The preparation and characterization of nanoparticles
Firstly, CaCO3 NPs loaded with mRNA (mRNA@CaCO3 NPs) were prepared through a reverse microemulsion method. Transmission electron microscopy (TEM) observed that CaCO3 NPs were spherical in shape with a size of about 60 nm (Fig. 1A). Then, cell membrane (CM) was derived from GL261 cells through repeated freeze–thaw process. To prepare cRGD-labeled CM (cRGD-CM), GL261 cells were pre-treated with N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz) to attach azide group on the cell surface[25]. After that, for the preparation of CM coated CaCO3 NPs (mRNA@CM-CaCO3 NPs), CM and mRNA@CaCO3 NPs were mixed and co-extruded by a 200 nm polycarbonate membrane. Finally, click reaction was used to modify cRGD on the surface of mRNA@CM-CaCO3 NPs, which was produced between the azide groups of cell surface and the alkyne groups of the pre-synthesized endo-bicyclo[6.1.0]nonyne(BCN)-cRGD (endo-BCN-cRGD). The successful production of endo-BCN-cRGD was verified through mass spectroscopy (Additional file 1: Fig. S1). After cRGD attached on the surface of mRNA@CM-CaCO3 NPs, mRNA@cRGD-CM-CaCO3 NPs were finally prepared. As shown in Fig. 1B, obvious core–shell structure (a visible shell layer of ≈ 8 nm) was observed in mRNA@cRGD-CM-CaCO3 NPs, indicating successful CM fusion. The CM coating was further verified through the size and zeta potential changes detected through dynamic light scattering (DLS). An increase of average hydrodynamic diameters from 104 nm (mRNA@CaCO3 NPs) to 157 nm (mRNA@cRGD-CM-CaCO3 NPs) was observed (Fig. 1C). The larger sizes measured through DLS than TEM could attribute to the surface hydration of NPs in DLS detections. The zeta potential was decreased from 14.1 mV to 3.5 mV after CM coated (Fig. 1D). Meanwhile, the CM coating and mRNA encapsulation can be also proved by elemental mapping (Fig. 1E), where the P element, as a representative element of CM and mRNA, was well distributed both inside and outside the Ca element. In addition, the encapsulation efficiency of mRNA in mRNA@cRGD-CM-CaCO3 NPs was approximately 70% at the loading capacity of nearly 2% (mRNA weight/mRNA@cRGD-CM-CaCO3 NPs weight). These results proved that CM was successfully coated in the surface of mRNA@CaCO3 NPs.
To confirm that mRNA@cRGD-CM-CaCO3 NPs could arrive in the tumor site before decomposition, the pH-dependent release experiment of mRNA from nanoparticles was performed. As shown in Fig. 2A, less amount of Cy3-labelled mRNA (Cy3-mRNA) was released from mRNA@cRGD-CM-CaCO3 NPs at neutral conditions, demonstrating that CaCO3 NPs were stable in the systemic circulation. By contrast, at pH 5.5 condition, faster release of mRNA was observed, and nearly 90% of mRNA was released after 72 h. These results demonstrated the pH-activated decomposition of CaCO3 NPs. The quantitative analysis of CO2 gas generation was further evaluated (Fig. 2B), almost no CO2 gas was produced from mRNA@cRGD-CM-CaCO3 NPs at neutral conditions. In contrast, a considerable amount of CO2 was generated at pH 5.5 condition.
Cellular uptake and transfection of mRNA@cRGD-CM-CaCO3 NPs
To test whether cRGD-CM-CaCO3 NPs could effectively deliver mRNA into brain tumor GL261 cells. Cellular uptake and luciferase transfection assay were performed. We first used Cy3-mRNA to detect the cellular uptake efficiency in different cell lines including HepG2, 4T1, CT26 and GL261 cells through flow cytometry (Fig. 2C). When comparison was made among all 4 cell lines, the mean fluorescence intensity of GL261 cells treated with mRNA@CM-CaCO3 NPs or mRNA@cRGD-CM-CaCO3 NPs were significantly stronger, suggesting that GL261 CM coating could assist CaCO3 NPs enter into GL261 cells through the homotypic targeting effect. Furthermore, when comparison was made in each cell line, the mean fluorescence intensity was stronger in cRGD-labeled group (mRNA@cRGD-CM-CaCO3 NPs) compared with CM coated only group (mRNA@CM-CaCO3 NPs), which proved that cRGD played a significant role in facilitating the cellular uptake of nanoparticles. Subsequently, we used mRNA encoding luciferase (Luc mRNA) to evaluate the transfection efficiency of mRNA@cRGD-CM-CaCO3 NPs. According to Fig. 2D, Luc mRNA@cRGD-CM-CaCO3 NPs were able to transfect GL261 cells with a high luminescence intensity. Additionally, the transfection efficiency of Luc mRNA was further improved after US irradiation (2776 Intellect Mobile Ultrasound Device, Chattanooga, USA), which might due to the enhanced gene delivery efficiency through US-mediated acoustic cavitation and sonoporation effect [26].
In Vitro immunogenic necroptosis effect of nanoparticles
Necroptosis is characterized by membrane rupture and cytoplasmic swelling [27]. To verify the cell death mechanism, the annexin V/propidium iodide (PI) assay was performed in GL261 cells [10, 28]. As illustrated by Fig. 3A, when the cells were only treated with US irradiation or cRGD-CM-CaCO3 NPs, no significant changes in the morphology and no fluorescent signals from annexin V/PI were observed, which proved that US irradiation or cRGD-CM-CaCO3 NPs alone did not cause obvious damage to GL261 cells. Whereas, cRGD-CM-CaCO3 NPs plus US-treated cells displayed a loss in their cell morphology, demonstrating that the cell membrane was damaged due to the US-mediated cavitation effect. Moreover, the membrane rupture induced the leakage of membrane fragments, cytosolic components and chromatins [29]. These results validated that the combination of cRGD-CM-CaCO3 NPs and US irradiation could induce necroptosis of GL261 cells. Encouraged by the above data, released DAMPs (including HMGB1 and ATP) were also evaluated. cRGD-CM-CaCO3 NPs + US group significantly improved the extracellular secretion of HMGB1 and ATP compared with other groups. As a result, the DCs maturation frequency of cRGD-CM-CaCO3 NPs + US group was highest and up to 49.6% (Fig. 3B).
Next, cRGD-CM-CaCO3 NPs were loaded with IL-12 mNRA and the in vitro cytotoxicity was performed by methyl thiazolyl tetrazolium (MTT) assay. As expected (Additional file 1: Fig. S2), no significant cytotoxic effect was observed in cRGD-CM-CaCO3 NPs group. In contrast, cRGD-CM-CaCO3 NPs + US group and IL-12 mRNA@cRGD-CM-CaCO3 NPs group exhibited moderate cytotoxic effect, which was attributed to acoustic cavitation or the efficacy of IL-12 mRNA respectively. Notably, IL-12 mRNA@cRGD-CM-CaCO3 NPs + US group showed the strongest cytotoxicity and killed nearly 70% of the cells, implying that the combination treatment of IL-12 mRNA@cRGD-CM-CaCO3 NPs and US irradiation could increase in vitro antitumor effect.
Overall, these results proved that US-mediated necroptosis leaded to the release of DAMPs, which induced the DCs maturation and enhanced antitumor immunity.
In vivo imaging and safety evaluation
To verify the brain tumor-targeted of cRGD-CM-CaCO3 NPs in vivo, an intracranial orthotopic glioblastoma (GL261) mice model was used. Luc mRNA@ CaCO3 NPs, Luc mRNA@CM-CaCO3 NPs or Luc mRNA@cRGD-CM-CaCO3 NPs were intravenously injected at an mRNA dose of 0.25 mg/kg. After 6 h, we measured the bioluminescence signals by a IVIS imaging system (Fig. 4A). Most of the Luc mRNA@ CaCO3 NPs accumulated in the liver, once coated with CM, part of the Luc mRNA@CM-CaCO3 NPs were found in the brain tumor site. More importantly, after cRGD decorated, Luc mRNA@cRGD-CM-CaCO3 NPs displayed nearly 1.6-fold higher bioluminescence signal intensity than CM coated alone group (Luc mRNA@CM-CaCO3 NPs) in the glioma area (Fig. 4B). These results demonstrated that CM coated contribute to brain tumor targeting, and the cRGD modification can further enhance the targeting capability.
Next, we evaluated the toxicity of the nanoparticles in healthy C57BL/6 mice. The measurement of blood biochemistry parameters and HE staining of major organs were performed after treated with PBS, IL-12 mRNA@CaCO3 NPs, IL-12 mRNA@CaCO3 NPs + US, IL-12 mRNA@cRGD-CM-CaCO3 NPs or IL-12 mRNA@cRGD-CM-CaCO3 NPs + US. Blood urea nitrogen (BUN) is commonly used for assessing renal function [30]. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are effective predictors of liver pathology [31]. As shown in Additional file 1: Fig. S3, there was no obvious differences in BUN, AST and ALT levels among all the groups, proving that no significant renal and liver toxicity after nanoparticles treating. Moreover, no apparent histopathological changes were found in the major organs through HE staining (Additional file 1: Fig. S4). For further verifying the safety of US irradiation to the brain, HE staining of brain was performed after treated with PBS or US irradiation in healthy C57BL/6 mice. And no apparent histopathological changes in brain were found after US irradiation (Additional file 1: Fig. S5), which proving that therapeutic US irradiation was safe for the normal brain tissues. All these results demonstrated that CaCO3 NPs plus US irradiation can serve as a safe strategy for tumor therapy.
In vivo anti-glioma activity
Encouraged by the excellent antitumor effects in vitro and brain-targeting ability in vivo of cRGD-CM-CaCO3 NPs, we investigated antitumor efficacy of the nanoparticles in vivo by an orthotopic GL261-Luc glioma mouse model. As illustrated by Fig. 5A, B, IVIS Spectrum showed that rapid tumor growth in the PBS or IL-12 mRNA@CaCO3 NPs treated group. Whereas moderately restricted cancer growth was observed in IL-12 mRNA@CaCO3 NPs + US group and IL-12 mRNA@cRGD-CM-CaCO3 NPs group. Furthermore, the bioluminescence signals of IL-12 mRNA@cRGD-CM-CaCO3 NPs + US group was obviously weaker than any other group, indicating the strongest antitumor effect. In addition, survival study also proved that the combination of IL-12 mRNA@cRGD-CM-CaCO3 NPs and US irradiation can extend mice survival and lead to a 40% durable cure rate (Fig. 5C). The body weight of mice was greatly affected by different therapies, which is analogous to the trend of survival rate (Additional file 1: Fig. S6).
Importantly, IL-12 mRNA@cRGD-CM-CaCO3 NPs treatment plus US significantly increased the expression of IL-12 in brain tumor sections, as well as the IFN-γ production, which is induced by IL-12 directly (Fig. 5D) [32]. Moreover, IL-12 mRNA@cRGD-CM-CaCO3 NPs + US group had the largest proportion of CD8 + T cells in tumors compared with other groups (Fig. 6). Altogether, these results indicated that the anti-glioma immune response by IL-12 mRNA@cRGD-CM-CaCO3 NPs could be amplified through US-mediated necroptosis.