Compound Amphiphilic Peptide Nanomicelle, Preparation and Use Thereof

Abstract

Disclosed is a composite amphiphilic peptide nanomicelle, the preparation method and application, as to a novel integrin .sub.v.sub.3-targeted amphiphilic peptide nanomicelles, the applications in fluorescence imaging, photodynamic therapy, sonodynamic therapy and combined treatments. Based on the prominent properties of integrin .sub.v.sub.3-targeted amphiphilic peptide nanomicelles such as great biocompability, fluorescence imaging of encapsulated materials, photodynamic therapy and photothermal therapy, it is promising to be widely used in the field of labeling and tracing in vivo, biomedical imaging, early detection and treatment for tumor. It has good economic and social benefits in terms of life health and personalized medicine.

Claims

1. A compound amphiphilic peptide nanomicelle containing a fluorescent substance selected from the group consisting of indocyanine green (ICG), Rose Bengal (RB), methylene blue (MB), and doxorubicin (DOX).

2. The compound amphiphilic peptide nanomicelle of claim 1, wherein a main body of said compound amphiphilic peptide nanomicelle is a C.sub.18-GRRRRRRRRGDS (C.sub.18GR.sub.7RGDS) amphiphilic peptide containing an arginine-glycine-aspartate (RGD) tripeptide sequence.

3. The compound amphiphilic peptide nanomicelle of claim 2, wherein the compound amphiphilic peptide nanomicelle has a diameter of 10 to 100 nm and a potential of 20 to 40 mV.

4. A method for preparing the compound amphiphilic peptide nanomicelle according to claim 1, comprising following steps: a. dissolving the amphiphilic peptide C.sub.18GR.sub.7RGDS in ultrapure water to prepare an amphiphilic peptide solution with a concentration of 10 g/mL; b. dissolving the fluorescence substance in ultrapure water to prepare a fluorescence substance solution with a concentration of 10 g/mL; c. mixing the amphiphilic peptide solution and the fluorescence substance solution at 2:1 or 1:1 volume ratios followed by sonication at a frequency of 5-35 kHz, at 10-30 C. for 10-40 min with avoiding light to synthesize the compound amphiphilic peptide nanomicelle; d. transferring the compound amphiphilic peptide nanomicelle to a dialysis bag with a molecular weight cutoff of 500-1500 Dalton to obtain an integrin .sub.v.sub.3-targeted compound amphiphilic peptide nanomicelle targeted after dialysis for 48-72 h.

5. The method of claim 4, wherein the fluorescence substance is Rose Bengal

6. Use of the compound amphiphilic peptide nanomicelle of claim 1 for preparation of photothermal and photoacoustic agent or photodynamic therapy (PDT) and sonodynamic therapy (PTT) agent in melanoma and cervical cancer.

7. The compound amphiphilic peptide nanomicelle of claim 1, wherein said compound amphiphilic peptide nanomicelle is a new photodynamic therapy agent for nasopharyngeal carcinoma.

8. The compound amphiphilic peptide nanomicelle of claim 1, wherein said compound amphiphilic peptide nanomicelle is a new sonodynamic therapy agent for nasopharyngeal carcinoma.

9. The compound amphiphilic peptide nanomicelle of claim 1, wherein said compound amphiphilic peptide nanomicelle is a new combined therapeutic agent for photodynamic and sonodynamic therapy for nasopharyngeal carcinoma.

10. The compound amphiphilic peptide nanomicelle of claim 1, wherein said compound amphiphilic peptide nanomicelle is injected intravenously or intratumoratively, and used in a plurality of treatment methods for nasopharyngeal carcinoma.

11. The method of claim 4, wherein the dialysis bag in step d has a molecular weight cutoff of 500-1000 Dalton.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0029] FIG. 1 shows a general structure and process of the targeted compound amphiphilic peptide nanomicelles above mentioned.

[0030] FIGS. 2A-2B show the stability of nanomaterials for novel targeted complex amphiphilic peptide nanomicelle fluorescent probe (FIG. 2A) and the drug loading efficacy of Rose Bengal (FIG. 2B).

[0031] FIGS. 3A-3C show the cytotoxicity of novel targeted complex amphiphilic peptide nanomicelle fluorescent probe in melanoma cells (B16) (FIG. 3A), cervical cancer cells (Hela) (FIG. 3B) and fibroblasts (L929) (FIG. 3C).

[0032] FIGS. 4A-4D show the photoacoustic effect of targeted compound amphiphilic peptide nanoparticles applied to melanoma cells (B16) (FIG. 4A) and cervical cancer cell (Hela) (FIG. 4C), respectively. And the photoacoustic effect of targeted compound amphiphilic peptide nanoparticles applied to melanoma cells (B16) (FIG. 4B) and cervical cancer cells (Hela) (FIG. 4D) at different times.

[0033] FIGS. 5A-5C show the photothermal effect and photoacoustic effect of the targeted compound amphiphilic peptide nanomicelles at different times/concentrations; The yield of ROS from PARN (peptide amphiphile-rose bengal nanomicelles) at different time periods (1, 3, 5 min) under US or Laser (FIG. 5A); The yield of ROS from PARN/RB of different RB concentrations (0, 2, 4, 6, 8, 10 g/mL) under US (FIG. 5B); The yield of ROS from PARN/RB of different RB concentrations (0, 2, 4, 6, 8, 10 g/mL) under Laser (FIG. 5C).

[0034] FIG. 6 shows the cytotoxicity of the targeted compound amphiphilic peptide nanomicelles above mentioned in CNE-2Z cells.

[0035] FIG. 7 shows the changes in the control of tumor growth from SDT and PDT in nasopharyngeal carcinoma mice after intravenous administration of compound amphiphilic peptide nanomicelles.

[0036] FIG. 8 shows the changes in the control of tumor growth from SDT and PDT in nasopharyngeal carcinoma mice after intratumoral administration of compound amphiphilic peptide nanomicelles.

[0037] FIG. 9 is a photograph of tumor changes in nasopharyngeal carcinoma mice from SDT and PDT during the treatment by intravenous administration of compound amphiphilic peptide nanomicelles.

[0038] FIG. 10 is a photograph of tumor changes in nasopharyngeal carcinoma mice from SDT and PDT during the treatment by intratumoral administration of compound amphiphilic peptide nanomicelles.

DETAILED DESCRIPTION

[0039] The present disclosure is specifically described by the following examples, which are only used to further illustrate the present disclosure, and are not to be construed as limiting the scope of the present disclosure. The person having ordinary skill in the art may make some non-essential improvements to the present disclosure according to the contents of the present disclosure.

[0040] The present application is specifically described by the following examples, which are only used to further illustrate the present application, and are not to be construed as limiting the scope of the present application. The person having ordinary skill in the art may make some non-essential improvements to the present application according to the contents of the present application.

[0041] The raw materials used in the preparation of the application are all obtained by the commodity.

EXAMPLE 1

[0042] Mix 2 ml of the amphiphilic peptide C.sub.18GR.sub.7RGDS solution (Ningbo Kangbei Biochemical Co., Ltd., model: 817870, batch number: 17040701) (10 mg/mL) and 1 mL of methylene blue solution (2 mg/mL). Then the mixed solution was placed in the ultrasonic cleaner with avoiding light, and the conditions were set at 25 C., 30 min, and 28 kHz. After washing dialysis bag (molecular weight cutoff: 1000 Daltons) with ultrapure water, transfer the mixed solution into a dialysis bag, place a beaker containing ultrapure water dialysate and float it in the dialysate (the volume of dialysate is 2000 mL, pH=7.4). Place the magneton in the beaker and place the beaker on the magnetic stirrer (rotation speed: 120 rpm, room temperature). When the dialysis was carried out for 2 hours, the color of the dialysate was blue, the mixed solution in the dialysis bag became transparent, and the dialysis continued for 48 hours. There is no characteristic absorption peak of methylene blue after measuring the mixed solution by UV-Vis spectrophotometer, indicating that methylene blue is not encapsulated into the amphiphilic peptide nanomicelle. This also indicates that it is difficult to encapsulate the methylene blue with the amphiphilic peptide sequence synthesized by the present application to form the final product.

EXAMPLE 2

[0043] Mix 2 mL of the amphiphilic peptide C.sub.18GR.sub.7RGDS solution (10 mg/mL) and 1 mL of curcumin (2 mg/mL, dissolved in acetic acid), and place the mixed solution in the ultrasonic cleaner with avoiding light (25 C., 30 min, and 28 kHz). After washing dialysis bag (molecular weight cutoff: 1000 Daltons) with ultrapure water, transfer the mixed solution into a dialysis bag, place a beaker containing ultrapure water dialysate and float it in the dialysate (the volume of dialysate is 2.0 L, pH=7.4). Place the magneton in the beaker and place the beaker on the magnetic agitator (rotation speed: 120 rpm, room temperature). After 3 hours of dialysis, yellow precipitates were found in dialysis bag and gradually increased with the increase of dialysis time. This was because ultrapure water enters the mixture through a dialysis bag, and curcumin was insoluble in water and precipitated. This also shows that the amphiphilic peptide sequence of the application does not encapsulate curcumin well.

EXAMPLE 3

[0044] Mix 2 mL of the amphiphilic peptide C.sub.18GR.sub.7RGDS solution (Ningbo Kangbei Biochemical Co., Ltd., model: 817870, batch number: 17040701) (10 mg/mL) and 1 mL of rose bengal solution (2 mg/mL) and place the mixed solution in the ultrasonic cleaner with avoiding light (25 C., 30 min, and 28 kHz). After washing dialysis bag (molecular weight cutoff: 1000 Daltons) with ultrapure water, transfer the mixed solution into a dialysis bag, place a beaker containing ultrapure water dialysate and float it in the dialysate (the volume of dialysate is 2000 ml, pH=7.4). Place the magneton in the beaker and place the beaker on the magnetic stirrer (rotation speed: 120 rpm, room temperature). To replace dialysate every 4 hours and dialysis for 48 hours to get fluorescent amphiphilic peptide self-assembled nanomicelles. After testing, it was found that most of the rose bengal was successfully encapsulated into the polypeptide sequence of the application and formed self-assembled nanomicelles.

[0045] The particle size was measured by a Zetasiser every two days. It was found that the complex amphiphilic peptide was stable, and the positive charge was favorable for cell uptake (FIG. 2A).

[0046] The loading ratio of rose bengal in amphiphilic peptide nanomicelles was calculated through OD value measured by ultraviolet spectrophotometer, and the loading ratio tends to be stable as the concentration of rose bengal increase (FIG. 2B).

[0047] Melanoma and cervical cancer cells (.sub.v.sub.3.sup.+), and fibroblasts were plated in 96-well plates at 110.sup.4 cells per well respectively, and incubated at 37 C. for 48 hours. After the cells were full, these cells were incubated with different concentrations of the compound amphiphilic peptide nanomicelles (synthesized according to the present example) and the corresponding concentrations of rose bengal (control group) separately. The different concentrations reagents used were configured by medium. After 24 hours of culture, the medium was removed, and the cell viability was measured at 450 nm with a microplate reader after treating by CCK-8. As shown in FIGS. 3A-3C, as the concentration of rose bengal increase, the cytotoxicity of compound amphiphilic peptide nanomicelles was significantly higher than that of pure rose bengal. The compound amphiphilic peptide nanomicelles are also less toxic to normal cells such as fibroblasts. At a concentration of 20 g/mL, the cell viability of fibroblasts is over 80%. This indicates that the compound amphiphilic peptide nanomicelles have good biocompatibility and will also use this concentration as the optimum concentration for subsequent experiments.

EXAMPLE 4

[0048] Mix 3 mL of the amphiphilic peptide C.sub.18GR.sub.7RGDS solution (10 mg/mL) and 2 mL of rose bengal solution (2 mg/mL) and place the mixed solution in the ultrasonic cleaner with avoiding light (25 C., 40 min, and 28 kHz). After washing dialysis bag (molecular weight cutoff: 1000 Daltons) with ultrapure water, transfer the mixed solution into a dialysis bag, place a beaker containing ultrapure water dialysate and float it in the dialysate (the volume of dialysate is 2000 ml, pH=7.4). Place the magneton in the beaker and place the beaker on the magnetic stirrer (rotation speed: 120 rpm, room temperature). To replace dialysate every 4 hours and dialysis for 60 hours to get fluorescent amphiphilic peptide self-assembled nanomicelles.

[0049] Melanoma and cervical cancer cells (.sub.v.sub.3.sup.+) were plated in 96-well plates at 110.sup.4 cells per well respectively, and incubated at 37 C. for 48 hours. When the cells were full, 20 g/mL of compound amphiphilic peptide nanomicelles (synthesized according to the present example) and corresponding concentrations of rose bengal (control group) were added respectively to replace the former medium. After 4 h of incubation, each well of cells was supplied with fresh medium, and subsequently treated with ultrasound/laser irradiation for 3 min. The cells were cultured for 24 hours and treated by CCK-8 method. Then the cell viability was measured at 450 nm with a microplate reader. As shown in FIGS. 4A-4D, the photoacoustic and photothermal effects of amphiphilic peptide nanomicelles on melanoma and cervical cancer cells are illustrated.

EXAMPLE 5

[0050] Mix 1 mL of the amphiphilic peptide C.sub.18GR.sub.7RGDS solution (10 mg/mL) and 1 mL of rose bengal solution (2 mg/mL) and place the mixed solution in the ultrasonic cleaner with avoiding light (25 C., 20 min, and 28 kHz). After washing dialysis bag (molecular weight cutoff: 1000 Daltons) with ultrapure water, transfer the mixed solution into a dialysis bag, place a beaker containing ultrapure water dialysate and float it in the dialysate (the volume of dialysate is 2000 ml, pH=7.4). Place the magneton in the beaker and place the beaker on the magnetic stirrer (rotation speed: 120 rpm, room temperature). After 72 hours dialysis, fluorescent amphiphilic peptide self-assembled nanomicelles were obtained.

[0051] 2 mL compound amphiphilic peptide self-assembled nanomicelles and rose bengal solutions with rose bengal concentrations (20 g/mL) were prepared and mixed with 20 L 1,3-diphenyl isobenzofuran (DPBF) (2 mg/mL in acetonitrile), respectively. The OD values were measured at 548 nm with a microplate reader. Then the mixed solution was treated by laser (808 nm, 1.5 W/cm.sup.2, 0/1/3/5 min) or ultrasound (1 W/cm.sup.2, 0/1/3/5 min) and measured the OD value at 548 nm again. Results as shown in FIGS. 5A-5C, the OD value of the compound nanomicelles decreased with the increase of time, and 3 minutes was selected as the optimal treatment time for the follow-up experiment.

[0052] 2 mL different concentrations compound amphiphilic peptide self-assembled nanomicelles and corresponding concentrations of rose bengal were prepared and mixed with 20 L DPBF, respectively. After measuring the OD values of mixed solution at 548 nm with a microplate reader, the mixed solution was treated by laser (808 nm, 1.5 w/cm.sup.2, 3 min) or ultrasound (1 W/cm.sup.2, 3 min) and measured the OD value at 548 nm again. As a result (FIGS. 5A-5C), the absorption peak at 548 nm of DPBF is reduced after oxidation, which can be used to quantify the reactive oxygen species (ROS) produced by photosensitizers and sonosensitizers after laser irradiation or ultrasound. It is concluded that the compound nanomicelle produces more active oxygen.

[0053] FIG. 1 shows a general structure and process of the compound amphiphilic peptide nanomicelles synthesized according to present application. FIGS. 2A-2B show the stability of nanomaterials and the change in the loading rate of Rose Bengal. As shown in FIGS. 2A and 2B, the material has good stability, with no significant change in particle size within 30 days ranging from 10 to 40 nanometers, which makes it easier for nanoscale substances to enter the cell through the cell membrane. The loading rate of rose bengal tends to saturation with the increase of the quality of rose bengal. The prepared amphiphilic peptide nanoparticles have good biocompatibility and safety (FIGS. 3A-3C). The compound amphiphilic peptide nanomicelles have good sonodynamic and photodynamic effects in melanoma and cervical cancer cells (FIGS. 4A-4D), and the compound amphiphilic peptide nanomicelles have perfect photodynamic and sonodynamic (FIGS. 5A-5C).

[0054] The compound amphiphilic peptide targeting melanoma and cervical cancer integrin .sub.v.sub.3 as a fluorescent imaging, SDT and PDT material cannot only enrich the ways of non-invasive (or minimally invasive) treatment of melanoma and cervical cancer. Moreover it is of great significance for the development of new cancer diagnosis and treatment mode, reducing the side effects of current clinical treatment, reducing the damage to normal tissues, and improving the efficacy and accuracy of cancer diagnosis. At the same time, it has important practical value for promoting the clinical transformation of the amphiphilic peptide as a novel melanoma and cervical cancer targeted diagnosis and treatment.

EXAMPLE 6

[0055] First of all, the designed concentration (0, 20, 40, 60, 80 and 100 g/mL) solution of nanomicelle and rose bengal were prepared with 1640 medium. CNE-2Z cells were seeded into a 96-well player at a density of 210.sup.4 cells per well. Each sample with a certain concentration was added to 5 wells. The cytotoxicity of the pure rose bengal and the nanomicelle solution at different concentrations was compared. After culturing for 48 hours, the cells were grown to 80%, and the medium containing different volumes of nanomicelle and rose bengal solution was added instead of the original medium according to the experimental group setting. After an additional incubation for 24 h at 37 C. in the dark, fresh medium (100 L) together with CCK-8 (10 L) was added followed by fluorescence analysis on a microplate reader at a wavelength of 450 nm. Cell viability was calculated according to the formula, with the maximum and minimum of OD values excluded:

Cell Viability=average of (ODOD.sub.blk)/average of (OD.sub.0OD.sub.blk)100%

[0056] As shown in FIG. 6, the pure rose bengal is less toxic to CNE-2Z cells and has no rising tendency with the increase of concentration. However, the nanomicelles we synthesized are more toxic to NE-2Z cells, and with the increase of concentration, the cell viability is lower and lower, 20 micrograms per milliliter, the survival rate has been reduced to 50%. The nanomicelles we synthesized are more toxic to CNE-2Z cells, and with the increase of concentration, the cell survival rate is lower and lower. And the survival rate has dropped to 50% at 20 g/mL.

EXAMPLE 7

[0057] 20 nude mice bearing CNE-2Z tumors were randomly divided into five groups: control group, untreated group (PARN), PDT group (PARN+Laser), SDT group (PARN+US), combination therapy group (PARN+Laser+US). Then 200 L of PARN was injected into mice via the tail vein except control group. For PDT group, the tumors of mice were treated by the 808 nm laser at 1.5 W/cm.sup.2 for 3 min after 5 h after vein administration of PARN, the distance between tumor and light source is 2 cm. As to SDT group, the tumors of mice were conducted by US in the pre-set condition (frequency: 3 MHz, power density: 3 W/cm.sup.2, duty cycle: 50%, time: 3 min). For combination therapy group, ultrasound treatment after laser irradiation was acted on tumor, the treatment conditions are the same as above. The groups of intratumoral injection treatment were similar to the intravenous groups. In addition to the control group, 150 L of PARN was injected into tumor tissue in each group, and the laser or ultrasound was applied to the tumor site instantly, the treatment conditions are the same as those described above.

[0058] As shown in FIG. 7 and FIG. 9, the combined treatment group (PARN+Laser+US) had a significant inhibitory effect on CNE-2Z tumors after intravenous injection. It was observed that the growth of the tumor was significantly inhibited in PARN+Laser, PARN+US and PARN+Laser+US when PARN was administrated by intratumoral injection.

[0059] The present application has been described using specific examples to explain the principles and embodiments of the present application. The description of the above examples is only for helping to understand the method and the central idea of the present application. It should be noted that those skilled in the art can make various improvement and modifications to the present application without departing from the theory of the application, and these improvement and modifications also fall within the protection of the claims of the present application.