CN112007155B - Photothermal immune nanofiber, and preparation method and application thereof - Google Patents
Photothermal immune nanofiber, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a photothermal immune nanofiber, a preparation method and application thereof. The photothermal immune nanofiber comprises an immune active peptide and a photothermal reagent, wherein the molar ratio of the immune active peptide to the photothermal reagent is (4-50). The photothermal immune nanofiber is formed by mixing an immune active peptide solution and a photothermal reagent solution, and regulating and assembling a photothermal reagent by using immune active molecules under the interaction of hydrogen bonds, static electricity and the like. The photothermal immune nanofiber provided by the invention can simultaneously exert the advantages of photothermal treatment and immunoregulation, has the advantages of good photothermal conversion effect, mild and lasting immunoregulation effect and adjustable absorption wavelength, and can be used for preparing photothermal immune combined treatment medicines for tumors.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a photothermal immune nanofiber, and a preparation method and application thereof.
Background
In recent years, the tumor immunotherapy is rapidly developed, and a powerful weapon is provided for the treatment and the attack of malignant tumors. However, at present, the mild regulation effect can only exert 20% of treatment effect in clinical application, so that the combination of the immune regulation and other treatment modes becomes a main strategy for improving the clinical treatment effect. The photothermal therapy is a simple-to-operate and biologically safe therapy mode, and the treatment method utilizes a photothermal reagent to rapidly convert heat of absorbed light energy to cause local high temperature of tumor focus and realize necrosis or apoptosis of tumor cells. Based on this, the photothermal-immune combination shows unique advantages in the treatment of malignant tumors. On the one hand, phototherapy can achieve rapid ablation of a subject tumor; on the other hand, the immune regulation can play a long-acting inhibiting role in the processes of tumor later-stage diffusion, metastasis, recurrence and the like, and the two treatment modes are combined, so that the life cycle of a patient can be prolonged, the life quality of the patient can be improved, and even the tumor is expected to be radically treated.
The selection of photothermal agents in combined photothermal and immune therapy is one of the core problems. For example, the preparation and application of a biliverdin-based photothermal reagent (CN 109224073A) are disclosed, and the biliverdin organic micromolecules with biological endogenous and near infrared absorption verify the effectiveness of the biliverdin organic micromolecules in photothermal therapy, but for deeper tumor therapy, the treatment wavelength of the pigment molecule needs to be further modified to prolong.
Another central issue in combined photothermal and immune therapy is the choice and design of the immune molecules. In clinical preclinical experiments at present, the selection of immunoregulatory molecules is often protein macromolecules with complex structures, and the macromolecules have the problems of poor cell penetrability, high immunogenicity, unclear side effects, complex synthesis and the like in the application process. Therefore, the search for small molecules with high activity, clear structure and clear action path is a scientific problem to be solved urgently; in addition, for the coupling of immune molecules and other treatment modes, different active medicines are often encapsulated or injected in a front-back separated mode, and the lower medicine encapsulation concentration and the large auxiliary material dosage and the complicated injection and treatment modes limit further application.
Therefore, in the field, it is expected to search for photothermal and immune small molecules with high efficacy, and adopt a simple method to realize organic integration of the photothermal and immune small molecules, and finally achieve the purpose of combined enhancement of curative effects in the process of tumor treatment.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a photothermal immune nanofiber, a preparation method and application thereof. The preparation method provided by the invention is simple, the assembly efficiency is high, the prepared nano-fiber can simultaneously play the advantages of photothermal treatment and immunoregulation, and has the advantages of good photothermal conversion effect, mild and lasting immunoregulation effect and adjustable absorption wavelength.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a photothermal immune nanofiber comprising an immunologically active peptide and a photothermal agent in a molar ratio of 4-50 (e.g. can be 4.
The invention enables the immunoactive peptide and the photothermal reagent to be self-assembled to form a fibrous nano material through the interaction of hydrogen bonds, static electricity and the like between the immunoactive peptide and the photothermal reagent, has photothermal effect and immunocompetence at the same time, can exert the advantages of photothermal treatment and immunoregulation at the same time, has the advantages of good photothermal conversion effect and mild and lasting immunoregulation effect, and can be used for photothermal-immune combined treatment of tumors.
The nano-fiber provided by the invention enhances the photo-thermal conversion capability of photo-thermal reagent molecules at the treatment wavelength; meanwhile, the resistance of the drug to photodegradation is improved by the assembly effect. Moreover, the absorption wavelength (treatment penetration depth) of the nano-fiber can be adjusted by changing the proportion of the immune active peptide and the photothermal agent, so that the nano-fiber is suitable for treating tumors with different depths.
In the invention, if the molar ratio of the immunoactive peptide to the photothermal reagent is too low, the formation of a fiber structure is not facilitated; if the molar ratio of the immunoactive peptide to the photothermal agent is too high, the immunoactive peptide forms an assembly structure due to weak intermolecular interaction, which is not favorable for co-assembly with the photothermal agent.
In a preferred embodiment of the present invention, the immunologically active peptide contains 2 to 30 (e.g., 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, 20, 22, 25, 28, or 30) amino acid fragments.
Preferably, the immunologically active peptides are selected from any one of thymic peptides, histidine decarboxylase inhibitor peptides, antigenic peptides and immune checkpoint blocking peptides or a combination of at least two thereof. The immunologically active peptide is more preferably a thymosin peptide and/or a histidine decarboxylase inhibitor peptide, in view of the structure and effect thereof being clear.
Preferably, the thymosin is thymosin alpha 1 and/or thymopentin, and further preferably thymopentin.
Preferably, the histidine decarboxylase inhibitor peptide is a peptide comprising an N-terminal intact histidine-phenylalanine dipeptide structure.
Preferably, the histidine decarboxylase inhibitor peptide is selected from any one of or a combination of at least two of histidine-phenylalanine dipeptide, histidine-phenylalanine-leucine tripeptide and histidine-phenylalanine-isoleucine tripeptide, and is further preferably histidine-phenylalanine dipeptide.
As a preferred technical scheme of the invention, the absorption wavelength of the photo-thermal reagent is more than or equal to 680nm; for example, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, etc. may be mentioned.
The photo-thermal reagent with longer absorption wavelength is selected, which is beneficial to improving the treatment penetration depth and treating the tumor with deeper position.
Preferably, the photothermal agent is selected from any one of indocyanine green, near-infrared cyanine IR140, near-infrared cyanine IR806, melanin and melanin derivatives, or a combination of at least two thereof. Indocyanine green, which is an agent that has been approved by the U.S. food and drug administration for clinical imaging, is not only derived from living organisms but also has a longer near infrared absorption wavelength, and is an excellent photothermal molecule, and thus the photothermal agent is further preferably indocyanine green.
As a preferred technical scheme of the invention, the diameter of the photothermal immune nanofiber is 2-200nm; for example, it may be 2nm, 5nm, 10nm, 15nm, 20nm, 25nm, 30nm, 40nm, 50nm, 60nm, 80nm, 100nm, 120nm, 150nm, 180nm or 200 nm.
In a second aspect, the present invention provides a method for preparing the photothermal immune nanofiber as described in the first aspect, the method comprising the steps of:
(1) Preparing an immunologically active peptide solution and a photothermal reagent solution;
(2) Mixing the immunoactive peptide solution and the photothermal agent solution, wherein the molar ratio of the immunoactive peptide to the photothermal agent is 4-50;
(3) And (3) ageing the mixed solution obtained in the step (2) in a dark place to obtain the photothermal immune nanofiber.
The photothermal immune nanofiber is realized by regulating and assembling (mainly hydrogen bonds and electrostatic action) of the photothermal reagent by the immune active molecules, the method is simple to operate and high in assembling efficiency, the prepared nanofiber is good in photothermal conversion effect, and the immune regulation action is mild and lasting.
As a preferable embodiment of the present invention, the concentration of the photothermal reagent solution in step (1) is 0.1 to 4mg/mL, and may be, for example, 0.1mg/mL, 0.2mg/mL, 0.5mg/mL, 0.8mg/mL, 1mg/mL, 1.2mg/mL, 1.5mg/mL, 1.8mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, or 4 mg/mL; further preferably 0.1 to 2mg/mL.
Preferably, the immunoactive peptide is thymosin, and the solvent of the immunoactive peptide solution in step (1) is water.
Preferably, the immunoactive peptide is a histidine decarboxylase inhibitor peptide, and the solvent of the immunoactive peptide solution in the step (1) is an aqueous NaOH solution with a molar concentration of 0.01-1 mol/L.
As a preferable technical scheme of the invention, in the mixed solution in the step (2), the concentration of the photothermal reagent is 0.1-2mg/mL; for example, it may be 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.5mg/mL, 0.6mg/mL, 0.8mg/mL, 1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.5mg/mL, 1.6mg/mL, 1.8mg/mL, 2mg/mL or the like.
In the present invention, the concentration of the photo-thermal agent in the mixed solution is maintained within the above range, which contributes to the formation of nanofibers. If the concentration of the photothermal reagent is too low, the intermolecular hydrogen bonding acting force is low, and the immunoactive peptide and the photothermal reagent are not easy to assemble into fibers and may form a spheroidal structure; if the concentration of the photothermal agent is too high, self-aggregates or irregular aggregates may be formed due to the self-aggregation effect of the photothermal agent molecules.
The concentration of the immunoactive peptide in the mixed solution is not particularly limited, and the molar ratio of the immunoactive peptide to the photothermal agent is kept in the range of 4-50.
Preferably, the immunoactive peptide is thymosin, and the pH of the mixed solution in the step (2) is 6.8-7.3; for example, it may be 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, etc.
Preferably, the immunoactive peptide is a histidine decarboxylase inhibitor peptide, and the pH of the mixed solution in the step (2) is 6.5-7.5; for example, it may be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, etc.
As a preferred embodiment of the present invention, the temperature of the aging in the step (3) is 0 to 25 ℃ and may be, for example, 0 ℃,1 ℃,2 ℃, 3 ℃, 4 ℃, 5 ℃,8 ℃,10 ℃, 12 ℃, 15 ℃, 18 ℃, 20 ℃, 22 ℃ or 25 ℃ or the like; further preferably 0 to 4 ℃.
Preferably, the aging time in step (3) is 4-72h, such as 4h, 5h, 6h, 8h, 10h, 12h, 15h, 20h, 25h, 30h, 35h, 40h, 50h, 60h, 70h or 72 h; further preferably 12 to 24 hours.
In a third aspect, the present invention provides a use of the photothermal immune nanofiber as described in the first aspect, wherein the photothermal immune nanofiber is used for preparing a combined photothermal immune therapeutic drug for tumors, preferably for preparing a combined photothermal immune therapeutic drug for esophageal tumor, bladder tumor, pancreatic tumor or melanoma.
Compared with the prior art, the invention has the following beneficial effects:
1. the nano-fiber provided by the invention enhances the photo-thermal conversion capability of photo-thermal reagent molecules at the treatment wavelength; meanwhile, the assembly effect improves the resistance of the drug to photodegradation. Moreover, the absorption wavelength of the nanofiber can be adjusted by changing the proportion of the immunoactive peptide and the photothermal agent, so that the treatment penetration depth can be adjusted.
2. The photothermal immune nanofiber provided by the invention has photothermal effect and immunocompetence at the same time, wherein the photothermal agent can rapidly kill a main tumor, and the immunocompetent component has long-acting regulation effects on elimination of residual tumor cells and inhibition of tumor recurrence and metastasis after photothermal ablation of the main tumor, and has unique advantages on treating tumors which are deep in body position and easy to recur and metastasize.
3. The preparation method adopted by the invention is assembly regulation, the whole preparation process is green and simple, no auxiliary material component is required to be added, the active component completely participates in the structure formation, and the efficacy is high.
Drawings
FIG. 1 is a schematic representation of the thymopentin-indocyanine green nanofiber solution obtained in example 1;
FIG. 2 is a TEM image of the thymopentin-indocyanine green nanofibers obtained in example 2;
FIG. 3 is a graph showing an ultraviolet-visible absorption spectrum of a sample obtained in example 3;
FIG. 4 is a graph showing photothermal conversion of the histidine decarboxylase inhibitor peptide-indocyanine green nanofiber, indocyanine green monomer and control group obtained in example 4;
FIG. 5 is a graph showing the photo-thermal transformation of histidine decarboxylase inhibitor peptide-indocyanine green nanofibers, indocyanine green monomers, and saline control groups obtained in example 4;
fig. 6 is a fluorescent imaging photograph of a pancreatic cancer mouse model treated with thymopentin-indocyanine green nanofibers obtained in example 1, indocyanine green monomers, and saline control groups;
fig. 7 is a graph showing the immunocytokine content of a melanoma mouse model treated with histidine decarboxylase inhibitor peptide-indocyanine green nanofibers, indocyanine green monomers, and saline control groups obtained in example 4;
FIG. 8 is a TEM micrograph of histidine decarboxylase inhibitor peptide-NIR cyanine IR806 nanofibers obtained in example 8;
FIG. 9 is a TEM image of a spheroid-like assembly structure formed by thymopentin-indocyanine green obtained in comparative example 1;
FIG. 10 is a TEM photograph of irregular aggregate structure formed by thymopentin-indocyanine green obtained in comparative example 2;
fig. 11 is a schematic diagram of the histidine decarboxylase inhibitor peptide-indocyanine green mixed sediment solution obtained in comparative example 3.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
The embodiment provides a thymopentin-indocyanine green nanofiber, which is prepared by the following method:
respectively preparing thymopentin aqueous solution with volume mass concentration of 8mg/mL and indocyanine green aqueous solution with volume mass concentration of 0.4 mg/mL; adding 500 mu L of thymopentin aqueous solution into 500 mu L of indocyanine green aqueous solution, mixing and uniformly mixing, and adjusting the pH value of the mixed solution to 7.2 by using 0.1mol/L NaOH aqueous solution; and (3) ageing for 24h in a dark place at 4 ℃ to obtain the thymopentin-indocyanine green nanofiber, wherein the molar ratio of the thymopentin to the indocyanine green is 23.3.
Fig. 1 is an optical photograph of the thymopentin-indocyanine green nanofiber solution obtained in the present example, and it can be seen from fig. 1 that the obtained nanofibers are uniformly dispersed and have good opalescence.
Example 2
The embodiment provides a thymopentin-indocyanine green nanofiber, which is prepared by the following method:
respectively preparing a thymopentin aqueous solution with the volume mass concentration of 16mg/mL and an indocyanine green aqueous solution with the volume mass concentration of 4 mg/mL; mixing 500 mu L of thymopentin aqueous solution and 500 mu L of indocyanine green aqueous solution, and adjusting the pH value of the mixed solution to 7.2 by using 1mol/L NaOH aqueous solution; and (3) ageing for 12h in a dark place at 25 ℃ to obtain the thymopentin-indocyanine green nanofiber, wherein the molar ratio of the thymopentin to the indocyanine green is 4.7.
The thymopentin-indocyanine green nanofiber provided in this embodiment is characterized by a transmission electron microscope, and as a result, as shown in fig. 2, it can be seen that the nanofiber has a uniform morphology, and a fiber diameter is less than 50nm.
Example 3
Respectively preparing 8mg/mL thymopentin aqueous solution and 0.2mg/mL indocyanine green aqueous solution;
sample 1: mixing 500 mu L of thymopentin aqueous solution and 500 mu L of indocyanine green aqueous solution, and adjusting the pH value of the mixed solution to 7.2 by using 0.1mol/L NaOH aqueous solution; after 24h of light-shielding aging at 4 ℃, samples 1 of thymopentin 4 mg/mL-indocyanine green 0.1mg/mL (thymopentin to indocyanine green molar ratio of 46.5.
Sample 2: mixing 20 mu L of thymopentin aqueous solution and 500 mu L of indocyanine green aqueous solution, adjusting the pH value of the mixed solution to 7.2 by using 0.1mol/L NaOH aqueous solution, and fixing the volume to 1mL by using ultrapure water and uniformly dispersing; aging in the dark at 25 ℃ for 12h to obtain sample 2 with thymopentin 0.16 mg/mL-indocyanine green 0.1mg/mL (thymopentin to indocyanine green molar ratio of 1.9;
sample 3: mu.L of an aqueous indocyanine green solution was mixed with 500. Mu.L of ultrapure water to obtain a control group of an aqueous indocyanine green solution of 0.1 mg/mL.
The ultraviolet-visible absorption spectrum characterization test is performed on 3 groups of samples obtained in this example, and the spectra are shown in fig. 3, it can be seen that, in sample 2, the molar ratio of thymic gland pentapeptide to indocyanine green is < 2; while the molar ratio of the thymine pentapeptide to the indocyanine green in the sample 1 is 46.5, the spectrum of the indocyanine green shows obvious red shift, which indicates that a fibrous assembly is formed, and the red-shifted wavelength is beneficial to improving the subsequent treatment penetration depth.
Example 4
The embodiment provides a peptide-indocyanine green nanofiber as a histidine decarboxylase inhibitor, and the preparation method comprises the following steps:
preparing an indocyanine green aqueous solution with the volume mass concentration of 1 mg/mL; preparing a histidine-phenylalanine dipeptide solution with the volume mass concentration of 8mg/mL by taking a NaOH aqueous solution with the molar concentration of 1mol/L as a solvent; mixing and uniformly dispersing 500 mu L of histidine-phenylalanine dipeptide solution and 500 mu L of indocyanine green aqueous solution, and adding 1mol/L of HCl aqueous solution to adjust the pH value of the mixed solution to 7.2; and (3) aging for 24h at 4 ℃ in the dark to obtain the histidine decarboxylase inhibitor peptide-indocyanine green nanofiber, wherein the molar ratio of the histidine decarboxylase inhibitor peptide/indocyanine green is 20.9.
mu.L of an aqueous indocyanine green solution was mixed with 500. Mu.L of ultrapure water to obtain a control group of 0.5mg/mL aqueous indocyanine green solution.
The histidine decarboxylase inhibitor peptide-indocyanine green nanofiber obtained in the embodiment was subjected to photothermal testing, an aqueous solution of indocyanine green and ultrapure water were used as a control group, and the wavelength of 808nm and the power of 1W/cm were selected 2 The laser light continuously irradiates the three groups of samples for 10min respectively, and a temperature rise curve is drawn, and as shown in fig. 4, it can be seen that the photothermal conversion of the indocyanine green molecules is not affected after the indocyanine green is adjusted by the histidine-phenylalanine dipeptide to form the nanofiber.
Example 5
The histidine decarboxylase inhibitor peptide-indocyanine green nanofiber obtained in example 4 was subjected to ultrafiltration concentration: adding 200 mu L of nanofiber solution into a 10kDa ultrafiltration tube, performing 1000rpm and 10min ultrafiltration concentration, wherein the volume of the concentrated nanofiber is about 100 mu L, and performing a living body level photothermal conversion effect test.
The live tumor model was established as follows: c57BL/6 Male mouse in situ pancreatic cancer model, cultured and resuspended Pan02 mouse pancreatic cancer cells were expressed as 1X 10 6 The density of individual cells was inoculated at the mouse pancreatic site when the tumor volume was 50. + -. 10mm 3 At the time of injection, the mice were injected with the drugs in situ (50. Mu.L after ultrafiltration concentration)Histidine decarboxylase inhibitor peptide-indocyanine green nanofiber, 50 mu L of indocyanine green monomer solution with concentration of 1mg/mL and 50 mu L of normal saline, and after the drug is diffused for 25min, the mouse is irradiated by laser (808nm, 0.6W/cm) 2 10 min) while monitoring the mouse in situ tumor temperature using a thermal infrared imager.
The test results are shown in fig. 5: the nanofiber formed by using the histidine-phenylalanine dipeptide to regulate the indocyanine green has a better in-vivo temperature rise effect than an indocyanine green monomer group, because the formed fiber structure can enhance the absorption of the indocyanine green at a laser position of 808nm, and the formed fiber structure can enhance the retention concentration of a drug at a tumor lesion, so that the nanofiber has higher efficient photo-thermal conversion efficiency.
Example 6
The thymopentin-indocyanine green nanofibers obtained in example 2 were concentrated by ultrafiltration: adding 200 μ L of nanofiber into 10kDa ultrafiltration tube, performing 1000rpm and 10min ultrafiltration concentration, wherein the volume of the concentrated nanofiber is about 100 μ L, and performing in-vivo photothermal therapy effect test.
The live tumor model was established as follows: c57BL/6 male mouse in situ pancreatic cancer model, pan02 mouse pancreatic cancer cells are marked by Luciferase (Luciferase, luc) to obtain Pan02-Luc cells, and the cultured and resuspended cells are expressed according to 1 × 10 6 The density of individual cells was inoculated at the mouse pancreatic site when the tumor volume was 50. + -. 10mm 3 When the preparation method is used, the mice are subjected to laparotomy and in-situ drug injection (50 mu L of thymopentin-indocyanine green nanofiber solution subjected to ultrafiltration concentration, 50 mu L of 0.2mg/mL indocyanine green monomer solution and 50 mu L of normal saline), and after the drugs are diffused for 25min, the mice are subjected to laser irradiation (808nm, 0.6W/cm) 2 10 min) and mice were monitored for tumor changes at stage 21d. The change of the size of the mouse tumor can be judged by the fluorescence intensity, and the injected potassium luciferin is used as a substrate, and the potassium luciferin emits light under the oxidation action of ATP and luciferase, so that the change of the tumor intensity is used as a mark.
The test results are shown in fig. 6: the fluorescence of the mouse tumor of the thymopentin-indocyanine green nanofiber group disappears, the fluorescence intensity of the indocyanine green monomer group is centered, and the fluorescence intensity of the tumor of the physiological saline control group is strongest, so that the tumor inhibition effect of the nanofibers is the best, and the reasons include the enhancement of the photothermal conversion effect and the anti-tumor immunoregulation of later organisms.
Example 7
The immunization effect of the histidine decarboxylase inhibitor peptide-indocyanine green nanofiber obtained in example 4 was verified, and indocyanine green monomer and PBS phosphate buffer solution at the same concentration were used as a control group.
The live tumor model was established as follows: establishing a female mouse subcutaneous melanoma model, and culturing and resuspending B16-F10 mouse melanoma cells according to 5 multiplied by 10 6 The density of each cell is inoculated on the left lower back of the mouse, and the tumor volume is 100 +/-30 mm 3 The photothermal immunotherapy of mice was performed at the time: injecting 200 μ L volume of medicine (histidine decarboxylase inhibitor peptide-indocyanine green nanofiber solution, indocyanine green monomer solution, PBS phosphate buffer solution) into tail vein, and irradiating tumor with laser (808nm, 0.4/W cm) after 8 hr 2 10 min), taking the spleen of the mouse after 21d, performing erythrocyte lysis, centrifuging, cleaning, dispersing the spleen cells in a culture medium, incubating for 24h, performing specific antibody staining after the incubation is finished, and testing the content of interleukin 4 and interferon gamma by using a flow cytometer, wherein the interleukin 4 is used for indicating CD4 + Activation of cells, interferon gamma for CD8 + Activation of the cells.
The test results are shown in fig. 7: the interleukin 4 and the gamma interferon of the histidine decarboxylase inhibitor peptide-indocyanine green nanofiber group are higher than those of the other two groups of control groups, so that the immune system of a mouse injected with the nanofiber group is activated, and a better anti-tumor treatment effect is exerted.
Example 8
This example provides a histidine decarboxylase inhibitor peptide-near infrared cyanine IR806 nanofiber, which was prepared as follows:
preparing a near-infrared cyanine IR806 aqueous solution with the volume mass concentration of 2mg/mL; preparing a histidine-phenylalanine-isoleucine tripeptide solution with the volume mass concentration of 4mg/mL by using a NaOH aqueous solution with the molar concentration of 0.1mol/L as a solvent; mixing and uniformly dispersing 500 mu L of histidine-phenylalanine-isoleucine tripeptide solution and 500 mu L of near-infrared cyanine IR806 aqueous solution, and adding 0.1mol/L of HCl aqueous solution to adjust the pH value of the mixed solution to 7.2; and aging the mixture at 4 ℃ in the dark for 24 hours to obtain the histidine decarboxylase inhibitor peptide-near infrared cyanine IR806 nanofiber, wherein the molar ratio of the histidine decarboxylase inhibitor peptide to the near infrared cyanine IR806 is 3.4.
The histidine decarboxylase inhibitor peptide-near infrared cyanine IR806 nanofiber provided in this example was characterized by transmission electron microscopy, and the results are shown in fig. 8, which shows that the nanofiber has uniform morphology and a fiber diameter of less than 50nm.
Comparative example 1
Respectively preparing thymopentin aqueous solution with volume mass concentration of 2mg/mL and indocyanine green aqueous solution with volume mass concentration of 0.1 mg/mL; adding 500 mu L of thymopentin aqueous solution into 500 mu L of indocyanine green aqueous solution, mixing and uniformly mixing (the concentration of the indocyanine green is 0.05 mg/mL), and adjusting the pH value of the mixed solution to 7.2 by using 0.1mol/L NaOH aqueous solution; and (3) ageing for 24h at 4 ℃ in a dark place to obtain a thymopentin-indocyanine green mixed solution, wherein the molar ratio of thymopentin to indocyanine green is 23.3.
The thymopentin-indocyanine green mixed solution provided in comparative example 1 was characterized by a transmission electron microscope, and the result is shown in fig. 9, it can be seen that a sphere-like assembly structure is formed and a fibrous structure is not formed because the concentration of indocyanine green is lower than 0.1mg/mL, the hydrogen bonding sites with thymopentin are low, and the hydrophobic effect is strong.
Comparative example 2
Respectively preparing 24mg/mL thymopentin aqueous solution and 6mg/mL indocyanine green aqueous solution; adding 500 mu L of thymopentin aqueous solution into 500 mu L of indocyanine green aqueous solution, mixing and uniformly mixing (the concentration of the indocyanine green is 3 mg/mL), and adjusting the pH value of the mixed solution to 7.2 by using 0.1mol/L NaOH aqueous solution; and (3) aging for 12h at 25 ℃ in a dark place to obtain a thymopentin-indocyanine green mixed solution, wherein the molar ratio of thymopentin to indocyanine green is 4.7.
The thymopentin-indocyanine green mixed solution provided in comparative example 2 was characterized by a transmission electron microscope, and as a result, as shown in fig. 10, it can be seen that due to the fact that the indocyanine green concentration is too high (> 2 mg/mL), the self-polymerization effect is obvious, and most of the dispersed system is irregular aggregates, and no fiber structure is formed.
Comparative example 3
Preparing an indocyanine green aqueous solution with the volume mass concentration of 0.2 mg/mL; preparing a histidine-phenylalanine-leucine tripeptide solution with the volume mass concentration of 10mg/mL by taking a NaOH aqueous solution with the molar concentration of 0.1mol/L as a solvent; mixing and uniformly dispersing 500 mu L of histidine-phenylalanine-leucine tripeptide solution and 500 mu L of indocyanine green aqueous solution, and adding 0.1mol/L HCl aqueous solution to adjust the pH value of the mixed solution to 7.2; and aging the mixture at 4 ℃ in the dark for 24 hours to obtain a histidine decarboxylase inhibitor peptide-indocyanine green mixed solution, wherein the molar ratio of the histidine decarboxylase inhibitor peptide/the indocyanine green is 125.4.
Fig. 11 is an optical photograph of a mixed solution of histidine decarboxylase inhibitor peptide and indocyanine green obtained in this comparative example, and it can be seen from fig. 11 that, due to the excessively high molar ratio of the histidine decarboxylase inhibitor peptide to the photothermal agent, the peptide molecules form precipitates due to their weak interactions, such as hydrophobic interaction, which are not favorable for the co-assembly with the photothermal agent.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.
Claims (21)
1. A photothermal immune nanofiber, wherein the photothermal immune nanofiber comprises an immunologically active peptide and a photothermal agent, and the molar ratio of the immunologically active peptide to the photothermal agent is 4-50;
the photothermal agent is selected from any one or a combination of at least two of indocyanine green, near-infrared cyanine IR140 or near-infrared cyanine IR 806;
the immune active peptide is selected from thymic peptide and/or histidine decarboxylase inhibitor peptide;
the photothermal immune nanofiber is prepared by the following method, and the method comprises the following steps:
(1) Preparing an immunologically active peptide solution and a photothermal reagent solution;
(2) Mixing the immune active peptide solution and the photothermal reagent solution, wherein the molar ratio of the immune active peptide to the photothermal reagent is 4-50;
(3) Aging the mixed solution obtained in the step (2) in a dark place to obtain photothermal immune nanofibers;
the concentration of the photothermal reagent solution in the step (1) is 0.1-4 mg/mL;
in the mixed solution in the step (2), the concentration of the photo-thermal reagent is 0.1-2mg/mL.
2. The photothermal immune nanofiber according to claim 1, wherein the immunologically active peptide comprises 2-30 amino acid fragments.
3. The photothermal immune nanofiber according to claim 1, wherein the thymosin peptide is thymosin alpha 1 and/or thymopentin.
4. The photothermal immune nanofiber according to claim 3, wherein the thymosin peptide is thymopentin.
5. The photothermal immune nanofiber according to claim 1, wherein said histidine decarboxylase inhibitor peptide is a peptide comprising a histidine-phenylalanine dipeptide structure having an N-terminal integrity.
6. The photothermal immune nanofiber according to claim 5, wherein the histidine decarboxylase inhibitor peptide is selected from any one of or a combination of at least two of histidine-phenylalanine dipeptide, histidine-phenylalanine-leucine tripeptide, and histidine-phenylalanine-isoleucine tripeptide.
7. The photothermal immune nanofiber according to claim 6, wherein said histidine decarboxylase inhibitor peptide is histidine-phenylalanine dipeptide.
8. The photothermal immune nanofiber according to claim 1, wherein the photothermal agent has an absorption wavelength of 680nm or more.
9. The photothermal immune nanofiber according to claim 1, wherein the photothermal agent is indocyanine green.
10. The photothermal immune nanofiber according to claim 1, wherein the diameter of the photothermal immune nanofiber is 2-200 nm.
11. A method for preparing the photothermal immune nanofiber according to any one of claims 1 to 10, wherein the preparation method comprises the steps of:
(1) Preparing an immunologically active peptide solution and a photothermal reagent solution;
(2) Mixing the immune active peptide solution and the photothermal reagent solution, wherein the molar ratio of the immune active peptide to the photothermal reagent is 4-50;
(3) Aging the mixed solution obtained in the step (2) in a dark place to obtain the photothermal immune nanofiber;
the concentration of the photothermal reagent solution in the step (1) is 0.1-4 mg/mL;
in the mixed solution in the step (2), the concentration of the photo-thermal reagent is 0.1-2mg/mL.
12. The method of claim 11, wherein the concentration of the photothermal agent solution in step (1) is 0.1-2mg/mL.
13. The method of claim 11, wherein the immunoactive peptide is thymosin and the solvent of the solution of the immunoactive peptide in step (1) is water.
14. The method of claim 11, wherein the immunoactive peptide is a histidine decarboxylase inhibitor peptide, and the solvent of the solution of the immunoactive peptide in step (1) is an aqueous solution of NaOH with a molar concentration of 0.01 to 1 mol/L.
15. The method according to claim 11, wherein the immunoactive peptide is thymosin, and the pH of the mixed solution in the step (2) is 6.8 to 7.3.
16. The method of claim 11, wherein the immunologically active peptide is a histidine decarboxylase inhibitor peptide, and the pH of the mixed solution in step (2) is 6.5-7.5.
17. The production method according to claim 11, wherein the temperature of the aging in the step (3) is 0 to 25 ℃.
18. The method according to claim 16, wherein the temperature of the aging in the step (3) is 0 to 4 ℃.
19. The method according to claim 11, wherein the aging time in the step (3) is 4 to 72 hours.
20. The method of claim 18, wherein the aging time in step (3) is 12 to 24 hours.
21. Use of the photothermal immune nanofiber according to any one of claims 1 to 10 for the preparation of a combined photothermal immune therapy for pancreatic tumor or melanoma.
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| CN111544588A (en) * | 2020-05-12 | 2020-08-18 | 中国科学院过程工程研究所 | Immunity active peptide-biliverdin conjugate, preparation method and application thereof |
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| CN111544588A (en) * | 2020-05-12 | 2020-08-18 | 中国科学院过程工程研究所 | Immunity active peptide-biliverdin conjugate, preparation method and application thereof |
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