Novel Gd-Loaded Silicon Nanohybrid: A Potential Epidermal Growth

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Novel Gd-Loaded Silicon Nanohybrid: A Potential EGFR Expressing Cancer Cell Targeting MRI Contrast Agent Sougata Sinha, Wing Yin Tong, Nathan H Williamson, Steven James Peter McInnes, Simon Puttick, Anna Cifuentes-Rius, Richa Bhardwaj, Sally E Plush, and Nicolas H. Voelcker ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14538 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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ACS Applied Materials & Interfaces

Novel Gd-Loaded Silicon Nanohybrid: A Potential EGFR Expressing Cancer Cell Targeting MRI Contrast Agent Sougata Sinha,† Wing Yin Tong,†,‡ Nathan H. Williamson,† Steven J. P. McInnes,† Simon Puttick,†,§,# Anna Cifuentes-Rius,†,‡ Richa Bhardwaj,† Sally E. Plushψ and Nicolas H. Voelcker*,†,‡,#,∥,⊥

†

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Future

Industries Institute, University of South Australia, South Australia, Australia ‡

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,

Monash University, 381 Royal Parade, Parkville, Victoria, Australia §

Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St

Lucia, Brisbane, Australia ψ

Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia,

Adelaide, SA 5000, Australia. #

Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, VIC,

Australia ∥

Melbourne Centre for Nanofabrication, Victorian Node of the Australian National

Fabrication Facility, Clayton, VIC, Australia ⊥ Monash

Institute of Medical Engineering, Monash University, Clayton, Victoria, Australia

1 ACS Paragon Plus Environment


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ABSTRACT: Continuing our research efforts in developing mesoporous silicon nanoparticle based biomaterials for cancer therapy, here we employed porous silicon nanoparticles as a nanocarrier to deliver contrast agents to diseased cells. Nanoconfinement of small molecule Gd-chelates (L1-Gd) enhanced the T1 contrast dramatically compared to distinct Gd-chelate (L1-Gd) by virtue of its slow tumbling rate, increased number of bound water molecules and their occupancy time. The newly synthesized Gd-chelate (L1-Gd) was covalently grafted on silicon nanostructures and conjugated to an antibody specific for epidermal growth factor receptor (EGFR) via a hydrazone linkage. The salient feature of this nanosized contrast agent is the capability of EGFR targeted delivery to cancer cells. Mesoporous silicon nanoparticles were chosen as the nanocarrier because of their high porosity, high surface area and excellent biodegradability. This type of nanosized contrast agent also performs well in high magnetic fields.

KEYWORDS: MRI, T1 contrast agents, Gd, porous silicon nanoparticles, EGFR


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INTRODUCTION Magnetic resonance imaging (MRI) is one of the most predominant diagnostic imaging modalities capable of producing high resolution images in a non-invasive manner.1-6 Furthermore, the acquisition can be tailored to produce many different contrast mechanisms which provides unparalleled diagnostic power for many diseases including cancer. The contrast in an MR image can be further tuned through addition of an exogenous contrast agent. MRI contrast agents are divided in two sub-classes, namely T1 and T2 contrast agents.5 Paramagnetic metal ions such as gadolinium (Gd3+) and manganese (Mn2+) are known to produce positive contrast in a T1 weighted image by decreasing the T1 of nearby protons thus enhancing the T1-weighted image signal intensity. Gd3+, having seven unpaired electrons, the highest number of any metal ion, possesses a very high magnetic moment (7.9 µB) which makes it favourable as a contrast agent over Mn2+.7 However, Gd3+ as a free metal ion is inherently toxic and thus should only be used in a chelated state. Linear and macrocyclic chelators such as diethylene-triamine-pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and dipyridoxyl-di-phosphate (DPDP) are known to produce thermodynamically and kinetically stable Gd-complexes that retain high T1 contrast enhancement.4,8 Most of the clinically approved contrast agents such as Magnevist®, Gadovist® and ProHance® are composed of one of these chelates, however, there are a number of drawbacks associated with these small molecule contrast agents.8 Firstly, the small molecule nature of these contrast agents leads to a high rotational correlation time (τc) which, results in a relatively low relaxivity. As a result, high concentrations of contrast agents are required to afford sufficient contrast. Secondly, the molecular design affords no specific disease targeting and contrast enhancement is largely limited to the vascular network and renal clearance pathways. As such, the primary use of small molecule MRI contrast agents is in diagnosing abnormal vasculature and often lacks specificity. According to the 3 ACS Paragon Plus Environment


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Solomon−Bloembergen−Morgan (SBM) theory, relaxivity largely depends on the rotational correlation time (τc) of the system, the number of bound water molecules and their occupancy time.9-11 These requirements can be satisfied by loading Gd-chelates on nanostructures either covalently or non-covalently.3,12-24 In the context of cancer diagnosis, another major advantage of a nanosized contrast agent is the possibility to exploit the enhanced permeability and retention (EPR) effect which allows passive accumulation of nanoparticles in tumor tissues across the leaky tumor vasculature. Furthermore, preferential accumulation, increase in local concentration and precise delivery of contrast agent within the diseased cells can be achieved using an active targeting approach by conjugating the nanoparticles to high affinity ligands such as antibodies, peptides or DNA-aptamers that recognise cognate receptors on the target cell surface.25 A further disadvantage of small molecule contrast agents that is linked to their high τc, is their decreasing efficiency at higher magnetic field strengths. In the drive for higher signal-tonoise ratios and higher spatial resolution, advances in MRI hardware have led to the development of high field (3 T) and ultra-high field (7 T) clinical scanners which have resulted in a decrease of efficiency of currently approved contrast agents.5,26 As such, there is an ongoing endeavour to develop highly efficient T1-weighted contrast agents which are fitfor-purpose for deployment at higher magnetic fields. Epidermal growth factor receptor (EGFR) is over-expressed in many types of cancer and plays an important role in oncogenesis.27,28 50 to 70% of non-small-cell lung cancer (NSCLC), colon and breast carcinomas express EGFR or ErbB-3.29,30 Since elevated EGFR expression in tumor biopsies as compared to normal tissues were identified in over 10 cancer types reported in various studies, EGFR was suggested to be a strong prognostic indicator.31 For example, over-expression of EGFR is a feature of glioblastoma (GBM) but not found in low grade gliomas.32 A certain proportion of GBMs (31%) over-express EGFR along with its


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mutant EGFRvIII,33 which plays a key role in gliomagenesis.34,35 However, EGFRvIII expression without elevated EGFR expression is rarely observed,36 indicating that EGFR over-expression on the GBM cells surface is a feature that could be utilized for GBM homing and targeted chemotherapy. Apart from the modulation of molecular pathways, antibodies have also been utilized for targeted delivery of chemotherapeutics to tumour tissue.37,38 Small particles decorated with antibodies for targeting achieve maximal uptake via both receptormediated and caveolae-mediated endocytosis, and thus they improve the delivery of payloads.39 For EGFR-targeted delivery in GBM, Cetuximab conjugated lipid based nanoparticles delivering doxorubicin are now in a Phase 1 clinical trial, highlighting the promise of antibody-assisted targeted delivery using nanoparticles in cancer treatments.40 Judicial selection of nanoparticles and their size is also crucial in developing MRI contrast agents. It is well known that the size of nanoparticles largely dictates the biodistribution. Small nanoparticles (~5 nm) are readily cleared through the renal route and thereby reduce circulation times,41 while large nanoparticles (>200 nm) are prone to accumulate in the spleen owing to the size of interendothelial cell slits.42 Even larger particles, at the micron scale, are likely to be captured within capillaries of lungs.43 Therefore, it is generally agreed that nanoparticles ranging from 100-200 nm in diameter possess a reasonable half-life in circulation, suitable for effective preferential accumulation and retention in the tumor tissues.44,45 By virtue of their remarkably high porosity, high surface area and excellent biodegradability, porous silicon nanoparticles (pSiNPs) have attracted the attention of the scientific community and have been extensively used as an implantable drug delivery system in both clinical and pre-clinical settings.46-53 pSiNPs offer many advantages as nanomedicines including their large surface-to-volume ratio and key structural features such as size, shape and control over degradation kinetics. Furthermore, simple modification is afforded by tuning the fabrication parameters to achieve the desired loading, accumulation and controlled



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release of various therapeutic agents at biological target sites.54 There have now been a number of reports detailing the development of MRI contrast agents utilizing porous silicon microparticles (pSiMPs) for geometric confinement of small molecule Gd-chelates.3,12,13 Ananta et al. have reported the strong positive impact of geometric confinement on longitudinal relaxivity (r1) of clinically approved low relaxivity contrast agents.3 Such a dramatic enhancement is an outcome of decreased water mobility and rotational motion of the Gd-chelate inside the nanopores. However, in this report, a non-specific adsorption strategy was used to load Gd-chelates into the mesoporous silicon structure and the stability of covalently grafted Gd-chelates system has since been shown to be favourable.13 The above mentioned reports on porous silicon mainly deal with nano-sized pores in silicon micro particles. However, to the best of our knowledge, utilization of Gd-loaded pSiNPs has not been explored for developing contrast agents. In line with our interest in developing pSi based smart materials for therapeutic applications, 48-53,55-58

in this report, we present our recent study in developing porous silicon

nanostructure-based T1-weighted MRI contrast agents for EGFR targeted delivery to cancer cells. We report the synthesis, characterization and potential ability of a Gd-loaded silicon nanohybrid to act as a cancer-cell-targeting MRI contrast agent. Our focus was to develop a small molecule linker (L1) that enables us to simultaneously attach an antibody and a Gd chelator to the nanoparticle surface (see Scheme 1 and Figure 1). The Gd-complex (L1-Gd) of L1 is composed of a Gd-chelate coupled to two equally spaced hydrazide groups via an aromatic linkage. L1-Gd was covalently grafted on the pSiNP surface by conventional 1ethyl-3-[3-dimethylaminopropyl]carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling followed by covalent conjugation of an EGFR specific antibody (see Figure 1) via a hydrazone linkage. Antibody conjugation allows L1-Gd-pSiNPs to target cancer cells which


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present EGFR on their cell surface, thereby increasing the efficacy of cancer specific imaging.

EGFR specific antibody

EGFR extracellular domain

Cancer cell membrane

Nucleus

Figure 1. Proposed structure and working principle of L1-Gd-pSiNPs-EGFR based cancer cell targeting T1-weighted MRI contrast agent.

EXPERIMENTAL Materials. Solvents and chemicals were purchased from either Sigma-Aldrich (Australia) or Merck

(Australia),

unless

otherwise

stated

and

used

as

received.

1,4,7,10-

tetraazacyclododecane was purchased from Strem Chemicals USA. HPLC grade solvents were used for synthesis. Hydrofluoric Acid (HF 48%) was purchased from Scharlau (Australia). Boron doped p-type silicon wafers (

Novel Gd-Loaded Silicon Nanohybrid: A Potential Epidermal Growth

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