CN115531555B - Application of mannose-modified nanoparticles in preparation of medicines for treating osteosarcoma - Google Patents

Abstract

The invention relates to an application of mannose-modified nanoparticles in preparation of a drug for treating osteosarcoma, wherein the mannose-modified nanoparticles contain regorafenib and alpha-difluoromethylornithine. The mannose modified nano-particles containing regorafenib and alpha-difluoromethylornithine prepared by the invention can promote polarization of macrophages from an M2 phenotype to an M1 phenotype, inhibit angiogenesis, produce synergistic anti-tumor effect and have negligible systemic toxicity. In addition, macrophage repolarization promotes immune cell activation (e.g., increases CD8 ⁺ T cells infiltrate and reduce Treg cells), helping to reprogram the tumor microenvironment of osteosarcoma.

Description

Application of mannose-modified nanoparticles in the preparation of drugs for the treatment of osteosarcoma

Technical field

The present invention relates to the field of biomedicine technology, and in particular to the use of mannose-modified nanoparticles in the preparation of drugs for the treatment of osteosarcoma.

Background technique

Osteosarcoma (OS) is a primary malignant bone tumor originating from mesenchymal tissue and most commonly affects children and adolescents. The success of immunotherapy in cancer treatment relies on beneficial interactions between tumor cells and the host immune response. However, osteosarcoma is considered a relatively "cold tumor" and is less responsive to immune checkpoint inhibitors PD-1 and PD-L1 due to low expression of PD-L1. Low tumor-infiltrating lymphocytes (TILs), including a lack of CD8 ⁺ T cells, and high immunosuppressive cell populations, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and macrophages, may limit the benefit of immunotherapy , resulting in poor prognosis of osteosarcoma.

Tumor-associated macrophages (TAMs), as the main infiltrating immune cells in the osteosarcoma microenvironment, are closely related to tumor angiogenesis, immune suppression, progression and metastasis. According to different polarization states and functions, TAMs are divided into anti-tumor M1 phenotype (termed TAM1) and pro-tumor M2 phenotype (termed TAM2). TAM1 can release cytotoxic chemicals such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) to enhance immune responses and attack tumor cells. TAM2 infiltration tends to promote tumor angiogenesis and immune evasion, ultimately leading to tumor progression and metastasis. Conversely, angiogenesis can also modulate immune responses by suppressing the immune system by polarizing macrophages of the M2 phenotype.

Blocking the VEGF/VEGFR signaling pathway to inhibit neovascularization using multikinase inhibitors and anti-vascular endothelial growth factor monoclonal antibodies is a therapeutic strategy to affect immune cell populations in the TME. Regorafenib is an FDA-approved multikinase inhibitor that inhibits tumor progression by targeting oncogenic kinases involved in regulating cell signaling and tumor angiogenesis. More importantly, regorafenib can convert M2-type TAMs into M1-type, induce T cell activation, and combine with anti-PD-1-sensitized tumors to achieve synergistic immunomodulatory effects. However, due to the blockade of VEGF/VEGFR signaling, regorafenib has the adverse effect of inhibiting physiological angiogenesis. In a phase II clinical trial, regorafenib significantly improved progression-free survival in patients with advanced osteosarcoma compared with placebo, but there was no statistically significant difference in overall survival. Therefore, the shift from single-target to multi-target therapeutic approaches is critical to trigger the host immune system and provide long-term efficacy.

Anti-angiogenic treatments have been shown to have some impact on the tumor microenvironment, but other pharmacological treatments have recently been shown to modulate the immune system. In particular, polyamine blockade therapy (PBT), involving the inhibition of polyamine biosynthesis and transport, significantly inhibited tumor progression in immunocompetent tumor-bearing mice but not in T cell-deficient nude mice. Polyamine levels are significantly elevated in tumors and are critical for tumor survival, which contributes to tumor immunosuppression, angiogenesis, and cell proliferation. Alpha-difluoromethylornithine (DFMO) is an FDA-approved inhibitor of ornithine decarboxylase (ODC) that blocks polyamine biosynthesis. In immune-competent mice, DFMO treatment has been shown to inhibit tumor growth by increasing CD8 ⁺ T cell infiltration and reducing MDSCs activity. Therefore, PBT combined with anti-angiogenic therapy may be a feasible solution to improve the effect of immunotherapy, reduce drug side effects, and reshape the microenvironment of osteosarcoma.

Based on the abundant expression of mannose receptors on TAMs and osteosarcoma cells, there is an urgent need for mannose-modified nanoparticles to be used in the preparation of drugs for the treatment of osteosarcoma.

Contents of the invention

The purpose of the present invention is to provide an application of mannose-modified nanoparticles in the preparation of drugs for the treatment of osteosarcoma in view of the deficiencies in the prior art.

In order to achieve the above objects, the technical solutions adopted by the present invention are:

A first aspect of the present invention is to provide an application of mannose-modified nanoparticles in the preparation of drugs for treating osteosarcoma. The mannose-modified nanoparticles contain regorafenib and α-difluoromethylornithine.

The second aspect of the present invention is to provide a method for preparing mannose-modified nanoparticles, the mannose-modified nanoparticles containing regorafenib and α-difluoromethylornithine, and the steps include:

S1. Add the aqueous solution of α-difluoromethylornithine to the dichloromethane solution of mannose-modified PLGA-PEG, and slowly add the dimethyl sulfoxide solution of regorafenib under stirring. In the mixed solution, ultrasonicate on ice to obtain a w/o emulsion;

S2. Add the w/o emulsion obtained in step S1 to the first polyvinyl alcohol aqueous solution, and ultrasonically homogenize in an ice bath to obtain a w/o/w emulsion;

S3. Add the w/o/w emulsion obtained in step S2 to the second polyvinyl alcohol aqueous solution, slowly stir at room temperature overnight, wash with deionized water several times, and freeze-dry to obtain the mannose. Modified nanoparticles.

Preferably, the concentration of the regorafenib solution in dimethyl sulfoxide is 10 mg/mL.

Preferably, the preparation steps of the mannose-modified PLGA-PEG include:

S1-1. Dissolve PLGA-PEG nanoparticles in 2-(N-morpholine)ethanesulfonic acid buffer, and add excess N-hydroxysuccinimide and 1-(3-dimethylaminopropyl) )-3-ethylcarbodiimide, after stirring for a period of time, add 4-phenyl isothiocyanate-A-D-mannoside to the mixed solution, and stir at room temperature overnight;

S1-2. Use cold ether and methanol to precipitate mannose-modified PLGA-PEG, remove unreacted 4-phenyl isothiocyanate-A-D-mannoside and excess reactants, and then vacuum dry it.

Preferably, the concentration of the mannose-modified PLGA-PEG solution in dichloromethane is 100 mg/mL.

Preferably, the concentration of the aqueous solution of α-difluoromethylornithine is 50 mg/mL.

Preferably, the concentration of the first polyvinyl alcohol aqueous solution is 1.0%, w/v.

Preferably, the concentration of the second polyvinyl alcohol aqueous solution is 0.3%, w/v.

The third aspect of the present invention is to provide a mannose-modified nanoparticle prepared by the aforementioned preparation method.

The present invention adopts the above technical solution and has the following technical effects compared with the existing technology:

The mannose-modified nanoparticles containing regorafenib and α-difluoromethylornithine prepared by the present invention can promote the polarization of macrophages from M2 phenotype to M1 phenotype, while inhibiting the formation of blood vessels and producing synergy. Anti-tumor effect with negligible systemic toxicity. In addition, macrophage repolarization promotes immune cell activation (such as increased CD8 ⁺ T cell infiltration and decreased Treg cells), which helps to reprogram the tumor microenvironment of osteosarcoma.

Description of drawings

Figure 1 shows the results of characterization of the prepared mannan-modified PLGA-PEG nanoparticles using transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques;

Figure 2 shows the results of confocal laser scanning microscopy;

Figure 3 shows the results of experimental research on inhibiting K7 cell activity;

Figure 4A shows the results of CD206 protein expression on the surface of M2 macrophages;

Figure 4B is a graph showing the results of IL-10 secretion;

Figure 5 shows the results of in vitro angiogenesis inhibition after treatment in each group;

Figure 6A is a graph showing the results of tumor volume measured every three days during the drug administration period;

Figure 6B shows the weight results of the peeled tumors in each group;

Figure 6C is a graph showing the results of tumor inhibition rate (TGI%) in each group;

Figure 6D shows the survival time results of tumor-bearing mice;

Figure 6E shows the body weight results of nude mice measured every three days during the drug administration period;

Figure 7 shows the flow cytometry results.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without any creative work fall within the scope of protection of the present invention.

It should be noted that, as long as there is no conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but shall not be used as a limitation of the present invention.

Material

Regorafenib, α-difluoromethylornithine hydrochloride (DFMO) and coumarin-6 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). PEG _5K -PLGA _10K - _NH2 was obtained from Shandong Academy of Pharmaceutical Sciences, China. Polyvinyl alcohol (PVA, 87%-89% hydrolyzed, molecular weight [Mw] 31000-50000), lipopolysaccharide (LPS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide ( EDC) and IR 780 iodide were from Sigma-Aldrich (St. Louis, MO, USA). 4-Phenyl isothiocyanate-AD-mannoside was purchased from Shanghai Bid Pharmaceutical Co., Ltd., China. Sulfo- n -hydroxysuccinimide (NHS) was purchased from Aladdin Holding Group Co., Ltd., Beijing, China. Mouse recombinant mouse macrophage colony-stimulating factor (M-CSF) and recombinant mouse interleukin-4 (IL-4) were provided by PeproTech (Rocky Hill, NJ, USA).

Mannose receptor (CD206), CD31 and β-actin primary antibodies were purchased from Abcam (Cambridge, UK). Bcl-2, Bcl-xl, Cleaved-caspase-3, c-Myc, p-STAT3, NF-κB, VEGFR2, p-VEGFR2, p-MAPK, p-ERK, p-mTOR, Ki-67 and GAPDH Primary antibodies were purchased from Cell Signaling Technology (Boston, USA). Fixable reactive stain 620 and APC-Cy7 anti-mouse CD45 were purchased from R&D Company (USA). FITC anti-mouse/human CD11b, PerCP-Cy5.5 anti-mouse F4/80, Pacific Blue ^TM anti-mouse CD86, PE/Cy7 anti-mouse CD206, FITC anti-mouse CD45, APC-Fire ^TM 750 anti-mouse CD3, PE-Cy7 Anti-mouse CD4, PerCP-Cy5.5 anti-mouse CD8α, PE anti-mouse CD25, Alexa Fluor 647 anti-mouse Foxp3 and AF700 anti-mouse granzyme B were purchased from BioLegend (San Diego, CA, USA). Mouse VEGFA, IL-10, IFN-γ and TNF-α ELISA kits were from Lianke Biotechnology Co., Ltd. (Hangzhou, China). Other chemicals were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

Example 1

This embodiment provides a method for preparing mannose-modified nanoparticles. The mannose-modified nanoparticles contain regorafenib and α-difluoromethylornithine. The steps include:

S0. Dissolve 100 mg of PLGA-PEG nanoparticles in 10 mL of 2-(N-morpholine)ethanesulfonic acid (MES) buffer (pH5.0), and add 10 mg of N-hydroxysuccinimide (NHS) and 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). After stirring for 2 h, 100 mg of 4-phenyl isothiocyanate-A-D-mannoside was added to The mixed solution was stirred at room temperature overnight. After 12 hours, use cold ether and methanol to precipitate the mannose-modified PLGA-PEG, remove unreacted 4-phenyl isothiocyanate-A-D-mannoside and excess reactants, and dry in a vacuum to obtain mannose-modified PLGA-PEG. PLGA-PEG;

S1. Add the aqueous solution (100 μL) of α-difluoromethylornithine (5 mg) to the dichloromethane solution (3 mL) of mannose-modified PLGA-PEG (300 mg), and add regorafenib (1 mg). The dimethyl sulfoxide solution (100 μL) was slowly added dropwise to the mixed solution under stirring, and ultrasonicated on ice for 30 s to obtain a w/o emulsion;

S2. Add the w/o emulsion obtained in step S1 to the first 1.0% (w/v) polyvinyl alcohol aqueous solution (15 mL), and ultrasonically homogenize in an ice bath for 30 minutes to obtain a w/o/w emulsion;

S3. Add the w/o/w emulsion obtained in step S2 to the 0.3% (w/v) second polyvinyl alcohol aqueous solution (50 mL), slowly stir at room temperature (RT) overnight, and add deionized water After washing three times, it is freeze-dried to obtain the mannose-modified nanoparticles.

The chemical structure of the prepared mannose-modified nanoparticles was detected by hydrogen nuclear magnetic resonance spectroscopy ( ^1H -NMR) in CDCl ₃ ; the mannose-modified nanoparticles were measured by dynamic light scattering (DLS) technology (Malvern, UK). Particle size, zeta potential, and polydispersity index (PDI); the morphology of mannose-modified nanoparticles was observed by transmission electron microscopy (TEM) (H-7650; Hitachi, Japan). High-performance liquid chromatography was used to determine the encapsulation efficiency and drug loading capacity of mannose-modified nanoparticles, and calculated according to the following formula:

Encapsulation rate (%) = (M _{encapsulated drug} )/(M _{added drug} ) × 100%;

Drug loading capacity (%) = (M _{encapsulated drug} )/(M _{nanoparticles} ) × 100%.

method

The methods involved in the following embodiments include:

The preparation method and detection method of loading coumarin-6 or IR780 nanoparticles are similar to Example 1.

Stability and in vitro drug release of nanoparticles: Nanoparticles were dispersed in PBS containing 10% FBS to evaluate stability. The size changes of the nanoparticles were examined at each time point. In vitro cumulative drug release from mannose-modified nanoparticles was measured using dialysis method. Mannose-modified nanoparticles were put into dialysis bags (MWCO, 8 kDa-14 kDa), suspended in PBS containing 0.05% sodium dodecyl sulfate at different pH (7.4 and 6.5), and incubated at 37 °C at 100 rpm. Shake gently. At designated time points, take 1 mL of release medium, measure the concentration of regorafenib by HPLC, and supplement with an equal amount of fresh medium.

Cell culture: Mouse osteosarcoma cell line K7, mouse macrophage cell line RAW264.7, and human umbilical vein endothelial cells (HUVEC) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). K7 cells and RAW264.7 were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Human umbilical vein endothelial cells were cultured in ECM containing 5% FBS, 1% ECG, and 1% penicillin/streptomycin. All cell lines were maintained in an incubator containing 5% _CO2 and 95% air at 37 °C.

Collection and polarization of bone marrow-derived macrophages (BMDM): Bone marrow cells were collected from the femur and tibia of C57BL/6 mice. Cells were cultured in RPMI 1640 supplemented with 10% FBS and 20 ng/mL M-CSF for 5 days to promote differentiation into bone marrow-derived macrophages (BMDM). After differentiation, BMDM were differentiated into M1 phenotype by 100ng/mL LPS stimulation, or differentiated into M2 phenotype by 20ng/mL IL-4 stimulation for 24 hours. Macrophage phenotypic markers were further detected using qRT-PCR, western blot and ELISA.

In vitro cellular uptake studies: K7 cells and polarized macrophages were cultured for 24 hours in 6-well plates and 35 mm confocal dishes at 2 × ¹⁰ cells per well or 1 × ¹⁰ cells per dish. Coumarin-6 loaded nanoparticles and mannose-modified nanoparticles were added to the cells and cultured for 4 hours respectively. Coumarin-6 was quantified by fluorescence spectrophotometer (Hitachi, Japan) at an excitation wavelength of 420 nm and an emission wavelength of 506 nm. In parallel groups, cells were pretreated with mannose (100 mM) for 4 hours and then incubated with mannose-modified nanoparticles. Subsequently, the cellular uptake efficiency was determined by flow cytometry (BD Biosciences, USA). In addition, cells in the confocal culture dishes were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with DAPI, and observed using a confocal laser scanning microscope (CLSM; TCS-SP8, Leica, Germany).

In vitro cytotoxicity study: K7 cells were seeded in a 96-well plate at a density of 4,000 cells per well and cultured for 24 hours. Cells were treated with free regorafenib, DFMO, DFMO combined with regorafenib (DR), nanoparticles or mannose-modified nanoparticles. After 48 hours of culture, cell viability was evaluated using the CCK-8 kit (Dojindo Molecular Technology Co., Ltd., Japan) according to the instructions.

Preparation of Transwell co-culture model: A Transwell system (Corning, USA) with a 0.4 μm pore membrane was used to construct a co-culture model in order to realistically simulate the physiological conditions of the tumor microenvironment in which all cells are exposed to drugs. K7 cells were seeded in the lower layer, and TAM1 or TAM2 were seeded in the upper layer of the Transwell system, and cultured together for 12 hours. Cells were then treated with free regorafenib (2 μM), DFMO (1 mM), DR, nanoparticles, or mannose-modified nanoparticles for 24 h. K7 cells were collected for further experiments. In contrast, if TAM1 or TAM2 are used for examination, they are seeded in the lower layer to facilitate the collection process, while K7 cells are seeded in the upper layer. Similarly, TAM1 or TAM2 was seeded into the upper layer of the Transwell system, and human umbilical vein endothelial cells (2 × 10 ⁴ ) were seeded into the lower layer using Matrigel matrix pretreatment. Cells were then treated with the above drugs and cultured at 37°C for 12 hours. Capillary networks were imaged using a fluorescence microscope (Leica, Germany).

Flow cytometry analysis: In order to quantitatively analyze the apoptosis rate of K7 cells after drug treatment with or without co-culture, according to the instructions, cells were collected after 72 hours of co-culture, washed, and analyzed by Annexin V-FITC/ PI apoptosis detection kit (Thermo Fisher, Waltham, USA) was used for further staining. All samples were analyzed by flow cytometry. Meanwhile, to evaluate the in vivo polarization ability of mannose-modified nanoparticles, flow cytometry was used to quantitatively analyze the proportions of M1/M2 phenotype macrophages and CD4 ⁺ and CD8 ⁺ T cells in tumors and spleens. Fresh tumor and spleen tissue sections were digested with type IV collagenase, hyaluronidase and DNase, and then red blood cells were separated using red blood cell lysis buffer. Cells are extracted with lymphocyte separation fluid for T cell sorting and incubated with specific biomarkers of immune cells such as CD86, CD206, Foxp3 and granzyme B. Filter the cell suspension and detect using FACS-Calibur.

Western blotting: After treatment, cells and tumor tissues were collected and lysed in RIPA buffer with protease and phosphatase inhibitor cocktail (Sigma-Aldrich, USA) for 30 min on ice. After centrifugation at 12000×g for 25 min at 4°C, the supernatant was collected and determined by BCA analysis (Beyotime, China). Protein samples were separated by SDA-PAGE and transferred to PVDF membrane (Millipore, USA). The membranes were incubated with TBST containing 5% nonfat dry milk for 1 h at room temperature and with primary antibodies overnight at 4°C. The protein bands are then incubated with HRP-conjugated secondary antibodies according to standard protocols. Finally, expose using ECL developer and use GAPDH or β-action as a control.

qRT-PCR and ELISA: Total RNA was extracted from macrophages using TRIzol reagent (Thermo, USA) and reverse transcribed into cDNA using TaKaRa PrimeScript RT kit (TaKaRa, Japan) according to the instructions. Real-time quantitative PCR was then performed using SYBR Premix ^DimerTM (TaKaRa, Japan) according to the instructions. β-action was used as a control to normalize mRNA levels. Use ELISA kits according to the instructions to detect cytokine levels (VEGFA, IL-10, IFN-γ and TNF-α) in culture fluid or tumor tissue.

In vivo imaging and biodistribution study: K7 cells (1×10 ⁶ cells) were subcutaneously injected into the back of BALB/c mice (female, 5-6 weeks old) to establish an osteosarcoma xenograft model. When tumors grew to ⁵⁰⁰ mm, mice were randomly divided into two groups. Each mouse was injected into the tail vein with an equal amount of IR780-loaded nanoparticles or mannose-modified nanoparticles. The biodistribution of nanoparticles in mice was examined at different time points using an IVIS imaging system (Xenogen). After 24 hours, the mice were euthanized, and tumors and major organs were surgically collected for in vitro imaging. For colocalization studies, tumor tissues from osteosarcoma models were also collected for cryosectioning. Sections were incubated with anti-CD206 antibody overnight at 4°C, then immunofluorescently stained with Alexa-Fluor488-conjugated goat secondary antibody for 1 hour at room temperature, and finally stained with DAPI. Observe colocalization using CLSM.

In vivo anti-tumor efficacy and safety evaluation: A subcutaneous osteosarcoma model was established in BALB/c mice. ^When the tumor size reached approximately 100 mm, the tumor-bearing mice were randomly divided into six groups (6 mice per group). Each group was intravenously injected with PBS (control), regorafenib, DFMO, DR, nanoparticles, and mannose-modified nanoparticles every other day at a dose of 5 mg/kg regorafenib and 20 mg/kg DFMO for approximately two weeks. . The tumor volume and body weight of each mouse were measured separately to evaluate the anti-tumor efficacy and safety in vivo. Tumor volume was calculated using the following formula: V (mm ³ ) = (length × width ² ) ÷ 2. When the tumor volume reached ²⁰⁰⁰ cm, the mice were euthanized. Tumor tissue and major organs were collected for further analysis. The tumor growth inhibition rate (TGI%) was calculated according to the following formula: TGI (%) = (1-W _treat / W _control ) × 100% (W _treat and W _control represent the tumor weight of the treatment group and control group, respectively). All experiments and procedures were ethical and approved by the Animal Ethics Committee of Shanghai First People's Hospital.

Statistical analysis: All data were analyzed using Graphpad Prism 8 and IBM SPSS 18. Data are expressed as mean ± standard deviation, and each experiment was repeated three times. To compare differences between the two groups, a corrected t test was used. When more than two groups were involved, one-way analysis of variance (ANOVA) was used. Log-rank (Mantel-Cox) test was used to compare the results of animal survival. Statistically significant differences are described as *P<0.05, **P<0.01 and ***P<0.001.

Example 2: Characterization and characterization of mannose-modified nanoparticles

The mannose receptor, also known as CD206, is one of the most commonly used receptors targeted by TAMs due to its high expression in TAM2. Notably, they are also expressed in K7 cells. This result provides a physiological basis for the rational design of mannose-modified nanoparticles targeting TAMs and osteosarcoma cells. In the current study, through specific recognition of mannose receptors, mannose was combined with PLGA-PEG as a targeting ligand for TAM2 and osteosarcoma cells. Mannose-modified nanoparticles containing regorafenib and DFMO were successfully synthesized via a double-emulsion solvent evaporation method. ¹ H-NMR proton spectrum was used to identify the mannose-coupled PLGA-PEG copolymer. The average particle size of mannose-modified nanoparticles is 146 nm (Figure 1), while that of unmodified PLGA-PEG nanoparticles (called nanoparticles) is 125 nm. Due to the partial neutralization of negative charges after mannose modification, the zeta potential of the nanoparticles changed from -23.3mV to -10.6mV. Mannose-modified nanoparticles showed good stability in 10% FBS, making them suitable for their biomedical applications. The PDI of mannose-modified nanoparticles and nanoparticles were 0.15 and 0.16, respectively, indicating a narrow molecular weight distribution. The encapsulation efficiencies of mannose-modified nanoparticles for regorafenib and DFMO were 74.6% and 77.68%, respectively, and the drug loading efficiencies were 0.6% and 2.9%, respectively. Mannose-modified nanoparticles exhibited sustained release mode. To examine whether mannose-modified nanoparticles are preferentially taken up by K7 cells and TAM2, BMDM or Raw264.7 macrophages were first polarized into TAM1 or TAM2 and then compared with coumarin-6-loaded mannose-modified nanoparticles. Incubate with nanoparticles for 4 hours. For flow cytometry analysis, the average fluorescence intensity of mannose-modified nanoparticles taken up by K7 cells and TAM2 was stronger than that of unmodified nanoparticles. The cellular uptake efficiency of mannose-modified nanoparticles was significantly reduced when cells were pretreated with free mannose. Furthermore, due to the low expression of CD206, the uptake of mannose-modified nanoparticles and unmodified nanoparticles by TAM1 was almost the same. The results for confocal laser scanning microscopy (CLSM) were consistent with those of flow cytometry (Figure 2), further confirming the enhanced mannose receptor-mediated cellular uptake of mannose-modified nanoparticles. Collectively, these data suggest that the mannose receptor plays a critical role as a target in mediating drug delivery.

Example 3: In vitro cytotoxicity

Increased uptake of mannose-modified nanoparticles by K7 cells should improve therapeutic efficacy. To verify this, the anticancer activity of mannose-modified nanoparticles was evaluated using CCK-8 assay. Treatment with mannose-modified nanoparticles significantly inhibited the proliferation of K7 cells compared with free regorafenib, DR, or unmodified nanoparticles (Figure 3), with the lowest IC50. However, approximately 90% of TAM1 and TAM2 were unaffected by mannose-modified nanoparticles and nanoparticle treatment and were well tolerated. Studies have shown that regorafenib inhibits the growth of osteosarcoma cells by inducing apoptosis, which is mediated through the inactivation of the AKT and ERK signaling pathways. DFMO prevents the progression of esophageal cancer by inhibiting the MAPK/ERK and AKT/mTOR/p70S6k signaling pathways. Mannose-modified nanoparticles can significantly inhibit the expression of p-AKT, p-MAPK, p-ERK and p-mTOR, which are signaling pathways for cell survival and growth, and therefore exhibit powerful anti-tumor efficacy. In addition, the anti-apoptotic proteins Bcl-xl and Bcl-2 were significantly down-regulated and the expression of cleaved caspase3 increased after treatment with mannose-modified nanoparticles. In addition, the apoptosis of K7 cells by mannose-modified nanoparticles was further evaluated. Mannose-modified nanoparticles produced higher apoptosis rates in K7 cells compared with other treatments. Vascular endothelial growth factor A (VEGFA) is one of the important cytokines in TME. VEGFA is a member of the VEGF family and is mainly secreted by cancer cells and TAMs to promote tumor neovascularization and vascular permeability. Compared with unmodified nanoparticles, VEGFA secretion was significantly reduced after treatment with mannose-modified nanoparticles. In order to truly simulate the physiological conditions of all cells exposed to drugs in the tumor microenvironment, the Transwell co-culture model was used to study the effects of TAMs on tumor cells. In the Transwell co-culture system of K7 cells and TAM2, mannose-modified nanoparticles also increased the expression of apoptosis-related proteins and caused a higher apoptosis rate and less VEGFA secretion compared with other groups.

Example 4: In vitro polarization and repolarization of macrophages

TAMs have multiple functions, including participating in tumor growth, metastasis, angiogenesis, and immunosuppression, and are highly dynamic and plastic, with reversible polarization between M1 and M2 phenotypes. A large amount of evidence shows that TAM2 is significantly associated with poor prognosis and lung metastasis in patients with osteosarcoma. Therefore, reprogramming TAMs is a potential strategy to eliminate immunosuppression in osteosarcoma treatment. BMDMs were induced into M1 and M2 phenotype macrophages. Macrophage phenotype was analyzed by western blot of CD206 (M2 marker). The expression of CD206 was significantly reduced after treatment with mannose-modified nanoparticles, which means that mannose-modified nanoparticles are able to reverse TAM2 polarization. In the Transwell co-culture system of TAMs and K7 cells, mannose-modified nanoparticles reversed the polarization of TAM2 by downregulating CD206 (Figure 4A). To further validate this result, RT-qPCR was used to detect the mRNA expression of macrophage biomarkers including CD86, TNF-α, CD206, and TGF-β. Compared with untreated cells, mannose-modified nanoparticles significantly increased the expression of CD86 and TNF-α (M1 marker) in TAM1 and TAM2, while reducing the expression of CD206 and TGF-β (M2 marker). IL-10 produced by TAMs can inhibit anti-tumor effects by inhibiting the function of APC, thus hindering the capacity of cytotoxic T cell effectors. Treatment with mannose-modified nanoparticles reduced the level of IL-10 in tumor cells (Figure 4B). IL-10 is a stimulator of STAT3. Phosphorylation of STAT3 is a key step in TAM2 polarization, and inhibition of the STAT3 signaling pathway can cause macrophage phenotype to change from M2 to M1. The results showed that mannose-modified nanoparticles inhibited the phosphorylation of STAT3 in TAM1 and TAM2, which is consistent with previous studies on the effect of DFMO on macrophage polarization through the STAT3 pathway. Pro-inflammatory NF-κB, which plays a key role in immune and inflammatory responses, was enhanced by mannose-modified nanoparticles in the TAMs/K7 Transwell co-culture system due to STAT3 inhibition. In summary, mannose-modified nanoparticles significantly promote the repolarization of TAM2 and reshape the tumor immune microenvironment.

Example 5: In vitro anti-angiogenic effects of mannose-modified nanoparticles

Tumor angiogenesis provides necessary nutrients and oxygen to rapidly expanding malignant tumors. Multiple studies have shown that TAMs, especially M2 macrophages (TAM2), are critical for the formation of abnormal blood vessels in tumors due to their increased VEGF release. During tumor development, VEGF interacts with the VEGF receptor (VEGFR) on endothelial cells to promote endothelial cell proliferation and migration, as well as angiogenesis near the tumor site. Therefore, a Matrigel tubule formation assay was used to detect anti-angiogenesis in a transwell co-culture system of HUVECs and TAMs. VEGFA secretion is mainly produced by TAM2. TAM2 was co-cultured with HUVEC. VEGFA secretion was significantly reduced after drug treatment. In capillary-like networks, TAM1 inhibits tubule formation, whereas TAM2 promotes tubule formation. This promotion was blocked by mannose-modified nanoparticles due to the repolarization of TAM2 toward TAM1 (Fig. 5). Furthermore, mannose-modified nanoparticles showed anti-angiogenic effects by downregulating the expression of VEGFR2 and p-VEGFR2 in HUVECs. These results indicate that mannose-modified nanoparticles can inhibit the VEGFA/VEGFR2 signaling pathway and regulate macrophage polarization, thereby inhibiting tumor angiogenesis.

Example 6: In vivo tissue biodistribution

To evaluate the in vivo tumor targeting ability of mannose-modified nanoparticles, the tissue biodistribution of IR780-loaded nanoparticles was studied in a K7 osteosarcoma tumor-bearing model. Both nanoparticles and mannose-modified nanoparticles successfully distributed to the tumor site, but mannose-modified nanoparticles exhibited higher fluorescence intensity in tumors compared with unmodified nanoparticles at all time points evaluated. In vitro fluorescence images of excised tumors further revealed higher tumor accumulation of mannose-modified nanoparticles. Additionally, CD206 and mannose-modified nanoparticles were colocalized by immunofluorescence staining to assess intratumoral penetration. Immunofluorescence imaging showed that the distribution of mannose-modified nanoparticles within the tumor was basically consistent with the expression of CD206. The above results show that based on the mannose receptor delivery system, the targeting function of mannose-modified nanoparticles acts on cancer cells and TAMs.

Example 7: In vivo anti-tumor efficacy and safety evaluation

A mouse K7 osteosarcoma subcutaneous tumor model was used to evaluate the inhibitory effect of mannose-modified nanoparticles on tumor growth in vivo. As expected, the tumor growth inhibition rates in the regorafenib and DFMO groups were 39.0% and 22.7%, respectively, and the tumor growth inhibition rate in the DR group was 56.8%. The mannose-modified nanoparticle group had the strongest anti-tumor effect, with a tumor inhibition rate of 69.8%, while the unmodified nanoparticle group was 59.9% (Figures 6A-6C). Furthermore, among tumor-bearing mice, the mannose-modified nanoparticle group had the longest survival time (Figure 6D). In addition, Ki67 staining analysis of tumor sections also confirmed that the cell proliferation ability was the lowest after treatment with mannose-modified nanoparticles, which was consistent with tumor growth inhibition. This illustrates the value of targeted delivery of mannose-modified nanoparticles to improve anticancer effects. Furthermore, the preliminary biosafety of mannose-modified nanoparticles was evaluated. During the treatment period, the weight of tumor-bearing mice in each group did not decrease significantly (Figure 6E), and there were no major pathological changes in the main organs (heart, liver, spleen, lungs, and kidneys). These results indicate that mannose-modified nanoparticles are nontoxic to these organs and exhibit significant biosafety.

Example 8: Remodeling TIME and inhibiting angiogenesis in vivo

The in vivo repolarization of TAMs was evaluated by measuring the ratio of M1/M2 phenotypes, cytokine secretion, and expression profiles of biomarker proteins to explore the immunotherapeutic effect of mannose-modified nanoparticles. Flow cytometry was used to detect the proportion of TAMs phenotypes in tumor tissues. In the PBS group, intratumoral TAMs were mainly composed of M2 type (F4/80 ⁺ CD206 ⁺ ), while M1 type macrophages (F4/80 ⁺ CD86 ⁺ ) were rarely expressed, which may contribute to rapid tumor growth. Regorafenib and DFMO, alone or in combination, also increased the proportion of M1 phenotype macrophages and decreased the proportion of M2 phenotype macrophages, while the mannose-modified nanoparticle treatment group showed more obvious changes (Figure 7A and Figure 7B). By analyzing the M1/M2 ratio related to the polarization effect, the in vivo polarization ability of mannose-modified nanoparticles can be more accurately estimated. The number of total TAMs remained relatively constant among the groups, but the M1(CD86 ⁺ )/M2(CD206 ⁺ ) ratio was significantly higher in the mannose-modified nanoparticles treatment group than in the other treatment groups, suggesting that due to Targeted drug delivery, mannose-modified nanoparticles effectively reprogram macrophages from M2 to M1 phenotype in vivo. It is known that TAM2 can exert immunosuppressive functions by inhibiting the activity of tumor-infiltrating lymphocytes. CD4 ⁺ helper T cells and CD8 ⁺ cytotoxic T cells were further quantitatively analyzed to assess anti-tumor immune response activation after various treatments. As shown in Figure 7C and Figure 7D, mannose-modified nanoparticle treatment significantly increased the number of cytotoxic CD8 ⁺ granzyme B ⁺ T lymphocytes in tumors to 29.4% compared to 6.89% in the PBS group, while intratumoral The proportion of infiltrating CD4 ⁺ Foxp3 ⁺ T cells (Tregs) is significantly reduced, which is associated with immune evasion and poor prognosis in tumor patients. Furthermore, in the spleen, an increase in CD8 ⁺ T lymphocytes and a decrease in CD4 ⁺ T cells were detected after treatment with mannose-modified nanoparticles. Both TAM1 and CD8 ⁺ T cells produce immunogenic cytokines, such as IFN-γ and TNF-α, to enhance the immune response against tumors. Therefore, ELISA was used to detect intratumoral cytokine levels. Due to the remodeling of TAMs and the activation of CD8 ⁺ T cells in tumor tissues, the levels of anti-tumor IFN-γ and TNF-α were significantly increased after treatment with mannose-modified nanoparticles. In addition to analyzing the ratio of M1/M2 macrophages and cytokine secretion, macrophage surface proteins (CD206 for M2 markers, CD86 for M1 markers) and angiogenesis marker proteins ( VEGFR2, HIF-1α and CD31) expression profiles to further confirm the polarization ability and anti-angiogenic ability of mannose-modified nanoparticles. After treatment with mannose-modified nanoparticles, CD206 expression in tumors was down-regulated, while CD86 expression was up-regulated, which was consistent with the results of the M1/M2 macrophage ratio. Importantly, expression of the angiogenic proteins HIF-1α and VEGFR2 was downregulated, resulting in reduced tumor vessel density (CD31 staining). These data indicate that mannose-modified nanoparticles can reprogram the TME by enhancing polarization of TAMs, immune cell infiltration, and inhibiting angiogenesis, thereby improving anti-tumor activity in vivo.

In summary, the mannose-modified nanoparticles containing regorafenib and α-difluoromethylornithine prepared by the present invention can promote the polarization of macrophages from M2 phenotype to M1 phenotype, while inhibiting vascular generation, producing synergistic anti-tumor effects with negligible systemic toxicity. In addition, macrophage repolarization promotes immune cell activation (such as increased CD8 ⁺ T cell infiltration and decreased Treg cells), which helps to reprogram the tumor microenvironment of osteosarcoma.

The above are only preferred embodiments of the present invention, and do not limit the implementation and protection scope of the present invention. Those skilled in the art should be able to realize that any equivalents made by using the description and illustrations of the present invention Solutions resulting from substitutions and obvious changes should be included in the protection scope of the present invention.

Claims (6)

1. A method for preparing mannose-modified nanoparticles, the mannose-modified nanoparticles containing regorafenib and α-difluoromethylornithine, characterized in that the steps include: S1. Add the aqueous solution of α-difluoromethylornithine to the dichloromethane solution of mannose-modified PLGA-PEG, and slowly add the dimethyl sulfoxide solution of regorafenib under stirring. In the mixed solution, ultrasonicate on ice to obtain a w/o emulsion; S2. Add the w/o emulsion obtained in step S1 to the first polyvinyl alcohol aqueous solution, and ultrasonically homogenize in an ice bath to obtain a w/o/w emulsion; S3. Add the w/o/w emulsion obtained in step S2 to the second polyvinyl alcohol aqueous solution, slowly stir at room temperature overnight, wash with deionized water several times, and freeze-dry to obtain the mannose. modified nanoparticles; The preparation steps of the mannose-modified PLGA-PEG include: S1-1. Dissolve PLGA-PEG nanoparticles in 2-(N-morpholine)ethanesulfonic acid buffer, and add excess N-hydroxysuccinimide and 1-(3-dimethylaminopropyl) )-3-ethylcarbodiimide, after stirring for a period of time, add 4-phenyl isothiocyanate-A-D-mannoside to the mixed solution, and stir at room temperature overnight; S1-2. Use cold ether and methanol to precipitate mannose-modified PLGA-PEG, remove unreacted 4-phenyl isothiocyanate-A-D-mannoside and excess reactants, and then vacuum dry to obtain; The concentration of the first polyvinyl alcohol aqueous solution is 1.0%, w/v; The concentration of the second polyvinyl alcohol aqueous solution is 0.3%, w/v. 2. The preparation method according to claim 1, characterized in that the concentration of the dimethyl sulfoxide solution of regorafenib is 10 mg/mL. 3. The preparation method according to claim 1, characterized in that the concentration of the dichloromethane solution of the mannose-modified PLGA-PEG is 100 mg/mL. 4. The preparation method according to claim 1, characterized in that the concentration of the aqueous solution of α-difluoromethylornithine is 50 mg/mL. 5. A mannose-modified nanoparticle prepared by the preparation method according to any one of claims 1 to 4. 6. Application of the mannose-modified nanoparticles as claimed in claim 5 in the preparation of drugs for the treatment of osteosarcoma.