Elsevier

Biomaterials

Volume 35, Issue 2, January 2014, Pages 856-865
Biomaterials

The genotype-dependent influence of functionalized multiwalled carbon nanotubes on fetal development

https://doi.org/10.1016/j.biomaterials.2013.10.027Get rights and content

Abstract

In many cases cancer is caused by gene deficiency that is being passed along from generation to generation. Soluble carbon nanotubes (CNTs) have shown promising applications in the diagnosis and therapy of cancer, however, the potential relationship between cancer-prone individuals and response to CNT exposure as a prerequisite for development of personalized nanomedicine, is still poorly understood. Here we report that intravenous injections of multi-walled carbon nanotubes into p53 (a well-known cancer-susceptible gene) heterozygous pregnant mice can induce p53- dependent responses in fetal development. Larger sized multi-walled carbon nanotubes moved across the blood-placenta barrier (BPB), restricted the development of fetuses, and induced brain deformity, whereas single-walled and smaller sized multi-walled carbon nanotubes showed no or less fetotoxicity. A molecular mechanism study found that multi-walled carbon nanotubes directly triggered p53-dependent apoptosis and cell cycle arrest in response to DNA damage. Based on the molecular mechanism, we also incorporated N-acetylcysteine (NAC), an FDA approved antioxidant, to prevent CNTs induced nuclear DNA damage and reduce brain development abnormalities. Our findings suggest that CNTs might have genetic background-dependent toxic effect on the normal development of the embryo, and provide new insights into protection against nanoparticle-induced toxicity in potential clinical applications.

Introduction

Carbon nanotubes (CNTs) have attracted increased attention since their discovery because of their unique physical and chemical properties [1]. However, information concerning the potential health hazards of CNTs remains inadequately explored. Previous reports have shown that, under certain inhalation exposures, CNTs either activate cyclooxygenase enzymes in the spleen to suppress systemic immune function [2] or induce subpleural fibrosis [3]. Although inhalation is the most relevant method for determining the toxicity of CNTs, more researchers pay attention to the acute and chronic toxicity of water-soluble, functionalized CNTs when the CNTs enter the bloodstream. This is so for a number of reasons: (i) poor controllability of inhalation exposure; (ii) less cytotoxicity of water-soluble CNTs than non-functionalized particles [4]; and more importantly, (iii) functionalized CNTs have shown exciting bioapplications. For example, engineered carbon nanotubes (CNTs) have been extensively utilized as cancer theranostics [1], serving as composite nanomaterials for cancer imaging [5], drug loading [6], [7], and photothermal therapy [8], [9].

All cancers arise as a result of alternations that have occurred in the DNA sequence of the genomes of cancer cells [10]. The tumor suppressor p53 gene acts as a major defense against cancer; and it is well-established that over half of all human cancers bear a p53 gene mutation [11]. Information concerning the potential hazards in p53-deficient individuals caused by CNTs exposure is still unclear, despite demonstrations in previous reports that p53-dependent responses occur with other exogenous factors such as radiation [12], [13], [14] and environmental pollutant [15]. The p53 heterozygous (p53+/−) mouse model is a cancer-prone model, and was originally developed to facilitate the understanding of the role of p53 in protecting cells from exogenous factors induced DNA damage [16]. In addition, embryonic development is extremely sensitive to chemical toxins in water, food and drug formulations [17]. Here, we have chosen fetuses with different p53 genotypes as a model system of cancer-susceptible gene, derived from p53 heterozygous (p53+/−) mothers that were injected with surface-modified CNTs during pregnancy, to evaluate potential differences in fetotoxic effects of the nanotubes on fetal development and growth. To investigate the biodistribution and fetotoxicity of particles, short single-walled CNTs (SWCNTs) and short multiwalled CNTs (MWCNTs) of similar surface potential and length--but with different outer diameters of <8 nm (MWCNT-8), 20–30 nm (MWCNT-20) and ∼50 nm (MWCNT-50), respectively—were functionalized with PL-PEG-NH2 by following a previously reported procedure [18], then administered to the pregnant mice.

Section snippets

Amine-functionalized single-walled and multi-walled carbon nanotubes (CNTs)

Amine-functionalized CNTs were prepared by reacting CNTs with PL-PEG-NH2 (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], Avanti Polar Lipids) as previously reported [5]. Briefly, 20 mg CNTs (Cheap tube Inc, VT) was added to 3-fold excess of PL-PEG-NH2 in 5 ml distilled water. Then, the solution was dispersed for 1 h via probe sonication using a VCX-750 ultrasonic processor (Sonics & Materials, Newtown, CT). The probe was driven at 40% of the instrument's

Effect of functionalized CNTs on fetal development and survival

The CNT particles dispersed well in aqueous solutions (Fig. 1). The properties and purities of the particles are characterized in Table 1 and S1. To elucidate the fetotoxicity of SWCNTs and MWCNTs on fetuses of differing p53 genetic backgrounds, p53+/− pregnant mice were prepared by mating p53+/− male and p53+/− female mice prior to injection of particles (Scheme S1). A repeated dose was firstly used to explore whether SWCNTs and MWCNTs affect fetal development during the organogenesis period

Discussion

The role of diameter in the toxicity of CNTs has been considered in previous studies. Wang et al. reported MWCNTs with smaller diameter showed less toxicity than the larger ones toward alveolar macrophages [35]. Similarly, Yamashita et al. examined the biological effects of different sized MWCNTs and SWCNTs, and found a higher potency to induce DNA damage in A549 epithelial cells and inflammatory response in the lung of mice for long and thick MWCNTs than for short and thin ones while similar

Conclusion

This study demonstrates for the first time the link between fetotoxicity of functionalized CNTs and p53-related genetic background. Of the CNT materials studied, MWCNT-50 directly induced body weight changes in p53 wild type (p53+/+) fetuses and brain deformities in p53 knockout (p53−/−) fetuses, showing a p53 status-dependent manner of fetotoxicity (Fig. 7A). The mechanism of action appears to involve MWCNT-50 induced DNA damage of fetuses that triggered p53-dependent molecular function (Fig. 7

Acknowledgments

This work was supported by the Intramural Research Program, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health. We thank Dr. Henry S. Eden for proof-reading the manuscript and Ms. Myungsun Lee for drawing the scheme.

References (63)

  • Z. Liu et al.

    In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice

    Nat Nanotechnol

    (2007)
  • Z. Liu et al.

    Drug delivery with carbon nanotubes for in vivo cancer treatment

    Cancer Res

    (2008)
  • Z. Liu et al.

    Supramolecular stacking of doxorubicin on carbon nanotubes for in vivo cancer therapy

    Angew Chem Int Ed Engl

    (2009)
  • H.K. Moon et al.

    In vivo near-infrared mediated tumor destruction by photothermal effect of carbon nanotubes

    ACS Nano

    (2009)
  • A. Burke et al.

    Long-term survival following a single treatment of kidney tumors with multiwalled carbon nanotubes and near-infrared radiation

    Proc Natl Acad Sci U S A

    (2009)
  • M.R. Stratton et al.

    The cancer genome

    Nature

    (2009)
  • A.J. Levine et al.

    The p53 tumour suppressor gene

    Nature

    (1991)
  • T. Norimura et al.

    p53-dependent apoptosis suppresses radiation-induced teratogenesis

    Nat Med

    (1996)
  • S. Baatout et al.

    Developmental abnormalities induced by X-irradiation in p53 deficient mice

    In Vivo

    (2002)
  • Y. Kubota et al.

    Radiation-induced tissue abnormalities in fetal brain are related to apoptosis immediately after irradiation

    Int J Radiat Biol

    (2000)
  • C.J. Nicol et al.

    A teratologic suppressor role for p53 in benzo[a]pyrene-treated transgenic p53-deficient mice

    Nat Genet

    (1995)
  • D.R. Green et al.

    Cytoplasmic functions of the tumour suppressor p53

    Nature

    (2009)
  • K. Yamashita et al.

    Silica and titanium dioxide nanoparticles cause pregnancy complications in mice

    Nat Nanotechnol

    (2011)
  • Z. Liu et al.

    Preparation of carbon nanotube bioconjugates for biomedical applications

    Nature Protoc

    (2009)
  • S. Parrinello et al.

    Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts

    Nat Cell Biol

    (2003)
  • M.L. Dumble et al.

    Generation and characterization of p53 mutant mice

    Methods Mol Biol

    (2003)
  • H. Gao et al.

    PET of glucagon like peptide receptor upregulation after myocardial ischemia or reperfusion injury

    J Nucl Med

    (2012)
  • J.H. Santos et al.

    Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells

    Methods Mol Biol

    (2006)
  • K.G. Peters et al.

    Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse

    Development

    (1992)
  • V. Menezes et al.

    Nanoparticulate drug delivery in pregnancy: placental passage and fetal exposure

    Curr Pharm Biotechnol

    (2011)
  • X.P. Zhang et al.

    Cell fate decision mediated by p53 pulses

    Proc Natl Acad Sci U S A

    (2009)
  • Cited by (65)

    • Developmental toxicity of engineered nanomaterials

      2022, Reproductive and Developmental Toxicology
    • Breakthrough of ZrO<inf>2</inf> nanoparticles into fetal brains depends on developmental stage of maternal placental barrier and fetal blood-brain-barrier

      2021, Journal of Hazardous Materials
      Citation Excerpt :

      Instead, inconsistent reports are widely spread in literatures from rodent studies (Chu et al., 2010; Huang et al., 2014; Sadauskas et al., 2007). For example, nanoparticles have been shown to both cross placental barrier causing fetal developmental toxicity (Huang et al., 2014; Philbrook et al., 2011a; Yamashita et al., 2011) and not cross placental barrier inducing no toxicity (Sadauskas et al., 2007; Kulvietis et al., 2012). A possible reason is that most such studies focused on effects of consecutive exposures on embryotoxicity without considering the development of maternal placental barrier and fetal biological barriers in this process (Philbrook et al., 2011a; Hougaard et al., 2010).

    • Positron emission tomography and nanotechnology: A dynamic duo for cancer theranostics

      2017, Advanced Drug Delivery Reviews
      Citation Excerpt :

      The quantitative nature, limitless tissue penetration and sensitivity of PET can afford excellent means for long-term tracking of radiolabeled nanoparticles in living subjects. For instance, using 64Cu-DOTA labeled MWNTs, Huang et al. demonstrated that CNTs might induce genetic background-dependent toxic effects on the normal development of the embryo [310]. Larger-sized MWNTs could move across the blood–placenta barrier, restricted fetal development, and induced brain deformity, while SWNTs and smaller MWNTs showed reduced fetotoxicity.

    View all citing articles on Scopus
    View full text