A delivery vehicle for targeted immunization and methods of making and using the same

Abstract

The present application relates to a kind of delivery carrier targeted to immune, which is formed by the covalent coupling of small molecule immunostimulant with Toll-like receptor 7 agonistic activity selected from SZU-102, CZM-1021, CZM-1022, GY106 or SZU-251 and polyamide-amine dendrimer PAMAM, and the general structure is PAMAM-(TLR7a) n , TLR7a is the structural unit of TLR7 small molecule immunostimulant, and n is an integer from 1 to 64.The present application also provides a preparation method of the delivery carrier and a delivery system comprising the delivery carrier and nucleic acid molecules.The delivery system can target the delivery of nucleic acid molecules to dendritic cells for the preparation of antitumor drugs.Nanocomposites constructed using the delivery carrier of the present application can efficiently transfect dendritic cells, have good immune activation effect and clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical and biomedical engineering, and relates to a functionalized delivery carrier based on dendritic polyamide-amine (PAMAM), particularly to a cationic nanocomposite capable of targeting dendritic cells and delivering immunomodulatory genes, and its application in immunotherapy and antitumor drug preparation. Background Technology

[0002] In recent years, immunotherapy has shown great potential in the treatment of cancer, chronic infections, and autoimmune diseases. Dendritic cells (DCs), as the body's most powerful professional antigen-presenting cells, play a central role in initiating and regulating adaptive immune responses. Efficiently and targetedly delivering antigens or immunomodulatory genes to DCs, while simultaneously promoting DC maturation, is one of the key strategies for cancer immunotherapy and vaccine development.

[0003] Currently used drug delivery carriers, such as liposomes, polymer micelles, and inorganic nanoparticles, while improving drug pharmacokinetic behavior to some extent, still face many challenges. For example, traditional lipid carriers generally tend to passively accumulate in the liver, making it difficult to achieve extrahepatic targeting, especially for specific immune cell subsets. Furthermore, achieving specific cell targeting often requires complex bioengineering modifications to the carrier or the introduction of targeting ligands, significantly increasing the difficulty and complexity of formulation preparation.

[0004] Polyamide-amine (PAMAM) dendritic polymers are a class of highly branched, monodisperse cationic polymers with unparalleled molecular uniformity, narrow molecular weight distribution, well-defined size and shape characteristics, and multifunctional end surfaces. These nanoscale polymers consist of an ethylenediamine core, a repeating branched amide-amine internal structure, and primary amine end surfaces. Commercial PAMAMs have iterative products, with fourth-generation and above exhibiting spherical or globular morphologies.

[0005] Based on its highly branched three-dimensional structure and abundant surface amino-modifiable sites, PAMAM has been widely used as a gene and drug delivery vector. However, unmodified PAMAM vectors have two inherent drawbacks: First, the numerous primary amine groups on its surface protonate under physiological conditions to form a dense positive charge, which, while beneficial for compressing nucleic acids, makes it difficult for them to cross the lipid bilayer of the cell membrane, resulting in low endocytosis efficiency; Second, natural PAMAM lacks the ability to actively recognize specific immune cell subsets, making it difficult to selectively enrich dendritic cells (DCs), thus limiting the expression efficiency of its delivered genes in antigen-presenting cells.

[0006] Therefore, how to endow the PAMAM vector with immune targeting and biocompatibility without sacrificing its delivery efficiency is a key research focus at present.

[0007] The Toll-like Receptor (TLR) family is an important member of the innate immune pattern recognition receptor family, with TLR7 primarily expressed in plasmacytoid dendritic cells and myeloid dendritic cells. Small molecule agonists of TLR7 can specifically bind to and activate the TLR7 signaling pathway, inducing not only dendritic cell maturation and cytokine secretion but also acting as "built-in" targeting ligands to guide the enrichment of TLR7-positive cells. Based on this, to endow the PAMAM vector with immune targeting, researchers attempted to couple a Toll-like receptor agonist as a ligand to the surface of PAMAM.

[0008] Xu. et al. (Resiquimod-Induced Nanovaccine (RINV) for Personalized CancerImmunotherapy. Angewandte Chemie International Edition (e2025, e202507902.) R848 was covalently linked to G5-PAMAM via disulfide linkers to construct a reducible-responsive nanovaccine carrier, G5-R848. Redox-triggered intracellular R848 release was utilized to promote antigen cross-presentation. Tong et al. (A size-switchable nanocluster remodels the immunosuppressive microenvironment of tumor and tumor-draining lymph nodes for improved cancer immunotherapy.) Biomaterials (2025, 315, 122910.) pH-sensitive nanoclusters SPN-R848 were formed by the self-assembly of R848-coupled PAMAM derivatives. CN114712493A also reported a method for preparing PAMAM-R848 covalently coupled compounds by Michael addition reaction of the amino groups on the PAMAM surface with the double bonds of R848 disulfide derivatives.

[0009] However, all of the above studies share a common technical deficiency. Research indicates that the amino group at position 1 of the R848 molecule is one of its key active sites for binding to the TLR7 receptor. If R848 is coupled to a carrier through this site, chemical modification directly interferes with the crucial hydrogen bond interaction between R848 and the TLR7 receptor, leading to a significant reduction or even complete loss of its immune-activating activity. For example, data published in WO 2023057564A1 shows that after chemical coupling, R848's TLR7 activation of EC...50 The activity was reduced by more than 80% when the free state concentration increased from 182 nM to over 1000 nM. In addition, this type of amidation coupling introduces a strongly polar amide bond (-NH-CO-), which further enhances the overall polarity of the conjugate compared to unmodified PAMAM, thereby exacerbating the transmembrane barrier and hindering the carrier from entering the cell. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of existing delivery vectors, such as difficulty in actively targeting dendritic cells, lack of immune activation function, and complex preparation process, and to provide a targeted immune delivery vector composed of PAMAM dendritic polymer covalently modified with TLR7 small molecule immune agonist.

[0011] Another object of the present invention is to provide a method for preparing the delivery vector and its application in the delivery of functional nucleic acids.

[0012] Another object of the present invention is to provide the use of the delivery carrier in the preparation of antitumor drugs, vaccine adjuvants or immunomodulatory drugs.

[0013] To achieve the above objectives, the present invention first provides a delivery carrier for targeted immunity, wherein the delivery carrier is composed of a small molecule immune agonist TLR7 with Toll-like receptor 7 agonist activity covalently coupled with a polyamide-amine dendritic polymer PAMAM, and its general structural formula is PAMAM-(TLR7a). n .

[0014] Wherein, TLR7a is a structural unit of a TLR7 small molecule immune agonist, and the TLR7 small molecule immune agonist is selected from any one of SZU-102, CZM-1021, CZM-1022, GY106, or SZU-251, and their specific structural formulas are as follows: , , , , .

[0015] Wherein, n represents the average number of TLR7a structural units coupled to each PAMAM molecule, and n is an integer from 1 to 64.

[0016] Therefore, the delivery carrier described in this invention is composed of TLR7a structural units linked to PAMAM via thiourea bonds, specifically formed by the reaction of isothiocyanate groups on the TLR7 small molecule immune agonist with primary amine groups on the surface of PAMAM.

[0017] Furthermore, in the delivery carrier described in this invention, the PAMAM can be any generation selected from the first to the tenth generation.

[0018] Furthermore, in the delivery carrier of the present invention, the PAMAM is preferably a fifth-generation polyamide-amine dendritic polymer, PAMAM-G5.

[0019] More preferably, the delivery carrier provided by the present invention is composed of a small molecule immune agonist SZU-102 covalently coupled with a polyamide-amine dendritic polymer PAMAM-G5, having the general structural formula shown in formula (I):

[0020] Where n is an integer from 1 to 64.

[0021] Furthermore, in the delivery carrier represented by the above structural formula (I), n is preferably 13.

[0022] In this invention, the delivery carrier formed by the covalent coupling of SZU-102 and PAMAM-G5 is referred to as PA102.

[0023] Furthermore, when the TLR7 small molecule immune agonist is selected from CZM-1021, the prepared delivery carrier is designated as PA103; when selected from CZM-1022, it is designated as PA104; when selected from GY106, it is designated as PA105; and when selected from SZU-251, it is designated as PA106, all of which constitute analogs of PA102.

[0024] Furthermore, the small molecule immunostimulant used in this invention for covalently coupling with the polyamide-amine dendritic polymer (PAMAM) is not limited to the aforementioned TLR7 small molecule immunostimulant. Any other immunostimulant capable of coupling with the primary amino groups on the PAMAM surface can also be used, such as immunostimulants with carboxylic acid or activated ester groups, or immunostimulants with groups or atoms that can be substituted by amino groups, such as halogens, or immunostimulants capable of forming azo compounds with amino groups. Additionally, a linker chain can be formed between the immunostimulant and PAMAM, and this linker chain can be cleaved by proteases or dipeptidases within cells to release genes or the product of the immunostimulant.

[0025] This invention achieves the goal of targeting dendritic cells (DCs) by chemically coupling the TLR7 small molecule immune agonist to PAMAM material. The delivery vector constructed by this invention can both load antigens and be a biocompatible material that can target and activate DCs, thus making it an ideal carrier for targeted immunization and vaccine manufacturing.

[0026] Secondly, the present invention also provides a method for preparing the delivery carrier, specifically by mixing PAMAM with the TLR7 small molecule immune agonist in an organic solvent system, so that the primary amine groups on the surface of PAMAM undergo a coupling reaction with the isothiocyanate groups to form thiourea bonds.

[0027] Furthermore, in the preparation method of the present invention, the molar ratio of PAMAM to TLR7 small molecule immune agonist is preferably 1:20 to 64.

[0028] Specifically, the preparation method described in this invention is preferably carried out in an anhydrous DMSO solvent system at room temperature.

[0029] Furthermore, the present invention also provides a delivery system comprising the above-described delivery carrier and a therapeutic nucleic acid molecule loaded thereon, wherein the delivery carrier and the therapeutic nucleic acid molecule are combined through electrostatic interactions to form a nanocomposite.

[0030] Furthermore, the therapeutic nucleic acid molecule is selected from at least one of plasmid DNA, mRNA, siRNA, or miRNA.

[0031] Specifically, the nanocomposite can be obtained by mixing the delivery carrier described in this invention with a negatively charged therapeutic nucleic acid molecule in nuclease-free water and then incubating at room temperature.

[0032] Tests showed that the nanocomposite constructed using the delivery carrier of this invention exhibits uniform spherical nanoparticles with a particle size distribution between 50 and 100 nm, which is beneficial for cell uptake.

[0033] As a preferred embodiment, the therapeutic nucleic acid molecule of the present invention is plasmid DNA encoding an anti-PD-L1 antibody.

[0034] Therefore, based on the above delivery system, the present invention further provides the application of the delivery system in the preparation of a medicament for delivering therapeutic nucleic acid molecules to dendritic cells or immune cells.

[0035] Furthermore, the drug may include, but is not limited to, cancer treatment drugs, infectious disease treatment drugs, or vaccine adjuvants.

[0036] Furthermore, the tumor treatment drug described in this invention is more preferably a drug for treating colon cancer.

[0037] This invention designs a novel molecular coupling strategy, which utilizes the isothiocyanate group of the TLR7 small molecule immune agonist to chemically couple with the primary amine group on the surface of the PAMAM carrier to generate a thiourea bond, thereby developing a novel delivery platform with both targeting and immune activation functions.

[0038] 1) This invention utilizes TLR7 small molecule immune agonist as a specific targeting ligand to achieve active recognition and enrichment of dendritic cells (DCs) by the carrier, thereby improving delivery efficiency; at the same time, TLR7 small molecule immune agonist exerts an immune adjuvant effect while targeting, promoting DC maturation and enhancing subsequent antigen-specific T cell responses.

[0039] 2) The coupling site of the coupling method designed in this invention does not involve the key functional groups necessary for TLR7 receptor binding and does not participate in the TLR7 receptor binding domain, so the immune agonistic activity after coupling is completely preserved.

[0040] 3) PAMAM, as a highly polar polyamino material, forms polycations under physiological conditions, making it difficult for it to enter cells through cellular lipids. The thiourea bonds formed by coupling in this invention have lower polarity, resulting in a lower overall polarity of the delivery carrier compared to PAMAM. This facilitates crossing the lipid bilayer of the cell membrane and improves the entry capacity of the delivery carrier into cells.

[0041] 4) The delivery carrier of this invention uses stable covalent bonds to ensure the structural integrity of the delivery carrier before it reaches the target cell; at the same time, the nucleic acid drug is effectively compressed by the cationic PAMAM backbone to form a stable nanocomplex, which protects the nucleic acid from degradation and promotes endocytosis and intracellular release.

[0042] Therefore, the delivery vector described in this invention can flexibly load different types of therapeutic genes such as antibody genes and cytokine genes, achieving good synergistic anti-tumor effects. Attached Figure Description

[0043] Figure 1 These are transmission electron micrographs of PA102, PA102 / pDNA, and PA102 / pDNA in anhydrous conditions.

[0044] Figure 2 This is the particle size distribution and zeta potential diagram of PA102 / pDNA.

[0045] Figure 3 The effects of PA102 / pDNA on the transfection efficiency and 72h cell viability of BMDCs under different N / P ratios were investigated.

[0046] Figure 4 These are fluorescence micrographs of PA102 / pDNA-eGFP transfected BMDCs cells at different N / P ratios.

[0047] Figure 5 These are the results of immune-inducing activity tests on different test substances.

[0048] Figure 6 The different treatment groups showed the effect of inhibiting tumor growth in CT26 tumor-bearing mice. Implementation

[0049] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It should be noted that the embodiments are merely illustrative and are intended to provide a thorough understanding of the technical solutions of the present invention and to provide guidance for those skilled in the art to implement and apply the present invention. It should be understood that these descriptions do not constitute any limitation on the scope of protection of the present invention.

[0050] Unless otherwise expressly stated, the production processes, experiments, tests or analysis methods involved in the embodiments of the present invention are all considered to be conventional methods known to those skilled in the art, and only need to be implemented in accordance with conventional conditions or relevant product instructions. The steps and names involved are also generally clear and unambiguous in the art.

[0051] The instruments, equipment, raw materials, reagents, or samples used in the embodiments are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels or prepared by known methods, and their source does not have a substantial impact on the implementation results of the present invention.

[0052] Unless otherwise expressly defined, the scientific and technical terms used in this invention have the meanings commonly understood by one of ordinary skill in the art. In case of any conflict, the definitions in this specification shall prevail.

[0053] The terms “comprising,” “including,” “having,” etc., used in this invention should be understood as open-ended, meaning “including but not limited to.” The term “and / or” includes any and all combinations of one or more of the associated listed items. Quantitative terms such as “a,” “one,” etc., do not exclude multiples; “multiple” or “a variety” refers to quantities greater than or equal to two.

[0054] The terms "preferred", "better", and "exemplary" used in this invention are only used to describe specific solutions or effects and are not intended to limit the necessary scope of the solution or the scope of protection.

[0055] This invention relates to the description of numerical parameters (such as quantity, concentration, temperature, time, etc.), and it should be understood that reasonable deviations naturally exist due to measuring instruments, operational errors, statistical fluctuations, etc. The range of such deviations should be within limits acceptable to those skilled in the art based on common sense.

[0056] All animal experimental procedures in this invention embodiment were approved by the Laboratory Animal Ethics Committee. Example

[0057] Example 1: Synthesis of SZU-102

[0058] Compound SZU-142 (145 mg, 0.42 mmol) was dissolved in 1 mL of anhydrous DMF, and carbon disulfide (76 µL, 1.26 mmol) and triethylamine (174 µL, 1.26 mmol) were added sequentially. The mixture was stirred overnight at room temperature. The next day, p-toluenesulfonyl chloride (87 µL, 0.46 mmol) was added, and the mixture was stirred at room temperature for another 5 h.

[0059] After the reaction was complete, the mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (C1000-C1000 HPLC). 18 The target peak was collected by column elution with acetonitrile / water gradient, and lyophilized to obtain 84.4 mg of white solid SZU-102, with a yield of 52%.

[0060]

[0061] ESI-MS (m / z): 387.4 [M+H]⁺.

[0062] Example 2: Synthesis and Characterization of PA102

[0063] Weigh PAMAM-G5 (28.8 mg, 1.0 µmol) and SZU-102 (7.7 mg, 20 µmol), dissolve them in 2 mL of anhydrous DMSO, and stir at room temperature for 12 h.

[0064] The reaction solution was freeze-dried to remove the solvent. The residue was dissolved in 20 mL of pure water and transferred to a 1 kDa ultrafiltration tube. The solution was ultrafiltered three times by centrifugation at 4000 rpm, with fresh pure water added each time. The retentate in the ultrafiltration tube was collected, freeze-dried, and a white, loose solid, PA102, was obtained.

[0065]

[0066] The molecular weight measured by MALDI-TOF mass spectrometry is 33849.60. Among them, the molecular weight of PAMAM-G5 is 28826, and the n=13 of PA102 is calculated.

[0067] The coupling ratio of PA102 was verified using ultraviolet spectroscopy.

[0068] SZU-102 standard solutions with concentrations of 5, 10, 15, 20, 25, and 30 µg / mL were prepared, and the absorbance was measured at a wavelength of 280 nm to obtain the standard curve equation. y =0.0385 x -0.0559 (R) 2 =0.9999).

[0069] A detection solution of 100 µg / mL PA102 was prepared, and the absorbance was measured to be 0.521. Substituting these values ​​into the standard curve equation, the concentration of SZU-102 was calculated to be 14.98 µg / mL, corresponding to a molar concentration of 0.03878 µM / mL. The molar concentration of the PA102 detection solution was 0.002954 µM / mL, and the coupling ratio of PA102 was calculated to be approximately 13.

[0070] Example 3: Preparation and characterization of the PA102 / pDNA complex

[0071] Take the PA102 prepared in Example 2 and prepare a solution with a concentration of 1 mg / mL using nuclease-free water.

[0072] plasmid DNA encoding PD-L1 antibody ( J Immunother Cancer . 2022 Oct; 10(10):e004590. doi: 10.1136 / jitc-2022-004590) as pDNA, prepared as a 0.5µg / µL solution.

[0073] With an N / P molar ratio of 40 for amino groups in PA102 to phosphate groups in pDNA, the corresponding volume of PA102 solution was slowly added to the pDNA solution, vortexed to mix, and incubated at room temperature for 30 min to prepare the PA102 / pDNA nanocomposite.

[0074] The retrieval of pDNA in the complex was detected by 1% agarose gel electrophoresis (TAE buffer, 90V, 45min). The results showed that the pDNA band was completely blocked in the sample well, indicating that PA102 and pDNA formed a stable nanocomplex.

[0075] Transmission electron microscopy (FEI Tecnai G2 F20 S-Twin, 100kV) was used to characterize the physical morphology of the carrier PA102, the PA102 / pDNA nanocomposite, and the PA102 / pDNA nanocomposite in its anhydrous state, as detailed below. Figure 1 As shown, the complex consists of uniform spherical particles.

[0076] The particle size and zeta potential of the PA102 / pDNA nanocomposite were measured using a dynamic light scattering system (DLS, Malvern Zetasizer Nano ZS, USA). Figure 2 The results showed that the PA102 / pDNA nanocomposite had a particle size distribution between 50 and 100 nm and a weakly negative Zeta potential.

[0077] Example 4: Effects of different N / P ratios on transfection efficiency and cytotoxicity of the PA102 / pDNA complex

[0078] In gene transfection, the nitrogen-to-phosphorus ratio (N / P) is a core parameter, which is significant in quantifying the charge interaction between cationic carrier materials and negatively charged nucleic acids (such as plasmid DNA).

[0079] Using the plasmid pDNA-eGFP encoding enhanced green fluorescent protein (eGFP) as a reporter gene, PA102 and pDNA-eGFP were mixed at different N / P ratios (10, 20, 40, 60, 80, 100) to prepare PA102 / pDNA-eGFP complexes, which were then transfected into mouse bone marrow-derived dendritic cells (BMDCs).

[0080] The experiment used PEI / pDNA-eGFP as a positive control, and also set up a blank control group, a naked pDNA group, and an empty vector control group.

[0081] Two hours after transfection, eGFP expression was detected by flow cytometry. Figure 3 The results showed that no eGFP expression was observed in the blank control group, naked pDNA group, and empty vector control group, while the PEI / pDNA-eGFP positive control group exhibited high transfection activity. In the PA102 / pDNA-eGFP complex group, except for N / P=10 and 20, the transfection efficiency was higher than that of PEI, and the transfection efficiency reached its highest at 2h when N / P=100.

[0082] Cell viability was detected by CCK-8 assay 72 h after transfection. Figure 3 The results showed that cell viability remained above 90% when N / P = 10, 20, and 40; however, cell viability began to decline when N / P = 60, indicating that cytotoxicity began to increase when N / P exceeded 40.

[0083] Figure 4 The 72-hour fluorescence microscopy observation results showed the same results: the number of fluorescent positive cells was significantly greater than that of PEI when N / P=40, and the cell density was good; when N / P=100, although the number of cells in the bright field was greater, the density of fluorescent positive cells was significantly reduced.

[0084] The above experimental results show that the introduction of PAMAM with high positive charge density and SZU-102 modification can significantly improve the gene transfection DC ability of plasmid DNA.

[0085] Example 5: Assay of the immune-inducing activity of PA102

[0086] BALB / c mice were randomly divided into 4 groups and administered different test substances via intraperitoneal injection. The in vivo immune activation effect of PA102 vector itself and its safety when combined with TGF-β receptor inhibitors were evaluated to prevent potential cytokine storm risks.

[0087] PAMAM-G5 group (negative control group): PAMAM-G5 8.5 mg / kg was injected to evaluate the autoimmunogenicity of the unmodified PAMAM vector.

[0088] PA102 group (experimental group): PA102 10mg / kg was injected to evaluate the immune-activating activity of PA102.

[0089] PA102+SD-208 group (combination group 1): PA102 10 mg / kg and SD-208 2.5 mg / kg were injected to evaluate the regulatory effect of SD-208 on PA102-induced cytokines.

[0090] PA102+Vactosertib group (combination group 2): PA102 10 mg / kg and Vactosertib 2.5 mg / kg were injected to evaluate the regulatory effect of Vactosertib on PA102-induced cytokines, and were used as parallel validation with SD-208.

[0091] Blood samples were collected from each group 2 hours and 6 hours after drug administration. Serum was separated and the concentrations of IL-6 and TNF-α in peripheral blood were detected by ELISA.

[0092] The results are as follows Figure 5 As shown, no IL-6 and TNF-α concentrations were detected in the serum of the PAMAM-G5 group at 2 h and 6 h after administration, indicating that the unmodified PAMAM carrier itself does not induce significant cytokine release. In the PA102 group, the concentrations of IL-6 and TNF-α were significantly increased at both 2 h and 6 h, indicating that PA102, after in vivo administration, can effectively activate the immune system and induce acute cytokine release, confirming that its TLR7 agonist activity is retained after conjugation.

[0093] Furthermore, the concentrations of IL-6 and TNF-α in the PA102+SD-208 group and the PA102+Vactosertib group at 2h and 6h were significantly lower than those in the PA102 group. Both SD-208 and Vactosertib, two TGF-β receptor inhibitors, significantly reduced the cytokine levels produced by PA102, indicating that they can effectively inhibit PA102-induced cytokine release.

[0094] The above experimental results demonstrate that PA102 has good in vivo immune activation activity and can significantly induce the release of acute cytokines such as IL-6 and TNF-α. TGF-β receptor inhibitors (SD-208 and Vactosertib) can effectively reduce the cytokine levels induced by PA102, suggesting that the strategy of combining with TGF-β inhibitors has good safety regulation potential, providing a safety basis for in vivo anti-tumor experiments of PA102 / pDNA combined with SD-208.

[0095] Example 6: Animal experiment on the treatment of colon cancer with PA102 / pDNA complex

[0096] TGF-β is a key immunosuppressive cytokine in the tumor microenvironment, which can promote tumor immune escape by inducing regulatory T cell differentiation and inhibiting the activity of CD8+ T cells and natural killer cells. SD-208 is a selective TGF-β receptor type I kinase inhibitor that can block the TGF-β / Smad signaling pathway, reverse TGF-β-mediated immunosuppression, and restore anti-tumor immune responses.

[0097] This embodiment evaluated the in vivo antitumor effect of PA102 / pDNA-delivered PD-L1 antibody plasmid and its synergistic effect with the TGF-β receptor antagonist SD-208 in the treatment of CT26 colon cancer in mice by constructing a BALB / c mouse subcutaneous xenograft model.

[0098] Female BALB / c mice aged 6–8 weeks were subcutaneously inoculated with CT26 mouse colon cancer cell line cells (5 × 10⁻⁶ cells) in the right axilla. 5 (each tumor), until the tumor volume reaches approximately 80mm. 3 At that time, tumor-bearing mice were randomly divided into 5 groups and administered the drug via intraperitoneal injection once a week for a total of 3 times.

[0099] PBS group (blank control group): Injected with an equal volume of PBS to assess the natural growth of tumors.

[0100] PA102 group (empty vector control group): 10 mg / kg PA102 was injected to exclude the non-specific effects of the vector's own immunostimulatory effect on tumor growth.

[0101] SD-208 group (TGF-β receptor antagonist monotherapy group): 2.5 mg / kg SD-208 was injected to evaluate the therapeutic effect of blocking the TGF-β signaling pathway alone on CT26 tumors.

[0102] PA102 / pDNA group (treatment group): 15 mg / kg PA102 / pDNA complex was injected to evaluate the in vivo antitumor effect of PA102 in delivering PD-L1 antibody plasmid.

[0103] PA102 / pDNA+SD-208 group (combination therapy group): 15 mg / kg PA102 / pDNA + 2.5 mg / kg SD-208 were injected to evaluate the synergistic effect of the combination therapy.

[0104] Tumor length and short diameters were measured daily during the drug administration period, and the volume was calculated. Mice were sacrificed at the end of the experiment, and tumor tissue was dissected and weighed. Specific results are as follows: Figure 6 show.

[0105] Figure 6 In the tumor growth curves of (a), the tumors in the PBS group showed progressive growth, with a continuous increase during the administration period. The tumor growth curve of the SD-208 group was basically the same as that of the PBS group, with only a slight decrease at the terminal time point, indicating that blocking the TGF-β signaling pathway alone did not have a significant inhibitory effect on CT26 tumor growth. The tumor growth in the PA102 group was reduced to some extent compared with the PBS group, which is presumably related to the non-specific immune activation induced by the TLR7 agonist activity of PA102 itself. The tumor growth in the PA102 / pDNA group was significantly inhibited, and the growth curve was significantly lower than that of the PA102 group, indicating that the PD-L1 antibody plasmid delivered by PA102 was successfully expressed in vivo and generated a specific anti-tumor immune response. The PA102 / pDNA+SD-208 group had the most significant tumor inhibition effect, with the lowest growth curve among all the administration groups, further superior to the PA102 / pDNA group.

[0106] Figure 6 (b) The tumor weight statistics are consistent with its growth curve. As can be seen from the figure, compared with each control group, the PA102 / pDNA group has a significant tumor inhibition effect. The PA102 / pDNA+SD-208 group further improved the tumor inhibition rate, indicating that the combined strategy of immune checkpoint blockade and reversal of the TGF-β immunosuppressive microenvironment has a synergistic effect.

[0107] The above embodiments of the present invention do not exhaustively describe all the details of the present invention, nor do they limit the present invention to the embodiments described above. Therefore, various changes, modifications, substitutions, and variations made by those skilled in the art to the above embodiments without departing from or deviating from the principles and spirit of the present invention should be included within the protection scope of the present invention.

Claims

1. A delivery carrier for targeted immunity, comprising a small molecule TLR7 immunostimulant with Toll-like receptor 7 agonist activity covalently coupled with a polyamide-amine dendritic polymer PAMAM, having the general structural formula PAMAM-(TLR7a). n ; in, TLR7a is a structural unit of a TLR7 small molecule immune agonist, wherein the TLR7 small molecule immune agonist is selected from any one of SZU-102, CZM-1021, CZM-1022, GY106 or SZU-251; n is an integer from 1 to 64; The TLR7a structural unit is connected to PAMAM via a thiourea bond, which is formed by the reaction of the isothiocyanate group on the TLR7 small molecule immune agonist with the primary amine group on the surface of PAMAM.

2. The delivery carrier according to claim 1, characterized in that: The PAMAM is selected from any generation from the first to the tenth generation.

3. The delivery carrier according to claim 1, characterized in that: The PAMAM is PAMAM-G5.

4. The delivery carrier according to claim 1, characterized in that: Composed of the small molecule immune agonist SZU-102 and the polyamide-amine dendritic polymer PAMAM-G5, it has the general structural formula shown in formula (I): ； Where n is an integer from 1 to 64.

5. The delivery carrier according to claim 4, characterized in that: n is 13.

6. The method for preparing the delivery carrier according to any one of claims 1 to 5 is to mix PAMAM with the TLR7 small molecule immune agonist in an organic solvent system, so that the primary amine groups on the surface of PAMAM undergo a coupling reaction with the isothiocyanate groups to form thiourea bonds.

7. The method for preparing the delivery carrier according to claim 6, characterized in that: The molar ratio of PAMAM to the TLR7 small molecule immune agonist is 1:20 to 64.

8. A delivery system comprising a delivery carrier according to any one of claims 1 to 6 and a therapeutic nucleic acid molecule loaded thereon, wherein the delivery carrier and the therapeutic nucleic acid molecule are bonded together by electrostatic interaction to form a nanocomposite.

9. The delivery system according to claim 8, characterized in that: The therapeutic nucleic acid molecule is selected from at least one of plasmid DNA, mRNA, siRNA, or miRNA.

10. The delivery system according to claim 8 or 9, characterized in that: The therapeutic nucleic acid molecule is plasmid DNA encoding an anti-PD-L1 antibody.

11. Use of the delivery system according to any one of claims 8 to 10 in the preparation of a medicament for delivering therapeutic nucleic acid molecules to dendritic cells or immune cells.

12. The application according to claim 11, characterized in that: The drug is a tumor treatment drug, an infectious disease treatment drug, or a vaccine adjuvant.

13. The application according to claim 11 or 12, characterized in that: The aforementioned tumor treatment drug is used to treat colon cancer.

Images