Exfoliated Black Phosphorus Promotes in Vitro Bone Regeneration

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Functional Nanostructured Materials (including low-D carbon)

Exfoliated black phosphorus promotes in vitro bone regeneration and suppresses osteosarcoma progression through cancer-related inflammation inhibition Maria Grazia Raucci, Ines Fasolino, Maria Caporali, Manuel SerranoRuiz, Alessandra Soriente, Maurizio Peruzzini, and Luigi Ambrosio ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21592 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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ACS Applied Materials & Interfaces

Exfoliated Black Phosphorus Promotes In Vitro Bone Regeneration and Suppresses Osteosarcoma Progression Through Cancer-Related Inflammation Inhibition

Maria Grazia Raucci,1*# Ines Fasolino,1* Maria Caporali,2 Manuel Serrano-Ruiz,2 Alessandra Soriente,1 Maurizio Peruzzini,2 Luigi Ambrosio1 1Institute

of Polymers, Composites and Biomaterials – National Research Council (IPCB-CNR) Mostra

d’Oltremare pad.20 - Viale J.F. Kennedy 54, 80125 Naples, Italy 2Institute

of Chemistry of Organometallic Compounds – National Research Council (ICCOM-CNR), via

Madonna del Piano 10, 50019 Sesto Fiorentino, Italy

*These

authors equally contributed to the work.

#Corrisponding Author: Dr. Maria Grazia Raucci e-mail: [email protected] phone: +39 081 2425945 fax: +39 081 2425932

Keywords: few-layer black phosphorus, bone cancer treatment, bone regeneration, biomaterials, bone cancer related inflammation, tissue regeneration.

1 ACS Paragon Plus Environment

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Abstract Nowadays chemotherapy is the main treatment for osteosarcoma disease, even if limited by the lack of selectivity between healthy and cancer cells during the inhibition of cell division. Herein, we propose the use of few-layer black phosphorous (2D bP) as an alternative tool for osteosarcoma treatment and report how 2D bP can stimulate newly forming bone tissue generation after osteosarcoma resection. In our study, we have developed an in vitro model to evaluate the efficacy of 2D bP material with and without near-infrared light irradiation treatment and we have demonstrated that the presence of 2D bP without treatment inhibits the metabolic activity of osteosarcoma cells (SAOS-2), while inducing both the proliferation and the osteogenic differentiation of human pre-osteoblast cells (HOb) and mesenchymal stem (hMSC) cells. Furthermore, we also propose an in vitro co-culture model (SAOS-2 and HOb cell lines) in order to study the effect of 2D bP on inflammatory response related to cancer. On this co-culture model, 2D bP may increase antinflammatory cytokines generation (i.e. interleukin10) and inhibit proinflammatory mediators synthesis (i.e. interleukin-6), thus suggesting the opportunity to prevent cancer-related inflammation. Finally, we have demonstrated that 2D bP represents a promising candidate for future regenerative medicine and anticancer applications.


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1. Introduction Currently, osteosarcoma is the most common bone cancer which mainly affects young people.1 Surgical resection of tumor followed by chemotherapy for micro-metastasis inhibition constitutes the current standard procedure to cure 65% of osteosarcoma cases. However, osteosarcoma often is refractory to standardized chemotherapy regime. Chemotherapy, on the other hand, uses chemotherapeutic drugs with the effect of blocking cell proliferation, without any distinction between healthy and cancer cells.2 In fact, systemic chemotherapy may also cause toxicity to healthy cells that are not affected by cancer. Thus, many efforts have been undertaken to identify novel strategies for the osteosarcoma treatment. Besides the new pharmacological findings of novel drugs such as Sorafenib (Nexavar®)3, current approaches are aimed at investigating the use of nanomedicines for osteosarcoma therapy. Several studies are based on the opportunity to release chemotherapeutic drugs in local site creating the potential to improve both the safety and efficacy of cancer chemotherapy.1 In particular, the introduction of a suitable material shows the advantage to improve the benefit of surgery by minimizing the systemic toxicity that is usually associated with standard pharmacological treatments. The possibility to promote the anticancer drug release by using external stimuli4 such as magnetic5, electrical stimuli6 or by using a hyperthermia treatment is well known in literature.7, 8 However, local release of an anticancer drug doesn’t overcome the drawback related to the distinction of cancer from healthy cells. In recent years, several studies have been focused on the use of Photodynamic Therapy (PDT) as a minimally invasive therapeutic procedure that can apply a selective cytotoxic activity toward cancer cells.9,10 On this respect, a recently discovered 2D material, named phosphorene (the monolayer of bP), being the P-counterpart of graphene and obtained by exfoliation of crystals of the black allotrope of the element11, has attracted the attention of scientists working in the biomedical field intrigued by its very low (or absent) toxicity towards various cells.12 Although, the 2D nanomaterial usually used in the chemical, physical and biomedical applications, is the few-layer black phosphorus (2D bP). Recent studies have shown the effectiveness of 2D bP as photodynamic therapy agent for cancer treatment.13 This activity has been ascribed to the capability of bP to generate singlet oxygen14 and to act as photosensitizer that, in presence of reactive oxygen species (ROS) and infrared light irradiation, constitutes an essential component of PDT. This photodynamic activity makes 2D bP a promising tool for the design of multifunctional nanomaterials endowed with therapeutic and diagnostic functions. In addition, phosphorus (P) is an essential element involved in the development of vertebrate bones and as known phosphorus deficiency in the



diet causes a reduction in the mineral content of bone. Conversely, an excess of this element in the diet induces matrix deposition and an accelerated mineralization. Furthermore, P-deficiency increases the number of osteoclasts, thus causing bone resorption.15 In the present study, we propose the in vitro use of thermo-irradiated (Near-IR light) and not irradiated 2D bP as a strategy to inhibit the progression of osteosarcoma. At the same time, we have investigated the osteogenic effect of these phosphorus nanosheets as in vitro model of new forming bone tissue. Starting from the anti-tumoral effect of thermo-irradiated (Near-IR light) and not irradiated 2D bP on osteosarcoma, the relation between inflammation and cancer development has been also analyzed. According to current epidemiological investigations, chronic inflammation increases the risk of developing various cancer types.16 Among the triggers of chronic inflammation, there are inflammatory cells and inflammatory mediators such as chemokines, cytokines and prostaglandins. Such inflammatory mediators are produced during microbial infections, autoimmune and inflammatory diseases (i.e. inflammatory bowel disease associated with colon cancer) that constitute the origin of several tumors.17 Advances in understanding molecular pathways involved in cancer-inflammation processes, have identified some key molecules responsible for cancer-related inflammation. These include transcription factors (such as NF-κB and signal transducer and activator of transcription 3, i.e. STAT3) and inflammatory cytokines (IL-1β, IL-6, IL-23 and TNF-ɑ).18 The NF-κB plays a pivotal role in the development and progression of inflammation by activating the Toll-like receptor (TLR)-MyD88, TNF-α and IL-1β signaling pathways. Moreover, NF-κB promotes the expression of genes which codify enzymes involved in the prostaglandin-synthesis pathway (such as COX2), inducible nitric oxide synthase (iNOS) and angiogenic factors.16 We also have investigated how cancer-related inflammation can be inhibited in the presence of 2D bP. For this purpose, inflammatory marker expression was tested on in vitro co-culture models made of osteosarcoma cells (SAOS-2) and healthy osteoblast cells (HOb) stimulated by lipopolysaccharide (LPS).


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2. Results and Discussion 2.1 Anticancer and osteoinductive properties of 2D bP The cytotoxicity of black phosphorus has attracted recently a remarkable interest in cancer therapy due to its ability to destroy human lung carcinoma epithelial cells (A549) without killing healthy cells.12 Thus 2D bP may be used in the fabrication of devices for biomedical applications also because of its natural in vivo biodegradability.19 In fact bP is readily biodegradable inside the human body and produces nontoxic intermediates, such as phosphate, resulting a safe material for in vivo applications.20 However, the main goal in cancer therapy is to inhibit the progression of tumor by preserving healthy cells. Red phosphorus, which is another allotrope of phosphorus, largely used as functional material in semiconductors, batteries and recently as photocatalyst for bacterial inactivation, was also found to be non-toxic.21 In the present study, we verified and compared also the cytotoxic effect of bulk red and black phosphorous (at different concentration, in the range from 10 to 500 µg/ml) by using murine fibroblast cell line (L929) and human mesenchymal stem cell (hMSC) (1x104 cells/well) (Figures S3-S4). The results demonstrated that bulk red and black phosphorus don’t show any negative effect on murine fibroblast cells proliferation. Meanwhile a different behaviour was observed for hMSC; in fact, a reduction in cell proliferation was obtained for bulk red phosphorous starting from concentration 50 µg/mL to 500 µg/mL after 48 hrs of incubation. By contrast, bulk black phosphorous increased hMSC cell proliferation by increasing its concentration from 50 to 500 µg/mL after 48 hrs of exposure. Furthermore, the few-layer black phosphorous (2D bP) did not show cytotoxicity on human mesenchymal stem cells (Figure S5). Taking into account no negative action of 2D bP, we have investigated the effect of 2D bP on in vitro model of osteosarcoma using SAOS-2 cancer cells. In an osteosarcoma microenvironment, cancer cells are surrounded by healthy cells. For this reason, the most important challenge in osteosarcoma treatment is to specifically reduce cancer cells while avoiding the damage of surrounding healthy bone tissue. On this basis, we have evaluated the effect of 2D bP on in vitro proliferation of SAOS-2 cells compared to the same effect on healthy osteoblast cell line. SAOS-2 cell line was chosen because induces higher hydroxyapatite production levels22 than other cell lines such as MG-6323. Besides, the effect of 2D bP on ALP expression (an early marker of osteogenesis) was also studied. The results showed that 2D bP as substrate significantly increases HOb cell proliferation over culture time compared to control consisting in cells seeded on a polystyrene plate surface (Figure 1A). In addition, 2D bP induced a higher ALP



expression than control; in particular a plateau effect on ALP expression at day 14 of cell culture was observed (Figure 1B).

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Figure 1. Alamar blue (A) and alkaline phosphatase activity (B) results after 3, 7, 14 and 21 days of healthy HOb cell cultures.

Different behaviour of 2D bP as substrate was observed for osteosarcoma cells (SAOS-2), since in the presence of 2D bP, the cell proliferation significantly decreased over culture time compared to control (Fig. 2A). Moreover, we have found that the presence of 2D bP was able to inhibit the ALP expression in SAOS2 cells (Figure 2B), thus indicating the ability of 2D bP as substrate to block the metabolic activity of osteosarcoma derived cancer cells.

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SAOS-2

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Figure 2. Alamar blue (A) and alkaline phosphatase activity (B) results after 3, 7, 14 and 21 days of osteosarcoma SAOS-2 cell cultures.


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Tumor ablation is the first approach for the osteosarcoma treatment; in this context our results suggest that 2D bP not only contributes to inhibit cancer progression but also induces new bone tissue formation due to its oxidation to phosphate anions that, combined with calcium cations, represent hydroxyapatite (the major component of bone) precursors.24 Thus, after cancer resection, 2D bP exerts antiproliferative effects on cancer cells and simultaneously promotes induction of newly forming bone tissue development by stimulating pre-osteoblast differentiation. On this basis, the photo-thermal activity of 2D bP was analyzed by comparing its effects on cell proliferation with and without excitation induced by two near-infrared irradiation cycles. In literature, there are very few studies about the use of 2D bP in photo-thermal therapy for breast cancer treatment.25, 26 To date exfoliated black phosphorus (bP), as a novel two-dimensional nano-material, has attracted great attention owing to its anticancer properties in cancer photo-thermal therapy (PTT). Indeed, bP is able to kill cancer cells inducing hyperthermia processes.27 Notably it is reported that after a near-infrared light irradiation of ultrathin bP nanosheets, singlet oxygen is generated that serves as effective photodynamic therapy (PDT) in cancer treatment.27 Hence in this study, we have also analyzed the influence of 2D bP after near-infrared irradiation (NIR). Our investigations suggested that NIR had no effect on HOb proliferation because 2D bP without NIR stimulation promoted the HOb viability, thus inducing higher proliferation values than control (Figure 3A). Conversely, 2D bP with and without NIR, induced a significant reduction in SAOS-2 proliferation (Figure 3B).



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24 hrs (SAOS-2)

Figure 3. Alamar blue results after 2 cycles of infrared treatment on HOb (A) and SAOS-2 (B). Our results on cell proliferation pointed out that 2D bP can inhibit cell proliferation on in vitro model of osteosarcoma, even without photo-thermal treatment. Moreover, the data obtained on HOb cells suggested that this substrate might also exert a protective effect on healthy precursors of bone mature cells. This intriguing result pushed us to investigate the mechanism of action of 2D bP. In this respect, the use of 2D bP in photodynamic therapy for cancer treatment has been reported6 because of its ability to induce a high production of singlet oxygen species that kill cancer cells. The different behaviour of 2D bP on reactive oxygen species production when used as cell substrate, confirmed our previous results on cell proliferation. Indeed, the measurement of ROS production after 24 hours due to the stimulation caused by the Fenton reagent, indicated that on healthy HOb the presence of 2D bP without irradiation reduced the ROS production, suggesting its potential antioxidant properties (Figure 4A). In contrast, near-infrared stimulation had not significant effects on ROS generation (Figure 4B). In the case of osteosarcoma-derived cells (SAOS-2), the presence of 2D bP doubled the ROS production compared to the reference experiment and the ROS generation increased further in combination with NIR (Figures 4C-D). The overdose of ROS in SAOS-2 induced by 2D bP with and without NIR irradiation has activated different pathways involved in cell death.


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24 hrs (SAOS-2)

Figure 4. Reactive Oxygen Species (ROS) production from HOb (A-B) and SAOS-2 (C-D) cells with and without infrared stimulation. Here, cell viability assessment in presence of 2D bP was also performed using SEM and immunofluorescence analysis. The latter, after 7 days of cell culture on 2D bP substrate, showed a good morphology for HOb (Figures 5A, 5B). Conversely, after the same time, SAOS-2 appeared to lose their characteristic morphology as suffering cells in the presence of 2D bP (Figures 5C, 5D).



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Figure 5: Confocal images of HOb (A, B) and SAOS-2 cells on 2D bP substrates (C, D) obtained using immunofluorescence analysis after 7 days of culture. The cytotoxic effects on SAOS-2 were confirmed by SEM images, where the initial characteristic morphology of the cells disappeared and was replaced by a typical shape of apoptotic cells (Figures 6D-F). By contrast, SEM images of HOb (Figures 6A-C) showed a good cell spreading and the polygonal morphology of differentiating cells.

Figure 6. SEM images after seven days of cell culture by using HOb (A-C) and SAOS-2 (D-F) cells on 2D bP substrate.


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Conversely, 2D bP possesses a good biocompatibility in terms of survival of healthy cell lines, thus representing a promising candidate for future regenerative medicine applications. Indeed, our previous studies demonstrated that bulk bP in dimethyl sulfoxide (DMSO) solution at different concentrations did not exert cytotoxic effects also on L929 fibroblasts (Figure S3) and hMSCs (Figure S4) after 24 and 48 hours. A comparison was carried out using red phosphorus in DMSO solution that reduces hMSC viability at higher concentrations (Figure S4). In this study, the preliminary effect of exfoliated 2D bP on human mesenchymal stem cells (see supporting information) was also investigated. The results demonstrated an opposite behaviour of cells seeded on its surface than control. Indeed, an increase of cell proliferation in the first three days followed by a decrease until day 14 was observed (Figure S5A). However, the decrease was due to an early expression of osteogenic marker with higher ALP values than control (Figure S5B). In this study, it was demonstrated the positive effect of 2D bP as substrate to stimulate the proliferation of healthy cells inducing as well osteogenic differentiation in human mesenchymal stem cells (hMSC). At the same time, 2D bP shows an opposite effect on cancer cells by reducing cell proliferation and cell maturation in mature osteoblasts. Thus, 2D bP as substrate for cell culture shows potential application not only for cancer treatment, but also as promoter for new bone tissue formation.

2.2 Effect of 2D bP on cancer related inflammation Cancer is closely related to chronic inflammation;16 the latter is characterized by a significant increase in pro-inflammatory cytokines production which creates a dynamic microenvironment favorable to cancer development and growth.16 Hence, we have evaluated the effect of 2D bP on inflammatory response through pro and anti-inflammatory cytokine investigations on co-culture models consisting of SAOS-2 and HOb. This experimental co-culture model better reproduces tumor microenvironment. On this co-culture LPS (1 μg/ml for 72 hours) stimulation caused a significant increase in IL-6 values (Figures 7A-B) but highest IL-6 values were observed for LPS without NIR stimulation (Figure 7B). IL-6 is a proinflammatory cytokine that promotes in vitro proliferation of SAOS-2.28 Here, the interaction between cells and 2D bP significantly decreased IL-6 levels induced by LPS after 3 days of stimulation as model of acute inflammation (Figures 7A, 7B). The reduction of IL-6 levels confirmed the previously described cytotoxic effect of 2D bP on SAOS-2 cells. The lower values of IL-6 in cells treated with LPS obtained with NIR stimulation is due to the antinflammatory properties of NIR



based therapy. Indeed, it has been demonstrated that the treatment with NIR laser reduces pain, inflammation and edema, promotes tissue wound healing and prevents tissue damage.29

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Cocultures

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Figure 7: Effect of irradiated and not irradiated phosphorene on interleukin-6 (IL-6) (A,B) levels in a co-culture in vitro model (SAOS-2 plus HOb) treated with lipopolysaccharide (LPS). Measurements were performed 3 days after LPS (1 µg/ml) stimulation. Results (expressed as picograms per ml of proteic extract) are mean ± SEM of 3-4 experiments. #p

Exfoliated Black Phosphorus Promotes in Vitro Bone Regeneration

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