LU504660A1

LU504660A1 - Rapeseed peptide and use thereof in preparation of nanocarrier for drug

Info

Publication number: LU504660A1
Application number: LU504660A
Authority: LU
Inventors: Jian Yuan; Zhigao Wang; Jiaqiang Huang; Mingjie Wang; Zhipeng Zou; Rong He; Xingrong Ju
Original assignee: Univ Nanjing Finance & Economics
Priority date: 2021-11-11
Filing date: 2021-11-15
Publication date: 2023-07-05
Also published as: CN114409729B; WO2023082243A1; CN114409729A; LU504660B1

Abstract

The present disclosure provides a rapeseed peptide and a use thereof in preparation of a nanocarrier for a drug, and belongs to the technical field of biomedical materials. The rapeseed peptide has a sequence shown in SEQ ID NO: 1, and can be used in preparation of a nanocarrier for a drug. The present disclosure also provides a method for preparing an antitumor drug with the rapeseed peptide as a carrier, including the following steps: dissolving the rapeseed peptide and hydroxycamptothecin (HCPT) successively in trichloromethane (TCM) to obtain a mixed solution A; adding the mixed solution A dropwise to phosphate-buffered saline (PBS), adding Tween 80, and thoroughly stirring a resulting mixture to obtain a mixed solution B; and removing the TCM in the mixed solution B to obtain the antitumor drug. The rapeseed peptide of the present disclosure is an excellent nanocarrier for an antitumor drug.

Description

DESCRIPTION LU504660

RAPESEED PEPTIDE AND USE THEREOF IN PREPARATION OF NANOCARRIER

FOR DRUG

TECHNICAL FIELD

The present disclosure relates to the technical field of biomedical materials, and in particular relates to a rapeseed peptide and a use of thereof in preparation of a nanocarrier for a drug.

BACKGROUND

In recent years, the drug therapy has always been a main therapy for many diseases. However, due to problems such as poor selectivity, large toxic and side effects, and uncontrollable drug release, many drugs lead to an unsatisfactory treatment effect.

In order to improve the efficacy of a drug, it is often necessary to increase a concentration and frequency of administration of the drug, but a large amount of the drug in vivo will damage normal tissues and organs, resulting in a poor long-term treatment effect.

With the development of nanomedicine technology and polymer materials, nanomaterial drug-delivery systems have gradually become a new field of nanomedicine, and some nanomaterials such as micelles, liposomes, hydrogels, and magnetic particles have been successively used in the field of drug delivery. However, hydrophilic traditional hydrogels lack ligands to interact with hydrophobic drugs, and have limited abilities to load a drug and control the release of a drug. In addition, polymer materials have disadvantages such as large toxic and side effects and poor biocompatibility. As a group of novel carrier materials, amphiphilic peptides have a small molecular weight, can be degraded and absorbed by the human body, exhibit prominent biocompatibility and excellent self-assembly performance, and avoid the shortcomings such as difficult degradation and large toxic and side effects of polymer materials.

Therefore, the amphiphilic peptides have become one of the hot spots fouso4660 international research on self-assembling materials. However, there is a lack of amphiphilic peptides that have an excellent drug-loading effect and can significantly reduce an ICso of a drug in the prior art.

SUMMARY

A first objective of the present disclosure is to provide a rapeseed peptide, which is an excellent nanocarrier for an antitumor drug. The rapeseed peptide has a high drug encapsulation rate, and can significantly reduce an ICso of a drug and reduce a dose of a drug.

A second objective of the present disclosure is to provide a use thereof in preparation of a nanocarrier for a drug.

The present disclosure also provides a method for preparing an antitumor drug with the rapeseed peptide as a carrier, which is simple. An antitumor drug prepared by the method has excellent pH and CathB dual responsiveness, a prominent drug-loading effect, a higher release rate of the nanocarrier in a lysosome and a tumor microenvironment (TME) than in a physiological environment, a moderate drug release rate, and excellent stability.

To achieve the above objectives, the present disclosure provides the following technical solutions:

The present disclosure provides a rapeseed peptide with a sequence shown in SEQ

ID NO: 1.

The present disclosure also provides a use thereof in preparation of a nanocarrier for a drug.

In the present disclosure, the drug is hydroxycamptothecin (HCPT).

In the present disclosure, a mass ratio of the rapeseed peptide to the HCPT is (5-15):1.

The present disclosure also provides a method for preparing an antitumor drug with the rapeseed peptide as a carrier, including the following steps: (1) dissolving the rapeseed peptide and HCPT successively in trichloromethane (TCM) to obtain a mixed solution A;

(2) adding the mixed solution A dropwise to phosphate-buffered saline (PBS)u504660 adding Tween 80, and thoroughly stirring a resulting mixture to obtain a mixed solution B; and (3) removing the TCM in the mixed solution B to obtain the antitumor drug.

In the present disclosure, a mass concentration of the rapeseed peptide in the mixed solution À is 5 mg/mL to 15 mg/mL.

In the present disclosure, a volume ratio of the PBS to the mixed solution A is 1:(8-15); and a mass percentage content of the Tween 80 in the mixed solution B is 0.5% to 1.5%.

In the present disclosure, in step (3), after the TCM in the mixed solution B is removed, a resulting system is filtered to obtain an antitumor nanodrug.

In the present disclosure, a filter membrane used for the filtration has a pore size of 0.4 um to 0.8 um.

The rapeseed peptide provided by the present disclosure is an excellent nanocarrier for an antitumor drug, which has a high drug encapsulation rate, and can significantly reduce an ICso of a drug and reduce a dose of a drug. An anti-tumor drug prepared with the rapeseed peptide SVIRPPL of the present disclosure has excellent pH and cathepsin

B (Cath B) dual responsiveness, high drug carrier specificity, a prominent drug-loading effect, a higher release rate of the nanocarrier in a lysosome and a TME than in a physiological environment, a moderate drug release rate, and excellent stability. The present disclosure creatively uses the rapeseed peptide SVIRPPL (an amphiphilic peptide) as a carrier material to avoid the aggregation of a protein nanocarrier in a liver, improve the tumor penetration of a nanocarrier, and reduce the biometric identification in vivo, thereby improving the efficiency of targeted transport.

BRIEF DESCRIPTION OF THE DRAWINGS LU504660

FIG. 1 shows a dynamic light scattering (DLS) particle size of an antitumor nanodrug prepared with a rapeseed peptide C during storage at 4°C, where an ordinate represents an average particle size, and an abscissa represents a time in a unit of d.

FIG. 2 shows a polydispersity index (PDI) change of an antitumor drug prepared with a rapeseed peptide C during storage at 4°C, where an abscissa represents a time in a unit of d.

FIG. 3 shows transmission electron microscopy (TEM) images of a rapeseed peptide C blank nanocarrier (A) without HCPT and an antitumor drug (B).

FIG. 4 shows impacts of antitumor drugs with different encapsulated HCPT concentrations and HCPT aqueous solutions with different concentrations on a survival rate of a HepG2 tumor cell, where HCPT represents an HCPT aqueous solution, and

SVIRPPL-HCPT/NP represents an antitumor drug; an abscissa represents a concentration of an HCPT aqueous solution or an encapsulated HCPT concentration in an antitumor drug; and the different letters indicate that there are significant differences.

FIG. 5 shows impacts of antitumor drugs with different encapsulated HCPT concentrations and HCPT aqueous solutions with different concentrations on a survival rate of an MKN-28 tumor cell, where HCPT represents an HCPT aqueous solution, and

SVIRPPL-HCPT/NP represents an antitumor drug; an abscissa represents a concentration of an HCPT aqueous solution or an encapsulated HCPT concentration in an antitumor drug, and an ordinate represents a survival rate of the MKN-28 tumor cell; and the different letters indicate that there are significant differences.

FIG. 6 shows impacts of antitumor drugs with different encapsulated HCPT concentrations and HCPT aqueous solutions with different concentrations on a survival rate of an A549 tumor cell, where HCPT represents an HCPT aqueous solution, and

SVIRPPL-HCPT/NP represents an antitumor drug; an abscissa represents a concentration of an HCPT aqueous solution or an encapsulated HCPT concentration in an antitumor drug, and an ordinate represents a survival rate of the A549 tumor cell; and the different letters indicate that there are significant differences.

FIG. 7 shows impacts of antitumor drugs with different encapsulated HCPT concentrations and HCPT aqueous solutions with different concentrations on a survival rate of an MCF-7 tumor cell, where HCPT represents an HCPT aqueous solution, and

SVIRPPL-HCPT/NP represents an antitumor drug; an abscissa represents a concentration of an HCPT aqueous solution or an encapsulated HCPT concentration in1504660 an antitumor drug, and an ordinate represents a survival rate of the MCF-7 tumor cell; and the different letters indicate that there are significant differences.

FIG. 8 shows HCPT release curves of SVIRPPL-HCPT/NP at different pH values with or without Cath B.

FIG. 9 shows pathological sections of a liver tumor tissue treated in each group, where saline represents a normal saline group, SVIRPPL NP represents an SVIRPPL

NP group, HCPT represents an HCPT aqueous solution group, and SVIRPPL-HCPT represents an SVIRPPL-HCPT/NP group.

FIG. 10 shows bioluminescence images of mice in each group before the first injection of D-luciferin (denoted as day 0) and on day 7 and day 15 after the first injection of D-luciferin, where saline represents a normal saline group, SVIRPPL NP represents an SVIRPPL NP group, HCPT represents an HCPT aqueous solution group, and

SVIRPPL-HCPT NP represents an SVIRPPL-HCPT/NP group.

FIG. 11 shows fluorescent quantification analysis results of liver tumors of mice in each group before the first injection of D-luciferin (denoted as day 0) and on day 7, day 15, and day 19 after the first injection of D-luciferin, where saline represents a normal saline group, SVIRPPL NP represents an SVIRPPL NP group, HCPT represents an

HCPT aqueous solution group, and SVIRPPL-HCPT NP represents an

SVIRPPL-HCPT/NP group; and the different letters indicate that there are significant differences.

FIG. 12 shows distribution conditions of an antitumor drug throughout the body and in in vitro organs at different time points after the injection of Cy5.5-labeled

SVIRPPL-HCPT/NP.

DETAILED DESCRIPTION OF THE EMBODIMENTS LU504660

The present disclosure is further described below in conjunction with specific examples and accompanying drawings, but the implementations of the present disclosure are not limited thereto.

Unless otherwise specified, the raw materials used in the following examples are commercially available, and the methods used are conventional operation methods well known by those skilled in the art.

Example 1

After a rapeseed protein was subjected to hydrolysis with an alkaline protease, a small peptide SVIRPPL (SEQ ID NO: 1) was discovered and isolated. The small peptide was an amphiphilic peptide and named rapeseed peptide C. The rapeseed peptide C was prepared by the Synpeptide Inc according to a conventional solid-state synthesis method and was used for the experiment in the present disclosure.

A method for preparing an antitumor drug with the rapeseed peptide C was provided, including the following steps: (1) 10 mg of a rapeseed peptide C powder was added to 1 mL of TCM, and a resulting mixture was stirred for thorough dissolution to obtain a solution of the rapeseed peptide C in TCM; and then 0.1 mL of an aqueous solution with 1 mg of HCPT was slowly added at a rate of 8 mL/h dropwise to the solution of the rapeseed peptide C in

TCM, and a resulting mixture was subjected to an ultrasonic treatment at a temperature of 15°C and a power of 70 kW in the dark until HCPT was completely dissolved to obtain a mixed solution A. (2) The mixed solution A obtained in step (1) was added at a rate of 8 mL/h dropwise to 10 mL of PBS with a concentration of 0.01 M and a pH of 7.4 (purchased from

Solarbio), then Tween 80 was added, and a resulting mixture was stirred for 8 h at a temperature of 4°C and a rotational speed of 600 r/min to obtain a homogeneous mixed solution B, where a volume percentage concentration of Tween 80 in the mixed solution

B was 1%; and during the dropwise addition, the PBS was magnetically stirred at 600 r/min.

(3) The mixed solution B obtained in step (2) was subjected to ultrasonic dispersians04660 for 1 min at a temperature of 4°C and a power of 300 KW and then stirred at a temperature of 25°C and a rotational speed of 100 r/min to remove TCM through volatilization, and a resulting system was filtered through a membrane with a pore size of 0.45 um to obtain the antitumor drug (abbreviated as SVIRPPL-HCPT/NP).

An HCPT-free rapeseed peptide C blank nanocarrier (abbreviated as SVIRPPL NP) was prepared according to the preparation method of the antitumor drug, except that 0.1 mL of water was used instead of the 0.1 mL of the aqueous solution with 1 mg of HCPT.

Example 2

In this example, the antitumor drug prepared in Example 1 was characterized.

A Malvern Zetasizer Nano ZS instrument (He-He as a laser: 633 nm; and scattering angle: 173°) was used to detect a DLS particle size and PDI of the antitumor drug prepared in Example 1 during storage at 4°C. A determination method of an encapsulation rate was as follows: 1 mL of the drug obtained after the TCM removal in step (3) of Example 1 was taken and centrifuged, and a resulting precipitate was collected to obtain unencapsulated HCPT, 1 mL of a TCM solution was added to the precipitate to prepare an HCPT solution, and a spectrophotometer was used to determine a concentration of HCPT at 367 nm; and a content of unencapsulated HCPT in the antitumor drug was calculated and used to calculate an encapsulation rate of

HCPT according to the following formula:

Total HCPT esntent - content of free

HOP ma solution 159

Encapsulatson rate (Fa) = Total HOPT content

Test results showed that the antitumor drug prepared in Example 1 was a transparent solution, and the antitumor drug had an average particle size of 178 nm and an average PDI value of 0.26 when it was just prepared. Storage experimental results were shown in FIG. 1 and FIG. 2, and it could be known that, after being stored at 4°C for d, the antitumor drug had an average particle size of 195 nm and a PDI value of less than 0.3, indicating that the antitumor drug can maintain excellent colloidal stability. It was tested that an HCPT encapsulation rate of the antitumor drug was 78.5%. 6 h after the preparation of the antitumor drug and the HCPT-free rapeseed peptide

C blank nanocarrier, the antitumor drug and the HCPT-free rapeseed peptide C blank nanocarrier each were subjected to TEM analysis, and analysis results were shown in1504660

FIG. 3. It can be seen from the figure that the blank nanocarrier and the antitumor drug each are approximately spherical, have a uniform particle distribution, and are spheres with a diameter of 160 nm to 180 nm, which are consistent with the DLS test results. It further indicates that the amphiphilic rapeseed peptide C can form a nanodrug carrier with high stability through self-assembly.

Example 3

In this example, antitumor activities of the antitumor drug prepared in Example 1 for four types of tumor cells were investigated.

According to the encapsulation rate calculated in Example 2, an antitumor drug with an encapsulated HCPT concentration of 7.8 uM was prepared with reference to the method of Example 1, and then diluted with PBS of 0.01 M and pH 7.4 to prepare six antitumor drug samples with different encapsulated HCPT concentrations of 0.01 pM, 0.05 uM, 0.1 uM, 0.25 uM, 0.5 pM, and 1 pM. An HCPT-free rapeseed peptide C blank nanocarrier was prepared with reference to the method of Example 1, except that 0.1 mL of water was used instead of the 0.1 mL of the aqueous solution with 1 mg of HCPT, that is, no HCPT was added; and the HCP T-free rapeseed peptide C blank nanocarrier was then diluted with PBS of 0.01 M and pH 7.4 to prepare rapeseed peptide C blank nanocarrier samples with encapsulated HCPT concentrations of 0.01 pM, 0.05 uM, 0.1 uM, 0.25 uM, 0.5 uM, and 1 uM corresponding to the antitumor drug samples. In addition,

HCPT was dissolved in PBS of 0.01 M and pH 7.4 to prepare HCPT aqueous solution samples with different concentrations of 0.01 uM, 0.05 uM, 0.1 uM, 0.25 uM, 0.5 uM, and 1 uM.

A liver cancer cell HepG2, a gastric cancer cell MKN-28, a lung cancer cell A548)504660 and a breast cancer cell MCF-7 (purchased from the Jiangsu Synthgene Biotechnology

Co., Ltd.) each were inoculated into a 96-well plate at a density of 5 x 10% cells/well, where some wells were not inoculated with cells and served as blank wells; and resulting four 96-well plates were incubated overnight at 37°C. In each of the 96-well plates inoculated with tumor cells, sample wells (including sample wells with the antitumor drug samples of different encapsulated concentrations, sample wells with the rapeseed peptide C blank nanocarrier samples, and sample wells with HCPT aqueous solution samples of different concentrations), control wells, and blank wells were set. 100 pL of a sample was added to each sample well (with cells). 100 pL of PBS of 0.01 M and pH 7.4 was added instead of a sample to each control well (with cells). In each blank well, there were no cells, and only 100 uL of PBS of 0.01 M and pH 7.4 was added. The four 96-well plates were incubated for 24 h in a 37°C CO: incubator; a resulting supernatant was discarded, and then the plates were washed with PBS of 0.01 M and pH 7.4 to remove a residual liquid; 120 uL of a 1 mg/mL methyl thiazolyl tetrazolium (MTT) solution was added to each well; the plates were further incubated for 4 h in a 37°C CO: incubator; a resulting supernatant was discarded, and 100 uL of dimethyl sulfoxide (DMSO) was added to each well; the plates were incubated for 20 min in a 37°C CO» incubator under shaking, and then the absorbance (OD value) of each well was determined at a wavelength of 490 nm; and a survival rate of a tumor cell intervened with each concentration of the antitumor drug, each concentration of the rapeseed peptide C blank nanocarrier, or each concentration of the HCPT aqueous solution was calculated according to the following formula:

OD value of a sample wall -

Tumor cell survival rate (Foi = LE SEE. sou we — 100

CF vaine of a blank well

Results were shown in FIG. 4 to FIG. 7. Both the HCPT aqueous solution and the antitumor drug exhibited a significant concentration-dependent inhibitory effect for proliferation of tumor cells. Compared with the HCPT aqueous solution, the antitumor nanodrug significantly enhanced inhibitory effects of HCPT for the above four types of tumor cells, indicating that the rapeseed peptide C is an excellent carrier, and can effectively improve the antitumor effect and bioavailability of HCPT.

According to calculation results, lCso values of the antitumor drug for the liver canceus04660 cell HepG2, the gastric cancer cell MKN-28, the lung cancer cell A549, and the breast cancer cell MCF-7 were 0.17 uM, 0.18 uM, 0.25 pM, and 0.27 pM, respectively; lCso values of the HCPT aqueous solution for the liver cancer cell HepG2, the gastric cancer cell MKN-28, the lung cancer cell A549, and the breast cancer cell MCF-7 were 0.45 uM, 0.37 uM, 0.46 uM, and 0.48 uM, respectively; and lCso values of the rapeseed peptide C blank nanocarrier for the liver cancer cell

HepG2, the gastric cancer cell MKN-28, the lung cancer cell A549, and the breast cancer cell MCF-7 were 0.57 uM, 0.81 uM, 0.97 uM, and 1.10 pM, respectively.

The above results indicate that the rapeseed peptide C as a nanocarrier can effectively reduce an ICso value of HCPT for tumor cells, effectively improve an inhibitory effect of the antitumor drug for tumor cells, and reduce the biotoxicity caused by a high

HCPT concentration.

Example 4

In this example, release curves of HCPT in the antitumor drug prepared in Example 1 at different pH values with or without Cath B were tested.

In vitro release characteristics of the antitumor drug prepared in Example 1 were investigated by a standard dialysis method. 5 mL of the antitumor drug prepared in

Example 1 was filled in each of four dialysis bags (molecular weight cut-off (MWCO): 500

Da), then the dialysis bags were placed in solutions 1, 2, 3, and 4 (Table 1), respectively, and a solution outside each dialysis bag was stirred at 37°C and 100 rpm/min to investigate release curves of HCPT in the antitumor drug prepared in Example 1 at different pH values with or without Cath B. At 1 h, 2h, 4h, 8h, 12h, 16 h, 24 h, 36 h, and 48 h, 2 mL of a solution outside each dialysis bag was collected, and then 2 mL of an original solution was added (the original solution here referred to a solution in an initial state outside each dialysis bag, that is, the solution in Table 1). An HCPT content in a solution collected each time was determined by an ultraviolet (UV) spectrophotometer at 367 nm, and a release kinetic curve of HCPT was plotted.

According to the preparation method in Example 1, the antitumor drug prepared 504660

Example 1 had an HCPT concentration of 100 pg/mL; and according to the encapsulation rate (78.5%) determined in Example 2, it could be calculated that 0.4 mg of HCPT was encapsulated per 5 mL of the antitumor drug. In order to prove that HCPT detected outside a dialysis bag was not caused by the dialysis bag itself, the following control was set: 0.4 mg of HCPT was dissolved in 5 mL of 0.01 M PBS, a resulting solution was filled in four dialysis bags (MWCO: 500 Da), and the dialysis bags were placed in solutions 1, 2, 3, and 4 (as shown in Table 1), respectively; and then release characteristics of HCPT in the aqueous solutions in the dialysis bags were measured by the same method as above, and a release kinetic curve of HCPT was plotted.

Table 1 Compositions of the solutions outside the dialysis bags il

Component dialysis bag

As shown in FIG. 8, the antitumor drug prepared in Example 1 exhibits pH-dependent release characteristics, release rates of the antitumor drug in a lysosome (pH 5.0, including CathB) and a TME (pH 6.5) are higher than a release rate of the antitumor drug in a physiological environment (pH 7.4), and the antitumor drug has slow release and exhibits excellent in vitro stability at pH 7.4; the release of an HCPT aqueous solution in a dialysis bag in each solution is not pH-dependent, and HCPT can rapidly pass through a dialysis bag, with a release rate as high as 70% within 5 h; and the addition of CathB at pH 5.0 will further accelerate the release of HCPT from the antitumor drug, and a release rate of HCPT from the antitumor drug in the presence of

CathB within 48 h is twice a release rate at pH 7.4, indicating that the acidic condition will destroy the bonding between the rapeseed peptide and the HCPT in the antitumor drug, resulting in rapid drug release. In view of the acid-dependent characteristics of CathB, especially in a very acidic lysosome and a weakly-acidic TME, the antitumor drug allows accelerated drug release through pH and CathB responses. LU504660

Example 5 Mouse experiment 1. Treatment experiment of SVIRPPL-HCPT/NP

Male NOD SCID mice (6 weeks old) were purchased from the Beijing Vital River

Laboratory Animal Technology Co., Ltd., and raised under pathogen-free conditions. The mice each were laparotomized along a left costal margin to expose a left liver lobe, and the left liver lobe was injected with 30 uL of a suspension including 1 x 108 HepG2 cells to construct a liver cancer in situ. When a tumor volume reached 100 mm? three weeks later, the treatment experiment was conducted.

In order to evaluate an in vivo therapeutic effect of SVIRPPL-HCPT/NP, tumor-bearing mice were randomly divided into the following four groups: a normal saline group, an HCPT aqueous solution group, an SVIRPPL NP (HCPT-free rapeseed peptide

C blank nanocarrier prepared in Example 1) group, and an SVIRPPL-HCPT/NP (prepared in Example 1) group. Mice in each group were administered through tail vein injection once every three days, and during a treatment process, the administration was conducted 4 times in total. A mode of administration each time was as follows: in the

HCPT aqueous solution group, the HCPT aqueous solution was administered at an

HCPT dose of 5.0 mg/kg; in the SVIRPPL-HCPT/NP group, the SVIRPPL-HCPT/NP (prepared in Example 1) was administered at an HCPT dose of 5.0 mg/kg; in the normal saline group, a same volume of normal saline was administered; and in the SVIRPPL NP group, a same volume of the HCPT-free rapeseed peptide C blank nanocarrier (prepared in Example 1) was administered.

After the last administration, a mouse was randomly selected from each group and sacrificed, and a tumor tissue was collected and subjected to section analysis. The collected tumor tissue was fixed in 10% neutral formalin, dehydrated with gradient ethanol solutions, then embedded with paraffin, sectioned by a tissue microtome, stained with hematoxylin and eosin (H&E), mounted with a resin, and finally observed under an optical microscope to determine a pathological change of the tumor tissue.

Results were shown in FIG. 9. The pathological section analysis of liver tumor tissues showed that the treatment with normal saline did not have a significant impact on a tumor tissue; and tumor tissues treated with the SVIRPPL NP, the HCPT aqueous solution, and the SVIRPPL-HCPT/NP underwent different degrees of necrosis. Because

HCPT was easily cleared in vivo, HCPT was not easily accumulated at a tumor.

According to staining results, a tumor tissue in the HCPT aqueous solution group)504660 only partly underwent necrosis, with a small necrosis range. According to staining results, a tumor tissue in the SVIRPPL-HCPT/NP group underwent necrosis in a large range, and a tumor tissue in the SVIRPPL NP group underwent necrosis in a small range. In the normal saline group, there was moderately-differentiated hepatocellular carcinoma, and cancer cells were flaky and diffusely arranged and underwent deep nucleoplasm staining.

In the SVIRPPL NP group and the HCPT aqueous solution group, there was mildly-differentiated hepatocellular carcinoma, clumpy cancer cells and adjacent normal tissues could be observed, and cancer cells underwent deep nucleus staining. In the

SVIRPPL-HCPT/NP group, only a small number of cancer cells were distributed, and there was a specified degree of inflammatory infiltration. The above results indicate that

SVIRPPL-HCPT/NP can be effectively transported to a tumor and inhibit the growth of tumor cells.

In order to fully understand a therapeutic effect of SVIRPPL-HCPT/NP in HepG2 liver tumor-bearing mice, after the last administration, mice in each group were injected with D-luciferin at 150 mg/kg once every seven days, with a total of 3 injections. The growth of liver cancer tumors in mice was observed by bioluminescence imaging.

Bioluminescence imaging results before the first injection of D-luciferin (denoted as day 0) and on day 7 and day 15 after the first injection of D-luciferin (FIG. 10) showed that

SVIRPPL-HCPT/NP could effectively inhibit the growth of liver cancer tumors, and a biofluorescence intensity in the SVIRPPL-HCPT/NP treatment group was significantly lower than a biofluorescence intensity in the normal saline treatment group. Fluorescent quantification analysis results of liver tumors (FIG. 11) showed that a fluorescence intensity at a tumor in a mouse on day 7 after the treatment with SVIRPPL-HCPT/NP did not increase significantly compared with a fluorescence intensity before treatment, but in the normal saline group, the HCPT aqueous solution group, and the SVIRPPL NP group, the fluorescence intensity increased significantly; and an average fluorescence intensity of the SVIRPPL-HCPT/NP group was significantly lower than average fluorescence intensities of other groups. The in vivo imaging results further indicate that

SVIRPPL-HCPT/NP has an excellent anti-tumor ability.

2. Research on biodistribution of SVIRPPL-HCPT/NP LU504660

Male NOD SCID mice (6 weeks old) were purchased from the Beijing Vital River

Laboratory Animal Technology Co., Ltd., and raised under pathogen-free conditions. The mice each were laparotomized along a left costal margin to expose a left liver lobe, and the left liver lobe was injected with 30 uL of a suspension including 1 x 108 HepG2 cells to construct a liver cancer in situ. When a tumor volume reached 200 mm°, an in vivo fluorescence imaging experiment and a biodistribution experiment were conducted.

Methods of the in vivo fluorescence imaging experiment and the biodistribution experiment were as follows: SVIRPPL-HCPT/NP (the antitumor drug prepared in

Example 1) was labeled with a Cy5.5 active ester (a near-infrared (NIR) fluorescent cyanine dye), and the distribution of the antitumor drug throughout the body and in in vitro organs was analyzed by an NIR imaging system IVIS LuminaXRII (ex/em = 680 nm/700 nm). Cy5.5-labeled SVIRPPL-HCPT/NP was injected at an HCPT dose of 5 mg/kg into tumor-bearing mice through tail veins, and at different time points after the injection, the mice were anesthetized with 2% isoflurane and then subjected to whole-body imaging by an IVIS LuminaXRII imager at ex/em = 680 nm/700 nm.

Experimental results were shown in FIG. 12. 2 h after the injection,

SVIRPPL-HCPT/NP could be monitored at a tumor; 8 h after the injection, the accumulation of SVIRPPL-HCPT/NP at a tumor was maximized; and 24 h after the injection, SVIRPPL-HCPT/NP could still be monitored at a tumor, which lasted until 48 h after the injection. These results indicate that SVIRPPL-HCPT/NP can effectively target a tumor and accumulate at the tumor.

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</INSDFeature> LU504660 </INSDSeq_feature-table> <INSDSeq_sequence>SVIRPPL</INSDSeq_sequence> </INSDSeq> </SequenceData> </ST26SequenceListing>

Claims

CLAIMS LU504660

1. A rapeseed peptide with a sequence shown in SEQ ID NO: 1.

2. A use of the rapeseed peptide according to claim 1 in preparation of a nanocarrier for a drug.

3. The use according to claim 2, wherein the drug is hydroxycamptothecin (HCPT).

4. The use according to claim 3, wherein a mass ratio of the rapeseed peptide to the HCPT is (5-15):1.

5. A method for preparing an antitumor drug with the rapeseed peptide according to claim 1 as a carrier, comprising the following steps: (1) dissolving the rapeseed peptide according to claim 1 and HCPT successively in trichloromethane (TCM) to obtain a mixed solution A; (2) adding the mixed solution À dropwise to phosphate-buffered saline (PBS), adding Tween 80, and thoroughly stirring a resulting mixture to obtain a mixed solution B; and (3) removing the TCM in the mixed solution B to obtain the antitumor drug.

6. The method according to claim 5, wherein a mass ratio of the rapeseed peptide to the HCPT is (5-15):1.

7. The method according to claim 6, wherein a mass concentration of the rapeseed peptide in the mixed solution À is 5 mg/mL to 15 mg/mL.

8. The method according to claim 7, wherein a volume ratio of the PBS to the mixed solution À is 1:(8-15); and a mass percentage content of the tween 80 in the mixed solution B is 0.5% to 1.5%.

9. The method according to claim 8, wherein in step (3), after the TCM in the mixed solution B is removed, a resulting system is filtered to obtain an antitumor nanodrug.

10. The method according to claim 9, wherein a filter membrane used for theJs04660 filtration has a pore size of 0.4 um to 0.8 um.