Neuroblastoma (NB) is one of the most common extracranial solid tumors in children and infants. NB occurs in approximately 25-50 cases per million and accounts for 10%-20% of pediatric malignancy deaths . High-risk NB (HRNB) is diagnosed when MYCN amplification is greater than stage 1 or age ≥ 18 months with stage 4 disease. Approximately 50% of NB cases are classified as HRNB . The MYCN gene is a critical regulator of cell growth and proliferation. Amplification of MYCN is a common genetic alteration in NB that is associated with poor prognosis, advanced disease stage, aggressive tumor behavior, and poor patient outcomes .
Treatment of NB involves three phases: induction; consolidation; and maintenance therapy. Treatment options include chemotherapy, surgery, high-dose chemotherapy with autologous stem cell rescue, radiotherapy, and immunotherapy . Low- and intermediate-risk patients have a 5-year survival rate of 85%-90% ; however, high-risk patients have a significantly lower 5-year survival rate, averaging 50% . Most HRNB patients relapse and die of drug-resistant disease despite initial chemotherapy . Therefore, new treatment options are urgently needed for HRNB patients.
Various immunotherapies have shown promise for treating NB, but T-cell infiltration in the tumor microenvironment (TME) remains a challenge for effective treatment . Although immune checkpoint blocking (ICB) antibodies targeting PD-L1 have shown efficacy for low- and intermediate-risk NB, the effectiveness is limited for high-risk cases due to the lack of T-cell infiltration in the TME . Monoclonal antibody (mAb) therapy targeting disialoganglioside (GD2), which is overexpressed on NB cells, has been shown to improve the prognosis of NB patients ; however, despite this improvement, the 5-year survival rate for HRNB remains < 50% . New immunotherapy approaches are being researched to address these challenges.
Stimulator of interferon genes (STING) agonists have captured wide attention with respect to immunotherapy of NB. Notably, the STING agonist response is not dependent on T cells within the TME . Microbial-released DNA or DNA shed by dying tumor cells, or STING agonists are detected by intracellular DNA sensors, such as cyclic GMP-AMP synthase (cGAS), leading to activation of the STING signaling pathway in dendritic cells . This results in phenotypic maturation. After activation, downstream signaling pathways, such as IRF3 and NF-κB, are activated, resulting in the production of type I interferons and other cytokines, such as CXCL10, which activate CD8+T cells through a cascade of events . STING agonists stimulate antigen presentation by activating dendritic cells and promote T cell activation as well . Previous studies have shown that STING agonists can be used in NB immunotherapy. Lihong Wang-Bishop et al.  reported that STING-NPs incorporating PEG-DBP co-polymer inhibit the growth of mouse MB, extend survival, and induce immune memory to prevent tumor recurrence . As a result, STING agonists exhibit great promise for the treatment of NB.
The main challenge in the use of STING agonists involves the in vivo delivery. For example, CDN is a negatively-charged hydrophilic small molecule that limits entry of STING agonists into the cytoplasm. CDN is easily degraded and inactivated by ectonucleotide pyrophosphatase/phosphodiesterase, which adversely affects its efficacy. To overcome these problems, researchers have designed various CDN delivery systems based on biomaterials, such as cationic silica nanoparticles for local drug delivery  and poly (β-amino ester) polymer nanoparticles . The clinical application of polymer nanoparticles, however, is limited based on the complex design and synthesis process, side effects induced by the added organic solvent, and insufficient biocompatibility .
By way of comparison, liposomes can effectively entrap water-soluble molecules, improve drug stability, and reduce drug toxicity. Moreover, the industrial production technology for liposomes is much more mature, and various liposomal drug delivery systems have been approved for clinical use. For example, liposome-encapsulated doxorubicin hydrochloride (Doxil) was approved for marketing by the US Food and Drug Administration (FDA) for the first time in 1995, the safety of which has been extensively verified .
We have developed a liposome delivery system for an active, natural, STING agonist (2′,3′-cGAMP) to improve cellular delivery efficiency. Specifically, we prepared liposomes loaded with 2′,3′-cGAMP by four methods: thin-film dispersion; ammonium sulfate gradient; calcium acetate gradient; and lipid calcium phosphate nanoparticle. Among these four methods, lipid calcium phosphate nanoparticles (LCP-NPs) achieved the highest encapsulation efficiency. Hence, more detailed physicochemical characterization was performed and in vitro experiments were conducted using LCP-NPs. LCP-NPs can maintain a stable particle size at 4°C for 48 h and the surface is positively charged. Compared with free cGAMP, LCP-NPs resulted in a slower release, enhanced cytotoxicity against tumor cells, greater activation of the cGAS-STING pathway, and increased expression of immune factors, such as CXCL9, TNF-α, and IFN-β. This study provides a perspective on the delivery of STING agonists.
2. MATERIALS AND METHODS
The following materials were purchased for the current study: 2′,3′-cGAMP (MedChemExpress, Shanghai, China); Igepal (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China); 1,2-dioleoyl-3-trimethylammoniumpropane, monochloride ([DOTAP]; Shanghai Kanglang Biotechnology Co., Ltd., Shanghai, China); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] ([DSPE-PEG2000]; A.V.T. Pharmaceutical Co., Ltd., Shanghai, China); Na2HPO4, (NH4)2SO4, CaCL2, egg phosphatidylcholine (EPC), cholesterol, and the Cell Counting Kit-8 [CCK8] (Solarbio Technology Co., Ltd., Beijing, China); TRIeasy™ Total RNA Extraction Reagent, Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR, and Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology Co., Ltd., Shanghai, China); fetal bovine serum ([FBS]; PAN-Biotech GmbH, Adenbach, Germany); and Quanti-Blue™ Solution (InvivoGen, San Diego, CA, USA).
2.2 Preparation of cGAMP liposomes using four different methods
2.2.1 Thin-film dispersion method
The recipe ingredients were accurately weighed and added to an eggplant-shaped bottle, followed by the addition of an appropriate amount of chloroform to ensure full dissolution. The solvent was then removed via rotary evaporation under reduced pressure at a 40°C water bath to produce a homogeneous film. Next, 330 μL of the aqueous cGAMP solution was added and the mixture was vigorously vortexed. The drug was loaded in a water bath at 70°C for 1 h. The mixture was ultrasonicated for 4 min until a light blue opalescence appeared. The obtained nanoparticles were abbreviated as TFD-NPs.
2.2.2 Ammonium sulfate gradient method
The recipe materials were accurately weighed and placed into an eggplant-shaped bottle. Chloroform was added to dissolve the materials, and the solvent was evaporated under reduced pressure in a 40°C water bath to form a uniform film. The film was hydrated with 1 mL of aqueous ammonium sulfate solution (120 mmol/L), followed by vortexing and ultrasound treatment until a light blue opalescence appeared. After cooling to room temperature, free ammonium sulfate was removed via a Sephadex G-50 gel column to obtain blank liposomes. The liposomes were then mixed with 330 μL of a cGAMP aqueous solution and loaded in a water bath at 70°C for 1 h. After the solution was cooled to room temperature, the free cGAMP was removed via a Sephadex G-50 gel column to obtain cGAMP-liposomes. The obtained nanoparticles were referred to as ASG-NPs.
2.2.3 Calcium acetate gradient method
Each recipe ingredient was accurately weighed and added to an eggplant-shaped bottle. Chloroform was added to achieve full dissolution, and the resulting solution was evaporated under reduced pressure in a 40°C water bath to form a uniform film. Next, a 130 mmol/L calcium acetate aqueous solution (adjusted to pH = 6 with glacial acetic acid) was added, followed by vortexing and hydration at 70°C for 15 min. The mixture was ultrasonicated for 4 min until a light blue opalescence appeared. Free calcium acetate was removed via a Sephadex G-50 gel column to obtain blank liposomes, which were then mixed with 330 μL of a cGAMP aqueous solution. The drug was loaded in a water bath at 70°C for 1 h. After the solution was cooled to room temperature, the free cGAMP was removed via a Sephadex G-50 gel column to obtain cGAMP-liposomes. The nanoparticles are abbreviated as CAG-NPs.
2.2.4 Lipid calcium phosphate nanoparticle method
Na2HPO4 (300 μL) was meticulously added to a mixture with 15 mL of cyclohexane/Igepal (71:29), followed by the addition of 200 μL of DOPA. The resulting mixture was sonicated to obtain the Na2HPO4 phase microemulsion. Similarly, 300 μL of CaCl2 containing cGAMP was added to 15 mL of cyclohexane/Igepal (71:29) mixture and sonicated. After the ultrasound, the CaCl2 phase microemulsion was obtained. The two-phase microemulsion was thoroughly mixed for 20 min. To break the demulsification, 30 mL of absolute ethanol was added. The mixture was then centrifuged at 16,000 g for 20 min and washed 2-3 times with ethanol. The resulting CaP pellets were dissolved in 1 mL of chloroform for later use. The recipe ingredients were meticulously weighed and mixed with the CaP-core, then the chloroform was evaporated at 40°C. Tris-HCl buffer was added, followed by ultrasonication for 4 min to obtain cGAMP-liposomes, which were named LCP-NPs [15–17]. The formulations for all four methods are presented in Table 1 .
2.3 Particle size and zeta potential analysis
The prepared liposomes were subjected to dynamic light scattering (DLS) analysis. Particle size and polydispersity were determined using an NKT-N9 nanoparticle size analyzer (NKT, Shandong Province, China). The wavelength of the NKT-N9 laser beam was set at 633 nm. The refractive index was 1.33. The polydispersity indicated the uniformity of particle size, and the smaller the polydispersity, the more uniform were the particles. Zeta potential was determined using a Malvern Zetasizer Nano ZS instrument (Worcestershire, England). The wavelength of the instrument laser beam was set at 633 nm. The angle between the incident and scattered beams was 90°. Each sample was measured for 15 cycle times, and the measurement temperature was set at 25°C.
2.4 Determination of liposome encapsulation efficiency
The content of the drug was determined by HPLC (Shimadzu, Kyoto, Japan). The liposomes were acurrately aspirated, then destroyed by adding 1% Triton X-100 solution, and depolymerized by vortexing to release the encapsulated cGAMP. The content of cGAMP was determined using reverse-phase HPLC. The following chromatographic conditions were used in the analysis: Agilent Zorbax SB-C18 column (4.6 × 250 mm, 5 μm; Little Falls Wilmington, DE, USA) with a detection wavelength of 260 nm; mobile phase, phase A (50 mM TEAA, pH=7.2) and phase B (Acetonitrile); and column temperature, 30°C. The gradient elution process was as follows [18, 19]:
2.5 Particle size stability of liposomes
The prepared liposomes were placed at 4°C for observation and photography within 48 h after preparation. The stability of the liposomes was investigated by measuring the particle size and polydispersity coefficient of the liposomes.
2.6 Microscopy characterization and in vitro release of LCP-NPs
The surface morphology of LCP-NPs was characterized by transmission electron microscopy ([TEM], JEM 2100; JEOL, Kyoto, Japan) after negative staining with 1% uranyl acetate solution at an accelerating voltage of 80 kV.
Free cGAMP or LCP-NP were added to activated dialysis bags (MWCO3500; BioDee, Beijing, China) for the in vitro release experiment. These bags were then placed into a conical flask containing the appropriate amount of release medium and shaken at a constant temperature of 37°C and 100 rpm/min using an oscillator. At specific time points (0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 24 h), 150 μL of the release solution was collected. Fresh release medium (150 μL) was added to the conical flask after each sampling and the mixture was continued to to be shaken. After 24 h, the dialysis clips clamped at both ends of the dialysis bag were loosened, and a sample was obtained after stirring for an additional 30 min. The cGAMP content was determined using reverse-phase HPLC and the cumulative drug release rate was calculated.
2.7 CCK8 assay of LCP-NPs
NB neuro-2a cells were seeded at a density of 1×105 cells/mL in 96-well plates (100 μL per well). To maintain a humid environment, 200 μL of PBS was added to the surrounding wells. Neuro-2a cells were placed in an incubator and cultured overnight. The culture medium was aspirated, and 200 μL of drug-containing serum-free medium was added to each well. The plate was then incubated for 8 h with different concentrations of the drug (0.5, 1, 5, 10, 50, and 100 μM). After removing the medium, 100 μL of the serum-free medium was added to each well, followed by 10 μL of CCK8 solution. The plates were incubated for 1 h in the incubator, and the absorbance at 450 nm was measured using a Spectra MAX 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability and the IC50 were calculated based on the results.
2.8 Evaluation of cGAS-STING pathway activation in reporter cells
RAW Blue ISG cells were seeded in 96-well plates. A series of drug concentration gradients were set to 0.1, 0.5, 1, 3, 10, and 30 μM. After adherence, the cells were incubated with LCP-NPs and free cGAMP for 2 h. After the incubation, the drug was discarded and replaced with a serum-containing medium for 22 h to generate secreted embryonic alkaline phosphatase (SEAP). To assess SEAP activity, 50 μL of cell supernatant from each well was transferred into a new 96-well plate. Then, 150 μL of Quanti-Blue™ Solution was added to each well, followed by incubation at 37°C in an incubator. After color development, the absorbance of the supernatant at 620 nm was measured using a microplate reader .
2.9 Gene expression analysis
RT-qPCR was used to determine the expression of the immune factors in DC2.4 cells treated with different nanoparticle preparations . DC2.4 cells were homogeneously seeded in 6-well plates and incubated overnight. The LCP-NPs and free cGAMP were diluted to 1 μg/mL in serum-free medium and the cells were treated for 6 h. Cellular mRNA was extracted according to the TRIeasy™ Total RNA Extraction Reagent instructions. The RNA concentration was determined using NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed to synthesize cDNA using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR according to the manufacturer’s instructions. Real-Time Quantitative PCR (RT-qPCR) was performed using an ABI 7500 Real-Time PCR (Applied Biosystems, Inc., Foster City, CA, USA). GAPDH was used as an internal reference. The relative expression changes were calculated using the ΔΔCt method [21, 22]. The primer sequences were as follows:
3. RESULTS AND DISCUSSION
3.1 Characterization of physicochemical properties of four liposomes
The high hydrophilicity of cGAMP presents challenges when formulating cGAMP into liposomes, such as suboptimal drug encapsulation efficiency and variable drug release kinetics . In addition, the size and distribution of liposomes can be influenced by the preparation method. To address these challenges, we evaluated four different methods for formulating cGAMP-loaded liposomes, with the goal of identifying the optimal method for achieving high-drug encapsulation efficiency. The methods tested included the thin-film dispersion, ammonium sulfate gradient, calcium acetate gradient, and lipid calcium phosphate nanoparticle methods. The corresponding obtained nanoparticles were abbreviated as TFD-NPs, ASG-NPs, CAG-NPs, and LCP-NPs, respectively. As shown in Figure 1 , the liposomes prepared by these four methods were translucent and slightly blue-opalescent.
The particle size and zeta potential analysis are shown in Table 2 and Figure 2 . The particle sizes of TFD-NPs and LCP-NPs were between 80 and 100 nm. The particle sizes of ASG-NPs and CAG-NPs were between 100 and 120 nm with a dispersibility < 0.200 and good preparation reproducibility. The zeta potential of ASG-NPs and CAG-NPs was negative, which might oppose cellular uptake due to the electrostatic repulsion to the cell membrane.
|Nanoparticles||Size (nm)||PDI||Zeta (mV)||Encapsulation efficiency (%)|
|TFD-NPs||85.54 ± 3.86||0.139 ± 0.02||58.1 ± 17.3||13.63 ± 1.02|
|ASG-NPs||119.07 ± 5.34||0.060 ± 0.01||-19.7 ± 6.8||5.84 ± 0.78|
|CAG-NPs||102.35 ± 4.62||0.071 ± 0.01||-24.7 ± 11.6||9.15 ± 0.92|
|LCP-NPs||82.57 ± 3.72||0.157 ± 0.03||15.9 ± 4.3||21.24 ± 1.28|
Liposome encapsulation efficiencies are listed in Table 2 . Water-soluble small molecules, such as 2′,3′-cGAMP (solubility of 50 mg/mL), generally exhibit a lower encapsulation efficiency because water-soluble small molecules leak out of the liposomes due to high solubility in water . LCP-NPs had the highest encapsulation among the four methods. As shown in Figure 3 , a pH-sensitive calcium phosphate (CaP) core was prepared using the lipid calcium phosphate nanoparticle method. In this study stable CaP cores were prepared using a water/oil microemulsion. The CaP cores were coated with an inner layer of DOPA and an outer layer of cationic lipids, DOTAP, cholesterol, and DSPE-PEG, thus forming a hollow spherical structure with a lipid bilayer. The hollow structure of the CaP core, which was formed in water droplets provides an opportunity to capture water-soluble drugs, at least in part. The lipid coating prevented the CaP core from aggregating during the centrifuge separation step and made it soluble in chloroform. After adding the outer lipid layer, the chloroform was evaporated, and finally the lipid bilayer was self-assembled in an aqueous solution.
It has been reported that the LCP-NP method has been used for the encapsulation of nucleic acid drugs in vivo. cGAMP is a small molecule of the cyclodinucleic acid class. This experiment demonstrated the feasibility of LCP-NP for the encapsulation of cGAMP.
Next, we investigated the particle size stability of liposomes at different time points after formulation. The results are shown in Figure 4 . Under storage conditions of 4°C for 48 h, the appearance of the solution remained unchanged, appearing as a clear, milky blue solution and the particle size of the liposomes remained stable. Predictably, liposomes remain stable during the subsequent cell experiments in which drug treatments were carried out within 48 h.
3.2 Microscopic characterization and in vitro release of LCP-NPs
LCP-NPs had good physicochemical properties and the encapsulation efficiency of LCP-NPs was the highest among the four liposomes. LCP-NPs were selected for detailed morphologic and in vitro release studies. The TEM photograph in Figure 5a shows that LCP-NPs are spherical and the particle size results are similar to those determined by DLS. The in vitro release curve is shown in Figure 5b . The free cGAMP solution released approximately 70% within 0.5 h, whereas LCP-NPs released approximately 50%. In contrast, LCP-NPs had a sustained-release effect. The cumulative release of LCP-NPs was 80% within 24 h.
3.3 Cytotoxicity of LCP-NPs
Given the previous literature reports, it has been documented that STING agonists exhibit immunomodulatory effects at low concentrations, while at high concentrations STING agonists induce tumor cell death . Our objective was to comprehensively investigate the immunomodulatory properties of LCP-NPs and the potential for inhibiting cell growth. Therefore, we examined the formulation inhibitory effects on neuro-2a cell growth at various concentrations. Our experimental findings showed a dose-dependent inhibitory effect of the free cGAMP and LCP-NPs on neuro-2a cell growth, which was consistent with tumor cell growth inhibition. The IC50 of the LCP-NPs was 2.16 μM and the IC50 of the free cGAMP solution was 16.98 μM. Calcium phosphate and DOTAP have been generally considered to be safe [26, 27]. We therefore speculated that the increased neuro-2a cell cytotoxicity of LCP-NPs compared to free cGAMP was due to increased cellular uptake of cGAMP, and likely resulted from the electrostatic interactions onthe surface of LCP-NPs . According to Wang-Bishop , STING agonists directly activate caspase-3 in neuro-2a cells and induce tumor cell death. Additionally, Sokolowska  highlighted the complexity of the STING pathway in cancer cells; specifically, the response of tumor cells to STING activation might exhibit inhibitory or growth-promoting effects. There is currently no consensus on whether STING activation in cancer cells leads to cell death, necessitating further study to determine the impact of STING activation on cancer cell viability.
3.4 cGAS-STING pathway activation by LCP-NPs
To explore the effect of nanoparticle formulations in stimulating type I interferon production, RAW-Blue ISG cells were used to investigate the impact of LCP-NPs on the cGAS-STING pathway because macrophages have a crucial role in cGAMP-mediated antitumor activity in the TME and secrete reporter SEAP upon stimulation of the cGAS-STING pathway by cGAMP. SEAP reacts with Quanti-Blue™ Solution to indicate the level of cGAS-STING pathway activation through a colorimetric assay, where a higher absorbance corresponds to an increased production of SEAP and a stronger activation of the cGAS-STING pathway. Figure 7 illustrates the dose-dependent effect of LCP-NPs on cGAS-STING pathway activation in RAW-Blue ISG. LCP-NPs effectively activate the cGAS-STING pathway in RAW-Blue ISG cells in a dose-dependent manner. Increasing concentrations of LCP-NPs led to a stronger activation of the cGAS-STING pathway. The inability of free cGAMP to activate the cGAS-STING pathway may be due to its limited cellular uptake. LCP-NPs, possessing a positively charged surface, readily penetrate into cells and trigger cGAS-STING pathway activation, as shown in Scheme 1 .
3.5 Immune factor expression
It has previously been reported that cGAMP activates the cGAS-STING pathway of APCs, resulting in increased expression of downstream immune factors. In this study we measured the levels of immune factor mRNA expression to further demonstrate the activation of the cGAS-STING pathway by LCP-NPs. After incubating DC2.4 cells with LCP-NPs or free drugs, the expression of immune factors was evaluated using RT-qPCR. The results are shown in Figure 8 . Compared to free cGAMP, LCP-NPs were shown to significantly upregulate the expression of several immune factors, such as CXCL9, TNF-α, and IFN-β. Specifically, LCP-NPs resulted in an 8-fold increase in CXCL9 expression, a 77-fold increase in TNF-α expression, and a 5-fold increase in IFN-β expression. CXCL9 is the key to recruiting effector T cells into tumors. TNF-α is a cytokine that induces rapid destruction of tumor blood vessels after STING agonist is delivered to tumors . IFN-β is essential for the generation of anti-tumor T cell responses. As Scheme 1 depicts, these findings suggest that LCP-NPs effectively deliver cGAMP into the cytoplasm of cells. After LCP-NPs enter cells, the STING agonist released into the cytoplasm enhances the activation of the cGAS-STING pathway in APCs, causing APCs to produce type I interferon and other cytokines, and finally activate CD8+T cells.
These results provide evidence for the potential use of LCP-NPs as an immunotherapeutic agent for the treatment of cancers. Corollary studies are warranted to assess the effectiveness and safety of LCP-NPs in vivo, as well as to determine the underlying mechanisms involved in the cGAS-STING pathway.
Liposomes are widely studied nanodrug carriers that are biocompatible and biodegradable. Liposomes effectively protect the encapsulated drugs from degradation and slowly release the drugs. In the current study, cGAMP-loaded liposomes were prepared using four different methods. Lipid calcium phosphate nanoparticles with the highest encapsulation efficiency was selected to perform detailed physicochemical characterization and cellular experiments. The obtained LCP-NPs exhibited ideal particle size and good stability. In vitro studies demonstrated that LCP-NPs increased cytotoxicity against neuro-2a cells, enhanced activation of the cGAS-STING pathway, and upregulated the expression of immune factors. These findings indicated the potential of LCP-NPs for the delivery of cGAMP. This system could provide a new prospect for NB immunotherapy. The in vivo effect is also worthy of further exploration.