellar solutions with amphiphilic polypeptide derivative concentration of 0.5 mg/mL and PpIX concentration of 0.1 mg/mL were prepared respectively, one of which was added with 5% fetal bovine serum (FBS) (purchased from American Thermo Fisher technology companies), and the other as a control group without FBS. The particle size change of the drug-loaded micelles was measured at 25° C. with a Malvern particle size analyzer (Malven Nano ZS90) on days 0, 1, 2, 3, 4, 5, 6, and 7, respectively.

Figure 5 is a graph of the particle size change of the micelles under different conditions within 7 days, Figure 5A is a graph of the particle size change of the compound 2 micelles, and Figure 5B is a graph of the particle size change of the compound 3 micelles. As shown in the figure, when 5% FBS was not added to the micellar solution, the particle size and particle size distribution did not change significantly with the increase of time; when 5% FBS was added, the micellar particle size did not change significantly or slightly increased, indicating that the micelles could also remain stable in the presence of proteins, which provided the possibility for their stable transport in the blood.

Example 11 Disulfide Bond Responsiveness Experiment

Weigh 10 mg of tris(2-chloroethyl)phosphate TCEP, add 1 mL of PBS to dissolve, adjust the pH of the solution to 7.4 with 1M Na ₂ CO ₃ and 1M HCl, and obtain a TCEP mother solution with a concentration of 10 mg/mL. Weigh 1 mg of the amphiphilic polypeptide derivative (Compound 3) and dissolve it in 1 mL of PBS (pH 7.4), and adjust the pH to 7.4 with 0.1 M NaOH/HCl to obtain two solutions with a concentration of 1 mg/mL. Add 11.35 μL of TCEP mother solution (1eq.) to one of the solutions, mix well, and place it for incubation at 37° C.; the other without TCEP solution is used as a control group. After 1 hour, take 200uL of the solution and add an equal volume of acetonitrile, and perform subsequent analysis by HPLC. The detection conditions are shown in Table 4 and Table 5:

Table 4 Liquid Chromatography Setting Conditions

Table 5 Liquid chromatography elution conditions

Fig. 6 is compound 3 disulfide bond response liquid chromatograms and mass spectrograms, Fig. 6A is the liquid chromatograms without TCEP, Fig. 6B is the liquid chromatograms with TCEP, Fig. 6C is the corresponding product (LND of Fig. 6B - Mass Spectrum of K(NBD)FFRFK-SH).

As shown in the figure, the peak elution time of compound 3 without TCEP was 11.084min. After adding TCEP, the original peak disappeared and a new peak appeared, and the peak elution time was 12.237min, indicating that the original compound was completely cut by TCEP and produced new substance. The mass spectrometry results of the new substance are: [M+H] ⁺ 1398.3, [M+2H] ⁺ 699.9, which is consistent with the molecular weight of the new substance produced by cleavage of TCEP in theory, proving that compound 3 can be cleaved by TCEP, and within 1h The cleavage is complete, indicating that the above compounds all have good disulfide bond responsiveness.

The measurement of embodiment 12 micelle drug release

Determination of drug-loaded micelles standard curve: Take 100 μL of drug-loaded micelles with compound 1 or compound 3 concentration of 0.5 mg/mL and PpIX concentration of 0.1 mg/mL respectively, add 900 μL PBS to obtain PpIX concentration of 10 μg/mL Drug-loaded micellar solution. The solution was serially diluted with PBS to obtain drug-loaded micellar solutions with PpIX concentrations of 6.67 μg/mL, 4.44 μg/mL, 2.96 μg/mL, and 1.97 μg/mL, and the above five concentrations were measured by a UV spectrophotometer. solution to be tested. With the concentration of PpIX as the abscissa and the UV absorbance of the micelles at 390 nm as the ordinate, a standard curve was drawn.

Determination of micellar drug release: by monitoring the changes in the UV absorption of micelles at 390nm, the amount of PpIX released was calculated. Drug-loaded micellar solutions with a concentration of 0.5 mg/mL of amphiphilic polypeptide derivatives (compound 1 and compound 3) and a concentration of PpIX of 0.1 mg/mL were prepared respectively. The micellar solution was divided into 400 μL tubes, and then GSH solution (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) was added to the micellar solution, and then placed in a 37° C. incubator for incubation. Take out a tube at 0, 1, 2, 4, 8, and 24 hours for centrifugation, so that the released hydrophobic PpIX precipitates, take the supernatant for UV detection, and bring the UV absorption value into the standard curve to calculate the PpIX value. cumulative release.

Figure 7 is the release curve of micellar drugs, Figure 7A is the release curve of compound 1 under different GSH concentration conditions, and Figure 7B is the release curve of compound 3 under different GSH concentration conditions.

As shown in Figure 7, in the absence of GSH, the release amount of PpIX was close to 0, indicating that the micellar structure was stable. Under the action of GSH, the micelles were destroyed and PpIX was released. When the GSH concentration was above 400 μM, PpIX could be completely released in about 4 hours, while when the GSH concentration was below 250 μM, the release amount in 24 hours was less than 20 %. This result indicated that when the micelles entered into the cancer cells, PpIX could be completely released under the action of the overexpressed enzymes in the cancer cells, so as to be used for the subsequent photodynamic therapy of tumors.

Embodiment 13 The measurement of micelle singlet oxygen (ROS) generation situation

Using 9,10-anthracendiyl-bis(methylene)dimalonic acid (ABDA, purchased from Shanghai Macklin Biochemical Technology Co., Ltd.) as a ROS capture agent, the generation of micellar ROS and light exposure were measured by UV spectrophotometer. time relationship. Prepare two drug-loaded micelle solutions with compound 1 or compound 3 at a concentration of 0.5 mg/mL and PpIX at a concentration of 0.1 mg/mL, one with 5 mM GSH solution and the other without, and then placed at 37 °C Incubator for incubation. After 1 h, the two micellar solutions were diluted with PBS so that the final concentration of PpIX was 5 μg/mL, 1 mL of the diluted micellar solution was taken, and 50 μ of LABDA solution was added thereto, and the sample solution was irradiated with a 650 nm laser (power: 1W/cm ² ). After 0, 2, 4, 6, 8, and 10 min of irradiation, the absorption spectrum of ABDA was detected with an ultraviolet spectrophotometer. Record the absorbance value of ABDA at 380nm. The absorbance value at 0 min is recorded as A ₀ , and the absorbance value at subsequent time points is recorded as A. With time as the abscissa and A/A ₀ as the ordinate, draw the relationship between ROS production and light time. relation chart.

Figure 8 is a diagram of micellar singlet oxygen (ROS) generation, Figure 8A is a diagram of micellar singlet oxygen (ROS) generation for compound 1, and Figure 8B is a diagram of compound 3 micellar singlet oxygen (ROS) generation.

As shown in the figure, when the micelles were not destroyed by GSH, the absorbance value of ABDA did not decrease significantly after 10 minutes of light irradiation; on the contrary, the absorbance value of the GSH group decreased significantly with the increase of light time, indicating that adding The GSH group produced a large number of ROS, indicating that PpIX can generate singlet oxygen under light after being released from the micelles. This result provides support for the drug-loaded micelles to be used in photodynamic therapy after entering cancer cells. .

Example 14 In vitro anti-tumor experiment

Test method: A549 cells or A375 cells (both purchased at ATCC, USA), inoculated cell suspension (30,000 A549 cells or A375 cells per 1 mL of 1640 medium or DMEM medium) in a 96-well plate, 100 μL per well, and the seeding density was 3000 cells/well. After incubating at 37°C (5% CO ₂ ) for 24 hours, add 100 μL of PpIX or PpIX+ compound (PpIX concentration gradient is 0.125, 0.25, 0.5, 1, 2 μg/mL or 0.5, 1, 2, 4, 8 μg /mL, the concentration gradient of compound 1 is 12.5, 25, 50, 100, 200 μg/mL, and the concentration gradient of compound 3 is 6.25, 12.5, 25, 50, 100 μg/mL), and 5 replicate wells are set for each concentration, with equal volume 1640 medium or DMEM medium was used as the control group, and the cells were incubated for 48 hours after treatment. After the incubation, 20 μL of MTT reagent (Biosharp, USA) was added to each well, and incubated at 37°C in the dark for 4 h, then discarded the upper culture solution, added 200 μL of DMSO to each well, and finally used a microplate reader (TeanSunrise, Austria) ) to measure the absorbance of each well at 490 nm. Each group of experiments was measured 3 times in parallel.

Fig. 9 is a graph showing the survival rate of A549 cells co-incubated with PpIX and PpIX+compound 1 for 48 hours.

Figure 10 is a graph of the survival rate of A375 cells co-incubated with PpIX and PpIX+compound 3 for 48 hours.

As shown in Figures 9 and 10, the cell viability of A549 and A375 decreased with the increase of the concentration of PpIX or PpIX+ compound, indicating that PpIX, compound 1 and compound 3 all had inhibitory effects on cancer cells; compared PpIX group and PpIX+ For the cell survival rate of the compound group, it was found that the cell survival rate of the PpIX+ compound group was always lower than that of the PpIX group, indicating that the joint action of PpIX and the drug on the compound can improve the anticancer effect and further enhance the inhibitory effect on tumor cells.

As stated above, while the invention has been shown and described with reference to certain preferred embodiments, this should not be construed as limiting the invention itself. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (9)

1. An amphiphilic polypeptide derivative of general formula I, whose sequence is R ₁ -Phe-Arg-Phe-Lys-R ₂ -R ₃ ,

in, R ₁ is selected from

_R2 is selected from

R ₃ is selected from PEG440, PEG1000 or PEG3400. 2. The amphiphilic polypeptide derivative according to claim 1, characterized in that it is selected from:

3. The preparation method of the amphiphilic polypeptide derivative described in any one of claim 1 or 2, is characterized in that, comprises the following steps: after dichloro resin is activated by dichloromethane, adds the amino acid of Fmoc protection, carries out the first After the reaction, remove the amino acid, clean the resin, and seal the resin with a blocking solution; after the sealing is completed, wash the resin with DMF, and use 20% piperidine/DMF solution to deprotect the Fmoc protecting group. Remove; after deprotection, wash the resin with DMF, and add the next Fmoc-protected amino acid to connect the next amino acid; repeat DMF cleaning, deprotection, DMF cleaning, and addition of Fmoc amino acid to extend the peptide chain; After all the amino acids are connected, trifluoroacetic acid is added to the resin for cutting; after the cutting is completed, the cutting liquid is collected, and the crude product is obtained by rotary evaporation, dissolved in water, and freeze-dried; the freeze-dried product is separated and purified to obtain the amphiphilic Sexual polypeptide derivatives. 4. The preparation method according to claim 3, wherein the blocking solution is methanol:N,N-diisopropylethylamine:DCM=15:5:80, V/V/V. 5. Use of the amphiphilic polypeptide derivative according to any one of claims 1 to 2 in the preparation of cancer therapeutic drugs. 6. A drug-loaded micelle, characterized in that it comprises the amphiphilic polypeptide derivative according to any one of claims 1-2 and the photosensitizer protoporphyrin IX. 7. the preparation method of drug-loaded micelles described in claim 6 is characterized in that, amphiphilic polypeptide derivative and photosensitizer protoporphyrin IX are respectively configured into DMSO mother liquor, get two kinds of DMSO mother liquors and mix, be dispersed in in the PBS solution, mixed evenly, and then subjected to ultrasonication or cell disruption to the solution to obtain the drug-loaded micelles. 8. The application of the drug-loaded micelle according to any one of claim 6 in cancer treatment. 9. The use according to claim 8, characterized in that the cancer is lung cancer and melanoma.

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