Use of annexin in improving tumor penetration efficiency of nanocarrier with phospholipid surface

Abstract

Provided is use of annexin in improving the tumor penetration efficiency of a nanocarrier with a phospholipid surface. The present invention innovatively explored a protein capable of promoting the tumor penetration efficiency of a phospholipid-based nanocarrier, and found that coating the phospholipid-based nanocarrier with annexin can improve the tumor penetration efficiency, thereby improving the bioavailability of a drug loaded with the nanocarrier and improving the therapeutic effect. In addition, it is also found that the efficiency-enhancing ability of annexin is positively correlated with the expression level of integrin α5 or β1. By means of pre-detecting and analyzing the expression level of integrin α5 or β1 in a patient, the ability of annexin coating technology to promote the transport of nanocarriers can be predicted, and the beneficiary population suitable for the use of the annexin coating technology can be screened.

Description

Application of annexin in improving tumor entry efficiency of nano-carriers with phospholipid surface TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to application of annexin in improving tumor entry efficiency of nano-carriers with phospholipid surface. BACKGROUND

[0002] Phospholipid-based nano-carriers are innovative treatment modes for tumors, and are increasingly widely used in clinical applications. Such carriers include liposomes, lipid nanoparticles (LNP), phospholipid-coated nano-carriers (such as phospholipid-coated, lipid prodrug-coated, and biomembrane-coated organic or inorganic inner core designs), and the like. By using these nano-carriers, the bioavailability and safety of active pharmaceutical ingredients (API) can be improved. However, the anti-tumor activity of these nano-drug carrier designs is limited, and in many tumor situations, the anti-tumor activity of nano-drugs is comparable to that of free drugs.

[0003] There are many reasons for the unsatisfactory anti-tumor efficacy of nano-drug delivery systems, including the fact that existing nano-designs lack sufficient consideration of pathological biological barriers (such as tumor vascular endothelial cell barriers, tumor stroma, and intratumoral pericytes). It is worth mentioning that the tumor enrichment ability of intravenously injected nano-drug delivery systems is often attributed to the interstitial space of tumor vascular endothelial cells, and the so-called EPR effect (enhanced permeability and retention effects) is used to explain the high concentration of nano-drug carriers in tumors. There is an important debate and new insight into the accurate explanation of the “enhanced permeability retention” (EPR) effect, which enables cancer nano-carriers to penetrate solid tumors. The traditional explanation of the EPR effect mainly considers the leakage of the tumor vascular system as the main factor controlling the delivery / accumulation of nanoparticles.

[0004] Although it is now recognized that the EPR effect is limited in PDAC and BC, the nano-formulations currently available for these cancers, including irinotecan (IRIN) and doxorubicin (DOX) loaded liposomes, are not intentionally designed to address the challenges posed by tumor ECs. On the contrary, these liposomes seem to rely on the putative EPR effect, which may not always be present or effective in PDAC and BC, at least. Therefore, there is an urgent need for an effective method to improve the performance of nano-carriers in cancer types with poor EPR effect.

[0005] More and more evidence shows that the EPR effect is not the only mechanism for tumor entry, and the transcytosis effect is another important mechanism for the nanocarrier to break through the vascular endothelial barrier, which is called the ETR effect (enhanced transcytosis and retention effect) by the inventor, and is a non-classical EPR effect. That is, the nanocarrier tumor entry mechanism is completed through multiple mechanisms such as EPR and ETR. In some high-stroma solid tumors (such as pancreatic cancer, triple-negative breast cancer, colorectal cancer, and some lung cancer, etc.), ETR is the main tumor entry mechanism, and its effect is greater than the EPR effect. Unlike the classical EPR effect, transcytosis does not depend on the endothelial gap of tumor blood vessels. From the morphological point of view, the interaction between nanoparticles and blood vessels can be simply understood as "going through", rather than "leaking out". However, there is no systematic study on the nanobiological nature, regulation mechanism and application behind the transcytosis effect.

[0006] In summary, it is of great significance to the field of tumor treatment to deeply study the tumor entry mechanism of nanocarriers and develop efficient tumor entry carriers. SUMMARY

[0007] In view of the deficiencies of the prior art and actual needs, the application provides an application of annexin in improving the tumor entry efficiency of nanocarriers with a phospholipid surface, excavates proteins capable of improving the tumor entry efficiency of phospholipid nanocarriers, and then improves the biodistribution of the loaded drugs in tumors, improves the bioavailability and curative effect of the drugs.

[0008] To achieve this purpose, the application adopts the following technical solutions:

[0009] In a first aspect, the application provides an application of annexin in improving the tumor entry efficiency of phospholipid nanocarriers.

[0010] In the application, based on the analysis of the protein corona on the surface of the phospholipid nanocarrier and the verification of the protein coating experiment, it is found that annexin can be combined with the surface of the phospholipid nanocarrier and enhance the transcytosis effect of the phospholipid nanocarrier, thereby improving the efficiency (tumor enrichment ability) of the phospholipid nanocarrier to reach the tumor site.

[0011] Preferably, the annexin includes any one of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, annexin A9 or a combination of at least two thereof.

[0012] Preferably, the annexin is of human origin.

[0013] It is understood that the phospholipid-based nanocarriers refer to the nanocarriers with phospholipid surface commonly used in the art, including any one of liposomes, lipid nanoparticles or phospholipid-coated nanoparticles (such as inorganic nanoparticles, organic nanoparticles, nanoparticles of natural origin, etc.) or a combination of at least two thereof.

[0014] Preferably, the application comprises modifying the annexin on the surface of the phospholipid-based nanocarriers.

[0015] Preferably, the liposomes comprise any one of doxorubicin liposomes, irinotecan liposomes, paclitaxel liposomes, mitoxantrone liposomes, daunorubicin cytarabine liposomes, mivazerol liposomes or amphotericin liposomes or a combination of at least two thereof.

[0016] Preferably, the method for modification comprises mixing and incubating the annexin with the phospholipid-based nanocarriers.

[0017] Preferably, the phospholipid-based nanocarriers comprise one, two or more of the following lipids:

[0018] Phospholipids: Egg phosphatidylcholine (EPC), Soy phosphatidylcholine (SPC), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), Dioleoyl-phosphatidylethanolamine (DOPE), Phosphatidylglycerol (PG), such as: DMPG, DSPG, Phosphatidylinositol (PI), Phosphatidic acid (PA), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), Hydrogenated soy phosphatidylcholine (HSPC);

[0019] Cholesterol and Analogs: Cholesterol, Cholesteryl hemisuccinate (CHEMS), Cholesteryl oleate, Sitosterol;

[0020] PEGylated Lipids: DSPE-PEG2000, (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]), DOPE-PEG2000, DMPE-PEG2000, DSPE-PEG-maleimide / biotin / amine, Cholesterol-PEG derivatives, and PEGylated lipids with similar structures, different molecular weights of PEG;

[0021] Ionizable & Cationic Lipids: Ionizable lipids, DLin-MC3-DMA, SM-102, ALC-0315, DODMA (Dimethyldioctadecylammonium), Dlin-DMA;

[0022] Permanent cationic lipids: DOTAP (Dioleoyltrimethylammonium propane), DOTMA (Dioleoyltrimethylammonium), DDAB (Dimethyldioctadecylammonium bromide), CTAB (Cetyltrimethylammonium bromide);

[0023] Anionic & Zwitterionic Lipids: DMPG / DSPG (Phosphatidylglycerol, anionic), DOPS / POPS (Phosphatidylserine, anionic), Cardiolipin, Sphingomyelin;

[0024] Targeting & Functional Lipids: DSPE-PEG-Maleimide, DSPE-PEG-Folate, DSPE-PEG-RGD, Glycolipids (e.g. GM1, GM3); and

[0025] Other Specialized Lipids: Monolaurin, Lysolipids, Squalene derivatives, Ceramides / Sphingolipids.

[0026] Preferably, the phospholipid-based nanoparticle comprises a phospholipid consisting of:

[0027] (i) Distearoylphosphatidylcholine (DSPC), Cholesterol (Chol) and (DSPE-PEG2000);

[0028] (ii) Distearoylphosphatidylcholine (DSPC), Sodium dioleoylphosphatidyl acid (DOPA), (2,3-Dioleoyl-propyl)-trimethylammonium-chloride salt (DOTAP): Cholesterol (Chol) and (DSPE-PEG2000);

[0029] (iii) Distearoylphosphatidylcholine (DSPC), Sodium dioleoylphosphatidyl acid (DOPA), Cholesterol (Chol) and (DSPE-PEG2000);

[0030] (iv) Hydrogenated soy phosphatidylcholine (HSPC), Cholesterol (Chol) and (DSPE-PEG2000);

[0031] (v) Distearoylphosphatidylcholine (DSPC) and Cholesterol (Chol);

[0032] (vi) Egg yolk phosphatidylcholine (EPC) and Cholesterol (Chol);

[0033] (vii) Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylglycerol (DPPG), Cholesterol (Chol) and Triolein;

[0034] (viii) Sphingomyelin (SM) and Cholesterol (Chol);

[0035] (ix) Egg yolk phosphatidylglycerol (EPG) and Dimyristoylphosphatidylcholine (DMPC);

[0036] (x) distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), cholesterol (Chol);

[0037] (xi) palmitoyloleylphosphatidylcholine (POPC) and dioleoylphosphatidylserine (DOPS);

[0038] (xii) diarachidoylphosphatidylcholine (DEPC), palmitoylphosphatidylglycerol (DPPG), cholesterol (Chol) and tricaprylin;

[0039] (xiii) egg yolk phosphatidylcholine (EPC) and sodium deoxycholate; or

[0040] (xiv) hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Chol) and distearoylphosphatidylglycerol (DSPG).

[0041] In a second aspect, the present application provides a use of a membrane-associated protein in the preparation of a delivery carrier, wherein the membrane-associated protein comprises any one of or a combination of at least two of the following: annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1 or annexin A9.

[0042] Preferably, the use comprises modifying the membrane-associated protein on the surface of the phospholipid-based nanocarrier.

[0043] Preferably, the phospholipid-based nanocarrier comprises any one of or a combination of at least two of the following: a liposome, a lipid nanoparticle or a phospholipid-coated nanoparticle.

[0044] In the present application, based on the membrane-associated protein having the transcytosis enhancement effect, the membrane-associated protein can be further applied to the preparation of a delivery carrier to obtain a delivery carrier with enhanced tumor entry efficiency.

[0045] In a third aspect, the present application provides a delivery carrier, comprising a phospholipid-based nanocarrier and a membrane-associated protein modified on the surface of the nanocarrier.

[0046] Preferably, the membrane-associated protein comprises any one of or a combination of at least two of the following: annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1 or annexin A9.

[0047] Preferably, the phospholipid-based nanocarriers include any one or a combination of at least two of liposomes, lipid nanoparticles, or phospholipid-coated nanoparticles.

[0048] In a fourth aspect, the present application provides an application of annexin in improving the drug biodistribution at a lesion site or in preparing a pharmaceutical composition, which includes modifying the annexin on the surface of a phospholipid-based nanocarrier, and loading the phospholipid-based nanocarrier modified with the annexin with a drug.

[0049] Preferably, the annexin includes any one or a combination of at least two of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, or annexin A9.

[0050] Preferably, the phospholipid-based nanocarriers include any one or a combination of at least two of liposomes, lipid nanoparticles, or phospholipid-coated nanoparticles.

[0051] Preferably, the lesion site includes a lesion site with high expression of α5 integrin and / or β1 integrin.

[0052] In the present application, the specific mechanism of annexin in improving the tumor entry efficiency of phospholipid-based nanocarriers is analyzed in depth, and it is found that the synergistic effect of annexin is realized through a “sandwich model” of phospholipid surface-annexin-integrin, that is, annexin can first bind to the surface of phospholipid-based nanocarriers, and then annexin can also interact with integrin (such as α5 integrin or β1 integrin) on the surface of cells to promote the “transcytosis” of phospholipid-based nanocarriers. Therefore, based on this mechanism, it is known that annexin can not only promote the enrichment of phospholipid-based nanoparticles at tumor sites, but also promote the enrichment of phospholipid-based nanoparticles at other lesion tissues with high expression of α5 and / or β1 subunits. Specifically, the tumor types include breast cancer, pancreatic cancer, liver cancer, ovarian cancer, osteosarcoma, prostate cancer, glioma, melanoma, myxofibrosarcoma, skin cancer, lung cancer, and gastric cancer, etc., and the non-tumor types include psoriasis and diabetes, etc.

[0053] In addition, the present application further verifies through experiments the influence of annexin on the bioavailability and efficacy of the drug loaded by the phospholipid-based nanocarriers, and it is found that the bioavailability of drug delivery and the optimization of efficacy can be achieved.

[0054] In a fifth aspect, the present application provides a nanodelivery system, which includes a phospholipid-based nanocarrier, annexin, and a drug, the annexin is modified on the surface of the nanocarrier, and the drug is loaded inside the phospholipid-based nanocarrier.

[0055] In the present application, based on the ability of annexin to improve the bioavailability of drug delivery and optimize the therapeutic effect, a nano delivery system is further developed, phospholipid nanocarriers are used to load drugs, and annexin is used to improve the delivery efficiency, thereby improving the bioavailability of drugs and the therapeutic effect.

[0056] It can be understood that the drugs in the present application that can be delivered by phospholipid nanocarriers include drugs commonly used in the art, such as antitumor drugs doxorubicin, irinotecan, paclitaxel, mitoxantrone, daunorubicin, cytarabine, and mivabumab.

[0057] Preferably, the annexin comprises any one or a combination of at least two of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, or annexin A9.

[0058] Preferably, the phospholipid nanocarrier comprises any one or a combination of at least two of liposomes, lipid nanoparticles, or phospholipid-coated nanoparticles.

[0059] In the present application, the safety of annexin is analyzed, and it is found that annexin has no biological toxicity effect, and annexin can spontaneously bind to the surface of phospholipid nanocarriers. Therefore, in actual application, the delivery carrier coated with annexin can be prepared in advance, or the phospholipid nanocarrier can be mixed with annexin and incubated before use.

[0060] In a sixth aspect, the present application provides a combination drug composition comprising a phospholipid nanocarrier loaded with a drug and annexin. The combination drug composition is a single complex preparation or a combination of two separate preparations.

[0061] Preferably, the annexin comprises any one or a combination of at least two of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, or annexin A9.

[0062] Preferably, the phospholipid nanocarrier comprises any one or a combination of at least two of liposomes, lipid nanoparticles, or phospholipid-coated nanoparticles.

[0063] In a seventh aspect, the present application provides the use of a reagent for detecting α5 integrin and / or β1 integrin in evaluating the ability of annexin to improve the efficiency of phospholipid nanocarriers into tumors and / or improve the drug biodistribution at the lesion site.

[0064] In the present application, based on the annexin potentiation mechanism "phospholipid surface-annexin-integrin" sandwich model, further quantitative analysis found that the potentiation effect of annexin was positively correlated with the expression level of a5 integrin and / or b1 integrin. In the integrin high expression model (for example, breast cancer, log2 of FPKM≧4; FPKM (fragments per kilobase per million mapped reads) represents the gene expression level), the phospholipid-based nanocarrier can significantly enhance the tumor efficiency, the drug bio-distribution and the therapeutic effect at the lesion site. In the integrin low expression model, the potentiation effect is generally low. Therefore, by detecting and analyzing the a5 or b1 integrin expression level of the patient in advance, the ability of annexin coating to promote the transportation of nanocarriers and improve the drug efficacy can be predicted, and the population that can apply the annexin coating technology can be screened, so as to further realize the precision treatment.

[0065] Preferably, the application comprises: detecting the expression level of a5 integrin and / or b1 integrin in the patient, and evaluating the tumor efficiency of the delivery carrier of the third aspect or evaluating the therapeutic effect of the nanometer delivery system of the fifth aspect or the combination drug composition of the sixth aspect based on the expression level.

[0066] In the eighth aspect, the present application provides a kit comprising

[0067] (a) a drug-loaded phospholipid-based nanocarrier; and

[0068] (b) annexin;

[0069] The annexin comprises any one or a combination of at least two of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1 or annexin A9;

[0070] The phospholipid-based nanocarrier comprises any one or a combination of at least two of liposomes, lipid nanoparticles or phospholipid-coated nanoparticles;

[0071] Preferably, the drug-loaded phospholipid-based nanocarrier is mixed and incubated with the annexin before use.

[0072] In the ninth aspect, the present application provides the use of annexin and phospholipid-based nanocarriers in the preparation of a medicament for treating diseases. This aspect can also be expressed as the annexin and phospholipid-based nanocarriers of the present application for treating diseases.

[0073] Preferably, the lesion site of the disease is a lesion site with high expression of α5 integrin and / or β1 integrin.

[0074] Preferably, the disease is selected from breast cancer, pancreatic cancer, liver cancer, ovarian cancer, osteosarcoma, prostate cancer, glioma, melanoma, myxofibrosarcoma, skin cancer, lung cancer, gastric cancer, psoriasis, diabetes, or a combination thereof.

[0075] In a tenth aspect, the present application provides a method for treating a disease, comprising administering the combination pharmaceutical composition of the present application or the kit of the present application to a subject in need.

[0076] Preferably, the lesion site of the disease is a lesion site with high expression of α5 integrin and / or β1 integrin.

[0077] In one embodiment, the disease is cancer, and the medicament is used for treating cancer; preferably, the cancer is selected from breast cancer, pancreatic cancer, liver cancer, ovarian cancer, osteosarcoma, prostate cancer, glioma, melanoma, myxofibrosarcoma, skin cancer, lung cancer, gastric cancer, or a combination thereof; preferably, the medicament comprises doxorubicin, irinotecan, paclitaxel, mitoxantrone, or a combination thereof.

[0078] In one embodiment, the disease is psoriasis, and the medicament is used for treating psoriasis; in another embodiment, the disease is diabetes, and the medicament is used for treating diabetes.

[0079] In one embodiment, the method for treating a disease of the present application further comprises a step of detecting the expression of α5 integrin and / or β1 integrin at a lesion site in a subject before administration.

[0080] Compared with the prior art, the present application has the following beneficial effects:

[0081] The present application creatively mines proteins capable of promoting the tumor entry efficiency of current phospholipid nanocarriers, finds that coating phospholipid nanocarriers with annexin can promote the “transcytosis” of nanocarriers, improve the tumor entry efficiency, and thus improve the bioavailability of nanocarrier-loaded drugs and the therapeutic effect. In addition, it is found that the synergistic ability of annexin is based on the “phospholipid surface-annexin-integrin” structure, and is positively correlated with the expression amount of α5 or β1 integrin. Therefore, it is known that pre-coating with annexin can also increase the biodistribution of phospholipid nanocarriers at lesion sites with high expression of α5 or β1 integrin, and the ability of annexin-coating technology to promote the transport of nanocarriers can be predicted by pre-detecting and analyzing the expression amount of α5 or β1 integrin in patients, and the benefit population that can apply the annexin-coating technology can be screened. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 shows the mechanism of nanoparticle tumor entry.

[0083] Figure 2 shows an established orthotopic KPC model, a stringent preclinical model for studying drug delivery. The luciferase gene was introduced into KPC cells. After orthotopic implantation of KPC-luc cells, subsequent necropsy and bioluminescence imaging found primary tumors growing within 1-2 weeks. Trichrome staining shows a primary tumor rich in stroma adjacent to normal pancreatic tissue.

[0084] Figure 3 is a topography map of probes F1, F2, F3, with a scale of 25 nm.

[0085] Figure 4 is a schematic diagram of the structure of gold core nanocarriers.

[0086] Figure 5 is a biodistribution map of different types of phospholipid nanoparticles in KPC orthotopic pancreatic cancer mice; ex vivo IVIS imaging of tumor-bearing mice receiving 50 mg / kg intravenous injection of DyLight680-labeled F1-F3 (n=4) is shown. Tumor tissues and major organs were collected 24 hours after injection. The fluorescence intensity in the tumor and liver was quantified using IVIS live image software for region of interest (ROI) analysis.

[0087] Figure 6 is a statistical chart of the biodistribution of different types of phospholipid nanoparticles in KPC orthotopic pancreatic cancer mice; F1 emits the highest particle signal at the tumor site. Data are expressed as mean ± standard deviation. Statistical significance was assessed by unpaired t-test *p<0.05.

[0088] Figure 7 is a schematic diagram of serum incubation of various particle variants F1 to F3, followed by mass spectrometry analysis.

[0089] Figure 8 is a graph of the results of nanocarrier surface protein corona analysis. The heatmap shows the most abundant proteins detected on the surface of the nanocarriers. We show 46 proteins that exhibit high abundance. The right panel is a Venn diagram of the identified proteins in F1-F3. The unique protein species specific to the best-performing particle (F1) in the biodistribution study is highlighted by the alpha zone (red). The beta zone (cyan) includes protein types detected in F1 that also appear in F2 and F3.

[0090] Figure 9 is a graph of the results of nanocarrier surface coating protein electrophoresis analysis. Based on the findings of Example 1, ANXA2, ANXA3, ANXA5, ANXA7, and ANXA8 were tested, as well as pure VTN, albumin, and GAPDH. After incubating F1 particles with these protein types, electrophoresis analysis confirmed the effective attachment of these proteins on F1.

[0091] Figure 10 is a schematic of the EC transwell apparatus used to assess the potential of nanoparticle transport across a HUVEC monolayer.

[0092] Figure 11 is a graph of the results of the analysis of the ability of different protein coated nanocarriers to cross epithelial cells; the transport activity of A2, A3, A5 and VTN pre-coated particles was enhanced compared to albumin pre-coated particles and uncoated LC-MSNPs (n=3).

[0093] Figure 12 shows the use of 70kD dextran to confirm tight junctions in the transwell system. After the formation of the cell monolayer membrane, FITC labeled dextran of 70kD molecular weight was introduced to the upper layer of the transport well. The fluorescence value of FITC leaked through the cell layer to the lower layer was assessed at 1 hour. This can be used as an indicator of the integrity of the cell monolayer membrane. The results demonstrate the consistency of the tightness of the cell layer in our study (n=3, data shown as mean ± standard deviation).

[0094] Figure 13 is a graph of the results of the distribution of different protein coated nanocarriers in KPC orthotopic pancreatic cancer mice; IVIS imaging revealed biodistribution in tumors and major organs (n=4). The semi-quantification of fluorescence intensity revealed about 2-fold enrichment of distribution in the A2-coated group in KPC tumors, while not interfering with the biodistribution in other organs.

[0095] Figure 14 shows the A2 binding stability check. A2 pre-coated F1 nanoparticles were incubated in saline supplemented with serum for 0h, 3h, 6h, 12h, 24h and 48h. Subsequently, electrophoresis analysis was performed to assess the remaining proteins attached to the surface of F1 after washing with PBS 4 times.

[0096] Figure 15 is a graph of the results of the distribution of annexin A2 coated nanocarriers in different tumor models; the A2-coating method was replicated in other orthotopic models, i.e. breast cancer (EMT6, Py8119 and 4T1) and cancer (CT26), n=4. The tumor access of EMT6-BC model was improved by about 6-fold after a single intravenous injection.

[0097] Figure 16 shows representative ex vivo NIR fluorescence imaging of tumors and organs 24h after intravenous injection of NIR-labeled LC-MSNPs with different protein coatings in tumor-bearing mice (particle dose of 50mg / kg). Note that this study includes multiple cancer models, and the ex vivo IVIS images represent a snapshot taken at a single time point. There are subtle differences in the normal organ distribution across groups, which can require further investigation in the future.

[0098] Figure 17 is a version of gold core-labeled F1 pre-coated with A2 protein prepared.

[0099] Figure 18 is a graph of nanocarrier intratumoral morphology features; tumor samples were collected at 1 hour and 6 hours post intravenous injection, followed by TEM studies of tumor tissue. EC: endothelial cell; VVO: vesicle vacuole organelle. The inset image confirms the presence of gold labeling within the particle. Green arrow: transcellular nanoparticle within EC vesicle.

[0100] Figure 19 shows TEM images confirming the presence of nanoparticles within cancer cells at 6 hours post intravenous injection. Data are expressed as mean ± standard deviation. Statistical significance was assessed by unpaired t-test *p < 0.05.

[0101] Figure 20 shows: a, additional TEM images of tumor mice 1 hour post intravenous injection of gold A2-embedded LC-MSNPs. Significant phenomena observed within 1 hour include cell membrane protrusions, filopodia, and nanoparticle internalization into endothelial cells. These additional pictures show that transcytosis is occurring with the nanoparticles. b, TEM images of control EMT6 tumors that were not treated with anything. No obvious transcytosis features were observed in the control tumors.

[0102] Figure 21 is a graph of Martini force field constructed structure.

[0103] Figure 22 is a graph of coarse-grained (CG) simulation analysis results.

[0104] Figure 23 is a graph of all-atom (AA) simulation analysis results.

[0105] Figure 24 is a graph of contact area results between annexin A2 and lipids.

[0106] Figure 25 is a graph of the “phospholipid surface-annexin-integrin” sandwich model.

[0107] Figure 26 shows that while the interaction between A2 and a5b1 integrin is controlled by hydrophobic interactions (a), the interaction between A2 and the nanosurface (lipid) is mediated by electrostatic interactions (c). The specific amino acids and their positions in the indicated proteins are shown in the inset boxes, b (for A2 and a5b1 integrin) and d (for A2 and lipid).

[0108] Figure 27 is a graph of knockdown of a5b1 integrin expression affects nanocarrier transport results; nanoparticle transcytosis experiments were performed on integrin a5 subunit knockdown HUVEC cells by transwell assay.

[0109] Figure 28 shows transwell monolayer integrity check in shRNA-mediated gene knockdown experiments. FITC-Dextran fluorescence values across the endothelial cell layer gap in the transwell in gene knockdown experiments. Using shRNA did not change the integrity of the monolayer.

[0110] Figure 29 shows the effect of the transport inhibitor genistein on the transport of A2 pre-coated nanocarriers across HUVEC monolayers, the results show that particle transport is inhibited by approximately 40%. Data are presented as mean ± SD, n = 4. Statistical significance was assessed by unpaired t-test. ****p < 0.0001.

[0111] Figure 30 shows the physicochemical characterisation and protein binding stability analysis of mutant A2 (A2-M) coated LC-MSNPs. a, Size, PDI and zeta potential measurements of A2-M coated LC-MSNPs. c, Electrophoretic analysis showing the stability of A2 (left) and A2-M (middle) binding to LC-MSNPs at the indicated time points. The grey values of A2 and A2-M protein bands in the electrophoresis were quantified using ImageJ software.

[0112] Figure 31 shows the six amino acid residues on mutant A2 that were replaced with alanine. The resulting A2 mutant (A2-M) was used to pre-coat particles with NIR label and then the nanoparticle distribution study was performed in an orthotopic EMT6 mouse model. A2 coating was used as a control.

[0113] Figure 32 shows the “zombie” model: To investigate the transport of nanoparticles at the tumour site, we established a “zombie” model using EMT6 orthotopic tumour mice following a published protocol.

[0114] Figure 33 is a graph showing the effect of using formalin-fixed tumour blood vessels on nanoparticle transport; in the “zombie” mice, the blood vessels of EMT6 tumour-bearing mice were fixed and blood containing A2-coated LC-MSNPs was circulated using a pump. Data are presented as mean ± SD. Statistical significance was assessed by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. By this method, we demonstrated that the nanoparticle abundance at the “zombie” tumour site was reduced by approximately 70%.

[0115] Figure 34 shows that pharmacological inhibition of α5β1 integrin hinders A2 pre-coated nanocarriers from entering the tumour. KPC orthotopic tumour mice were first intravenously injected with 1 mg / kg of α5β1 integrin inhibitor ATN-161, and then 2 hours later, they were intravenously injected with 50 mg / kg of near-infrared labelled A2 pre-coated F1 nanoparticles. The mice were sacrificed and the tumours were harvested 24 hours after the F1 injection for intratumoural fluorescence quantification. Data are presented as mean ± SD, n = 4. Statistical significance was assessed by unpaired t-test. *p < 0.05.

[0116] Figure 35 shows A2 pre-coating enhances drug delivery effect of LC-MSNPs and liposomes. a, Size and PDI of drug-loaded nanocarriers were fully characterized before and after A2 coating. b and c, orthotopic EMT-6 BC and KPC PDAC were treated with DOX and IRIN loaded LC MSNPs, respectively. A2 pre-coated carriers were injected intravenously twice a week, and control groups included drug-loaded LC-MSNPs and normal saline (n=6). Product limit method analysis indicated that A2 pre-coating improved the therapeutic efficacy of IRIN-LC-MSNPs in PDAC (log-rank test). Similar improvement was observed in EMT-6 BC model (n=6, *p<0.05, **p<0.01). d, We confirmed the non-biological A2 conjugation using liposomes. e and f, IRIN and DOX were loaded into liposomes synthesized in-house. Treatment regimen was similar to (b and c). The dose of IRIN and DOX was 40 mg / kg and 10 mg / kg, respectively. Kaplan-Meier analysis (log-rank test) indicated that using A2 pre-coated DOX-loaded liposomes can significantly improve the therapeutic efficacy in EMT6 orthotopic model (p<0.001), while using A2 pre-coated IRIN liposomes can improve the survival rate in PDAC. g, Body weight data in the therapeutic efficacy data of b, c, e and f groups. Note that the animal study to evaluate IRIN-loaded LC MSNPs and liposomes was conducted simultaneously, so the two groups shared the same saline control. For clarity, we plotted the data according to the carrier type.

[0117] Figure 36 shows tumor burden assessed by IVIS bioluminescence signal in orthotopic KPC-luc model receiving IRIN LC-MSNPs (left panel) and IRIN liposomes (right panel). For ease of data interpretation, although the therapeutic efficacy came from the same study, the data were presented in two panels separately. Data were presented as mean ± SD, n=6. Statistical significance was analyzed using paired t test. *p<0.05.

[0118] Figure 37 shows Flow cytometry measured the percentage of CD31+ a5+ integrin ECs among CD31+ ECs in the indicated organs. Data were presented as mean ± SD, n=5.

[0119] Figure 38 shows representative H&E images of major organs obtained in the therapeutic efficacy study of orthotopic KPC model and EMT6 model. Scale bar, 100 pm.

[0120] Figure 39 is a graph showing A2-mediated particle enrichment increases depend on a5b1 integrin expression; proof of A2-mediated particle pathway increase depends on a5b1 integrin expression in EMT6 tumor-bearing mice.

[0121] Figure 40 is a graph showing the fluorescence imaging results of drug tumor distribution in integrin high and low patient-derived breast cancer xenograft models after intravenous injection of A2-coated or non-coated doxorubicin-loaded liposomes (CD31 indicates tumor blood vessels, DOX indicates doxorubicin).

[0122] Figure 41 is a graph showing the quantification results of silicon tumor accumulation in integrin high and low patient-derived breast cancer xenograft models after intravenous injection of A2-coated or non-coated doxorubicin-loaded phospholipid-coated silicon-based nanocarriers; similar experiments were repeated using DOX LC-MSNPs, followed by silicon element analysis. Data are presented as mean ± s.d. Statistical significance was tested by unpaired t test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001.

[0123] Figure 42 shows the gating strategy of flow cytometry in Figure 39.

[0124] Figure 43 shows that to validate the hypothesis that a5b1 -dependent efficacy improves EMT6 model results, we advanced the study using BC PDX models. The a5b1 integrin expression data of two models are shown, as well as H&E and Trichrome staining of PDX tumors. b, The gene expression levels of integrin a5 and b1 were further validated by qPCR (ACTB was used as an internal control). The expression levels of integrin a5 and b1 in BR00164 model were normalized to those in BR00290, respectively. Data are presented as mean ± s.d. (n = 6). Primer pairs are as follows

[0125] ITGA5-F 5’-GGCTTCAACTTAGACGCGGAG-3’

[0126] ITGA5-R 5’-TGGCTGGTATTAGCCTTGGGT-3’

[0127] ITGB1-F 5’-CCTACTTCTGCACGATGTGATG-3’

[0128] ITGB1-R 5’-CCTTTGCTACGGTTGGTTACATT-3’

[0129] Figure 44 shows the analysis of the fluorescence intensity of DOX in randomly selected fields using ImageJ software. Representative images and semi-quantitative results of A2-mediated liposome enrichment in PDX are provided in the lower panel of Figure 21. Data are presented as mean ± s.d. Statistical significance was tested by unpaired t test. *p < 0.05.

[0130] Figure 45 shows the selection of BR00164 PDX with high a5b1 integrin expression and BR00290 PDX with low a5b1 integrin expression for comparison of A2-mediated transcellular activation. Intravenous injection of DOX-containing LC-MSNPs (with two A2 pre-coating) into BC PDX mouse model. Similar to liposome formulation, DOX (drug fluorescence), CD31 (IF staining) and nuclei (DAPI) were analyzed for color change. DOX fluorescence was observed with microscope, while Si content was analyzed with ICP-MS (Figure 39). Data are presented as mean ± SD. Statistical significance was assessed by unpaired t test. ***p < 0.001.

[0131] Figure 46 is a schematic diagram of the dosing regimen of the animal experiment.

[0132] Figure 47 shows the binding capacity of A2 protein on LC-MSNP model particles with different PEG densities. According to the design of PEG, various commercial liposomes were included in this figure. The PEG density of the commercial doxorubicin liposome (Doxil®, lot number 692240505) tested in Figure 6 is 5%. b, LC-MSNP model particles were incubated with A2 protein for 1 hour, followed by a washing step, and immediately analyzed using SDS-PAGE electrophoresis. c, Normalized gray value of A2 protein band at the PEG density shown in b.

[0133] Figure 48 shows: a-b, we evaluated the stability of A2 pre-coating on low and high density DSPE-PEG2000 LC-MSNPs. After A2 pre-coating and removal of unbound free protein, the particles were gently shaken on a shaker at designated time points, and the protein surface attachment was quantified. Data are presented as mean ± SD, n = 3. d-e, A2 was tested for its enhancing effect on LC-MSNPs containing 0% or 3% DSPE-PEG2000 in orthotopic EMT6 model. The results showed that the efficacy of A2 coating was affected by PEG density, i.e. 3% PEG but not 0% PEG could significantly improve the efficacy. We speculate that this is due to the higher “surface accessibility” of the bare surface, which can lead to the reduced retention of A2 on the surface over time during shaking. Data are presented as mean ± SD, n = 5. Statistical significance was assessed by unpaired t test, ****, P < 0.0001.

[0134] ​Figure 49 shows A2 coating enhances the anti-tumor effect of commercial DOX liposome in vivo. a, A2 coating protocol for commercial DOX liposome. b, Size, PDI and zeta potential of A2 coated commercial DOX liposome (n=3). c, A2 protein can be effectively attached to commercial DOX liposome. D, Size and PDI values of A2 pre-coated commercial DOX liposome were measured in serum-supplemented normal saline at different time points (n=3). e, Drug release profile of commercial DOX liposome without A2 coating when incubated with serum-supplemented normal saline (n=3). f, Mouse PK comparative study using intravenous injection of commercial DOX liposome w / wo A2. g, PK parameters were calculated using DAS2.0 software. h, A2 pre-coated commercial DOX liposome was intravenously injected every 4 days, the control group included commercial DOX-liposome without A2 coating, for a total of 5 doses. The dose of DOX was 5 mg / kg. i, Noodle plot of each treated animal from day 0-30. j, Using A2 pre-coated DOX liposome approved and commercialized by the US Food and Drug Administration significantly improved the efficacy compared to purchased liposome in EMT6 orthotopic model (n=8, paired t-test, ***p=0.0008). k, Based on Kaplan-Meier analysis and time-to-event test, it was observed that using A2 pre-coating significantly improved the survival outcome (**p=0.0056). Data are shown as mean ± standard deviation.

[0135] Figure 50 shows A2 mediated tumor pathway enhancement effect using size- controlled LC MSNPs. a. Electrophoretic analysis indicates that all LC MSNPs in this size range can effectively bind A2 (n=3). b. BALB / c mice bearing EMT6 tumors were intravenously injected with A2 coated LC MSNPs in this size range, each with NIR labeling, and sacrificed 24 hours post-injection. Tumors were excised for IVIS imaging (n=6). All A2 coated LC MSNPs in this size range behaved similarly. Data are shown as mean ± standard deviation. DETAILED DESCRIPTION

[0136] The annexin in the present application, i.e. Annexin class protein, is a family of calcium-dependent phospholipid binding proteins. Preferably, the annexin includes any one of annexin A2, annexin Al, annexin A10, annexin Al l, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, annexin A8L2 or annexin A9 or a combination of at least two thereof. The annexin is preferably of human origin, and the detailed sequences are shown in the table below. However, in addition to the humanized annexin, the annexin should also include monomers or combinations of homologous proteins across species.

[0137] [Note] In addition to humanized annexin, the annexin should also include monomers or combinations of homologous proteins across species (such as rodents, non-human primates, ungulates, birds, etc.).

[0138] We have demonstrated that intravenous (IV) LC-MSNPs can enter KPC-derived orthotopic PDAC models, in part through a mechanism mediated by transcytosis 3 (Figure 1). Based on this knowledge, our main goal was to investigate how the design of the particle’s lipid coating influences the abundance and mechanism of tumor biodistribution in PDAC. We tend to use LC-MSNPs as “model nanoparticles” because of their ease of particle visualization and the flexibility they offer for modulating the lipid composition. In addition, it also allows for data generation on non-supporting bilayers with LC-MSNPs, which can improve the performance of liposomes under certain conditions. To conduct biodistribution studies, we used KPC cells from spontaneous PDAC tumors in transgenic KrasLSL-G12D / +Trp53LSL-R172H / +Pdx1-Cre mice. This strict PDAC tumor model, which is rich in stroma, is very similar to human PDAC in terms of oncogene expression, growth characteristics, metastasis, histological characteristics, and development of dysplastic stroma, which can be evidenced by robust tumor growth and trichrome staining (Figure 2).

[0139] To further illustrate the technical means adopted by the present application and its effects, the present application will be further described below in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are only used to explain the present application, and not to limit the present application.

[0140] Unless otherwise specified in the examples, the techniques or conditions described in the literature in the art, or according to the product instructions, are used. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0141] Example 1

[0142] This example mines proteins that improve the tumor entry efficiency of phospholipid-based nanocarriers.

[0143] 1. Design phospholipid-based nanocarriers that can be traced to test tumor entry efficiency.

[0144] Nanocarrier synthesis process:

[0145] (1) Preparation of mesoporous silica (MSNPs) core:

[0146] 1) Preparation of 25% cetyltrimethylammonium chloride (CTAC): Weigh 10 g CTAC into 30 mL water and dissolve it by ultrasonic at 37 °C.

[0147] In a beaker, add 171 mL water, 9 mL 25% CTAC and stir by magnetic sub with 500 rpm and heat to 85 °C; weigh 0.72 g TEA-OL, add pure water to about 4 mL, shake and mix, and add it into the beaker at 85 °C; add 6 mL ethyl silicate (TEOS) after 30 min and react for 3 h. Measure the particle size and polydispersity index (PDI) by laser particle size analyzer.

[0148] 2) The above sample is named M0XX, and anhydrous ethanol is added according to alcohol: M0XX = 1:2, and it is left for a period of time. First, low-speed centrifugation (to concentrate the particles at the bottom of the centrifuge tube): 4500 rpm for 5 min; then high-speed centrifugation to compress the precipitated part: 10000 rpm for 10 min; discard the supernatant.

[0149] 3) Hydrochloric acid / ethanol washing

[0150] Add 25 mL of a mixed solution of hydrochloric acid / ethanol (v:v = 1:50) to the above precipitate, ultrasonic dispersion for 35 min, high-speed centrifugation for 10000 rpm for 35 min, discard the supernatant, and repeat three times.

[0151] 4) Ethanol washing

[0152] Add 25 mL of anhydrous ethanol to the above precipitate, ultrasonic dispersion for about 10 min, high-speed centrifugation for 35 min, and discard the supernatant. Repeat twice.

[0153] 5) Add anhydrous ethanol again, ultrasonic dispersion, 4000 rpm centrifugation for 5 min, and take the supernatant, which is MSNP.

[0154] (2) MSNP with dye:

[0155] 1) Amino-labeled MSNP (for example, with 10% amino modification ratio):

[0156] ① Add MSNP to the sample bottle according to 10% APTES (M001 120 mg + 12 μL APTES), heat in an 80 °C oil bath, and stir overnight.

[0157] ② After the reaction, wash it twice with anhydrous ethanol: transfer the above MSNP-NH2 liquid to a 1.5 mL centrifuge tube, centrifuge at 15000 rpm for 20 min at room temperature, discard the supernatant, add 950 μL of anhydrous ethanol, ultrasonic dispersion, and then centrifuge and disperse with solvent.

[0158] 2) MSNP-NH2 labeled dye

[0159] ①Prepare dye stock solution:

[0160] Add 1.2 mL dimethylformamide (DMF) to a 2 mL centrifuge tube, centrifuge at 13300 rpm (to prevent insoluble particles from drying agent), take 1 mL supernatant to dissolve dye dry powder, and prepare a stock solution of a certain concentration.

[0161] ②Add reaction sample bottle according to MSNP:dye = 1000:1 (mass ratio), cover with tin paper to avoid light, and stir overnight at room temperature.

[0162] ③Ethanol washing: centrifuge at 15000 rpm for 15 min, discard the supernatant, add 1 mL anhydrous ethanol and ultrasonic dispersion, repeat twice.

[0163] (3) MSNP encapsulation:

[0164] 1) Encapsulating neutral membrane

[0165] The required lipids for encapsulating neutral membrane are as follows (for a total mass of 40 mg):

[0166] 2) Encapsulating positively charged membrane

[0167] The required lipids for encapsulating positively charged membrane are as follows (for a total mass of 40 mg):

[0168] 3) Encapsulating negatively charged membrane

[0169] The required lipids for encapsulating negatively charged membrane are as follows (for a total mass of 40 mg):

[0170] 4) Heat the synthesized dye-labeled MSNP in a 65℃ metal bath;

[0171] 5) Dissolve the above lipid mixture in 80 μL anhydrous ethanol and heat to 65℃ for dissolution, use a pipette to take the dye-labeled MSNP and add it to the dissolved lipid mixture, and perform probe sonication (power 10%, mode is working for 10 s and stopping for 5 s).

[0172] 6) Centrifuge the liquid after probe sonication at 15000 rpm for 18 min, retain the supernatant (which is unencapsulated liposome), ultrasonic dispersion of the precipitate with 1 mL PBS, centrifuge at 15000 rpm for 18 min, ultrasonic dispersion of the precipitate with 1 mL PBS, and store at 4℃.

[0173] (4) Filtration: use a screw-in syringe to take the liquid in (3), install a filter head. First pass through a 0.45 μm filter, then pass through a 0.22 μm filter, and collect the filtrate.

[0174] (5) Before the biological experiment, the different nano-carriers will be systematically characterized, such as: morphology and size, hydration radius, surface properties, electrical properties, fluorescence labeling intensity, endotoxin content, impurities, etc. The results are shown in Table 1.

[0175] Table 1

[0176] (6) In the analysis of high-resolution biological electron microscopy, we also designed a nano-carrier containing a gold core (structure diagram as shown in Figure 4), and its phospholipid coating is similar to the design without gold core.

[0177] 2. Comparison of the biodistribution of different types of traceable phospholipid nano-carriers.

[0178] The KrasLSL-G12D / +Trp53LSL-R172H / +Pdx1-Cre (KPC) pancreatic cancer orthotopic mouse model was established, and the specific process included: first, female B6 / 129 mice (~8 weeks) were anesthetized with isoflurane, and anesthesia was maintained with intraperitoneal injection of ketamine 50 mg / kg and xylazine 10 mg / kg, the skin within 1 cm of the mouse surgical site and its margin was depilated, and was disinfected with alternating scrubbing with iodophor and 75% ethanol. Then, the mouse was placed on a heating pad for surgery, and sterile gauze was covered on the left side of the mouse's abdomen, a ~0.7 cm incision was made to expose the pancreas, and a 27G needle syringe was used to inject 2x10 6 KPC-luc cells in 50 μL of DMEM / Matrigel (1:1 v / v). Finally, the fascial layer was sutured with absorbable suture, the skin was sutured with non-absorbable suture, the mouse was placed on a warm pad until it fully woke up, then transferred to a clean cage, and artificial tear ointment was used to protect the mouse's eyes during the operation.

[0179] (1) The mice were inoculated with tumors for about 10 days, and received intravenous injection of nano-carriers. The tumor-bearing mice were randomly divided into 3 groups, 4 mice in each group, and each group was injected with the above-constructed nano-carriers into the tail vein, 0.5 mg per mouse. After 24 hours, the contents of different nano-carriers (F1, F2 and F3) in primary cancer and metastatic cancer were analyzed by small animal in vivo imaging instrument (IVIS) and near-infrared two-zone imaging.

[0180] (2) Biological electron microscopy analysis was performed to determine important structural features related to "transcytosis" effect, such as transcytosis vesicles, nano-carrier interaction with vascular endothelial cells, etc.

[0181] (3) The primary cancer and metastatic cancer tissues were collected, and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to evaluate the intratumoral spatial distribution of nano-carriers.

[0182] The comparison and ranking of the degree of "transcytosis" of silicon liposomes with different physicochemical properties were carried out, and the results are shown in Figures 5 and 6. It can be seen that the tumor entry efficiency of F1 nanocarriers is the highest.

[0183] 3. Analysis of different nanocarrier protein crowns

[0184] In addition to its chemical identity, the nanocarrier entering the blood circulation also acquires a biological identity, which is highly related to the nanobiocrown on the surface of the nanomaterial (such as a protein crown). The basic experimental process of protein crown analysis is shown in Figure 7, which includes:

[0185] (1) Nanocarriers F1, F2, and F3 (1 mg / mL, 50 μL) were pre-incubated with tumor-bearing mouse plasma (200 μL) at 37°C for 3 h, respectively;

[0186] (2) Centrifugation at 15000 rpm, 4°C for 45 min, three times, to remove residual plasma in the supernatant;

[0187] (3) Resuspend the precipitate with 10 times the concentration of PBS (pH = 7.4), centrifuge again at 15000 rpm, 4°C for 45 min, repeat three times, to remove the protein crown with weak adsorption;

[0188] (4) Collect the precipitate and wash it with PBS (pH = 7.4, 100 μL) three times;

[0189] (5) Denaturation: add 25 μL guanidine hydrochloride (6M) to the precipitate and sequentially add 50 μL Tris-HCl (pH = 6.8, 50 mM) and 25 μL tris(2-carboxyethyl)phosphine (TCEP, 50 mM), heat at 60°C in a metal bath for 45 min;

[0190] (6) Add 50 μL iodoacetamide (IAA, 50 mM) to the solution of step (5) and react in the dark at 25°C for 45 min;

[0191] (7) Heat at 70°C in a metal bath for 5 min, centrifuge at 25°C, 14000 rpm for 45 min, remove the precipitate and keep the supernatant;

[0192] (8) Enzymatic digestion: sequentially add 10 μL trypsin (0.1 mg / mL) and 6 μL Tris-HCl (pH = 8.8) to the supernatant of step (7) and incubate overnight at 37°C in a water bath;

[0193] (9) Desalting: ZipTip C18 column was used for desalting, 10 times of pure methanol elution, 10 times of pure acetonitrile elution, 10 times of 0.1% trifluoroacetic acid elution, direct adsorption of the supernatant after enzymatic hydrolysis, concentration for sample loading, 0.1% trifluoroacetic acid elution three times, desalting again, 70% acetonitrile + 0.1% trifluoroacetic acid solution, elution of the column sample;

[0194] (10) Freeze-drying;

[0195] (11) LC-MS / MS mass spectrometry to identify protein components.

[0196] The protein crown search range was determined by mass spectrometry analysis, and the differences of the protein crowns on the surfaces of different designed nanocarriers were compared, and the results are shown in Figure 8, aiming to determine the protein targets on the surface of the nanocarriers with obvious transcytosis effect. By using proteomics database and search engine and other means, unknown target proteins related to transcytosis were mined, and the results are shown in Tables 2 and 3. Through protein adsorption analysis, the protein types unique to F1, F2 and F3 or shared by them were distinguished, and through database analysis, Annexin was identified as being highly associated with F1 tumor entry, and its mechanism is related to transcytosis.

[0197] Table 2

[0198] Table 3

[0199] Example 2

[0200] This example further tests the influence of Annexin on the tumor entry efficiency of phospholipid-based nanocarriers

[0201] A series of Annexin-coated F1 nanocarriers were prepared using different Annexin subtypes (A2, A3, A5, A7 and A8). The specific preparation process is as follows:

[0202] (1) Nanocarrier F1 with strong transcytosis effect (1 mg / mL, 50 μL) was incubated with Annexin (0.5 mg / mL, 200 μL) at 37°C for 3 h.

[0203] (2) Centrifugation at 15000 rpm, 4°C for 45 min, three times, collect the precipitate and remove the residual protein in the supernatant.

[0204] (3) Resuspend the precipitate with 10 times concentration of PBS (pH = 7.4), centrifuge again (15000 rpm, 4°C, 45 min), repeat three times, remove the residual protein in the supernatant.

[0205] (4) Wash with PBS (pH = 7.4, 100 μL) three times to obtain Annexin-coated F1 nanocarriers.

[0206] w / o, plasma, vitronectin (VTN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or albumin (Alb) coated F1 nanocarriers were prepared according to the above method respectively

[0207] The electrophoresis analysis of the F1 nanocarriers coated with each protein was performed, and the results are shown in Figure 9. It can be seen that each protein can be firmly combined with the nanocarriers.

[0208] The characterization of the F1 nanocarriers coated with each protein was performed, and the results are shown in Table 4, including the size, PDI and charge in the cell culture medium (DMEM) and phosphate buffer (PBS). The particle size of the F1 nanocarriers coated with each protein is almost around 130 nm, and the potential in PBS is nearly neutral. Based on the endothelial cell monolayer transport model (transwell) experiment, the ability of each protein coated F1 nanocarrier to pass through the epithelial cells (EC cells) was tested, and the specific experimental process is shown in Figure 10, including: human vascular endothelial cells (HUVEC) were cultured on the transwell chamber until a tight monolayer membrane structure was formed to simulate the blood vessel barrier. And 70 kDa fluorescent labeled dextran was used to detect whether the tightness of different monolayers was consistent. Then the nanocarriers (Dy680 labeled) coated with different pre-coated proteins were prepared into a concentration of 200 ug / mL with DMEM medium, and added to the upper chamber to simulate the vascular side. After 24 h, the lower layer solution was taken out and the Dy680 fluorescence value was detected by a multifunctional enzyme label instrument to compare the number of different nanocarriers passing through the endothelial monolayer. The results are shown in Figure 11, which shows that the use of annexin A2, A3 or A5 coated nanocarriers can effectively improve the transcellular transport ability of the nanocarriers.

[0209] Table 4

[0210] The consistency of the EC monolayer integrity was confirmed by FITC-dextran assay (Figure 12), highlighting the accuracy of the differences observed in transcytosis induced by various surface pre-coatings.

[0211] Further, the F1 nanocarriers coated with each protein were intravenously injected into pancreatic cancer KPC mice at a dose of 50 mg / kg. After 24 h of injection, the organs of the mice were dissected, and each organ was placed in a small animal live imaging instrument to observe the fluorescence signal, and the biodistribution was compared by the fluorescence signal strength.

[0212] Results are shown in Figure 13. Experimental data demonstrated that Annexin A2 had the best synergistic result (about 2-fold); A3, A5 also had better synergistic results. We injected intravenously Dy680 labeled F1 particles coated with A2 into mice with orthotopic KPC tumors. After 24 hours, we sacrificed the animals and performed in vitro IVIS imaging on the transplanted tumors and major organs (such as heart, lung, liver, spleen and kidney). We observed that the KPC tumor signal in the A2 and A5 groups increased moderately but statistically (i.e. 2-fold) compared to various control groups. Interestingly, A5 pre-coating but not A2 resulted in increased liver signal (Figure 13). Furthermore, we also confirmed that A2 coating remained stable and unchanged for at least 48 hours after preparation before further testing of A2 (Table 5, Figure 14).

[0213] Table 5

[0214] Meanwhile, we also validated A2 coated nanocarriers in other various tumor cells (EMT6, Py8199, 4T1 and CT26) with injection dose of 50mg / kg. After 24h post-injection, we dissected the mouse organs and placed each organ in the small animal live imaging instrument to observe the fluorescence signal. Results are shown in Figures 15 and 16. It can be seen that the synergistic result in breast cancer EMT6 is as much as 6-fold. This can be substantial for preclinical anticancer efficacy studies, which usually require about 4-10 IV doses in mice. The Py8119 orthotopic model also showed about 2-fold increase.

[0215] In addition, the morphology of the nanocarriers in the tumor was observed based on tissue transmission electron microscopy (TEM). The specific process included cutting the removed fresh tumor tissue into pieces, soaking them in glutaraldehyde, and making paraffin sections, fixing them on a copper grid, and observing them under TEM. The results are shown in Figure 18. After 1 hour of circulation, pseudopod-like protrusions were found at the endothelial cells on the blood vessel side, and nanocarrier particles were aggregated. After 6 hours of circulation, nanocarrier particles were found to be aggregated inside the endothelial cells of the blood vessels, and a large number of vesicle structures appeared, and some nanocarrier particles were also enriched inside the vesicles. Here, we replicated the A2 coating experiment using a substitute version of the F1 particle, which has a 10 nm gold core while maintaining the same size and lipid coating. The morphology of the prepared gold-containing F1 is shown in Figure 17. This modification addresses the challenge of low electron density observed with nanosilica, making visualization in the heterogeneous tumor microenvironment easy. After 1 hour, we observed gold-labeled LC-MSNP model nanoparticles in the vascular lumen, the formation of filamentous pseudopods (Figure 18, upper panel, ROI #1), and evidence of EC intravesicular transport (Figure 18). Similar observations were made at 6 hours (Figure 18, lower panel, ROI #3), as well as particle deposition within the tumor stroma (Figure 18, lower panel, ROI #4). Notably, at 6 hours, we also identified A2-coated LC-MSNPs showing perinuclear distribution within cancer cells (Figure 19, ROI #5). The small inset box highlights the gold label on the LC-MSNP model particles, enabling confident identification of particles of interest at single-particle resolution. TEM photos of control EMT6 tumors and additional TEM evidence of A2-mediated transcellular delivery are shown in Figure 20.

[0216] In summary, the use of annexin in the present application can further improve the tumor entry efficiency of phospholipid nanocarriers.

[0217] Example 3

[0218] This example analyzes the mechanism of action of annexin in promoting the tumor entry of phospholipid nanocarriers, taking annexin A2 as an example.

[0219] First, molecular dynamics (MD) simulation was performed to analyze the interaction between A2 and lipid molecules. The structure of the system was constructed using the Martini force field (Figure 21), and a well-equilibrated system was obtained. The molecular dynamics simulation was studied by combining the use of coarse-grained (CG) and all-atom (AA) models. During the coarse-grained (CG) simulation, we observed that the A2 protein reached a stable position within 50 microseconds, and the root mean square deviation (RMSD) of the protein heavy atoms relative to their initial position was calculated to be 22 angstroms (Figure 22). Subsequently, the CG final structure became the initial structure for the all-atom (AA) simulation, and a 500 nanosecond MD analysis was performed. In this case, the average RMSD value of the Cα atoms of the A2 protein relative to its initial position fluctuated around 19 angstroms (Figure 23). In addition, we monitored the contact area between the A2 protein and the lipid throughout the simulation trajectory, and the average contact area remained stable at approximately 1000 square angstroms (Figure 24), indicating that the binding of A2 to the lipid was very stable during the AA simulation. That is, the RMSD value shows that the simulation system reaches a convergent state, and the interface contact area shows that the contact between A2 and the phospholipid molecules reaches a stable state, revealing the "phospholipid surface - annexin - integrin" sandwich model (Figure 25). The most promising binding site of A2 with α5β1 integrin is predicted to be located within the RGD binding pocket. This pocket was previously located at the center of the groove-like exposed surface on the top surface of α5β1 integrin, and is expected to be a key interaction site39. The binding sites of A2 with α5β1 integrin and lipid are marked in purple and red, respectively (Figure 25). Surface hydrophobic potential (Figure 26a, b) and electrostatic potential (Figure 26c, d) analysis further provides molecular insights into these interactions. Between A2 and α5β1 integrin, hydrophobic interactions play an important role, which is evidenced by the frequent occurrence of non-polar residues such as alanine in our analysis (Figure 26b). Between the lipid surface and A2, our analysis reveals the key role of the α11 helix in A2, which contains negatively charged residues such as D187, E189, D192, D194, D197, and D200. These residues further attach to the phospholipid head group, which has a local polarity with a slightly positive charge potential (Figure 26d).

[0220] To verify the rationality of the "phospholipid surface - annexin - integrin" sandwich model, integrin knockout experiments and the introduction of a zombie model were used to confirm that the above conditions can interfere with the synergistic results of A2.

[0221] The specific experimental process includes:

[0222] First, EMT6 tumor-bearing mice were perfused with PBS solution containing heparin sodium (1 mg / mL) through the heart, and the effluent blood was collected. Then the mice were fixed with fixative solution (4% formaldehyde, 0.5% glutaraldehyde, PBS) through the heart for 1 h. The fixative solution was washed away with PBS perfusion for 20 min. To mimic the blood environment, nanoparticles (1 mg / 1.5 mL) were added to the collected mouse blood. The nanoparticles were circulated in the fixed mice at physiological blood flow rate (5-7 mL / min) using a peristaltic pump for 2 h. Then the nanoparticles in the blood vessels were removed by PBS perfusion. The tumors were removed and imaged by IVIS. As a control, the same concentration of nanoparticles was injected intravenously into live mice. After 2 h, the blood was removed by PBS perfusion through the heart, the tumors were removed, and IVIS imaging was performed to analyze the nanoparticle enrichment

[0223] Results, as shown in Figures 27 and 33, knocking down a5b1 integrin expression or using formalin-fixed tumor vessels (zombie model) both interfered with the role of A2 in promoting nanoparticle transport.

[0224] Two shRNA sequences targeting mRNA targets (sh-ITGA5#1 and #2) were also tested. EC integrity was not changed during the disassembly experiment (Figure 28). In addition, we also utilized the transcytosis inhibitor genistein40to demonstrate the interference with A2-mediated LC-MSNP transcytosis. Our data showed that more than 40% of particle transport in HUVEC monolayers was reduced (Figure 29).

[0225] As shown in Figure 26d, since residues D187, E189, D192, D194, D197, and D200 play a crucial role, we introduced mutations in these six amino acid residues on A2 to reduce electrostatic interactions by replacing them with alanine. The corresponding genes were synthesized by the core facility and integrated into the PET28a plasmid. The resulting A2 mutant proteins (A2-M) were used to coat LC-MSNPs. The size, PDI, and zeta potential values of the two samples were comparable. However, the assessment of protein stability showed that the stability of the A2-M coating decreased over time compared to the wild-type A2 coating. In addition, microscale thermophoresis (MST) analysis showed that the binding affinity of A2 and A2-M to a5b1 integrin was 7.93 nM and 30.88 nM, respectively (Figure 30). Furthermore, A2-M was used to precoat NIR-labeled LC-MSNPs for biodistribution studies in EMT6 tumor-bearing mice in situ compared to particles coated with wild-type (WT) A2. The effect of A2 coating was significantly impaired, resulting in more than 50% reduction in signal at the tumor site (Figure 31). Based on these findings, we attribute the reduction in homing efficiency to the decrease in binding stability during circulation, which can compromise the efficient activation of a5b1 integrin and subsequent downstream signaling events. Kd Values were 7.93 nM and 30.88 nM, respectively (Figure 30). Furthermore, A2-M was used to precoat NIR-labeled LC-MSNPs for biodistribution studies in EMT6 tumor-bearing mice in situ compared to particles coated with wild-type (WT) A2. The effect of A2 coating was significantly impaired, resulting in more than 50% reduction in signal at the tumor site (Figure 31). Based on these findings, we attribute the reduction in homing efficiency to the decrease in binding stability during circulation, which can compromise the efficient activation of a5b1 integrin and subsequent downstream signaling events.

[0226] Since transcytosis is an energy-dependent process, we established a "zombie" model in EMT-6 orthotopic tumor-bearing mice according to published protocols (Figure 32). Vessel fixation inhibits the transcytosis of active particles, while experimentally maximizing the entry of particles through enlarged tumor pores. Then, we used the "zombie" mice to elucidate the effect of A2 pre-coating in the same BC model (Figure 33). After fixing the vasculature, A2-coated Fl particles were circulated in serum-containing solution for 2 hours. The data showed a blocking effect on transcytosis, resulting in an approximately 85% reduction of A2 pre-coating effect. In addition, the effect of A2 on tumor particle enrichment also showed a downward trend when treated with ATN-161, an inhibitor of α5β1 integrin (Figure 34). From the computer simulation and interference experimental data, our findings collectively demonstrate the important role of lipid-A2-α5β1 interaction and nanoparticle transcytosis in the tumor vasculature.

[0227] Based on the above findings, we further hypothesized that the efficacy of nanocarriers could be improved by activated transcytosis using A2 pre-coating. Therefore, we conducted a series of efficacy experiments in PDAC and BC tumor models. As a proof of concept, we utilized our established remote loading method to encapsulate DOX and IRIN, a weakly basic chemotherapeutic drug commonly used for BC and PDAC32,43. The drug-loaded LC-MSNP nanocarriers were fully characterized before use (Figure 35a). In the first set of efficacy data, treatment was initiated approximately 10 days after orthotopic implantation of EMT6 BC cells. EMT6 orthotopic tumor-bearing mice received intravenous injection of DOX-loaded LC-MSNP nanocarriers with or without A2 pre-coating (DOX at 5 mg / kg; equivalent to A2 dose of approximately 2 mg / kg). Our results showed that A2 pre-coating significantly enhanced the anti-BC effect of DOX LC-MSNP, with a p-value of 0.0243 (Figure 35b). We also conducted efficacy studies using IRIN LC-MSNP at an IRIN dose of 40 mg / kg, with or without A2 coating. For IRIN-LC-MSNP, A2 coating improved the survival outcome in the KPC orthotopic model, with a p-value of 0.0035 (IRIN-LC-MSNP vs A2 pre-coated IRIN-LC-MSNP) (Figure 35c). Note that tumor burden in the KPC model was assessed using IVIS, which can be affected by late-stage ascites, as shown in Figure 36.

[0228] Given the A2 binding domain's association with silica-supported lipid bilayers, we continued to hypothesize that the A2 pre-coating approach could improve the performance of liposome formulations (non-supported lipid bilayers) and potentially have broader implications for classical nanocarriers. Thus, we first repeated the electrophoresis experiments and obtained results (Figure 35d) highly similar to those shown in Figure 9, thereby confirming our hypothesis that the protein does not biologically bind to non-supported lipids. Thus, a second set of efficacy data was anti-cancer efficacy in EMT6 and KPC orthotopic models, in which mice received internally synthesized DOX- or IRIN-loaded liposomes. The A2 coating significantly enhanced the tumor-inhibitory effect of DOX liposomes, with a p-value of 0.0025 (Figure 35e). Moreover, the A2 coating improved overall survival compared to IRIN liposomes alone (log-rank test, p = 0.0223) (Figure 35f).

[0229] In addition to the improvement in efficacy, it was important to thoroughly assess the safety of the A2 pre-coating approach to determine its suitability for use in nanotherapeutics. The body weight data from the efficacy studies in Figures 35b-c, 35e-f, as shown in Figure 35g, indicated no major toxicity during treatment. Since A2 targets the a5b1 integrin, we also investigated the expression of a5b2 integrin in tumor and endothelial cells of various organs, including liver, heart, spleen, lung, kidney, and brain. While a5b1 integrin is abundantly expressed in liver ECs, the levels in other organs were relatively low (Figure 37). Thus, histological assessments showed no apparent abnormalities with A2-coated nanocarriers (Figure 38).

[0230] Based on the above, the mechanism by which annexin pre-coating facilitates nanocarrier transcytosis relies on a5 or b1 integrin, which is overexpressed in various tumors, and thus it can be known that annexin pre-coating can increase the biodistribution of phospholipid-based nanocarriers (such as liposomes, lipid nanoparticles, phospholipid-coated nanoparticles, etc.) at sites of lesions with high expression of a5 or b1 integrin.

[0231] In addition, the annexin A2 effect was repeated in the orthotopic EMT6 model, investigating differences between mice (n = 7) and analyzing the abundance of a5 integrin-positive endothelial cells (a5 + CD31 + ) using flow cytometry (Figure 42). Related studies revealed a significant positive correlation between the abundance of nanoparticles in tumors and the number of a5b1 + CD31 + double-positive cells, R 2The value is 0.6008 (Figure 39). We further obtained breast cancer patient-derived xenograft models (PDX) in immunodeficient SCID mice, which were pre-characterized for ITGA5 (a5 subunit) and ITGB1 (b1 subunit) fragments per million per kilobase (FPKM) information. We intentionally selected BR00164 and BR00290 PDX models because they have similar texture patterns and fibrosis levels revealed by H&E staining and trichrome staining (Figure 43). More importantly, the BR00164 model shows high expression of a5b1, while the BR00290 model shows moderate expression, and the results of differential biodistribution of liposomes and silicon-based carriers are summarized in Table 5. As can be seen from Table 5, in the a5b1 high expression model, the enrichment of drug-loaded liposomes and silicon-based carriers coated with A2 is increased by 21.1 times and 15.5 times, respectively, relative to the experimental group without A2 coating, while in the a5b1 low expression model, there is no significant difference in the enrichment of nanocarriers between the A2-coated nanocarrier group and the non-coated nanocarrier group. Both BR00164 and BR00290 models received a single injection of doxorubicin liposomes, and 24 hours after injection, tumor tissues were collected to prepare sections for doxorubicin (DOX) visualization (red fluorescence), CD31 immunofluorescence staining (green), and nuclear staining (blue). Annexin A2 pre-coating showed a significant increase in doxorubicin fluorescence in the BR00164 model (a5b1 integrin high PDX), but little increase in the BR00290 model (a5b1 integrin low PDX) (Figures 40, 44), and we also used phospholipid-coated silicon-based nanocarriers to carry out similar experiments in the above PDX models, and similar results were obtained (Figures 41, 45; Table 6).

[0232] Table 6

[0233] From the above results, it can be seen that the expression amount of a5 or b1 integrin can affect the effect of annexin-promoted transport, that is, by pre-detecting and analyzing the expression amount of a5 or b1 integrin in patients, the ability of annexin-coating technology to promote the transport of nanocarriers can be predicted, and the benefit population that can apply annexin-coating technology can be screened.

[0234] Example 4

[0235] This example further analyzes the effect of annexin on the therapeutic effect of drugs after being loaded by phospholipid-based nanocarriers, taking annexin A2 as an example for analysis.

[0236] Take two typical phospholipid nanocarriers, traditional liposomes and phospholipid-coated mesoporous silica, as examples, and deliver two typical antitumor drugs, doxorubicin and irinotecan, to triple-negative breast cancer and pancreatic cancer to verify the synergistic effect of annexin A2.

[0237] The specific experimental process includes:

[0238] Construct KPC orthotopic pancreatic cancer mouse model (refer to the construction method described above).

[0239] Construct EMT6 triple-negative breast cancer mouse model: resuspend 3x10 6 EMT6 cells in Matrigel and inject them into the left four mammary fat pads of 6-week-old Balb / c female mice.

[0240] Prepare doxorubicin-loaded liposome-coated mesoporous silica, and the capture agent is ammonium sulfate (NH4)2SO4.

[0241] Prepare irinotecan-loaded phospholipid-coated mesoporous silica, including capture agent TEA8SOS.

[0242] Incubate doxorubicin-loaded phospholipid-coated silicon-based model nanocarriers (1 mg / mL, 50 μL) and irinotecan-loaded phospholipid-coated silicon-based model nanocarriers (1 mg / mL, 50 μL) with A2 protein (0.5 mg / mL, 200 μL) at 37°C for 3 h for coating.

[0243] Perform animal experiments, and the flowchart is shown in Figure 46. Drug injection is performed one week after tumor-bearing mice, twice a week, and drug treatment is given every two or three days. The groups are doxorubicin-loaded phospholipid-coated silicon-based model nanocarriers with / without A2 coating (6 times of co-administration) and normal saline, and irinotecan-loaded phospholipid-coated silicon-based model nanocarriers with / without A2 coating (5 times of co-administration) and normal saline.

[0244] The results are shown in Figure 35. In KPC pancreatic cancer and EMT6 triple-negative breast cancer, A2 enhances the antitumor activity of doxorubicin-loaded phospholipid-coated silicon-based model nanocarriers and irinotecan-loaded phospholipid-coated silicon-based model nanocarriers compared with doxorubicin-loaded phospholipid-coated silicon-based model nanocarriers and irinotecan-loaded phospholipid-coated silicon-based model nanocarriers without A2 coating.

[0245] To further verify the universality of annexin, directly use liposomes to load doxorubicin and irinotecan, and coat annexin A2 to test the in vivo treatment effect.

[0246] Prepare doxorubicin-loaded liposomes (refer to Pharmaceutics. 2023 Mar; 15(3):893).

[0247] Liposomes loaded with irinotecan were prepared (ref. Cancer Res. 2006 Mar 15; 66(6):3271-7).

[0248] Liposomes loaded with doxorubicin (1 mg / mL, 50 μL) and liposomes loaded with irinotecan (1 mg / mL, 50 μL) were separately incubated with A2 protein (0.5 mg / mL, 200 μL) at 37 °C for 3 h for coating.

[0249] Animal experiments were performed, and the flowchart is shown in Figure 46. The mice were injected with drugs one week after tumor inoculation, twice a week, and the drug treatment was given every two or three days. The groups were A2-coated or non-coated liposomes loaded with irinotecan (a total of 6 times of drug administration) and normal saline, A2-coated or non-coated liposomes loaded with doxorubicin (a total of 5 times of drug administration) and normal saline, and the in vivo effect test was performed, and the results are shown in Figure 35. In EMT6 triple-negative breast cancer and KPC pancreatic cancer, annexin A2 enhanced the anti-tumor activity of liposomes loaded with doxorubicin and liposomes loaded with irinotecan.

[0250] In summary, the present application uses annexin-coated drug-loaded nanocarriers to effectively improve the bioavailability and efficacy of drugs.

[0251] Example 5

[0252] We sought to determine whether A2 pre-coating could similarly improve the performance of commercial liposomes. To make a reasonable choice, we first synthesized a series of LC-MSNP model nanoparticles with different PEG densities (0-8%). These particles were incubated with A2 and gently shaken to facilitate A2 attachment. The results showed that A2 pre-coating was effective at all PEG densities (Figure 47). We also evaluated the stability of A2 attachment on LC MSNPs with low (0%) and high (3%) PEG. Notably, the A2 coating remained highly stable on 3% PEG particles for 48 hours, while on LC MSNPs without PEG, A2 attachment was reduced by about 50%. We speculate that this difference is due to higher "surface accessibility" in low PEG samples, which can result in weaker retention of proteins on the surface. The decrease in A2 coating stability can lead to the weakening of the coating effect observed without PEG (Figure 48).

[0253] For commercial liposomes, 5% pegylated doxorubicin liposomes (brand name ) were tested in the BC model in vivo.

[0254] To mimic clinical practice, we first developed a bedside preparation protocol (Fig. 49a) in which A2 solution was mixed with commercial DOX liposomes and incubated at 37 °C for one hour before intravenous injection. The purchased DOX liposomes and their A2 pre-coating formulations were fully characterized before use (Fig. 49b). This included electrophoresis experiments confirming the efficient conjugation of A2 to commercial DOX liposomes (Fig. 49c). A2-coated commercial DOX liposomes dispersed well in serum-supplemented physiological saline and exhibited colloidal stability for at least 0-48 h (Fig. 49d). Importantly, both uncoated and A2-coated liposomes released less than 0.05% free DOX when incubated in serum-supplemented physiological saline at 37 °C for 24 h (Fig. 49e).

[0255] Pharmacokinetic (PK) studies were performed using A2-coated commercial DOX liposomes, with uncoated commercial DOX liposomes as a control (DOX, 5 mg / kg). Both uncoated and A2-coated liposomes exhibited similar PK profiles (Fig. 49f). The half-lives (t 1 / 2 ) of commercial DOX liposomes and A2-coated DOX liposome preparations were 23.9 and 20.26 h, respectively. Moreover, the key PK parameters, including area under the curve (AUC) and clearance, of the two formulations were comparable, indicating minimal systemic PK impact of A2 coating on commercial DOX liposomes (Fig. 49g).

[0256] We further evaluated the therapeutic enhancement of the A2 pre-coating strategy using commercial DOX liposomes in an orthotopic EMT6 model. Tumor-bearing mice received intravenous injections of A2-coated or uncoated DOX liposomes (DOX: 5 mg / kg; A2: ~1.25 mg / kg) every four days for a total of five doses (Fig. 49h). A2 pre-coating significantly enhanced the anti-cancer efficacy of commercial DOX liposomes in vivo (Fig. 49i-j, p = 0.0008). Notably, we also observed a significant improvement in survival outcome for the A2-coated group (p = 0.0056). Remarkably, two out of eight mice achieved long-term survival, extending up to 80 days post-treatment (Fig. 49k).

[0257] Discussion

[0258] Increasing evidence suggests that ATR effects, including transcytosis, can be independent of vascular leakage and can play a dominant role in nanodrug delivery in matrix-rich solid tumors. Through analysis of protein attachment, we determined that A2 modification is a convenient means of activating tumor transcytosis and improving the efficacy of nanocarriers in solid tumors. Annexin A2 is a calcium-dependent phospholipid-binding protein that plays an important role in various cellular processes, including membrane trafficking, endocytosis, and exocytosis. By targeting A2 or using it as a molecular bridge, we are able to improve the specificity and efficiency of drug delivery, especially for tumors that are difficult to access due to limited penetration of therapeutic agents. Although the A2 pre-coating technique showed efficacy, we did not observe significant particle entry into the brain in our cases. In addition, we have demonstrated that A2 itself does not change tumor growth. In addition, safety assessments showed that A2 coating was not toxic to major organs, including those expressing α5β1. Regarding the expression of α5β1, the different results observed in the BC PDX data suggest that it is feasible to consider a predictive approach to achieve potent enhancement of nanocarriers. We also wish to emphasize that A2-mediated enhanced transcytosis can have a broad impact on intravenously injected nanocarriers. In addition to PEG density, we also conducted a comparative study using A2-coated LC MSNPs with core sizes ranging from 50 nm to 150 nm. Notably, all LC MSNPs in this size range were effectively attached to A2 (Figure 50a). Similar enhancement of A2 uptake at tumor sites was observed (Figure 50b). In addition, we also investigated the effects of various physiological and pathological conditions, including age, sex, immunosuppression, and inflammatory status, on the effects of A2 pre-coating in mice. In vivo data showed no significant differences between these conditions.

[0259] In summary, the present invention creatively mines proteins that can promote the tumor entry efficiency of current phospholipid-based nanocarriers, discovers that coating phospholipid-based nanocarriers with annexin can promote the "transcytosis" of nanocarriers, improve tumor entry efficiency, and thus improve the bioavailability of nanocarrier-loaded drugs and treatment efficacy. In addition, it is found that the synergistic ability of annexin is based on the "phospholipid surface-annexin-integrin" structure, and is positively correlated with the expression of α5 or β1 integrin. Therefore, it can be known that annexin pre-coating can also increase the biodistribution of phospholipid-based nanocarriers at lesions with high expression of α5 or β1 integrin, and the ability of annexin coating technology to promote the transport of nanocarriers can be predicted by pre-detecting and analyzing the expression of α5 or β1 integrin in patients, and the benefit population that can apply annexin coating technology can be screened.

[0260] Applicants declare that the detailed method of the present application is illustrated by the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, i.e. it does not mean that the present application must rely on the above-mentioned detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. Use of annexin in improving the tumor entry efficiency of phospholipid-based nanocarriers.

2. The use of a phospholipid nanocarrier according to claim 1, wherein the phospholipid nanocarrier is a phospholipid nanocarrier comprising a phospholipid, a polymer, and a protein, and the protein is a phospholipid-binding protein. The annexin comprises any one of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, annexin A8L2 or annexin A9 or a combination of at least two thereof; Preferably, the phospholipid-based nanocarriers comprise any one of liposomes, lipid nanoparticles or phospholipid-coated nanoparticles or a combination of at least two thereof. Preferably, the liposomes comprise any one of doxorubicin liposomes, irinotecan liposomes, paclitaxel liposomes or mitoxantrone liposomes or a combination of at least two thereof.

3. The use of a method according to claim 1 for improving the intratumoral efficiency of phospholipid-based nanocarriers, characterized in that, The use comprises modifying the annexin on the surface of the phospholipid-based nanocarriers. Preferably, the method for modification comprises mixing and incubating the annexin with the phospholipid-based nanocarriers.

4. The use of annexin in improving the tumor entry efficiency of phospholipid-based nanocarriers according to claim 1, wherein the phospholipid-based nanoparticles comprise one, two or more of the following lipids: Phospholipids: Egg phosphatidylcholine (EPC), Soy phosphatidylcholine (SPC), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), Dioleoyl-phosphatidylethanolamine (DOPE), Phosphatidylglycerol (PG), such as: DMPG, DSPG, Phosphatidylinositol (PI), Phosphatidic acid (PA), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), Hydrogenated soy phosphatidylcholine (HSPC); Cholesterol and Analogs: Cholesterol, Cholesteryl hemisuccinate (CHEMS), Cholesteryl oleate, Sitosterol; PEGylated Lipids: DSPE-PEG2000, (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]), DOPE-PEG2000, DMPE-PEG2000, DSPE-PEG- maleimide / biotin / amine, Cholesterol-PEG derivatives, and PEGylated lipids with similar structures, different molecular weights of PEG; Ionizable & Cationic Lipids: Ionizable lipids, DLin-MC3-DMA, SM-102, ALC-0315, DODMA (Dimethyldioctadecylammonium), Dlin-DMA; Permanent cationic lipids: DOTAP (Dioleoyltrimethylammonium propane), DOTMA (Dioleoyltrimethylammonium), DDAB (Dimethyldioctadecylammonium bromide), CTAB (Cetyltrimethylammonium bromide); Anionic & Zwitterionic Lipids: DMPG / DSPG (Phosphatidylglycerol, anionic), DOPS / POPS (Phosphatidylserine, anionic), Cardiolipin, Sphingomyelin; Targeting & Functional Lipids: DSPE-PEG-Maleimide, DSPE-PEG-Folate, DSPE-PEG-RGD, Glycolipids (e.g., GM1, GM3); and Other Specialized Lipids: Monolaurin, Lysolipids, Squalene derivatives, Ceramides / Sphingolipids.

5. The use of the Annexin of claim 1 to improve the tumor entry efficiency of phospholipid-based nanocarriers, wherein the phospholipid-based nanoparticles comprise phospholipids consisting of: (i) distearoylphosphatidylcholine (DSPC), cholesterol (Chol), and (DSPE-PEG2000); (ii) distearoylphosphatidylcholine (DSPC), dioleoylphosphatidic acid sodium salt (DOPA), (2,3-dioleoyl-propyl)-trimethylammonium-chloride salt (DOTAP): cholesterol (Chol), and (DSPE-PEG2000); (iii) distearoylphosphatidylcholine (DSPC), dioleoylphosphatidic acid sodium salt (DOPA), cholesterol (Chol), and (DSPE-PEG2000); (iv) hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Chol), and (DSPE-PEG2000); (v) distearoylphosphatidylcholine (DSPC), and cholesterol (Chol); (vi) egg yolk phosphatidylcholine (EPC), and cholesterol (Chol); (vii) dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), cholesterol (Chol), and triolein; (viii) sphingomyelin (SM), and cholesterol (Chol); (ix) egg yolk phosphatidylglycerol (EPG), and dimyristoylphosphatidylcholine (DMPC); (x) distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), cholesterol (Chol); (xi) palmitoyloleylphosphatidylcholine (POPC), and dioleoylphosphatidylserine (DOPS); (xii) diarachidoylphosphatidylcholine (DEPC), dipalmitoylphosphatidylglycerol (DPPG), cholesterol (Chol), and tricaprylin; (xiii) egg yolk phosphatidylcholine (EPC), and sodium deoxycholate; or (xiv) hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Chol), and distearoylphosphatidylglycerol (DSPG).

6. Use of annexin in the preparation of a delivery vehicle, characterized in that, The Annexin comprises any one of Annexin A2, Annexin A1, Annexin A10, Annexin A11, Annexin A13, Annexin A3, Annexin A4, Annexin A5, Annexin A6, Annexin A7, Annexin A8, Annexin A8L1, Annexin A8L2, or Annexin A9, or a combination of at least two thereof. The use comprises modifying the Annexin on the surface of the phospholipid-based nanocarriers. The phospholipid-based nanocarrier comprises any one of a liposome, a lipid nanoparticle, or a phospholipid-coated nanoparticle, or a combination of at least two thereof; Preferably, the liposome comprises any one of a doxorubicin liposome, an irinotecan liposome, a paclitaxel liposome, a mitoxantrone liposome, a daunorubicin cytarabine liposome, a mivatril liposome, or an amphotericin liposome, or a combination of at least two thereof.

7. A delivery vehicle, characterized in that, The delivery carrier comprises a phospholipid-based nanocarrier and an annexin modified on the surface of the nanocarrier; The annexin comprises any one of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, annexin A8L2, or annexin A9, or a combination of at least two thereof. The phospholipid-based nanocarrier comprises any one of a liposome, a lipid nanoparticle, or a phospholipid-coated nanoparticle, or a combination of at least two thereof; Preferably, the liposome comprises any one of a doxorubicin liposome, an irinotecan liposome, a paclitaxel liposome, a mitoxantrone liposome, a daunorubicin cytarabine liposome, a mivatril liposome, or an amphotericin liposome, or a combination of at least two thereof.

8. Use of annexin for increasing the bio-distribution of a drug at a lesion site or for the manufacture of a pharmaceutical composition, characterized in that, The application comprises modifying the annexin on the surface of a phospholipid-based nanocarrier, and loading the phospholipid-based nanocarrier modified with the annexin with a drug; The annexin comprises any one of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1, or annexin A9, or a combination of at least two thereof. The phospholipid-based nanocarrier comprises any one of a liposome, a lipid nanoparticle, or a phospholipid-coated nanoparticle, or a combination of at least two thereof; Preferably, the liposome comprises any one of a doxorubicin liposome, an irinotecan liposome, a paclitaxel liposome, a mitoxantrone liposome, a daunorubicin cytarabine liposome, a mivatril liposome, or an amphotericin liposome, or a combination of at least two thereof. The lesion site comprises a lesion site with high expression of α5 integrin and / or β1 integrin; Preferably, the lesion site with high expression of α5 integrin and / or β1 integrin comprises a tumor-like lesion site and a non-tumor-like lesion site, the tumor-like lesion site comprises any one of breast cancer, pancreatic cancer, liver cancer, ovarian cancer, osteosarcoma, prostate cancer, glioma, melanoma, myxofibrosarcoma, skin cancer, lung cancer, or gastric cancer, or a combination of at least two thereof, and the non-tumor-like lesion site comprises psoriasis and / or diabetes.

9. A nanodelivery system, characterized in that, The nanodelivery system comprises a phospholipid-based nanocarrier, an annexin modified on the surface of the nanocarrier, and a drug loaded inside the phospholipid-based nanocarrier. the annexin comprises any one or a combination of at least two of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1 or annexin A9; the phospholipid-based nanocarrier comprises any one or a combination of at least two of a liposome, a lipid nanoparticle or a phospholipid-coated nanoparticle; preferably, the liposome comprises any one or a combination of at least two of a doxorubicin liposome, an irinotecan liposome, a paclitaxel liposome, a mitoxantrone liposome, a daunorubicin cytarabine liposome, a mivatril liposome or an amphotericin liposome.

10. A combination pharmaceutical composition, characterized in that, the combination drug composition comprises a phospholipid-based nanocarrier loaded with a drug and an annexin; the combination drug composition is a single complex preparation or a combination of two separate preparations; the annexin comprises any one or a combination of at least two of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1 or annexin A9; the phospholipid-based nanocarrier comprises any one or a combination of at least two of a liposome, a lipid nanoparticle or a phospholipid-coated nanoparticle; preferably, the liposome comprises any one or a combination of at least two of a doxorubicin liposome, an irinotecan liposome, a paclitaxel liposome, a mitoxantrone liposome, a daunorubicin cytarabine liposome, a mivatril liposome or an amphotericin liposome.

11. The composition of claim 10, wherein one, two or more of the following lipids are used in the phospholipid-based nanoparticle: Phospholipids: Egg phosphatidylcholine (EPC), Soy phosphatidylcholine (SPC), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), Dioleoyl-phosphatidylethanolamine (DOPE), Phosphatidylglycerol (PG), e.g. DMPG, DSPG, Phosphatidylinositol (PI), Phosphatidic acid (PA), Dipalmitoylphosphatidylcholine (DPPC), Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dimyristoylphosphatidylcholine (DMPC), Hydrogenated soy phosphatidylcholine (HSPC); Cholesterol and Analogs: Cholesterol, Cholesteryl hemisuccinate (CHEMS), Cholesteryl oleate, Sitosterol; PEGylated Lipids: DSPE-PEG2000, (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]), DOPE-PEG2000, DMPE-PEG2000, DSPE-PEG-maleimide / biotin / amine, Cholesterol-PEG derivatives, and PEGylated lipids with similar structures, different molecular weights of PEGylated lipids; Cholesterol and Analogs: Cholesterol, Cholesteryl hemisuccinate (CHEMS), Cholesteryl oleate, Sitosterol; PEGylated Lipids: DSPE-PEG2000, (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]), DOPE-PEG2000, DMPE-PEG2000, DSPE-PEG-maleimide / biotin / amine, Cholesterol-PEG derivatives, and PEGylated lipids with similar structures, different molecular weights of PEGylated lipids; Ionizable & Cationic Lipids: Ionizable lipids, DLin-MC3-DMA, SM-102, ALC-0315, DODMA (Dimethyldioctadecylammonium), Dlin-DMA; Permanent cationic lipids: DOTAP (Dioleoyltrimethylammonium propane), DOTMA (Dioleoyltrimethylammonium), DDAB (Dimethyldioctadecylammonium bromide), CTAB (Cetyltrimethylammonium bromide); Anionic & Zwitterionic Lipids: DMPG / DSPG (Phosphatidylglycerol, anionic), DOPS / POPS (Phosphatidylserine, anionic), Cardiolipin, Sphingomyelin; Targeting & Functional Lipids: DSPE-PEG-Maleimide, DSPE-PEG-Folate, DSPE-PEG-RGD, Glycolipids (e.g. GM1, GM3); and Other Specialized Lipids: Monolaurin, Lysolipids, Squalene derivatives, Ceramides / Sphingolipids.

12. The composition of claim 10, wherein the phospholipid-based nanoparticle comprises a phospholipid consisting of: (i) distearoylphosphatidylcholine (DSPC), cholesterol (Chol), and (DSPE-PEG2000); (ii) distearoylphosphatidylcholine (DSPC), dioleoylphosphatidic acid sodium salt (DOPA), (2,3-dioleoyl-propyl)-trimethylammonium-chloride salt (DOTAP): cholesterol (Chol), and (DSPE-PEG2000); (iii) distearoylphosphatidylcholine (DSPC), dioleoylphosphatidic acid sodium salt (DOPA), cholesterol (Chol), and (DSPE-PEG2000); (iv) hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Chol), and (DSPE-PEG2000); (v) hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Chol), and (DSPE-PEG2000); and (vi) hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Chol), and (DSPE-PEG2000). (v) distearoylphosphatidylcholine (DSPC) and cholesterol (Chol); (vi) egg yolk phosphatidylcholine (EPC) and cholesterol (Chol); (vii) dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), cholesterol (Chol) and triolein; (viii) sphingomyelin (SM) and cholesterol (Chol); (ix) egg yolk phosphatidylglycerol (EPG) and dimyristoylphosphatidylcholine (DMPC); (x) distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), cholesterol (Chol); (xi) palmitoyloleylphosphatidylcholine (POPC) and dioleoylphosphatidylserine (DOPS); (xii) diarachidoylphosphatidylcholine (DEPC), dipalmitoylphosphatidylglycerol (DPPG), cholesterol (Chol) and tricaprylin; (xiii) egg yolk phosphatidylcholine (EPC) and sodium deoxycholate; or (xiv) hydrogenated soy phosphatidylcholine (HSPC), cholesterol (Chol) and distearoylphosphatidylglycerol (DSPG).

13. Use of an agent that detects a5 integrin and / or b1 integrin in assessing the ability of annexin to improve the tumor entry efficiency and / or to improve the drug biodistribution at the lesion site of a phospholipid-based nanocarrier; Preferably, the application comprises: detecting the expression level of a5 integrin and / or b1 integrin in a patient, assessing the tumor entry efficiency of the delivery vehicle of claim 7 or assessing the therapeutic efficacy of the nanodelivery system of claim 9 or the combination pharmaceutical composition of claim 10 based on the expression level.

14. A kit comprising (a) a drug-loaded phospholipid-based nanocarrier; and (b) annexin; the annexin comprises any one of annexin A2, annexin A1, annexin A10, annexin A11, annexin A13, annexin A3, annexin A4, annexin A5, annexin A6, annexin A7, annexin A8, annexin A8L1 or annexin A9 or a combination of at least two thereof; the phospholipid-based nanocarrier comprises any one of a liposome, a lipid nanoparticle or a phospholipid-coated nanoparticle or a combination of at least two thereof; preferably, the drug-loaded phospholipid-based nanocarrier is mixed and incubated with the annexin before use.

15. Use of annexin and a phospholipid-based nanocarrier in the preparation of a medicament for treating a disease.

16. The use according to claim 15, wherein the lesion site of the disease is a lesion site with high expression of a5 integrin and / or b1 integrin; preferably, the disease is selected from breast cancer, pancreatic cancer, liver cancer, ovarian cancer, osteosarcoma, prostate cancer, glioma, melanoma, myxofibrosarcoma, skin cancer, lung cancer, gastric cancer, psoriasis, diabetes or a combination thereof. ​

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