Redox nanoparticle and live cell carrier and use thereof

Abstract

The application discloses a redox response type nanoparticle and a living cell carrier, so as to realize the reduction response release of the nanoparticle in a tumor environment and the effect of activating immune cells such as NK cells at a fixed point. The NK cell nanoengineering method of the application can simply and efficiently produce a large number of stable nanoparticles, and load the nanoparticles on the surface of living NK cells, and successfully solves the problem that exogenous cytokines cannot be transplanted around NK cells to produce high-dose accumulation to activate NK cells.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine, specifically relating to a novel redox nanoparticle and live cell carrier, particularly a method for preparing cytokine nanoparticles (ILNPs) and the construction and application of live NK cell carriers (NK-NPs). Background Technology

[0002] Natural killer (NK) cells play a crucial role in directly killing malignant tumor cells and regulating adaptive immune responses. NK cell immunotherapy can serve as a safe, off-the-shelf adoptive cell immunotherapy. NK cells can more quickly recognize and eliminate tumor cells without antigen sensitization or MHC restriction, playing a key role in early immune defense. Simultaneously, NK cells can regulate adaptive immune responses by secreting various cytokines and chemokines (such as IFN-γ and TNF-α), further stimulating T cell activation and leading to a cascade of immune stimulation responses. However, in the treatment of solid tumors, NK cells face challenges such as proliferation, persistence, and insufficient activation, significantly diminishing the effectiveness of theoretically effective NK cell immunotherapy.

[0003] NK cell development and function are regulated by multiple signaling pathways, including cytokines, activating receptors, and inhibitory receptors. Cytokines such as IL-21, IL-12, IL-15, IL-17, and IL-2 are crucial for activating NK cells and initiating transcription factors to regulate autoimmune responses, enhancing NK cell cytotoxicity, promoting NK cell proliferation and differentiation, and accelerating NK cell maturation and the expression of related receptors. However, exogenous cytokines cannot accumulate sufficiently around NK cells in vivo to create a high concentration zone sufficient to activate NK cell function, which significantly limits the application of cytokines.

[0004] Currently, "cocktail therapy," which combines multiple drugs and treats tumors through different mechanisms, has become an effective means of treating cancer, while the rapid development of nanotechnology has also provided new materials and new ideas for cancer treatment.

[0005] Nanocarriers are widely used due to their ability to improve drug solubility and stability, achieving low-toxicity, high-efficiency, and sustained-release antitumor effects. Nanoparticles, with their small size and high specific surface area, are the primary candidates for drug-release carriers. However, there is still much room for exploration in areas such as enhancing the targeting of nanoparticles, improving their response to the tumor microenvironment, achieving sustained and controlled release of delivered drugs, maintaining structural integrity under normal physiological conditions, releasing drugs only at the lesion site, thereby reducing damage to normal tissues and maximizing tumor-killing effects.

[0006] Linking NK cells with nanoparticles enables the nanoengineering of NK cells, endowing them with relevant functions, such as carrying chemotherapy nanoparticles to enhance anti-tumor effects. This provides a new clinical basis for modified NK cell immunotherapy that combines cells with exogenous cytokines.

[0007] In existing technologies, co-incubating free exogenous cytokine IL-21 with NK cells (Evelyn O Ojo et al., Sci Rep. 2019 Oct 17; 9(1):14916., doi:10.1038 / s41598-019-51287-6) can achieve in vitro expansion of NK cells. However, free cytokines lack target selectivity, are easily cleared by blood circulation, and cannot reach an effective stimulating concentration around NK cells. Furthermore, a large amount of free exogenous cytokines entering the body can easily trigger a cytokine storm. With the development of nanocarrier delivery technology, loading NK cells onto delivery carriers provides a solution to the above problems. Generally speaking, there are two methods for modifying the surface of nanoparticles: electrostatic adsorption and chemical bonding. Compared with electrostatic adsorption, chemical bonding makes the target substance more stable in binding to the particle and less likely to detach from the particle surface, such as CN113004418A. Maleimide groups are used to couple with thiol groups on NK cells to achieve selective modification of NK cells, such as CN113209019A. However, the newly generated CS bonds by this covalent bonding method are not stable enough under physiological conditions and will undergo reverse Michael addition, resulting in instability and low efficiency.

[0008] In order to solve the technical problems that have existed in the prior art for a long time, the present invention aims to overcome the shortcomings of free exogenous cytokine IL-21 being unable to aggregate around NK cells and the unstable, inefficient and poor specificity of nanoparticles attached to the surface of NK cells. The invention provides a novel redox-responsive nanoparticle and a live cell carrier that enables the nanoparticles to be stably and efficiently "carried" on the surface of NK cells. Summary of the Invention

[0009] To address the above problems, this invention provides a method for preparing redox-responsive nanoparticles (ILNPs), the method comprising the following steps:

[0010] 1) Dissolve a reduction-responsive, high-efficiency crosslinking agent in dimethyl sulfoxide (DMSO) solution to obtain a crosslinking agent solution;

[0011] 2) Dissolve the cytokine protein powder in phosphate buffered saline (PBS) to obtain a cytokine protein solution;

[0012] 3) Mix the cross-linking agent solution with the cytokine protein solution to obtain a mixture. Take an appropriate amount of the mixture and quantify the mixture with PBS buffer. Then, rotate and stir at 20℃-35℃ and 500-2500 rpm. Add the recognition group of live cells to the above solution and rotate and stir again at 20℃-35℃ and 500-2500 rpm. After ultrafiltration, redox-responsive nanoparticles (ILNPs) are obtained.

[0013] The reduction-responsive high-efficiency crosslinking agent is selected from any one or more of the following:

[0014] A.NPC-NH-SS-NH-NPC, its structural formula is as follows:

[0015]

[0016] B.NPC-O-SS-O-NPC, its structure is as follows:

[0017]

[0018] C.NHS-SS-NHS, its structural formula is as follows:

[0019]

[0020] Preferably, the reduction-responsive high-efficiency crosslinking agent is NPC-NH-SS-NH-NPC.

[0021] Further, the cytokine is an interleukin; preferably, the interleukin includes any one or more of the following: IL-21, IL-12, IL-15, IL-17, IL-2; more preferably, the cytokine is IL-21.

[0022] Further, the recognition group of the living cells includes any one or more of the following: Anti-CD45, Anti-CD56, NHS active ester, and Mal (maleimide) active ester; preferably, the recognition group of the living cells is Anti-CD45.

[0023] Furthermore, the cross-linking agent solution and the cytokine protein solution are mixed in a molar ratio of 0.1:1 to 10:1.

[0024] Furthermore, the stirring time is 10-60 min, preferably 30 min; the ultrafiltration conditions are 1-3 ultrafiltrations with an ultrafiltration tube, each ultrafiltration lasting 5-25 min, preferably 2 ultrafiltrations with an ultrafiltration tube, each ultrafiltration lasting 10 min.

[0025] Furthermore, the particle size of the ILNPs is approximately 50-300 nm, and more preferably, the particle size of the ILNPs is approximately 100-200 nm.

[0026] The present invention also provides redox-responsive nanoparticles (ILNPs) prepared by the above method.

[0027] This invention also provides a method for constructing a live cell carrier that combines nanoparticles with immune cells, comprising the following steps:

[0028] 1) Preparation of ILNPs

[0029] ILNPs were obtained using the preparation method described above;

[0030] 2) Culture and expansion of immune cells

[0031] Immune cells were collected and expanded using a suitable culture medium containing 10% fetal bovine serum.

[0032] 3) Construction of live cell carriers for immune cells

[0033] In a suitable culture medium, the immune cells amplified in step 2) are co-incubated with the ILNPs prepared in step 1) in proportion for 1-2 hours. After centrifugation to remove unconnected ILNPs, a live cell carrier of nanoparticles combined with immune cells is obtained.

[0034] Furthermore, the immune cells include modified immune cells or unmodified isolated immune cells, the modification including genetic modification and physicochemical modification, and the immune cells include NK cells, DC cells, CIK cells, DC-CIK cells, T cells and / or B cells.

[0035] Furthermore, the immune cells are NK cells, including modified NK cells or unmodified isolated NK cells, wherein the modification includes genetic modification and physicochemical modification, and the culture medium is 1640 culture.

[0036] The present invention also provides a live cell carrier of nanoparticles bound to immune cells prepared by the above method, preferably the live cell carrier of nanoparticles bound to immune cells is a live cell carrier (NK-NPs) of nanoparticles containing IL-21 bound to NK cells.

[0037] Furthermore, the NK cells include modified NK cells or unmodified isolated NK cells, and the modification includes genetic modification and physicochemical modification.

[0038] The present invention also provides a composition containing the ILNPs and / or the live cell carrier, preferably, the composition being a pharmaceutical composition and further containing a medically or pharmaceutically acceptable carrier or excipient.

[0039] The present invention also provides the use of the ILNPs and / or the live cell carrier in the preparation of medicaments for treating diseases, preferably including tumors, inflammation, Alzheimer's disease and / or cardiovascular and cerebrovascular diseases.

[0040] Further, the ILNPs, the drug, or the composition further includes an antitumor drug; preferably, the antitumor drug includes a broad-spectrum antitumor drug and / or a targeted antitumor drug; more preferably, the broad-spectrum antitumor drug is selected from one or more of camptothecin-based drugs, doxorubicin-based drugs, taxane-based drugs, or platinum-based drugs, and the targeted antitumor drug is selected from zanubrutinib, nilotinib, imatinib, vemodilamide, vemurafenib, tesimolimus, sunitinib, celitinib, and regorafenib. The following are one or more of the following: afatinib, trametinib, pontezomib, pazopanib, axitinib, romidesin, everolimus, ibrutinib, lenvatinib, dabrafenib, crizotinib, carfilzomib, ostinotinib, cabozantinib, carbitinib, gefitinib, vorinostat, vandetanib, alectinib, denosumab, sorafenib, bosutinib, belistat, olaparib, aflibercept, lapatinib, dasatinib, palbociclib, palbistat, or erlotinib.

[0041] Further, the ILNPs, the drug, or the composition further include polypeptides, the polypeptides comprising antigens or antibodies. More preferably, the antibody is selected from any one or more of adalimumab, cetuximab, teimomab, trastuzumab, nivolumab, daralimumab, ramucirumab, nexituzumab, pembrolizumab, pembrolizumab, ofamumab, bonatumab, bevacizumab, panitumab, oxetuzumab, bentuximab, denutucizumab, tosimomab, erlotuzumab, trastuzumab, or rituximab.

[0042] Furthermore, the tumor is selected from one or more of the following: basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, nasopharyngeal carcinoma, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.

[0043] Further, the ILNPs, the drug, or the composition are administered via an injectable dosage form; preferably, the injection includes one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymph node injection, intratumoral injection, or foot injection.

[0044] This invention discloses a method for constructing redox-responsive IL-21 nanoparticles and a live-cell carrier, enabling the nanoparticles to release in a reduction-responsive manner and activate NK cells at specific sites in a tumor environment, thus providing a method for the nanoengineering of NK cells. The method involves mixing IL-21 protein with a redox-reactive crosslinking agent NPC-NH-SS-NH-NPC (bis(4-nitrophenyl), 344741-95-3). Next, the live-cell recognition group Anti-CD45 is added to the surface of the nanoparticles, allowing them to be loaded onto the surface of NK cells via an antigen-antibody reaction, thus preparing spherical ILNP nanoparticles. This method can easily and efficiently produce a large number of stable nanoparticles. Subsequently, the ILNPs are co-incubated with NK cells, allowing the NK cells to "hitch a ride" on the nanoparticles, thus preparing the live-cell carrier NK-NPs, achieving efficient connection and synchronous transport of NK cells and nanoparticles.

[0045] The release of NK cell-nanocarriers (NK-NPs) at the lesion site in this invention: Under the stimulation of the microenvironment at the lesion site (including but not limited to tumors, such as inflammation, Alzheimer's disease, and other cardiovascular and cerebrovascular diseases), the reducing power of the NK cell surface increases, leading to the release of exogenous cytokines (including but not limited to IL-21, such as IL-12, IL-15, IL-17, IL-2, etc.) from the redox-responsive nanoparticles. This creates a high-cytokine-stimulated region around the NK cells, achieving in situ, controllable, and efficient stimulation of NK cell activation. This successfully solves the problem that exogenous cytokines cannot be transplanted to the vicinity of NK cells to generate high-dose accumulation for NK cell activation.

[0046] Compared with the prior art, the present invention has the following beneficial technical effects:

[0047] 1. This invention uses a two-step shaking and stirring method to prepare ILNP nanoparticles. A novel crosslinking agent, NPC-NH-SS-NH-NPC, is used to directly crosslink cytokines (including but not limited to IL-21, such as IL-12, IL-15, IL-17, IL-2, etc.) together to form a redox-responsive nanoparticle with obvious controllability and high specificity.

[0048] 2. A method for constructing nanoparticles based on proteins and cross-linking agents, characterized by mild, simple, efficient, rapid, and reproducible reaction conditions.

[0049] 3. Since the preparation of nanoparticles in this invention is based on the action of the novel cross-linking agent NPC-NH-SS-NH-NPC, the method of this invention can be applied to the encapsulation of various functional proteins and small molecule drugs, such as different cytokines, into nanoparticles.

[0050] 4. Since the construction of the NK cell live cell vector of the present invention is based on the protein specifically expressed on the surface of NK cells, we can utilize this feature to apply the construction concept of live cell vector to the engineering connection and modification of nanoparticles of other cells, bacteria, viruses and other organisms.

[0051] 5. For relatively complex biological systems such as blood and human tissues, this live-cell vector construction method exhibits significant antibody specificity, greatly avoiding the false signals caused by non-specific labeling based on amino and carboxyl group reactions in traditional methods. Furthermore, compared to positive and negative charge adsorption methods, the labeled products are more stable due to the specific binding of antigen and antibody.

[0052] 6. The method for constructing live cell vectors of the present invention is simple and easy to implement, and is convenient for operation and promotion. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of redox-responsive nanoparticles (ILNPs).

[0054] Figure 2 This is a Malvern particle size analyzer image of ILNP nanoparticles.

[0055] Figure 3 This is a schematic diagram of the live cell vector NK-NPs.

[0056] Figure 4 This is a schematic diagram illustrating the release of nanoparticles.

[0057] Figure 5 This is a scanning electron microscope image of nanoparticles on the surface of NK cells.

[0058] Figure 6 A laser confocal image successfully constructed to identify live cell carriers by labeling nanoparticles and NK cells. Detailed Implementation

[0059] The present invention will be further described in detail below through specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0061] Example 1: Preparation of ILNP nanoparticles

[0062] 0.05 μmol of NPC-NH-SS-NH-NPC was dissolved in 25 μl of dimethyl sulfoxide (DMSO) solution, and 0.0335 μmol of IL-21 protein powder was dissolved in 50 μl of PBS solution. Both solutions were placed in the same EP tube, and the mixture was diluted to 1 mL with PBS buffer. The mixture was then stirred at 25 °C and 1200 rpm for 30 min. Next, 10 μl of 1 mg / mL Anti-CD45 was added to the above solution, and the mixture was stirred again at 25 °C and 1200 rpm for 30 min. Ultrafiltration was performed twice, for 10 min each time, to obtain redox-responsive nanoparticles (ILNPs) with a particle size of approximately 100-200 nm. Figure 1 and Figure 2 The diagram shows a schematic of the nanoparticles and their hydrated particle size.

[0063] Example 2: Preparation of ILNP nanoparticles

[0064] 1 μmol of NPC-NH-SS-NH-NPC was dissolved in 25 μl of dimethyl sulfoxide (DMSO) solution, and 0.05 μmol of IL-21 protein powder was dissolved in 50 μl of PBS solution. Both were placed in the same EP tube, and the mixture was diluted to 2 ml with PBS buffer. The mixture was then stirred at 25 °C and 1200 rpm for 60 min. Next, 5 μl of 1 mg / ml Anti-CD45 was added to the above solution, and the mixture was stirred again at 25 °C and 1200 rpm for 60 min. Ultrafiltration was performed once, for 5 minutes each time, to obtain redox-responsive nanoparticles (ILNPs). Figure 3 This is a schematic diagram of nanoparticle release.

[0065] Example 3: Construction of a live cell vector

[0066] Take 500,000 NK cells, centrifuge and wash, replace with fresh 1640 medium, then add the prepared nanoparticles (10 μl, 10 μg / mL), incubate for 1 hour, then centrifuge to remove unlinked nanoparticles, thus obtaining the live cell carrier NK-NPs. Figure 3 A schematic diagram of a live cell carrier, as shown below. Figure 4 Scanning electron microscope and Figure 5 As shown in the flow cytometry analysis, reduction-responsive nanoparticles were successfully loaded onto the surface of NK cells.

[0067] Example 4: Construction and confocal observation of live cell carriers

[0068] Take 1 million NK cells, centrifuge and wash them, and then seed the NK cells evenly into 8-well culture plates. Nunc (USA), replace with fresh 1640 medium, adding 200 μL of medium to each well. After 24 hours, replace with 1640 medium containing 2 μl of 10 μg / mL nanoparticles per well. After 1 hour of incubation, wash twice with PBS, stain cell nuclei, and observe the 8-well plate under a laser scanning confocal microscope (Leica TCS SP5, Germany). Figure 6 As shown, the nanoparticles are distributed on the surface of the NK cell membrane. Blue represents the fluorescence of the cell nucleus Hoechest 33258, and red represents the fluorescence of ILNPs, indicating that the nanoparticles and ILNPs were successfully linked to the surface of NK cells, thus successfully constructing a live NK cell carrier.

[0069] 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 within the protection scope of the present invention.

Claims

1. A method for preparing redox-responsive nanoparticles (ILNPs), the method comprising the following steps: 1) Dissolve a reduction-responsive, high-efficiency crosslinking agent in dimethyl sulfoxide (DMSO) solution to obtain a crosslinking agent solution; 2) Dissolve the cytokine protein powder in phosphate-buffered saline (PBS) to obtain a cytokine protein solution; 3) Mix the cross-linking agent solution with the cytokine protein solution to obtain a mixture. Take an appropriate amount of the mixture and quantify it with PBS buffer. Then, rotate and stir at 20℃-35℃ and 500-2500 rpm. Add the recognition group of live cells to the above solution and rotate and stir again at 20℃-35℃ and 500-2500 rpm. After ultrafiltration, redox-responsive nanoparticles (ILNPs) are obtained. The reduction-responsive high-efficiency crosslinking agent is NPC-NH-SS-NH-NPC, and its structural formula is as follows: ； The ILNPs have a particle size of 50-300 nm; the cytokine is IL-21.

2. The method according to claim 1, wherein the recognition group of the living cell includes any one or more of the following: Anti-CD45, Anti-CD56, NHS active ester, and Mal (maleimide) active ester.

3. The method according to claim 1, wherein the cross-linking agent solution and the cytokine protein solution are mixed in a molar ratio of 0.1:1 to 10:

1.

4. The method according to claim 1, wherein the stirring time is 10-60 min; and the ultrafiltration conditions are ultrafiltration 1-3 times with an ultrafiltration tube, each time for 5-25 min.

5. The method according to claim 4, wherein the stirring time is 30 min, and the ultrafiltration conditions are ultrafiltration twice with an ultrafiltration tube, each time for 10 min.

6. The method according to claim 1, wherein the particle size of the ILNPs is 100-200 nm.

7. Redox-responsive nanoparticles (ILNPs) prepared by the method according to any one of claims 1-6.

8. A method for constructing a live cell carrier that combines nanoparticles with immune cells, comprising the following steps: 1) Preparation of ILNPs ILNPs were obtained according to the preparation method described in any one of claims 1-6; 2) Culture and expansion of immune cells Immune cells were collected and expanded using a suitable culture medium containing 10% fetal bovine serum; 3) Construction of live cell carriers for immune cells In a suitable culture medium, the immune cells amplified in step 2) are co-incubated with the ILNPs prepared in step 1) in proportion for 1-2 hours. After centrifugation to remove unlinked ILNPs, a live cell carrier of nanoparticles combined with immune cells is obtained. The live cell carrier of nanoparticles combined with immune cells is a live cell carrier of NK cells combined with nanoparticles containing IL-21.

9. The method according to claim 8, wherein the immune cells comprise modified immune cells or unmodified isolated immune cells, the modification comprising genetic modification and physicochemical modification, and the immune cells comprising NK cells, DC cells, CIK cells, DC-CIK cells, T cells and / or B cells.

10. The method according to claim 9, wherein the immune cell is an NK cell, the NK cell includes modified NK cells or unmodified isolated NK cells, the modification includes genetic modification and physicochemical modification, and the culture medium is 1640 culture medium.

11. A live cell carrier for binding immune cells to nanoparticles prepared by the method according to any one of claims 8-10.

12. The live cell carrier according to claim 11, wherein the live cell carrier for binding immune cells to nanoparticles is a live cell carrier (NK-NPs) for binding IL-21-containing nanoparticles to NK cells, wherein the NK cells include modified NK cells or isolated NK cells without modification, and the modification includes genetic modification and physicochemical modification.

13. A composition comprising the ILNPs of claim 7 and / or the live cell carrier of any one of claims 11-12.

14. The composition according to claim 13, wherein the composition is a pharmaceutical composition and further comprises a medically or pharmaceutically acceptable carrier or excipient.

15. The use of the ILNPs of claim 7 and / or the live cell carrier of any one of claims 11-12 in the preparation of a medicament for treating a disease, wherein the disease is a tumor.

16. The composition according to any one of claims 13-14 or the application according to claim 15, wherein the composition or the medicament further comprises an antitumor drug.

17. The composition or application according to claim 16, wherein the antitumor drug comprises a broad-spectrum antitumor drug and / or a targeted antitumor drug.

18. The composition or application according to claim 17, wherein the broad-spectrum antitumor drug is selected from one or more of camptothecin derivatives, doxorubicin derivatives, taxane derivatives, or platinum-based drugs, and the targeted antitumor drug is selected from one or more of zanubrutinib, nilotinib, imatinib, vemodilution, vemurafenib, tesimolimus, sunitinib, celitinib, regorafenib, afatinib, trametinib, saponinib, bortezomib, pazopanib, axitinib, romidesin, everolimus, ibrutinib, lenvatinib, dabrafenib, crizobinib, denosumab, sorafenib, bosutinib, belistat, olaparib, aflibercept, lapatinib, dasatinib, palbociclib, palbitalstat, or erlotinib.

19. The composition of claim 13 or the application of claim 15, wherein the composition or the drug further comprises a polypeptide.

20. The composition or application according to claim 19, wherein the polypeptide comprises an antigen or an antibody.

21. The composition or application according to claim 20, wherein the antibody is selected from any one or more of adalimumab, cetuximab, teimomab, trastuzumab, nivolumab, daralimumab, ramucirumab, nexituzumab, pembrolizumab, pembrolizumab, ofamumab, bonatumab, bevacizumab, panitumab, oxetuzumab, bonatuzumab, denutucizumab, tosimomab, erlotuzumab, trastuzumab, or rituximab.

22. The application according to claim 15, wherein the tumor is selected from one or more of basal cell carcinoma, squamous cell carcinoma, esophageal cancer, malignant glioma, bladder cancer, cervical cancer, breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal carcinoma, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma, and blastoma.

23. The ILNPs of claim 7, the composition of claim 13, or the application of claim 15, wherein the ILNPs, the drug, or the composition are injectables.

24. The application according to claim 23, wherein the injection comprises any one or more of subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, intralymphatic injection, intratumoral injection, or foot injection.

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