Preparation of nano self-assembly based on 8-FF-TPP and application of nano self-assembly in melanoma

Abstract

The invention provides a nanometer self-assembly body based on 8-FF-TPP and application of the nanometer self-assembly body in photo-thermal diagnosis and treatment of melanoma. According to the self-assembly body, a specific structure of an FF sequence and a porphyrin (TPP) group is introduced into polypeptide, so that the self-assembly body is self-assembled into nano-particles, and the nano-particles have good photothermal conversion efficiency and the characteristic of targeting melanoma cells. The nano self-assembly can generate a photothermal effect under irradiation of laser with a specific wavelength, has a killing effect on melanoma cells, and shows excellent photothermal stability. The invention also comprises a preparation method, the stable nano self-assembly body is obtained through the steps of solid-phase synthesis, HPLC purification, DMSO self-assembly and the like, and the nano self-assembly body is used for melanoma imaging and targeted therapy.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a nano self-assembly for photothermal therapy of melanoma and a preparation method thereof. The nano self-assembly can specifically target the tumor site and generate a photothermal effect through laser irradiation, thereby achieving photothermal therapy of melanoma. Background Art

[0002] Melanoma is a highly malignant skin cancer, and current treatments include surgery, chemotherapy, immunotherapy, etc. However, these treatments have problems such as large side effects and high recurrence rates. Therefore, it is of great significance to develop safe and effective targeted treatments. Photothermal therapy is an emerging treatment method that can achieve high-temperature killing at the tumor site through the photothermal conversion effect of nanomaterials. Studies have found that polypeptide materials with self-assembly properties can form stable nanoparticles, and polypeptides with targeting capabilities can be made into nanoparticles, which will have the potential to target cancer cells. Based on this, the present invention provides a nano self-assembly based on a polypeptide motif and a TPP porphyrin group, which can efficiently absorb light energy and convert it into heat energy for precise treatment of melanoma. Summary of the invention

[0003] The present invention aims to design a nano self-assembly of 8-FF-TPP, which forms nanoparticles through self-assembly characteristics, has good photothermal conversion efficiency and the ability to target melanoma cells. The present invention also provides a preparation method thereof and describes in detail its application in photothermal therapy of melanoma.

[0004] The technical solution of the present invention is as follows:

[0005] The structure of the 1.8-FF-TPP nano self-assembly includes a FF dipeptide structure and a porphyrin (TPP) group, which can effectively self-assemble to form nanoparticles. Its polypeptide has sialic acid that targets the cell surface. The partial sequence of the nanoparticle polypeptide is TPHGYQPMQGKTGPGYFFG.

[0006] The preparation method of 2.8-FF-TPP nanoparticles includes the steps of chemical synthesis, purification, dissolution, self-assembly and the like.

[0007] 3. The nanoparticles provided by the present invention are obtained by coupling a targeting peptide with GPGYFFG and porphyrin. Cell experiments have shown that the complex can specifically target sialic acid on the cell membrane surface, a melanoma tumor-bearing model has been established, and an in vivo imaging device has verified that the nanoparticles of the present invention can target the tumor site in vivo.

[0008] 4. The preparation method of the nanoparticles provided by the present invention is stable. The prepared complex is verified by RP-HPLC, with high purity and no interference peaks. The molecular weight is correct by mass spectrometry. The hydrated particle size of the nanoparticles assembled under different conditions is consistent by transmission electron microscopy, the solution is clear and transparent, and the stability is strong when diluted by different solutions. The continuous illumination cooling cycle study proves that it has good photothermal conversion stability.

[0009] 5. The nanoparticles prepared by the present invention have high purity, strong stability and good photothermal conversion stability.

[0010] 6. The application provided by the present invention has been confirmed through laser irradiation experiments that nanoparticles can significantly inhibit the growth of tumor cells and have good application prospects in tumor diagnosis and anti-tumor preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Purification and mass spectrometry identification of 8-FF-TPP, A. Chromatographic separation and purification of 8-FF-TPP, B. Mass spectrometry identification of 8-FF-TPP;

[0012] Figure 2 Characterization of 8-FF-NPs, A particle size of 8-FF-NPs, B transmission electron microscopy of 8-FF-NPs, C optical photograph of 8-FF-NPs;

[0013] Figure 3 Photothermal properties of 8-FF-NPs. A. 8-FF-NPs (300 μg mL -1 ) temperature change, B is irradiated with 638nm laser (1.5W cm -2 ) concentration-dependent temperature change of 8-FF-NPs, C 8-FF-NPs (300 μg mL -1 ) Temperature change curve after reaching the highest temperature and cooling, D 8-FF-NPs (200μg mL -1 ) during laser irradiation on / off cycles (1.5 W cm -2 ) during the period;

[0014] Figure 48-FF-NPs target sialic acid. A is the fluorescence image of HepG2 cells incubated with 10 μM 8-FF-NPs for 3 h, in which the control group was pretreated with sialidase overnight, DAPI stained the cell nucleus, DiO stained the cell membrane, and 8-FF-NPs were red under the microscope. B is the fluorescence quantitative image of A. C is the fluorescence image of B16-F10 cells incubated with 10 μM 8-FF-NPs for 3 h, in which the control group was pretreated with sialidase overnight, DAPI stained the cell nucleus, DiO stained the cell membrane, and 8-FF-NPs were red under the microscope. D is the fluorescence quantitative image of C. Averages±SEM, ****p<0.0001, **p<0.01;

[0015] Figure 5 8-FF-NPs are toxic to HepG2 cells;

[0016] Figure 6 In vivo imaging of 8-FF-NPs, A, distribution of 8-FF-NPs in mice at different time points, B, fluorescence distribution in mouse anatomy after 24 hours. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0018] Example 18 - Synthesis and purification of FF-TTP

[0019] 1.1 Experimental procedures

[0020] The peptide synthesis adopts F-moc solid phase synthesis, and the amino acids are coupled to the RinkAmide resin one by one in the order of synthesis from the C-terminus to the N-terminus. The specific steps are as follows:

[0021] (1) Activation of resin: Weigh 0.1 mmol of resin, add 3 ml of DMF, pour the resin into the synthesis column, and place it in a mixer for activation for 1 h;

[0022] (2) Resin washing: Wash the activated resin three times with DMF and drain;

[0023] (3) Deprotection: The dried resin was deprotected with 20% piperidine, the first reaction took 7 min and the second reaction took 8 min, then washed with DMF 8 times;

[0024] (4) Activation of amino acids: 0.5 mmol HOBT, 0.5 mmol HCTU and 0.4 mmol amino acid were dissolved in 3 mL 5% N-methylmorpholine solution and reacted for 15 min;

[0025] (5) First amino acid coupling: Add the activated amino acid to the resin and react for 1 h. After the reaction, drain the liquid and add DMF to wash 8 times;

[0026] (6) Second amino acid coupling: The amino acid coupled to the resin in (5) was deprotected with piperidine, and then the next activated amino acid was added and reacted for 1 h. After the reaction, the liquid was drained and washed with DMF 8 times;

[0027] (7) The subsequent amino acid coupling is carried out in the order of deprotection, activation, and coupling until the last amino acid coupling is completed.

[0028] (8) After the peptide synthesis is completed, take 0.04mmoL of resin, and then react with 20% piperidine for 15 minutes to remove the Fmoc protection of the peptide. Weigh 30mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 20mg of 1-hydroxybenzotriazole and 5mg of TPP-G. Dissolve 5mg of TPP-G in 2mL of anhydrous DMF, then add 30mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, blow and mix the solution, then add 20mg of 1-hydroxybenzotriazole and react at room temperature for 15min. After the reaction is completed, pour the reaction solution into the synthesis column resin and react at room temperature overnight in the dark. After the reaction is completed, wash the resin with DMF 8 times, drain the liquid, and then wash it with anhydrous methanol 8 times, and drain the liquid. Finally, add the lysis solution, the formula of the lysis solution is 90% TFA, 2.5% anisole, 2.5% anisole thioether and 5%

[0029] The sample was pyrolyzed for 2.5 hours, precipitated with ice ether, 30 mL of ether was added, and then mixed with the pyrolysis solution, centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and naturally dried to obtain 8-FF-TPP.

[0030] 1.2 Experimental Results

[0031] FF is a core self-assembly motif extracted from the β-amyloid peptide of Alzheimer's disease. FF components have been shown to have significant advantages, including easy production, versatility, biodegradability, biocompatibility and non-immunogenicity. In addition, FF can be adjusted through non-covalent interactions with various functional molecules. For example, FF can self-assemble to form nanostructures through hydrogen bonding and π-π stacking of aromatic groups. Co-assembly is a way to obtain nanomaterials with controllable morphology and good stability. SA-8, which was screened by the laboratory through phage display technology, has been shown to be able to bind well to sialic acid. Since photothermal therapy has the advantages of low invasiveness, simple operation, short treatment time and fast recovery, we modified porphyrin into SA-8 and introduced GPGYFFG in the modified sequence to make the assembled drug morphology controllable and have good photostability. Figure 1As shown, 8-FF-TPP was purified by RP-HPLC at 415 nm. We collected the sample with a peak at a retention time of 28 min. Mass spectrometry identification showed that it was the target sample. The molecular weight identified by mass spectrometry was 2798 Da. The results showed that the sample was successfully synthesized.

[0032] Example 2 Preparation of 8-FF-TPP Nano-Self-Assembly

[0033] 2.1 Experimental procedures

[0034] The synthesized 8-FF-TPP was assembled into nanoparticles. The peptide was dissolved in DMSO and then added dropwise to an aqueous solution. The volume ratio of DMSO to water in the solution was 1:9, and the final assembled concentration was 2 mg / mL. The assembled sample was dialyzed after 24 hours. 1 mL of the solution required 1 L of water for 24 hours. The aqueous solution needed to be replaced 3 times, and the molecular retention capacity of the dialysis bag was 5 kDa. The solution after the dialysis was centrifuged with an ultrafiltration tube, and the centrifuged solution was 8-FF-NPs.

[0035] 2.2 Experimental Results

[0036] 8-FF-TPP was self-assembled with a DMSO to water ratio of 1:9, an assembly concentration of 2 mg / mL, assembly for 24 h, and dialysis for 24 h. The assembled nanoparticles were characterized by TEM and dynamic light scattering, such as Figure 2 As shown, the assembled particles of 8-FF-NPs can form regular uniform spheres under transmission electron microscopy, with a diameter of about 35nm, and the hydrated particle size measured by dynamic light scattering is about 33.9±5.6nm. The optical image shows that the solution is clear and transparent, and the solution is uniform.

[0037] Example 3 Determination of photothermal conversion performance of 8-FF-NPs

[0038] 3.1 Experimental procedures

[0039] The photothermal conversion performance was determined by irradiating 8-FF-NPs dispersions with different concentrations (25, 50, 100, and 300 μg / mL) with 638 nm laser. To further analyze the effect of laser power density on the photothermal effect of nanoparticles, 638 nm lasers with different power densities (0.1, 1.0, and 1.5 W cm -2 ) was used to irradiate the 8-FF-NPs (0.3 mg / mL) dispersion and plot the temperature curve. Finally, the 8-FF-NPs were irradiated with laser “on-off” cycles for multiple times to test their photothermal stability.

[0040] 3.2 Experimental Results

[0041] In order to verify the photothermal capability of 8-FF-NPs, a 638 nm laser was used to irradiate the 8-FF-NPs suspension and the temperature change of the suspension was recorded. Figure 3 A shows that 300 μg mL -1 The temperature of 8-FF-NPs increases with the increase of laser power. -2 Increased to 1.5W cm -2 , the temperature rose by 25.4℃ after 10min of irradiation. Figure 3 B shows that the temperature of 8-FF-NPs at different concentrations increases with the increase of laser power. The laser power is 1.5 W cm -2 When the concentration of 8-FF-NPs increased from 25 μg mL -1 to 300 μg mL -1 , irradiation for 10 minutes, the temperature difference is 10.4℃. The results show that the nanoparticles exhibit a significant concentration-dependent and laser power density-dependent photothermal conversion process, so the temperature required for treatment can be achieved by adjusting the nanoparticle concentration or laser power. Figure 3 C shows the maximum temperature reached and the cooling curve. Figure 3 D. Through the study of the continuous illumination cooling cycle process, after 5 repeated cycles, the temperature rise trend of 8-FF-NPs did not show a significant weakening, and it has good photothermal stability.

[0042] Example 4 8-FF-NPs co-localization experiment

[0043] 4.1 Experimental procedures

[0044] 1×10 5B16-F10 or HepG2 were cultured overnight, and then the experimental group was changed to serum-free medium. The control group was incubated overnight with sialidase (100mU / mL) dissolved in serum-free medium. Sialidase can hydrolyze sialic acid on the cell surface. 8-FF-NPs were diluted to 10μM with serum-free medium. The cells were washed twice with PBS before adding the drug to the cells, and then the drug was added and incubated for 3h. After the drug incubation, the cells were washed 3 times with PBS, and the cell membrane was stained with DiO. 2.5μL DiO and 2.5μL staining enhancer were added to each milliliter of staining buffer. The membrane staining working solution was added to the cells and incubated at 37℃ in the dark for 15min. After the membrane staining was completed, the cells were washed three times with PBS, and 1mL 4% paraformaldehyde fixative was added to each dish and fixed for 15min. After the fixation was completed, the cells were washed 3 times with PBS, and 5mg / mL LDAPI prepared in PBS was added. After incubation at 37℃ in the dark for 15min, the cells were washed 3 times with PBS. Leica SP8 was used for fluorescence imaging, with the maximum excitation of DiO at 488 nm and the maximum emission at 501 nm, the maximum excitation of DAPI at 364 nm and the maximum emission at 454 nm, and the excitation light of 8-FF-NPs at 450 nm.

[0045] 4.2 Experimental Results

[0046] HepG2 and B16-F10 cells highly express sialic acid on their surfaces, so we explored the targeting of 8-FF-NPs to HepG2 and B16-F10 cells, and incubated the cells with 10 μM 8-FF-NPs for 3 hours. To prove that 8-FF-NPs can target sialic acid on the cell membrane, we pre-incubated HepG2 and B16-F10 with sialidase, allowing sialidase to hydrolyze sialic acid on the cell membrane surface. The cell nucleus was stained blue with DAPI, the cell membrane was stained green with DiO, and 8-FF-NPs showed red fluorescence. The results are shown in Figure 4 As shown in A, for HepG2 cells, in the 8-FF-NPs+sialidase group, the red fluorescence was significantly inhibited and almost invisible. In the 8-FF-NPs group, the red fluorescence was very obvious, and the red fluorescence and green fluorescence overlapped well. The results showed that 8-FF-NPs can specifically bind to sialic acid. Figure 4 As shown in B, the fluorescence intensity of the 8-FF-NPs+sialidase group differed by 2.5 times from that of the 8-FF-NPs group. The average fluorescence intensity data quantitatively demonstrated that 8-FF-NPs could specifically bind to sialic acid. Figure 4As shown in C, we used B16-F10 cells to prove that 8-FF-NPs can specifically bind to sialic acid. In the 8-FF-NPs+sialidase group, the red fluorescence was relatively weak and almost invisible. In the 8-FF-NPs group, the red fluorescence was very obvious, and the red fluorescence and green fluorescence overlapped very well. All these indicate that 8-FF-NPs can specifically bind to sialic acid. Figure 4 As shown in D, the fluorescence intensity of the 8-FF-NPs+sialidase group and the 8-FF-NPs group differed by about 2 times, indicating that 8-FF-NPs could specifically bind to sialic acid. The combined co-localization experiments of HepG2 and B16-F10 cells proved that 8-FF-NPs could target sialic acid.

[0047] Example 5 8-FF-NPs cytotoxicity experiment

[0048] 5.1 Experimental procedures

[0049] After the cells were digested with trypsin, the culture medium was used to terminate the digestion and centrifuged at 1000 rpm for 3 min. The cell density was adjusted and 5000 cells were added to each well of the 96-well plate in 100 μL. The added cells were cultured in the incubator for 24 h and then the culture medium was removed. The corresponding prepared drugs were added to each well, and each group was repeated for 3 wells. After the drug was incubated for 12 h, the culture medium was removed and replaced with serum-free culture medium. The 638 nm laser with a laser power of 0.5 W cm -2 Irradiate the cells for 180 seconds. The dark group was not irradiated with laser, and other treatments were the same as the laser group. After irradiation, the cells were cultured in an incubator for 12 hours before adding CCK-8, adding 10 μL to each well, continuing to incubate for 1 to 5 hours, and finally measuring the absorbance at a wavelength of 450 nm. The blank control was serum-free culture medium with the corresponding CCK-8 added. Cell viability (%) = (absorbance of the experimental group - absorbance of the blank control) / (absorbance of the experimental control group - absorbance of the blank control).

[0050] 5.2 Experimental Results

[0051] Depend on Figure 5 It can be seen that the cytotoxicity of 8-FF-NPs below 0.0625 μM is not different between dark and light conditions. Laser power 0.5 W cm -2 After 180s of irradiation, the cell proliferation of 0.625μM was inhibited by more than 80%, while the toxicity to cells was small under dark conditions, and it was obviously toxic to cells at 20μM, with a difference of more than 30 times. 50 The above results show that the material can kill cancer cells very well with good selectivity.

[0052] Example 6 Intratumoral accumulation of 8-FF-NPs drug

[0053] 6.1 Experimental procedures

[0054] B16-F10 cells were digested with trypsin, the digested cells were centrifuged to remove the medium, serum-free medium was added, and the cell density was adjusted to 6×10 7 / mL. Purchase 5-6 week old BALB / c mice and inject 100 μL into each mouse's subcutaneous tissue. Wait until the tumor volume reaches 100 mm 3 The subsequent experiments were conducted when 200 μL 8-FF-NPs (21.5 mg / kg) and 200 μL TPP (4.8 mg / kg) were injected into the mice through the tail vein, and the mice were scanned in the in vivo imaging device at different time points. After 24 h, the mice were dissected and the tissues and organs of the mice were imaged.

[0055] 6.2 Experimental Results

[0056] To further verify the targeting ability of 8-FF-NPs in vivo, the distribution of 8-FF-NPs and porphyrin was performed by fluorescence imaging after injection through the tail vein. Figure 6 A. When porphyrin alone enters the mouse body, the fluorescence intensity of each organ in the mouse body does not increase significantly over time, and there is no specific enrichment in the tumor site. The results show that porphyrin is rapidly metabolized in the body. 8-FF-NPs nanoparticles are increasingly enriched in the tumor site over time. It can be seen that there is obvious enrichment at 8h, and it is very obvious at 12h. Until 24h, the fluorescence of 8-FF-NPs in the tumor site is still very strong. Figure 6 B. We dissected mice after 24 hours and analyzed the fluorescence intensity of various organs and tumors in the two groups of experiments. The fluorescence intensity of 8-FF-NPs in the liver and tumor sites was very strong. The results show that 8-FF-NPs have better sustainability and targeting in the body, and have a slow metabolic rate in the body, which can make the nanoparticles have better therapeutic effects.

Claims

1. A nano self-assembly based on 8-FF-TPP, characterized in that: The self-assembly polypeptide sequence is TPHGYQPMQGKTGPGYFFG, and the porphyrin group (TPP) is modified at the polypeptide terminal. The porphyrin-modified polypeptide forms stable nanoparticles with good targeting and photothermal conversion performance. Under laser irradiation, the temperature of the self-assembly increases significantly, which can achieve targeted photothermal therapy of tumor cells.

2. The nano self-assembly according to claim 1, characterized in that: The particle size of the self-assembly is 10 to 100 nanometers, preferably 30 to 50 nanometers, to achieve optimal in vivo distribution and tumor targeting.

3. The nano self-assembly according to claim 1, characterized in that: The polypeptide sequence enhances the targeting binding ability of the self-assembly on the tumor cell membrane by binding to sialic acid.

4. A method for preparing 8-FF-TPP nano self-assembly, comprising the following steps: The molecules containing the polypeptide sequence and the porphyrin TPP group were prepared by solid phase synthesis and purified by high performance liquid chromatography (HPLC). The obtained molecules were dissolved in DMSO, and water was added to induce the formation of self-assembled nanoparticles to obtain a uniform nanoparticle suspension.

5. An application in the treatment of melanoma, characterized in that The 8-FF-TPP nano self-assembly described in any one of claims 1 to 2 is used for photothermal therapy, which comprises injecting the self-assembly into the body, achieving photothermal conversion under specific wavelength laser irradiation, and selectively killing tumor tissue.

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