Preparation of 8-FF-TPP-based nano-self-assembled structures and their application in melanoma

Abstract

This invention provides a self-assembled nanoparticle based on 8-FF-TPP and its application in photothermal therapy for melanoma. This self-assembled nanoparticle is formed by introducing an FF sequence and porphyrin (TPP) group into a polypeptide, exhibiting excellent photothermal conversion efficiency and targeting properties for melanoma cells. The self-assembled nanoparticle can generate a photothermal effect under specific wavelength laser irradiation, killing melanoma cells and demonstrating excellent photothermal stability. The invention also includes a preparation method that, through solid-phase synthesis, HPLC purification, and DMSO self-assembly, yields a stable self-assembled nanoparticle for melanoma imaging and targeted therapy.

Description

Technical Field

[0001] This invention relates to the medical field, specifically to a nano-self-assembled structure for photothermal therapy of melanoma and its preparation method. This nano-self-assembled structure can specifically target tumor sites and generate a photothermal effect through laser irradiation, thereby achieving photothermal therapy for melanoma. Background Technology

[0002] Melanoma is a highly malignant skin cancer, and current treatments include surgery, chemotherapy, and immunotherapy. However, these treatments suffer from significant side effects and high recurrence rates. Therefore, developing safe and effective targeted therapies is of great importance. Photothermal therapy is an emerging treatment method that utilizes the photothermal conversion effect of nanomaterials to achieve high-temperature killing of tumors. Research has found that peptide materials with self-assembly properties can form stable nanoparticles. Fabricating targeted peptides into nanoparticles has the potential to target cancer cells. Based on this, this invention provides a nano-self-assembled structure based on peptide motifs and TPP porphyrin groups, which can efficiently absorb light energy and convert it into heat energy for precise treatment of melanoma. Summary of the Invention

[0003] This invention aims to design an 8-FF-TPP nano-self-assembled structure that forms nanoparticles through self-assembly, exhibiting excellent photothermal conversion efficiency and the ability to target melanoma cells. The invention also provides a method for its preparation and details its application in photothermal therapy for melanoma.

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

[0005] The structure of the 1.8-FF-TPP nano-self-assembled structure includes an FF dipeptide structure and a porphyrin (TPP) group, which can effectively self-assemble into nanoparticles. Its polypeptide has sialic acid targeting the cell surface. The polypeptide sequence of the nanoparticle is TPHGYQPMQGKTGPGYFFG.

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

[0007] 3. The nanoparticles provided by this invention are obtained by coupling a targeting peptide with GPGYFFG and porphyrin. Cell experiments have demonstrated that the complex can specifically target sialic acid on the cell membrane surface. A melanoma-bearing model was established, and in vivo imaging verified that the nanoparticles described in this invention can target tumor sites in vivo.

[0008] 4. The method for preparing nanoparticles provided by this invention is stable. The prepared composite was verified by RP-HPLC, showing high purity and no interfering peaks. Mass spectrometry confirmed its correct molecular weight. Transmission electron microscopy showed that the hydrated particle size of the nanoparticles assembled under different conditions was consistent, and the solution was clear and transparent. The composite exhibited strong stability after dilution with different solutions. Continuous light-cooling cycle studies demonstrated its good photothermal conversion stability.

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

[0010] 6. The application provided by this invention, through laser irradiation experiments, has confirmed that nanoparticles can significantly inhibit the growth of tumor cells, and have good application prospects in tumor diagnosis and anti-tumor agents. Attached Figure Description

[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) with different laser powers -1 Temperature changes, B under 638nm laser irradiation (1.5W cm) -2 The concentration-dependent temperature change of 8-FF-NPs under the following conditions, C 8-FF-NPs (300 μg / mL) -1 The temperature change curves after reaching the maximum temperature and cooling are shown for D8-FF-NPs (200 μg mL). -1 ) During laser irradiation on / off cycles (1.5W cm -2 Temperature changes during the period;

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

[0015] Figure 5 8-FF-NPs are cytotoxic 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 mice after 24 hours of dissection. Detailed Implementation

[0017] To make the technical problems to be solved, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

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

[0019] 1.1 Experimental Procedure

[0020] Peptide synthesis was performed using F-moc solid-phase synthesis, with amino acids being coupled one by one to RinkAmide resin, starting from the C-terminus and proceeding to the N-terminus. The specific steps are as follows:

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

[0022] (2) Washing the resin: Wash the activated resin three times with DMF and then dry it.

[0023] (3) Deprotection: The dried resin was deprotected with 20% piperidine. The first reaction was carried out for 7 minutes and then dried. The second reaction was carried out for 8 minutes and then washed with DMF 8 times.

[0024] (4) Activation of amino acids: Weigh 0.5 mmol HOBT, 0.5 mmol HCTU and 0.4 mmol amino acids and dissolve them in 3 mL of 5% N-methylmorpholine solution and react for 15 min;

[0025] (5) First amino acid coupling: Add the activated amino acid to the resin and react for 1 hour. After the reaction is complete, dry the liquid and wash with DMF 8 times.

[0026] (6) Coupling of the second amino acid: Deprotect the amino acid coupled to the resin in (5) with piperidine, then add the next activated amino acid, react for 1 hour, after the reaction is completed, dry the liquid, add DMF and wash 8 times.

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

[0028] (8) After peptide synthesis, take 0.04 mmol of resin and react with 20% piperidine for 15 min to remove the peptide and attach Fmoc protection. Weigh 30 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 20 mg of 1-hydroxybenzotriazole, and 5 mg of TPP-G. Dissolve 5 mg of TPP-G in 2 mL of anhydrous DMF, then add 30 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, mix the solution by blowing, and then add 20 mg of 1-hydroxybenzotriazole. React at room temperature for 15 min. After the reaction, pour the reaction solution into the synthesis column resin and react overnight at room temperature in the dark. After the reaction, wash the resin 8 times with DMF, dry the liquid, then wash 8 times with anhydrous methanol and dry the liquid. Finally, add lysis buffer, which is formulated as 90% TFA, 2.5% anisole, 2.5% anisole sulfide, and 5%...

[0029] Dimercaptoethane. The sample was lysed for 2.5 h, precipitated with ice-cold diethyl ether, 30 mL of diethyl ether was added, and then mixed with the lysis buffer. The mixture was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the sample was air-dried to obtain 8-FF-TPP.

[0030] 1.2 Experimental Results

[0031] FF is a core self-assembling motif extracted from β-amyloid peptides used in Alzheimer's disease. FF components have demonstrated significant advantages, including ease of production, multifunctionality, biodegradability, biocompatibility, and non-immunogenicity. Furthermore, FF can be regulated through non-covalent interactions with various functional molecules; for example, FF can self-assemble into nanostructures through hydrogen bonding and π-π stacking of aromatic groups. Co-assembly is a method for obtaining morphologically controllable and stable nanomaterials. SA-8, screened using phage display technology in our laboratory, has been shown to bind well to sialic acid. Due to the advantages of photothermal therapy, such as low invasiveness, simple operation, short treatment time, and rapid recovery, we modified SA-8 with porphyrin and introduced GPGYFFG into the modified sequence to ensure controllable drug morphology and good photostability. Figure 1As shown, 8-FF-TPP was purified by RP-HPLC at 415 nm. We collected the sample with a retention time of 28 min and identified it as the target sample by mass spectrometry. The molecular weight identified by mass spectrometry was 2798 Da, indicating that the sample was successfully synthesized.

[0032] Example 2: Preparation of 8-FF-TPP nano-assemblies

[0033] 2.1 Experimental Procedure

[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 assembly concentration was 2 mg / mL. The assembled sample was dialyzed after 24 hours. 1 mL of solution required 1 L of water for dialysis over 24 hours, and the aqueous solution needed to be changed 3 times. The molecular weight cutoff of the dialysis bag was 5 kDa. After dialysis, the solution was centrifuged using an ultrafiltration tube. The centrifuged solution was the 8-FF-TPP.

[0035] 2.2 Experimental Results

[0036] 8-FF-TPP nanoparticles were self-assembled using a DMSO:water ratio of 1:9 and an assembly concentration of 2 mg / mL. Assembly was carried out over 24 hours, followed by dialysis for 24 hours. The assembled nanoparticles were characterized by TEM and dynamic light scattering, such as... Figure 2 As shown, 8-FF-NPs, when assembled under a transmission electron microscope, form spherical particles of uniform size with a diameter of approximately 35 nm. The hydrated particle size, measured by dynamic light scattering, is approximately 33.9 ± 5.6 nm. Optical images show that the solution is clear, transparent, and homogeneous.

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

[0038] 3.1 Experimental Procedure

[0039] The photothermal conversion performance was measured by irradiating 8-FF-NPs dispersions of different concentrations (25, 50, 100, 300 μg / mL) with a 638 nm laser. The effect of laser power density on the photothermal effect of nanoparticles was further analyzed using 638 nm lasers with different power densities (0.1, 1.0, 1.5 W / cm²). -2 The 8-FF-NPs (0.3 mg / mL) dispersion was irradiated, and temperature change curves were plotted. Finally, the 8-FF-NPs were subjected to multiple laser "on-off" cycle irradiations to test their photothermal stability.

[0040] 3.2 Experimental Results

[0041] To verify the photothermal capabilities of 8-FF-NPs, a 638nm laser was used to irradiate an 8-FF-NPs suspension, and the temperature changes of the suspension were recorded. Figure 3 A shows that 300 μg mL -1 The temperature of 8-FF-NPs increases with increasing laser power, especially when the laser power increases from 0.1 W / cm². -2 Increased to 1.5W cm -2 After 10 minutes of irradiation, the temperature rose by 25.4°C. For example... Figure 3 Figure B shows that for 8-FF-NPs at different concentrations, under the same laser power irradiation, the temperature increases with increasing concentration, with a laser power of 1.5 W cm⁻¹. -2 When the concentration of 8-FF-NPs decreased from 25 μg / mL -1 Up to 300 μg mL -1 After irradiation for 10 minutes, the temperature difference was 10.4℃. The results indicate that the nanoparticles exhibit a photothermal conversion process that is both concentration-dependent and laser power density-dependent. Therefore, the required treatment temperature can be achieved by adjusting the nanoparticle concentration or laser power. Figure 3 C shows the peak temperature and cooling curves. Figure 3 D. Through continuous light-cooling cycle study, after 5 cycles, the heating trend of 8-FF-NPs did not show a significant weakening, indicating that it has good photothermal stability.

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

[0043] 4.1 Experimental Procedure

[0044] Seed 1×10⁻⁶ seeds in a 20mm laser confocal glass dish. 5Cells were cultured overnight in B16-F10 or HepG2 medium. The experimental group was then cultured in serum-free medium, while the control group was incubated overnight with sialidase (100 mU / mL) dissolved in serum-free medium. Sialidase hydrolyzes sialic acid on the cell surface. 8-FF-NPs were diluted to 10 μM in serum-free medium. Before adding the drug to the cells, the cells were washed twice with PBS, then the drug was added, and incubated for 3 h. After drug incubation, the cells were washed three times with PBS, and the cell membranes were stained with DiO. 2.5 μL of DiO and 2.5 μL of staining enhancer were mixed per mL of staining buffer. The staining working solution was added to the cells and incubated at 37°C in the dark for 15 min. After staining, the cells were washed three times with PBS, and 1 mL of 4% paraformaldehyde fixative was added to each dish for fixation for 15 min. After fixation, the cells were washed three times with PBS, and 5 mg / mL LDAPI prepared in PBS was added. The cells were incubated at 37°C in the dark for 15 min, followed by three washes with PBS. For fluorescence imaging, a Leica SP8 excitation beam was used. The maximum excitation wavelength for DiO was 488 nm, and the maximum emission wavelength was 501 nm. The maximum excitation wavelength for DAPI was 364 nm, and the maximum emission wavelength was 454 nm. The excitation wavelength for 8-FF-NPs was 450 nm.

[0045] 4.2 Experimental Results

[0046] HepG2 and B16-F10 cells highly express sialic acid on their surface. Therefore, we explored the targeting ability of 8-FF-NPs to HepG2 and B16-F10 cells. 10 μM of 8-FF-NPs were incubated with cells for 3 hours. To demonstrate that 8-FF-NPs can target sialic acid on the cell membrane, we pre-incubated HepG2 and B16-F10 cells with sialidase, allowing the sialidase to hydrolyze the sialic acid on the cell membrane surface. The cell nuclei were stained blue with DAPI, the cell membrane was stained green with DiO2, and 8-FF-NPs showed red fluorescence. The results are as follows: Figure 4 As shown in Figure A, for HepG2 cells, the red fluorescence in the 8-FF-NPs+sialidase group was significantly inhibited, and the fluorescence was almost invisible. In contrast, the red fluorescence in the 8-FF-NPs group was very obvious, and the red and green fluorescence could overlap well. The results indicate that 8-FF-NPs can specifically bind to sialic acid. Figure 4 As shown in Figure B, the fluorescence intensity of the 8-FF-NPs+sialidase group differed from that of the 8-FF-NPs group by a factor of 2.5. The average fluorescence intensity data quantitatively demonstrates that 8-FF-NPs can specifically bind sialic acid. Figure 4As shown in C, we used B16-F10 cells to demonstrate that 8-FF-NPs can specifically bind to sialic acid. In the 8-FF-NPs+sialidase group, the red fluorescence was weak and almost invisible, while in the 8-FF-NPs group, the red fluorescence was very obvious, and the red and green fluorescence could overlap well. All of these indicate that 8-FF-NPs can specifically bind to sialic acid. Figure 4 As shown in Figure D, the fluorescence intensity of the 8-FF-NPs+sialidase group differed from that of the 8-FF-NPs group by approximately two-fold, indicating that 8-FF-NPs can specifically bind to sialic acid. Combined with co-localization experiments in HepG2 and B16-F10 cells, this demonstrates that 8-FF-NPs can target sialic acid.

[0047] Example 5: 8-FF-NPs Cytotoxicity Assay

[0048] 5.1 Experimental Procedure

[0049] After digesting the cells with trypsin, the digestion was stopped by adding culture medium. The cells were centrifuged at 1000 rpm for 3 min, and the cell density was adjusted. 100 μL of 5000 cells were added to each well of a 96-well plate. The cells were incubated for 24 h, after which the culture medium was removed. The corresponding prepared drug was added to each well, with three replicates per group. After 12 h of drug incubation, the culture medium was removed and replaced with serum-free medium. A 638 nm laser with a power of 0.5 W cm⁻¹ was used for further incubation. -2 Cells were irradiated for 180 seconds. The dark group was not irradiated with laser, but other treatments were the same as the laser group. After irradiation, the cells were incubated for 12 hours, then 10 μL of CCK-8 was added to each well, and incubation continued for 1–5 hours. Finally, the absorbance was measured at a wavelength of 450 nm. The blank control was serum-free medium with the corresponding amount of CCK-8 added. Cell viability (%) = (absorbance of experimental group - absorbance of blank control) / (absorbance of experimental control group - absorbance of blank control).

[0050] 5.2 Experimental Results

[0051] Depend on Figure 5 It can be seen that 8-FF-NPs show little difference in cytotoxicity under both dark and light conditions below 0.0625 μM. (Laser power: 0.5 W cm⁻¹) -2 After 180 seconds of irradiation, 0.625 μM irradiation inhibited more than 80% of cells. The toxicity to cells was low under dark conditions, but significant at 20 μM, with a difference of more than 30 times. The IC50 after laser irradiation was also significant. 50 The concentration was 0.147 ± 0.076 μM. These results indicate that the material can effectively kill cancer cells with good selectivity.

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

[0053] 6.1 Experimental Procedure

[0054] B16-F10 cells were digested with trypsin, and after digestion, the cells were centrifuged to remove the culture medium. Serum-free culture medium was added, and the cells were resuspended and the cell density was adjusted to 6 × 10⁶ cells / year. 7 / mL. Purchase 5-6 week old BALB / c mice and inject 100μL subcutaneously into each mouse. Wait until the tumor volume reaches 100mm². 3 Subsequent experiments were then conducted. 200 μL of 8-FF-NPs (21.5 mg / kg) and 200 μL of LTP (4.8 mg / kg) were injected into mice via the tail vein. The mice were scanned in an in vivo imaging system at different time points. After 24 hours, the mice were dissected, and their tissues and organs were imaged.

[0055] 6.2 Experimental Results

[0056] To further verify the targeting ability of 8-FF-NPs in vivo, fluorescence imaging was performed on the distribution of 8-FF-NPs and porphyrins after tail vein injection, as shown in the following figures. Figure 6 A. When porphyrin alone enters a mouse, the fluorescence intensity in various organs does not increase significantly over time, and no specific enrichment occurs at the tumor site, indicating that porphyrin is rapidly metabolized in vivo. With increasing time, 8-FF-NPs nanoparticles show increasing enrichment at the tumor site; significant enrichment is visible after 8 hours, very pronounced at 12 hours, and strong fluorescence remains at the tumor site until 24 hours. Figure 6 B. We dissected mice after 24 hours and analyzed the fluorescence intensity of various organs and tumors in the two experimental groups. 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 persistence and targeting in vivo and a slow metabolic rate in vivo, which allows the nanoparticles to have better therapeutic effects.

Claims

1. A nano-self-assembled structure based on 8-FF-TPP, characterized in that: The structure comprises a polypeptide sequence TPHGYQPMQGKTGPGYFFG and a porphyrin group TPP. The porphyrin group TPP is modified at the end of the polypeptide sequence to form a TPHGYQPMQGKTGPGYFFG-TPP structure. This structure is dissolved in DMSO, and water is added to induce the formation of a nano-self-assembled structure. The nano-self-assembled structure exhibits good tumor targeting and photothermal conversion properties. Under laser irradiation, the temperature of the nano-self-assembled structure increases significantly, achieving targeted photothermal therapy of tumor cells.

2. The nano-self-assembled structure according to claim 1, characterized in that: The particle size of the nano-self-assembled particles is 10 to 100 nanometers.

3. The nano-self-assembled structure according to claim 2, characterized in that: The particle size of the nano-self-assembled particles is 30 to 50 nanometers.

4. The nano-self-assembled structure according to claim 1, characterized in that: The polypeptide sequence enhances the targeted binding ability of the self-assembled structure to the tumor cell membrane by binding to sialic acid.

5. A method for preparing a nano-self-assembled body as described in any one of claims 1-4, comprising the following steps: The polypeptide sequence was prepared by solid-phase synthesis, with porphyrin TPP groups modified at the ends of the polypeptide sequence. The sequence was then purified by high-performance liquid chromatography. The purified substance was dissolved in DMSO and water was added to induce the formation of nano-self-assembled structures.

6. The use of the nano-self-assembled body according to any one of claims 1-4 in the preparation of a medicament for photothermal treatment of melanoma, characterized in that, The treatment involves injecting nano-self-assembled bodies into the body, achieving photothermal conversion under specific wavelength laser irradiation, and selectively killing tumor tissue.

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