Intracellular protein delivery carrier with immune enhancement and bone repair functions as well as preparation method and application of intracellular protein delivery carrier

Abstract

The invention discloses an intracellular protein delivery carrier with immune enhancement and bone repair functions as well as a preparation method and application thereof, and relates to the technical field of intracellular protein delivery carriers. Wherein the intracellular protein delivery carrier comprises a core formed from a calcium phosphorus inorganic phase, the inorganic phase being a manganese-containing calcium phosphate phase; the outer surface of the core is coated with the hydrophilic layer, and the hydrophilic layer is provided by a block copolymer polyethylene glycol-polyglutamic acid; the target protein is loaded in the core, and the target protein is co-encapsulated in the process of mineralizing to form an aqueous phase of the core and is not chemically modified. The invention reveals that the nano-carrier of calcium phosphate and polyethylene glycol-polyglutamic acid combined with manganese ions can enhance immunity, promote bone repair and deliver protein drugs into cells for the first time, and verifies that the nano-carrier activates a cGAS-STING pathway to achieve the ability of enhancing immunity and promoting bone repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of intracellular protein delivery carriers, in particular to an intracellular protein delivery carrier with immune enhancement and bone repair functions and a preparation method and application thereof. BACKGROUND

[0002] Protein drugs are rapidly extending to intracellular targets due to their high selectivity and clear action sites, but their transmembrane ability is weak and they are easily degraded by endosomes / lysosomes, resulting in limited effective delivery in the cytoplasm. Existing carriers such as high molecular micelles / vesicles, lipid nanoparticles, MOF / mesoporous silica, nanogels and cell penetrating peptides, although they can partially alleviate endocytosis retention through membrane fusion, proton sponge or membrane perforation, still have problems such as complex synthesis and formulation, toxicity and inflammatory response caused by strong cations or membrane disturbance, damage to protein activity due to chemical modification, and still low escape efficiency. On the other hand, tumor and infection-related immunotherapy emphasizes the activation of innate immunity, especially the enhancement of dendritic cell antigen presentation and type I interferon production through the cGAS-STING pathway. In recent years, it has been found that Mn 2+ can act as a functional cofactor of cGAS, significantly amplifying the signal of the pathway and showing the potential of "delivery-type immunoadjuvant". At the same time, the field of bone repair has confirmed that calcium phosphate materials (hydroxyapatite, tricalcium phosphate, amorphous calcium phosphate, etc.) have excellent biocompatibility and bone conduction / osteoinduction properties, and can release Ca 2+ and PO4 3− locally to promote osteogenesis-related gene expression and mineralization, which is an ideal inorganic substrate for the integration of "drug delivery + bone repair". However, there is still a lack of solutions that can efficiently deliver intracellular proteins and mediate immune enhancement by Mn 2+ and simultaneously exert the bone repair function of calcium phosphate on the same nanoplatform while considering safety and manufacturability.

[0003] The most similar existing solutions to the present application mainly have two types: one is the co-delivery system of manganese-based immunonanoadjuvant and protein / antigen, such as loading protein on MnO2, MnCO3 or Mn-doped MOF / mesoporous silica, and using acid / glutathione to trigger the release of Mn 2+The first is to activate STING, and some literatures superimpose a peroxide reaction to enhance endosome escape; the second is polycarboxylic acid or polypeptide template (such as polyaspartic acid, polyglutamic acid, and polyacrylic acid) induced calcium phosphate mineralization nanoparticles for mild encapsulation of proteins and rely on endosome acid lysis for release. The former can provide immune enhancement, but often involves a strong redox or transition metal framework, and there is a safety window pressure for long-term residual and metal ion dose control, and the protein often needs surface modification or loading under organic / high salt conditions, which easily affects the activity; at the same time, the Mn release timing and spatial accuracy are insufficient, and the endosome escape efficiency is limited. The latter has good biocompatibility and pH response, but usually does not integrate immune activation modules, and the physical and chemical damage to the endosome membrane is weak, making it difficult to achieve stable and efficient escape without introducing high cationic polymers. In addition, the existing two types of solutions are mostly not systematically considered for bone regeneration requirements, and lack of overall design of taking calcium phosphate as a bone repair functional carrier and achieving "immune activation-bone promotion" dual regulation in the immune microenvironment. In summary, how to simultaneously achieve protein unmodified high-activity loading, Mn 2+ precise release to enhance innate immunity, and calcium phosphate-mediated bone repair gain, constitutes a key point that the current technology needs to break through. SUMMARY

[0004] In view of this, the present application provides an intracellular protein delivery carrier with immune enhancement and bone repair functions and a preparation method and application thereof.

[0005] According to one aspect of the present disclosure, an intracellular protein delivery carrier with immune enhancement and bone repair functions is provided, comprising a core formed by a calcium-phosphorus inorganic phase, the inorganic phase being a manganese-containing calcium phosphate phase; a hydrophilic layer coated on the outer surface of the core, the hydrophilic layer being provided by a block copolymer polyethylene glycol-polyglutamic acid; a target protein loaded in the core, the target protein being co-encapsulated in the process of mineralization of the core in an aqueous phase, and not being chemically modified; wherein the carrier can be acid-sensitive dissolved and release the target protein and Mn 2+ .

[0006] According to another aspect of the present disclosure, a method for preparing an intracellular protein delivery carrier with immune enhancement and bone repair functions is provided, comprising the following steps: step 1, dissolving calcium chloride and manganese chloride in a Tris-HCl aqueous solution to obtain solution A; step 2, dissolving PEG-b-Glu and a target protein in a HEPES buffer containing disodium hydrogen phosphate to obtain solution B; step 3, slowly adding the solution B to the solution A, and stirring for a certain time to form a manganese-containing calcium phosphate mineralization core and co-encapsulate the target protein; and step 4, dialyzing and freeze-drying the obtained dispersion system to obtain the carrier.

[0007] According to a third aspect of the present disclosure, there is provided a use of an intracellular protein delivery carrier with immune enhancement and bone repair functions in the preparation of a medicament for enhancing an innate immune response, which includes inducing type I interferon production by activating the Mn-mediated cGAS-STING signaling pathway.

[0008] The present application has the following beneficial effects: the carrier of the present application can quickly dissolve and release the loaded protein drugs and Ca 2+ with Mn 2+ , achieving efficient escape and delivering the protein to the cytoplasm; and the controllable Mn 2+ exposure precisely activates the cGAS-STING pathway, promotes the maturation of dendritic cells and the production of type I interferon, thereby obtaining a stable immune enhancement effect under the premise of ensuring safety. At the same time, the calcium phosphate matrix locally releases Ca 2+ and PO4 3− , promotes osteogenic differentiation and mineralization, and endows the carrier with bone conduction / bone repair functions, forming a synergistic platform of “protein delivery + immune enhancement + bone repair”. By adjusting the PEG density, PGlu coordination strength and mineralization degree, the particle size, surface properties and release kinetics can be engineered and optimized, taking into account the in vivo stability, efficacy and large-scale manufacturing requirements. BRIEF DESCRIPTION OF DRAWINGS

[0009] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and not a limitation on the present disclosure.

[0010] Figure 1 A schematic diagram of a protein delivery carrier.

[0011] Figure 2 A dynamic light scattering (DLS) particle size distribution diagram of the carrier.

[0012] Figure 3 A dynamic light scattering (DLS) zeta potential characterization diagram of the carrier

[0013] Figure 4 A transmission electron microscope (TEM) morphology diagram of the carrier.

[0014] Figure 5 A protein accumulation release curve diagram of the carrier under different pH conditions (pH 7.4 / 6.5 / 5.0).

[0015] Figure 6 A Mn 2+ accumulation release curve diagram of the carrier under different pH conditions (pH 7.4 / 6.5 / 5.0).

[0016] Figure 7 Confocal laser scanning microscope (CLSM) images of endocytosis and lysosomal escape of cells.

[0017] Figure 8 Graph of the results of in vitro cytotoxicity evaluation of the vector on cell proliferation.

[0018] Figure 9 Graph of the tumor growth curve of a mouse xenograft tumor model.

[0019] Figure 10 Graph of flow cytometry analysis of the immune microenvironment of tumor tissue.

[0020] Figure 11 Graph of statistical analysis of flow cytometry results of the immune microenvironment of tumor tissue.

[0021] Figure 12 Micro-CT (micro-CT) three-dimensional reconstruction graph of a bone destruction model after tumor bone metastasis.

[0022] Figure 13 Graph of statistical analysis of micro-CT relative bone volume of a bone destruction model after tumor bone metastasis.

[0023] Figure 14 Graph of statistical analysis of micro-CT trabecular bone number of a bone destruction model after tumor bone metastasis.

[0024] Figure 15 Graph of statistical analysis of micro-CT relative trabecular bone thickness of a bone destruction model after tumor bone metastasis.

[0025] Figure 16 Graph of statistical analysis of the relative mRNA level of pSTING in the qPCR detection of the cGAS-STING activation of tumor cells by the material.

[0026] Figure 17 Graph of statistical analysis of the relative mRNA level of p-TBK1 in the qPCR detection of the cGAS-STING activation of tumor cells by the material.

[0027] Figure 18 Graph of statistical analysis of the relative mRNA level of IFN-β in the qPCR detection of the cGAS-STING activation of tumor cells by the material. DETAILED DESCRIPTION

[0028] In order to make the purposes, technical solutions and advantages of the embodiments of the present disclosure clearer, the following will be combined with the drawings of the embodiments of the present disclosure to clearly and completely describe the technical solutions of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present disclosure.

[0029] Unless otherwise defined, technical terms or scientific terms used in the present disclosure shall have the common meaning understood by a person of ordinary skill in the art to which the present disclosure belongs. The terms "first", "second", and similar terms used in the present disclosure do not denote any order, quantity, or importance, but are used to distinguish different components. The terms "include", "contain", and similar terms mean that the elements or objects before the terms encompass the elements or objects listed after the terms and their equivalents, and do not exclude other elements or objects. The terms "connect" or "connected" and similar terms are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. "Up", "down", "left", "right", and the like are used only to indicate relative positional relationships, and when the absolute positions of the described objects are changed, the relative positional relationships may also be changed accordingly. In order to keep the following description of the embodiments of the present disclosure clear and concise, the present disclosure omits the detailed description of some known functions and known components.

[0030] The term "treatment" refers to contacting (e.g., administering) a subject with a drug, composition, etc. based on the present disclosure after the subject has contracted a disease, so as to alleviate the symptoms of the disease compared with not contacting, and does not mean that the symptoms of the disease must be completely inhibited. Contracting a disease means that the body has symptoms of the disease.

[0031] The term "prevention" refers to contacting (e.g., administering) a subject with a drug, composition, etc. based on the present disclosure before the subject has contracted a disease, so as to alleviate the symptoms after contracting the disease compared with not contacting, and does not mean that the disease must be completely inhibited.

[0032] The present application aims to construct a cell protein delivery carrier with the characteristics of immune enhancement and bone repair function, and a preparation method thereof. The carrier can achieve efficient loading of various protein drugs, does not require chemical modification to maintain the natural activity of the protein throughout the process, and can enhance cell uptake efficiency through ligand modification. After the carrier enters the cell, the endosome / lysosome acidic environment (pH 5.0-6.0) triggers rapid dissolution of the calcium phosphate (CaP) skeleton, synchronously releasing the loaded protein drugs and Ca 2+ / Mn 2+ , achieving efficient escape of therapeutic proteins to the cytoplasm and functional exertion. In addition, the released Mn2+ The cGAS-STING pathway can be activated to drive dendritic cell (DC) maturation and secretion of type I interferon, significantly alleviate tumor microenvironment (TME) immunosuppression, and enhance protein efficacy, forming a drug delivery-immunomodulation dual-effect synergistic mechanism. The local release of Ca 2+ and PO4 3− promotes osteogenic differentiation and mineralization, endows the carrier with bone conduction / bone repair function, and forms a synergistic platform of "protein delivery + immune enhancement + bone repair".

[0033] In the embodiments of the present application, the intracellular protein delivery carrier with immune enhancement and bone repair functions comprises: a core formed by a calcium-phosphorus inorganic phase, the inorganic phase being a calcium phosphate phase containing manganese; a hydrophilic layer coated on the outer surface of the core, the hydrophilic layer being provided by a block copolymer polyethylene glycol-polyglutamic acid; a target protein loaded in the core, the target protein being co-encapsulated in the process of mineralization of the aqueous phase to form the core and not being chemically modified; wherein the carrier can be acid-sensitive dissolved and release the target protein and Mn 2+ under the acidic conditions of endosomes / lysosomes.

[0034] Specifically, the PEG-b-Glu block copolymer: the PEG segment provides a highly hydrophilic and protein-adsorption-resistant outer layer crown, significantly reduces serum protein crown formation and reticuloendothelial system clearance, and improves colloidal stability and in vivo circulation time; the Glu segment as a polyanionic skeleton with multiple carboxyl groups can coordinate with Ca 2+ / Mn 2+ precisely induce and limit the nucleation and growth of calcium phosphate, forming a dense and uniform inorganic composite core, thereby realizing the engineering controllability of the mineralization degree, particle size distribution and ion composition. The coordination-mineralization interface can also form a flexible microenvironment with proteins through weak interactions such as electrostatic / hydrogen bonds, complete efficient co-encapsulation under mild conditions in an all-aqueous phase, reduce interface denaturation, maximize protein activity, and inhibit early leakage through multiple points. Overall, PEG-b-Glu endows the system with excellent biocompatibility and manufacturability, long-term stability in serum environment, structured regulation ability of CaP mineralization and Mn 2+ integration, and mild loading properties to realize high loading and protection of activity without modifying the protein, laying a material foundation for subsequent efficient delivery, immune enhancement and bone repair functions.

[0035] Calcium phosphate (CaP) nanoparticles: Calcium phosphate is a biocompatible material with good stability and degradability in vivo, and has acid environment responsiveness. The present application uses calcium phosphate and PEG-b-Glu to form nanoparticles, which have higher drug loading capacity and good acid environment release control ability. In addition, calcium phosphate is the core component of the system that gives the system bone repair function, its composition is highly similar to that of natural bone mineral, has excellent biocompatibility and bone conductivity, can be used as a "mineralization template" for new bone formation, and provides a favorable microenvironment for osteoblast adhesion, proliferation and differentiation. Its controllable degradation releases Ca 2+ and PO4 3− As a key ion required for osteogenesis, it can up-regulate the expression of ALP, Runx2, OCN and other osteogenesis-related markers, promote the deposition of hydroxyapatite in the extracellular matrix and the formation of mature bone-like tissue, thereby accelerating the mineralization process and bone integration at the defect site. The surface structure and chemical characteristics of calcium phosphate at the nanoscale are conducive to simulating the hierarchical mineralization of bone tissue, enhancing the heterogeneous nucleation and growth of apatite in the body fluid, and shortening the "bridge from carrier to autologous bone" period; it can be physiologically absorbed by osteoclasts, and degradation and regeneration are synchronized, which helps to achieve a dynamic balance between construction and remodeling and obtain persistent mechanical integration. In addition, calcium phosphate has good regulatory potential for the immune microenvironment related to bone regeneration, which can reduce inflammatory osteoclastic response and promote the transformation to a pro-repair phenotype, thereby providing a more friendly matrix and signal support for osteoblasts and angiogenesis. As an inorganic co-loading platform for protein drugs, calcium phosphate can also form a synergistic "warehouse" of ions and proteins at the defect site, maintaining protein activity while providing ion and structural dual assistance for its pro-osteogenic effect, ultimately achieving dual enhancement of bone mass and bone quality and rapid functional reconstruction.

[0036] Manganese ions (Mn 2+ ): Mn 2+ can activate the cGAS-STING pathway, drive the maturation of dendritic cells and the secretion of type I interferons, significantly alleviate the immune suppression of the tumor microenvironment, and transform the "cold" microenvironment into a "hot" microenvironment, providing strong innate immune drive for the antitumor or anti-infective effect of protein drugs. In addition, relying on the multi-point coordination of PEG-b-Glu to Mn 2+ and the embedding and fixation of the calcium phosphate mineralization network, manganese ions can be stably loaded under neutral conditions and avoid excessive free exposure, and can be released on demand in the endosome / lysosome microenvironment after cell uptake, achieving time and space controllable immune activation while ensuring safety and obtaining stable and repeatable immune enhancement.

[0037] In some embodiments of the present application, the end group of polyethylene glycol is methoxy or hydroxy, and the end group of polyglutamic acid polymer is amino.

[0038] In some embodiments of the present application, the target protein is selected from at least one of enzymes, antibodies or fragments thereof, cytokines, receptor proteins, antigen proteins, transcription factors, gene editing related proteins or complexes thereof.

[0039] In some embodiments of the present application, the carrier is freeze-dried to obtain a powder preparation, and the preparation contains at least one of sucrose, trehalose or mannitol as a forming / protecting adjuvant.

[0040] The embodiments of the present application also provide a method for preparing an intracellular protein delivery carrier with immune enhancement and bone repair functions, comprising, Step 1: dissolving calcium chloride and manganese chloride in a Tris-HCl aqueous solution to obtain solution A; Step 2: dissolving PEG-b-Glu and the target protein in a HEPES buffer solution containing disodium hydrogen phosphate to obtain solution B; Step 3: slowly adding the solution B to the solution A, stirring for a certain time to form a manganese-containing calcium phosphate mineralization core and co-encapsulate the target protein; Step 4: dialyzing and freeze-drying the obtained dispersion system to obtain the carrier. Specifically, the dialysis and ultrafiltration method is used for purification, a 25mM HEPES saline buffer solution (pH 7.4, containing 140mM NaCl and 2mM CaCl2) is used as the buffer system, a corresponding size of dialysis membrane (MWCO = 3500-14000) is selected according to the size of the loaded protein, and dialysis is performed at room temperature for 12 hours to remove free ions and small molecular impurities, and finally the carrier dispersion is obtained.

[0041] In step 1, the synthesis method of the PEG-b-Glu block copolymer is as follows: S1: using NCA ring-opening polymerization method to synthesize polyethylene glycol-polyglutamic acid (PEG-b-Glu) block copolymer.

[0042] S2: by adjusting the ratio of polyethylene glycol (PEG) and polyglutamic acid (Glu) monomers, PEG-b-Glu block copolymers with different molecular weights and degrees of polymerization are obtained.

[0043] S3: the structure and molecular weight of the synthesis product are determined by 1H nuclear magnetic resonance (NMR) spectrum analysis.

[0044] In a buffer aqueous solution with pH 7.0-7.8, dissolving polyethylene glycol-polyglutamic acid and target protein includes dissolving polyethylene glycol-polyglutamic acid two-block copolymer and target protein in a HEPES buffer solution containing disodium hydrogen phosphate, and then adding calcium chloride and manganese chloride dissolved in Tris hydrochloride in advance.

[0045] In step S2, Ca 2+Calcium chloride (250 mM), Mn 2+ Manganese chloride (20 mM), Tris-HCl concentration of 1 mM, pH = 7.6. It should be noted that by adjusting the pH value and ion concentration, the particle size and morphology of the calcium phosphate nanoparticles can be controlled to ensure that they are suitable as drug carriers.

[0046] In some embodiments of the present application, the method further comprises step 5: freeze-drying the dispersion after mixing with a freeze-drying protective agent to obtain the carrier solid preparation.

[0047] The present application also provides the use of the carrier in the preparation of a medicament for enhancing the innate immune response, wherein the enhancement of the innate immune response includes the induction of type I interferon production by the activation of the Mn-mediated cGAS-STING signaling pathway; and the medicament is used to prepare a preparation for tumor immunotherapy, immune enhancement of infectious diseases, or vaccine adjuvant.

[0048] The present application first discloses that the nanocarrier of calcium phosphate combined with manganese ions of polyethylene glycol-polyglutamic acid can enhance immunity, promote bone repair, and deliver protein drugs into cells, and verifies its ability to activate the cGAS-STING pathway to achieve enhanced immunity and promote bone repair.

[0049] In example 1, polyethylene glycol-polyglutamic acid diblock copolymer (7.17 mg) and protein drug cytochrome C (1.8 mg) were dissolved in a HEPES buffer solution (50 mM, pH = 7.1) containing sodium phosphate dibasic (6 mM), and then calcium chloride (250 mM) and manganese chloride (20 mM) previously dissolved in Tris-HCl were added. After stirring (20℃, 1h), dialysis and freeze-drying, a new intracellular protein delivery carrier (denoted as CaPMn@CC) was obtained, which simultaneously encapsulated protein drugs and had the characteristics of immune enhancement effect and bone repair function.

[0050] In example 2, polyethylene glycol-polyglutamic acid diblock copolymer (7.17 mg) and drug molecule doxorubicin (1.8 mg) were dissolved in a HEPES buffer solution (50 mM, pH = 7.1) containing sodium phosphate dibasic (6 mM), and then calcium chloride (250 mM) and manganese chloride (20 mM) previously dissolved in Tris-HCl were added. After stirring (20℃, 1h), dialysis and freeze-drying, a new intracellular protein delivery carrier (denoted as CaPMn-DOX) was obtained, which simultaneously encapsulated protein drugs and had the characteristics of immune enhancement effect and bone repair function.

[0051] Example 3: Polyethylene glycol-polyglutamic acid diblock copolymer (7.17 mg) and protein molecule recombinant human bone morphogenetic protein 2 (BMP-2) (20 μg) were dissolved in HEPES buffer solution (50 mM, pH = 7.1) containing sodium phosphate dibasic (6 mM), followed by the addition of calcium chloride (250 mM) and manganese chloride (20 mM) previously dissolved in Tris hydrochloride. After stirring (20 °C, 1 h), dialysis, and lyophilization, a novel intracellular protein delivery carrier (denoted as CaPMn-BMP2) simultaneously encapsulating protein drugs and having the functions of immune enhancement and bone repair was obtained.

[0052] Example 4: Polyethylene glycol-polyglutamic acid diblock copolymer (7.17 mg) and protein molecule recombinant human osteoprotegerin (OPG) (10 μg) were dissolved in HEPES buffer solution (50 mM, pH = 7.1) containing sodium phosphate dibasic (6 mM), followed by the addition of calcium chloride (250 mM) and manganese chloride (20 mM) previously dissolved in Tris hydrochloride. After stirring (20 °C, 1 h), dialysis, and lyophilization, a novel intracellular protein delivery carrier (denoted as CaPMn-OPG) simultaneously encapsulating protein drugs and having the functions of immune enhancement and bone repair was obtained.

[0053] Example 5: Polyethylene glycol-polyglutamic acid diblock copolymer (7.17 mg) and protein molecule recombinant human bone morphogenetic protein 7 (BMP-7) (20 μg) were dissolved in HEPES buffer solution (50 mM, pH = 7.1) containing sodium phosphate dibasic (6 mM), followed by the addition of calcium chloride (250 mM) and manganese chloride (20 mM) previously dissolved in Tris hydrochloride. After stirring (20 °C, 1 h), dialysis, and lyophilization, a novel intracellular protein delivery carrier (denoted as CaPMn-BMP7) simultaneously encapsulating protein drugs and having the functions of immune enhancement and bone repair was obtained.

[0054] Example 6: Polyethylene glycol-polyglutamic acid diblock copolymer (7.17 mg) and protein molecule recombinant human bone morphogenetic protein 7 (BMP-7) (20 μg) were dissolved in HEPES buffer solution (50 mM, pH = 7.1) containing sodium phosphate dibasic (6 mM), followed by the addition of calcium chloride (250 mM) and manganese chloride (20 mM) previously dissolved in Tris hydrochloride. After stirring (20 °C, 1 h), dialysis, and lyophilization, a novel intracellular protein delivery carrier (denoted as CaPMn-BMP7) simultaneously encapsulating protein drugs and having the functions of immune enhancement and bone repair was obtained.

[0055] Example 7: Determination of drug loading and encapsulation efficiency of nanoparticles.

[0056] Take the purified nanometer particle suspension 100 μL of Example 1, add 100 μL of 0.1M HCl vortex, thoroughly decomposed CaP core release cytochrome C. 12,000 rpm centrifugation for 10 minutes, take the supernatant, using BCA protein quantitative kit, according to the instructions, at 562 nm wavelength to determine the absorbance. According to the standard curve to calculate the concentration of CC in the sample, and then calculate the drug loading (DLC) and encapsulation efficiency (EE%). Calculation formula: Drug loading (DLC, wt%) = (the mass of the CC loaded / total mass of nanoparticles) x 100%; Encapsulation efficiency (EE%) = (the mass of the CC loaded / total mass of CC fed) x 100%; The DLC of CaPMn@CC was measured to be 19.19 ± 0.7%, and the EE% was 72.3 ± 5.1%.

[0057] Example 8: Nanoparticle particle size, zeta potential and morphology characterization.

[0058] Take the nanoparticle suspension prepared in Example 1, dilute with ultrapure water, and use dynamic light scattering (DLS) particle size instrument to measure its hydrodynamic diameter and polydispersity index (PDI), the average particle size is 68.1 nm, PDI is 0.216 ( Figure 2 ). Using the same instrument laser Doppler electrophoresis module to determine its Zeta potential, the value is -4.26 mV ( Figure 3 ).

[0059] Take 10 μL of diluted nanoparticle suspension and drop it on a carbon support film copper mesh, let it stand for 2 minutes, then use filter paper to absorb the excess liquid, and dry naturally. Transmission electron microscope (TEM, JEOL JEM-1400 Flash, acceleration voltage 120 kV) is used to observe the morphology and take pictures. TEM images show that the nanoparticles are regular spherical or spherical, uniformly distributed, with a particle size of about 70 nm, which is consistent with the DLS result ( Figure 4 ).

[0060] Using ICP-MS, the Mn 2+ content is 1.645%, and the Ca 2+ content is 2.1%.

[0061] Example 9: pH-responsive drug release behavior of nanoparticles.

[0062] The release behavior of the nanoparticles under different pH conditions was investigated by dialysis method. 1 mL of the CaPMn@CC nanoparticle suspension (containing about 350 μg of CC) prepared in Example 1 was taken and placed in a dialysis bag (MWCO: 14,000 Da) pre-treated, and was placed in 50 mL of a release medium with different pH values (PBS, pH = 7.4, 6.5, 6.0, 5.5), respectively, and was placed in a constant temperature shaker at 37°C and 100 rpm for release experiment. 1 mL of the release medium was taken out at the predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72 h), and 1 mL of fresh release medium at the same temperature was supplemented at the same time. The sample taken out was determined for the concentration of the released CC by HPLC or BCA method, the cumulative release rate was calculated, and the release curve was drawn. The results showed that the cumulative release rate was 13.9% at pH 7.4 for 48 hours, and the release rate was more than 30% at pH 5.5 for 4 hours, which showed a significant pH response function characteristic. Figure 6 ).

[0063] The release behavior of the manganese ions in the CaPMn@CC nanoparticles was determined using the same method, and the sample taken out at the predetermined time points (0.5, 1, 2, 4, 8 h) was determined for the concentration of the released Mn 2+ by ICP test (inductively coupled plasma test), the cumulative release rate was calculated, and the release curve was drawn. The results showed that the cumulative release rate was 16.1% at pH 7.4 for 8 hours, and the release rate was more than 59.3% at pH 5.0 for 1 hour (the cumulative release rate was 59.3% at pH 5.0 for 1 hour) Figure 5 , which showed a significant pH response function characteristic and provided a basis for enhancing immune function of the nano-carrier.

[0064] Example 10: Evaluation of endocytosis and lysosome escape.

[0065] Breast cancer cells 4T1 were inoculated in a confocal dish (10,000 cells / well), and were cultured in a 37°C, 5% CO2 incubator for 24 hours. The cells were divided into two groups: the experimental group was added with FITC-labeled CaPMn@CC-FITC nanoparticles (prepared by the method of Example 1, and CC was pre-labeled with FITC), and the control group was added with the same amount of free FITC-CC. After 4 hours of incubation, the culture medium was discarded, and the cells were gently washed with PBS for three times. The culture medium containing 200 nM LysoTracker TM Red (lysosome red fluorescent probe) was added, and was incubated for 1 hour. After washing with PBS again, the culture medium containing 1 μg / mL Hoechst 33342 (nuclear blue fluorescent dye) was added and was incubated for 10 minutes. After washing with PBS for three times, fresh culture medium was added, and the laser confocal microscope (CLSM) was used to observe and collect images immediately. The CLSM images (Fig. 6) showed that the experimental group had a large number of nanoparticles in the cytoplasm, and the nanoparticles were not in the lysosomes, which indicated that the nanoparticles could escape from the lysosomes. Figure 7) shows that the green fluorescence (FITC) signal of the nanoparticle group is widely distributed in the cytoplasm and less coincides with the red lysosome signal compared with the free FITC-CC group (cytochrome C cannot actively enter the cell, so the fluorescence cannot be developed in the cell), indicating that the nanoparticles can effectively promote the entry of CC into the cell and promote its escape from the lysosome into the cytoplasm.

[0066] Example 11: In vitro cytotoxicity experiment (MTT method).

[0067] 4T1 cells (5,000 cells / well) were inoculated in a 96-well plate and cultured for 24 hours. A blank control group (only culture medium), a negative control group (empty nanoparticle CaPMn), a free CC group and a CaPMn@CC nanoparticle group (Example 1) were set. Multiple concentration gradients and duplicate wells were set for each group. Treatment solutions containing different concentrations of CC (0.1, 0.5, 1, 5, 10 μM) were added, and the culture was continued for 48 hours. 20 μL of MTT solution (5 mg / mL) was added to each well, and the incubation was continued for 4 hours. The culture medium in the well was carefully aspirated, 150 μL of DMSO was added to each well, and the crystal formazan was fully dissolved by oscillation for 10 minutes. The absorbance value (OD value) of each well was determined at a wavelength of 490 nm using a microplate reader. The cell survival rate (%) was calculated as follows: (OD value of the experimental group-OD value of the blank group) / (OD value of the negative control group-OD value of the blank group) x 100%. The results show that, under the same CC concentration, the proliferation inhibition rate of the CaPMn@CC nanoparticle group on 4T1 cells is significantly higher than that of the free CC group and the empty nanoparticle group (p<0.01) Figure 8 , proving that it significantly enhances the cytotoxicity of CC through efficient delivery.

[0068] Example 12, female BALB / c mice (n=6 in each group) were injected subcutaneously with 1x10 6 4T1 cells on the right flank, and when the tumor grew to 100 mm 3 in diameter, the novel intracellular protein delivery carrier CaPMn@CC prepared in Example 1 was injected intravenously on days 0, 2, 4, 6 and 8 (1.5 mg / kg of effective Mn), and an equal amount of CaPMn nanoparticle carrier was injected intravenously, and pure cytochrome C (CC) was used as a control group. The tumor volume was measured regularly during the treatment period, and the tumor growth curve is shown in Figure 9 . Figure 9 The tumor growth curves of the control group and the CaPMn@CC treated mice (ANOVA test, p<0.0001) show that CaPMn@CC can effectively inhibit tumor growth compared with pure CC, proving that CaPMn@CC successfully delivers protein drugs into the cytoplasm to exert the cytotoxicity of protein drugs.

[0069] Example 13, female BALB / c mice (n=6 per group) were injected subcutaneously with 1 x 10 6 4T1 cells in the right flank, when the tumor grew to 100 mm 3 in diameter, the mice were treated with the novel intracellular protein delivery vehicle CaPMn prepared in Example 1 (1.5 mg / kg of effective Mn concentration) by intravenous injection on days 0, 2, 4, 6 and 8, and the same amount of MnCl2 and PBS were used as control groups. After treatment, the tumor tissues were taken for flow cytometry analysis. The CD80 immune cells of CaPMn were significantly higher than those of the control groups (ANOVA test, p<0.05) (see Figure 10- Figure 11 ). It was proved that CaPMn could enhance the body's immune function against tumors.

[0070] Example 14, female BALB / c mice (n=6 per group) were injected intratibially with 1 x 10 6 4T1 cells in the right hind leg, when the tumor grew to 100 mm 3 in diameter, the mice were treated with the novel intracellular protein delivery vehicle CaPMn prepared in Example 1 (1.5 mg / kg of effective Mn concentration) by intravenous injection on days 0, 2, 4, 6 and 8, and the same amount of MnCl2 and PBS were used as control groups. After treatment, the tumor tissues were taken for flow cytometry analysis. The CD80 immune cells of CaPMn were significantly higher than those of the control groups (ANOVA test, p<0.05) (see Figure 12- Figure 15 ). It was proved that CaPMn had bone repair ability.

[0071] Example 15: qPCR method was used to evaluate the ability of the nano-carrier to activate the cGAS-STING pathway.

[0072] 4T1 cells were seeded in 12-well plates (2 x 10 5 cells per well) and cultured for 24 hours. PBS control group (only culture medium), control group (MnCl2), and empty nano-particle CaPMn were set up. Each group was added with treatment solution containing the same effective concentration of Mn (26 μg / mL), and after 24 hours of incubation, TRIzol TMTotal RNA was extracted from cells lysed using Reagent (Invitrogen), and its concentration and purity were determined using a PORABIOPoNo-550 ultra-micro spectrophotometer (A260 / A280 ratios were all between 1.8 and 2.1). Reverse transcription and qPCR analyses were then performed. The expression levels of cGAS-STING pathway-related genes such as pSTING, pTBK1, and IFN-β were detected using the SYBR Green assay, with Actb as an internal reference gene, and the relative expression levels were calculated using the 2–ΔΔCt method. The results showed that, compared with the control group, the mRNA expression levels of the above genes were significantly upregulated in the nanocarrier-treated group (p<0.01), indicating that the material successfully activated the cGAS-STING signaling pathway (e.g., pSTING, pTBK1, IFN-β). Figure 16- Figure 18 ).

[0073] Furthermore, while the operations are described in a specific order, this should not be construed as requiring these operations to be performed in the specific order shown or in a sequential order. In certain environments, multitasking and parallel processing may be advantageous. Similarly, while several specific implementation details are included in the above discussion, these should not be construed as limiting the scope of this disclosure. Certain features described in the context of individual embodiments may also be implemented in combination in a single embodiment. Conversely, various features described in the context of a single embodiment may also be implemented individually or in any suitable sub-combination in multiple embodiments.

[0074] Although the subject matter has been described using language specific to structural features and / or methodological logic, it should be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or actions described above. Rather, the specific features and actions described above are merely illustrative examples of implementing the claims.

[0075] The above description is only a specific embodiment of this disclosure, but the protection scope of this disclosure is not limited thereto. The protection scope of this disclosure should be determined by the protection scope of the claims.

Claims

1. An intracellular protein delivery carrier with immune-enhancing and bone-repairing functions, characterized in that: include: The core is formed by a calcium-phosphorus inorganic phase, wherein the inorganic phase is a manganese-containing calcium phosphate phase; A hydrophilic layer covering the outer surface of the core, the hydrophilic layer being provided by the block copolymer polyethylene glycol-polyglutamic acid; The target protein loaded in the core is co-encapsulated during the aqueous phase mineralization process to form the core without chemical modification; The carrier is capable of acid-sensitive dissolution and release of the target protein and Mn under acidic conditions in the endosome / lysosome. 2 + .

2. The intracellular protein delivery carrier with immune-enhancing and bone-repairing functions as described in claim 1, characterized in that: Polyethylene glycol has methoxy or hydroxyl end groups, while polyglutamic acid polymers have amino end groups.

3. The intracellular protein delivery carrier with immune-enhancing and bone-repairing functions as described in claim 1, characterized in that: The target protein is selected from at least one of enzymes, antibodies or fragments thereof, cytokines, receptor proteins, antigen proteins, transcription factors, gene editing-related proteins or complexes thereof.

4. The intracellular protein delivery carrier with immune-enhancing and bone-repairing functions as described in claim 1, characterized in that: The intracellular protein delivery carrier is freeze-dried to obtain a powder formulation, wherein the powder formulation contains at least one of sucrose, trehalose, or mannitol as a forming / protecting excipient.

5. A method for preparing the intracellular protein delivery carrier with immune-enhancing and bone-repairing functions according to any one of claims 1-4, characterized in that: include, Step 1: Dissolve calcium chloride and manganese chloride in Tris-HCl aqueous solution to obtain solution A; Step 2: Dissolve PEG-b-Glu and the target protein in HEPES buffer containing disodium hydrogen phosphate to obtain solution B; Step 3: Slowly add solution B to solution A, stir for a certain period of time to form a manganese-containing calcium phosphate mineralization core and co-encapsulate the target protein; Step 4: Dialyze the obtained dispersion system and freeze-dry it to obtain the carrier.

6. The method as described in claim 5, characterized in that: The stirring temperature is 15–25℃ and the stirring time is 0.5–2h.

7. The method as described in claim 6, characterized in that: The concentration of calcium chloride in solution A is 150mM–300mM, and the concentration of manganese chloride is 15mM–30mM; the concentration of HEPES buffer in solution B is 50mM, the pH is 7.0–7.5, and the concentration of disodium hydrogen phosphate is 6mM.

8. The method as described in claim 7, characterized in that: The concentration of Tris-HCl in solution A is 0.8 mM–1.2 mM, and the pH is 7.3–7.

8.

9. Use of an intracellular protein delivery carrier as described in any one of claims 1–4 in the preparation of a medicament for enhancing innate immune responses, characterized in that: The enhanced innate immune response includes inducing type I interferon production through activation of the Mn-mediated cGAS-STING signaling pathway.

10. The use as described in claim 9, characterized in that: The drug is used to prepare formulations for use in tumor immunotherapy, immune enhancement of infectious diseases, or as vaccine adjuvants.

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