Glucose oxidase and L-arginine functionalized black phosphorus nanosheets for multimodal targeted therapy of glioblastoma
Introduction
In the past decades, traditional strategies including surgical resection, chemotherapy and radiation therapy have been applied for the treatment of glioblastoma (GBM), which is one of the most common primary brain cancers with devastating prognosis [1]. However, innovative treatments are urgently needed because recurrence and metastasis of GBM frequently occur owing to the unsatisfactory effect of traditional mono-modal therapies [2], [3], [4], [5]. Numerous evidences have suggested that combination of different approaches for cancer treatment improves overall survival compared to mono-modal therapy in GBM [6], [7], [8], [9], [10]. However, the combination of routine treatments such as chemotherapy, radiotherapy or immune check point inhibitors often cause drug resistance and systematic toxicity [2], [3], [7]. Therefore, exploring a combination of innovative treatment paradigm is highly desired for GBM management.
Recently, the photothermal, tumor cell starvation and gas therapies have attracted enormous attentions in preclinical research [11]. These novel treatment modalities have been suggested to be promising candidates for treatment of multiple tumors due to their minimal invasiveness toward surrounding normal tissues and less side effect [12], [13], [14], [15], [16]. Photothermal therapy (PTT), which depends on the efficient conversion of near-infrared (NIR) light energy into heat by photo-absorbing agents and irradiation at target sites, could destruct tumor cells without damaging normal tissues [17], [18], [19], [20], [21]. However, employing PTT alone is hard to eliminate cancer cells completely because the heat distribution within tumor tissue is heterogeneous and hyperthermia might kill immune cells that have antitumor activity [12], [22]. Therefore, it is necessary to seek highly efficient antitumor strategies to synergize with PTT. Cancer cell starvation has been demonstrated as a novel “green” therapy that could directly cut off the nutrients supplied to tumor cells resulting in cell death [14]. The starving-like agent glucose oxidase (GOx) exerts more effective therapeutic efficacy than conventional starvation treatment through assisting the conversion of glucose into gluconic acid and H2O2, which subsequently oxidize L-arginine (Arg) into nitric oxide (NO) for gas therapy [23]. NO has been reported to have antitumor activity with various mechanisms including directly inducing cell apoptosis at high concentrations, reversing multidrug resistance by inhibiting P-Glycoprotein expression [24], reprogramming tumor immune microenvironments and more [25], [26]. Therefore, developing an integrated platform to realize the combination of PTT, GOx and NO therapy could exert a synergistic antitumor effect in GBM.
In this study, we utilized black phosphorus nanosheets (BP) as a photothermal agent to explore the combined antitumor activity of PTT, GOx and NO. BP has been demonstrated to have a high surface to volume ratio for facilitating drug-loading [27], [28]. BP exhibits excellent biocompatibility and biodegradability, low biotoxicity and high photothermal conversion efficiency under NIR irradiation [29], [30], [31], [32], [33]. However, BP lacks stability when exposes to air and moisture, that limits its clinical application in PTT and drug delivery [34], [35], [36], [37]. To address this issue, we introduced a functional molecule Arg to the surface of BP (BPA) to serve as a NO donor via esterification reaction between the carboxyl group of Arg and the hydroxyl group (P-OH) derived from the preliminary oxidation of BP, which could also prevent BP from further oxidative degradation. To stimulate the release of NO, GOx was linked to Arg by amidation to construct the multimodal nanodrug (BPAG). BPAG was able to release H2O2 via oxidizing glucose using the catalysis of GOx followed by NO generation from the oxidization of Arg. Moreover, the release of H2O2 and NO could be significantly accelerated by 808 nm NIR irradiation. Furthermore, we coated BPAG with the macrophage membrane to facilitate the penetration through blood–brain barrier (BBB) and improve GBM-targeting ability [38], [39]. The membrane coated BPAG (M@BPAG) was subsequently administered by intravenous injection and actively targeted to GBM tissues exhibiting strong antitumor activity via the combinational effect of PTT, GOx and NO without systematic toxicity. In short, an integrated nanodrug with potential clinical application for GBM therapy is engineered and characterized by BBB permeability, GBM targeting, rapidly cascaded H2O2-NO release, and PTT effect, which exhibits excellent antitumor performance in vivo.
Section snippets
Materials and reagents
Black phosphorus nanosheets (BP) dispersion was purchased from Kunming Black Phosphorus Technology Co., Ltd. (Kunming, China). L-arginine (Arg) was purchased from Yuanye Biological Technology Co., Ltd. (Shanghai, China). Glucose oxidase (GOx) was purchased from Jiuding Chemical Technology Co., Ltd. (Chengdu, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro (EDC), N-Hydroxy succinimide (NHS) and 4-Dimethylaminopyridine (DMAP) were purchased from Energy Chemical Co., Ltd. (Shanghai,
Preparation and characterization of M@BPAG
The Arg functionalized black phosphorus nanosheets (BPA) was prepared by the esterification reaction between the carboxyl (–COOH) group of Arg and the hydroxyl group (P-OH) formed from the preliminary oxidation on the surface of BP [43], [44]. The carboxyl group on GOx was utilized to conjugate BPA under the catalysis of EDC and NHS to form multifunctional BPAG (Fig. 1A). Both the XPS and Raman spectra were performed to demonstrate the reaction on the surface of BP. XPS and Raman results
Conclusion
In summary, we have successfully developed an integrated nanodrug (M@BPAG) that combined PTT, GOx with NO for GBM targeted therapy based on GOx and Arg functionalized BP. The mild chemical modification empowered BP with superior stability and cascaded release of H2O2 and NO in the presence of glucose, which could be accelerated by NIR irradiation. The M@BPAG with NIR irradiation converted cold tumors to hot ones by elevating the infiltration of CD8+ T cells and M1 macrophages while decreasing
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Fundamental Research Funds for the Central Universities (2632020ZD13), the National Natural Science Foundation of China (NSFC 31800154, 51903088), the China Postdoctoral Science Foundation (2021M693953), the Jiangsu Province Postdoctoral Science Foundation (2021K385C), and the Natural Science Foundation of Jiangsu Province (BK20211221).
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These authors contributed equally to this work.