Preparation of a self-assembled nanoparticle and its application in melanoma targeted imaging and photothermal therapy

Abstract

This invention discloses a self-assembled nanoparticle for targeted imaging and photothermal therapy of melanoma, aiming to address the problems of poor targeting, low photothermal efficiency, and poor biocompatibility in existing photothermal therapy methods for melanoma. This invention designs a self-assembled nanoparticle based on a specific peptide (SA-7), aromatic amino acid (FF), and porphyrin (TPP). The self-assembled nanoparticle can efficiently target melanoma cells and generate a strong photothermal effect under 638nm laser irradiation. Simultaneously, the nanoparticle exhibits good biocompatibility and metabolic properties, making it suitable for precise imaging and treatment of melanoma.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and nanomaterials technology, and in particular to a nanoparticle material based on the self-assembly of peptides and porphyrins for imaging and photothermal therapy targeting melanoma. Background Technology

[0002] Melanoma is a highly malignant skin cancer that is prone to spreading and metastasizing. Conventional treatments (such as chemotherapy and radiotherapy) are ineffective in inhibiting the invasion of melanoma cells and easily damage normal cells. In recent years, photothermal therapy (PTT) has shown promising application prospects in cancer treatment as a novel tumor treatment method. However, existing photothermal therapeutic agents, such as inorganic nanomaterials (gold nanoparticles, graphene, etc.), suffer from problems such as lack of targeting, poor biocompatibility, and difficulty in metabolism, making their clinical application difficult.

[0003] Therefore, there is an urgent need to develop novel organic photothermal therapy materials with good targeting, biocompatibility, and photothermal conversion efficiency to improve treatment efficacy. Porphyrin compounds are considered promising photothermal agents due to their photosensitivity and good biocompatibility, but their targeting is insufficient when used alone. Therefore, developing porphyrin nanoparticles that are self-assembled with specific peptides holds promise as a highly efficient targeted photothermal therapy for melanoma. Summary of the Invention

[0004] The purpose of this invention is to provide a self-assembled organic nanoparticle material, which is designed into nanoparticles by targeting peptides and polypeptides and porphyrin monomers containing FF, which can specifically target melanoma cells and generate a highly efficient photothermal effect under laser irradiation, thus realizing an integrated application in melanoma imaging and treatment.

[0005] The self-assembled nanoparticles provided by this invention comprise the following components:

[0006] 1. Peptide sequence SA-7 (AMDPRQSNRLYS): It can specifically recognize sialic acid receptors on the surface of melanoma cells, enhancing targeting;

[0007] 2. Aromatic amino acid FF: It forms a nanoparticle core structure through hydrophobic interactions and π-π stacking, which enhances the stability of the particles;

[0008] 3. Porphyrin unit TPP-G: Porphyrin molecules used for photothermal effects, which generate heat energy under 638nm laser irradiation to destroy tumor cells.

[0009] 4. The nanoparticles provided by this invention have excellent anti-tumor effects in in vitro cell experiments.

[0010] 5. In vitro hemolysis experiments showed that the nanoparticles had very low toxicity.

[0011] 6. Animal experiments have shown that nanoparticles have excellent targeting properties to tumor sites. Attached Figure Description

[0012] Figure 1 TPP-G mass spectrum and NMR spectrum, mass spectrum of purified ATPP-G, and 1H NMR spectrum of purified TPP-G. 1 HNMR(500MHz,DMSO-d6)δ10.46(s,1H),8.89(d,J=4.7Hz,2H),8.83(s,6H),8.22(d,J=6.7Hz,6H),8.18 (d,J=8.1Hz,2H),8.13(d,J=8.1Hz,2H),7.82(d,J=7.2Hz,9H),4.36(s,2H),4.34(s,2H),-2.92(s,2H);

[0013] Figure 2 7-FF-TPP purification and mass spectrometry identification: A. Chromatographic separation and purification of 7-FF-TPP, with the arrow indicating the target peak; B. 7-FF-TPP mass spectrometry identification.

[0014] Figure 3 Characterization of 7-FF-NPs;

[0015] Figure 4 Photothermal properties of 7-FF-NPs: A) Temperature changes of 7-FF-NPs (200 μg mL⁻¹) with different laser powers; B) Irradiation with a 638 nm laser (1.5 W cm⁻¹). -2 The concentration-dependent temperature change of 7-FF-NPs under the following conditions, C 7-FF-NPs (200 μg mL) -1 The temperature change curves after reaching the maximum temperature and cooling are shown for D7-FF-NPs (300 μg mL). -1 ) During laser irradiation on / off cycles (1.5W cm -2 Temperature changes during the period. Figure 5 7-FF-NPs target sialic acid. A: Fluorescence image of HepG2 cells incubated with 10 μM 7-FF-NPs for 3 h, where the control group was pretreated with sialylase overnight. B: Quantitative fluorescence image of A. C: Fluorescence image of B16-F10 cells incubated with 10 μM 7-FF-NPs for 3 h, where the control group was pretreated with sialylase overnight. DAPI staining of cell nuclei and DiO staining of cell membranes. 7-FF-NPs appear red under a microscope. D: Quantitative fluorescence image of C. Averages ± SEM. ****p<0.0001, **p<0.01;

[0016] Figure 67-FF-NPs exhibit different cytotoxic effects; A7-FF-NPs exhibit cytotoxicity against HepG2 cells; laser power 0.5 W / cm². -2 Laser irradiation for 180 seconds showed the cytotoxicity of B7-FF-NPs against LO2 and B16-F10 cells at a laser power of 0.3 W / cm². -2 Laser irradiation time: 90 seconds;

[0017] Figure 7 In vivo imaging of 7-FF-NPs: A. Distribution of 7-FF-NPs in mice at different time points; B. Fluorescence distribution in mice after 24 hours of dissection. Detailed Implementation

[0018] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0019] Example 1: Synthesis of TPP-G

[0020] 1.1 Experimental Procedure

[0021] Weigh 113 mg of porphyrin (4-(10,15,20-triphenylporphyrin-5-yl)aniline) and 55 mg of diethanol anhydride, dissolve them in 4 mL of anhydrous DMF, and react for 48 h. After the reaction, add saturated saline solution to the sample, extract three times with dichloromethane, then add anhydrous Na₂SO₄ to remove water from the organic phase, and evaporate the organic solvent to dryness. Add 10 mL of dichloromethane to the evaporated product, and slowly add the solution dropwise to 100 mL of petroleum ether. Mix well and let stand for 4 h; a large amount of flocculent precipitate is observed to form. Filter the solution with filter paper, evaporate the solid precipitate to dryness, and obtain pure TPP-G.

[0022] 1.2 Experimental Results

[0023] Experimental results are as follows Figure 1 As shown, we purified the porphyrin by reacting it with diethanol anhydride to obtain the product TPP-G, as follows. Figure 1 A shows that the mass spectrometry detected a molecular weight of 746 Da. The porphyrin ring large π-conjugated system forms a strong shielding region in an external magnetic field; the two H atoms on the pyrrole N atom are generally around -3.0; the aromatic hydrogens surrounding the porphyrin ring are generally between 7.0 and 9.0. For example... Figure 1 As shown in Figure B, the peak with chemical shift δ = -2.92 represents two hydrogen atoms at position f; the four hydrogen atoms at positions with chemical shifts δ = 4.34 and δ = 4.36 represent hydrogen atoms on the acid anhydride (a, b), and the hydrogen atom at chemical shift δ = 10.46 represents hydrogen atoms on the amide bond (c). Mass spectrometry and NMR spectra indicate that TPP-G was successfully synthesized. 1HNMR(500MHz,DMSO-d6)δ10.46(s,1H),8.89(d,J=4.7Hz,2H),8.83(s,6H),8.22(d,J=6.7Hz,6H),8.18 (d,J=8.1Hz,2H),8.13(d,J=8.1Hz,2H),7.82(d,J=7.2Hz,9H),4.36(s,2H),4.34(s,2H),-2.92(s,2H).

[0024] Example 27 - Synthesis of FF-TTP

[0025] 2.1 Experimental Procedure

[0026] SA-7 was synthesized using F-moc solid-phase synthesis, with amino acids being coupled one by one to RinkAmide resin, starting from the C-terminus and working towards the N-terminus. The specific steps are as follows:

[0027] (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;

[0028] (2) Washing the resin: Wash the activated resin three times with DMF and then dry it.

[0029] (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.

[0030] (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;

[0031] (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.

[0032] (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.

[0033] (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.

[0034] (8) In order to regulate the nanoparticles, GFFYGPG was added between SA-7 and porphyrin.

[0035] (9) 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, drain the liquid, then wash 8 times with anhydrous methanol and drain the liquid. Finally, add lysis buffer, which is formulated as 90% TFA, 2.5% anisole, 2.5% anisole sulfide, and 5%...

[0036] 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 7-FF-TPP.

[0037] 2.2 Experimental Results

[0038] Experimental results are as follows Figure 2 As shown, 7-FF-TPP was purified by RP-HPLC at 415 nm, and the peak eluted at 30 min was the target sample. Subsequently, we used mass spectrometry to analyze the sample collected at 30 min, and the molecular weight identified by mass spectrometry was 2891 Da, indicating that the sample was successfully synthesized.

[0039] Example 3: Preparation of Nanoscale Self-Assembly

[0040] 3.1 Experimental Procedure

[0041] The synthesized 7-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 7-FF-NPs.

[0042] 3.2 Experimental Results

[0043] Experimental results are as follows Figure 3 As shown, 7-FF-TPP self-assembled with DMSO and water at a ratio of 1:9 and an assembly concentration of 2 mg / mL. After 24 hours of assembly, the nanoparticles were dialyzed for 24 hours. The assembled nanoparticles were characterized by TEM and dynamic light scattering, as shown... Figure 3As shown, 7-FF-NPs, when assembled under a transmission electron microscope, form uniformly sized nanoparticles with a diameter of approximately 50 nm. Dynamic light scattering analysis determined the hydrated particle size to be 231 ± 89 nm. Optical images show a clear, transparent, and homogeneous solution.

[0044] Example 4: Photothermal conversion performance determination

[0045] 4.1 Experimental Procedure

[0046] The photothermal conversion performance was measured by irradiating 7-FF-NPs dispersions of different concentrations (50, 100, 200, 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 7-FF-NPs (0.2 mg / mL) dispersion was irradiated, and temperature change curves were plotted. Finally, the 7-FF-NPs were subjected to multiple laser "on-off" cycle irradiations to test their photothermal stability.

[0047] 4.2 Experimental Results

[0048] To verify the photothermal capabilities of 7-FF-NPs, a 638nm laser was used to irradiate a 7-FF-NPs suspension, and the temperature changes of the suspension were recorded. Figure 4 Images A and B show that 7-FF-NPs exhibit a significant concentration-dependent and laser power density-dependent photothermal conversion process, therefore the required treatment temperature can be achieved by adjusting the nanoparticle concentration or laser power. (200 μg / mL) -1 7-FF-NPs exhibit an increase in temperature as laser power increases, especially when the laser power decreases from 0.1 W / cm². -2 Increased to 1.5W cm -2 After 10 minutes of irradiation, the temperature rose by 29.9°C. For example... Figure 4 B shows that for 7-FF-NPs at different concentrations, the temperature increases with increasing concentration, and the laser power is 1.5W cm⁻¹. -2 7-FF-NPs concentration from 50 μg / mL -1 Up to 300 μg mL -1 Irradiate for 10 minutes with a temperature difference of 20℃. Figure 4 C shows the peak temperature and cooling curves. Furthermore, Figure 4 The study of the continuous light-cooling cycle was conducted, and the temperature of 7-FF-NPs did not decrease significantly after five cycles, indicating that it has good photothermal stability.

[0049] Example 5 Co-location Experiment

[0050] 5.1 Experimental Procedure

[0051] Seed 1×10⁻⁶ seeds in a 20mm laser confocal glass dish. 5 B16-F10 or HepG2 cells were cultured overnight. 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. 7-FF-NPs were diluted to 10 μM in serum-free medium. The drug was added to the cells, and the cells were washed twice with PBS before incubation 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 7-FF-NPs was 450 nm.

[0052] 5.2 Experimental Results

[0053] Many studies have reported high expression of sialic acid in tumor cells, such as HepG2 and B16-F10 cells. We explored the targeting of 7-FF-NPs to HepG2 and B16-F10 cells by incubating 10 μM 7-FF-NPs with cells for 3 hours. To demonstrate that 7-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 sialic acid on the cell membrane surface. The cell nucleus was stained blue with DAPI, the cell membrane was stained green with DiO, and 7-FF-NPs showed red fluorescence. The results are as follows: Figure 5 As shown in Figure A, for HepG2 cells, the red fluorescence of the 7-FF-NPs+sialidase group was significantly inhibited, while the red fluorescence of the 7-FF-NPs group was very obvious. The red and green fluorescence could overlap well. These results show that 7-FF-NPs can specifically bind sialic acid. Figure 5 As shown in Figure B, the fluorescence intensity of the 7-FF-NPs+sialidase group differed from that of the 7-FF-NPs group by a factor of 5. The average fluorescence intensity data quantitatively demonstrates that 7-FF-NPs can specifically bind sialic acid. Figure 5As shown in Figure C, B16-F10 cells were used to demonstrate that 7-FF-NPs can specifically bind to sialic acid. The red fluorescence in the 7-FF-NPs+sialidase group was relatively weak, while the red fluorescence in the 7-FF-NPs group was very obvious. The red and green fluorescence overlapped well, indicating that 7-FF-NPs can specifically bind to sialic acid. Figure 5 As shown in Figure D, the fluorescence intensity of the 7-FF-NPs+sialidase group differed from that of the 7-FF-NPs group by approximately two-fold, indicating that 7-FF-NPs can specifically bind to sialic acid. Co-localization experiments in HepG2 and B16-F10 cells further demonstrated that 7-FF-NPs can target sialic acid.

[0054] Example 6 Cytotoxicity Experiment

[0055] 6.1 Experimental Procedure

[0056] After trypsin digestion, the digestion was stopped by adding culture medium. 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. After culturing for 24 h, the culture medium was removed, and the corresponding prepared drug was added to each well. Each group was repeated in triplicate. After 12 h of drug incubation, the culture medium was removed and replaced with serum-free medium. Cells were then irradiated with the appropriate laser power at 638 nm. After irradiation, cells were incubated for 12 h, and then 10 μL of CCK-8 was added to each well. Incubation continued for 1-5 h, and the absorbance was measured at a wavelength of 450 nm. The blank control consisted of serum-free medium and the corresponding CCK-8. Cell viability (%) = (Experimental group absorbance - Blank control absorbance) / (Experimental control absorbance - Blank control absorbance).

[0057] The protocol for laser-free cytotoxicity assays is as follows: Cells are digested with trypsin, digestion is stopped by adding culture medium, and the cells are centrifuged at 1000 rpm for 3 min. Cell density is adjusted, and 100 μL of 5000 cells are added to each well of a 96-well plate. After incubation for 24 h, the culture medium is removed, and the corresponding prepared drug is added to each well. Each group is repeated in triplicate. After 12 h of drug incubation, the culture medium is removed and replaced with serum-free medium. Cells are incubated for another 12 h, and then 10 μL of CCK-8 is added to each well. Incubation continues for 1-5 h, and the absorbance is measured at a wavelength of 450 nm. The blank control is prepared with serum-free medium and the corresponding CCK-8.

[0058] 6.2 Experimental Results

[0059] To assess the in vitro antitumor effect of 7-FF-NPs, two different cell types were used to test the therapeutic efficacy of 7-FF-NPs. For example... Figure 6First, 7-FF-NPs were tested to assess their therapeutic effect on HepG2 cells at a range of concentrations. In the dark reaction, even at a concentration of 20 μM, no toxicity was observed against cancer cells. Under laser irradiation, the IC50 value was [not specified]. 50 The concentration was 0.4 ± 0.188 μM; secondly, the cytotoxicity of 7-FF-NPs was tested using melanoma cells, and under laser irradiation, the IC50 value was 0.4 ± 0.188 μM. 50 The IC50 value was 0.945 ± 0.339 μM. Finally, we tested the cytotoxicity using normal LO2 cells. Under the same laser power conditions as melanoma, the cytotoxic IC50 value for LO2 cells was 0.945 ± 0.339 μM. 50 The concentration was 1.695 ± 0.417 μM. Cytotoxicity results showed that 7-FF-NPs were more cytotoxic to B16-F10 cells than to LO2 cells. This is presumably because 7-FF-NPs themselves target sialic acid, leading to greater accumulation of 7-FF-NPs on B16-F10 cells. In conclusion, 7-FF-NPs show great potential for tumor treatment.

[0060] Example 7: Intratumoral accumulation of drugs

[0061] 7.1 Experimental Procedure

[0062] 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 conducted. 200 μL of 7-FF-NPs (22.2 mg / kg) and 200 μL of LTPP (4.8 mg / kg) were injected into mice via the tail vein. The mice were scanned in vivo at different time points. After 24 hours, the mice were dissected, and their tissues and organs were collected for imaging.

[0063] 7.2 Experimental Results

[0064] 7-FF-NPs and TPP were administered via tail vein injection, and their distribution was characterized by fluorescence imaging, such as... Figure 7 A. 7-FF-NPs nanoparticles accumulated more and more at the tumor site over time. Accumulation was visible after 4 hours, became very significant after 12 hours, and remained strong at the tumor site even after 24 hours. Figure 7 B. Next, we dissected the mice that had been running for 24 hours and analyzed the fluorescence intensity of various organs and tumors. 7-FF-NPs showed strong fluorescence intensity in the liver and tumor sites, indicating that 7-FF-NPs have good targeting properties in vivo and are metabolized slowly in vivo, which can make them more effective.

[0065] The above results indicate that the porphyrin-modified polypeptide nanoparticles of the present invention can be effectively targeted to tumor sites, enabling tumor imaging and photothermal therapy, and have promising applications in tumor diagnosis and photothermal therapy.

Claims

1. A self-assembled nanoparticle for targeted imaging and photothermal therapy of melanoma, characterized in that, The nanoparticles are composed of a targeting peptide sequence SA-7, an aromatic amino acid GFFYGPG sequence, and a porphyrin unit TPP coupled together. The SA-7 sequence is AMDPRQSNRLYS. The preparation method of the self-assembled nanoparticles includes the following steps: First, the porphyrin unit TPP-G is synthesized, and the structural formula of TPP-G is shown in Formula 1. Secondly, a polypeptide with the amino acid sequence AMDPRQSNRLYSGFFYGPG was synthesized by F-moc protected solid-phase synthesis. Finally, TPP-G was coupled with the AMDPRQSNRLYSGFFYGPG polypeptide by Fmoc solid-phase synthesis to form 7-FF-TPP. The 7-FF-TPP formed a stable nanoparticle structure through self-assembly and was used to target melanoma cells.

2. The self-assembled nanoparticles according to claim 1, characterized in that, The nanoparticles are between 50 and 100 nm in size and have melanoma-specific binding ability.

3. A method for preparing the self-assembled nanoparticles according to claim 1, comprising the following steps: a. Synthesis of porphyrin unit TPP-G; b. TPP-G was coupled with AMDPRQSNRLYSGFFYGPG peptide via Fmoc solid-phase synthesis to form a 7-FF-TPP peptide chain. c. Dissolve the 7-FF-TPP peptide chain in a DMSO-water mixture and obtain nanoparticles through self-assembly.

4. The application of the self-assembled nanoparticles according to claim 1 in the preparation of drugs for targeted melanoma imaging.

5. The application of the self-assembled nanoparticles according to claim 1 in the preparation of a drug for photothermal therapy of melanoma, characterized in that... A localized thermal effect on melanoma is achieved by irradiating nanoparticles with laser light.

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