PH response type dengue virus bionic drug delivery system as well as preparation method and application thereof

Abstract

The invention provides a pH response type dengue virus bionic drug delivery system as well as a preparation method and application thereof, and relates to the technical field of biological medicines. According to the dengue virus bionic drug delivery system, virus-like particles are used as delivery carriers, STING agonists are loaded into VLP through an electroporation method, DSPE-Hyd-PEG modification is further conducted on the VLP, and finally the dengue virus bionic drug delivery system capable of achieving pH response is obtained. According to the drug delivery system provided by the invention, the delivery efficiency of the VLP-mediated STING agonist and the strength of antigen-specific immune response are remarkably improved, and the drug delivery system has relatively high bioavailability and relatively good biological safety, has more advantages on the inhibition effect on tumor growth, can realize intravenous injection drug delivery, and is suitable for clinical application. The non-specific effect and potential side effects on normal tissues during systematic administration are reduced, the limitation of STING agonist intratumor injection is broken through, and a new strategy is provided for STING pathway targeted therapy of metastatic tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pH-responsive dengue virus biomimetic drug delivery system, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy has achieved significant clinical breakthroughs in recent years, with immune checkpoint blockade (ICB) therapy targeting PD-1 / PD-L1 and CTLA-4 being particularly prominent. However, the complex immunosuppressive microenvironment of solid tumors limits the benefits of immunotherapy. Increasing clinical evidence confirms that the efficacy of ICB is closely related to pre-existing anti-tumor T-cell responses within the tumor, and that T-cell infiltration levels are positively correlated with the prognosis of immunotherapy. Notably, the initiation and maintenance of T-cell responses depend on the synergistic effect of the innate immune system, providing an important direction for optimizing immunotherapy strategies.

[0003] Activation of the interferon gene-stimulating factor (STING) pathway is a key mechanism regulating innate immunity, and its agonists, cyclic dinucleotides (CDNs), have shown significant therapeutic potential. Furthermore, their induction of type I interferon can inhibit sodium and calcium channel function through IFN-I-IFNAR1 signaling, reducing peripheral neuronal excitability and alleviating bone cancer pain. However, the clinical application of CDNs is limited by drug delivery barriers, including lack of cell-target specificity, low cytoplasmic delivery efficiency, and dose-limiting toxicity. Simultaneously, non-specific activation of STING may induce apoptosis of T cells and B cells, negatively impacting tumor treatment. Recent research indicates that STING-specific activation of dendritic cells (DCs) is crucial for initiating antigen-specific anti-tumor immunity, providing a theoretical basis for the development of precision delivery systems.

[0004] Virus-like particles (VLPs) are hollow structures lacking viral nucleic acids. Their morphology, size, and surface antigenicity are highly similar to natural viral particles, but they lack infectivity and replication capacity due to the absence of genetic material. This characteristic allows them to retain viral immunogenicity while avoiding potential safety risks, making them a hot research topic in the biomedical field. With their high immunogenicity, non-infectiousness, and controllable structure, VLPs have achieved significant results in vaccine development, drug delivery, and diagnostics, and are continuously expanding into gene therapy, nanotechnology, and other areas. With advancements in genetic engineering and protein assembly technologies, the application potential of VLPs will be further explored, providing crucial support for human health and the development of biotechnology. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] Existing STING agonists, when administered systemically, are prone to triggering systemic immune storms and have poor clinical safety. Intratumoral injection has limited applicability. Free STING agonists are difficult to target and deliver to dendritic cells (DCs), the key "sentinel" cells of the immune system, and are easily degraded by lysosomes. Conventional nanocarriers lack DC-specific targeting ability and lysosomal escape function. To solve this technical problem, this invention utilizes the characteristic that dengue virus (DENV) first infects immature DCs after invading the body, and combines it with pH-responsive surface modification technology to provide a pH-responsive dengue virus biomimetic drug delivery system that can achieve efficient and low-toxicity systemic delivery of STING agonists.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] Firstly, this invention provides a pH-responsive dengue virus biomimetic drug delivery system capable of delivering a STING agonist, the drug delivery system containing dengue virus-like particles with a surface modified DSPE-Hyd-PEG and a STING agonist.

[0010] Furthermore, the pH-responsive dengue virus biomimetic drug delivery system, abbreviated as CDN@VLP-H-PEG, is prepared according to the following steps:

[0011] 1. Construct a lentiviral expression vector expressing the DENA-prM / E gene.

[0012] A lentiviral expression plasmid containing 5'LTR-ΔU3, a promoter, DENV-prM-E protein, WPRE element, and 3'LTR-ΔU3 was constructed. The lentiviral expression plasmid and helper plasmid were transfected into packaging cells (HEK293T) via liposomes. In the packaging cells, the DNA fragment located between two long terminal repeat sequences was transcribed into RNA, which was then packaged into lentiviral particles by viral proteins expressed by the helper plasmid and placed in HBSS buffer for later use.

[0013] 2. Constructing a HEK293T cell line expressing the DENA-prM / E gene

[0014] HEK293T cells were seeded into six-well plates and cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 18-20 h at 37°C and 5% CO2 concentration to achieve 30%-50% confluence of HEK293T cells during transduction. The lentiviral expression plasmid prepared in step 1 was added to the medium and gently mixed. The initial medium was aspirated, and 1 mL of medium containing the lentiviral expression plasmid was added to each well. The cells were cultured overnight at 37°C and 5% CO2 concentration. The next day, the medium containing the lentiviral expression plasmid was aspirated, and fresh complete culture medium was added. The cells were cultured overnight at 37°C and 5% CO2 concentration to obtain the HEK293T cell line expressing the DENA-prM / E gene.

[0015] 3. Extraction of DENA virus-like particles (VLPs)

[0016] HEK293T cells expressing the DENA-prM / E gene were cultured using exosome-free fetal bovine serum and the cell supernatant was collected. The harvested cell supernatant was centrifuged first at 2000g for 30 min at 4°C to remove cell debris, and then centrifuged at 10000g for 30 min at 4°C to remove microvesicles or protein aggregates. The sample was then filtered through a 0.22 μm filter to remove apoptotic bodies and microvesicles. Finally, the sample was centrifuged at 30000rpm for 4 h at 4°C using an SW32Ti rotor, and the precipitate containing VLP was resuspended in 1 mL of PBS and stored at -20°C for later use.

[0017] 4. Preparation of CDN@VLP

[0018] 10 9 One virus particle was mixed with 1-15 μg of CDN in 100 μL of electroporation buffer and electroporated under the following conditions: voltage, 500 V; pulse length, 10 ms; number of pulses, 5. After electroporation, the resulting solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 6000 rpm for 10 min. The supernatant was the prepared biomimetic delivery system CDN@VLP.

[0019] 5. Preparation of CDN@VLP-H-PEG

[0020] Add 25-150 μL of a 20 mg / mL DSPE-Hyd-PEG solution to 2 mL of a 5 mg / mL CDN@VLP dispersion, then gently blow the mixture dry. After incubating at room temperature for 1 hour, transfer the dispersion to a dedicated centrifuge tube and centrifuge at 120,000 g and 4 °C for 70 min. Aspirate the upper part of the solution to remove unbound DSPE-Hyd-PEG and obtain CDN@VLP-H-PEG.

[0021] Furthermore, the aforementioned virus-like particles originated from any one of the four dengue virus serotypes DEVN1-4.

[0022] Furthermore, in step 1, the lentiviral vectors were named V1, V2, V3, and V4 according to the differences in dengue virus subtypes; the differences among the four lentiviral vectors were only reflected in the differences in the prM-E protein sequences of different dengue virus subtypes.

[0023] Furthermore, the STING agonist is a cyclic dinucleotide CDN agonist.

[0024] Furthermore, the CDN agonist is ADU-S100.

[0025] This invention also provides the application of the above-mentioned pH-responsive dengue virus biomimetic drug delivery system in the preparation of antitumor drugs or drugs for relieving bone cancer pain.

[0026] (III) Beneficial Effects

[0027] This invention utilizes virus-like particles (VLPs) as delivery carriers, employs electroporation to encapsulate the STING agonist into the VLPs, and further modifies them with DSPE-Hyd-PEG to obtain a pH-responsive dengue virus biomimetic drug delivery system, abbreviated as CDN@VLP-H-PEG. The pH-responsive dengue virus biomimetic drug delivery system provided by this invention has the following advantages:

[0028] 1. Compared with free CDN and CDN@VLP, CDN@VLP-H-PEG has better biosafety and bioavailability in vivo, broadening the therapeutic window of STING agonists and providing a new strategy for STING pathway targeted therapy of metastatic tumors.

[0029] 2. By utilizing PEG long-cycle modification and acid-responsive shedding design, CDN@VLP2-H-PEG can achieve precise regulation of "systemic drug delivery → tumor-targeted activation", significantly improving the delivery efficiency of VLP-mediated STING agonists and the intensity of antigen-specific immune responses, and has a more advantageous inhibitory effect on tumor growth.

[0030] 3. The CDN@VLP2-H-PEG provided by this invention can be administered via intravenous injection. The enhanced targeting and intelligent activation characteristics help to limit the activity of STING agonists mainly to the tumor site, reduce non-specific effects and potential side effects on normal tissues when administered systemically, and break the limitations of intratumoral injection of STING agonists. Attached Figure Description

[0031] Figure 1 Transmission electron microscopy image of VLP2

[0032] Figure 2 (A) Hydrated particle size distribution of CDN@VLP and CDN@VLP-H-PEG, (B) Quantitative particle size distribution and (C) Potential characterization.

[0033] Figure 3 CDN Standard Curve

[0034] Figure 4 Blood biochemical parameters of mice in different treatment groups were measured: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and gamma-glutamyl transferase (GGT); compared with the Saline group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0035] Figure 5 Flow cytometry results and quantitative analysis of different preparations taken up by DC cells in vitro; ns P>0.05, ****P<0.0001.

[0036] Figure 6 CLSM results of CDN@VLP2 endosome escape at different time points.

[0037] Figure 7 (A) Tumor growth curves of mice in different groups treated with different doses of CDN and CDN@VLP2 during the treatment period; (B) Expression of CD8+T in tumors after treatment with different doses of CDN and CDN@VLP2.

[0038] Figure 8 (A) Tumor growth curves of mice in different groups during treatment with different formulations; (B) Tumor weight after treatment; *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] Preparation and characterization of a pH-responsive dengue virus biomimetic drug delivery system (abbreviated as CDN@VLP-H-PEG).

[0042] I. Preparation of CDN@VLP-H-PEG:

[0043] 1. Construct a lentiviral expression vector expressing the DENA-2prM / E gene.

[0044] The DENA-2prM / E gene lentiviral expression vector contains 5'LTR-ΔU3, a promoter, DENV-prM-E protein, WPRE element, and 3'LTR-ΔU3. The plasmid was designed and synthesized by Yunzhou Biotechnology (Guangzhou) Co., Ltd. The synthesized plasmid, along with the helper plasmid, was transfected into packaging cells (HEK293T) via liposomes. In the packaging cells, the DNA fragment located between two long terminal repeat sequences is transcribed into RNA, which is then packaged into lentiviral particles by viral proteins expressed by the helper plasmid and placed in HBSS buffer for later use.

[0045] 2. Constructing a HEK293T cell line expressing the DENA-2prM / E gene

[0046] 3×10 5 HEK293T cells were seeded into six-well plates and cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 18-20 hours at 37°C and 5% CO2 to achieve 30%-50% confluence at transduction. The lentiviral solution was then thawed on ice, and the dissolved lentiviral particles were gently mixed by pipetting. An appropriate amount of lentiviral solution (MOI 3) was added to the DMEM medium containing 10% FBS and gently mixed. The initial medium was aspirated, and 1 mL of medium containing lentiviral solution was added to each well. The plate was gently shaken to ensure the virus solution covered all cells, and the plates were cultured overnight. The next day, the virus medium was aspirated, and fresh DMEM high-glucose medium containing 10% FBS was added. The plates were cultured overnight at 37°C and 5% CO2.

[0047] Resistance screening: On day three, prepare fresh culture medium containing 2 μg / mL puromycin, change the medium, and set up one well with cells without virus as a control. When the control cells completely die, the drug screening ends to obtain the HEK293T cell line expressing the DENA-prM / E gene.

[0048] 3. Extraction of DENA-2 virus-like particles (VLP2)

[0049] HEK293T cells that had been stably transformed were cultured using exosome-free fetal bovine serum, and the cell supernatant was collected. The harvested cell supernatant was centrifuged, first at 2000g for 30 min at 4°C to remove cell debris, and then at 10000g for 30 min at 4°C to remove microvesicles or protein aggregates. The sample was then filtered through a 0.22μm filter to remove apoptotic bodies and microvesicles. Finally, the sample was centrifuged at 30000rpm for 4 h at 4°C using an SW32Ti rotor, and the precipitate containing VLP2 was resuspended in 1 mL of PBS and stored at -20°C for later use.

[0050] 4. Preparation of CDN@VLP

[0051] 10 9 One virus particle was mixed with 1-15 μg of CDN in 100 μL of electroporation buffer and electroporated under the following conditions: voltage, 500 V; pulse length, 10 ms; number of pulses, 5. After electroporation, the resulting solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 6000 rpm for 10 min. The supernatant was the prepared biomimetic delivery system CDN@VLP. Preferably, the amount of CDN used was 10 μg, and the CDN was ADU-S100.

[0052] 5. Preparation of CDN@VLP-H-PEG

[0053] Add 25-150 μL of a 20 mg / mL DSPE-Hyd-PEG solution to 2 mL of a 5 mg / mL CDN@VLP dispersion, then gently blow the mixture dry. After incubating at room temperature for 1 hour, transfer the dispersion to a dedicated centrifuge tube and centrifuge at 120,000 g and 4 °C for 70 min. Aspirate the upper part of the solution to remove unbound DSPE-Hyd-PEG and obtain CDN@VLP-H-PEG. Preferably, the volume of DSPE-Hyd-PEG solution used is 100 μL.

[0054] II. Characterization of CDN@VLP-H-PEG

[0055] 1. Characterization of virus-like particles VLP2

[0056] After diluting VLP2 to an appropriate concentration with ultrapure water, 10 μL of sample was dropped onto a copper grid and allowed to stand for 1 min. Excess solution was then gently blotted away with filter paper, and this process was repeated three times. The sample was then stained with 2% phosphotungstic acid. After drying overnight, the morphological characteristics of the sample were observed and photographed using a transmission electron microscope (TEM).

[0057] The results are as follows Figure 1As shown, transmission electron microscopy reveals that VLP2 is a spherical structure with a diameter of about 50 nm, which is highly consistent with the physical properties of natural viral particles, providing a basic condition for its use as a drug carrier.

[0058] 2. Characterization of CDN@VLP and CDN@VLP-H-PEG

[0059] CDN@VLP and CDN@VLP-H-PEG were diluted to appropriate concentrations with ultrapure water, and their particle size distribution and zeta potential were determined by dynamic light scattering (DLS).

[0060] The results are as follows Figure 2 As shown, the potential of CDN@VLP is -21.3±1.0mV. Due to the negative charge of PEG itself, the electronegativity of CDN@VLP-H-PEG is more pronounced, with a potential of -24.5±0.6mV. The negative charge on the surface of the nanoparticles is beneficial to their stability during blood circulation. The particle size of CDN@VLP is 55.6±3.2nm. After modification with DSPE-Hyb-PEG, the particle size of CDN@VLP-H-PEG increases to 82.7±3.7nm, indicating successful modification.

[0061] 3. Determination of CDN drug loading in CDN@VLP-H-PEG

[0062] (1) CDN standard curve and linear relationship

[0063] A 1 mM standard CDN solution was serially diluted to 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 2.5 μM and 1 μM. The peak area of ​​the solutions at 260 nm absorbance was measured by high performance liquid chromatography with 20% acetonitrile. A standard curve of CDN concentration (μM)-peak area (mAU·s) was established.

[0064] (2) Determination of CDN drug loading

[0065] 10 9 Viral particles were mixed with 1 μg, 2 μg, 5 μg, 8 μg, 10 μg, 12 μg, and 15 μg of CDN in 100 μL of electroporation buffer, and electroporated under the following conditions: voltage, 500 V; pulse length, 10 ms; number of pulses, 5. After electroporation, the resulting solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 6000 rpm for 10 min. The upper layer was the prepared CDN@VLP, and the lower layer was free CDN. The lower layer was collected, and the free CDN was quantified using high-performance liquid chromatography (HPLC).

[0066] Subsequently, based on the CDN standard curve, the encapsulation efficiency for the different feed ratios was calculated according to formula (3.1). Where W 总 W represents the total amount of CDN deployed. 游 This represents the free CDN content in the lower layer of liquid.

[0067] Encapsulation rate % = (W 总 -W 游 W 总 ×100% Formula 1

[0068] The drug loading rate at the optimal encapsulation ratio was calculated according to formula (3.2). Where W 总 W represents the total amount of CDN deployed. 游 W represents the content of free CDN. P The quality of VLP2.

[0069] Drug loading % = (W 总 -W 游 ) / (W 总 -W 游 +W P )×100% Formula 2

[0070] The results are as follows Figure 3 As shown, a standard curve was established with the concentration of CDN standard as the abscissa and the peak area as the ordinate, and the linear regression equation was obtained as: y = 10.322x - 2.7768, where R2 = 0.9997. When the mass of CDN is 10 μg, the encapsulation efficiency is 75.72% and the drug loading is 16.82%.

[0071] Example 2

[0072] CDN@VLP-H-PEG in vivo safety assessment

[0073] B16-F10 mouse melanoma cells were cultured to the logarithmic growth phase, processed and collected, and the resulting cell pellet was resuspended in serum-free cell culture medium to prepare a density of 1×10⁶ cells / mL. 7 To establish a mouse melanoma model, approximately 100 μL of tumor cell suspension per cell / mL was subcutaneously injected into the right hind limb of each mouse. The tumor was allowed to grow to 100 mm. 3After approximately 7 days, the mice with the established model were randomly divided into 5 groups: (1) Saline group (physiological saline), (2) 20 μg CDN group, (3) 20 μg CDN@VLP group, (4) 20 μg CDN@VLP-H-PEG, and (5) 30 μg CDN@VLP-H-PEG, with 6 mice in each group. The drugs were administered via tail vein in each group, once every 3 days, for a total of 3 administrations over a period of 9 days. On the 10th day of treatment, 4 mice from each group were randomly selected for ocular blood sampling to assess the in vivo safety of CDN@VLP-H-PEG.

[0074] Detection indicators: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and gamma-glutamyl transferase (GGT).

[0075] The results are as follows Figure 4 As shown, compared with the Saline group, the livers of mice in the 20 μg CDN group showed obvious pathological phenomena, and some mice in the 20 μg CDN@VLP group had abnormal ALT levels. However, after the same dose and increased dose, there were no statistically significant differences in blood biochemical indicators in the CDN@VLP-H-PEG group. This indicates that CDN@VLP-H-PEG has good biosafety under systemic administration conditions, broadens the therapeutic window of STING agonists, and provides a new strategy for STING pathway-targeted therapy of metastatic tumors.

[0076] Example 3

[0077] Investigation of the ability of DC cells to take up CDN@VLP-H-PEG

[0078] In a 6-well plate, each well is inoculated with 3 × 10⁶ seeds. 5 Seven groups of dendritic cells (DCs) were divided into seven groups and cultured in 1640 medium with 10% FBS at pH 7.4. One group of DCs was inoculated with mannan to block CD209 antibody. All cells were cultured overnight. After 24 hours, the medium was discarded, and the cells were washed twice with PBS. Then, 1 mL of medium containing PBS, free CDN, CDN@VLP (pre-blocked with CD209 in this group of DCs), CDN@VLP blocked with E protein, CDN@VLP, CDN@VLP-H-PEG (with 0.1M hydrochloric acid added to adjust the pH to 6.5), and CDN@VLP-H-PEG (all using Cy5-labeled CDN: 100 ng / mL) was added to each group. The cells were co-incubated at 37°C and 5% CO2 for 12 hours. Finally, the drug-containing medium was discarded, the cells were washed three times with PBS, and the cells were collected by trypsin digestion and diluted with PBS to appropriate concentrations. Flow cytometry was used to detect the uptake of each agent by DCs or CD209-blocked DCs.

[0079] The results are as follows Figure 5 As shown, when the CD209 receptor on dendritic cells (DCs) or the E protein receptor-binding domain on VLP2 is blocked, the uptake of CDN@VLP2 by DCs is significantly reduced. This indicates that the uptake of CDN@VLP2 by DCs is achieved through the recognition of the CDN@VLP2 surface E protein receptor-binding domain by the DC-specific adhesion receptor CD209, followed by receptor-mediated endocytosis that allows viral particles to enter the cell. Furthermore, at pH 7.4, CDN@VLP2-H-PEG, due to its DSPE-Hyd-PEG modification, effectively shields the VLP surface, reducing VLP adsorption by plasma proteins and clearance by immune cells, thus preventing DC recognition and uptake. At pH 6.5, CDN@VLP2-H-PEG, utilizing a PEG acid-responsive shedding design, maintains its uptake capacity by DCs, achieving precise regulation of "systemic drug delivery → tumor-targeted activation," improving drug biosafety and bioavailability in vivo, and preventing systemic immune storms.

[0080] Example 4

[0081] Intranuclear escape analysis

[0082] DC cells were seeded in 15 mm confocal microplates (1×10⁻⁶ cells / mL). 5 Cells were cultured in confocal microarrays at appropriate densities using a small dish (e.g., 1 / v dish) and incubated in a cell culture incubator. CDN@VLP2 (using Cy5-labeled CDN: 100 ng / mL) was added to each confocal microarray. After 1, 3, and 6 hours of uptake, the culture medium was discarded, and the cells were washed three times with PBS. Cells were then stained with Lyso Tracker green (75 nM) for 15 min and Hoechst 33342 for 15 min. The cells were protected from light and placed in a small amount of PBS under CLSM for observation and imaging.

[0083] The results are as follows Figure 6 As shown, the overlap between the cytoplasmic localization of CDN and the Lyso Tracker signal gradually decreased over time, indicating that VLP2 mediates the migration of CDN to STING-expressing cell bodies. After co-incubating CDN@VLP2 with DC cells for 1 hour, CDN@VLP2 and the endosome dye showed high co-localization in yellow, indicating that CDN@VLP2 had been internalized into the cells at 1 hour, but had not yet escaped from the endosome. After co-incubation for 3 hours, some red fluorescence of the formulation was observed to diffuse out from the green fluorescence of the endosome, proving that CDN@VLP2 had partially escaped from the endosome. After co-incubation for 6 hours, the red fluorescence of the formulation completely escaped from the endosome, indicating that CDN@VLP2 has good endosome escape performance. These results suggest that VLP2 is a good carrier for delivering STING agonists.

[0084] Example 5

[0085] Analysis of the in vivo antitumor efficacy of CDN and CDN@VLP after local administration

[0086] 1 Experimental Methods

[0087] B16-F10 mouse melanoma cells were cultured to the logarithmic growth phase, processed and collected, and the resulting cell pellet was resuspended in serum-free cell culture medium to prepare a density of 1×10⁶ cells / mL. 7 To establish a mouse melanoma model, approximately 100 μL of tumor cell suspension per mL was subcutaneously injected into the right hind limb of each mouse.

[0088] CDN administration group: When the tumor grows to 100mm 3 After approximately 7 days, the drugs were administered. The mice with the established model were randomly divided into 6 groups: (1) Saline group, (2) 1 μg CDN group, (3) 5 μg CDN group, (4) 10 μg CDN group, (5) 20 μg CDN group, and (6) 40 μg CDN group. There were 3 mice in each group. The drugs were administered via intratumoral injection, once every 3 days, for a total of 3 administrations over a period of 9 days.

[0089] CDN@VLP administration group: When the tumor grows to 100mm 3 After approximately 7 days, the mice were administered the drug. The mice with the established model were randomly divided into 6 groups: (1) Saline group, (2) 1 μg CDN@VLP group, (3) 5 μg CDN@VLP group, (4) 10 μg CDN@VLP group, (5) 20 μg CDN@VLP group, and (6) 40 μg CDN@VLP group. Each group consisted of 3 mice. The drug was administered via intratumoral injection, once every 3 days, for a total of 3 administrations over a 9-day period.

[0090] The size of the mouse tumor was measured every two days, and the tumor volume was set to the preset endpoint of 1200 mm. 3 The mice were euthanized. The length and width of the tumors were measured using digital calipers, and the volume was determined according to the formula: Volume = L × W 2 The tumor volume is calculated using L / 2, where L represents the maximum length of the tumor and W represents the maximum width of the tumor.

[0091] Tumor tissue obtained after treatment was fixed overnight in 4% paraformaldehyde solution and then prepared into tumor sections. The level of CD8+ T cells (CD8: green) in the tumor tissue was detected by multicolor immunofluorescence.

[0092] 2 Experimental Results

[0093] Using ADU-S100 as a representative STING agonist of CDN, the dose-dependent and antitumor effects of free CDN and CDN@VLP2 were evaluated in a mouse melanoma model.

[0094] like Figure 7 As shown, in the CDN-treated groups, the tumors in the Saline group mice continued to grow, while the CDN group showed a dose-dependent tumor growth inhibition effect within the dosage range of 5-20 μg / mouse. However, all mice in the intratumoral administration of free CDN showed rapid tumor recurrence after drug withdrawal, suggesting that free CDN cannot induce a long-term immune memory effect. The high-dose group (40 μg / mouse) showed a significant trend of diminishing efficacy, and the fluorescence intensity of yellow fluorescence (CD8+ T cells) in this group was significantly lower than that in the Saline group, indicating that high-dose CDN may actually lead to a reduction in CD8+ T cell infiltration.

[0095] In contrast, low-dose (5 μg / animal) CDN@VLP produced a significant tumor-suppressive effect, and tumor growth almost stopped after drug withdrawal, indicating that low-dose CDN@VLP2 can induce a durable anti-tumor response. The high-dose group (40 μg CDN@VLP) also showed a significant tumor-suppressive effect. Immunofluorescence staining was used to assess CD8+ in tumor tissue. + T cell infiltration. Compared with the saline group, all treatment groups showed increased intratumoral expression representing CD8. + The green fluorescence intensity of T cells was significantly enhanced. Notably, a low dose of CDN@VLP2 (5 μg) could induce CD8+. + T cell activation. Even with high-dose CDN@VLP2 (40 μg), CD8... + T cells were still significantly activated, indicating that the VLP biomimetic delivery system enhances the in vivo immunotherapy effect of STING agonists.

[0096] Example 6

[0097] Evaluation of the in vivo antitumor efficacy of CDN@VLP-H-PEG after intravenous administration

[0098] 1. Experimental Methods

[0099] B16-F10 mouse melanoma cells were cultured to the logarithmic growth phase, processed and collected, and the resulting cell pellet was resuspended in serum-free cell culture medium to prepare a density of 1×10⁶ cells / mL. 7 To establish a mouse melanoma model, approximately 100 μL of tumor cell suspension per mL was subcutaneously injected into the right hind limb of each mouse.

[0100] When the tumor grows to 100mm 3After approximately 7 days, the mice were administered the drug. The mice with the established model were randomly divided into 5 groups: (1) Saline group, (2) 20 μg VLP group, (3) 20 μg CDN group, (4) 20 μg CDN@VLP group, and (5) 20 μg CDN@VLP-H-PEG group, with 3 mice in each group. The drug was administered via the tail vein, once every 3 days, for a total of 3 administrations over a period of 9 days.

[0101] The size of the mouse tumor was measured every two days, and the tumor volume was set to the preset endpoint of 1200 mm. 3 The mice were euthanized. The length and width of the tumors were measured using digital calipers, and the volume was determined according to the formula: Volume = L × W 2 The tumor volume is calculated using L / 2, where L represents the maximum length of the tumor and W represents the maximum width of the tumor.

[0102] 2 Experimental Results

[0103] like Figure 8 As shown, compared with the Saline group, the VLP group showed no statistically significant difference in tumor growth. Similarly, the tumor volume in the free CDN group mice still showed a significant increasing trend, suggesting that the systemic delivery efficiency of free CDN was insufficient. The CDN@VLP group and the 20μg and 30μg CDN@VLP-H-PEG groups all showed significant inhibitory effects on tumor growth. Compared with the CDN@VLP group, the 20μg CDN@VLP-H-PEG group had a more significant inhibitory effect on tumor growth (P<0.001). The tumor volume in the 20μg CDN@VLP-H-PEG group was 2.1 times smaller than that in the CDN@VLP group, and this reduction was dose-dependent.

[0104] The above results demonstrate that CDN@VLP-H-PEG exhibits excellent antitumor effects and good safety when administered via tail vein.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pH-responsive dengue virus biomimetic drug delivery system, characterized in that... The drug delivery system contains dengue virus-like particles with a surface modified DSPE-Hyd-PEG and a STING agonist.

2. The method for preparing a pH-responsive dengue virus biomimetic drug delivery system according to claim 1, characterized in that, The drug delivery system is referred to as CDN@VLP-H-PEG, and CDN@VLP-H-PEG is prepared according to the following steps: Step 1: Construct a lentiviral expression vector expressing the DENA-prM / E gene A lentiviral expression plasmid containing 5'LTR-ΔU3, a promoter, DENV-prM-E protein, WPRE element, and 3'LTR-ΔU3 was constructed. The lentiviral expression plasmid and helper plasmid were transfected into packaging cells (HEK293T) via liposomes. In the packaging cells, the DNA fragment located between two long terminal repeat sequences was transcribed into RNA, which was then packaged into lentiviral particles by viral proteins expressed by the helper plasmid and placed in HBSS buffer for later use. Step 2: Construct a 293T cell line expressing the DENA-prM / E gene HEK293T cells were seeded into six-well plates and DMEM medium containing 10% fetal bovine serum (FBS) was added. The cells were cultured at 37°C and 5% CO2 for 18-20 hours to achieve 30%-50% confluence during transduction. The lentiviral expression plasmid prepared in step 1 was added to the medium and gently mixed. The initial medium was aspirated, and 1 mL of medium containing the lentiviral expression plasmid was added to each well. The cells were cultured overnight at 37°C and 5% CO2. The next day, the medium containing the lentiviral expression plasmid was aspirated, and fresh complete culture medium was added. The cells were cultured overnight at 37°C and 5% CO2 to obtain the HEK293T cell line expressing the DENA-prM / E gene. Step 3: Extraction of DENA virus-like particles (VLPs) HEK293T cell lines expressing the DENA-prM / E gene were cultured using exosome-free fetal bovine serum and cell supernatant was collected. Centrifuge the harvested cell supernatant at 2000g for 30 minutes at 4°C to remove cell debris. Centrifuge at 10000g for 30 min at 4℃ to remove microvesicles or protein aggregates; then filter the sample with a 0.22μm filter to remove apoptotic bodies and microvesicles; finally, centrifuge at 30000rpm for 4 h at 4℃ using an SW32Ti rotor, then resuspend the precipitate containing VLP in 1mL PBS and store at -20℃ for later use. Step 4: Preparation of CDN@VLP 10 9 One virus particle was mixed with 1-15 μg of CDN in 100 μL of electroporation buffer and electroporated under the following conditions: voltage, 500 V; pulse length, 10 ms; number of pulses, 5. After electroporation, the resulting solution was transferred to a 30 kDa ultrafiltration tube and centrifuged at 6000 rpm for 10 min. The supernatant was the prepared biomimetic delivery system CDN@VLP. Step 5: Preparation of CDN@VLP-H-PEG Add 25-150 μL of a 20 mg / mL DSPE-Hyd-PEG solution to 2 mL of a 5 mg / mL CDN@VLP dispersion, then gently blow the mixture dry. After incubating at room temperature for 1 hour, transfer the dispersion to a dedicated centrifuge tube and centrifuge at 120,000 g and 4 °C for 70 min. Aspirate the upper part of the solution to remove unbound DSPE-Hyd-PEG and obtain CDN@VLP-H-PEG.

3. The pH-responsive dengue virus biomimetic drug delivery system according to claim 1, characterized in that, The dengue virus-like particles are derived from any one of the four dengue virus serotypes DEVN1-4.

4. The pH-responsive dengue virus biomimetic drug delivery system according to claim 1, characterized in that, The STING agonist is a cyclic dinucleotide CDN agonist.

5. The preparation method of the pH-responsive dengue virus biomimetic drug delivery system according to claim 2, characterized in that, Lentiviral vectors were named V1, V2, V3, and V4 according to the differences in dengue virus subtypes; the differences among the four lentiviral vectors are only reflected in the differences in the prM-E protein sequences of different dengue virus subtypes.

6. The application of the pH-responsive dengue virus biomimetic drug delivery system as described in claim 1 in the preparation of antitumor drugs or drugs for relieving bone cancer pain.

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