Preparation of nano self-assembly particles and application of nano self-assembly particles in melanoma targeted imaging and photo-thermal therapy

Abstract

The invention discloses self-assembled nanoparticles for melanoma targeted imaging and photo-thermal therapy, and aims to solve the problems of poor targeting, low photo-thermal efficiency and poor biocompatibility in the existing method for photo-thermal therapy of melanoma. The invention designs a self-assembled nanoparticle based on specific polypeptide (SA-7), aromatic amino acid (FF) and porphyrin (TPP). The nanoparticles formed by self-assembly can efficiently target melanoma cells, and generate a strong photothermal effect under the irradiation of 638nm laser. Meanwhile, the nanoparticles have good biocompatibility and metabolic performance, and can be used for accurate imaging and treatment of melanoma.

Description

Technical Field

[0001] The invention relates to the field of biomedical engineering and nanomaterial technology, and in particular to a nanoparticle material based on self-assembly of polypeptides and porphyrins, which is used for imaging and photothermal therapy of targeted melanoma. Background Art

[0002] Melanoma is a highly malignant skin cancer that is easy to spread and metastasize. Conventional treatments (such as chemotherapy and radiotherapy) are difficult to effectively inhibit the invasion of melanoma cells and are prone to damage normal cells. In recent years, photothermal therapy (PTT), as a new type of tumor treatment, has shown good application prospects in cancer treatment. However, existing photothermal therapeutic agents such as inorganic nanomaterials (gold nanoparticles, graphene, etc.) have problems such as lack of targeting, poor biosafety, and metabolic difficulties, making them difficult to apply in clinical practice.

[0003] Therefore, there is an urgent need to develop a new type of organic photothermal therapy material with good targeting, biocompatibility and photothermal conversion efficiency to improve the therapeutic effect. Porphyrin compounds are considered to be very promising photothermal agents due to their photosensitivity and good biocompatibility, but their targeting is insufficient when used alone. Therefore, the development of porphyrin nanoparticle materials that are combined with specific peptides and self-assembled is expected to become an efficient melanoma targeted photothermal therapy solution. Summary of the invention

[0004] The purpose of the present invention is to provide an organic nanoparticle material based on self-assembly, which is designed into nanoparticles by targeting peptides and polypeptides containing FF and porphyrin monomers, can specifically target melanoma cells, produce efficient photothermal effect under laser irradiation, and realize integrated application in melanoma imaging and treatment.

[0005] The self-assembled nanoparticles provided by the present invention include the following components:

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

[0007] 2. Aromatic amino acid FF: forms the core structure of nanoparticles through hydrophobic interaction and π-π stacking, enhancing the stability of the particles;

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

[0009] 4. The nanoparticles provided by the present invention have good anti-tumor effects in in vitro cell experiments.

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

[0011] 6. Animal experiments have shown that nanoparticles have good targeting to tumor sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 TPP-G mass spectrum and NMR, A mass spectrum of ATPP-G after purification, B NMR hydrogen spectrum of TPP-G after purification, 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 Purification and mass spectrometry identification of 7-FF-TPP, A chromatographic separation and purification of 7-FF-TPP, arrows indicate the target peak, B mass spectrometry identification of 7-FF-TPP;

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

[0015] Figure 4 Photothermal properties of 7-FF-NPs. A shows the temperature change of 7-FF-NPs (200 μg mL-1) with different laser powers. B shows the temperature change of 7-FF-NPs (200 μg mL-1) with different laser powers. -2 ) concentration-dependent temperature change of 7-FF-NPs, C 7-FF-NPs (200 μg mL -1 ) Temperature change curve after reaching the highest temperature and cooling, D 7-FF-NPs (300μg mL -1 ) during laser irradiation on / off cycles (1.5 W cm -2 ) during the temperature change. Figure 5 7-FF-NPs target sialic acid, A is the fluorescence image of HepG2 cells incubated with 10μM 7-FF-NPs for 3h, in which the control group was pretreated with sialidase overnight, B is the fluorescence quantitative image of A, C is the fluorescence image of B16-F10 cells incubated with 10μM 7-FF-NPs for 3h, in which the control group was pretreated with sialidase overnight, DAPI stains the cell nucleus, DiO stains the cell membrane, 7-FF-NPs are red under the microscope, D is the fluorescence quantitative image of C, averages±SEM, ****p<0.0001, **p<0.01;

[0016] Figure 6Toxicity of 7-FF-NPs to different cells, toxicity of A7-FF-NPs to HepG2 cells, laser power 0.5 W cm -2 , laser irradiation for 180 s, cytotoxicity of B7-FF-NPs to LO2 and B16-F10 cells, laser power 0.3 W cm -2 , laser irradiation time 90s;

[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 mouse anatomy after 24 hours. DETAILED DESCRIPTION

[0018] 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 the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] Example 1 Synthesis of TPP-G

[0020] 1.1 Experimental procedures

[0021] Weigh 113 mg of porphyrin (4-(10,15,20-triphenylporphyrin-5-yl)aniline) and 55 mg of diglycolic anhydride, dissolve in 4 mL of anhydrous DMF, and react for 48 hours. Add saturated brine to the reacted sample, extract with dichloromethane three times, then add anhydrous Na2SO4 to remove the water in the organic phase, and spin dry the organic solvent. Add 10 mL of dichloromethane to the spin-dried product, slowly add the solution dropwise to 100 mL of petroleum ether, mix well and let stand for 4 hours, and a large amount of flocculent precipitation is found. Filter the solution with filter paper, spin dry the solid precipitate, and obtain pure TPP-G.

[0022] 1.2 Experimental Results

[0023] The experimental results are as follows Figure 1 As shown, we react porphyrin with diglycolic anhydride and purify it to obtain the product TPP-G, as shown Figure 1 A shows that the mass spectrometer detected a molecular weight of 746Da. The large π-conjugated system of the porphyrin ring will form a strong shielding area in an external magnetic field. The two H atoms on the pyrrole N atom are generally around -3.0; the aromatic hydrogen around the porphyrin ring is generally 7.0-9.0. Figure 1 As shown in B, the peak with chemical shift δ=-2.92 is the two hydrogen atoms at f; the four hydrogen atoms at chemical shifts δ=4.34 and δ=4.36 are hydrogen atoms on the anhydride (a, b), and the chemical shift δ=10.46 is the hydrogen atom on the amide bond (c). According to the mass spectrometry and NMR spectra, 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 procedures

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

[0027] (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;

[0028] (2) Resin washing: Wash the activated resin three times with DMF and drain;

[0029] (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;

[0030] (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;

[0031] (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;

[0032] (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;

[0033] (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.

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

[0035] (9) 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%

[0036] 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 7-FF-TPP.

[0037] 2.2 Experimental Results

[0038] The experimental results are as follows Figure 2 As shown, 7-FF-TPP was purified by RP-HPLC at 415 nm, and the peak at 30 min was the target sample. Then we used mass spectrometry to detect 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 Nano-Self-Assembly

[0040] 3.1 Experimental procedures

[0041] The synthesized 7-FF-TPP was assembled into nanoparticles. The polypeptide 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 solution after centrifugation was 7-FF-NPs.

[0042] 3.2 Experimental Results

[0043] The experimental results are as follows Figure 3 As shown, 7-FF-TPP was self-assembled, the ratio of DMSO to water was 1:9, the assembly concentration was 2 mg / mL, and the assembly was dialyzed for 24 hours after 24 hours. The assembled nanoparticles were characterized by TEM and dynamic light scattering, as shown in Figure 3As shown, the assembled particles of 7-FF-NPs formed regular and uniform nanoparticles under transmission electron microscopy, with a diameter of about 50nm, and the hydrated particle size measured by dynamic light scattering was 231±89nm. The optical image showed that the solution was clear and transparent, and the solution was uniform.

[0044] Example 4 Photothermal conversion performance measurement

[0045] 4.1 Experimental procedures

[0046] The photothermal conversion performance was determined by irradiating 7-FF-NPs dispersions with different concentrations (50, 100, 200, 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 ) irradiated 7-FF-NPs (0.2 mg / mL) dispersion and plotted the temperature change curve. Finally, 7-FF-NPs were irradiated with laser "on-off" cycles for multiple times to test its photothermal stability.

[0047] 4.2 Experimental Results

[0048] In order to verify the photothermal capability of 7-FF-NPs, a 638 nm laser was used to irradiate the 7-FF-NPs suspension and the temperature change of the suspension was recorded. Figure 4 A and B show that 7-FF-NPs 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. -1 The temperature of 7-FF-NPs increases with the increase of laser power. -2 Increased to 1.5W cm -2 , the temperature rose by 29.9℃ after 10min of irradiation. Figure 4 B shows that the temperature of 7-FF-NPs at different concentrations increases with the increase of concentration. The laser power is 1.5 W cm -2 , 7-FF-NPs concentration increased from 50 μg mL -1 to 300 μg mL -1 , irradiation for 10 minutes, temperature difference is 20℃. Figure 4 C shows the highest temperature reached and the cooling curve. Further, Figure 4 D Through the continuous illumination cooling cycle process, the temperature of 7-FF-NPs did not show a significant decrease after five repeated cycles, and the results showed that it has good photothermal stability.

[0049] Example 5 Colocalization Experiment

[0050] 5.1 Experimental procedures

[0051] 1×10 5 B16-F10 or HepG2 cells 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. 7-FF-NPs were diluted to 10μM with serum-free medium. The drug was added to the cells and the cells were washed twice with PBS first, 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 with 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 7-FF-NPs at 450 nm.

[0052] 5.2 Experimental Results

[0053] Many literatures have reported that tumor cells highly express sialic acid, such as HepG2 and B16-F10 cells. We explored the targeting of 7-FF-NPs to HepG2 and B16-F10, and incubated the cells with 10μM 7-FF-NPs for 3h. To prove that 7-FF-NPs can target sialic acid on the cell membrane, we pre-incubated HepG2 and B16-F10 cells 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 7-FF-NPs showed red fluorescence. The results are shown in Figure 5 As shown in A, for HepG2 cells, the red fluorescence of the 7-FF-NPs+sialidase group was significantly inhibited, and the red fluorescence of the 7-FF-NPs group was very obvious. The red fluorescence and green fluorescence overlapped well. These results showed that 7-FF-NPs can specifically bind to sialic acid. Figure 5 As shown in B, the fluorescence intensity of the 7-FF-NPs+sialidase group differed by 5 times from that of the 7-FF-NPs group. The average fluorescence intensity data quantitatively demonstrated that 7-FF-NPs could specifically bind to sialic acid. Figure 5As shown in C, B16-F10 cells were used to prove that 7-FF-NPs can specifically bind to sialic acid. The red fluorescence of the 7-FF-NPs+sialidase group was relatively weak, while the red fluorescence of the 7-FF-NPs group was very obvious. The red fluorescence and green fluorescence overlapped well. The experimental results showed that 7-FF-NPs can specifically bind to sialic acid. Figure 5 As shown in D, the fluorescence intensity of the 7-FF-NPs+sialidase group and the 7-FF-NPs group differed by about 2 times, indicating that 7-FF-NPs can specifically bind to sialic acid. The co-localization experiment of HepG2 and B16-F10 cells proved that 7-FF-NPs can target sialic acid.

[0054] Example 6 Cytotoxicity Experiment

[0055] 6.1 Experimental procedures

[0056] After the cells were digested with trypsin, the culture medium was used to stop digestion, and the cells were centrifuged at 1000rpm for 3min. The cell density was adjusted, and 5000 cells were added to each well of the 96-well plate with 100μL. The added cells were cultured in the incubator for 24h, and then the culture medium was aspirated. The corresponding prepared drugs were added to each well, and each group was repeated for 3 wells. After the drug was incubated for 12h, the culture medium was aspirated and replaced with serum-free culture medium, and the cells were irradiated with the corresponding laser power at 638nm. After the irradiation, the cells were cultured in the incubator for 12h and then CCK-8 was added. 10μL was added to each well, and the incubation continued for 1-5h. Finally, the absorption value was measured at a wavelength of 450nm. The blank control was added with serum-free culture medium and the corresponding CCK-8. Cell viability (%) = (absorbance value of the experimental group - absorbance value of the blank control) / (absorbance value of the experimental control group - absorbance value of the blank control).

[0057] The protocol for the cytotoxicity experiment without laser irradiation is as follows: After the cells are digested with trypsin, the culture medium stops digestion, centrifuges at 1000rpm for 3min, adjusts the cell density, adds 100μL to each well of a 96-well plate to seed 5000 cells, and incubates the added cells in an incubator for 24h before aspirating the culture medium. The corresponding prepared drugs are added to each well, and each group is repeated for 3 wells. After the drug is incubated for 12h, the culture medium is aspirated and replaced with serum-free culture medium. After 12h of incubation in an incubator, CCK-8 is added, 10μL is added to each well, and the cells are incubated for 1-5h. Finally, the absorbance is measured at a wavelength of 450nm. The blank control is the addition of serum-free culture medium and the corresponding CCK-8.

[0058] 6.2 Experimental Results

[0059] Regarding the in vitro anti-tumor effect of 7-FF-NPs, two different cell types were used to test the therapeutic effects of 7-FF-NPs. Figure 6First, the therapeutic effect of 7-FF-NPs on HepG2 cells was tested. A series of concentrations were tested. In the dark reaction, even at a concentration of 20 μM, it had no toxicity to cancer cells. Under laser irradiation, IC 50 The cytotoxicity of 7-FF-NPs was tested on melanoma cells. Under laser irradiation, IC 50 is 0.945±0.339μM; Finally, we tested the cytotoxicity of normal LO2 cells. Under the same laser power as melanoma, the cytotoxic IC 50 The cytotoxicity results showed that 7-FF-NPs were more toxic to B16-F10 cells than to LO2 cells. The possible reason is that 7-FF-NPs have targeting properties to sialic acid, which leads to more enrichment of 7-FF-NPs on B16-10 cells. In short, 7-FF-NPs have great potential in treating tumors.

[0060] Example 7 Intratumoral Accumulation of Drugs

[0061] 7.1 Experimental procedures

[0062] 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 3 200 μL 7-FF-NPs (22.2 mg / kg) and 200 μL TPP (4.8 mg / kg) were injected into the mice through the tail vein, and the mice were scanned with a live imaging device at different time points. After 24 hours, the mice were dissected and the tissues and organs of the mice were imaged.

[0063] 7.2 Experimental Results

[0064] Fluorescence imaging of the distribution of 7-FF-NPs and TPP was performed after injection through the tail vein. Figure 7 A, 7-FF-NPs nanoparticles are increasingly enriched in the tumor site as time goes by. Enrichment can be seen at 4h, and it is very obvious at 12h. Until 24h, the fluorescence of 7-FF-NPs in the tumor site is still very strong. Figure 7 B. Next, we dissected mice that had run for 24 hours and analyzed the fluorescence intensity of various organs and tumors. 7-FF-NPs had strong fluorescence intensity in the liver and tumor sites. The results showed that 7-FF-NPs had good targeting in the body and were metabolized slowly in the body, which enabled them to have better therapeutic effects.

[0065] The above results show that the nanoparticles of the porphyrin-modified polypeptide of the present invention can be well targeted to the tumor site, can achieve tumor imaging and photothermal therapy, and have good application prospects 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 (AMDPRQSNRLYS), an aromatic amino acid GFFYGPG sequence and a porphyrin unit TPP, and form a stable nanoparticle structure through self-assembly for targeting melanoma cells.

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

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. coupling TPP-G with the polypeptide by Fmoc solid phase synthesis to form a 7-FF-TPP peptide chain; c. Dissolve the 7-FF-TPP peptide chain in a DMSO-water mixed solution and obtain nanoparticles through self-assembly.

4. Use of the self-assembled nanoparticles according to claim 1 in the preparation of targeted melanoma imaging.

5. The use of the self-assembled nanoparticles according to claim 1 in photothermal therapy of melanoma, characterized in that By irradiating the nanoparticles with laser, a local thermal effect on melanoma is achieved.

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