A pka-controllable nanochaperone system and preparation and application thereof

Abstract

The application discloses a kind of nano-chaperone system with controllable pKa and its preparation and application;By self-assembly method, the complex micelles formed by three kinds of block copolymer PEG-b-PCL, phenylboronic acid modified PBA-PEG-b-PCL and PCL-b-P(D) AE are prepared, can load heat denaturation cytokine and chemotherapy drug paclitaxel and be composed of nano-chaperone system as in-situ cancer vaccine;The nano-chaperone IL-12 / PTX@P-nChap is injected intravenously in mouse body, can significantly reduce the immunotoxicity caused by IL-12 and prolong blood circulation time;In the tumor microenvironment, the refolding and release of IL-12 and the competitive loading of tumor-related antigens produced by tumor immunogenic cell death effectively induce cancer immune response, have significant inhibitory effect on late large "cold" tumor, have obvious distant effect on distal tumor and very good inhibit the recurrence and metastasis of tumor after operation.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a method for constructing a nano-molecular chaperone based on composite micelles that can regulate different acid dissociation constants (pKa). In particular, it also relates to the application of this nano-system to enhance the efficacy of cancer immunotherapy by selectively refolding and denaturing cytokines. Background Technology

[0002] Therapeutic cancer vaccines aim to deliver tumor antigens and adjuvants, activating the immune system to recognize and eliminate tumor cells, demonstrating significant efficacy in inhibiting primary tumor growth and preventing tumor recurrence and metastasis. In recent years, highly customizable, biocompatible, and biodegradable polymeric nanoparticles have been widely used in the preparation of nanovaccine formulations, capable of encapsulating and delivering various antigens and adjuvants, such as polylactic-co-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyethyleneimine (PEI), and poly-β-amino ester (PAE). Despite the success and great potential of nanovaccines, the clinical translation of therapeutic cancer vaccines remains challenging because they typically only elicit significant therapeutic responses when treating early-stage or highly inflammatory (hot) tumors, but are less effective when treating large, advanced, "cold" tumors. This is because cancer vaccines lack immunogenicity, which is primarily determined by antigenicity and adjuvant properties. Compared to nanovaccines that load antigens in vitro, in situ vaccination therapy utilizes the tumor itself as an antigen source to drive the immune response, effectively overcoming the inherent heterogeneity of tumors and enhancing the antigenicity of cancer vaccines.

[0003] Cytokines, as novel molecular adjuvants, play a central role in regulating immune responses. Despite their immense potential, the clinical application of cytokines in cancer therapy has been limited by severe dose-limiting toxicity resulting from systemic immune activation. To date, various approaches, including cytokine engineering (such as supercytokines, immune cytokines, and conditionally active cytokines), cytokine mimics, and targeted delivery systems, have been developed to reduce systemic toxicity and improve efficacy. However, these approaches rarely avoid toxicity caused by the activation of circulating immune cells at high doses. This is because the cytokines delivered in these methods are correctly folded, which inevitably activates immune cells. Another reason is that current systemic administration strategies cannot completely avoid off-target release of the carrier. Furthermore, the immunosuppressive environment of solid tumors raises the immune activation threshold of cytokines, thus limiting the efficacy of single-cytokine immunotherapy.

[0004] Interleukin (IL)-12 is one of the most promising anti-tumor therapeutic cytokines in clinical trials. It can stimulate both the innate and adaptive immune systems and elicit antigen-specific immune responses. As a pleiotropic cytokine, IL-12 can directly activate CD8+.+ Recombinant IL-12 plays a crucial role in the effector functions of T cells and natural killer (NK) cells, inducing the secretion of cytokines, particularly interferon-γ (IFN)-γ, thereby coordinating anti-tumor defense. However, due to its instability in vivo, short half-life, and high dosage, systemic administration of recombinant IL-12 can lead to severe dose-limiting immune-related adverse events (irAEs), such as cytokine release syndrome and liver injury caused by high concentrations of circulating IFN-γ. Therefore, it has not yet been successfully translated into clinical applications. Thus, based on the principle that protein structure determines its biochemical function and mimicking the function of natural molecular chaperones, it is essential to design a strategy that effectively avoids the targeted toxicity and non-tumor toxicity of cytokines by delivering a denatured form of IL-12. Although nanocomposite micelle delivery platforms cannot completely prevent off-target release, they can control the refolding of denatured protein carriers under both normal and tumor physiological conditions. Similar to natural chaperones that regulate protein folding through adenine triphosphate (ATP) hydrolysis, optimized nanomolecular chaperones utilize protonation isomerism to control the refolding of denatured proteins. Denatured proteins can only effectively refold into their native state in the tumor immune microenvironment (pH = 6.5), while in normal tissues (pH = 7.4), even if released, they remain misfolded and non-toxic. Furthermore, combining cytokine delivery with in situ vaccination can further enhance the efficacy of cancer immunotherapy. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides a nano-molecular chaperone system based on composite micelles with tunable pKa and its applications. The specific technical solution is as follows:

[0006] The present invention provides a method for constructing a nano-molecular chaperone system with tunable pKa, comprising the following steps:

[0007] 1) Polymer PEG-b-PCL and phenylboronic acid-modified PBA-PEG-b-PCL were synthesized by ring-opening polymerization (ROP) of ε-CL; PCL-bP(D)AE was synthesized by Michael addition polymerization of ε-CL with different proportions of 1,6-hexanediol diacrylate (HDD) and 1,10-decanediol diacrylate (DDD) and 4,4'-trimethylenedipiperidine (TDP).

[0008] 2) A series of single micelles and composite micelles with microphase separation structures were prepared by self-assembly, including single micelles PM and P-PM prepared by PEG-b-PCL or PEG-b-PCL and PBA-PEG-b-PCL; composite micelles MSPM prepared by PEG-b-PCL and PCL-bP(D)AE with different pKa values; nano molecular chaperone P-nChap prepared by PEG-b-PCL, PBA-PEG-b-PCL and PCL-bP(D)AE, wherein the pKa of PCL-bP(D)AE is 6.51; and nano molecular chaperones IL-12@P-nChap, PTX@P-nChap and IL-12 / PTX@P-nChap prepared by PEG-b-PCL, PBA-PEG-b-PCL, PCL-bP(D)AE, wherein the pKa of PCL-bP(D)AE is 6.51, thermally denatured IL-12 or the chemotherapeutic drug PTX.

[0009] The method for preparing the block copolymer of the present invention includes the following:

[0010] 1) Synthesis of PEG-b-PCL: PEG-b-PCL was synthesized via ROP of ε-CL monomer using PEG-OH as a macromolecular initiator and Sn(Oct)2 as a catalyst; PEG-OH was dried under vacuum before use; PEG-OH and ε-CL monomer were dissolved in an appropriate amount of anhydrous toluene, and then stirred at 110℃. After liquid nitrogen freezing-vacuuming-thawing, the solution was precipitated with excess ice-cold diethyl ether and filtered to obtain white powder product PEG-b-PCL.

[0011] 2) PBA-PEG-b-PCL: PBA-PEG-b-PCL is synthesized by reductive amination of PBA and NH2-PEG-b-PCL. NH2-PEG-b-PCL is synthesized via ROP using ε-CL as the monomer and BOC-NH-PEG-OH as the macromolecular initiator. BOC-NH-PEG-OH and ε-CL are dissolved in anhydrous toluene, and then reacted with stirring at 110°C. After a liquid nitrogen freezing-vacuuming-thawing process, the solution is precipitated with excess ice-cold diethyl ether and filtered to obtain a white powder product, BOC-NH-PEG-b-PCL. The product is dissolved in an appropriate amount of dichloromethane, and then trifluoroacetic acid is added. The reaction was carried out with stirring at 25°C. The reaction solution was dried by rotary evaporation, and an appropriate amount of anhydrous methanol was added to dissolve the crude product. The solution was then dried by rotary evaporation to remove excess trifluoroacetic acid. Subsequently, the residual trifluoroacetic acid was neutralized by adding an appropriate amount of DMSO and triethylamine. The white product NH2-PEG-b-PCL was precipitated with ice-cold diethyl ether, filtered, and dried under vacuum. Then, NH2-PEG-b-PCL and PBA were dissolved in an appropriate amount of DMF. After stirring at room temperature, sodium borohydride was added in batches until no bubbles were generated, and the reaction was continued to be stirred at room temperature. Subsequently, the product PBA-PEG-b-PCL was obtained by dialysis and freeze-drying.

[0012] 3) Synthesis of PCL-bP(D)AE with different pKa values: PCL-bP(D)AE with different pKa values ​​was synthesized by ROP and Michael addition polymerization of ε-CL. ε-CL and 2-hydroxyethyl acrylate were dissolved in anhydrous toluene, and after a process of liquid nitrogen freezing-vacuuming-thawing, the mixture was stirred and reacted at 110°C. Then, it was precipitated with ice-cold diethyl ether, filtered, and vacuum dried to obtain the product PCL-A. Next, PCL-A, TDP, and HDD, or PCL-A, TDP, and different molar ratios of HDD and DDD (1:4, 2:3, 3:2, 4:1), or PCL-A, TDP, and DDD were dissolved in anhydrous chloroform and stirred and reacted at 55°C. Finally, it was precipitated with ice-cold diethyl ether, filtered, and vacuum dried to obtain the product PCL-bP(D)AE.

[0013] The present invention describes a series of single micelles, composite micelles with different pKa values, and a method for preparing nanomolecular chaperones as personalized in situ vaccines, comprising the following steps:

[0014] 1) Preparation of single micelles PM and P-PM: PEG-b-PCL, or PEG-b-PCL and PBA-PEG-b-PCL at a mass ratio of 4:1, were fully dissolved in anhydrous DMF. The solution was then dropped into PBS and stirred with a magnetic rod. After dialysis, single micelles PM and P-PM were obtained.

[0015] 2) Preparation of composite micelles (MSPM) with different pKa values: PEG-b-PCL and PCL-bP(D)AE with different pKa values ​​were fully dissolved in anhydrous DMF at a mass ratio of 1:1. The solution was then added dropwise to acidic water at pH 5.0. After stirring with a magnetic rod, different MSPMs were obtained by dialysis.

[0016] 3) Preparation of nano-molecular chaperones as personalized in situ vaccines: PEG-b-PCL, PBA-PEG-b-PCL, and PCL-bP(D)AE (pKa = 6.51) were fully dissolved in anhydrous DMF at a mass ratio of 3:2:5. The solution was then added dropwise to acidic water (pH = 5.0), 10 mL, stirred with a magnetic rod, and dialyzed to obtain the nano-molecular chaperone P-nChap. To obtain IL-12@P-nChap, an appropriate amount of IL-12 was added to P-nChap, heated to 70°C, then cooled to 4°C, stirred, ultrafiltered, centrifuged, and resuspended in PBS to obtain IL-12@P-nChap. PTX@P-nChap was prepared by dissolving PEG-b-PCL, PBA-PEG-b-PCL, PCL-bP(D)AE (pKa = 6.51), and PTX in anhydrous DMF at a mass ratio of 3:2:5:1. The solution was then added dropwise to acidic water (pH = 5.0) for 10 mL. After stirring with a magnetic rod, the solution was dialyzed to completely remove DMF and excess PTX. Finally, the solution was ultrafiltered, centrifuged, and resuspended in PBS to obtain PTX@P-nChap. Subsequently, IL-12 was added to PTX@P-nChap, heated to 70°C, cooled to 4°C, stirred, ultrafiltered, centrifuged, and resuspended in PBS to obtain IL-12 / PTX@P-nChap.

[0017] The present invention also provides a nano-molecular chaperone system with tunable pKa prepared by the method described above.

[0018] This invention also provides the application of a tunable pKa nanochaperone system in the preparation of in situ cancer vaccines. The tunable pKa nanochaperone is IL-12 / PTX@P-nChap.

[0019] The beneficial effects of this invention are:

[0020] 1. This invention designs a novel nanomolecular chaperone (IL-12 / PTX@P-nChap) by fine-tuning the pKa of P(D)AE to 6.51, thereby reducing the toxicity of systemic cytokine therapy by delivering denatured forms of cytokines using the nanomolecular chaperone. Complete deprotonation of P-nChap under normal physiological conditions (pH=7.4) can load and protect denatured IL-12 and inhibit its off-target refolding to avoid toxicity and prolong blood circulation; partial protonation occurs in the tumor microenvironment (pH=6.5), releasing taxane PTX to induce ICDs and generate TAAs, and promoting the refolding and release of IL-12; subsequently, P-nChap acts as a personalized in situ vaccine to capture TAAs and transport them to dendritic DCs, and then undergoes complete protonation in lysosomes (pH=5.0) to achieve lysosomal escape, thereby promoting antigen cross-presentation to enhance the efficacy of cancer immunotherapy.

[0021] 2. This invention provides a simple and universal drug delivery strategy to address one of the biggest challenges in cytokine therapy (undesirable systemic activity) and provides a robust platform for personalized cancer vaccination that can be widely applied to cancer immunotherapy.

[0022] 3. A composite micelle-based nanochaperone (P-nChap) with a specific pKa value (6.51) was prepared. The extended PEG chain on the outer layer exhibits high water dispersibility, ensuring the stability of the nanochaperone in aqueous solution. The functional group PBA modified at the end of the PEG chain can improve protein loading efficiency and reduce off-target release. Partial protonation of the P(D)AE chain in the tumor microenvironment (pH=6.5) induced isomerization of the nanochaperone, promoting the refolding and release of the protein client.

[0023] 4. A nanomolecular chaperone (IL-12 / PTX@P-nChap) capable of serving as a personalized in situ cancer vaccine was prepared. Under normal physiological conditions, it can load and protect denatured IL-12 and inhibit its off-target refolding to avoid toxic side effects and prolong blood circulation. In the tumor microenvironment (pH=6.5), it undergoes partial protonation, releasing PTX to induce ICD and generate TAAs, promoting the refolding and release of IL-12. It can also capture TAAs in situ and transport them to DCs, and then undergo complete protonation in lysosomes (pH=5.0) to achieve lysosomal escape, thereby promoting antigen cross-presentation and activating tumor immunity.

[0024] 5. This invention provides the application of a nano-molecular chaperone system with a specific pKa in anti-tumor immunity, which has a significant inhibitory effect on advanced large "cold" tumors, a significant remote effect on distant tumors, and can effectively prevent tumor recurrence and metastasis after surgery. Attached Figure Description

[0025] Figure 1 This is the synthetic route for different block polymers in Example 1;

[0026] (a) Synthetic route of PEG-b-PCL;

[0027] (b) Synthetic route of PBA-PEG-b-PCL;

[0028] (c) Synthetic route of PCL-bP(D)AE;

[0029] Figure 2 This is a characterization of the titration pKa value and transmittance (T%) of different PCL-bP(D)AE in Example 2;

[0030] (a) Titration curves of six different PCL-bP(D)AE;

[0031] (b) Transmittance of six different PCL-bP(D)AEs;

[0032] Figure 3 This is a schematic diagram of the preparation process of single micelle P-PM, composite micelle MSPM, and nanomolecular chaperone P-nChap and IL-12 / PTX@P-nChap in Example 3;

[0033] (a) Schematic diagram of the preparation process of P-PM and MSPM;

[0034] (b) Schematic diagram of the preparation process of P-nChap;

[0035] (c) Schematic diagram of the preparation process of IL-12 / PTX@P-nChap;

[0036] Figure 4 This is a characterization of the adsorption and release effects of the nano-molecular chaperone P-nChap on the model protein horseradish peroxidase (HRP) and its competitive loading effect on TAAs in Example 3.

[0037] (a) Quantitative capture of HRP by P-PM, nChap and P-nChap (left three columns), and HRP release in the presence of TAAs (middle three columns) and competitive adsorption of TAAs (right three columns);

[0038] (b) Fluorescence intensity of PM / ARS, P-PM / ARS, P-PM / ARS+HRP and P-PM / ARS+TAAs at 586 nm;

[0039] (c) Fluorescence spectra of Cy3-HRP in the presence or absence of P-nChap of Cy5-P(D)AE-b-PCL at pH 7.4 and pH 6.5;

[0040] Figure 5 The cumulative release curves and enzyme activity recovery of thermally denatured HRP at different times when co-incubated with the nanomolecule chaperone P-nChap in Example 3 under (a) pH 7.4 and (b) pH 6.5 conditions are characterized.

[0041] Figure 6 This is a characterization of the nano-molecular chaperone P-nChap in Example 3, which prolongs IL-12 blood circulation and reduces systemic toxicity;

[0042] (a) Pharmacokinetic curves of mice after intravenous injection of free IL-12 and IL-12 / PTX@P-nChap;

[0043] (b) Pharmacokinetic curves of mice after intravenous injection of free PTX and IL-12 / PTX@P-nChap;

[0044] (c) In vitro fluorescence images of tumors and major organs in B16F10 tumor mice 24 h after intravenous injection of (G1)Cy5-IL-12, (G2)Cy5-IL-12 / PTX@P-nChap, (G3)Cy5-PTX and (G4)IL-12 / Cy5-PTX@P-nChap;

[0045] Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), and blood urea nitrogen (BUN) in mice treated with (dg) PBS, IL-12, and IL-12@P-nChap, respectively.

[0046] Figure 7 This is the application of tunable pKa nanomolecule chaperone systems in cancer immunotherapy;

[0047] (a) Survival rate of mice with advanced large tumors after different treatments;

[0048] (b,c) Mean tumor growth volume curves of mice with primary tumor (b) and distal tumor (c) after different treatments;

[0049] (d) Representative images of tumor recurrence in mice after different treatments;

[0050] (e) Representative images of lung tumor metastatic nodules in mice after different treatments. Detailed Implementation

[0051] Example 1

[0052] 1) Synthesis of PEG-b-PCL in the aforementioned tunable pKa nanomolecule chaperone:

[0053] PEG 114 -OH groups are dried under vacuum before use. PEG... 114 -OH (2 g, 0.4 mmol), ε-CL monomer (3.65 g, 32 mmol) and one drop of Sn(Oct)2 were dissolved in 10 mL of toluene. The mixture was stirred at 110 °C for 12 h, and after three cycles of liquid nitrogen freezing-vacuuming-thawing, the solution was precipitated with excess ice-cold ether and filtered to obtain a white powder product, PCL-b-PEG.

[0054] 2) Synthesis of PBA-PEG-b-PCL in the aforementioned tunable pKa nanomolecule chaperone:

[0055] BOC-NH-PEG 114 -OH (2 g, 0.4 mmol), ε-CL (3.65 g, 32 mmol), and one drop of Sn(Oct)₂ were dissolved in 10 mL of toluene. The mixture was stirred at 110 °C for 12 h, and after three cycles of liquid nitrogen freezing-vacuuming-thawing, the solution was precipitated with excess ice-cold diethyl ether and filtered to obtain a white powder product, BOC-NH-PEG-b-PCL. Next, BOC-NH-PEG-b-PCL (2 g, 0.14 mmol) was dissolved in 5 mL of dichloromethane, and then 5 mL of trifluoroacetic acid was added. The mixture was stirred at 25 °C for 24 h. Subsequently, the reaction solution was evaporated to dryness using a rotary evaporator, and the crude product was dissolved in anhydrous methanol and then evaporated to dryness again to remove excess trifluoroacetic acid. Then, 1 mL of DMSO and 100 μL of triethylamine were added to the mixture, and the reaction was carried out at room temperature for 4 h to neutralize the residual trifluoroacetic acid. Finally, the mixture was precipitated with ice-cold diethyl ether, filtered, and vacuum dried to obtain the white product NH₂-PEG-b-PCL. Then, NH2-PEG-b-PCL (2.82 g, 0.2 mmol), PBA (60 mg, 0.4 mmol), and triethylamine (50 μL) were dissolved in 10 mL of N,N-dimethylformamide (DMF) and reacted at room temperature for 48 h. After the reaction was complete, the reaction flask was placed in an ice bath, and sodium borohydride was added in portions until no more bubbles were generated, and then reacted at room temperature for 12 h. Subsequently, the reaction solution was dialyzed against deionized water for 3 days (molecular weight cutoff = 5000). Finally, the PBA-PEG-b-PCL product was obtained by freeze-drying.

[0056] 3) Synthesis of PCL-bP(D)AE with different pKa values ​​in the aforementioned tunable pKa nanomolecule chaperone:

[0057] ε-CL (3.92 g, 34.4 mmol), HEA (50 mg, 0.43 mmol) and one drop of Sn(Oct)2 were dissolved in 10 mL of anhydrous toluene. The mixture was subjected to three cycles of liquid nitrogen freezing-vacuuming-thawing. The mixture was stirred at 110 °C for 12 h. The product was then precipitated with ice-cold diethyl ether, filtered, and vacuum dried to obtain PCL-acrylate (PCL-A). Next, PCL-A (0.3 g, 0.033 mmol), TDP (0.14 g, 0.66 mmol), and HDD (0.15 g, 0.66 mmol), or PCL-A (0.3 g, 0.033 mmol), TDP (0.14 g, 0.66 mmol), and different molar ratios of HDD / DDD (1:4, 2:3, 3:2, 4:1, 0.66 mmol), or PCL-A (0.3 g, 0.033 mmol), TDP (0.14 g, 0.66 mmol), and DDD (0.18 g, 0.66 mmol) were dissolved in 10 mL of anhydrous chloroform. The reaction mixture was stirred at 55 °C for 3 days. Precipitation was achieved with ice-cold diethyl ether, filtered, and dried under vacuum to obtain the product PCL-bP(D)AE.

[0058] Example 2

[0059] pKa values ​​and transmittance (T%) were tested for different PCL-bP(D)AE samples in Example 1:

[0060] Acid-base titration curve ( Figure 2 a) and transmittance curve ( Figure 2 b) shows the pH buffering capacity of all PCL-bP(D)AEs, with pKa values ​​adjusted for the range of 6.29 to 6.80.

[0061] Example 3

[0062] 1) Preparation of single micelles PM and P-PM in the tunable pKa nanomolecule chaperone described above:

[0063] PEG-b-PCL (5 mg / mL) or PEG-b-PCL and PBA-PEG-b-PCL (5 mg / mL, w:w = 4:1) were thoroughly dissolved in anhydrous DMF. The solution was then added dropwise to PBS (pH = 7.4, 10 mL) every 30 seconds. After stirring with a magnetic rod for 2 hours, the solution was dialyzed against PBS (pH = 7.4) for 3 days (molecular weight cutoff = 5000) to obtain single micelle PM and P-PM.

[0064] 2) Preparation of composite micelles (MSPM) with different pKa values ​​in the aforementioned tunable pKa nanomolecule chaperone:

[0065] PEG-b-PCL and PCL-bP(D)AE (5 mg / mL, w:w = 1:1) with different pKa values ​​were fully dissolved in anhydrous DMF. The solution was then added dropwise to acidic water (pH = 5.0, 10 mL) every 30 seconds. After stirring with a magnetic rod for 2 hours, the solution was dialyzed in PBS (pH = 7.4) for 3 days (molecular weight cutoff = 5000) to obtain different MSPMs.

[0066] 3) Preparation of the nanomolecular chaperones P-nChap, IL-12@P-nChap, PTX@P-nChap, and IL-12 / PTX@P-nChap in the aforementioned tunable pKa nanomolecular chaperone:

[0067] PEG-b-PCL, PBA-PEG-b-PCL, and PCL-bP(D)AE (pKa = 6.51) (5 mg / mL, w:w:w = 3:2:5) were fully dissolved in anhydrous DMF. The solution was then added dropwise to acidic water (pH = 5.0, 10 mL) every 30 seconds. After stirring with a magnetic rod for 2 h, the solution was dialyzed in PBS (pH = 7.4) for 3 days (molecular weight cutoff = 5000) to obtain the nanomolecule chaperone P-nChap. To obtain IL-12@P-nChap, IL-12 (20 μg / mL) was added to P-nChap (0.5 mg / mL, 1 mL, pH 7.4), and the mixture was heated at 70 °C for 1 h. The mixture was then cooled to 4°C and stirred for 2 hours. It was then centrifuged at 8000 rpm for 10 minutes using an ultrafiltration tube (molecular weight cutoff = 150 kDa), and finally resuspended in PBS to obtain IL-12@P-nChap. To obtain PTX@P-nChap, PEG-b-PCL, PBA-PEG-b-PCL, PCL-bP(D)AE (pKa = 6.51), and PTX (w:w:w:w = 3:2:5:1) were dissolved in anhydrous DMF and added dropwise to acidic water (pH = 5.0, 10m) every 30 seconds. L), after stirring with a magnetic rod for 2 hours, dialyze with PBS (pH=7.4) for 3 days (molecular weight cutoff=5000) to completely remove DMF and excess PTX, finally obtaining PTX@P-nChap, which is used after ultrafiltration; IL-12 (20 μg / mL) is added to PTX@P-nChap (0.5 mg / mL, 1 mL), heated at 70 °C for 1 hour, then cooled to 4 °C and stirred for 2 hours, centrifuged with an ultrafiltration tube (molecular weight cutoff=150 kDa), and resuspended in PBS to obtain IL-12 / PTX@P-nChap.

[0068] Example 4

[0069] The adsorption and release effects of P-PM, nChap, and P-nChap in the tunable pKa nanomolecule chaperone prepared in Example 3, as well as their competitive loading effects on TAAs, were tested. The method steps and related evaluations are as follows:

[0070] TAAs were extracted from B16F10 cells using a protein extraction reagent, followed by centrifugation at 8000 rpm (molecular weight cutoff = 150 kDa) for 10 min to remove large or aggregated proteins. Cy5-labeled HRP solution (0.8 mg / mL, 50 μL) was added to different micelle solutions (0.5 mg / mL, 950 μL, pH 7.4) and incubated at 85 °C for 1 h. The solution was then cooled to 4 °C and incubated for another 1 h, followed by centrifugation using ultrafiltration tubes for 10 min (molecular weight cutoff = 150 kDa) to separate unadsorbed proteins. The adsorption amount was calculated using fluorescence spectroscopy. The HRP@micelles were then resuspended in PB (pH = 6.5) to a concentration of 0.5 mg / mL. Excess TAA solution (8 mg / mL) was added to the HRP@micelle solution, and the cells were incubated at 37 °C for 12 h. The amount of HRP released from the dialysate was calculated using fluorescence spectroscopy, and the total protein content in the dialysate was determined using the BCA method. The formula for calculating the amount of TAAs not adsorbed by micelles is: Amount of TAAs not adsorbed by micelles = Total protein detected by BCA - Amount of HRP released. Finally, the competitive loading capacity of TAAs can be calculated using the following formula: Competitive loading capacity of TAAs = Total amount of TAAs - Amount of TAAs not adsorbed by micelles.

[0071] Due to the dynamic covalent interaction between PBA and glycoproteins, PBA was modified at the PEG end of nanomicelles to capture glycoproteins by detecting the interaction between PBA and HRP (or TAA) glycosyl groups. P-PM (0.5 mg / mL), Alizarin Red S solution (ARS, 5 mg / mL), HRP solution (5 mg / mL), and TAAs (5 mg / mL) were prepared. Then, 1 mL of P-PM solution was placed in a quartz cell, and fluorescence emission intensity was measured in the 500-800 nm range with an excitation wavelength of 469 nm. Next, 1 μL of ARS solution was added to the above-mentioned fluorescence cell containing P-PM solution each time, and incubation was performed at room temperature for 10 min, continuing to monitor fluorescence intensity until the fluorescence intensity at the maximum emission wavelength (586 nm) could no longer increase. Finally, 1 μL of HRP solution or TAAs was added sequentially to the mixture of P-PM and ARS, and incubation was performed for 10 min. Fluorescence intensity was monitored until no change in fluorescence intensity at 586 nm.

[0072] Using HRP as a protein model, the capture of proteins by the nChap hydrophobic microdomains (P(D)AE) was investigated using the fluorescence resonance energy transfer (FRET) effect of Cy3-Cy5. P-nChap containing Cy5-labeled P(D)AE and Cy3-labeled HRP were incubated in PBS (10 mM, pH 7.4 or 6.5) for 1 h, followed by excitation at 515 nm using a fluorescence spectrophotometer. Fluorescence emission spectra in the 550-750 nm range were recorded using the fluorescence spectrophotometer.

[0073] The results showed that P-nChap had better adsorption and release effects on HRP and better competitive loading effects on TAAs than P-PM and nChap. This is because PBA has a chaperone effect, which can recognize and capture glycoproteins on the surface of glycoproteins through the reversible covalent bond between the borate group and the cis-1,2-diol group. Figure 4 a). The fluorescence intensity of the P-PM / ARS solution decreased at 586 nm after the addition of HRP or TAAs, confirming the capture of glycoproteins by the PBA group. Figure 4 b). Furthermore, FRET assays using Cy3-labeled HRP and Cy5-labeled P(D)AE confirmed that HRP was loaded into hydrophobic microdomains. Figure 4 c).

[0074] Example 5

[0075] The cumulative release curves and enzyme activity recovery of P-nChap and thermally denatured HRP in the tunable pKa nanomolecule chaperone prepared in Example 3 were measured at different times under conditions of pH 7.4 and pH 6.5 with excess TAAs. The method steps and related evaluations are as follows:

[0076] The release of HRP in P-nChap at different time points was investigated. First, the pH of KH₂PO₄ buffer solution (0.1M) was adjusted to 7.4 and 6.5 using NaOH solution (1M) to simulate the microenvironments of blood, normal tissue, and tumor. Then, HRP@P-nChap was transferred to dialysis tubes (molecular weight cutoff = 150 kDa), and the tubes were immersed in 5 mL of either of the aforementioned buffer solutions. The tubes were then continuously oscillated at 100 rpm at 37°C. At predetermined time intervals, 1 mL of the released dialysate was removed and replaced with the same volume of fresh dialysate. HRP was pre-labeled with Cy₅, and the cumulative release of HRP from the dialysate was calculated using fluorescence spectroscopy.

[0077] Next, denatured HRP was co-incubated with P-nChap under different pH conditions with or without TAAs to perform refolding experiments. HRP solution (1 mg / mL, 50 μL) was added to P-nChap solution (0.5 mg / mL, 950 μL, pH 7.4) and heated at 85 °C for 1 h. Then, HRP was allowed to refold under pH 7.4 or pH 6.5 conditions with or without TAAs. The enzyme activity of HRP at different time points was measured using a UV-Vis spectrophotometer. 20 μL of sample and 480 μL of guaiacol solution (15.5 μL guaiacol dissolved in 50 mL PBS and 52 μL 30% H2O2) were added to a quartz cell. The rate of decrease in absorbance at 436 nm over time was measured using a UV-Vis spectrophotometer as a measure of HRP activity. Relative enzyme activity is defined as the slope of the sample in the first 15 seconds divided by the slope of native HRP in the first 15 seconds.

[0078] Based on our previous findings, phase transitions in hydrophobic microdomains are similar to ATP-driven conformational changes in natural molecular chaperones, playing a crucial role in assisting protein refolding. Therefore, we hypothesized that even if denatured HRP is occasionally released at pH 7.4, it remains misfolded. To verify this, we examined the release and restored enzyme activity of HRP in the presence of P-nChap at pH 7.4 without TAAs or at pH 6.5 with TAAs. The results showed that at pH 7.4, even when a small amount of HRP is released from P-nChap, most of it is misfolded and lacks enzymatic activity. Figure 5 a); while at pH 6.5, in the presence of TAAs, approximately 80% of HRP is released from P-nChap, of which approximately 94% of the HRP correctly refolds within 24 hours. Figure 5 b). These results clearly demonstrate the great potential of nanomolecular chaperone delivery platforms in avoiding the toxicity of off-target cytokine release.

[0079] Example 6

[0080] The application of a pKa-tunable nanomolecule chaperone (P-nChap) in reducing IL-12-induced systemic toxicity and prolonging blood circulation, the method steps and related evaluations are as follows:

[0081] To investigate the pharmacokinetics of IL-12 or PTX, C57BL / 6 mice were intravenously injected with either free Cy5-IL-12 and Cy5-IL-12 / PTX@P-nChap (100 μL, IL-12 dose: 0.25 mg / kg) or free Cy5-PTX and IL-12 / Cy5-PTX@P-nChap (100 μL, PTX dose: 2 mg / kg), and blood samples (30 μL) were collected at different time points. Blood clearance kinetics were assessed by detecting the residual concentrations of Cy5-IL-12 or Cy5-PTX according to standard curves for each group, and the concentrations of all samples were quantified by detecting the fluorescence intensity excited at 633 nm at a wavelength of 670 nm.

[0082] In addition, B16F10 cells (5 × 10⁶ cells per mouse) were subcutaneously injected into the right side of the mice. 5 (Number of cells) to generate tumor mice. When the tumor volume reaches approximately 300 mm²... 3 Mice were randomly divided into different groups and then intravenously injected with free Cy5-IL-12 and Cy5-IL-12 / PTX@P-nChap (100 μL, IL-12 dose: 0.25 mg / kg) or free Cy5-PTX and IL-12 / Cy5-PTX@P-nChap (100 μL, PTX dose: 2 mg / kg). After 24 hours, the mice were euthanized, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected for in vitro fluorescence image evaluation.

[0083] like Figure 6 As shown in a and b, IL-12 / PTX@P-nChap exhibited a longer elimination half-life compared to free IL-12. P-nChap also significantly improved the pharmacokinetics of PTX. Biodistribution studies using B16F10 tumor mice showed that P-nChap significantly enhanced the accumulation of IL-12 and PTX in tumors; at 24 h, the fluorescence intensity of IL-12 / PTX@P-nChap in tumors was significantly higher than that of free IL-12 and PTX. Figure 6 c). Blood biochemical analysis showed that the levels of ALT, AST, BUN, and CR in the blood of mice treated with free IL-12 were significantly upregulated. However, there was no significant difference in these blood biochemical parameters between IL-12@P-nChap and PBS. Figure 6 dg).

[0084] Example 7

[0085] Application of the aforementioned tunable pKa nanomolecule chaperone (IL-12 / PTX@P-nChap) system in cancer immunotherapy:

[0086] Although therapeutic cancer vaccines have shown promising clinical efficacy, they typically elicit a significant immunostimulatory response only when treating early-stage or highly inflammatory tumors, and not when treating advanced, large tumors. Here, we used the B16F10-OVA tumor C57BL / 6 mouse model to evaluate the growth-inhibiting effect of IL-12 / PTX@P-nChap on advanced, large tumors. Figure 7 As shown in figure a, compared with other groups, IL-12 / PTX@P-nChap treatment significantly inhibited the growth of B16F10-OVA tumors and prolonged the survival time of mice by 50 days, indicating that the ICD effect of PTX and the immune activation effect of refolded IL-12 synergistically enhanced the immune response, thus exhibiting optimal tumor suppression ability. Subsequently, the promoting effect of IL-12 / PTX@P-nChap as a personalized nanovaccine synergistic with immune checkpoint blockade (ICB) therapy on distant tumors was investigated. Figure 7 As shown in b and c, compared with PBS and IL-12+PTX treatment, IL-12 / PTX@P-nChap treatment significantly inhibited both primary tumors (administered) and distant tumors (unadministered) in mice, demonstrating significant anti-tumor efficacy and remote effects. Furthermore, the efficacy of IL-12 / PTX@P-nChap combined with anti-PDL1 antibody (aPDL1) treatment (IL-12 / PTX@P-nChap+aPDL1) was further improved.

[0087] Finally, to explore the potential of IL-12 / PTX@P-nChap nanovaccine combined with ICB therapy in inducing long-term immune memory effects to prevent tumor recurrence and metastasis, mice carrying B16F10 tumors were first treated and then surgically removed. Fourteen days later, the tumor was re-challenged on the contralateral side of the primary tumor, and tumor recurrence and longest survival time were monitored. Additionally, a lung metastasis model was established by tail vein injection of B16F10 cells to evaluate the efficacy of the nanovaccine against tumor metastasis. Results showed that IL-12 / PTX@P-nChap and IL-12 / PTX@P-nChap+αPDL1 pretreatment significantly delayed the growth of secondary B16F10 tumors compared to other treatments, and the tumor recurrence and growth were slowest in mice pretreated with IL-12 / PTX@P-nChap+αPDL1. Figure 7 d) indicates that the combination therapy of IL-12 / PTX@P-nChap with ICB may induce a durable anti-tumor immune response, thereby achieving long-term tumor regression. Furthermore, lung imaging showed that no obvious metastatic tumor nodules were observed on the lung surface of mice in the IL-12 / PTX@P-nChap+αPDL1 pretreatment group. Figure 7 e).

[0088] The tunable pKa nano-molecular chaperone system described in this invention is simple to synthesize. In in vitro experiments, its ability to efficiently help the model protein HRP refold and restore enzyme activity was successfully demonstrated. In mouse model experiments, it was successfully demonstrated that it significantly prolongs the blood circulation time of IL-12 and PTX and greatly reduces the immunotoxicity caused by IL-12 after systemic administration. In addition, the nano-molecular chaperone delivery platform effectively combines cytokine delivery and in situ vaccination, which has a significant inhibitory effect on advanced large "cold" tumors, a significant remote effect on distant tumors, and can effectively inhibit tumor recurrence and metastasis after surgery.

[0089] It should be noted that this invention addresses therapeutic strategies involving the delivery of cytokines while reducing their immunotoxicity and in situ vaccination. It presents a nano-molecular chaperone system with tunable pKa, aiming to effectively avoid the targeting toxicity and non-tumor toxicity of cytokines in the blood, while simultaneously restoring the immune activation of cytokines at the tumor site and achieving in situ vaccination. The technical solutions disclosed and proposed in this invention can be modified or recombinated by those skilled in the art without departing from the content, spirit, and scope of this invention to achieve the final preparation technology. It is particularly important to point out that this invention is not limited to the embodiments described herein; any obvious improvements and modifications made by those skilled in the art based on the disclosure of this invention should be within the protection scope of this invention.

Claims

1. A controllable pKa The method for constructing a nano-molecular chaperone system is characterized by, Comprehensive steps: S1: Synthesis of polymer PEG- via ε-CL ROP method b -PCL and phenylboronic acid modified PBA-PEG- b -PCL; PCL- was synthesized via Michael addition polymerization through ROP of ε-CL and the addition of HDD, DDD, and TDP. b -PAE; S2: A series of nanomolecule chaperones with microphase-separated structures were prepared by self-assembly, including those composed of PEG- b -PCL and has adjustable pKa PCL value b -PAE-prepared nanomolecular chaperone MSPM, wherein the MSPM is selected from PEG- b -PCL, PBA-PEG- b -PCL and PCL- b -PAE, where PCL- b -PAE pKa = 6.51, the nano-molecular chaperone P-nChap prepared; and PEG- b -PCL, PBA-PEG- b -PCL, PCL- b -PAE, where PCL- b -PAE pKa = 6.51, at least one of the following nanomolecular chaperones IL-12@P-nChap, PTX@P-nChap, and IL-12 / PTX@P-nChap prepared from thermally denatured IL-12 and / or the chemotherapeutic drug PTX:

2. An adjustable one as described in claim 1 pKa The method for constructing nano-molecular chaperone systems is characterized by, PEG-block copolymer b The synthesis of ε-PCL includes the following steps: using PEG-OH as a macromolecular initiator and Sn(Oct)2 as a catalyst, PEG-... is synthesized via ROP of ε-CL monomers. b -PCL; PEG-OH was dried under vacuum before use; PEG-OH and ε-CL monomers were dissolved in anhydrous toluene, and then reacted with stirring at 110°C. After liquid nitrogen freezing-vacuuming-thawing, the solution was precipitated with excess ice-cold ether and filtered to obtain a white powder product, PEG- b -PCL.

3. An adjustable one as described in claim 1 pKa The method for constructing nano-molecular chaperone systems is characterized by block... copolymer PBA-PEG- b The synthesis of PCL includes the following steps: PBA-PEG- b -PCL is composed of PBA and NH2-PEG- b -PCL reductive amination synthesis, NH2-PEG- b -PCL was synthesized via ROP using ε-CL as the monomer and BOC-NH-PEG-OH as the macromolecular initiator. BOC-NH-PEG-OH and ε-CL were dissolved in anhydrous toluene, and then reacted with stirring at 110°C. After a liquid nitrogen freezing-vacuuming-thawing process, the solution was precipitated with excess ice-cold diethyl ether and filtered to obtain a white powder product, BOC-NH-PEG-. b -PCL; The product was dissolved in dichloromethane, and then trifluoroacetic acid was added. The reaction mixture was stirred at 25°C. The reaction solution was dried by rotary evaporation, and the crude product was dissolved in anhydrous methanol and then evaporated to remove excess trifluoroacetic acid. Subsequently, the residual trifluoroacetic acid was neutralized by adding dimethyl sulfoxide and triethylamine. The final product was a white NH2-PEG- b -PCL was obtained by precipitation with ice-cold ether, followed by filtration and vacuum drying; Then, NH2-PEG- b PCL and PBA were dissolved in N,N-dimethylformamide; after stirring at room temperature, sodium borohydride was added in portions until no more bubbles were generated, and the reaction was continued to be stirred at room temperature; subsequently, the product PBA-PEG- was obtained by dialysis and freeze-drying. b -PCL.

4. An adjustable one as described in claim 1 pKa The method for constructing nano-molecular chaperone systems is characterized by, Different pKa PCL value b -PAE was synthesized via ROP and Michael addition polymerization of ε-CL; it has different pKa PCL value b The synthesis of ε-PAE includes the following steps: ε-CL and 2-hydroxyethyl acrylate are dissolved in anhydrous toluene, subjected to a liquid nitrogen freezing-vacuuming-thawing process, and then reacted with stirring at 110°C. Precipitation is then carried out with ice-cold diethyl ether, filtered, and vacuum dried to obtain product PCL-A. Next, PCL-A, TDP, and HDD, or PCL-A, TDP, and HDD and DDD in a molar ratio of 1:4, 2:3, 3:2, or 4:1, or PCL-A, TDP, and DDD, are dissolved in anhydrous chloroform and reacted with stirring at 55°C. Finally, precipitation is carried out with ice-cold diethyl ether, filtered, and vacuum dried to obtain product PCL-A. b -PAE.

5. An adjustable one as described in claim 1 pKa The method for constructing nano-molecular chaperone systems is characterized by, Different pKa The preparation method of MSPM, a nanomolecular chaperone composed of composite micelles, is as follows: PEG- b -PCL and have different pKa PCL value b -PAE was fully dissolved in anhydrous DMF at a 1:1 mass ratio, and then the solution was added dropwise to acidic water at pH = 5.

0. After stirring with a magnetic rod, different MSPMs were obtained by dialyzing.

6. An adjustable one as described in claim 1 pKa The method for constructing nano-molecular chaperone systems is characterized by, The preparation method of the nano-molecular chaperone as a personalized in situ vaccine is: PEG- b -PCL, PBA-PEG- b -PCL and PCL-b-PAE, where PCL-b-PAE pKa = 6.51, dissolved completely in anhydrous DMF at a mass ratio of 3:2:5, then the solution was added dropwise to acidic water (pH = 5.0), 10 mL, stirred with a magnetic rod, and dialyzed to obtain the nano-molecular chaperone P-nChap; to obtain IL-12@P-nChap, an appropriate amount of IL-12 was added to P-nChap, heated to 70°C, then cooled to 4°C, stirred, ultrafiltered, centrifuged, and resuspended in PBS to obtain IL-12@P-nChap; to obtain PTX@P-nChap, PEG- b -PCL, PBA-PEG- b -PCL, PCL- b - PAE and PTX were dissolved in anhydrous DMF in a mass ratio of 3:2:5:1, where PCL- b -PAE pKa = 6.51, added dropwise to acidic water, pH = 5.0, 10 mL, stirred with a magnetic rod, dialyzed to completely remove DMF and excess PTX, and finally ultrafiltered, centrifuged and resuspended in PBS to obtain PTX@P-nChap; then, IL-12 was added to PTX@P-nChap, heated to 70°C and cooled to 4°C with stirring, ultrafiltered, centrifuged and resuspended in PBS to obtain IL-12 / PTX@P-nChap.

7. The adjustable [property] prepared by the method according to any one of claims 1-6 pKa Nanomolecular chaperone system.

8. The adjustable capability of claim 7 pKa Application of nano-molecular chaperone systems in the preparation of in situ cancer vaccines.

9. The application according to claim 8, characterized in that, The adjustable pKa The nanomolecule chaperone is IL-12 / PTX@P-nChap.

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