Targeted albumin spherical nucleic acid medicine as well as preparation method and use method thereof

Abstract

The invention belongs to the field of research and development of tumor drugs, and provides a targeted albumin spherical nucleic acid drug for delivering a hydrophobic drug, aiming at solving the problems that the existing traditional albumin drug is poor in targeted enrichment, strong in systemic side effect and the like, and the targeted albumin spherical nucleic acid drug is characterized in that a hydrophobic molecule is wrapped in albumin (HSA), and meanwhile, the hydrophobic molecule is used as a spherical nucleic acid drug for delivering the hydrophobic drug. Albumin with a plurality of hydrophobic structural domains on the surface and a plurality of aptamer sequences (Apt) with hydrophobic groups at the 5'end are mutually non-covalently modified and combined through hydrophobic interaction to form a spherical nucleic acid structure, and the aptamer has specific targeting property. The invention also provides a preparation method and a use method of the spherical nucleic acid medicine. The drug provided by the invention has excellent targeting property and stability, can realize specific targeting delivery of different types of tumors, improves the treatment effect of the drug, reduces toxic and side effects, and is suitable for large-scale industrial application.

Description

Technical Field

[0001] The present invention relates to the field of tumor drug research and development, and in particular to the research and development of albumin-targeted drugs related to clinical tumor treatment. Background Art

[0002] Human serum albumin (HSA), one of the main components of serum proteins, has a rich functional group and a natural amphiphilic structure, which allows it to be covalently or non-covalently linked to drug molecules. Furthermore, due to its advantages such as wide availability, low cost, long half-life, and good biocompatibility, it is widely used in drug carrier research. For example, albumin-bound paclitaxel (Abraxane) has been approved by the FDA for the clinical treatment of multiple late-stage tumors. However, existing albumin-delivered drugs rely solely on the enhanced permeability and retention effect (EPR) of solid tumors, unable to target drugs to the tumor site and protect normal tissues or cells from damage. This results in strong drug toxicity and severe side effects, limiting its further clinical application.

[0003] Aptamers are functional oligonucleotide sequences that can specifically recognize targets such as proteins, cells, bacteria, and viruses. Aptamers can act not only as direct protein blockers but can also be coupled with small molecule drugs, nanoparticles, or cells for targeted delivery. However, linear aptamer chains suffer from poor stability, weak affinity, and susceptibility to off-target effects, limiting their clinical application in nanomedicine delivery.

[0004] Current cancer treatment drugs have some limitations: for example, tumor photothermal therapy is a non-invasive treatment method that induces cell apoptosis and necrosis through local laser hyperthermia, and its clinical application is hindered by both physical and physiological barriers. From the perspective of physical barriers, as the key to photothermal conversion in photothermal therapy, conventional photothermal agents are difficult to effectively achieve active targeting of tumors. At the same time, the penetration depth of near-infrared lasers is limited and the spatiotemporal resolution is low, making it impossible to completely ablate large or deep tumors. From the perspective of physiological barriers, the local high temperature (50°C) generated by photothermal therapy can cause severe inflammatory reactions in the body and irreversible damage to surrounding organs. However, due to the thermal damage repair effect of intracellular heat shock proteins, mild photothermal (below 43°C) can significantly inhibit the therapeutic effect of tumors.

[0005] For example, pancreatic cancer is a highly aggressive malignant tumor of the digestive system and is known as the "king of cancer." Since pancreatic cancer develops insidiously and progresses rapidly, most patients are already in the middle or late stages when they seek medical treatment. Traditional treatment options are limited and the prognosis is poor. Copper is an essential trace element in the living system, and its levels are strictly regulated in the human body. Recently, a new form of cell death related to copper ions, cuprotosis, has been discovered. It relies on copper ion carriers to transport copper into cancer cells, leading to cell death. However, existing copper ion carriers are small molecules with a short blood half-life, making it difficult to transport enough copper into cancer cells. Elesclomol (ES), as a copper ion carrier, can bind to Cu 2+ To induce copper cell death in tumor cells, however, existing medical technologies cannot achieve efficient induction of copper death in pancreatic cancer. Summary of the Invention

[0006] To address the shortcomings of existing tumor treatment drugs, the present invention provides a targeted albumin spherical nucleic acid drug, aiming to achieve specific targeted delivery for different tumor types, improve drug treatment efficacy, and reduce toxic side effects. The drug of the present invention is characterized by the internal encapsulation of hydrophobic molecules within albumin (HSA). Simultaneously, albumin with multiple hydrophobic domains on its surface and multiple nucleic acid aptamer sequences (Apt) with hydrophobic groups at their 5' ends are non-covalently modified and bound to each other through hydrophobic interactions, forming a spherical nucleic acid structure. The nucleic acid aptamer has specific targeting properties.

[0007] Preferably, the hydrophobic molecules include one or more of paclitaxel (PTX), elisemol (ES), Ce6, IR1061, and resveratrol (Res). It is understood that for those skilled in the art, all hydrophobic drugs that can be delivered via albumin as a carrier in the prior art are included within the scope of the "hydrophobic molecules encapsulated within albumin (HSA)" defined in the present invention.

[0008] Particularly preferably, the aptamer sequence specifically targets tumor cells, and the hydrophobic molecules include IR1061 and resveratrol (Res), forming the IR / Res@HSA-Apt albumin spherical nucleic acid drug. It is understood that, for those skilled in the art, all prior art aptamers capable of targeting tumor cell-specific molecules, with the exception of SYL3C, are within the scope of the aptamers that can be selected in the IR / Res@HSA-Apt drug of the present invention.

[0009] Particularly preferably, the nucleic acid aptamer sequence is SYL3C, which can specifically target the epithelial cell adhesion molecule EpCAM.

[0010] In addition, preferably, the nucleic acid aptamer sequence can also specifically target mitochondria, and the hydrophobic drug is elisemol (ES), so as to form ES@HSA-Apt albumin spherical nucleic acid drug.

[0011] Preferably, catalase (CAT) is added to the ES@HSA-Apt conjugate to form an ES@HSA / CAT-Apt albumin spherical nucleic acid drug.

[0012] Particularly preferably, the nucleic acid aptamer sequence specifically targets the cytochrome c molecule (Cyt c) on the surface of mitochondria.

[0013] Particularly preferably, the hydrophobic group at the 5' end of the nucleic acid aptamer sequence is an octadecyl group. Compared with other lipid-modified nucleic acid aptamers, octadecyl aptamers have lower costs and more mature synthesis strategies.

[0014] The present invention also provides a method for preparing a targeted albumin spherical nucleic acid drug, comprising the following steps:

[0015] Step 1: Dissolve the hydrophobic drug;

[0016] Step 2: Take an appropriate volume of albumin solution, slowly add the hydrophobic drug solution to the albumin solution in a predetermined ratio while stirring, and stir for a certain period of time in the dark;

[0017] Step 3: Centrifuge after stirring to remove free molecules;

[0018] Step 4: After centrifugation, the supernatant is dialyzed to obtain the albumin-bound drug (drug@HSA);

[0019] Step 5: Add a nucleic acid aptamer sequence with a hydrophobic group at the 5' end and incubate with constant temperature stirring to obtain a spherical nucleic acid structure with albumin drug as the core (drug@HSA-Apt).

[0020] The present invention also includes a method for using the targeted albumin spherical nucleic acid drug, that is, using the aforementioned targeted albumin spherical nucleic acid drug for the preparation of drugs related to tumor targeted therapy.

[0021] Beneficial effects

[0022] 1. Compared with the complex nucleic acid-protein coupling strategy of achieving covalent linkage through cross-linking agents, click chemistry and other methods, the present invention can achieve simple and rapid nucleic acid modification on the albumin surface. Based on the protein spherical nucleic acid (SNA) strategy, the present invention introduces octadecyl as a hydrophobic structure into the 5' end of the nucleic acid aptamer, cleverly utilizing the multiple natural hydrophobic domains on the albumin surface to non-covalently link multiple nucleic acid aptamers at one time, and introduces a high-density oligonucleotide layer on the albumin drug surface through simple stirring and incubation, constructing a spherical nucleic acid delivery platform with albumin drug as the core, thereby achieving precise delivery of various hydrophobic drugs to tumors or intracellular regions, effectively solving the problems that traditional albumin drugs cannot solve, such as poor target enrichment and strong systemic side effects.

[0023] 2. Based on this approach, we verified that a variety of hydrophobic drugs (paclitaxel, resveratrol, ilisimol, Ce6, IR1061, etc.) can be hydrophobically bound to albumin to form albumin drugs (Drug@HSA). After coupling with nucleic acid aptamers, they can form a stable hydrophobic drug delivery system (Drug@HSA-Apt), which has demonstrated excellent targeting ability, stability and therapeutic effects beyond expectations in mild photothermal therapy of tumors and copper death therapy.

[0024] In the practice of mild photothermal therapy for tumors, this invention's technical solution, based on a spherical nucleic acid-based targeted albumin drug, precisely co-delivers IR1061 and resveratrol for mild photothermal therapy of tumors. IR1061, as a near-infrared (NIR) II photothermal agent, provides a deeper photothermal conversion, while resveratrol, as a heat shock protein inhibitor, inhibits thermal damage repair by downregulating heat shock proteins in tumor cells, thereby achieving mild photothermal therapy for targeted tumors.

[0025] In the practice of copper death treatment for pancreatic cancer, the technical solution of the present invention is the first to deliver ilisimol to intracellular mitochondria based on a targeted albumin drug with a spherical nucleic acid structure, thereby sensitizing copper death-induced immunogenic cell death by improving the hypoxic environment.

[0026] 3. Compared to existing nucleic acid-protein coupling strategies, this one-step modification of albumin drugs based on hydrophobic interactions has been demonstrated by this invention to offer a simple preparation process, high stability, and strong versatility. It can conveniently and efficiently transform commercial albumin drugs into effective and stable targeted drugs, thus addressing the current pain points of insufficient targeting and stability in cancer treatment. Furthermore, the manufacturing and synthesis process of this product is easily controlled, promising the industrialization and large-scale production of this invention's targeted albumin spherical nucleic acid drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1: (A) Preparation method of targeted albumin spherical nucleic acid drug and its (B) schematic diagram for mild photothermal therapy of tumors and (C) copper-death tumor treatment.

[0028] Figure 2 : Transmission electron microscopy (TEM) images and particle size distribution of targeted albumin spherical nucleic acid drugs loaded with different hydrophobic drugs.

[0029] Figure 3 : Lyophilization-reconstitution characterization and large-scale preparation of albumin-targeted spherical nucleic acid drugs.

[0030] (a) Particle size characterization of PTX@HSA-Apt before and after freeze-drying and reconstitution.

[0031] (b) Gel electrophoresis of PTX@HSA-Apt before and after freeze-drying and reconstitution.

[0032] (c) and (d) are photos of the large-scale preparation of 100 mL PTX@HSA-Apt before and after freeze-drying, respectively.

[0033] Figure 4 : IR / Res@HSA-Apt has stable photothermal performance.

[0034] (a) Temperature rise curves of IR / Res@HSA-Apt with different IR1061 concentrations at 1064 nm.

[0035] (b) Temperature changes of IR / Res@HSA-Apt during three cycles under 1064 nm laser irradiation, showing that the material has stable photothermal conversion performance.

[0036] Figure 5 : Flow cytometry results showed that the drug IR / Res@HSA-Apt of the present invention can effectively target tumor cells and improve the specific recognition ability of albumin drugs.

[0037] Figure 6 : Lysosomal escape performance of ES@HSA / CAT-Apt.

[0038] Figure 7 : Mitochondrial targeting performance of ES@HSA / CAT-Apt.

[0039] Figure 8 : The targeted albumin spherical nucleic acid drug of the present invention has significant tumor targeting and killing performance.

[0040] (a) Cell proliferation and cytotoxicity (CCK-8 assay) showed the comparison of the effects of IR@HSA, Res@HSA, IR / Res@HSA, and IR / Res@HSA-Apt on tumor cell viability under light conditions.

[0041] (b) Flow cytometry detection of cell apoptosis (Annexin V / PI method) showed the comparison of the killing effects of Control, IR@HSA, Res@HSA, IR / Res@HSA, and IR / Res@HSA-Apt on tumor cells under no light or light conditions.

[0042] (c) Cell proliferation and cytotoxicity (CCK-8 method) showed the comparison of the effects of tumor cell viability under ES, ES@HSA, ES@HSA / CAT, and ES@HSA / CAT-Apt treatment.

[0043] Figure 9 : IR@HSA-Apt has excellent targeting performance in vivo.

[0044] (a) In vivo imaging results of 4T1 tumor-bearing mice showed that IR@HSA-Apt still had significant tumor targeting and retention after 36 hours in vivo.

[0045] (b) Fluorescence statistics of tumor sites at different time points in 4T1 tumor-bearing mice.

[0046] Figure 10 : The thermal imaging results of IR@HSA-Apt in 4T1 tumor-bearing mice showed that the material can achieve mild photothermal conversion in vivo.

[0047] Figure 11 :Experimental results of mild photothermal therapy of tumors using IR / Res@HSA-Apt.

[0048] (a) Tumor growth curve of 4T1 tumor-bearing mice.

[0049] (b) Body weight change curve of 4T1 tumor-bearing mice.

[0050] (c) Fluorescence image of heat shock protein expression in tumor tissue.

[0051] Figure 12 : Copper-killing tumor therapy by ES@HSA / CAT-Apt.

[0052] (a) Body weight change curve of Panc02 tumor-bearing mice.

[0053] (b) Tumor growth curve of Panc02 tumor-bearing mice.

[0054] (c) Statistical graph of tumor weight in Panc02 tumor-bearing mice.

[0055] (d) HE sections of tumors in Panc02 tumor-bearing mice after treatment with different drug administration methods. DETAILED DESCRIPTION

[0056] The following specific examples of the present invention are used to describe in detail the preparation method, structural characterization, in vitro performance, in vivo targeting, stability, safety and therapeutic effects of the drugs involved in the present invention.

[0057] 1. Preparation of albumin-targeted spherical nucleic acid drugs

[0058] Without changing the traditional albumin drug synthesis method, we designed a new albumin drug with active targeting capabilities. The preparation method of the targeted albumin spherical nucleic acid drug is as follows:

[0059] (1) Prepare a 40 mg / mL human serum albumin (HSA) solution (ddH2O) and ultrasonically disperse it for 3 minutes to completely dissolve the albumin.

[0060] (2) Hydrophobic drug molecules dissolved in an organic solution, such as paclitaxel (PTX), elisemol (ES), Ce6, IR1061, or resveratrol (Res), are added to an albumin solution in a certain proportion. Since albumin contains a large number of natural hydrophobic domains, under hydrophobic interactions, the albumin aggregates to form nanoparticles that encapsulate the hydrophobic drug, forming an albumin-bound drug.

[0061] (3) Using octadecyl covalent modification at the 5' end of the nucleic acid aptamer (Apt) sequence, a hydrophobic structure is introduced into the nucleic acid sequence to form a C 18 -The structure of Apt;

[0062] (4) Through hydrophobic interaction, C 18 -Apt was non-covalently modified on the surface of albumin nanodrug to obtain a stable Drug@HSA-Apt structure.

[0063] The following describes in detail the preparation methods of different targeted albumin drugs.

[0064] 1. Preparation of IR / Res@HSA-Apt

[0065] First, resveratrol (Res) was dissolved in methanol to prepare a 20 mg / mL Res solution, and IR1061 was dissolved in DMSO to prepare a 10 mg / mL IR1061 solution;

[0066] Place an appropriate volume of serum albumin (HSA) solution in a sample bottle and stir on a magnetic stirrer at 800 rpm. Slowly add Res and IR1061 solutions to the HSA solution while stirring, so that the final ratio of the components in the solution is HSA:IR1061:Res = 2:1:3. Stir in the dark for 12 hours or more.

[0067] After stirring, the mixture was centrifuged at 4000 rpm for 3 minutes to remove free molecules, and the supernatant was dialyzed in a dialysis bag (MWCO = 14 kDa) for 24 hours to obtain IR / Res@HSA albumin-bound drug.

[0068] Then add C 18 -SYL3C (SYL3C, a nucleic acid aptamer that specifically targets the epithelial cell adhesion molecule EpCAM) was added to a final aptamer concentration of 1 μM. The mixture was then incubated on an Eppendorp Thermomixer at 37°C, 300 rpm for 4 hours to obtain IR / Res@HSA-Apt. The product can be temporarily stored at 4°C until further use.

[0069] 2. Preparation of ES@HSA-Apt

[0070] First, elismol (ES) was dissolved in methanol to prepare a 20 mg / mL solution. An appropriate volume of serum albumin (HSA) solution was placed in a sample bottle and stirred on a magnetic stirrer at 800 rpm. ES solution was slowly added to the HSA solution while stirring to achieve a final ratio of HSA:ES = 5:1. The solution was stirred overnight in the dark. Free molecules were removed by centrifugation at 4000 rpm, and the supernatant was dialyzed against PBS for 24 hours (MWCO = 14 kDa) to obtain ES@HSA albumin-bound drug. C was added to the solution. 18 -Targeting Cyt c nucleic acid aptamer (for specific sequence, refer to Shamsipur, Mojtaba, et al. "Impedimetric monitoring of apoptosis using cytochrome-aptamerbioconjugated silver nanocluster." Biosensors & Bioelectronics (2016): 195-202, Experimental Materials Section Anti-Cyt c aptamers C1 sequence) until the final concentration of the aptamer is 1 μM, and then incubate on an Eppendorp Thermomixer at 37°C, 300 rpm for 4 hours to finally obtain ES@HSA-Apt, which can be temporarily stored at 4°C for later use.

[0071] 3. Preparation of ES@HSA / CAT-Apt

[0072] First, elismol (ES) was dissolved in methanol to prepare a 20 mg / mL solution. 36 g of serum albumin (HSA) and 4 g of catalase (CAT) were weighed and dissolved in deionized water. The solution was stirred at 800 rpm on a magnetic stirrer. ES solution was slowly added to the above solution while stirring, so that the final ratio of the components in the solution was HSA:ES:CAT = 45:9:5. The solution was stirred overnight in the dark. Free molecules were removed by centrifugation at 4000 rpm. The supernatant was dialyzed against PBS (MWCO = 14 kDa) for 24 hours to obtain ES@HSA / CAT albumin-bound drug. C was added to the solution. 18 -Targeting Cyt c nucleic acid aptamer, until the final concentration of the aptamer is 1 μM, and then incubated on an Eppendorp Thermomixer at 37°C, 300 rpm for 4 hours to finally obtain ES@HSA / CAT-Apt. The product can be temporarily stored at 4°C for later use.

[0073] 2. Detection of properties of albumin drug spherical nucleic acid:

[0074] 1. Structural Characterization of Albumin-Targeted Spherical Nucleic Acid Drugs

[0075] Targeted albumin-based spherical nucleic acid drugs loaded with different hydrophobic drugs were characterized by transmission electron microscopy to observe the morphology and distribution of their nanostructures. Furthermore, the aqueous solution of the albumin-based spherical nucleic acid drug was diluted tenfold, and the drug particle size was measured using a nanoparticle size analyzer and compared with that of the albumin drug without nucleic acid aptamer modification.

[0076] The experimental results are as follows Figure 2 , transmission electron microscopy results show that our synthetic strategy has a wide range of versatility and can be combined with a variety of hydrophobic drugs to form spherical structures under hydrophobic interaction. Figure 2 The curve graph shows the hydrated particle size measurement results of the corresponding albumin drug solution. It can be seen that after the surface is modified with nucleic acid aptamers, the particle size is slightly increased compared with the albumin drug without nucleic acid aptamer modification.

[0077] 2. Structural Characterization of Albumin Spherical Nucleic Acid Drugs Before and After Lyophilization and Reconstitution

[0078] Freeze the prepared albumin spherical nucleic acid drug and remove it after solidification. Seal the solidified albumin drug with parafilm or plastic wrap, leaving air holes. Place in a freeze dryer for 48 hours, until the sample is a powder or flake solid. After freeze-drying, reconstitute the albumin drug solution in PBS or ddH2O and store at 4°C until needed.

[0079] To demonstrate the stability of non-covalent modification between aptamers and albumin drugs before and after lyophilization, C 18Samples of PTX@HSA-Apt, PTX@HSA-Apt before lyophilization and reconstitution, and PTX@HSA-Apt after lyophilization and reconstitution were sequentially loaded onto a 15% polyacrylamide gel and subjected to gel electrophoresis in 1× TBE buffer. After electrophoresis, the gel was removed and placed on a shaker. The gel was stained with an appropriate amount of 1× GelRed nucleic acid dye for 30 minutes. After destaining with deionized water, the gel was imaged and observed. Next, the gel was boiled in deionized water and then stained with Coomassie Brilliant Blue Rapid Stain for 10 minutes. After destaining with deionized water, the position and color development of the blue bands were observed and photographed.

[0080] The experimental results are as follows Figure 3 Figures ad, c, and d are photos of the large-scale preparation of 100 mL of PTX@HSA-Apt before and after lyophilization; the nanoparticle size and band color after lyophilization and reconstitution are shown in the attached figure. Figure 3 a, 3b, The results show that the albumin globular nucleic acid structure did not degrade and the non-covalent hydrophobic linkage had good stability.

[0081] 3. Photothermal stability of IR / Res@HSA-Apt

[0082] IR / Res@HSA-Apt nanoparticle dispersions with different IR1061 concentrations were heated at 1 W / cm 2 The power density was 500 nm and the laser was exposed to 1064 nm wavelength (with high penetration and high resolution) for 5 minutes. Deionized water treated in the same way was used as a blank control. The temperature was measured with a digital temperature detector at 30-second intervals to observe the heating effect of IR / Res@HSA-Apt. The results are shown in the attached figure. Figure 4 As shown in a, it can be seen that the concentration of IR1061 in IR / Res@HSA-Apt has a significant effect on the sensitivity of the heating curve. As the concentration of IR1061 in IR / Res@HSA-Apt increases, the heating rate of the solution also accelerates.

[0083] In addition, IR / Res@HSA-Apt was repeatedly exposed to laser irradiation, and the temperature rise and fall of the material were recorded every 30 seconds until the material temperature returned to the initial temperature before the next laser irradiation was started to observe the photothermal conversion ability of IR / Res@HSA-Apt. The experimental results are shown in the attached figure. Figure 4 As shown in b, it can be seen that IR / Res@HSA-Apt has stable photothermal conversion performance.

[0084] 3. Targeting and therapeutic effects of albumin-drug spherical nucleic acids:

[0085] 1. In vitro targeting ability detection of aptamer-mediated albumin spherical nucleic acid drugs

[0086] (1) In vitro tumor targeting ability of IR / Res@HSA-Apt

[0087] Flow cytometry was used to further verify the targeting ability of the aptamer. 4T1 cells were plated at 5.0×10 4 The cells were incubated at a density of 100 μg / mL. After digestion, the cells were washed with binding buffer and then loaded with random DNA (rDNA)-modified IR / Res@HSA-rDNA and aptamer-modified IR / Res@HSA-Apt, respectively. The 3' end of the nucleic acid sequence was labeled with FITC. After incubation at 4°C for 2 hours in the dark, unbound aptamers were removed by centrifugation three times at 600 g. The cells were then harvested in the FITC channel and analyzed by flow cytometry.

[0088] The experimental results are shown in the attached Figure 5 , IR / Res@HSA-Apt linked to the SYL3C aptamer was able to clearly bind to the surface of 4T1 cells, demonstrating high targeting ability. However, modified random sequences failed to help albumin-binding drugs recognize 4T1 cell markers.

[0089] (2) Lysosomal escape ability of ES@HSA / CAT-Apt

[0090] First, PANC02 cells (mouse pancreatic cancer cells) were seeded in 24-well plates (3×10 4 Cells were cultured overnight at 37°C in a cell culture incubator containing 5% CO2. The mitochondrial targeting aptamer Cyt c-Apt-modified ES@HSA-Apt-cy5 was then added and incubated in the dark at 37°C for 1, 4, and 8 hours, respectively. The cells were then gently washed three times with PBS to remove excess drug. The cells were then incubated with a green fluorescent-labeled lysosomal probe working solution at 37°C for 15 minutes, after which the Lyso-TrackerGreen staining solution was removed. After three washes with PBS, all cells were imaged for colocalization with lysosomes using a Leica SP8 laser scanning confocal microscope to verify their colocalization with lysosomes at different times.

[0091] The results are shown in the attached Figure 6 ES@HSA / CAT-Apt enters the cytoplasm after 1 hour, colocalizes significantly with lysosomes after 4 hours, and escapes from lysosomes and relocalizes in the cytoplasm after 8 hours. The gray value in the lower row indicates the colocalization of the two fluorescent signals. A higher degree of overlap on the overlapping line indicates stronger colocalization, demonstrating excellent lysosomal escape ability. Effective lysosomal escape can reduce the probability of drug degradation and clearance by lysosomes, allowing it to accumulate more in mitochondria to exert its effect.

[0092] (3) In vitro mitochondrial targeting ability of ES@HSA / CAT-Apt

[0093] First, PANC02 cells (mouse pancreatic cancer cells) were seeded in 24-well plates (3×10 4 Cells were cultured overnight at 37°C in a cell culture incubator containing 5% CO2. Mitochondrial targeting aptamers Cyt c-Apt-modified ES@HSA-Apt-cy5 and ES@HSA-rDNA-cy5 with arbitrary sequence modifications were then added, incubated in the dark at 37°C for 4 hours, and then gently washed three times with PBS to remove excess drug. The cells were then incubated with a green fluorescent-labeled mitochondrial probe working solution at 37°C for 15 minutes, after which the Mito-Tracker Green staining solution was removed. After three washes with PBS, all cells were imaged for colocalization using a Leica SP8 laser scanning confocal microscope to verify their targeting ability.

[0094] The results are shown in the attached Figure 7 : Compared with the control group and the random sequence modification group, the fluorescence signal of ES@HSA / CAT-Apt colocalized more obviously with the mitochondrial probe (mito-tracker). The Gray value in the lower row indicates the colocalization of the two fluorescence signals. A higher degree of overlap on the overlapping line we drew indicates stronger colocalization, showing excellent targeting performance.

[0095] 2. In vitro cytotoxicity assay of albumin-drug spherical nucleic acids

[0096] (1) Cytotoxicity assay of IR / Res@HSA-Apt

[0097] 4T1 cells were seeded in 96-well plates (1×10 4 After incubation with cells for 4 hours at different concentrations of IR@HSA, Res@HSA, IR / Res@HSA and IR / Res@HSA-Apt (with IR1061 drug concentration as the dosage unit), each well was exposed to 1064 nm laser at 1 W / cm 2 The cells were irradiated with a power density of 10 minutes. The cells were then placed in a cell culture incubator and incubated for 6-8 hours. The cell viability was determined using a standard CCK-8 assay.

[0098] The cell apoptosis was determined using Annexin V-FITC apoptosis detection kit (Biyuntian, C1062M). 4T1 cells were seeded in 24-well plates (5 × 10 4The cells were incubated in PBS (400 cells / well) for 24 hours. After 2-4 hours of administration, the cells were washed with PBS and the culture medium was replaced to remove unbound drugs. Each group of materials received two treatments: illumination or non-illumination. After incubation for 6-8 hours, all cells were collected, centrifuged at 600g for 5 minutes, the supernatant was discarded, and 195μl Annexin V-FITC binding solution was added to resuspend the cells. 5μl Annexin V-FITC and 10μl propidium iodide staining solution were added to each group of cells and gently mixed. Incubated in the dark at 25℃ for 20 minutes, washed to remove unbound fluorescent dye, and flow cytometry was used to detect cell apoptosis in each group.

[0099] The experimental results are attached. Figure 8 a: The results of cell proliferation and cytotoxicity tests showed that when the drug concentration reached 20 μg / mL, IR / Res@HSA and IR / Res@HSA-Apt could significantly reduce the number of viable cancer cells under light conditions, and the therapeutic effect was significant; we used the Annexin V / PI method ( Figure 8 b) Further analysis revealed that IR / Res@HSA-Apt induced a significant apoptosis effect under light conditions, showing an excellent tumor-targeted killing effect compared with IR / Res@HSA.

[0100] (2) Cytotoxicity assay of ES@HSA / CAT-Apt

[0101] PANC02 cells were seeded in 96-well plates (1×10 4 Cells were incubated with PBS and different concentrations of ES, ES@HSA, ES@HSA / CAT, and ES@HSA / CAT-Apt for 24 hours, and cell viability was determined using a standard CCK-8 assay.

[0102] The experimental results are as follows Figure 8 As shown in Figure c, compared with ES and ES@HSA, both ES@HSA / CAT and ES@HSA / CAT-Apt exhibited significant tumor-killing effects. Among them, the albumin drug ES@HSA / CAT-Apt, modified with aptamers, showed the strongest killing effect compared with ES@HSA / CAT without aptamer modification, indicating that the targeted recognition of aptamers in ES@HSA / CAT-Apt plays an important role in tumor copper death and can significantly improve the therapeutic effect of copper death.

[0103] 3. Detection of the in vivo targeting ability of aptamer-mediated albumin spherical nucleic acid drugs

[0104] We further investigated the targeting and retention of IR@HSA-Apt at the tumor site using an in vivo mouse intravenous injection model. We injected IR@HSA and IR@HSA-Apt into the tail vein of 4T1 tumor-bearing mice and imaged the tumors using a near-infrared second-zone imager at 1, 2, 4, 8, 12, 24, and 36 hours to observe fluorescence accumulation.

[0105] The experimental results are shown in the attached Figure 9 a, b, The results showed that compared with the IR@HSA group, the IR@HSA-Apt group had a more accurate target area ( Figure 9 (a) The fluorescence signal is well-focused, with no diffusion in the peripheral region. Furthermore, the fluorescence signal is retained persistently and even intensifies over time, without the dissipation seen in the control group.

[0106] This experiment shows that compared with the traditional albumin drug control group, the albumin spherical nucleic acid drug formed by nucleic acid aptamer modification can significantly enhance the targeted accumulation and retention of albumin drugs at the lesion site, far exceeding expectations in the drug's targeting and retention capabilities, making the drug of the present invention have a targeting property that significantly surpasses traditional drugs.

[0107] 4. In vivo photothermal effect experiments of IR / Res@HSA-Apt

[0108] In order to determine the local temperature increase of the tumor caused by photothermal therapy during the high accumulation of IR@HSA-Apt, we used a thermal imager to monitor the temperature changes in the tumor site of 4T1 tumor-bearing mice.

[0109] When the tumor is approximately 50 mm 3 200 μL PBS and IR@HSA-Apt were injected intravenously into 4T1 tumor-bearing mice. 24 hours later, the irradiation was carried out at 1 W / cm 2 The tumor site of the mouse was irradiated with a 1064 nm laser for 20 minutes, and the temperature of the tumor site was recorded at 30 second intervals.

[0110] The experimental results refer to the attached Figure 10 For mice injected with IR@HSA-Apt, the surface temperature of the tumor rapidly increased to about 43°C and remained stable, indicating that IR@HSA-Apt still has good photothermal conversion performance in vivo.

[0111] 5. Mild photothermal therapy of tumors based on IR / Res@HSA-Apt

[0112] Mouse breast cancer cells 4T1 (1×10 6 A tumor-bearing mouse model was constructed with 10 cells / mouse. After 3 days, the tumor of the mouse reached about 50 mm.3 The mice were randomly divided into 5 groups for treatment: PBS group, Res@HSA+1064nm light group, IR / Res@HSA+1064nm light group, IR / Res@HSA-Apt no light group and IR / Res@HSA-Apt+1064nm light group, with at least 5 mice in each group. During the treatment process, 200 μL of the corresponding drug was injected through the tail vein on the third day after tumor modeling. 24 hours after administration, the tumor site of each mouse that needed light treatment was illuminated with 1064nm laser at 1W / cm 2 The power density was irradiated for 10 minutes, and the tumor size of the mice was monitored every other day for 16 consecutive days (V = length × width 2 On day 16, the mice were euthanized, and the main organs and tumors were collected from the sacrificed mice for hematoxylin-eosin (H&E) staining and various immunofluorescence analyses.

[0113] The experimental results refer to the attached Figure 11 ac: Compared with the PBS group, Res@HSA L+ and IR / Res@HSA-Apt L- had no significant inhibitory effect on tumor growth; and because the unmodified aptamer albumin drug had a low accumulation level and a short retention time in tumor tissue, IR / Res@HSA L+ could only partially delay tumor growth. In contrast, the spherical nucleic acid drug of the present invention (IR / Res@HSA-Apt L+) showed a significant tumor growth inhibitory effect under light conditions due to its simultaneous tumor targeting ability and mild photothermal conversion ability. Its therapeutic effect was significantly better than any other treatment group ( Figure 11 a). At the same time, the body weight of mice in each group remained at a stable level during the treatment period, and no obvious biological toxicity was shown ( Figure 11 b).

[0114] In addition, we conducted in vivo experiments to investigate the regulation of heat shock proteins in tumor cells during mild photothermal therapy. Tumor-bearing mice were divided into four groups: PBS, IR@HSA-Apt L+, IR / Res@HSA-Apt L-, and IR / Res@HSA-Apt L+. Treatments were performed as described above. Immunofluorescence analysis of tumors from each group revealed a significant increase in HSP70 and HSP90 levels in tumor tissues treated with IR@HSA-Apt L+ compared to PBS. Following injection of IR / Res@HSA-Apt, the levels of HSP70 and HSP90 were significantly decreased compared to the IR@HSA-Apt L+ or PBS groups, with or without illumination. This demonstrates Res's inhibitory effect on heat shock proteins in tumor cells in vivo, effectively promoting the photothermal therapy process within tumor cells.

[0115] 6. Tumor Copper Death Therapy Based on ES@HSA / CAT-Apt

[0116] Six-week-old female nude mice were injected subcutaneously with mouse pancreatic cancer cells Panc02 (1×10 6 A tumor-bearing mouse model was established with 10 cells / mouse. After 18 days, the tumor of the mouse reached about 90 mm. 3 The mice were randomly divided into 6 groups for treatment: PBS group, ES group, ES@HSA group, ES@HSA-Apt group, ES@HSA / CAT group, and ES@HSA / CAT-Apt group, with at least 5 mice in each group. During the treatment, 100 μL of the corresponding drug was injected through the tail vein on days 18, 21, and 24 after tumor modeling. The tumor size (V = length × width) of the mice was monitored every other day. 2 On day 40, the mice were euthanized, and the main organs and tumors were collected from the sacrificed mice for hematoxylin-eosin (H&E) staining analysis.

[0117] The experimental results refer to the attached Figure 12 Compared with the PBS group, the ES group, ES@HSA group, and ES@HSA-Apt group could only partially delay tumor growth. Since catalase (CAT) was added to the ES@HSA / CAT group to improve the hypoxic environment of the tumor, it could effectively enhance the copper death process that depends on mitochondrial respiration, showing a significant tumor growth inhibitory effect. In addition, the ES@HSA / CAT-Apt group added a mitochondrial-targeting aptamer, which could make the drug more concentrated in the mitochondria where copper death occurs, showing a better tumor growth inhibitory effect than other groups ( Figure 12 a / c / d). Meanwhile, the body weight of mice in each group remained stable during the treatment period, and no obvious biological toxicity was observed ( Figure 12 b).

[0118] Based on the above experimental results, the present invention introduces an octadecyl group as a hydrophobic structure into the 5' end of the nucleic acid aptamer, utilizing the naturally superior hydrophobic properties of albumin to bind multiple nucleic acid aptamers at once in vitro, thereby constructing a targeted albumin spherical nucleic acid drug. This can specifically deliver various hydrophobic drugs to tumors or other lesion tissues, effectively solving the problems that traditional albumin drugs cannot solve, such as poor targeting enrichment and strong systemic side effects.

[0119] In order to demonstrate in detail the excellent specific recognition and therapeutic effects of the albumin-targeted spherical nucleic acid drug of the present invention, the present invention verified the in vitro and in vivo performance of IR / Res@HSA-Apt used in tumor photothermal therapy and ES@HSA / CAT-Apt used in copper death treatment.

[0120] The drug IR / Res@HSA-Apt of the present invention can precisely deliver resveratrol and IR1061 to tumor tissue, rather than to adjacent areas of the cancer. Its drug targeting is precise, its aggregation effect is excellent, and the drug is not easily diffused or degraded over time. Compared with general albumin drugs, the drug's stability, aggregation density, and retention time all exceed expectations. In tumor tissues in vivo, due to the excellent targeting and stability of the drug IR / Res@HSA-Apt, the two drugs IR1061 and Res are able to collaborate efficiently at the tumor site: utilizing the excellent tissue penetration ability of the near-infrared second zone laser, IR1061 can achieve photothermal conversion at the tumor site under 1064nm laser irradiation. Res-induced downregulation of HSP70 and HSP90 can overcome the thermal resistance of tumor cells, triggering high levels of tumor apoptosis at relatively low temperatures or PTT (43°C). In vitro and in vivo experiments have demonstrated tumor-targeted therapeutic effects that significantly exceed those of other drugs.

[0121] At the same time, we applied ES@HSA / CAT-Apt to the copper death treatment of pancreatic cancer. The enhanced permeability and retention effect (ERP) of solid tumors can achieve passive targeting of drugs, enriching ES@HSA / CAT-Apt at the tumor site. After the drug enters the tumor cells, the copper ion carrier ES is actively targeted to the site of copper death - mitochondria using mitochondrial-targeted nucleic acid aptamers. In vitro, we studied whether the drug ES@HSA / CAT-Apt of the present invention can efficiently achieve lysosomal escape (i.e., it will not be engulfed and degraded by intracellular lysosomes), effectively targeting mitochondria to induce copper death in tumor cells. In in vivo experiments, we verified that the drug ES@HSA / CAT-Apt of the present invention can significantly and effectively induce tumor cell death in a subcutaneous pancreatic cancer model in mice.

[0122] It is worth mentioning that compared with existing commercial albumin-binding drugs (such as Abraxane), the targeted albumin spherical nucleic acid drug of the present invention can, based on existing preparation methods, non-covalently link albumin to multivalent nucleic acid aptamers through a one-step method, achieving an efficient transformation from traditional albumin drugs to albumin-targeted drugs. At the same time, in addition to the above-mentioned significant in vivo targeting and stability, the drug of the present invention has also been demonstrated to be able to maintain a stable HSA-Apt composite structure after freeze-drying and reconstitution. This excellent property facilitates the production and storage of the drug, making the large-scale industrial production of the albumin spherical nucleic acid drug of the present invention possible, and is expected to be directly applied to current clinical trials and commercial production.

[0123] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A targeted albumin spherical nucleic acid drug, characterized in that: The albumin contains hydrophobic molecules. At the same time, albumin (HSA) with multiple hydrophobic domains on its surface and multiple nucleic acid aptamer sequences (Apt) with hydrophobic groups at the 5' end are non-covalently modified and bound to each other through hydrophobic interactions to form a spherical nucleic acid structure. The nucleic acid aptamer has specific targeting.

2. The albumin-targeted spherical nucleic acid drug according to claim 1, wherein: The hydrophobic molecules include one or more of paclitaxel (PTX), elisimol (ES), Ce6, IR1061 and resveratrol (Res).

3. The albumin-targeted spherical nucleic acid drug according to claim 2, wherein: The nucleic acid aptamer sequence can specifically target tumor cells, and the hydrophobic molecules include IR1061 and resveratrol (Res) to form IR / Res@HSA-Apt albumin spherical nucleic acid drugs.

4. The albumin-targeted spherical nucleic acid drug according to claim 3, wherein: The nucleic acid aptamer is SYL3C, which can specifically target the epithelial cell adhesion molecule EpCAM.

5. The albumin-targeted spherical nucleic acid drug according to claim 2, wherein: The nucleic acid aptamer sequence specifically targets mitochondria, and the hydrophobic drug is elisemol (ES), so as to form ES@HSA-Apt albumin spherical nucleic acid drug.

6. The albumin-targeted spherical nucleic acid drug according to claim 5, characterized in that: The nucleic acid aptamer sequence specifically targets cytochrome c molecules (Cyt c) on the surface of mitochondria.

7. The albumin-targeted spherical nucleic acid drug according to claim 5, characterized in that: Catalase (CAT) was added to the ES@HSA-Apt conjugate to form the ES@HSA / CAT-Apt albumin spherical nucleic acid drug.

8. The albumin-targeted spherical nucleic acid drug according to claim 1, wherein: The hydrophobic group at the 5' end of the nucleic acid aptamer sequence is an octadecyl group.

9. A method for preparing a targeted albumin spherical nucleic acid drug: Step 1: Dissolve the hydrophobic drug; Step 2: Take an appropriate volume of albumin solution, slowly add the hydrophobic drug solution to the albumin solution in a predetermined ratio while stirring, and stir for a certain period of time in the dark; Step 3: Centrifuge after stirring to remove free molecules; Step 4: After centrifugation, the supernatant is dialyzed to obtain the albumin-bound drug (drug@HSA); Step 5: Add a nucleic acid aptamer sequence with a hydrophobic group at 5', incubate with constant temperature stirring, and obtain a targeted albumin spherical nucleic acid drug (drug@HSA-Apt).

10. A method for using a targeted albumin spherical nucleic acid drug: using the targeted albumin spherical nucleic acid drug according to claims 1-8 for the preparation of drugs related to tumor targeted therapy.

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