Dual-targeting nano-micelle and preparation method thereof

Abstract

The preparation method comprises the following steps: modifying and connecting IL-15 and PD-1 protein molecules by using biotin, modifying PLGA-PEG by using avidin, and finally preparing the double-targeting nano-micelle through specific binding of the biotin and the avidin and a self-assembly effect of the PLGA-PEG. The dual-targeting nano-micelle IL-15 / PD-1 / PEG-PLGA, which can efficiently target and activate NK cells and target surface high-expression PD-L1 tumor cells, is synthesized and prepared. The mole number of IL-15 and PD-1 loaded on the surface of the dual-targeting nano-micelle is close to 1: 1, and the dual-targeting nano-micelle is regular in form, uniform in dispersion and in the shape of a sphere with the particle size of 254.76 + / -28.02 nm. Meanwhile, it is verified that the dual-targeting nano-micelle IL-15 / PD-1 / PEG-PLGA can effectively improve the recognition and killing efficiency of the NK cells on tumor cells in vivo and in vitro.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry, and particularly relates to a double-targeting nanomicelle and a preparation method thereof. BACKGROUND

[0002] Cancer is the second leading cause of death worldwide, and the incidence and mortality of cancer will rise by about 50% in the future, so it is imminent to develop more advanced and effective cancer treatment methods. Compared with the commonly used treatment methods such as chemotherapy, radiotherapy and surgical treatment, immunotherapy does not directly kill cancer cells with toxic drugs or radiation, but fights against tumor cells by enhancing the activity of immune cells in the tumor microenvironment and surrounding lymphoid tissues. This anti-tumor immune response is efficient and persistent, can establish long-term immune surveillance, and has relatively low toxicity. The current main tumor immunotherapy strategies include immune checkpoint inhibitors, cytokine therapy, adoptive T cell therapy, etc. In the late 1980s, Rosenberg et al. found that IL-2 (Interleukin-2, IL-2) can activate immune cells to treat cancer. However, the treatment with free cytokines alone faces the problem of toxic side effects at high doses and loss of therapeutic effect due to drug concentration reduction. Later, people developed related checkpoint inhibitor antibodies, which combined with key inhibitory receptors to relieve immune suppression and restore the function of immune cells. Natural killer cells (NK cells) are one of the most important natural immune cells in anti-viral infection and anti-tumor. In clinical experiments, the adoptive treatment of NK cells in hematological malignancies has achieved satisfactory results, but due to the immunosuppressive effect of the tumor microenvironment, the number of NK cells infiltrating the tumor tissue is small, and the killing activity is significantly inhibited, but the effect in solid tumors is not satisfactory. How to efficiently activate NK cells and improve their directional aggregation in tumor sites is a key problem that needs to be solved in NK cell immunotherapy. SUMMARY

[0003] The purpose of the present application is to avoid the shortcomings of the prior art, and to provide a double-targeting nanomicelle IL-15 / PD-1 / PEG-PLGA which can effectively improve the recognition and killing efficiency of NK cells on tumor cells and a preparation method thereof.

[0004] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: a preparation method of a double-targeting nanomicelle, characterized by comprising the following steps:

[0005] Step one, synthesis of PLGA-PEG-SA, specifically:

[0006] First, dissolve streptavidin in PBS to prepare a streptavidin aqueous solution with a concentration of 1-4 mg / ml;

[0007] Then, PLGA10k-PEG5k-NHS at a concentration of 5 to 10 mg / ml is added dropwise to a streptavidin aqueous solution for reaction. The resulting reaction solution is dialyzed in pure water. The dialyzed solution is frozen and dried to obtain PLGA-PEG-SA having a molecular weight greater than that of the streptavidin raw material.

[0008] The molar ratio of PLGA10k-PEG5k-NHS to streptavidin is 5 to 10:1.

[0009] Step 2: Use biotin to modify proteins IL-15 and PD-1, specifically:

[0010] Add protein IL-15 to an ultrafiltration tube, then add biotin and pipette. After the biotin and protein IL-15 are evenly mixed, place in an incubator in the dark for incubation, and then centrifuge. Discard the lower layer solution, add labeling buffer, and centrifuge multiple times to remove unbound biotin molecules. Biotin-labeled protein IL-15 is obtained in the upper layer liquid of the ultrafiltration tube.

[0011] The amount of biotin added is calculated according to the molar ratio of biotin: protein IL-15 = 10 to 20:1;

[0012] The same method was used to obtain biotin-labeled protein PD-1, wherein the amount of biotin added was calculated according to a molar ratio of biotin: protein PD-1 = 10-20:1;

[0013] Step 3: Synthesize dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA, specifically:

[0014] PLGA-PEG-SA was dissolved in DMSO and reacted with biotin-labeled proteins IL-15 and PD-1. The resulting reaction solution was placed in a dialysis bag and dialyzed against PBS overnight to remove DMSO. The unloaded proteins were then removed to obtain the dual-targeted nanomicelle IL-15 / PD-1 / PEG-PLGA.

[0015] Among them, PLGA-PEG-SA was dissolved in DMSO and prepared at a ratio of 10 mg PLGA-PEG-SA dissolved in 1 mL DMSO;

[0016] The molar ratio of the sum of the labeled proteins IL-15 and PD-1 to PLGA-PEG-SA was 1:1-10;

[0017] The molar ratio of biotin-labeled protein IL-15 to biotin-labeled protein PD-1 is 1:1-3.

[0018] Furthermore, in step 1, the concentration of the reactant streptavidin aqueous solution is 2 mg / ml, the concentration of PLGA10k-PEG5k-NHS is 10 mg / ml, and the molar ratio of PLGA10k-PEG5k-NHS to streptavidin is 5:1.

[0019] Furthermore, in step 1, the reaction time of adding PLGA10k-PEG5k-NHS dropwise to the streptavidin aqueous solution is 4-8 hours; and the dialysis bag used for dialysis has a specification of MWCO8000-14000.

[0020] Furthermore, the amount of biotin added in step 2 is calculated according to the molar ratio of biotin: protein IL-15 = 20:1; biotin: protein PD-1 = 20:1.

[0021] Furthermore, in step 2, the temperature in the incubator is 37° C., the light-proof incubation time is 30 min, the centrifugal force during centrifugation is 12,000×g, and the centrifugation time is at least 10 min.

[0022] Furthermore, step 2 also includes taking out the biotin-labeled proteins IL-15 and PD-1, adding them to the preservation solution at a ratio of 1:1, and storing them at a temperature of -20°C.

[0023] Furthermore, the reaction in step 3 is carried out at a temperature of 37° C. and a stirring speed of 500 rpm, and the stirring reaction time in the dark is at least 4 hours;

[0024] The dialysis bag specification is MWCO8000-14000;

[0025] The removal of unloaded proteins is performed by placing the reaction solution after DMSO removal into an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and then centrifuging the tube at a centrifugal force of 5000×g and a temperature of 4° C. for at least 10 minutes.

[0026] Furthermore, in step 3, the molar ratio of the sum of the biotin-labeled proteins IL-15 and PD-1 to PLGA-PEG-SA is 1:5; the molar ratio of the biotin-labeled protein IL-15 to the labeled protein PD-1 is 1:2.

[0027] Furthermore, the loading rates of IL-15 and PD-1 in the dual-targeted nanomicelles were 62.05±3.53% and 33.35±4.59%, respectively; the molar ratio of IL-15 and PD-1 loaded on the surface of the dual-targeted nanomicelles was close to 1:1.

[0028] Furthermore, the nanomicelles have regular morphology, uniform dispersion, and a spherical shape with a particle size of 254.76±28.02 nm, a zeta potential of -7.64±2.64 mV, and a PDI of IL-15 / PD-1 / PEG-PLGA of 0.18.

[0029] In the above technical solution, PLGA10k-PEG5k-NHS is polylactic acid-co-glycolic acid-polyethylene glycol-succinimide ester; PLGA-PEG-SA is polylactic acid-co-glycolic acid-polyethylene glycol-streptavidin; DMSO is dimethyl sulfoxide; and PBS is phosphate buffered saline.

[0030] The beneficial effects of the present invention are: biotin is used to modify and connect IL-15 and PD-1 protein molecules, avidin is used to modify PLGA-PEG, and finally, through the specific binding of biotin and avidin and the self-assembly effect of PLGA-PEG, a dual-targeted nanomicelle IL-15 / PD-1 / PEG-PLGA is synthesized and prepared, which can efficiently target and activate NK cells and target tumor cells with high surface expression of PD-L1. At the same time, it is verified that the dual-targeted nanomicelle IL-15 / PD-1 / PEG-PLGA can effectively improve the recognition and killing efficiency of NK cells against tumor cells in vitro and in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the SDS-PAGE image of PLGA-PEG-SA;

[0032] Figure 2 The loading rate (a) and drug loading capacity (b) of IL-15 in dual-targeting nanomicelles at different ratios of IL-15+PD-1 and PLGA-PEG-SA

[0033] Figure 3 The loading rate (a) and drug loading (b) of PD-1 in dual-targeting nanomicelles at different ratios of IL-15+PD-1 and PLGA-PEG-SA

[0034] Figure 4 The loading rate (a) and drug loading (b) of IL-15 in dual-targeted nanomicelles at different ratios of IL-15 and PD-1

[0035] Figure 5 The loading rate (a) and drug loading (b) of PD-1 in dual-targeted nanomicelles at different ratios of IL-15 and PD-1

[0036] Figure 6The loading rate (a) and drug loading capacity (b) of IL-15 in dual-targeted nanomicelles at different concentrations of total protein and polymer

[0037] Figure 7 PD-1 loading rate (a) and drug loading capacity (b) in dual-targeting nanomicelles at different concentrations of total protein and polymer

[0038] Figure 8 is the molar number of IL-15 and PD-1 in the dual-targeting nanomicelles

[0039] Figure 9 Transmission electron microscopy images of pure polymer micelles and dual-targeted nanomicelles;

[0040] Figure 10 is the particle size distribution diagram of pure polymer micelles and dual-targeted nanomicelles;

[0041] Figure 11 Stability of dual-targeted nanomicelles

[0042] Figure 12 Effects of dual-targeting nanomicelles on NK cell proliferation

[0043] Figure 13 is the expression level of PD-L1 on the surface of different tumor cells;

[0044] Figure 14 Effects of dual-targeted nanomicelles on NK cell cytotoxicity

[0045] Figure 15 is the effect of dual-targeting nanomicelles and fusion protein on IFN-γ levels in NK cells (x±s, n=3);

[0046] Figure 16 This is the ELISA colorimetric result of the in vitro binding of dual-targeting nanomicelles to PD-L1;

[0047] Figure 17 In vitro binding characteristics of dual-targeted nanomicelles with CAKI-1 cells that highly express PD-L1

[0048] Figure 18 The tumor inhibition effect of dual-targeted nanomicelles DETAILED DESCRIPTION

[0049] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0050] Example 1:

[0051] 1.1 Preparation of double-targeted nanomicelle IL-15 / PD-1 / PEG-PLGA, the specific steps are as follows:

[0052] Step one, synthesis of polylactic acid-glycolic acid copolymer-polyethylene glycol-streptavidin PLGA-PEG-SA, specifically:

[0053] (1) 10 mg of streptavidin was dissolved in 5 mL of PBS, and 5 mg of PLGA10k-PEG5k-NHS was dissolved in 0.5 mL of DMF;

[0054] (2) PLGA10k-PEG5k-NHS was added dropwise to the streptavidin aqueous solution, and the reaction was carried out at room temperature for 4 h;

[0055] (3) The reaction solution was transferred to a dialysis bag with a MWCO of 8000-14000, and after dialysis in pure water overnight, the dialysate was collected and freeze-dried to obtain the product PLGA-PEG-SA;

[0056] (4) The results are shown in Figure 1 (Fig. M: protein Marker; A: PLGA-PEG-SA;

[0057] B: SA) shows that the molecular weight of PLGA-PEG-SA product is greater than that of streptavidin raw material, which indicates that PLGA-PEG-SA synthesis is successful.

[0058] Step two, biotin modification of protein IL-15 and PD-1, specifically:

[0059] (1) 50 g of protein IL-15 was added to an ultrafiltration tube, and 0.5 mL of labeled buffer was added, and centrifuged at 12000 x g for 10 min; 0.5 L of 20 mM biotin was added to the above ultrafiltration centrifuge tube, and the final volume was made to 0.5 mL by adding labeled buffer, and then mixed uniformly by gentle blowing, and then placed in a 37°C incubator, avoiding light, and incubated for 30 min;

[0060] (2) Centrifuge at 12000 x g for 10 min, discard the lower solution, add 400 L of labeled buffer to the ultrafiltration tube, mix gently, centrifuge at 12000 x g for 10 min, and centrifuge several times to remove unbound biotin molecules; At this time, the biotin-labeled protein IL-15 is in the upper liquid of the ultrafiltration tube, and 1:1 is taken to save in -20°C;

[0061] Biotin-labeled protein PD-1 was obtained by the same method as above.

[0062] Step three, synthesis of double-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA, specifically:

[0063] (1) Take 607.5 μg of the synthesized PLGA-PEG-SA, dissolve it in DMSO, and add 22.5 μg of biotin-labeled IL-15 and 37.8 μg of PD-1 drop by drop, stirring at the same time. Stir at 37°C, 500 rpm, and avoid light for 4 h;

[0064] (2) Collect the reaction product, put it in a dialysis bag with a MWCO of 8000-14000, and dialyze it in PBS overnight to remove DMSO;

[0065] (3) Collect the sample after dialysis, put it in a 100 kDa ultrafiltration centrifuge tube, and centrifuge at 5000 x g, 4°C for 10 min to remove the unloaded protein. Collect the liquid in the centrifuge tube, which is the double-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA.

[0066] 1.2 Determination of biotin labeling efficiency of protein molecules:

[0067] (1) 2-(4-Hydroxyphenylazo)benzoic acid (HABA) can specifically bind to avidin to form a yellow or orange complex with a maximum absorbance at 500 nm. However, the binding capacity of HABA to avidin is much worse than that of biotin, so in the presence of biotin, biotin will compete with HABA, leading to dissociation of HABA and a decrease in absorbance. Based on this principle, the amount of biotin-labeled protein (molar ratio of labeled protein to biotin) can be calculated according to the change in absorbance at 500 nm. Take 180 μL of HABA-Avidin mixture and add it to a 96-well plate. Measure the absorbance at 500 nm and record the measured value as A500(H-A). Measure multiple times and take the average.

[0068] (2) Add 20 μL of the biotin-labeled protein to be tested to a 96-well plate, shake to mix, and then measure the OD value at 500 nm. Measure at least 20 s or more, and record as A500(H-A-BP). Preferably, measure 3 times and take the average.

[0069] (3) Calculate the labeling efficiency. The calculation formula is as follows:

[0070]

[0071] (4) The biotin labeling efficiency of IL-15 and PD-1 was calculated to be 100%.

[0072] 1.3 Synthesis and preparation optimization of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA:

[0073] (1) Drug loading is generally evaluated by Entrapped Efficiency (EE) and Drug Loading (DL). The Entrapped Efficiency is the ratio of the loaded drug to the input drug, and the drug loading refers to the mass of the drug loaded per unit mass of the drug loading system. The total system is set to 100 μL, the total protein concentration of IL-15+PD-1 is controlled to be 111.11 nM, IL-15:PD-1=1:1 remains unchanged, and IL-15+PD-1:PLGA-PEG-SA=1:1, 1:5, 1:10 are set. By detecting the Entrapped Efficiency and Drug Loading of IL-15 and PD-1 at different ratios, the optimal molar ratio between protein and pure polymer is explored.

[0074] (2) After determining the optimal molar ratio between total protein and pure polymer, change the ratio between IL-15 and PD-1 proteins, setting IL-15:PD-1 = 1:1, 1:2, and 1:3, and explore the effects of different ratios between IL-15 and PD-1 on the loading rate and drug loading of the two proteins. At the same time, it is also necessary to consider the need to maintain consistency with the ratio of IL-15 and PD-1 (1:1) in the subsequent fusion protein to screen the optimal molar ratio between the two.

[0075] (3) In order to further improve the overall loading efficiency of the two proteins, the determined IL-15+PD-1:PLGA-PEG-SA and IL-15:PD-1 were kept unchanged, and the input amount (molar concentration) of total protein and pure polymer in the system was increased, that is, the total input amount of IL-15+PD-1 was set to 133.32nM (44.44nM, 88.88nM), 166.67nM (55.55nM, 111.11nM), 250nM (83.33nM, 166.67nM), 333.33nM (111.11nM, 222.22nM), corresponding to the input amount of PLGA-PEG-SA of 666.66nM, 833.35nM, 1.25μM, and 1.67μM, respectively, to explore whether the change in the total input amount would affect the loading efficiency of the two proteins, thereby determining the optimal molar concentration of the total protein in the system.

[0076] (4) The prepared different proportions of double-targeted nanomicelles were placed in 100 kDa ultrafiltration centrifuge tubes, centrifuged at 5000 x g at 4°C for 10 min, and the supernatant and lower filtrate were taken out, diluted to the appropriate concentration, and the concentrations of IL-15 and PD-1 in the supernatant and filtrate were detected by ELISA method, and the corresponding loading rate and drug loading of the double-targeted nanomicelles were calculated.

[0077] (5) The results are as follows, as shown in Figure 2 and Figure 3 (As shown in the figure, * indicates a significant difference (p < 0.01); *** indicates a very significant difference (p < 0.001); **** indicates a very significant difference (p < 0.0001)), when the molar ratio of IL-15+PD-1:PLGA-PEG-SA increased from 1:1 to 1:5, the loading rate of IL-15 increased from 42.74 ± 1.68% to 59.63 ± 4.45% (p < 0.001), the drug loading was 3.58 ± 0.49%, the loading rate of PD-1 increased from 12.58 ± 0.91% to 20.2 ± 1.06% (p < 0.001), and the drug loading was 0.94 ± 0.05%; when the ratio increased to 1:10, the loading rate of IL-15 increased from 59.63 ± 4.45% to 71.0 ± 1.84% (p < 0.001), the loading rate of PD-1 increased from 20.2 ± 1.06% to 24.48 ± 1.57% (p < 0.05), and the corresponding drug loading also decreased, which was 1.84 ± 0.18% (p < 0.0001) and 0.57 ± 0.04% (p < 0.05), respectively.

[0078] Considering the loading rate and drug loading results, especially the loading rate reflects the molar ratio of drugs that can be loaded into the micelles, it is determined that the optimal input ratio of IL-15+PD-1:PLGA-PEG-SA is 1:5.

[0079] To further improve the loading rate of PD-1 while ensuring the molar ratio of IL-15 and PD-1 loaded on the surface of the dual-targeting nanomicelles is 1:1, based on the determination of IL-15+PD-1:PLGA-PEG-SA=1:5, the total protein input in the system was controlled unchanged, and the ratio between IL-15 and PD-1 was changed. When IL-15:PD-1 increased from 1:1 to 1:2, the loading rate of IL-15 decreased from 59.63±4.45% to 56.67±9.72%(p>0.05); but the loading rate of PD-1 increased from 20.2±1.06% to 26.8±2.19%(p>0.05), when the input of PD-1 continued to increase, i.e. IL-15:PD-1=1:3, the loading rate of IL-15 decreased from 56.67±9.72% to 48.07±0.78%(p>0.05), while the loading rate of PD-1 increased from 26.8±2.19% to 38.78±5.27%, increased by about 12%(p<0.05).

[0080] It can be seen that increasing the molar ratio of PD-1 in the total protein can indeed significantly improve its loading rate. At the same time, the drug loading also changes accordingly, in general, the increase of the input ratio of PD-1 significantly improves the drug loading of PD-1(p<0.01). It is worth noting that, as Figures 4-5 (As shown in the figure, * and ** represent significant difference(p<0.05, p<0.01); *** represents extremely significant difference(p<0.001)) When IL-15:PD-1=1:2, the molar number of IL-15 and PD-1 is close to 1:1, therefore, considering the loading rate and the need to meet the final IL-15:PD-1=1:1, IL-15:PD-1=1:2 is determined.

[0081] As Figure 6 (As shown in the figure, * and ** represent significant difference(p<0.05, p<0.01)) and Figure 7As shown, in order to improve the overall load rate of protein, we also investigated the effect of the change of total protein and polymer input on the load rate and drug loading amount. In the same volume, the higher the protein concentration, the higher the probability of collision reaction. When the concentration of IL-15+PD-1 was increased from 133.33 nM (IL-15 input 2 ug, PD-1 input 3.38 ug) to 250 nM, the load rate of IL-15 and PD-1 was increased from 50.97 ± 4.07% to 62.05 ± 3.53% (p < 0.05) and 26.8 ± 2.19% to 33.35 ± 4.59% (p > 0.05), respectively. The calculated molar concentration of IL-15 and PD-1 loaded in the micelles was 51.71 ± 2.94 nM and 55.54 ± 7.68 nM, respectively, with a ratio close to 1:1, and the drug loading amount was relatively high, at 2.75 ± 0.35% and 2.0 ± 0.34%, respectively. When the concentration of IL-15+PD-1 was increased from 250 nM to 333.33 nM, the load rate of IL-15 and PD-1 did not increase significantly (p > 0.05).

[0082] In addition, in order to ensure that the molar number of IL-15 and PD-1 loaded on the surface of the double-targeting nanomicelles is close to 1:1, we calculated the molar number of IL-15 and PD-1 loaded at different ratios, as shown in Figure 8 (FIG. a: IL-15 and PD-1 at different ratios; b: different total protein concentrations).

[0083] Therefore, the double-targeting nanomicelles PLGA-PEG-IL-15 / PD-1 were finally prepared under the condition of IL-15+PD-1:PLGA-PEG-SA = 1:5, IL-15:PD-1 = 1:2, and the total concentration of IL-15+PD-1 being 250 nM in a total system of 100 μL. Under this condition, the load rate of IL-15 and PD-1 was 62.05 ± 3.53% and 33.35 ± 4.59%, respectively, and the molar ratio was close to 1:1.

[0084] 1.4 Characterization of the properties of double-targeting nanomicelles IL-15 / PD-1 / PEG-PLGA:

[0085] (1) Morphology detection of double-targeting nanomicelles:

[0086] The morphology of pure polymer micelles and double-targeting nanomicelles was observed by transmission electron microscopy (TEM). The pure polymer micelles or double-targeting nanomicelles were dropped onto a carbon-coated copper grid and dried at room temperature. The operation was carried out using an acceleration voltage of 200 kV.

[0087] The results are shown in Figure 9(Figure: a: pure polymer micelles; b: dual-targeted nanomicelles) The dual-targeted nanomicelles loaded with IL-15 and PD-1 exhibited regular, spherical morphology and were uniformly dispersed. After loading with IL-15 and PD-1, the particle size of the dual-targeted nanomicelles significantly increased from 59.21±2.35nm to 144.52±6.7nm, preliminarily demonstrating the successful preparation of the dual-targeted nanomicelles.

[0088] (2) Dual-targeted nanomicelle particle size and zeta potential test:

[0089] Dynamic light scattering (DLS) was used to measure the particle size and zeta potential of the dual-targeted nanomicelles. 1 mL of pure polymer micelles (PLGA-PEG-M) and dual-targeted nanomicelles (PLGA-PEG-IL-15 / PD-1) without protein loading were placed in a measuring dish. The test conditions were 25°C and a scattering angle of 173°. Each sample was tested at least three times.

[0090] The results are shown in Table 1 and Figure 10 (In the figure, a: pure polymer micelles; b: dual-targeted nanomicelles), wherein Table 1 is a table of particle sizes and ζ potentials of pure polymer micelles and dual-targeted nanomicelles.

[0091] The particle size of PLGA-PEG-SA was 170.02±13.30nm, and that of PLGA-PEG-IL-15 / PD-1 was 254.76±28.02nm. The larger the size of the nanomicelles after protein loading, the more successful the loading of IL-15 and PD-1. The zeta potential decreased from -3.72±0.89mV for PLGA-PEG-SA to -7.64±2.64mV, due to the negative charge of both protein molecules, which significantly reduced the potential of the dual-targeted nanomicelles. Furthermore, the PDI of LGA-PEG-IL-15 / PD-1 was 0.18, and the particle size distribution was uniform, indicating good dispersibility.

[0092] Table 1

[0093]

[0094] (3) Stability test of dual-targeted nanomicelles:

[0095] DLS was used to measure changes in the particle size and zeta potential of the dual-targeted nanomicelles to assess their stability. Specifically, the dual-targeted nanomicelles were placed in a culture medium containing 10% serum and incubated at 37°C at 80 rpm on an oscillating platform to simulate the in vivo blood environment. Simultaneously, the dual-targeted nanomicelles were placed in a 4°C PBS (pH 7.4) environment to simulate the storage environment. Samples were taken on days 1, 7, and 14 to measure changes in particle size and zeta potential. The measurement methods were the same as above.

[0096] The results are as follows Figure 11 (where a: particle size change in 37°C serum environment; b: ζ potential change in 37°C serum environment; c: particle size change in 4°C solvent environment; d: ζ potential change in 4°C solvent environment) shows that within the set 14 days, the particle size and ζ potential of the dual-targeted nanomicelles did not change significantly, whether in simulated serum or PBS environment, which preliminarily indicates that the dual-targeted nanomicelles have good stability in vitro.

[0097] Example 2:

[0098] To investigate the in vitro activity of dual-targeting nanomicelles IL-15 / PD-1 / PEG-PLGA.

[0099] 2.1 Effect of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA on the proliferation activity of NK cells in vitro

[0100] (1) NK-92 cells were plated into 96-well plates at a density of 10,000 per well, and the PBS group, PEG-PLGA group, IL-15+PD-1 group, IL-15-PEG-PLGA+PD-1 group, and IL-15 / PD-1 / PEG-PLGA group were set up.

[0101] (2) After 24 hours of culture, CCK-8 was added. After 2 hours of incubation, the absorbance at 450 nm was measured and the proliferation rate of NK cells was calculated. The calculation formula is as follows:

[0102]

[0103] Wherein, ODc: OD450 of NK-92 cells in the control group;

[0104] ODt: OD450 of NK-92 cells in the experimental group.

[0105] (3) Figure 12(In the figure, * and ** indicate significant differences (p<0.05 and p<0.01), as shown in the table, compared with the Control group, the IL-15+PD-1 group, the IL-15-PEG-PLGA+PD-1 group, and the IL-15 / PD-1 / PEG-PLGA group can significantly stimulate the proliferation of NK cells, with increases of 33.93±3.83% (p<0.05), 59.18±17.86% (p<0.01), and 57.73±16.40% (p<0.01), respectively.

[0106] 2.2 Determination of PD-L1 expression on the surface of different tumor cells:

[0107] (1) Take 1 million CAKI-1, 786-O, RPMI-8226, K562, Hela, and MCF-7 cells in the logarithmic growth phase, collect the cells by centrifugation, and wash them once with PBS.

[0108] (2) Resuspend the cells in 100 μL PBS, add 5 μL FITC-PD-L1 antibody to each group, and incubate at 4°C in the dark for 1 h.

[0109] (3) Wash once with PBS, resuspend the cells in 300 μl PBS, filter once with a mesh, add to the flow cytometer, test on the flow cytometer, save and analyze the data.

[0110] (4) Figure 13 As shown in the figure, compared with RPMI-8226, K562, Hela, and MCF-7 cells, 786-O cells and CAKI-1 cells highly express PD-L1 on their surfaces, with CAKI-1 cells expressing a higher level.

[0111] 2.3 Effects of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA on the cytotoxic activity of NK cells in vitro

[0112] (1) NK-92 cells were used as effector cells and CAKI-1 cells were used as target cells. They were plated into a 96-well plate at a ratio of 300,000 NK-92 cells: 100,000 target cells.

[0113] (2) PBS group, M-PEG-PLGA group, IL-15+PD-1 group, IL-15-PEG-PLGA+PD-1 group, and IL-15 / PD-1 / PEG-PLGA group were added and incubated for 4 h.

[0114] (3) Add CCK-8, 20 μL per well, incubate for 2 h, measure the absorbance at 450 nm, and calculate the NK cell killing rate. The calculation formula is as follows:

[0115]

[0116] Among them, OD e : OD450 of NK-92 cells;

[0117] OD t : OD450 of CAKI-1 cells;

[0118] OD e+t : OD450 of NK-92 cells+CAKI-1.

[0119] (4) Figure 14 (A: NK cell killing results under the microscope; B: Effect of dual-targeted nanomicelles on NK cell killing rate; * and ** indicate significant differences (p<0.05 and p<0.01)) It can be seen that compared with the Control group, the NK cell killing activity against CAKI-1 cells in the free IL-15+PD-1 group, IL-15-PEG-PLGA+PD-1 group, and IL-15 / PD-1 / PEG-PLGA group was significantly enhanced. Compared with the Control group, the free IL-15+PD-1, IL-15-PEG-PLGA+PD-1, and IL-15 / PD-1 / PEG-PLGA groups all increased the NK cell killing activity against CAKI-1 cells, which increased by 28.11±2.43% (p<0.05), 34.89±2.1% (p<0.05), and 30.27±0.79% (p<0.05), respectively.

[0120] 2.4 Effect of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA on IFN-γ secretion levels of NK cells in vitro

[0121] (1) Remove the kit and equilibrate to room temperature. After the killing is completed, aspirate the supernatant from the co-culture wells of NK cells and target cells in different groups, rotate at 2000 r / min for 10 minutes, and then aspirate the supernatant as the test sample.

[0122] (2) After adding 50 μL of IFN-γ standards of different concentrations and supernatants of NK cells from different groups into the wells, 100 μL of HRP-labeled detection antibody was added to each well. Each group was repeated at least 3 times. After sealing, the wells were incubated at 37°C for 1 hour.

[0123] (3) Drain the liquid and add 350 μL of the previously prepared washing solution to each well. Wash the plate three times. Prepare the colorimetric solution with a 1:1 volume ratio of substrate A to substrate B. Mix well and add 100 μL to each well. Incubate at 37°C in the dark for 15 min.

[0124] (4) Terminate the reaction, add 50 μL to each well, and measure the absorbance at 450 nm within 20 min.

[0125] (5) The results are as follows Figure 15 (As shown in the figure, ** indicates significant differences, and *** indicates extremely significant differences (p<0.01 and p<0.001)), compared with the Control group, the free IL-15+PD-1 group, IL-15-PEG-PLGA+PD-1 group, and IL-15 / PD-1 / PEG-PLGA group all significantly increased the secretion of NK cell interferon-γ, which increased by 30.81±4.82% (p<0.01), 38.37±14.4% (p<0.001), and 37.52±4.53% (p<0.001), respectively, which is similar to the trend of the killing rate.

[0126] 2.5 In vitro binding characteristics of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA and free PD-1:

[0127] (1) Dilute PD-L1 from 100 μg / mL to 20 μg / mL using the prepared coating buffer. Coat 100 μL per well of the enzyme-labeled strip and incubate overnight at 4°C. Add 300 μL / well of wash buffer and soak for 1 minute. Pat the plate dry and repeat the wash five times.

[0128] (2) Add 300 μL / well of blocking buffer and incubate at room temperature for 60 min. Add 300 μL / well of washing solution and soak for 1 min. Pat the plate dry.

[0129] (3) Add the sample to be tested, diluted to the appropriate multiple, 100 μL / well, and incubate at room temperature for 2 h. Add 300 μL / well of washing solution and soak for 1 min. Pat the plate dry and repeat the wash five times.

[0130] (4) Dilute 0.2 mg / mL of HRP-labeled mouse anti-human PD-1 antibody to 0.3 μg / mL using the prepared dilution buffer and incubate at room temperature for 60 min. Add 300 μL / well of wash buffer and soak for 1 min. Pat the plate dry and repeat the wash five times.

[0131] (5) Prepare the colorimetric solution 15 minutes in advance with a 1:1 ratio of substrate A to substrate B. Mix and store in the dark. Add 200 μL / well and incubate in the dark at room temperature for 20 minutes. Add 50 μL / well of stop solution and mix thoroughly.

[0132] (6) The results are as follows Figure 16As shown, PD-1 in the dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA can bind to PD-L1. This demonstrates that PD-1 is successfully loaded onto the surface of the dual-targeted nanomicelles. It also indicates that PD-1 on the dual-targeted nanomicelles, modified with biotin and linked to streptavidin, retains its ability to recognize and bind to PD-L1, and does not lose its function due to biotin modification. To eliminate the possible influence of IL-15, IL-15-PEG-PLGA nanomicelles loaded only with IL-15 were prepared. ELISA results showed that these nanomicelles had no ability to bind to PD-L1, further demonstrating the in vitro binding characteristics of the dual-targeted nanomicelles with PD-L1.

[0133] 2.6 In vitro binding characteristics of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA with CAKI-1 cells that overexpress PD-L1:

[0134] A cell invasion assay was used to investigate the ability of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA to enhance the ability of NK cells to specifically target tumor cells. The specific steps are as follows:

[0135] (1) CAKI-1 cells that highly express PD-L1 were used as target cells, and MCF-7 cells were used as negative control cells. 200,000 cells were seeded per well in a 24-well plate and cultured overnight.

[0136] (2) NK cells were incubated with Dye eFlour 670 at 37°C in the dark for 10 min, and cold culture medium containing 10% FBS was added to terminate labeling.

[0137] (3) The cells were incubated on ice for 5 minutes and washed three times with complete medium. The labeled NK cells were added to the chamber, which was placed in a 24-well plate seeded with target cells. IL-15-PEG-PLGA and IL-15 / PD-1 / PEG-PLGA were added, respectively. After incubation at 37°C for 6 hours, the chamber was removed and the number of NK cells targeting tumor cells was observed and counted under a fluorescence inverted microscope.

[0138] (4) Figure 17 (In the figure: ns indicates no significant difference (p>0.05); ** indicates significant difference (p<0.01)). Compared with the control group and IL-15-PEG-PLGA, IL-15 / PD-1-PEG-PLGA significantly promoted NK cell migration toward CAKI-1 cells that highly expressed PD-L1, increasing the effect by approximately 2.5-fold (p<0.01). However, this effect was not evident in MCF-7 cells that lowly expressed PD-L1.

[0139] Example 3:

[0140] To investigate the in vivo antitumor activity of dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA.

[0141] (1) 5×10 6 CAKI-1 cells were resuspended in serum-free medium and Matrigel (1:1).

[0142] (2) When the tumor volume reaches 50 mm 3 Mice were randomly divided into four groups: no treatment (PBS), IL-15 + PD-1, IL-15-PEG-PLGA + PD-1, and IL-15 / PD-1 / PEG-PLGA. Each group received a tail vein injection once daily for five consecutive days, followed by a two-day drug-free period, for two consecutive weeks. The daily IL-15 injection dose was 0.75 μg (referring to the clinical cytokine therapy dose). Tumor volume was calculated as follows:

[0143]

[0144] Where: a: tumor length;

[0145] b: Tumor width.

[0146] (3) Tumor volume was measured every 2 days using a vernier caliper. When the tumor volume reached 1000 mm 3 The mice were sacrificed at 4 hr. The tumor masses were removed, weighed, and their length and width were measured. The relative tumor volume (RTV), relative tumor proliferation rate (T / C), and tumor growth inhibition rate were calculated based on the measurement results. The calculation formulas are as follows:

[0147]

[0148] Wherein: V0: tumor volume measured at the time of cage administration;

[0149] V t : The volume of the tumor at each measurement.

[0150]

[0151] Where: T RTV : relative tumor volume of treatment groups;

[0152] C RTV : Relative tumor volume of the control group.

[0153]

[0154] Where: W c : Average tumor weight of the control group;

[0155] W t : Average tumor weight of treatment groups.

[0156] (4) The results are as follows Figure 18 As shown (A: tumor growth curve; B: tumor size; C: tumor weight; D: tumor inhibition rate; ns indicates no significant difference (p>0.05); * and ** indicate significant differences (p<0.05 and p<0.01); *** and **** indicate extremely significant differences (p<0.001 and p<0.0001)).

[0157] The IL-15+PD-1, IL-15-PEG-PLGA+PD-1, and IL-15 / PD-1 / PEG-PLGA groups all effectively inhibited tumor growth. Compared to the other treatment groups, the dual-targeted polymer micelle group was more significantly able to inhibit tumor growth and reduce tumor volume. Furthermore, based on the tumor weight of each treatment group, the tumor inhibition rate was 53.25±4.55% in the IL-15+PD-1 group, 61.04±2.83% in the IL-15-PEG-PLGA+PD-1 group, and significantly increased to 71.65±4.56% in the IL-15 / PD-1 / PEG-PLGA group (p<0.01).

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing dual-targeted nanomicelles, characterized in that: The steps include: Step 1: Synthesize PLGA-PEG-SA, specifically: First, dissolve streptavidin in PBS to prepare a streptavidin aqueous solution with a concentration of 1-4 mg / ml; Then, PLGA10k-PEG5k-NHS at a concentration of 5 to 10 mg / ml is added dropwise to a streptavidin aqueous solution for reaction. The resulting reaction solution is dialyzed in pure water. The dialyzed solution is frozen and dried to obtain PLGA-PEG-SA having a molecular weight greater than that of the streptavidin raw material. The molar ratio of PLGA10k-PEG5k-NHS to streptavidin is 5-10:

1. Step 2: Use biotin to modify proteins IL-15 and PD-1, specifically: Add protein IL-15 to an ultrafiltration tube, then add biotin and pipette. After the biotin and protein IL-15 are evenly mixed, place in an incubator in the dark for incubation, and then centrifuge. Discard the lower layer solution, add labeling buffer, and centrifuge multiple times to remove unbound biotin molecules. Biotin-labeled protein IL-15 is obtained in the upper layer liquid of the ultrafiltration tube. The amount of biotin added is calculated according to the molar ratio of biotin: protein IL-15 = 10 to 20:1; The same method was used to obtain biotin-labeled protein PD-1, wherein the amount of biotin added was calculated according to a molar ratio of biotin: protein PD-1 = 10-20:1; Step 3: Synthesize dual-targeted nanomicelles IL-15 / PD-1 / PEG-PLGA, specifically: PLGA-PEG-SA was dissolved in DMSO and reacted with biotin-labeled proteins IL-15 and PD-1. The resulting reaction solution was placed in a dialysis bag and dialyzed against PBS overnight to remove DMSO. The unloaded proteins were then removed to obtain the dual-targeted nanomicelle IL-15 / PD-1 / PEG-PLGA. Among them, PLGA-PEG-SA was dissolved in DMSO and prepared at a ratio of 10 mg PLGA-PEG-SA dissolved in 1 mL DMSO; The molar ratio of the sum of biotin-labeled proteins IL-15 and PD-1 to PLGA-PEG-SA is 1:1-10; the molar ratio of biotin-labeled protein IL-15 to biotin-labeled protein PD-1 is 1:1-3.

2. The method for preparing a dual-targeted nanomicelle according to claim 1, wherein: In step 1, the concentration of the reactant streptavidin aqueous solution is 2 mg / ml, the concentration of PLGA10k-PEG5k-NHS is 10 mg / ml, and the molar ratio of PLGA10k-PEG5k-NHS to streptavidin is 5:

1.

3. The method for preparing a dual-targeting nanomicelle according to claim 1 or 2, wherein: In step 1, the reaction time of adding PLGA10k-PEG5k-NHS dropwise to the streptavidin aqueous solution is 4-8 hours; the dialysis bag used for dialysis has a specification of MWCO8000-14000.

4. The method for preparing a dual-targeted nanomicelle according to claim 1, wherein: The amount of biotin added in step 2 is calculated according to the molar ratio of biotin: protein IL-15 = 20:1; Biotin: protein PD-1 = 20:

1.

5. The method for preparing a dual-targeting nanomicelle according to claim 1 or 4, wherein: The temperature in the incubator in step 2 is 37° C., and the incubation time in the dark is 30 min. The centrifugal force during centrifugation is 12,000×g, and the centrifugation time is at least 10 min.

6. The method for preparing a dual-targeting nanomicelle according to claim 1, wherein: Step 2 also includes taking out the biotin-labeled proteins IL-15 and PD-1, adding them to the preservation solution at a ratio of 1:1, and storing them at a temperature of -20°C.

7. The method for preparing a dual-targeting nanomicelle according to claim 1, wherein: The reaction in step 3 is carried out at a temperature of 37° C. and a stirring speed of 500 rpm, and the stirring reaction time is at least 4 hours in the dark; The dialysis bag specification is MWCO8000-14000; The removal of unloaded proteins is performed by placing the reaction solution after DMSO removal into an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and then centrifuging the tube at a centrifugal force of 5000×g and a temperature of 4° C. for at least 10 minutes.

8. The method for preparing a dual-targeting nanomicelle according to claim 1, wherein: In step 3, the molar ratio of the sum of the biotin-labeled proteins IL-15 and PD-1 to PLGA-PEG-SA is 1:5; the molar ratio of the biotin-labeled protein IL-15 to the labeled protein PD-1 is 1:

2.

9. A dual-targeting nanomicelle obtained by the preparation method according to claims 1-8, characterized in that: The loading rates of IL-15 and PD-1 in the dual-targeted nanomicelles were 62.05±3.53% and 33.35±4.59%, respectively; the molar ratio of IL-15 and PD-1 loaded on the surface of the dual-targeted nanomicelles was close to 1:

1.

10. The dual-targeting nanomicelle according to claim 9, wherein The nanomicelles have regular morphology, uniform dispersion, and a spherical shape with a particle size of 254.76±28.02 nm, a ζ potential of -7.64±2.64 mV, and a PDI of IL-15 / PD-1 / PEG-PLGA of 0.18.

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