CN110499349B

CN110499349B - First small molecule peptide with oxygen carrying potential, second small molecule peptide capable of generating active oxygen and preparation method thereof

Info

Publication number: CN110499349B
Application number: CN201910716784.0A
Authority: CN
Inventors: 秦四勇; 冯嘉茹; 张爱清
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2019-08-05
Filing date: 2019-08-05
Publication date: 2021-04-02
Anticipated expiration: 2039-08-05
Also published as: CN110499349A

Abstract

The invention relates to a first small molecule peptide with oxygen carrying potential, a second small molecule peptide capable of generating active oxygen and a preparation method thereof. The first small molecule peptide with oxygen carrying potential has the sequence C₁₇H₃₁-CONH-VRGDS-COOH, in which the unsaturated double bond functional group can be oxidized by lipoxygenase to form conjugated double bond and the side group can produce hydroperoxide, the method for constructing the second small molecule peptide by using the first small molecule peptide is enzymatic method, and the structure of the second small molecule peptide is C₁₇H₃₁O₂-CONH-VRGDS-COOH. The method for generating active oxygen by using the second small molecular peptide is Fenton-like reaction C₁₇H₃₁O₂-CONH-VRGDS-COOH on Fe²⁺Generates active oxygen with cytotoxicity under the catalysis of (2).

Description

First small molecule peptide with oxygen carrying potential, second small molecule peptide capable of generating active oxygen and preparation method thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a first small molecule peptide with oxygen carrying potential, a second small molecule peptide capable of generating active oxygen and a preparation method thereof.

Background

Tumors are the first disease threatening the health of the whole human. In 2019, 1 month, the statistical data of national cancers published by the national cancer center in China show that the number of new malignant tumor cases in 2015 is about 392.9 ten thousands, the number of new malignant tumor cases in 2015 is about 233.8 thousands of new malignant tumor cases, and more than 1 million of new malignant tumor cases are diagnosed as the cancers on average every day. The conventional means for cancer therapy mainly include surgical resection, radiotherapy, chemotherapy, and the like. In order to effectively adopt personalized treatment aiming at different cancer patients and reduce side effects in the conventional treatment process, various novel treatment modes such as photodynamic treatment, photothermal treatment, ultrasonic treatment, immunotherapy and the like are developed in recent years. Among them, photodynamic therapy (PDT), an emerging cancer treatment strategy, is favored by researchers in the fundamental research of tumor therapy due to its unique manipulative properties that effectively reduce toxic side effects on normal tissues. PDT is primarily a treatment of malignant tumors by laser irradiation of specific wavelengths to excite photosensitizers in the tumor tissue, which in the excited state transfer their energy to the surrounding oxygen to generate cytotoxic reactive Radicals (ROS). It is clear that the tumor treatment effect of PDT depends on the photosensitizer concentration, the light intensity and the oxygen content of the tumor area at the same time. In order to achieve tumor-targeted delivery of photosensitizers, exogenous vectors are often used as vehicles. The synthesis of the carrier is complex, and the potential biological toxicity cannot be guaranteed. Meanwhile, human tissues have strong absorption to light, and the tissue penetration capability of a general light source is low, so that the light intensity reaching a tumor region is not high. Near infrared light may be selected as the light source for PDT in order to improve the light penetration capability. However, near infrared energy is low and the efficiency of activating the photosensitizer is not ideal. In addition, the hypoxic environment of the tumor area also limits the therapeutic efficacy of PDT.

In view of the principle of PDT, the generation of active free radicals provides a new idea for the treatment of malignant tumors. In order to overcome the problems in the PDT process, a self-carrying nano system is constructed, an exogenous carrier and a light source are not needed, and active oxygen free radicals can be controllably generated under the condition of not depending on the oxygen concentration of a tumor area, so that the effect of treating malignant tumors is effectively improved. Therefore, the construction of the self-carrying nano-therapeutic system which has good biocompatibility and simple and convenient synthesis and can efficiently generate singlet oxygen has important significance.

Disclosure of Invention

In order to solve the technical problems, the invention provides a first small molecule peptide with oxygen carrying potential, a second small molecule peptide capable of generating active oxygen and a preparation method thereof.

According to one aspect of the invention, a first small molecule peptide with oxygen carrying potential is provided, which has a sequence consisting of a hydrophobic alkyl chain, a hydrophobic amino acid, a hydrophilic amino acid and an N-terminal amino end-capping in sequence.

Preferably, based on the above scheme, the hydrophobic alkyl chain contains cis-unsaturated double bonds

A functional group, the cis-unsaturated double bond functional group is oxidized to form a conjugated double bond under the catalysis of lipoxygenase, and the side group generates hydroperoxide (-OOH), namely

On the basis of the scheme, preferably, the hydrophobic amino acid is one or more of valine and glycine.

On the basis of the scheme, the preferable hydrophilic amino acid is one or more of aspartic acid, serine and arginine.

Based on the scheme, preferably, the first small molecule peptide with oxygen carrying potential has a structural formula C₁₇H₃₁-CONH-VRGDS-COOH, wherein V is valine, R is arginine, G is glycine, D is aspartic acid, S is serine.

Preferably, based on the scheme, the preparation of the first small molecule peptide with oxygen carrying potential adopts dichlorotrityl chloride resin as a carrier, and the small molecule peptide chain segments are sequentially extended from the C end to the N end on the carrier through an FMOC polypeptide solid phase synthesis method. The synthesis method of the first small molecular peptide with oxygen carrying potential is mature and simple, the reaction condition is mild, the obtained small molecular peptide has amphipathy and tumor cell targeting, and the cis-unsaturated double bond contained in the small molecular peptide is

The functional group is oxidized to form conjugated double bond under the catalysis of lipoxygenase, and the side group generates hydroperoxide (-OOH), has potential oxygen carrying function, and can be self-assembled in aqueous solution to form the nano spiral fiber with good biocompatibility.

The invention also provides a second small molecular peptide capable of generating active oxygen, which is obtained by adopting the first small molecular peptide with oxygen carrying potential as a raw material through an enzymatic method, and the structural formula of the second small molecular peptide capable of generating active oxygen is C₁₇H₃₁O₂-CONH-VRGDS-COOH, and V is valine, R is arginine, G is glycine, D is aspartic acid, S is serine. The preparation reaction conditions of the small molecular peptide are simple and mild, and the obtained small molecular peptide has amphipathy, tumor cell targeting property and self-oxygen carrying function, can efficiently carry active oxygen source-hydroperoxide (-OOH) and generate active oxygen with cytotoxicity.

The invention also provides a method for preparing the second small-molecule peptide capable of generating active oxygen, which comprises the following steps:

s1, weighing the first small molecule peptide (C)₁₇H₃₁-CONH-VRGDS-COOH) solid powder in a containerAdding 0.1M PBS (phosphate buffer solution) with the pH value of 9.0 for dissolution, and injecting air bubbles for 20 minutes under uniform stirring;

s2, weighing lipoxygenase, putting the lipoxygenase into a container, and adding 0.1M PBS buffer solution with the pH value of 9.0 for dissolving;

s3, adding the solution in the S2 into the solution in the S1, keeping the temperature at 5 ℃, reacting for 2 hours, and sealing the obtained solution for low-temperature storage.

The reaction condition for preparing the second small molecular peptide capable of generating active oxygen from the first small molecular peptide with the oxygen carrying potential is simple and mild, and the obtained second small molecular peptide capable of generating active oxygen has amphipathy, tumor cell targeting property and self oxygen carrying function, can efficiently carry an active oxygen source-hydroperoxide (-OOH) and generate active oxygen with cytotoxicity.

The invention also provides a method for generating active oxygen by using the second small molecular peptide capable of generating active oxygen, and FeCl is added into the second small molecular peptide₂Solution of the second small molecule peptide in FeCl₂Fe in solution²⁺Active oxygen with cytotoxicity is generated under the action of the active oxygen.

The second small molecular peptide capable of generating active oxygen provided by the invention is in Fe²⁺In the presence of (A), a cytotoxic singlet oxygen can be produced¹O₂). In order to overcome the problems in the PDT process, the invention takes the hydroperoxide as the generation source of active oxygen, and combines with functional peptide to construct a nano system for self-carrying hydroperoxide (-OOH), does not need an external carrier and a light source, can controllably generate active oxygen free radicals under the condition of not depending on the oxygen concentration of a tumor region, and can provide a new idea for the treatment of malignant tumors. The method for generating the second small molecular peptide capable of generating the active oxygen is simple and convenient and has good biocompatibility.

The invention effectively avoids the biosafety problem caused by aggregation possibly occurring in the using process of the traditional inorganic nano particle carrier. At the same time, to overcome the generation of active oxygen during PDT, oxygen and H are applied to the tumor area₂O₂Concentration dependence problem, the first fraction with oxygen carrying potential designed by the present inventionThe sub-peptide and the second small molecular peptide capable of generating active oxygen can controllably generate active oxygen free radicals without external carriers and light sources and independent of the oxygen concentration in a tumor area.

Drawings

FIG. 1 is a diagram of a first small molecule peptide having oxygen carrying potential provided in example 1 of the present invention;

FIG. 2 is a mass spectrum of a first small molecule peptide with oxygen carrying potential provided in example 1 of the present invention;

FIG. 3 is a Transmission Electron Microscope (TEM) image of a nanospiral fiber provided in example 1 of the present invention;

FIG. 4 is an Atomic Force Microscope (AFM) image of a nanospiral fiber provided in example 1 of the present invention;

FIG. 5 is a structural diagram of a second small molecule peptide capable of generating active oxygen according to example 2 of the present invention;

FIG. 6 is a mass spectrum of a second small molecule peptide capable of generating active oxygen according to example 2 of the present invention;

FIG. 7 shows that the second small molecule peptide capable of generating active oxygen provided in example 3 of the present invention generates active oxygen and Fe²⁺Oxidation to Fe³⁺A color change map of (a);

FIG. 8 is a graph showing the trend of fluorescence intensity changes of a second small-molecule peptide capable of generating active oxygen and a corresponding control group, which are provided in example 3 of the present invention, in generating active oxygen within 140 min;

FIG. 9 is a fluorescence detection graph of a second small molecule peptide capable of generating active oxygen and a corresponding control group at 12 hours according to example 3 of the present invention;

FIG. 10 shows that the second small-molecule peptide capable of generating reactive oxygen species and the corresponding control group provided in example 3 of the present invention generate cytotoxic singlet oxygen (ii) ((ii))¹O₂) A fluorescence detection map of (a);

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

The following examples illustrate the source routes of the various feedstocks: dichlorotrityl chloride resin (degree of substitution of resin 0.97mmol/g), valine (FMOC-Val-OH) having an amino group protected by N-fluorene-9-methoxycarbonyl, glycine (FMOC-Gly-OH) having an amino group protected by N-fluorene-9-methoxycarbonyl, serine (FMOC-Ser (tBu) -OH) having an amino group and a pendant carboxyl group protected by N-fluorene-9-methoxycarbonyl and t-butyl, respectively, arginine (FMOC-Arg (Pbf) -OH) having an amino group and a pendant amino group protected by N-fluorene-9-methoxycarbonyl and 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl, aspartic acid (FMOC-Asp (OtBu) -OH) having an amino group and a pendant carboxyl group protected by N-fluorene-9-methoxycarbonyl and t-butoxy, respectively, and a salt thereof, benzotriazole-N, N, N ', N' -tetramethyluronium fluorophosphate (HBTU) and 1-Hydroxybenzotriazole (HOBT) were purchased from Gill Biochemical (Shanghai) Co., Ltd.

Linoleic acid, piperidine (Piperiding), ninhydrin, N-Dimethylformamide (DMF), methanol, Dichloromethane (DCM) were purchased from the national drug group.

Trifluoroacetic acid (TFA), N-Diisopropylethylamine (DIEA) were purchased from Aladdin.

Triisopropylsilane (TIS) was purchased from Sahn chemical technology (Shanghai) Inc.

Lipoxidases are purchased from sigma (sigma).

For convenience of description, the first small molecule peptide with oxygen carrying potential is named OPA1, the second small molecule peptide capable of generating active oxygen is named OPA2, phosphate buffer is abbreviated as PBS, and N, N-dimethylformamide is abbreviated as DMF.

Example 1

This example provides a first small molecule peptide having oxygen-carrying potentialThe structural formula is C₁₇H₃₁-CONH-VRGDS-COOH, the specific synthesis steps are as follows:

1) weighing a certain amount of resin, placing the resin in a polypeptide solid phase synthesis column, washing the resin with DMF three times, emptying the solvent, swelling the resin with DMF for 1 hour, and emptying the solvent.

2) A suitable amount of FMOC-Ser (tBu) -OH in 3 molar equivalents and DIEA in 6 equivalents in DMF was added to the column and stirred slowly for 2 h.

3) The FMOC protecting group in the first amino acid serine is removed. Adding 20% Peperiding/DMF (V/V) deprotection solution into a solid phase synthesis column, stirring for 30min, washing with DMF three times, and emptying the solvent.

4) A solution of 2 molar equivalents of FMOC-Asp (OtBu) -OH, 2.4 molar equivalents of HBTU, HOBT and 6 equivalents of DIEA in DMF was added to the column and stirred slowly for 2 h.

5) Taking a small amount of resin in the step 4), putting the resin into a methanol solution (with the concentration of 0.1mg/mL) of ninhydrin, heating and boiling for 3-5min, and checking color, wherein if the color is not changed, the amino acid coupling is successful.

6) Removing FMOC protecting group in FMOC-Asp (OtBu) -OH. Adding a 20% deprotection solution of Piperiding/DMF (V/V) into a solid phase synthesis column, stirring for 30min, washing with DMF three times, and emptying the solvent.

7) A proper amount of DMF solution of 2-fold molar equivalent of FMOC-Gly-OH, 2.4-fold molar equivalent of HBTU, HOBT and 6-fold equivalent of DIEA was added into the synthesis column, and the mixture was stirred slowly for 2 hours.

8) Taking a small amount of resin in 7), putting the resin into a methanol solution (0.1mg/mL) of ninhydrin, heating and boiling for 3-5min, and checking color if the color is not changed, which indicates that the amino acid is successfully coupled.

9) Removing FMOC protecting group in FMOC-Gly-OH. Adding a 20% deprotection solution of Piperiding/DMF (V/V) into a solid phase synthesis column, stirring for 30min, washing with DMF three times, and emptying the solvent.

10) A suitable amount of DMF solution of 2-fold molar equivalents of FMOC-Arg (Pbf) -OH, 2.4-fold molar equivalents of HBTU, HOBT and 6-fold equivalents of DIEA was added to the synthesis column and stirred slowly for 2 h.

11) Taking a small amount of peptide resin in the step 10), putting the peptide resin into a methanol solution (0.1mg/mL) of ninhydrin, heating and boiling for 3-5min, and checking color if the color is not changed, which indicates that the amino acid is successfully coupled.

12) And removing the FMOC protecting group in Fmoc-Val-OH. Adding a 20% deprotection solution of Piperiding/DMF (V/V) into a solid phase synthesis column, stirring for 30min, washing with DMF three times, and emptying the solvent.

13) An appropriate amount of 3-fold molar equivalents of linoleic acid, HBTU, HOBT and 6-fold equivalents of DIEA in DMF was added to the synthesis column and stirred slowly for 8 h.

14) Taking a small amount of resin in the step 13), putting the resin into a methanol solution (0.1mg/mL) of ninhydrin, heating and boiling for 3-5min, and checking color, wherein if the color is not changed, the fact that the linoleic acid is grafted on the last amino acid is shown.

15) The resin was washed with DMF, methanol and DCM, respectively, and dried under vacuum at ambient temperature to give a dry resin for use.

16) And (4) cutting off the polypeptide. Adding a volume of TFA/H to the dry resin synthesis column obtained in 15)₂Stirring the mixed liquid of O/TIS (V/V/V is 95%/2.5%/2.5%) at normal temperature for 2h, collecting the cut liquid, evaporating and concentrating to obtain viscous liquid, adding the viscous liquid into cold ether dropwise for precipitation, centrifuging, removing the supernatant, drying at normal temperature for 12h to obtain peptide powder, and freeze-drying the final product at low temperature for later use.

Detecting the first small molecule peptide (OPA1) with oxygen carrying potential obtained in this example, wherein FIG. 1 is a structural diagram of OPA1 obtained in this example; fig. 2 is a mass spectrum of OPA1 obtained in this example, wherein m/z represents the mass-to-charge ratio on the abscissa and the relative intensity of the peak on the abscissa. The results show that: the actual molecular weight of OPA1 was consistent with the theoretical molecular weight. Fig. 3 is a Transmission Electron Microscope (TEM) image of OPA1 self-assembled nanospiral fibers provided in this example; fig. 4 is an Atomic Force Microscope (AFM) image of the OPA1 self-assembled nanospiral fiber provided in this example; the results show that: the self-assembled morphology of OPA1 is a helical nanofiber.

Example 2

The present example provides a method for producing a second small molecule peptide (OPA2) capable of generating active oxygen, which comprises the following steps:

1) weigh the appropriate amount of OPA1 (C)₁₇H₃₁-CONH-VRGDS-COOH) solid powder in a container, adding a suitable amount of PBS (0.1M, pH 9.0) buffer solution to dissolve, and injecting air bubbles for 20 minutes under stirring;

2) weighing appropriate amount of lipoxygenase, placing in a container, adding appropriate amount of PBS (0.1M, pH 9.0) buffer solution, and dissolving;

3) adding the solution in the step 2) into the solution in the step 1), keeping the temperature at 5 ℃, reacting for 2h, and sealing the obtained solution for low-temperature storage.

Detecting the second small molecule peptide (OPA2) capable of generating active oxygen obtained in this example, wherein FIG. 5 is a structural diagram of the second small molecule peptide provided in example 2 of the present invention; FIG. 6 is a mass spectrum of a second small molecule peptide according to example 2 of the present invention, wherein the abscissa m/z represents the mass-to-charge ratio and the ordinate represents the relative intensity of the peak corresponding to the abscissa; the results show that: the actual molecular weight of the second small molecular peptide is consistent with the theoretical molecular weight, and the second small molecular peptide is C₁₇H₃₁O₂-CONH-VRGDS-COOH。

Example 3

This example provides a method for generating active oxygen by using a second small-molecule peptide capable of generating active oxygen, which is based on Fenton-like reaction, i.e., in Fe²⁺In the presence of (a), the hydroperoxide (-OOH) in the second small molecule peptide structure that can generate reactive oxygen species generates Reactive Oxygen Species (ROS) that are cytotoxic.

FIG. 7 is a graph showing that the second small molecule peptide capable of generating active oxygen provided in example 3 of the present invention generates active oxygen and then Fe²⁺(concentration used was 100. mu.M) Oxidation to Fe³⁺A color change map of (a); the results show that the compound is in OPA2 (C)₁₇H₃₁O₂-CONH-VRGDS-COOH) in the presence of Fe²⁺Can be oxidized into Fe by it³⁺，FeCl₂The solution changed from colorless, clear and transparent to yellow, clear and transparent.

To examine the effect of OPA2 on the generation of active oxygen, experiments were performed with the materials in the following table to examine the fluorescence intensity of the second small molecule peptide capable of generating active oxygen according to the present invention for generating active oxygen within 140 min.

Detection of OPA2 (C) Using ROS fluorescence indicator DCFH-DA₁₇H₃₁O₂CONH-VRGDS-COOH) in Fe²⁺The experimental results are shown in fig. 8, and fig. 8 is a graph showing the trend of the fluorescence intensity of the second small molecule peptide provided in example 3 of the present invention and the corresponding control group in generating reactive oxygen species within 140 min. If. represents the fluorescence intensity of DCFH produced by oxidation of Reactive Oxygen Species (ROS) by DCFH, indirectly demonstrating the generation of Reactive Oxygen Species (ROS); in the figure, experimental group 5 represents the second small molecule peptide provided in example 3 in Fe²⁺The trend of the fluorescence intensity of the generated Reactive Oxygen Species (ROS) within 140min, and the experimental group 1 represents the trend of the fluorescence intensity of the reactive oxygen species indicator DCFH within 140 min. The results show that: in Fe²⁺Under the catalysis of the compound, the OPA2 can rapidly and efficiently generate the ROS in an initial short time, so that the fluorescence intensity of the reactive oxygen species indicator is obviously increased, compared with the experimental group 1, the generated Reactive Oxygen Species (ROS) can increase the fluorescence intensity of the reactive oxygen species indicator by about 250 times in 140min, and the result shows that the second small molecular peptide (OPA2) provided by the embodiment 3 of the invention is Fe²⁺Active oxygen is rapidly generated under the catalysis.

To further examine the effect of the second small molecule peptide on the generation of active oxygen, experiments were conducted with the materials in the following table to examine the fluorescence intensity of the second small molecule peptide capable of generating active oxygen according to the present invention after generating active oxygen at 12 hours.

Fig. 9 is a fluorescence detection graph of reactive oxygen species generated at 12 hours by the second small-molecule peptide capable of generating reactive oxygen species and the corresponding control group provided in example 3 of the present invention, in which the abscissa Wavelength represents the meaning of Wavelength, and the ordinate dcf.if. represents the fluorescence intensity of DCFH generated by oxidation of the reactive oxygen species indicator DCFH by the Reactive Oxygen Species (ROS), thereby indirectly demonstrating the generation of the Reactive Oxygen Species (ROS).

Detection of OPA2 (C) Using ROS fluorescence indicator DCFH-DA₁₇H₃₁O₂-CONH-VRGDS-COOH) by Fe²⁺Reactive Oxygen Species (ROS) generated after catalysis, Vitamin C (VC) as a reactive oxygen species inhibitor, and the graph of experiment group 5 shows OPA2 in Fe²⁺Generates Reactive Oxygen Species (ROS) and increases the fluorescence intensity of the reactive oxygen species indicator to about 325 degrees when the reaction time reaches 12 hours, and when the co-culture of the active oxygen species inhibitor vitamin c (vc) added to the experimental group 5 (i.e., the experimental group 4), the fluorescence intensity of the experimental group 4 is significantly decreased and the generation of reactive oxygen species is inhibited; the results show that: over time and in Fe²⁺In the presence of OPA2, the active oxygen (ROS) with cytotoxicity can be generated for a long time.

To demonstrate that the reactive oxygen species generated by the second small molecule peptide are cytotoxic singlet oxygen (ii)¹O₂) The following materials in the table were used to test singlet oxygen generated by the second small molecule peptide capable of generating active oxygen provided by the present invention¹O₂)。

FIG. 10 shows that the second small-molecule peptide capable of generating reactive oxygen species and the corresponding control group provided in example 3 of the present invention generate cytotoxic singlet oxygen: (¹O₂) The abscissa (wavelength (nm) in the graph represents the wavelength and the ordinate represents the wavelength¹O₂IF. represents the production of cytotoxic singlet oxygen (¹O₂) The fluorescence intensity of (2).

Using singlet oxygen with a single selectivity (¹O₂) Detection of OPA2 (C) by Green fluorescence marker SOSG₁₇H₃₁O₂CONH-VRGDS-COOH) in Fe²⁺Singlet oxygen produced in the presence of (¹O₂) Vitamin C (VC) as an active oxygen inhibitor.

The experimental results show that: in Fe²⁺In the presence of a second small molecule peptide and a singlet oxygen with single selectivity: (¹O₂) The green fluorescence indicator SOSG co-cultured (experiment group 5 in the figure) showed a significant fluorescence enhancement at 0.05 h and a continuous fluorescence intensity enhancement within 12h (experiment group 9 in the figure), and when the active oxygen inhibitor Vitamin C (VC) was added to the experiment group 5 (experiment group 4), the fluorescence intensity of the experiment group 4 and the experiment group 8 was correspondingly significantly reduced and the generated singlet oxygen was significantly inhibited within 0.05 h and 12h compared with the experiment group 5 and the experiment group 9, confirming that OPA2 was Fe-doped²⁺Efficiently catalyzing and generating cytotoxic singlet oxygen¹O₂)。

In summary, the second small-molecule peptide capable of generating active oxygen is constructed in the embodiment of the invention, and is used in Fe²⁺In the presence of (A), active oxygen having cytotoxicity can be efficiently and continuously produced within 12 hours, and the active oxygen is singlet oxygen having cytotoxicity: (¹O₂)。

In order to overcome the problems in the PDT process, the embodiment of the invention takes the hydroperoxide as the generation source of the active oxygen, and combines with the functional peptide to construct the nano system for self-carrying the hydroperoxide (-OOH), so that no external carrier and light source are needed, and the active oxygen free radicals can be controllably generated under the condition of not depending on the oxygen concentration of a tumor area, thereby effectively improving the effect of treating malignant tumors. The embodiment of the invention not only avoids the biosafety problem caused by aggregation of inorganic nano particles in the in-vivo use process, but also effectively overcomes the defect that the generation of active oxygen causes oxygen and H in a tumor area₂O₂The dependence of the concentration.

Finally, the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The first small molecule peptide with oxygen carrying potential is characterized by being formed by sequentially blocking a hydrophobic alkyl chain, a hydrophobic amino acid, a hydrophilic amino acid and an N-terminal amino group; the structural formula of the first small molecular peptide is C17H31-CONH-VRGDS-COOH, wherein V is valine, R is arginine, G is glycine, D is aspartic acid, and S is serine.

2. The first small molecule peptide having an oxygen-carrying potential of claim 1, wherein the-RGD-sequence in the sequence of the first small molecule peptide balances hydrophilicity and hydrophobicity in the peptide structure and further targets tumor cells.

3. The first small molecule peptide with oxygen carrying potential of claim 2, wherein the first small molecule peptide is obtained by using dichlorotrityl chloride resin as a carrier resin and sequentially extending small molecule peptide chain segments from C end to N end on the carrier resin by FMOC polypeptide solid phase synthesis.

4. A second small-molecule peptide capable of generating active oxygen, wherein the second small-molecule peptide is obtained by an enzymatic method by using the first small-molecule peptide as claimed in any one of claims 1 to 3 as a raw material, and the structural formula of the second small-molecule peptide is C₁₇H₃₁O₂-CONH-VRGDS-COOH, and V is valine, R is arginine, G is glycine, D is aspartic acid, S is serine.

5. A method for preparing the second small molecule peptide capable of generating active oxygen according to claim 4, comprising the steps of:

s1, weighing C₁₇H₃₁-CONH-VRGDS-COOH solid powder in containerAdding 0.1M PBS (phosphate buffer solution) with the pH value of 9.0 into the container for dissolving, and injecting air bubbles for 20 minutes under the condition of uniform stirring;

s2, weighing lipoxygenase, putting the lipoxygenase into a container, and adding 0.1M PBS (phosphate buffer solution) with the pH value of 9.0 for dissolving;

6. A method for generating active oxygen by using a second small-molecule peptide, which comprises adding FeCl to the second small-molecule peptide according to claim 4₂Solution of the second small molecule peptide in Fe²⁺Active oxygen with cytotoxicity is generated under the action of the active oxygen.