Advanced Techniques in Biology & Medicine
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Antineoplastic Activities of Functionalized Gd@C82 Nanomaterials via Targeting Tumor Vasculature

Xue Li, Mingming Zhen and Chunru Wang^{* *}Correspondence: Chunru Wang, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 100190, China, Tel: 010-62652120, Email:

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Introduction

Tumor vasculature is the foundation of tumor development, growth and metastatic dissemination, as tumor blood vessels could provide a steady supply of nutrients and oxygen for the rapid propagation of tumor cells [1,2]. In the absence of adequate vasculature, tumors will undergo growth inhibition and cell death [3]. Therefore, tumor blood vessels could be a promising cancer therapeutic target. In recent years, there are a large number of investigational drugs associated with tumor vascular-targeting agents (VTAs) appeared, some could inhibit the formation of new vessels, called tumor angiogenesis inhibitors (TAIs); and some could occlude the pre-existing tumor blood vessels, called vascular disrupting agents (VDAs) [4-6]. These VTAs possess much more advantages than conventional cancer chemotherapy, such as low toxicity, inaccessibility to drug resistance and wide application for different tumors [7].

With the rapid development of nanotechnology, engineered nanoparticles, including carbon nanomaterials, are emerging as excellent platforms for biomedical application, including effective cancer therapy [8-10]. Fullerenes, a unique class of carbon allotropes, have attracted a deal of attention and been studied intensively since their discovery because of the superior physiochemical properties

derived from their unique structures and rich modification [11,12]. Gadofullerene as an endohedral metallofullere developed recently for biomedical applications after functionalized with various functional groups like hydroxy, carboxyl, amino acid and so on [13,14]. Specially, hydroxylated Gd@C₈₂ is the most common derivative of gadofullerene with superior water-solubility and biocompatibility. And it has inspired considerable investigations on tumor angiogenesis inhibition and tumor vascular disruption by Gd@C₈₂(OH)_n nanoparticles and (Gd@C₈₂)m(OH)_n nanocrystals, respectively [15]. The current review examines the highly effective antitumor activities of functionalized Gd@C₈₂ via inhibiting angiogenesis and disrupting tumor vasculature, as a promising tumor vascular targeting agent with high biocompatibility and biosafety.

Fullerenes And Metallofullerenes

Fullerene is a new kind of carbon allotrope with closed spherical or ellipsoidal cage structure, composed by many different numbers of carbon atoms [16]. The structures of fullerenes are featured by symmetric cages with either 5- or 6-membered rings on the surface [17]. Metallofullerene is formed when many metal atoms are put inside fullerenes [13]. Derived from the unique structures, fullerenes/metallofullerenes possess unique properties, such as the excellent stability, the large surface area, the unsaturated double bands on the surface and the special surface easy to be functionalized [18]. It has been reported that fullerenes could be modified by physical modification with polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), cyclodextrin and so on, as well as chemical reactions by functional groups [14,19]. Functionalized fullerenes have attracted wide attention in the field of biomedicine. Importantly, Gd@C₈₂ is a common metallofullerene, consisting of a core of gadolinium (Gd) atom and a closed outer sheath of 82 carbon atoms [13]. After modified by hydroxy, carboxyl, amino acid and so on, Gd@C₈₂ could be a promising anti-tumor vascular agent via inhibiting angiogenesis or disrupting pre-existing tumor blood vessels (Figure 1).

Figure 1: The different antineoplastic mechanisms of functionalized Gd@C.

Antineoplastic activities of Functionalized Gd@C₈₂ via inhibiting Angiogenesis

Considering the essential of neovasculature for tumor growth, survival and metastasis, angiogenesis inhibition is emerging as a potential antitumor strategy [20]. In terms of the process of angiogenesis, TAIs always work by inhibiting angiogenic stimulators, endothelial proliferation, proteolysis of extracellular matrix (ECM) and integrins [21].

In 2005, the functionalized Gd@C₈₂ nanoparticles, Gd@C₈₂(OH)₂₂ (f-NPs), were synthesized via a liquid-liquid reaction, which could prevent tumor growth in ₂₂ hepatocellular carcinoma [22]. The f-NPs, whose size is approximately 70 nm, consist of an innermost core of gadolinium (Gd) atom, an outer carbon cage, and 22 outermost hydroxyl groups (Figure 2a and 2b) [23]. And this kind of functionalized Gd@C₈₂ nanoparticles showed high efficacy of anti-angiogenesis. As shown in Figure 2c, f-NPs could inhibit tumor angiogenesis by the simultaneous downregulation of more than 10 angiogenic factors, resulting in significantly reduced microvessel density and insufficient tumor blood perfusion, and thus, yielding a marked tumor growth inhibition with a tumor inhibitory rate much higher than the common tumor chemotherapeutic drug paclitaxel [23]. Meanwhile, the normal vessels were not interfered by f-NPs treatment without detectable abnormalities such as disfigurements, gaps, or breaches [23]. What’s more, Gd@ C₈₂(OH)₂₂ could also inhibit tumor angiogenesis by impairing matrix metalloproteinase (MMPs) activities as activated MMPs (specifically, MMP-2 and MMP-9) are responsible for proteolysis of the ECM, which is a prerequisite for angiogenesis [24,25]. It was validated that Gd@C₈₂(OH)₂₂ could not only depress MMPs expressions but also inhibit their activities by indirectly interacting with MMPs by a special binding mode, and thus suppress tumor growth (Figure 2d) [26]. All these findings indicted that Gd@ C₈₂(OH)₂₂ nanoparticles showed highly efficient antineoplastic effects, resulting from the inhibition of angiogenic factors and the suppression of MMPs activities.

Figure 2: The antitumor effects of Gd@C82(OH)22 (f-NPs) via inhibiting tumor angiogenesis. (a) The synthesis and structure of f-NPs. (b) SEM image of f-NPs. (c) The downregulation of angiogenic factors and anti-angiogenesis effects of the f-NPs. (d) Inhibition of fullerene on MMP-2 and MMP-9 in tumor tissues and tumor growth [23,26].

Antineoplastic activities of Functionalized Gd@C₈₂ via disrupting Vasculature under radiofrequency

In contrast to normal vessels, tumor vasculatures possess peculiar pathophysiological features, including chaotic architecture, high vascular permeability, non-uniform surface markers and the lack of lymphatic drainage [27,28]. These characters, known as the enhanced permeability and retention (EPR) effects, provide us the opportunities to disrupt the unmatured tumor vasculature, and thus lead to tumor ischemic necrosis. Accordingly, some VDAs like CA4P, ZD6126 and DMXAA have been clinically applied, but they also show serious side-effects such as tumor pain and cardiotoxicity [29].

Figure 3: The antitumor effects of (Gd@C₈₂)m(OH)_n (GFNPs) via disrupting pre-existing tumor blood vessels. (a) The structure of GFNPs. (b) Cryo-TEM micrograph of GFNCs. The inset is the SAED pattern. (c) The tumor vascular disrupting mechanisms by RF-assisted GFNPs [26].

Importantly, a new kind of functionalized Gd@C₈₂ nanocrystals, (Gd@C₈₂)m(OH)_n (GFNCs), were synthesized via a novel solidliquid reaction. Different from the previous hydroxylated Gd@C₈₂ nanoparticles, GFNCs consist of a cluster of Gd@C₈₂ crystal and further modification by outer functional groups, and the diameter is about 140 nm (Figure 3a and 3b). What is more, GFNCs exhibit superior properties such as expansion by phase transition, size controllability and so on [14]. Based on these characterizations of GFNCs, a new anticancer technique was presented in 2015, which could disrupt abnormal tumor blood vessels under the assistance of radiofrequency (RF) [30]. This opens the gates to realize disruption of tumor vasculatures by functionalized gadofullerene. Under RF assistance, this nanocrystal shows excellent anticancer activities in different kind of tumors like H22, 4T1, HepG2 and so on [30,31]. After RF-assisted GFNCs treatment, the tumor was observed to induce a rapid and extensive necrosis, and finally serious inner collapses occurred in 24 h [30]. Calculated by hematoxylin-eosin (HE) staining of tumor tissues, the tumor inhibition rate was up to 88.7% [31]. The disruption of tumor blood vessels was investigated in detail. To real-time monitor tumor vascular disruption during the treatment by RF-assisted GFNCs, dorsal skin flap chamber (DSFC) model was developed, by which the vascular structure and blood flow could be observed visually [32]. Tumor blood vessels were broken and hemorrhaged 20 minutes after treatment, leading to remarkable collapse and fragmentation of the whole tumor vascular net. As time goes by, the areas of bleeding were continually increased, the shape of the vessels became unrecognizable from DSFC window, and last the vessels got fuzzy and invisible. However, normal vessels remained undamaged after treated by RF-assisted GFNCs [32].

Besides, dynamic contrast enhanced (DCE)-MRI, a “gold standard” method to evaluate functional characteristics of the vascular net, was performed. The blood perfusion and the K_trans values (the rate of transfer of the contrast agent from the blood to the interstitial space) of tumors were greatly and persistently reduced after treatment, indicating the considerable destruction of capillary function of the tumor [32]. Furthermore, it was reported that VE-cadherin, the specific transmembrane adhesion proteins in endothelial cells located at intercellular junctions of tumor blood vessels, was down-regulated by RF-assisted GFNCs, indicating the disruption of tumor vascular endothelium [31]. Overall, the mechanisms of tumor vascular disruption by RF-assisted GFNCs were investigated profoundly as shown in Figure 3c [30]. After iv injection, GFNCs would rapidly flow along the normal blood vessels and penetrate the leaks of tumor vascular endothelial cells. With the properties of size-expansion by a phase transition, GFNCs would undergo explosive volume expansion and the possible rotation when the RF is applied. And thus, the networks of endothelia cells could be destroyed and abscised from the basilemma.

With further research, a new β-alanine functionalized gadofullerene nanocrystals, (Gd@C₈₂)m(Ala)_n, were developed and applied to vascular disruption [33]. Compared with GFNCs, this new nanoparticle presented superior physicochemical properties, which could improve the antitumor effects as vascular disrupting agents. The technique based on RF-assisted functionalized gadofullerene as a new tumor vascular disrupting strategy brings a new dawn for cancer treatment.

Low Toxicity Of Functionalized Gd@C₈₂

Although the potential applications of functionalized Gd@C₈₂ nanomaterials have been exploited in antitumor therapy as vascular targeting agents, it should also not be neglected in considering their biocompatibility and toxicity for clinical application. Numerous experiments have been explored to evaluate the low cytotoxicity of functionalized Gd@C₈₂ nanomaterials either in vitro or in vivo, which may be derived from the water-soluble modifications on the surface of carbon cage [34]. Recently, studies revealed that functionalized Gd@C₈₂ nanomaterials showed no reduction of cell abilities, but promotion of cell proliferation, such as immune cells (B cells, T cells or macrophages) [35], hepatoma cells (HepG2) [31], human umbilical vein endothelial cells (HUVECs) [31], human epidermal keratinocytes alpha (HEKα) [36], human embryonic lung fibroblast cells (MRC-5) [37], A549 and brain capillary endothelial cells (rBCECs) [38] and so forth.

Similarly, the toxicities in vivo were also studied by many researchers. After uptake of Gd@C₈₂(OH)₂₂, tumor-bearing mice remained normal body weight compared with mice in saline groups, suggesting no detectable toxicity [39]. To evaluate the liver and kidney function, the common indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), alkaline phosphatase (ALP) and creatinine (Cr) were monitored and tended to levels in healthy mice, indicating no toxicity of functionalized Gd@C₈₂ nanomaterials [23,33]. Furthermore, the nanomaterials could be mainly delivered to liver, spleen, kidney and bone of mice; however, no abnormal pathological changes were observed in these organs and tissues [30]. Notably, functionalized Gd@C₈₂ nanomaterials could restore the damaged liver, kidney and bone barrow induced by radiation or chemotherapeutic agents [8,40]. In addition, studies using the nematode Caenorhabditis elegans (C. elegans) as a model revealed that Gd@C₈₂(OH)₂₂ nanoparticles exhibited no detectable toxicity on growth, reproductivity and lifespan of C. elegans [41]. All these studies demonstrated that functionalized Gd@C₈₂ nanomaterials exhibits high biosafety, thus laying the foundations for further biomedical application.

Conclusion

In short, functionalized Gd@C₈₂ nanomaterials exhibit highpotential in treating tumor effectively and rapidly via targeting tumor blood vessels. In particular, Gd@C₈₂(OH)_n nanoparticles could inhibit angiogenesis not only by down-regulating many angiogenic factors but also by suppressing the expressions and activities of MMPs. Differently, (Gd@C₈₂)m(OH)_n nanocrystals could disrupt the pre-existing tumor blood vessels with the help of RF, deriving from their size-expansion when passing through tumor vascular endothelial cells. And no disruption of normal blood vessels occurred after injecting Gd@C₈₂ nanomaterials. Importantly, no detectable toxicity is observed in vitro or in vivo. With high-efficacy and low toxicity, functionalized Gd@C₈₂ nanomaterials could be a promising antineoplastic agent in clinic.

Acknowledgements

This work is supported by the National Major Scientific Instruments and Equipments Development Project (ZDYZ2015-2).

References

1. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407(6801):249-257.
2. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature. 2005;438(7070):967-974.
3. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285(21):1182-1186.
4. Patterson DM, Rustin GJ. Vascular damaging agents. Clin Oncol (R Coll Radiol). 2017;19(6):443-456.
5. Siemann DW, Bibby MC, Dark GG, Dicker AP, Eskens FA, Horsman MR, et al. Differentiation and definition of vascular-targeted therapies. Clin Cancer Res. 2005;11(2 Pt 1):416-420.
6. Thorpe PE. Vascular targeting agents as cancer therapeutics. Clin Cancer Res. 2004;10(2):415-427.
7. Pilat MJ, Lorusso PM. Vascular disrupting agents. J Cell Biochem. 2006;99(4):1021-1039.
8. Lin Y. What's happened over the last five years with high-throughput protein crystallization screening? Expert Opin Drug Dis. 2018;13(8):691-695.
9. Yan L, Zhao F, Li S, Hu Z, Zhao Y. Low-toxic and safe nanomaterials by surface-chemical design, carbon nanotubes, fullerenes, metallofullerenes, and graphenes. Nanoscale. 2011;3(2):362-382.
10. Kunjachan S, Detappe A, Kumar R, Ireland T, Cameron L, Biancur DE, et al. Nanoparticle mediated tumor vascular disruption: a novel strategy in radiation therapy. Nano Lett. 2015;15(11):7488-7496.
11. Yadav J. Fullerene: properties, synthesis and application. Res Rev J Phys. 2017;6(3):1-6.
12. Zhou Y, Li J, Ma H, Zhen M, Guo J, Wang L, et al. Biocompatible [60]/[70] fullerenols:potent defense against oxidative injury induced by reduplicative chemotherapy. ACS Appl Mater Inter. 2017;9(41):35539-35547.
13. Popov AA, Yang S, Dunsch L. Endohedral fullerenes. Chem Rev. 2013;113(8):5989-6113.
14. Li J, Wang T, Feng Y, Zhang Y, Zhen M, Shu C, et al. A water-soluble gadolinium metallo- fullerenol:facile preparation, magnetic properties and magnetic resonance imaging application. Dalton T. 2016;45(21):8696-8699.
15. Li Y, Tian Y, Nie G. Antineoplastic activities of [email protected]82(OH)22 nanoparticles:tumor microenvironment regulation. Sci China Life Sci. 2012;55(10):884-890.
16. Krätschmer W, Lamb LD, Fostiropoulos K, Huffman DR. Solid C60: a new form of carbon. Nature. 1990;347:354-358.
17. Anilkumar P, Lu F, Cao L, Luo PG, Liu JH, Sahu S, et al. Fullerenes for applications in biology and medicine. Curr Med Chem. 2011;18(14):2045-2059.
18. Jensen AW, Wilson SR, Schuster DI. Biological applications of fullerenes. Bioorg Med Chem. 1996;4(6):767-779.
19. Andersson T, Nilsson K, Sundahl M, Westman G, Wennerström O. C60 embedded in ?-cyclodextrin: a water-soluble fullerene. J Chem Soc Chem Commun. 1992;8:604-606.
20. Viallard C, Larrivée B. Tumor angiogenesis and vascular normalization: alternative therapeutic targets. Angiogenesis. 2017;20(4):409-426.
21. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med. 1995;333:1757-1763.
22. Chen C, Xing G, Wang J, Zhao Y, Li B, Jia G, et al. Multihydroxylated [[email protected]82(OH)22]n nanoparticles: antineoplastic activity of high efficiency and low toxicity. Nano Lett. 2005;5(10):2050-2057.
23. Meng H, Xing G, Sun B, Zhao F, Lei H, Li W, et al. Potent angiogenesis inhibition by the particulate form of fullerene derivatives. ACS Nano. 2010;4(5):2773-2783.
24. Kang SG, Araya-Secchi R, Wang D, Wang B, Huynh T, Zhou R. Dual inhibitory pathways of metallofullerenol [email protected]82(OH)22 on matrix metalloproteinase-2:molecular insight into drug-like nanomedicine. Sci Rep. 2014;4:4775-4782.
25. Chen SH, Kang SG, Luo J, Zhou R. Charging nanoparticles:increased binding of [email protected]82(OH)22 derivatives to human MMP-9. Nanoscale. 2018;10(12):5667-5677.
26. Kang SG, Zhou G, Yang P, Liu Y, Sun BY, Huynh T, et al. Molecular mechanism of pancreatic tumor metastasis inhibition by [email protected]82(OH)22 and its implication for de novo design of nanomedicine. Proc Natl Acad Sci USA. 2012;109:15431-15436.
27. Danhier F, Feron O, Preat V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release. 2010;148(2):135-146.
28. Danquah MK, Zhang XA, Mahato RI. Extravasation of polymeric nanomedicines across tumor vasculature. Adv Drug Deliv Rev. 2011;63(8):623-639.
29. Cooney MM, van Heeckeren W, Bhakta S, Ortiz J, Remick SC. Drug insight:vascular disrupting agents and angiogenesis—novel approaches for drug delivery. Nat Clin Pract Oncol. 2006;3(12):682-692.
30. Zhen M, Shu C, Li J, Zhang G, Wang T, Liu Y, et al. A highly efficient and tumor vascular-targeting therapeutic technique with size-expansible gadofullerene nanocrystals. Sci China Mater. 2015;58(10):799-810.
31. Li X, Zhen M, Deng R, Yu T, Li J, Zhang Y, et al. RF-assisted gadofullerene nanoparticles induces rapid tumor vascular disruption by down-expression of tumor vascular endothelial cadherin. Biomaterials. 2018;163:142-153.
32. Deng R, Wang Y, Zhen M, Li X, Zou T, Li J, et al. Real-time monitoring of tumor vascular disruption induced by radiofrequency assisted gadofullerene. Sci China Mater. 2018(8);61:1101-1111.
33. Zhou Y, Deng R, Zhen M, Li J, Guan M, Jia W, et al. Amino acid functionalized gadofullerene nanoparticles with superior antitumor activity via destruction of tumor vasculature in vivo. Biomaterials. 2017;133:107-118.
34. Meng J, Liang X, Chen X, Zhao Y. Biological characterizations of [[email protected]82(OH)22]n nanoparticles as fullerene derivatives for cancer therapy. Integr Biol. 2013;5(1):43-47.
35. Liu Y, Jiao F, Qiu Y, Li W, Lao F, Zhou G, et al. The effect of [email protected]82(OH)22 nanoparticles on the release of Th1/Th2 cytokines and induction of TNF-alpha mediated cellular immunity. Biomaterials. 2009;30(23-24):3934-3945.
36. Li J, Guan M, Wang T, Zhen M, Zhao F, Shu C, et al. [email protected]82-(ethylenediamine)8 Nanoparticle: A New High-Efficiency Water-Soluble ROS Scavenger. ACS Appl Mater Interfaces. 2016;8(39):25770-25776.
37. Zhou Y, Zhen M, Ma H, Li J, Shu C, Wang C. Inhalable gadofullerenol/[70] fullerenol as high-efficiency ROS scavengers for pulmonary fibrosis therapy. Nanomed Nanotechnol Biol Med. 2018;14(4):1361-1369.
38. Yin J, Lao F, Fu P, Wamer WG, Zhao Y, Wang PC, et al. The scavenging of reactive oxygen species and the potential for cell protection by functionalized fullerene materials. Biomaterials. 2009;30(4):611-621.
39. Pan Y, Wang L, Kang SG, Lu Y, Yang Z, Huynh T, et al. Gd-metallofullerenol nanomaterial suppresses pancreatic cancer metastasis by inhibiting the interaction of histone deacetylase 1 and metastasis-associated protein 1. ACS Nano. 2015;9(7):6826-6836.
40. Zhang Y, Shu C, Zhen M, Li J, Yu T, Jia W, et al. A novel bone marrow targeted gadofullerene agent protect against oxidative injury in chemotherapy. Sci China Mater. 2017;60(9):866-880.
41. Zhang W, Sun B, Zhang L, Zhao B, Nie G, Zhao Y. Biosafety assessment of [email protected]82(OH)22 nanoparticles on Caenorhabditis elegans. Nanoscale. 2011;3(6):2636-2641.