Tumor microenvironment pH-responsive size-variable peptide self-assembled drug carrier and application thereof in tumor specific treatment

Abstract

The invention discloses a tumor microenvironment pH-responsive size-variable peptide self-assembled drug carrier and an application thereof in tumor specific treatment. The peptide Ac-AAAFFHH-NH2 capable of being self-assembled in multiple stages and an anti-tumor drug are self-assembled into a drug-loaded nano-micelle, and the nano-micelle is subjected to secondary self-assembly to form an aggregate, namely the peptide self-assembled drug carrier. The drug carrier keeps a large aggregate size in a normal tissue neutral environment, and can be prevented from being swallowed by normal cells; when the nano-micelle is in a weakly acidic tumor microenvironment (TME), the pH-dependent size change occurs, and the aggregate is dissociated into the nano-micelle with positive charges. The nano-micelles can easily enter tumor cells, so that TME unique pH-triggered tumor specific treatment is realized. According to the invention, tumor treatment is carried out based on the pH difference between TME and a normal tissue microenvironment, so that damage to normal cells is avoided, and specific treatment of tumors is achieved.

Description

Technical Field

[0001] The invention belongs to a specific drug delivery system in tumor treatment in the biological field, and relates to a method for delivering chemotherapeutic drugs based on peptide self-assembly technology. Background Art

[0002] Targeted drug carriers, which can selectively deliver therapeutic drugs to tumor sites, thereby improving treatment efficiency and reducing side effects on healthy tissues and organs, have become an important way to reduce cancer mortality. Although there are currently a variety of drug carriers such as inorganic nanoparticles, liposomes, and polymer nanoparticles, they each have shortcomings that are difficult to overcome. Inorganic nanoparticles are not biodegradable and may persist and accumulate in the patient's body, eventually leading to chronic inflammatory responses. Liposomes have poor stability in vivo and in vitro, and certain liposome components or solvents used in the preparation process may cause adverse reactions such as fever, vomiting, and anaphylactic shock in patients. Most polymer nanoparticles have complex chemical compositions and modifications, which may trigger immune responses in the body, and their degradation products have potential long-term toxicity.

[0003] In contrast, peptides and their derivative materials, with their simple chemical composition, relatively stable structure, easy biodegradability and low toxicity, provide a safer and more effective way for cancer treatment and have become a key trend in therapeutic innovation. Currently, there are more than 60 peptides and peptide-derived materials among the therapeutic drugs approved by the U.S. Food and Drug Administration (FDA), of which about 18% are specifically used for cancer treatment, far more than inorganic nanoparticles, liposomes and polymer nanoparticles.

[0004] However, almost all existing targeted drug carriers achieve their targeting by selectively binding to receptors on tumor cells through surface-modified ligands. Although they are effective in treating specific types of tumors, there may be significant differences between different tumor cell types and subtypes due to the genetic characteristics and phenotypic diversity of tumor cells. In addition, even in the same type of tumor, the expression levels of tumor cell markers can vary between different patients. More importantly, the receptors of tumor cells are usually substances overexpressed in tumor cells, and these receptors are also expressed in normal cells, so they are not absolutely specific. This limits the application of carriers designed based on the principle of specific binding to tumor receptors and may cause potential damage to healthy cells.

[0005] The tumor microenvironment (TME) is a unique feature of tumors, usually characterized by hypoxia, acidification, and increased interstitial pressure, which is in sharp contrast to normal tissues. In terms of acidification, the enhanced metabolic activity of tumor cells and the acceleration of anaerobic glycolysis lead to the accumulation of acidic metabolites such as lactic acid in the tumor matrix, thus forming a mildly acidic microenvironment (pH 6.0-6.8), while the pH value of normal tissue is close to neutral (about 7.4). Therefore, the development of peptide drug carriers based on the unique pH in the TME may be a wise tumor-specific therapeutic strategy that takes advantage of the commonalities of tumors and the differences between tumors and normal cells, thereby avoiding damage to normal tissues. However, existing pH-responsive peptide drug carriers usually need to be at extreme pH values ​​(5.0 or 5.5) to work, which is not suitable for tumor-specific treatment based on TME. Summary of the invention

[0006] The purpose of the present invention is to provide a size-variable peptide self-assembly drug carrier based on the pH response of the tumor microenvironment, so as to solve the problems of low specificity and large side effects of current tumor targeted therapy.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention constructs a peptide capable of multi-level self-assembly, characterized in that the peptide is Ac-AAAFFHH-NH2. The peptide exhibits responsiveness to a unique pH in the TME, and can spontaneously arrange from bottom to top and form an ordered structure through non-covalent interactions between molecules (such as hydrogen bonds, van der Waals forces, electrostatic interactions, hydrophobic interactions, etc.). It consists of three basic units: a hydrophobic unit located at the N-terminus, which can provide the hydrophobic force required for self-assembly; a structurally stable unit located in the middle, which uses π-π stacking forces to ensure unidirectional self-assembly; and a hydrophilic unit located at the C-terminus, which relies on charge conversion to modify its self-assembly structure. Simple amino and acetylation modifications are performed at both ends of the peptide, which are commonly present in organisms and therefore do not cause potential harm to them, to enhance stability.

[0009] After ultrasonic treatment under a neutral environment (about pH 7.4), the peptide monomer can self-assemble with the drug into drug-loaded nanomicelles (~100nm), and the nanomicelles can self-assemble again under a neutral environment to form aggregates (~1.56μm), which are peptide self-assembled drug carriers. These aggregates maintain their larger particle size in normal tissues to avoid being phagocytosed by normal cells; when they are in a weakly acidic tumor microenvironment, pH-dependent size changes occur, and the aggregates dissociate into positively charged nanomicelles, which are then taken up by tumor cells and eventually kill tumor cells.

[0010] Preferably, the peptide and the anti-tumor drug are prepared into a mixed solution, ultrasonically treated and allowed to stand to allow the peptide to self-assemble, and then centrifuged to obtain a peptide self-assembly drug carrier.

[0011] Preferably, the pH of the mixed solution is 7.4±0.1, more preferably 7.35-7.45.

[0012] Preferably, the ultrasonic treatment is performed at a power of 100-200 W, for 2-10 minutes, and then allowed to stand for 10-30 minutes.

[0013] The preparation method of the above-mentioned peptide self-assembly drug carrier comprises the following steps:

[0014] (1) preparing a solution of anti-tumor drugs to be loaded in PBS;

[0015] (2) dissolving the peptide in the solution of (1) above and allowing to stand for 2-10 minutes after ensuring that it is fully dissolved;

[0016] (3) treating the solution in (2) with ultrasound at a frequency of 40±10 kHz and a power of 100-200 W for 5±2 minutes, followed by allowing to stand for 20±5 minutes;

[0017] (4) The solution was centrifuged at 3000±1000 rpm for 5±2 minutes, washed with PBS, centrifuged and freeze-dried to obtain a peptide self-assembly drug carrier.

[0018] Preferably, the concentration of the peptide is 1.5±0.5 mg / mL; the anti-tumor drugs include 5-fluorouracil, paclitaxel, tamoxifen citrate, cyclophosphamide, etc.

[0019] The peptide or peptide self-assembly drug carrier is used in tumor-specific treatment.

[0020] Preferably, the peptide self-assembled drug carrier is located in the microenvironment of normal tissue, and remains as a large-size aggregate to avoid being taken up by normal cells; when it is located in the tumor microenvironment, a pH-dependent size change occurs, and its weakly acidic pH environment causes the protonation of the hydrophilic unit, resulting in electrostatic repulsion and causing the aggregate to dissociate into positively charged nanomicelles. At the same time, these highly protonated nanomicelles interact with the negatively charged tumor cell membrane through electrostatic attraction, promoting their engulfment by tumor cells. After engulfment, the nanomicelles are broken down into amino acids in the lysosomes, and the internal drugs are released to kill cancer cells.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Can be loaded with a variety of chemotherapeutic drugs. St-PSAPs can be loaded with a variety of chemotherapeutic drugs because St-PSAPs are non-covalently bound to the drugs. St-PSAPs loaded with different drugs can be prepared simply by replacing the solution containing different drugs during the preparation process.

[0023] (2) It can achieve tumor-specific treatment. St-PSAP@drug treats tumors based on the pH difference between the tumor microenvironment (TME) and the normal tissue microenvironment, thus avoiding damage to normal cells.

[0024] (3) Compared with existing drug delivery systems, St-PSAPs@drug is composed of amino acids and simple modifications in the body and is biodegradable, thus avoiding the inflammatory response caused by accumulation in the body and the potential side effects of metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Molecular structure of peptide Ac-AAAFFHH-NH2 (left) and interaction diagram (right).

[0026] Figure 2 Schematic diagram of the self-assembly of peptide Ac-AAAFFHH-NH2 to form St-PSAPs.

[0027] Figure 3 Schematic diagram of the self-assembly of peptide Ac-AAAFFHH-NH2 and drug to form St-PSAPs@drug.

[0028] Figure 4 Schematic diagram of St-PSAPs@drug used to achieve pH-responsive tumor microenvironment (TME) tumor-specific therapy.

[0029] Figure 5 Schematic diagram of St-PSAPs loaded with the chemotherapy drug pentafluorouracil (5-FU) to form St-PSAP@5FU.

[0030] Figure 6 Electron micrographs and particle size distribution of St-PSAPs at pH 7.4 (left) and 6.4 (middle); the right figure is a locally enlarged electron micrograph of St-PSAPs at pH 7.4.

[0031] Figure 7 Electron micrographs and particle size distribution of St-PSAP@5FU at pH 7.4 (left) and 6.4 (middle); the right figure is a locally enlarged electron micrograph of St-PSAP@5FU at pH 7.4.

[0032] Figure 8 Particle size variation of St-PSAPs (left) and St-PSAP@5FU (right) with pH.

[0033] Fig. 9 Concentration stability curve of St-PSAPs particle size (left), time stability curve (middle), and degradation curve of St-PSAPs by proteinase K (right).

[0034] Fig.10 Concentration stability curve of St-PSAP@5FU particle size (left), time stability curve (middle), and degradation curve of St-PSAP@5FU by proteinase K (right).

[0035] Fig.11 EDS energy spectrum analysis image.

[0036] Fig.12 The drug release ability of St-PSAP@5FU was tested using UV-visible spectrophotometer.

[0037] Fig.13 Schematic diagram of the force analysis process of pH response using NaCl, SDS, and Urae.

[0038] Fig.14 Effects of NaCl, SDS, and Urae on electrostatic, hydrophobic, and hydrogen bonding interactions in St-PSAPs (left) and St-PSAP@5FU (right).

[0039] Fig.15 The secondary structures of St-PSAPs (left) and St-PSAP@5FU (right) were elucidated using infrared spectroscopy.

[0040] Fig.16 Infrared spectroscopy was used to analyze the secondary structure content and proportion in St-PSAPs (left) and St-PSAP@5FU (right).

[0041] Fig.17 Circular dichroism spectroscopy was used to verify the β-fold structure in St-PSAPs (left) and St-PSAP@5FU (right).

[0042] Fig.18 The existence of β-fold was further verified by using the specific interaction of thioflavin T with β-fold structure.

[0043] Fig.19 St-PSAP was loaded with the fluorescent molecule calcein to form St-PSAP@Calcein, thereby verifying that St-PSAPs can be taken up by cells.

[0044] Fig. 20 Uptake results of St-PSAP@Calcein by normal cells (a) and different tumor cells (bd).

[0045] Fig.21 The CCK-8 method verified the biocompatibility of St-PSAP@5FU and its specific therapeutic ability against different tumor cells; Fig.21 a is a normal cell, Fig.21 bd is a tumor cell.

[0046] Fig. 22 Calcein AM / PI cell live and dead observation; Fig. 22 a is a normal cell, Fig. 22 bd is a tumor cell. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0048] Eukaryotic cells HeLa, MCF-7, 4T1, and 293T cells were obtained from the cell bank of the Chinese Academy of Sciences. The peptide Ac-AAAFFHH-NH2 was synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. Ultrasonication was performed using a Xinzhi XZ-6DTD ultrasonic cleaning machine.

[0049] Example 1

[0050] Preparation of St-PSAPs: The peptide Ac-AAAFFHH-NH2 was dissolved in PBS solution at 1.5 mg / ml, and allowed to stand for 2 min after being fully dissolved. Subsequently, the solution was sonicated at 40 kHz, 90% power (about 162 W) for 5 min at room temperature. Then, the solution was allowed to stand for 20 min. Then, the solution was centrifuged at 3000 rpm for 5 min to obtain St-PSAPs. Finally, St-PSAPs were rinsed twice with PBS, centrifuged and freeze-dried for 24 h, and kept at 4°C for use.

[0051] Preparation of St-PSAP@5FU loaded with the chemotherapy drug 5-FU: Prepare a PBS solution of 10 mg / ml pentafluorouracil, dissolve the peptide Ac-AAAFFHH-NH2 in the solution at 1.5 mg / ml, and let it stand for 2 minutes after it is fully dissolved. Subsequently, the solution is ultrasonically treated at 40 kHz and 90% power (about 162 W) for 5 minutes at room temperature. Then, the solution is allowed to stand for 20 minutes. Then, the solution is centrifuged at 3000 rpm for 5 minutes to obtain drug-loaded St-PSAPs, namely St-PSAP@5FU. Finally, St-PSAP@5FU is rinsed twice with PBS, lyophilized for 24 hours after centrifugation, and kept at 4°C for use.

[0052] Example 2

[0053] St-PSAPs morphology and particle size distribution: The sample morphology was observed on a field emission scanning electron microscope [ZEISS Ultra55]. The basic ImageJ software (1.53t) was used to analyze the size of typical St-PSAPs nanoparticles in TEM images, and the data was processed using Origin software to quickly obtain its accurate distribution. The results are shown in Figure 6 As shown in Figure 2, SEM verified that St-PSAPs were uniformly dispersed and regular spherical at pH 7.4 and pH 6.4 without aggregation, and the average sizes were 1.72±0.40μm and 0.16±0.07μm, respectively.

[0054] Example 3

[0055] St-PSAP@5FU and morphology and particle size distribution: The sample morphology was observed on a field emission scanning electron microscope [ZEISS Ultra55]. The size of typical St-PSAP@5FU nanoparticles in TEM images was analyzed using basic ImageJ software (1.53t), and the data was processed using Origin software to quickly obtain its accurate distribution. The results are shown in Figure 7 As shown in Figure 2, SEM verified that St-PSAP@5FU was uniformly dispersed in regular spheres at pH 7.4 and pH 6.4 without aggregation, with average sizes of 1.56±0.36μm and 0.10±0.04μm, respectively.

[0056] Example 4

[0057] Dynamic light scattering was used to analyze the changes in the particle size of St-PSAPs and St-PSAP@5FU with pH: St-PSAPs and St-PSAP@5FU were suspended in PBS with different pH values ​​and then transferred to a measuring dish. The particle size measurement mode of Zetasizer Nano-ZS was selected to obtain the hydrated particle size at different pH values. The results are shown in Figure 2. Figure 8 As shown in the figure, the hydrated particle size of St-PSAPs gradually decreases from about 1.7 μm to about 300 nm as the pH decreases, and the hydrated particle size of St-PSAP@5FU gradually decreases from about 1.3 μm to about 300 nm as the pH decreases, showing obvious pH response characteristics.

[0058] Example 5

[0059] Dynamic light scattering was used to analyze the stability of St-PSAPs particle size as a function of concentration: the particle size measurement mode of Zetasizer NanoZS was selected to obtain the hydrated particle size of St-PSAPs at different dilution multiples ( Fig. 9 Left). The particle size of St-PSAPs remains stable at different concentrations.

[0060] The temporal stability of St-PSAPs was determined by UV-visible spectrophotometer (UV-2600): St-PSAPs were quickly diluted to a final concentration of 0.02 wt% with PBS buffer, and then the pH of the diluent was adjusted to 7.4, 7.0, 6.4, and 6.0, respectively. The diluent was immediately transferred to a 10 mm cuvette and its stability was monitored by measuring the absorbance at a wavelength of 202 nm. The results were recorded every 5 minutes for 300 minutes. The results showed that St-PSAPs maintained a stable size over a long period of time ( Fig. 9 middle).

[0061] The in vitro degradation characteristics of St-PSAPs were analyzed using static light scattering: The in vitro decomposition of St-PSAPs was measured using static light scattering. The excitation wavelength and emission wavelength were both set to 350nm, and the slit width was kept at 2nm. The fluorescence spectrometer (JASCO FP 8500, USA) was used for measurement in continuous mode. A graph of the relationship between light scattering intensity and time was plotted. The results showed that St-PSAPs could be effectively decomposed by the addition of proteinase K at the 45th minute ( Fig. 9 right).

[0062] Example 6

[0063] Dynamic light scattering was used to analyze the stability of St-PSAP@5FU particle size as it changes with concentration: ZetasizerNano ZS particle size measurement mode was selected to obtain the hydrated particle size of St-PSAP@5FU at different dilution multiples. The particle size of St-PSAP@5FU remained stable at different concentrations ( Fig.10 Left).

[0064] The temporal stability of St-PSAP@5-FU was determined by UV-visible spectrophotometer (UV-2600): St-PSAP@5-FU was quickly diluted to a final concentration of 0.02wt% with PBS buffer, and then the pH value of the diluent was adjusted to 7.4, 7.0, 6.4 and 6.0, respectively. The diluent was immediately transferred to a 10mm cuvette and its stability was monitored by measuring the absorbance at a wavelength of 202nm. The results were recorded every 5 minutes for 300 minutes. The results showed that St-PSAP@5-FU maintained a stable size over a long period of time ( Fig.10 middle).

[0065] The in vitro degradation characteristics of St-PSAP@5FU were analyzed by static light scattering: The in vitro decomposition of St-PSAPs and St-PSAP@5-FU was measured by static light scattering. The excitation wavelength and emission wavelength were both set to 350nm, and the slit width was kept at 2nm. The fluorescence spectrometer (JASCO FP 8500, USA) was used for measurement in continuous mode. The relationship between light scattering intensity and time was plotted. The results showed that with the addition of proteinase K at the 45th minute, St-PSAP@5FU could be effectively decomposed ( Fig.10 right).

[0066] Example 7

[0067] Loading of St-PSAP@5-FU: EDS spectrum analysis of St-PSAP@5-FU was performed with fluorine as the characteristic element to verify whether 5-FU was loaded by St-PSAP. The results are shown in Fig.11 As shown, the Fu element (red) exists in the energy spectrum image of St-PSAP@5-FU, that is, it is effectively loaded.

[0068] Example 8

[0069] Loading rate of St-PSAP@5-FU: Prepare a 10 mg / ml 5-fluorouracil PBS solution for standby use. Then, take 1 ml of the solution and dilute it with 0.1 M hydrochloric acid solution to make a solution containing about 10 μg of 5-fluorouracil per 1 ml (1000 times). Then, measure the absorbance at a wavelength of 265 nm by UV-visible spectrophotometry, and calculate the 5-FU concentration in the standby solution based on the absorption coefficient of 5-FU being 552.

[0070] 5-FU concentration in the reserve solution (C0):

[0071]

[0072] Where A represents absorbance, a is the absorption coefficient (L / g·cm-1), and b is the optical path length (cm).

[0073] Subsequently, St-PSAP@5FU was prepared and the mass of St-PSAP@5FU was weighed. The remaining solution was diluted with 0.1M hydrochloric acid solution to prepare a solution containing about 10 μg of pentafluorouracil per 1 ml (1000 times). Subsequently, the absorbance was measured at a wavelength of 265 nm by UV-visible spectrophotometry, and the 5-FU concentration in the remaining solution was calculated based on the absorption coefficient of 5-FU being 552.

[0074] 5-FU concentration in the residual solution (C1):

[0075]

[0076] Subsequently, the loading capacity (%) of St-PSAP@5FU was calculated.

[0077]

[0078] Where M represents the mass of St-PSAP@5FU (mg), and L represents the loading capacity of St-PSAP@5FU (%). Experimental results show that the loading capacity of St-PSAP@5FU is as high as 47.75%.

[0079] Drug release of St-PSAP@5-FU: After 2 mg of St-PSAP@5FU was suspended in 2 ml of PBS, the suspension was placed in a dialysis bag (molecular weight cutoff 8.0 kDa) and divided into two groups, one group added with 1 ul of proteinase K, and the other group added with the same volume of PBS. Then, the two groups of dialysis bags were immersed in 48 ml of PBS at pH 7.4 and pH 6.4, respectively, protected from light, and carried out under gentle shaking at 37 ° C. To detect the release of 5-Fu from St-PSAP@5FU, 2 ml of dialysate was accurately drawn at 1, 2, 4, 6, 8, 10, 12, and 24 h after dialysis, and the absorbance of the dialysate was detected and recorded using a UV-visible spectrophotometer. After each sampling, the same volume of PBS with the same pH should be added to supplement the original system.

[0080] Concentration of 5-FU in sample C2 (mg / ml):

[0081]

[0082] Where t is the dilution factor of the sample solution.

[0083] Drug release rate

[0084]

[0085] Where v is the volume of the dialysis system and Ψ is the total amount of 5-FU in all previous samples.

[0086] The results are as follows Fig.12As shown in the figure, St-PSAP@5-FU exhibited similar release behaviors in PBS at pH 7.4 and 6.4, with maximum drug release rates of approximately 25.93% and 32.87% after 24 h, respectively. This relatively low release rate indicates that St-PSAPs can effectively control the release of 5-FU outside cells. The slightly higher release rate at pH 6.4 compared to pH 7.4 may be due to the fact that 5-FU was partially retained in the interstitial space of the aggregates during the formation of nanomicelles. By adding proteinase K to the dialysis system, 5-FU could be rapidly released at both pH levels with a maximum release rate of approximately 90%, indicating that St-PSAPs have high biodegradability and effective drug release properties inside cells.

[0087] Example 9

[0088] pH-responsive interaction force analysis of St-PSAPs and St-PSAP@5-FU: Stock solutions of sodium chloride (to break electrostatic interactions), 1,6-hexanediol (to break hydrophobic interactions), and urea (to break hydrogen bonds and van der Waals forces) were prepared. Titration experiments were performed on St-PSAPs and St-PSAP@5-FU at pH 7.4 using static light scattering at 350 nm. The samples were titrated sequentially with increasing concentrations of sodium chloride (50-150 mM), 1,6-hexanediol (2-15% w / v), and urea (0.5-2 M). After adding each concentration of additive, light scattering was recorded at 30 s using a fluorescence spectrometer.

[0089] like Fig.14As shown in the figure, at pH 7.4 and 6.4, after adding 1,6-hexanediol and urea, the light scattering values ​​of St-PSAPs and St-PSAP@5-FU decreased severely with the increase of concentration, indicating that the secondary structures of the aggregates formed by St-PSAPs and St-PSAP@5-FU under neutral conditions and the nanomicelles formed under acidic conditions are mainly controlled by hydrogen bonds and hydrophobic interactions. With the increase of salt concentration, the light scattering values ​​of St-PSAPs and St-PSAP@5-FU remained relatively stable at pH 7.4, but increased at pH 6.4, indicating that the aggregated structure formed under neutral conditions was basically unaffected by the ion concentration, while under acidic conditions, the salt ions gradually neutralized the surface charge of the nanomicelles, causing them to aggregate. It further shows that St-PSAPs and St-PSAP@5-FU have almost no electrostatic repulsion in a neutral environment to form stable aggregates, while under acidic conditions, they exist in the form of nanomicelles due to electrostatic interactions. As the pH of St-PSAPs and St-PSAP@5-FU changes from 7.4 to 6.4, the histidine residues on their surfaces are gradually protonated from a near-neutral charge state, which increases the electrostatic repulsion between the particles. This process is reversible, as shown by the results of salt titration experiments, indicating that the pH-dependent size change is mainly driven by electrostatic interactions.

[0090] Example 10

[0091] The secondary structure information of St-PSAPs and St-PSAP@5-FU at different pH values ​​was collected by infrared spectroscopy: the St-PSAPs and St-PSAP@5-FU solutions at different pH values ​​were freeze-dried into powder in a freeze dryer, and the samples were added to potassium bromide powder and ground into fine powder to form a uniform and transparent sample film. The spectrum was at 4000-400cm -1 The spectra of the amide I region were deconvoluted using PeakFit software (version 4.12, Systat Software, Inc.) to determine the content and changes of secondary structure. The results showed that at pH 7.4 and pH 6.4, St-PSAPs ( Fig.15 Left) and St-PSAP@5-FU( Fig.15 Right) The values ​​of each subpeak are basically consistent, indicating that the aggregates and nanomicelles have similar secondary structures.

[0092] Embodiment 11

[0093] Determine the content and proportion of secondary structure: Use PeakFit to perform second-order derivative zero baseline correction and Gaussian function fitting on the spectrum, and use the determination coefficient r 2>0.99. By normalizing the vector of these derivatives, the peak intensity and ratio can be calculated. It can be clearly seen that ( Fig.16 ), the content of each secondary structure remained almost unchanged in aggregates and nanomicelles, and the β-sheet dominated the secondary structure, accounting for 63% and 60% in St-PSAPs and 45% and 41% in St-PSAP@5-FU, respectively.

[0094] Example 12

[0095] Circular dichroism spectroscopy verified the β-folded structure of St-PSAPs and St-PSAP@5-FU: CD spectra were obtained on a spectrometer (JASCO J-815, JOSCO, Tokyo). St-PSAPs and St-PSAP@5-FU were diluted to a final concentration of 0.02wt% with PBS buffers of different pH values ​​and transferred to a quartz cell with a 10mm optical path for measurement. The CD spectra of the samples were recorded at a constant temperature of 25°C, with a scanning wavelength of 260-190nm and a step size of 1nm. All scanning results were background subtracted and corrected using PBS buffer of the corresponding pH. The results showed that ( Fig.17 ), St-PSAPs and St-PSAP@5-FU showed significant positive peaks at about 195 nm (195-200 nm) at pH 7.4 and 6.4, showing strong β-folding signals. This indicates that the aggregates and nanomicelles in St-PSAPs and St-PSAP@5-FU have β-folding structures, which is consistent with the findings of infrared spectroscopy.

[0096] Embodiment 13

[0097] The specific interaction of thioflavin T with the β-fold structure was used to further verify the stable existence of the β-fold: ThT was dissolved in ultrapure water to prepare a 6 mM stock solution, which was filtered with a 0.22 μm syringe filter. The reserve solution was then added to the original St-PSAPs and St-PSAP@5-FU solutions to make a working ThT concentration of 40 μM. The solution was incubated at room temperature for a specified time, then transferred to a 10 mm cuvette, and the emission fluorescence signal of 460 to 600 nm with an excitation wavelength of 450 nm was measured using a fluorescence spectrometer FLS1000. Fig.18As shown, although the aggregates in pH 7.4 showed stronger fluorescence signals than the nanomicelles in pH 6.4, the continued presence of fluorescence from pH 7.4 to 6.0 confirmed that the β-sheet structure in St-PSAPs and St-PSAP@5-FU remained intact despite the size changes with pH. After the addition of proteinase K, the fluorescence signal disappeared, indicating the disappearance of the β-sheet structure. This observation is consistent with the findings of CD spectroscopy and IR spectroscopy, reinforcing the view that St-PSAPs and St-PSAP@5-FU maintain a stable β-sheet structure in response to pH changes.

[0098] Embodiment 14

[0099] Schematic diagram of verifying the cell uptake capacity of St-PSAPs and St-PSAP@5-FU: First, St-PSAP@calcein is prepared. Calcein is a compound that can emit green fluorescence at a wavelength of 521nm under 488nm light excitation. Fig.19 The chemical structure of calcein, its self-assembly with peptide monomers to form St-PSAP@Calcein, and its subsequent uptake by cells are shown. Notably, calcein alone cannot enter cells unless it is integrated into St-PSAPs to form St-PSAP@Calcein. At pH 6.4, St-PSAP@Calcein dissociates into positively charged nanomicelles, which adsorb on the negatively charged cell surface through electrostatic interactions, thereby promoting cellular uptake. Once inside the cell, the peptides on the micelle surface are hydrolyzed by proteases in the lysosomes, releasing calcein, causing the cells to emit green fluorescence under excitation. In contrast, at pH 7.4, St-PSAP@Calcein forms large aggregates that are difficult to be internalized by cells, and the cells remain unaffected and do not emit green fluorescence.

[0100] Embodiment 15

[0101] Cellular uptake capacity of St-PSAPs and St-PSAP@5-FU: A 50 μM calcein PBS solution was prepared, the peptide Ac-AAAFFHH-NH2 was dissolved in the solution at a concentration of 1.5 mg / mL, and allowed to stand for 2 minutes after complete dissolution. The solution was then sonicated at 40 kHz and 90% power for 5 minutes at room temperature. The solution was allowed to stand for 20 minutes and then centrifuged at 3000 rpm for 5 minutes to obtain St-PSAP@calcein. St-PSAP@calcein was washed twice with PBS, centrifuged and freeze-dried for 24 hours, and then stored at 4°C for later use.

[0102] Subsequently, normal cells 293T and tumor cells HeLa, MCF-7 and 4T1 were inoculated in confocal culture dishes and cultured for 24 hours, and then replaced with fresh culture medium (4T1 cells were cultured in RPMI-1640 containing 10% FBS, and other cells were cultured in DMEM containing 10% FBS). HeLa, MCF-7, 4T1, and 293T cells were all from the Cell Bank of the Chinese Academy of Sciences. St-PSAP@calcein was added to the culture medium to a final concentration of 0.15 mg / mL and incubated with the cells for another 4 hours. The culture medium was then removed and washed several times with PBS. The cells were observed using a confocal laser scanning microscope under the 488nm channel. 293T cells were tested only with a culture medium at pH 7.4, while tumor cells were tested with culture medium at pH 7.4 and 6.4.

[0103] The results are as follows Fig. 20 To evaluate the uptake in normal tissues, St-PSAP@Calcein was co-cultured with human embryonic kidney 293T cells. Confocal microscopy results showed only a small amount of fluorescence, indicating that normal cells had limited uptake. Similarly, under pH 7.4 conditions, human cervical cancer cells (HeLa), human breast cancer cells (MCF-7), and mouse breast cancer cells (4T1) showed only weak green fluorescence under 488nm excitation, indicating that the extent of internalization of St-PSAP@Calcein aggregates was limited. In contrast, under pH 6.4 conditions, these cells showed obvious green fluorescence accompanied by a unique regional distribution pattern. These results indicate that cells can effectively take up St-PSAPs only in a low pH environment similar to the tumor microenvironment (TME).

[0104] Example 16

[0105] The pH value of the normal cell environment is about 7.4. To verify the biocompatibility of St-PSAPs and St-PSAP@5-FU, these compounds and 5-FU were co-cultured with 293T cells for 24 hours. Subsequently, the cell activity was measured using the cell counting kit-8 (CCK-8) method. Fig.21As shown in a, St-PSAPs are non-toxic to cells. The cell activity in the St-PSAPs group was generally higher than that in the control group, which may indicate that it has a stimulating effect on cell growth. The toxicity of 5-FU to cells is obvious and dose-dependent. When the concentration of 5-FU was increased to 300 μg / mL, the cell activity was finally reduced to 48.07%. The activity of 293T cells treated with St-PSAP@5-FU (where the net content of 5-FU was consistent with that of the 5-FU treatment group) was only slightly decreased, indicating that under pH 7.4, St-PSAP@5-FU has little effect on normal cells and its damage to cells depends on the dose, which may be due to the gradual release of trace amounts of 5-FU in St-PSAP@5-FU. These results show that St-PSAPs have good biocompatibility, and St-PSAP@5-FU can effectively encapsulate 5-FU, prevent it from being taken up by normal cells, and reduce its toxicity to normal cells.

[0106] The tumor microenvironment is characterized by mild acidity. To confirm the inhibitory effect of St-PSAP@5-FU on cancer, we used CCK-8 assay to compare the cytotoxicity of St-PSAPs, 5-FU and St-PSAP@5-FU on cancer cells under neutral (pH 7.4) and weakly acidic (pH 6.4) conditions. Fig.21 As shown in Figures bd, treatment with St-PSAPs did not affect the viability of HeLa, MCF-7, or 4T1 cells at different pH levels, further demonstrating its excellent biocompatibility and inherent non-toxicity. 5-FU treatment resulted in a significant decrease in the viability of HeLa, MCF-7, and 4T1 cells, showing significant cytotoxicity. Notably, the viability of HeLa and 4T1 cells was dose-dependent on 5-FU, while MCF-7 cells did not show a significant dose-dependency and were highly sensitive to very low doses. After treatment with St-PSAP@5-FU, different trends emerged at the two pH levels. In a neutral environment (pH 7.4), St-PSAP@5-FU was essentially nontoxic to HeLa and 4T1 cells, while the viability of MCF-7 cells decreased significantly, which may be due to their sensitivity to the trace amount of 5-FU released from St-PSAP@5-FU. In contrast, under weakly acidic conditions (pH 6.4), St-PSAP@5-FU exhibited strong inhibitory effects against all three tumor cell types, outperforming 5-FU monotherapy in terms of inhibition rate. These findings highlight that St-PSAP@5-FU is able to effectively deliver 5-FU into cancer cells and exploit the pH difference between the tumor microenvironment and normal tissues to amplify its cytotoxic effects.

[0107] Embodiment 17

[0108] To visually evaluate the distribution of live and non-live cells, 293T cells were exposed to 5-FU, St-PSAPs, and St-PSAP@5-FU under neutral conditions for 24 h and subsequently stained with Calcein AM / PI solution ( Fig. 22 a). 5-FU treatment significantly reduced the cell density, indicating its inhibitory effect on the cells. In contrast, St-PSAPs and St-PSAP@5-FU treatment did not cause obvious cell death or density reduction in 293T cells, further demonstrating their non-toxicity to 293T cells under normal cell environments. Similarly, HeLa, MCF-7, and 4T1 cells were exposed to 5-FU, St-PSAPs, and St-PSAP@5-FU at two different pH levels for 24 h and subsequently stained with Calcein AM / PI solution, as shown in Figure 5. Fig. 22 bd. After 5-FU treatment, significant cell death was observed under both pH conditions. In contrast, cells treated with St-PSAPs alone at both pH values ​​showed no significant damage in all three cell types. In contrast, at pH 7.4, St-PSAP@5-FU treatment caused negligible damage to HeLa and 4T1 cells, but showed significant damage in MCF-7 cells, which is consistent with the cytotoxicity results and may be due to the high sensitivity of MCF-7 cells to the trace amount of 5-FU released from St-PSAP@5-FU. At pH 6.4, all three cancer cell types underwent significant cell death under St-PSAP@5-FU treatment. These findings confirm the advantages of St-PSAPs as a biocompatible carrier. In addition, St-PSAP@5-FU effectively enhanced the therapeutic effect of the chemotherapeutic drug 5-FU and alleviated its adverse reactions.

[0109] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A peptide capable of multi-stage self-assembly, characterized in that: The peptide is Ac-AAAFFHH-NH2.

2. A tumor microenvironment pH-responsive size-variable peptide self-assembly drug carrier, characterized in that: The peptide of claim 1 and the anti-tumor drug self-assemble into drug-loaded nano-micelles, and the nano-micelles undergo secondary self-assembly to form aggregates, which are peptide self-assembled drug carriers.

3. The peptide self-assembling drug carrier according to claim 2, characterized in that: The peptide self-assembled drug carrier maintains its size in normal tissues; when placed in a tumor microenvironment, a pH-dependent size change occurs, and the aggregates dissociate into positively charged nano-micelles.

4. The method for preparing the peptide self-assembly drug carrier according to claim 2 or 3, characterized in that: The peptide and the anti-tumor drug are prepared into a mixed solution, subjected to ultrasonic treatment and allowed to stand to allow the peptide to self-assemble, and then centrifuged to obtain a peptide self-assembly drug carrier.

5. The preparation method according to claim 4, characterized in that: The pH of the mixed solution is 7.4±0.

1.

6. The preparation method according to claim 5, characterized in that: The ultrasonic treatment conditions are as follows: power 100-200W, treatment for 2-10 minutes, and standing for 10-30 minutes.

7. The method for preparing the peptide self-assembling drug carrier according to any one of claims 2 to 6, characterized in that: The steps include: (1) preparing a solution of anti-tumor drugs to be loaded in PBS; (2) dissolving the peptide in the solution of (1) above and allowing to stand for 2-10 minutes after ensuring that it is fully dissolved; (3) treating the solution in (2) with ultrasound at a frequency of 40±10 kHz and a power of 100-200 W for 5±2 minutes, followed by allowing to stand for 20±5 minutes; (4) The solution was centrifuged at 3000±1000 rpm for 5±2 minutes, washed with PBS, centrifuged and freeze-dried to obtain a peptide self-assembly drug carrier.

8. The preparation method according to claim 7, characterized in that: The concentration of the peptide was 1.5 ± 0.5 mg / mL.

9. Use of the peptide according to claim 1, the peptide self-assembling drug carrier according to claim 2, 3 or 4, or the peptide self-assembling drug carrier prepared by the method according to any one of claims 5 to 8 in tumor-specific treatment.

10. The use according to claim 9, characterized in that: When the peptide self-assembled drug carrier is located in the neutral microenvironment of normal tissue, it remains as a large-size aggregate to avoid being taken up by normal cells. When it is located in the tumor microenvironment, it undergoes pH-dependent size changes to form positively charged nanomicelles, which are then taken up by tumor cells and ultimately kill the tumor cells.

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