TWI718528B - Method for manufacturing immune checkpoint blockade particles and use of the immune checkpoint blockade particles - Google Patents

Abstract

A method for manufacturing an immune checkpoint blockade particles includes mixing a plurality of functional monomers, a template peptide and a solvent to form a molecularly imprinted polymer by embedding the template peptide when polymerizing the plurality of functional monomers in the solvent. A plurality of recognition sites corresponding to the template peptide are formed on the surface of the molecularly imprinted polymer. The template peptide on the surface of the molecularly imprinted polymer is then removed. Moreover, the template peptide includes an amino acid sequence set forth as SEQ ID NO: 1, 2, 3, 5, 6 or 7. Accordingly, the immune particle can block the specific interaction of programmed cell-death protein 1 (PD-1) and programmed cell-death ligand 1 (PD-L1), and thus can be applied to immunotherapy.

Description

Preparation method of immune blocking particles and application of the immune blocking particles

The present invention relates to a method for preparing particles, especially a method for preparing immune blocking particles. The present invention also relates to the use of the immune blocking particles.

Cancer immunotherapy refers to the method of treating cancer by artificially stimulating the immune system to improve the immune system's ability to fight cancer. For example, the specific binding of programmed cell-death protein 1 (PD-1) and programmed cell-death ligand 1 (PD-L1) can be Suppresses the immune checkpoint pathway related to the activation of T cells.

Therefore, if a drug specific to PD-1 can be developed, it can be used as an immune checkpoint inhibitor (ICI), which can block the self-protection mechanism of cancer cells and make natural killer cells (natural killer cells). Cells, NK cells for short) are no longer regulated by immune checkpoints and can restore their ability to fight cancer cells.

In order to solve the above-mentioned problems, the purpose of the present invention is to provide a method for preparing immune blocking particles, which is used to prepare immune blocking particles that can be applied to cancer immunotherapy.

The second objective of the present invention is to provide a method for preparing immune blocking particles, which is used to prepare immune blocking particles that can be applied to photodynamic therapy.

Another object of the present invention is to provide a method for preparing immune blocking particles, which can prepare immune blocking particles that are easy to separate and purify.

Another object of the present invention is to provide a use of immune blocking particles for preparing drugs for treating liver cancer.

The preparation method of immune blocking particles of the present invention includes: mixing several functional monomers, a template peptide, a photosensitizer, a magnetic particle and a solvent, so that the several functional monomers are in the solvent During polymerization, the template peptide, the photosensitizer, and the magnetic particles are coated to form a molecular rubbing polymer, wherein each of the functional monomers is polyvinyl vinyl alcohol, and the photosensitizer can be a part Anthocyanins, the magnetic susceptibility of the magnetic particles is greater than ±0.1emu/g, and the surface of the magnetic particles is modified with (3-aminopropyl)triethoxysilane; and the molecular rubbing polymer is removed The template peptide on the surface to obtain the immune blocking particle, the surface of the immune blocking particle has a number of identification holes corresponding to the template peptide; wherein, the template peptide contains as shown in SEQ ID NO: 4 or 8. Show the amino acid sequence.

Accordingly, the method for preparing immune blocking particles of the present invention can obtain immune blocking particles with identification holes capable of recognizing PD-1 protein on the surface. Therefore, when the immune blocking particles are administered to a desired individual, that is, It can block the self-protection mechanism of cancer cells (that is, the specific combination of programmed cell death receptor 1 and programmed cell death ligand 1) in the body of the desired individual, and promote the activity of natural killer cells. The broken particles can be applied to cancer immunotherapy, which is the effect of the present invention. In addition, the several functional monomers can be polymerized to form a stable structure, and the immune blocking particle has good biocompatibility, thereby avoiding the immune blocking particle from being administered to the organism. The body has an adverse reaction. Furthermore, after the immune blocking particles are administered to the desired individual, a light energy can be administered to the desired individual, so that the photosensitizer is exposed to light with a wavelength of 540nm. Activate to effectively produce free radicals that can induce phototoxicity and promote the death of cancer cells. In addition, the worker can separate and purify the immune blocking particles by applying an external magnetic field (for example, using magnets for adsorption), so as to improve the convenience of separation and purification of the immune blocking particles.

In the preparation method of immune blocking particles of the present invention, each of the functional monomers is polyvinyl vinyl alcohol with a ethylene mole percentage of 5-50 mol%. In this way, the immune blocking particles have a better rubbing effect.

In the method for preparing immune blocking particles of the present invention, the solvent is water. In this way, the production cost of the immune blocking particles can be reduced.

The use of the immune blocking particles of the present invention is applied to the preparation of drugs for the treatment of liver cancer, wherein the immune blocking particles are prepared by the method for preparing immune blocking particles as described above.

Accordingly, the immune blocking particles of the present invention can block the specific binding of PD-1 and PD-L1, and thus can be used as an active component of cancer immunotherapy, which is the effect of the present invention.

The use of the immune blocking particles of the present invention, wherein the immune blocking particles are administered to a desired individual in the manner of oral administration, intravenous injection, intramuscular injection, and intraperitoneal injection. In this way, by adjusting the route of administration, the immune blocking particles can effectively enter the body of the desired individual.

The use of the immune blocking particle of the present invention, wherein the immune blocking particle is administered to the desired individual at a dose of 0.001 to 1000 mg per kilogram of the desired individual per week. In this way, by adjusting the dosage, the immune blocking particle can effectively exert its biological activity and promote the death of cancer cells.

The use of the immune blocking particles of the present invention, wherein the immune blocking particles are mixed with natural killer cells and then jointly administered to the desired individual. In this way, when the immune blocking particles and natural killer cells are mixed, the natural killer cells can be activated first, so that the activated natural killer cells administered to the desired individual can quickly exert their biological activity and promote the death of cancer cells .

The use of the immune blocking particle of the present invention, wherein after the immune blocking particle is administered to the desired individual, the affected area of the desired individual is irradiated with light with a wavelength of 460 to 580 nm for 0.01 to 30 minutes. In this way, the photosensitizer can be activated by exposure to light to generate free radicals that can induce phototoxicity and promote the death of cancer cells.

[Figure 1a] In experiment (A), a histogram of the average particle size of the nanoparticles in groups A1 to A3.

[Figure 1b] In experiment (A), the particle size distribution diagram of the A0~A3 groups of nanoparticles.

[Picture 2] In experiment (B), the histogram of the adsorption capacity of the template peptides of the B0~B1 groups of nanoparticles.

[Figure 3] In the experiment (C), the change curve of the adsorption amount of the template peptide of the immune blocking particle mAIP.

[Figure 4] In the experiment (D), the change curve of the adsorption amount of the template peptide of the immune blocking particle mAIP.

[Figure 5] In experiment (E), the change curve of the template peptide adsorption capacity of the E0~E1 group of nanoparticles.

[Figure 6] In experiment (F), the magnetic flux variation curve of the F0~F1 group of nanoparticles.

[Figure 7] In experiment (G), the histogram of the specific surface area of the G1~G2 group of nanoparticles.

[Figure 8] In experiment (H), the cell viability of H1~H4 groups of liver cancer cells.

[Figure 9a] In experiment (I), the cell survival rate of liver cancer cells in groups I1-0~I1-2.

[Figure 9b] In experiment (I), the cell survival rate of liver cancer cells in groups I2-0~I2-2.

[Figure 10] In experiment (J), in the J0~J2 liver cancer cells, the main proteins in the apoptosis pathway are caspase-8 (caspase-8, referred to as CASP8) and caspase -3(caspase- 3. The relative gene expression level of the nuclear factor activated B cell kappa light chain enhancer p105 subunit (nuclear factor NF-kappa-B p105 subunit, referred to as NFKB1).

[Figure 11] In experiment (K), the cell survival rate of liver cancer cells in the K1 and K2 groups.

In order to make the above and other objects, features and advantages of the present invention more obvious and understandable, the following is a detailed description of the preferred embodiments of the present invention with the accompanying drawings: Immunization of one embodiment of the present invention The preparation method of blocking particles is to make several functional monomers and a template peptide together to form a molecular rubbing polymer, and then removing the template polymer on the surface of the molecular rubbing polymer. The peptide is obtained.

In detail, the functional monomers can be polymerized, and the stencil can be coated while polymerizing to form the molecular rubbing polymer. The surface of the molecular rubbing polymer will form several identifications. Recognition site, each recognition site corresponds to the three-dimensional structure of the template peptide, so the surface of the immune blocking particle obtained after removing the template peptide located on the surface of the molecularly printed polymer is Will have this identification hole. For example, the functional monomer can be poly(ethylene-co-vinyl alcohol) (EVAL), chitin, poly(hydroxymethyl 3,4-ethylenedioxythiophene) ( poly(hydroxymethyl 3,4-ethylene dioxythiophene, poly(aniline-co-metanilic acid), poly(p-phenylene sulfide)), poly(thiophene) ). In this embodiment, the functional monomer is polyvinyl ethyl vinyl alcohol, and its ethylene molar percentage is between 5-50 mol%, which can form a stable structure and has good biocompatibility .

In order to enable the immune blocking particle to be used as an immune checkpoint inhibitor, in this embodiment, the template peptide can be a partial fragment of the PD-1 protein, so that the surface of the immune blocking particle can identify the PD-1 protein. Identify the hole position, and then administer the immune blocking particle to a desired individual At this time, the self-protection mechanism of cancer cells (that is, the specific combination of PD-1 and PD-L1) can be blocked, so that natural killer cells are no longer regulated by immune checkpoints. In the first embodiment of the present invention, the template peptide is a partial fragment of human PD-1 protein, which contains the amino acid sequence shown in SEQ ID NO: 1, 2 or 3. Preferably, the template The peptide is composed of the amino acid sequence shown in SEQ ID NO: 1, 2, 3 or 4; in the second embodiment of the present invention, the template peptide is a partial fragment of the mouse PD-1 protein , Which contains the amino acid sequence shown in SEQ ID NO: 5, 6 or 7, preferably, the template peptide is composed of the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8. Constituted.

Preferably, the functional monomers and the template peptide can be added to a solvent, so that the functional monomers are polymerized in the solvent while coating the template peptide, for example, At a temperature of 0-25°C, the mixture containing the functional monomers and the template peptide is added to the solvent, and stirred with a magnet for 1-30 minutes to facilitate the formation of the molecular rubbing polymer. The solvent can provide a good polymerization environment. For example, the solvent can be water, isopropanol, or a mixed solvent containing water and isopropanol.

In addition, after the molecular printing polymer is formed, the molecular printing polymer can be cleaned with a cleaning solution to expose the identification holes on the surface of the molecular printing polymer, and the immune blocking particles can be obtained. For example, the cleaning solution can be acetone, ethanol, methanol, water, or a mixed liquid containing the foregoing solvents.

It is worth noting that the several functional monomers can also be coated with a photosensitizer while polymerizing, so that the prepared immune blocking particles contain the photosensitizer, and the photosensitizer can be activated by light to produce Induces phototoxic free radicals and promotes the death of cancer cells.

Specifically, the worker can mix the functional monomers, the template peptide, the photosensitizer, and the solvent, so that when the functional monomers are polymerized in the solvent, the template The peptide and the photosensitizer are coated to form the molecular rubbing polymer together. For example, the photosensitizer can be hematoporphyrin, metalloporphyrin, porphycene, pheophorbide, purpurin, chlorin ( chlorin, protoporphrin, phthalocyanine and other porphyrin photosensitizers or psoralean, anthracene, chalacogenopyrylium dye ), nitro-bis-methane rearrangement (ADPM), cyanine, phenothiazinium dye and other non-porphyrin photosensitizers. In this example, the The photosensitizer is merocyanine (merocyanine 540). When the immune blocking particles are exposed to light with a wavelength of 540nm, they can effectively generate free radicals that can induce phototoxicity.

In addition, in order to facilitate the separation and purification of the immune blocking particles, it is preferable to coat the magnetic particles while polymerizing the several functional monomers, so that the prepared immune blocking particles contain the Magnetic particles (that is, mixing the functional monomers, the template peptide, the magnetic particles, and the solvent, that is, when the functional monomers are polymerized in the solvent, the magnetic particles can be coated at the same time ). The saturation magnetic susceptibility of the magnetic particles can be greater than ±0.1emu/g, for example, can be superparamagnetism (superparamagnetism) triiron tetroxide (Fe ₃ O ₄ ), iron oxide (FeO) or ferric oxide (Fe ₂ O ₃ ). For example, an aqueous solution containing iron ions is provided, and the aqueous solution containing iron ions contains trivalent iron ions (Fe ³⁺ ) and divalent iron ions (Fe ²⁺ ) with a molar ratio of 2:1. After adding a precipitation agent to the iron ion aqueous solution, the ferric ion and the ferric ion are co-precipitated to form ferroferric oxide at a temperature of 60 to 95°C; in this embodiment, chlorination is used Iron (FeCl ₃ ) and ferrous chloride (FeCl ₂ ) are jointly prepared to form the iron ion-containing aqueous solution, and the precipitation agent is an aqueous sodium hydroxide solution (NaOH _(aq) ) or an aqueous ammonium hydroxide solution (NH ₄ OH _{(aq )),} and the hydroxyl group (OH contained in the precipitant ^-) number of moles of trivalent iron ion (Fe ³⁺⁾ ions in the aqueous solution containing iron ratio of 4: 1.

In the case that the molecular rubbing polymer contains both the magnetic particles and the photosensitizer, the surface of the magnetic particles can preferably be modified, for example, the surface of the magnetic particles can be modified with 3-aminopropyltriethoxysilane (3-aminopropyltriethoxysilane). , Abbreviated as APTES) for modification, at this time, the photosensitizer (for example, merocyanidin) can be carried on the magnetic particles via the silyl group of (3-aminopropyl)triethoxysilane. In detail, in this example, ferric oxide (0.2~5g) is mixed with ethanol (10~250mL, the ethanol concentration is 99.5%), and (3-aminopropyl)triethoxy is added to it Silane (10~1000μL) is mixed for 10~90 minutes, and then heated to 100~300℃ for heat treatment for 20~120 hours to obtain a modified magnetic particle.

The modified magnetic particles (5~1000mg) are mixed with anthocyanins (10~1000μg/mL) for 1~30 minutes, the upper layer solution is removed, and then washed to obtain a composite magnetic nanoparticle.

Mix the composite magnetic nanoparticles (2~100mg) with polyvinyl ethyl vinyl alcohol solution (10~2000μL, dissolved in dimethyl sulfoxide, concentration of 0.001~2wt%) and the template peptide (2~500μL) ) To obtain a mixed solution, drop the mixed solution into deionized water at 0-25°C, and stir with a magnet for 1-30 minutes to form the molecular rubbing polymer.

The immune blocking particles prepared by the above method have identification holes on the surface that can recognize the PD-1 protein. Therefore, when the immune blocking particles are administered to the desired individual, they can be blocked in the body of the desired individual. The self-protection mechanism of cancer cells (ie, the specific combination of PD-1 and PD-L1) promotes the activity of natural killer cells.

The immune blocking particles can induce the immune system, and thus can be used as an active ingredient of cancer immunotherapy. Preferably, the immune blocking particles can be used to prepare drugs for the treatment or auxiliary treatment of liver cancer. The immune blocking particles can be combined with pharmaceutically acceptable carriers or excipients to form a pharmaceutical composition, or additional active ingredients that help promote the death of cancer cells are added to the pharmaceutical composition ( For example, natural killer cells or T cells), wherein the immune blocking particle The daughter system can be prepared into any form that is convenient for administration to a desired individual, such as lozenges, capsules, powders, granules or liquids.

In addition, the immune-blocking particles can be administered to the desired individual by various suitable administration routes. For example, the immune-blocking particles can be administered parenterally or orally. The desired individual, for example, the immune blocking particles can be administered by intravenous injection (IV injection), intramuscular injection (IM injection), intraperitoneal injection (IP injection), etc. Give the required individual.

Furthermore, the immune-blocking particles can be administered to the desired individual at a dosage of 0.001 to 1000 mg/kg/week; however, the aforementioned dosage should vary depending on the individual and the route required. This is not restricted.

Moreover, when the immune blocking particle contains the photosensitizer, after the immune blocking particle is administered to the desired individual, a light energy (for example, with a wavelength of 460 to 580 nm) can be administered to the desired individual. Light is irradiated to the affected part of the individual for 0.01-30 minutes), and the photosensitizer is activated by light to generate free radicals that can induce phototoxicity and promote the death of cancer cells.

In addition, since the immune blocking particles further include the magnetic particles, after the immune blocking particles are prepared, they can be separated and purified by applying an external magnetic field (for example, magnets can be used for adsorption, which is common in the art). The knowledgeable person can understand and will not repeat them), so the immune blocking particles can be easily separated and purified.

In order to optimize the preparation method of the immune blocking particles of the present invention, the following experiments were carried out:

(A) Change in particle size of immune blocking particles

This experiment is shown in Table 1, with a template peptide composed of the amino acid sequence shown in SEQ ID NO: 8 (at a concentration of 100 μg/mL), mixed with polyethylene containing different percentages of ethylene mol Vinyl alcohol and the composite magnetic nanoparticle to form the molecular extension of group A1 together. After the polymer is printed, water is used as the cleaning solution to clean the molecular rubbing polymer to remove the template peptide to obtain group A2 immune blocking particles (mAIP for short); then mix the immune blocking particles mAIP And a re-adsorption solution (containing a template peptide at a concentration of 100 μg/mL), so that the template peptide can be adsorbed again to the identification hole on the surface of the immune blocking particle mAIP. For the convenience of subsequent explanation, the identification hole The immune blocking particle mAIP bound with the template peptide is called the re-adsorbed particle (group A3).

Please refer to Figure 1a, before removing the template peptide, the particle size (diameter) of the molecular printing polymer of group A1 increases with the percentage of ethylene mole of polyvinyl ethyl vinyl alcohol. They are 196.9±6.9nm (27mol%), 241.5±42.5nm (32mol%), 253.9±56.9nm (38mol%) and 501.4±57.5nm (44mol%), respectively. After removing the template peptide, the particle size of the immune blocking particles mAIP in group A2 was larger than that before removal, which were 207.1±13.8nm (27mol%), 196.6±12.1nm (32mol%), 302.8± The reason for 15nm (38mol%) and 312.4±28.9nm (44mol%) may be that after the hydrophilic template peptide is removed, the immune blocking particle mAIP is more hydrophobic, resulting in agglomeration. In addition, the particle sizes of the re-adsorbed particles of the A3 group after re-adsorption are 188±40.9nm (27mol%) and 189.4±26.5, respectively. nm (32mol%), 200±32.5nm (38mol%) and 146.5±22.3nm (44mol%), showing that the hydrophilic template peptide can be adsorbed again to the identification hole on the surface of the immune blocking particle mAIP, making Its hydrophilicity can be improved.

In addition, observe the particle size of the molecular rubbing polymer of group A1, the immune blocking particle mAIP of group A2, and the re-adsorbed particles of group A3 obtained by using polyvinyl ethyl vinyl alcohol with a ethylene molar percentage of 32 mol%. The change in the distribution and the change _{in the size distribution of the modified magnetic particles (Fe 3} O ₄ @APTES) in the A0 group. The results are shown in Figure 1b. The A2 group after the template peptide is removed The particle size distribution of the immune blocking particles mAIP is wider, and the particle size of the re-adsorbed particles of the A3 group after re-adsorption can become more uniform.

(B) Effect of ethylene mole percentage

Please refer to Table 2. In this experiment, the immune blocking particles mAIP prepared by polyvinyl ethyl vinyl alcohol containing different ethylene mol percentages are used as the B1 group, and the polyvinyl mol percentages of the aforementioned different ethylene mol percentages are also used as group B1. Vinyl ethyl vinyl alcohol, but the non-imprinted polymer nanoparticles (NIPs) prepared by the template peptide were not added as the B0 group.

Measure the amount of template peptides that can be adsorbed by the non-immune blocking particles NIP of group B0, and the amount of template peptides that can be adsorbed by the immune blocking particles mAIP of group B1. The results are shown in Figure 2 , And according to (Equation 1) to calculate polyethylene ethyl ethyl containing different percentages of ethylene mol The rubbing efficiency α of the immune blocking particle mAIP prepared by enol.

The results showed that the immuno-blocking particles mAIP prepared from polyvinyl ethyl vinyl alcohol with ethylene mole percentages of 27 mol%, 32 mol%, 38 mol%, and 44 mol% had rubbing efficiencies of 0.74, 1.76, 1.13, and 1.33, respectively. It can be known that the immuno-blocking particles mAIP prepared by using polyvinyl ethyl vinyl alcohol with a ethylene molar percentage of 32 mol% has the best rubbing efficiency. Therefore, all subsequent tests use polyethylene with a ethylene molar percentage of 32 mol%. Ethyl vinyl alcohol.

(C) The influence of the concentration of the template peptide

Next, test the effect of using different concentrations of template peptides on the adsorption capacity of the obtained immune blocking particles mAIP when preparing the immune blocking particles mAIP. At this time, fix the weight percent concentration of the polyvinyl vinyl alcohol It is 0.1%, and the concentration of the template peptide in the re-adsorption solution is 50 μg/mL.

Please refer to Figure 3, the adsorption capacity of the immune blocking particles mAIP obtained from the template peptides with the concentration of 0.1μg/mL, 1μg/mL, 10μg/mL, 50μg/mL and 100μg/mL are 27.6± respectively. 2.6μg/mg, 29.2±2.5μg/mg, 31.6±4.1μg/mg, 25.3±2.8μg/mg and 20.9±0.5μg/mg.

It is worth noting that when the concentration of the template peptide is between 0.1 and 10 μg/mL, the adsorption capacity of the immune blocking particle mAIP is directly proportional to the concentration of the template peptide. Under the condition of higher than 10 μg/mL, it may be possible that the number of peptides in the template is too large, which makes it impossible to form a complete identification hole on the surface of the immune blocking particle mAIP, which affects the recognition effect of the immune blocking particle mAIP. All subsequent experiments were performed at a concentration of 10μg/mL template peptide.

(D) The effect of adsorption time

In addition, the adsorption time between the immune blocking particle mAIP and the template peptide in the re-adsorption solution was tested, and the effect on the adsorption capacity of the immune blocking particle mAIP was tested. When the time is 1, 5, and 10 minutes, the adsorption capacity of the immune blocking particle mAIP is 20.5±0.2μg/mg, 31.6±0.2μg/mg and 33.9±13.3μg/mg, and the subsequent increase in adsorption time will affect the adsorption capacity. There was no obvious change, so all subsequent experiments were performed with an adsorption time of 10 minutes.

(E) The influence of the concentration of the resorption solution

Please refer to Table 3, mix the immune blocking particle mAIP (group E1) with the re-adsorption solution containing template peptides of different concentrations. After 10 minutes of adsorption time, the immune blocking particle mAIP of group E1 As shown in Figure 5, when the re-adsorption solution contains template peptides at concentrations of 50μg/mL, 100μg/mL, 150μg/mL and 200μg/mL, the adsorption capacity of the immune blocking particles mAIP are respectively 33.9±1.3μg/mg, 79.6±2.8μg/mg, 97.5±10.7μg/mg and 109.3±5μg/mg.

In addition, the adsorption curves of the non-immune blocking particles NIP (group E0) and the re-adsorption solution containing different concentrations of template peptides were tested. The results are also shown in Figure 5, where the re-adsorption solution contains a concentration of 50 μg /mL, 100μg/mL, 150μg/mL and 200μg/mL template peptide, the non-immune blocking particle NIP can also adsorb the template peptide in the re-adsorption solution. The adsorption capacity is 13.4±6.2μg/mg, 33.5±0.2μg/mg, 35.6±0.8μg/mg and 62.5±0.3μg/mg. The possible reason is that it is used to prepare the immune blocking particle mAIP. Ethylene ethyl vinyl alcohol and the template peptide have sufficient affinity for the template peptide to be adsorbed on the surface of the immune blocking particle mAIP, and the adsorption amount is related to the concentration of the template peptide in the adsorption solution .

In addition, according to the above (Equation 1), the rubbing efficiency of the immune blocking particle mAIP to the resorption solution containing different concentrations of template peptides is 2.5, 2.4, 2.7, and 1.7, respectively, indicating that the resorption solution contains 50 When the template peptide is ~150μg/mL, the adsorption amount of the immune blocking particle mAIP in the E1 group is proportional to the adsorption amount of the non-immune blocking particle NIP in the E2 group, so that the conversion efficiency of the immune blocking particle mAIP is obtained. Similar, when the concentration of the template peptide in the re-adsorption solution is 200 μg/mL, the adsorption amount of the immune blocking particles mAIP in the E1 group tends to be saturated, which leads to a decrease in the printing efficiency.

(F) Hysteresis curve of immune blocking particles

In this experiment, as shown in Table 4, a superconducting quantum interference device (SQUID) was used to measure the modified magnetic particles (group F0) and the molecular rubbing polymer (group F1, migration). Except for the template peptide) and the immune blocking particle mAIP (group F2, after removing the template peptide) hysteresis curve.

Please refer to Figure 6, whether it is the modified magnetic particles of the F0 group, the molecular imprinting polymer of the F1 group, or the immune blocking particle mAIP of the F2 group, all have superparamagnetism.

In addition, the magnetic flux of the modified magnetic particles of the F0 group is 55.8 emg/g, and the molecular imprinting polymer of the F1 group also contains the functional monomer (EVAL) and the template peptide, resulting in non-toxicity. The increase in the proportion of magnetic substances reduces the magnetic flux to 51.6 emg/g, while the immune blocking particle mAIP of group F2 is due to the removal of the template peptide, which increases the proportion of magnetic substances again, and the magnetic flux rises to 54.5 emg/g.

(G) Specific surface area of immune blocking particles

In this experiment, as shown in Table 5, a specific surface area and porosimetry analyzer (BET) was used to determine the molecular rubbing polymer (group G1, before removing the template peptide) and the immune resistance. The specific surface area of the particle mAIP (group G2, after removing the template peptide).

Please refer to Figure 7, the specific surface area of the molecular printing polymer of group G1 is about 301.4±23.9m ² /g, and the immune blocking particle mAIP of group G2 is more than the recognition hole formed on the surface. The surface area rises to 337.7±35.4 mg ² /g.

In order to confirm that the immune blocking particles prepared by the preparation method of the immune blocking particles of the present invention can indeed be applied to cancer immune blocking therapy, and thereby effectively promote the death of liver cancer cells, the following experiments were carried out:

(H) Cytotoxicity

In order to exclude the magnetic particles (ferric oxide), the modified magnetic particles (ferric oxide modified by APTES, Fe ₃ O ₄ @APTES for short) and the photosensitizer (merocyanidin) have cellular effects on cells The possibility of toxicity is shown in Table 6, the magnetic particles (group H1), the modified magnetic particles (group H2), the modified magnetic particles and the photosensitizer mixture (the H3 Group), and the nanoparticles obtained by polymerizing the functional monomers (EVAL) and coating the modified magnetic particles and the photosensitizer (group H4) are added to the human liver cancer cell line (HepG2 cells) , To test the survival rate of HepG2 cells.

Please refer to Figure 8. No matter adding the nanoparticles in groups H1~H4, they will not affect the cell viability of HepG2 cells, indicating that the magnetic particles, the modified magnetic particles and the photosensitizer are not cytotoxic .

(I) Application in cancer immuno-blocking therapy

This test is based on a template peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, mixing the several functional monomers (EVAL), the photosensitizer (merocyanidin) and the modified The magnetic particles (Fe ₃ O ₄ @APTES) of the first embodiment are prepared to obtain the immune blocking particles (hAIP for short), and the functional monomers (EVAL) and the photosensitizer (merocyanidin) are mixed together. ) And the modified magnetic particles (Fe ₃ O ₄ @APTES) (that is, no template peptide is added) to prepare a non-imprinted polymer nanoparticles (NIP), and As shown in Table 7, the immune blocking particle hAIP, the non-immune blocking particle NIP and natural killer cells (Jurkat cells) of the first embodiment were added to HepG2 cells to test the cell survival rate of HepG2 cells.

Please refer to Figure 9a. The survival rate of HepG2 cells in groups I1-0~I1-2 decreased with the concentration of nanoparticles added, but the HepG2 cells in groups I1-1 and I1-2 had The survival rate is lower than that of the HepG2 cells of the I1-0 group, showing that the immune blocking particle of the first embodiment is formed by the template peptide composed of the amino acid sequence shown in SEQ ID NO: 4 It does enhance the activity of natural killer cells.

In addition, a template peptide consisting of the amino acid sequence shown in SEQ ID NO: 8 is used to mix the several functional monomers (EVAL), the photosensitizer (the merocyanidin) and the modified magnetic Particles (Fe ₃ O ₄ @APTES) to prepare the immune blocking particles (abbreviated as mAIP) of the second embodiment, and further mix the several functional monomers (EVAL), the photosensitizer (merocyanidin) and The modified magnetic particles (Fe ₃ O ₄ @APTES) (that is, the template peptide is not added) to prepare the non-immune blocking particle NIP, and as shown in Table 8, the second embodiment The immune blocking particle mAIP, the non-immune blocking particle NIP and natural killer cells (CTLL-2 cells) are added to HepG2 cells together to test the cell survival rate of HepG2 cells.

Please refer to Figure 9b. The survival rate of HepG2 cells in the I2-0~I2-2 groups all decreased with the concentration of nanoparticles added, but the HepG2 cells in the I2-1 and I2-2 groups The survival rate is lower than that of the HepG2 cells of the I2-0 group, showing that the immune blocking particle of the second embodiment is formed by a template peptide composed of the amino acid sequence shown in SEQ ID NO: 8 It does enhance the activity of natural killer cells.

In addition, as shown in Figures 9a and 9b, the survival rate of HepG2 cells in group I1-2 is lower than that of HepG2 cells in group I1-1, and the survival rate of HepG2 cells in group I2-2 is lower than The survival rate of the HepG2 cells of group I2-1 shows that it is better to mix the immune blocking particles with natural killer cells, so that the natural killer cells are first activated by the immune blocking particles, and then co-administered to the desired individual , So that the activated natural killer cells can quickly exert their biological activity, thus having a better anti-cancer effect.

(J) Molecular mechanism that causes cancer cell death

In this test, as shown in Table 9, the immune blocking particle mAIP of the second embodiment was added to HepG2 cells, or the immune blocking particle mAIP of the second embodiment, the non-immune blocking particle Particle NIP and natural killer cells (CTLL-2 cells) are added to HepG2 cells to test the apoptosis pathway of HepG2 cells by quantitative real-time polymerase chain reaction (Q-PCR) The expression levels of the main genes such as CASP8, CASP3 and NFKB1 in ), and the relative expression levels of the genes such as CASP8, CASP3 and NFKB1 in HepG2 cells without the immune blocking particles and/or natural killer cells added.

Please refer to Figure 10, the expression levels of genes such as CASP8, CASP3 and NFKB1 in the C1 group where the immune blocking particles mAIP of the second embodiment and natural killer cells (CTLL-2 cells) are added together into HepG2 cells The biggest change is the relative gene expression of CASP3. The C0 group is 165.9±25.3%, the C1 group is 282±32.8, and the C2 group is 224.6±21.7%. It shows that the immune blocking particles of the present invention can promote cells. Progress of the apoptotic pathway.

(K) Comparison of the immune blocking particle hAIP of the first embodiment and the immune blocking particle mAIP of the second embodiment

In this experiment, as shown in Table 10, the immune blocking particle hAIP of the first embodiment and natural killer cells (Jurkat cells) were added to HepG2 cells, or the immune blocking particle mAIP of the second embodiment was combined with natural killer cells. Cells (CTLL-2 cells) join HepG2 cells together After medium, the cell viability of HepG2 cells was tested.

Please refer to Fig. 11, the survival rate of HepG2 cells in the K1 and K2 groups all decreased with the concentration of nanoparticle added. Among them, the immune blocking particle hAIP of the first embodiment has a better effect.

In summary, the preparation method of immune blocking particles of the present invention can obtain immune blocking particles with identification holes capable of recognizing PD-1 protein on the surface. Therefore, when the immune blocking particles are administered to a desired individual , That is, it can block the self-protection mechanism of cancer cells in the body of the desired individual and promote the activity of natural killer cells. The immune blocking particles can be applied to cancer immune blocking therapy, which is the effect of the present invention.

Furthermore, the immune blocking particle of the present invention may also contain the photosensitizer at the same time. Therefore, after the immune blocking particle is administered to the desired individual, the light energy can be administered to the desired individual to make the photosensitizer It is activated by light to generate free radicals that can induce phototoxicity and promote the death of cancer cells. The immune blocking particles can be applied to both cancer immune blocking therapy and photodynamic therapy, which is the effect of the present invention.

In addition, the method for preparing immune blocking particles of the present invention can obtain molecular rubbing polymers containing the magnetic particles. Therefore, the worker can separate and purify the immune blocking particles by applying an external magnetic field (for example, using magnets for adsorption). ), to improve the isolation of the immune-blocking particles The effect of the convenience of the time of change.

In addition, the immune blocking particles of the present invention can block the specific binding of PD-1 and PD-L1, and thus can be used as an active ingredient of cancer immune blocking therapy, which is the efficacy of the present invention.

Although the present invention has been disclosed using the above-mentioned preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with the art without departing from the spirit and scope of the present invention may make various changes and modifications relative to the above-mentioned embodiments. The technical scope of the invention is protected. Therefore, the scope of protection of the invention shall be subject to the scope of the attached patent application.

<110> National Kaohsiung University

<120> Preparation method of immune blocking particles and use of the immune blocking particles

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Homo sapiens (human)

<400> 1

<210> 2

<211> 8

<212> PRT

<213> Homo sapiens (human)

<400> 2

<210> 3

<211> 9

<212> PRT

<213> Homo sapiens (human)

<400> 3

<210> 4

<211> 15

<212> PRT

<213> Homo sapiens (human)

<400> 4

<210> 5

<211> 9

<212> PRT

<213> Mus musculus (mice)

<400> 5

<210> 6

<211> 8

<212> PRT

<213> Mus musculus (mice)

<400> 6

<210> 7

<211> 10

<212> PRT

<213> Mus musculus (mice)

<400> 7

<210> 8

<211> 18

<212> PRT

<213> Mus musculus (mice)

<400> 8

Claims (8)

A method for preparing immune blocking particles includes: mixing several functional monomers, a template peptide, a photosensitizer, a magnetic particle and a solvent, so that when the several functional monomers are polymerized in the solvent , The template peptide, the photosensitizer and the magnetic particles are coated to form a molecular rubbing polymer, wherein each of the functional monomers is polyvinyl vinyl alcohol, and the photosensitizer can be merocyanidin , The magnetic susceptibility of the magnetic particle is greater than ±0.1emu/g, and the surface of the magnetic particle is modified with (3-aminopropyl)triethoxysilane; and the template located on the surface of the molecular rubbing polymer is removed Peptide to obtain the immune blocking particle, the surface of the immune blocking particle has a number of identification holes corresponding to the template peptide; wherein, the template peptide contains the amine shown in SEQ ID NO: 4 or 8 Base acid sequence. According to the method for preparing immune blocking particles of claim 1, wherein each of the functional monomers is polyvinyl vinyl alcohol with a ethylene mole percentage of 5-50 mol%. According to claim 1, the method for preparing immune blocking particles, wherein the solvent is water. A use of immune blocking particles is applied to the preparation of drugs for the treatment of liver cancer, wherein the immune blocking particles are prepared by the method for preparing immune blocking particles according to any one of claims 1 to 3. The use of the immune blocking particle according to claim 4, wherein the immune blocking particle is administered to a desired individual by oral administration, intravenous injection, intramuscular injection, or intraperitoneal injection. The use of the immune blocking particle of claim 5, wherein the immune blocking particle is administered to the desired individual at a dose of 0.001 to 1000 mg per kilogram of the desired individual per week. The use of the immune blocking particle of claim 5 or 6, wherein the immune blocking particle is mixed with natural killer cells and then jointly administered to the desired individual. The use of the immune blocking particle of claim 5 or 6, wherein, after the immune blocking particle is administered to the desired individual, light with a wavelength of 460 to 580 nm is irradiated to the desired individual’s disease Department 0.01~30 minutes.

Images