Glioblastoma (GBM) is the most aggressive brain tumor, which owns the characteristics of high recurrence, low survival rate and poor prognosis because of the existence of blood brain barrier (BBB) and complicated brain tumor microenvironment. Currently, immunotherapy has attracted much attention on account of favorable therapeutic effect. In this study, we designed a cRGD-modified cancer cell membrane (CM) coated calcium carbonate nanoparticle to deliver interleukin-12 messenger RNA (IL-12 mRNA@cRGD-CM-CaCO3 NPs). The cRGD-modified CM as the shell can endow the nanoparticles with BBB crossing and tumor homing/homotypic targeting effect in the brain tumor microenvironment. IL-12 mRNA-loaded calcium carbonate nanoparticles as the core allow synergistic immunotherapy of necroptosis-induced immune response and IL-12 mRNA transfection under ultrasound irradiation. The as-prepared biomimetic nanoparticles showed superior target and immunotherapeutic outcomes, suggesting that this biomimetic nanoplatform provides a feasible strategy for promoting BBB-penetrating and antitumor immunity.
Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, with a median survival less than 2 years . The standard GBM therapy include maximal surgical resection later on with chemical therapy and radiation therapy. Unfortunately, GBM always grow into normal brain tissue, so it is almost impossible to remove the entire tumor . More important, the presence of blood–brain barrier (BBB) prevent majority of drugs into the brain . Based on these, most of the GBM patients will eventually relapse though suffering repeated surgical resection and chemo/radiotherapy . Therefore, novel and efficient strategies are urgently needed to improve the treatment outcome of GBM.
Nowadays, immunotherapy has achieved promising clinical outcomes in a diversity of solid tumors, such as advanced melanoma and non-small cell lung cancer . It is believed that successful immunotherapy requires a self-sustaining “cancer-immunity cycle”, that is, immunogenic cell death (ICD) and activated dendritic cells (DCs) initially induce T-cell responses in draining lymph nodes. Then these T cells and other immune cells migrate to the tumor site, where promoting continued tumor cell killing and remodeling the tumor microenvironment [6, 7]. Hence, a variety of interlinked events are necessary for initiating this cycle, such as ICD induction, DCs activation, immune cells recruitment, proinflammatory factors generation and so on. Among them, the key is to induce ICD. For ICD induction, the most common method is utilizing certain chemical drugs to cause immunogenic apoptosis, which release tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) to activate tumor-specific immune response [8, 9]. However, the immunogenicity of released TAAs and DAMPs would significantly decrease during the apoptosis process, due to this process is often accompanied by intracellular oxidation and proteolysis, ultimately leading to limited antitumor immunity [10, 11]. As a consequence, it is urgent to develop efficient ICD inducers that could avoid the degradation of released TAAs and DAMPs.
Necroptosis, characterized by plasma membrane disintegration, is induced through specific stimulus such as mechanical stress and temperature variation . In contrast to apoptosis, necroptosis process does not cause intracellular oxidation and proteolysis, so the released TAAs and DAMPs could keep their biological activity . As a result, necroptosis could active immune response more effectively than immunogenic apoptosis. Previous studies have reported that calcium carbonate nanoparticles (CaCO3 NPs) could generate CO2 bubbles in tumoral lysosome acidic condition, and induce cavitation-mediated necroptosis under ultrasound (US) irradiation [14, 15]. Besides, CaCO3 NPs themselves are suitable vehicles for the delivery of small molecule drugs, genes and proteins [16, 17]. Considering messenger RNA (mRNA) represents a new type of therapeutics, and interleukin-12 (IL-12) is a promising candidate for cancer immunotherapy through activating T cell functions but lack of intravenous delivery approaches [18, 19]. Hence, we hypothesized that using CaCO3 NPs encapsulating mRNA encoded IL-12 and combined with US irradiation, could be a promising synergetic strategy to enhance antitumor immunity.
How to deliver CaCO3 NPs across the BBB and into the tumor cells is another challenge for effective GBM immunotherapy. Cyclic Arg-Gly-Asp (cRGD), as a well characterized peptide that could bind to αvβ3 integrin overexpressed in GBM neovasculature, has been widely used for the BBB penetrating . Nevertheless, complicated brain tumor microenvironment (containing not only brain tumor cells, but also fibroblasts, astrocytes and microglias) still hinder the brain tumor cells accumulation of cRGD-modified nanoparticles [21, 22]. Recently, cell membrane (CM) coating nanotechnology has garnered much attention for constructing biomimetic nanoparticles, which could assist GBM cell target through tumor homing and homotypic targeting capacities because of the complete replication of surface antigens from the GBM CM [21, 23, 24]. Thus, it is envisioned that combined of cRGD modification and CM coating in CaCO3 NPs might further improve the GBM cell targeted ability after crossing the BBB, thereby ensuring the necroptosis induction by in situ CO2 bubbles production under US and IL-12 mRNA translation in brain tumor cells.
Herein, we designed a CM coated CaCO3 NPs, which CaCO3 NPs loaded with IL-12 mRNA as the core and cRGD-labeled GBM CM as the shell (named as IL-12 mRNA@cRGD-CM-CaCO3 NPs, Scheme 1A). After intravenous injection, IL-12 mRNA@cRGD-CM-CaCO3 NPs directly entered GBM cell through BBB crossing and tumor homing/homotypic targeting capabilities (Scheme 1B). Firstly, CaCO3 NPs decomposed and generated CO2 gas in lysosome environment. Subsequently, CO2 bubbles collapsed and induced necroptosis of GBM cells by cavitation effect under US irradiation. Then, necroptosis released TAAs and DAMPs can be taken up and processed by DCs. Once activation, mature DCs would present antigens to T cells and trigger subsequent antitumor immunity. Meanwhile, loaded IL-12 mRNA was translated to IL-12 in the cytoplasm, which could stimulate the proliferation and activation of cytotoxic T lymphocytes (CTLs), as well as the production of cytokines. Based on the in vitro and in vivo results, we revealed that IL-12 mRNA@cRGD-CM-CaCO3 NPs were able to traverse through the BBB and target GBM cells. Furthermore, strong synergistic immunotherapy was achieved through the combination of acoustic cavitation-mediated necroptosis and IL-12 mRNA transfection.
Results and discussion
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. 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 .
In Vitro immunogenic necroptosis effect of nanoparticles
Necroptosis is characterized by membrane rupture and cytoplasmic swelling . 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 . 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 . Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are effective predictors of liver pathology . 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) . 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.
In summary, we have developed a novel biomimetic nanoparticle, including a shell of cRGD-modified cell membrane and a core of CaCO3 NPs loaded with IL-12 mRNA (IL-12 mRNA@cRGD-CM-CaCO3 NPs). Such a design of shell conferred BBB crossing and tumor homing/homotypic targeting abilities to nanoparticles, thus further promoting the brain tumor targeting. When exposed to US, the CaCO3 NPs core could induce necroptosis by CO2 bubbles-mediated cavitation effect, resulting in DAMPs released and DCs maturation. Combined with IL-12 mRNA, a superior antitumor activity against GBM was induced both in vitro and in vivo, which was attributed to the enhanced tumor targeting and DCs maturation, as well as the CTLs stimulation. Taken together, our strategy provides a platform for ultrasound-immune synergistic therapy of brain tumors.
Availability of data and materials
All data generated or analyzed during this study are included in this manuscript.
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